Assisted Reproduction Techniques’ Effects on Sperm Physiology of the Freshwater Fish,
Sauger (Sander canadensis)
Dissertation
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Bryan Joseph Blawut
Graduate Program in Comparative and Veterinary Medicine
The Ohio State University
2020
Dissertation Committee
Marco Coutinho da Silva, Advisor
Barbara A. Wolfe
Stuart A. Ludsin
Christopher Premanandan
Gustavo M. Schuenemann
Copyrighted by
Bryan Joseph Blawut
2020
2
Abstract
Assisted reproduction techniques (ARTs) are used frequently during the production recreationally and economically important food and sportfish. These techniques, however, are often associated with poor fertilization which limits the technique’s efficacy at large-scales. A perfect example of this phenomenon is in the use of ARTs during the production of the recreational sportfish Saugeye (Sander vitreus × S. canadensis). A limited overlap between the breeding seasons of male Sauger (S. canadensis) and Walleye (S. vitreus) necessitates the use of testicular harvest and sperm storage techniques, sperm cryopreservation, to maximize reproductive potential of the limited number of available male Sauger broodstock. While these ARTs often result in spermatozoa demonstrating reasonable motility characteristics, they unfortunately have exceedingly low fertilization ability compared to fresh milt. The cause of this reduced fertilization potential is likely the result of sub-lethal damage and impaired sperm physiology, but traditional sperm quality assessment cannot confirm this notion. More specifically, our current understanding of Sauger sperm physiology is inadequate, limiting our ability to diagnose and correct the perturbations associated with testicular harvest and sperm cryopreservation. To address this knowledge gap, we sought to address four central questions: (1) Do Sauger sperm demonstrate seasonal and main sperm duct maturation?, (2) How does sperm physiology change as a result of motility activation?,
ii
(3) Does testicular harvest result in the collection of physiologically immature cells?, and
(4) What parameters are most impacted by the freeze-thaw process and can these markers be related to fertilization potential?
To answer these questions, we addressed sperm physiology traits that have known connections to fertilization potential in other taxa, but have been poorly described, if at all, in fish spermatozoa. First, we used a combination of spermatozoa and testis structural analysis to assess seasonal and intra-testis sperm maturation potential (Chapter 2).
Second, we assessed and compared several aspects of sperm physiology potentially relevant to fertilization (e.g., the glycocalyx; Chapter 3), the plasma membrane (Chapter
4), and intracellular signaling (Chapter 5) in testicular, stripped, and cryopreserved sperm to identify critically impacted structures.
Sauger sperm collected via strip-spawning were a dynamic cell that responded to the activation signal of hypo-osmotic shock. Motility activation was associated with a 2- fold increase in Ca2+ (Ch 5), an increase in threonine phosphorylation (Ch 5), N-acetyl- glucosamine (GlcNAc) redistribution to the apical ridge of ~ 50% of cells (Ch 3), and fast-nonlinear motility patterns (Ch 5). Additionally, lipid rafts identified and localized to the midpiece (Ch 4). From these variables, GlcNAc was found to be essential to efficient fertilization (80% reduction when blocked using lectins, Ch 3).
Testicular harvest resulted in the collection of immature and mature spermatozoa simultaneously. Sauger reproductive tracts displayed seasonal, as well as intra-ductal, maturation potential as evidenced by differences in ductal morphology (Ch 2). In support of this notion, we found a subpopulation of testicular sperm we believed to be less mature
iii as evidenced by lower glycoprotein content (Ch 3), altered motility characteristics and evidence of lipid raft coalescence (Ch 4). These differences were believed to be the result of lack of exposure to the main sperm duct (seminiferous tubules vs. main sperm duct).
Despite the differences seen between stripped and testicular sperm, fertilization potential was only slightly reduced. Thus, testicular harvest resulted in a sufficient number of competent, mature sperm to achieve practical levels of fertilization (Appendix A).
Cryopreservation resulted in spermatozoa that were unable to respond to the activation stimulus and displayed signs of early apoptosis. Sperm membranes demonstrated a lack of stability, dissociation of lipid rafts, and changes to the lipid composition associated with lipid peroxidation (Ch 4). The glycocalyx no longer exhibited GlcNAc redistribution (Ch 3). Instead, α-mannose content (an early marker of apoptosis) was significantly increased. And lastly, elevated and static Ca2+, reduced serine and tyrosine phosphorylation, as well as a lack of fast-nonlinear sperm were also reported (Ch 5). While all physiology characteristics were altered, the lack of GlcNAc redistribution was determined to be an impairment to fertilization ability (Ch 3).
Collectively, these results provided an understanding of Sauger reproductive biology, a detailed analysis of ART effects on sperm physiology, and several new biomarkers for further assessment of Sauger sperm quality. Additionally, we provide a
Sauger sperm best management practices step-by-step guide to collecting, assessing, and utilizing Sauger sperm (Appendix B). Our results can be used to inform further refinement of ARTs by identifying conditions to prevent apoptotic changes and low fertility. Application of these analysis metrics should help to strengthen our
iv understanding of cryobiology and large scale-applicability of cryopreservation and testicular harvest. Because testicular harvest and cryopreservation are widely utilized in aquaculture, we believe these results to be relevant to a majority of fish (specifically freshwater fish) where these techniques are used.
v
Dedication
For my wife, Angela.
vi
Acknowledgments
Frist, I would like to thank my wife, Angela, and my family for their support during my completion of the graduate program.
Thank you Dr. Marco Coutinho da Silva, at The Ohio State University College of
Veterinary Medicine, for being a great mentor and role model during my stay at the. I could not have made it through a single breeding season without your support. I would also like to thank all of my committee members, Barbara Wolfe, Chris Premanandan,
Gustavo Schuenneman, and Stuart Ludsin for their support.
We would not have been able to complete this project without the support of the
Ohio Department of Natural Resources-Division of Wildlife (ODNR-DOW). Thank you to Rich Zweifel, Kevin Kayle, Doug Sweet, Brian Kitchen, Mort Pugh and the hatchery staff for your technical support during broodstock collection, fertilization, and egg incubation. Your support and expertise were invaluable during this research project.
Financial support for this project was provided by the Federal Aid in Sport Fish
Restoration Program (F-69-P, Fish Management in Ohio, project FADR72), administered jointly by the U.S. Fish and Wildlife Service and the Ohio Department of Natural
Resources-Division of Wildlife (ODNR-DOW).
vii
Vita
October 1st, 1991 ...... Born, Wheeling, WV
May 5th, 2014 ...... B.S. Wildlife & Fisheries and Animal Nutritional Sciences, West Virginia University
August 2014 to 2017 ...... Graduate Research Assistant, Department of
Veterinary Preventive Medicine, The Ohio
State University
2017 to present …………………………….Graduate Research Assistant, Department of
Veterinary Clinical Science, The Ohio State
University
Publications
Blawut, B., Wolfe, B., Moraes, C.R., Premanandan, C., Ludsin, S.A., Schuenneman, G., & Coutinho da Silva, M.A.C. (In Review). Changes to The Spermatozoa Glycocalyx and Its Role in Fertilization in Sauger (Sander canadensis). Aquaculture.
Fiamengo, T. E., Runcan, E. E., Premanandan, C., Blawut, B., & Coutinho da Silva, M. A. (2020). Evaluation of biofilm production by Escherichia coli isolated from clinical cases of canine pyometra. Topics in Companion Animal Medicine, 100429. https://doi.org/10.1016/j.tcam.2020.100429
Blawut, B., Wolfe, B., Moraes, C. R., Ludsin, S. A., & Silva, M. A. C. da. (2020). Use of Hypertonic Media to Cryopreserve Sauger Spermatozoa. North American Journal of Aquaculture, 82(1), 84–91. https://doi.org/10.1002/naaq.10125
Blawut, B., Wolfe, B., Moraes, C. R., Sweet, D., Ludsin, S. A., & Coutinho da Silva, M. A. (2020). Testicular collections as a technique to increase milt availability in Sauger viii
(sander canadensis). Animal Reproduction Science, 212, 106240. https://doi.org/10.1016/j.anireprosci.2019.106240
Moraes, C. R., Runcan, E. E., Blawut, B., & Coutinho da Silva, M. A. (2019). Technical Note: The use of iSperm technology for on-farm measurement of equine sperm motility and concentration. Translational Animal Science, 3(4). https://doi.org/10.1093/tas/txz115
Blawut, B., Wolfe, B., Moraes, C. R., Ludsin, S. A., & Coutinho da Silva, M. A. (2018). Increasing Saugeye (S. vitreus × S. canadensis) production efficiency in a hatchery setting using assisted reproduction technologies. Aquaculture, 495, 21–26. https://doi.org/10.1016/j.aquaculture.2018.05.027
Field of Study
Major Field: Comparative and Veterinary Medicine
ix
Table of Contents
Abstract ...... ii Dedication ...... vi Acknowledgments...... vii Vita ...... viii Table of Contents ...... x List of Tables ...... xiii List of Figures ...... xv Chapter 1. Introduction ...... 1 Seasonal Reproductive Development ...... 6 Sperm Activation ...... 8 The Glycocalyx ...... 9 The Plasma Membrane ...... 11 Intracellular Signaling ...... 14 Assisted Reproduction Techniques ...... 16 Testicular Harvest ...... 17 Cryopreservation ...... 18 Literature Cited ...... 23 Chapter 2: Spatial and Temporal Changes in Testis Morphology and Sperm Ultrastructure of the Sportfish Sauger (Sander canadensis)...... 44 Abstract ...... 44 Introduction ...... 45 Materials and methods ...... 47 Results ...... 53 x
Discussion ...... 58 Conclusions ...... 63 References ...... 64 Chapter 3: Changes to The Spermatozoa Glycocalyx and Its Role in Fertilization in Sauger (Sander canadensis) ...... 78 Abstract ...... 78 Introduction ...... 80 Materials and Methods ...... 82 Results ...... 90 Discussion ...... 94 Conclusion ...... 99 References ...... 101 Chapter 4: Effect of Assisted Reproduction Techniques on Sauger (Sander canadensis) Spermatozoa Plasma Membrane Physiology ...... 111 Abstract ...... 111 Introduction ...... 112 Materials and Methods ...... 117 Results ...... 131 Discussion ...... 136 Conclusions ...... 143 References ...... 144 Chapter 5. Assisted Reproduction Techniques’ Effects on Spermatozoa Calcium Homeostasis, Protein Phosphorylation, and Motile Subpopulations ...... 166 Abstract ...... 166 Introduction ...... 167 Materials and Methods ...... 171 Results ...... 182 Discussion ...... 187 Conclusions ...... 192 References ...... 194 Chapter 6. Conclusion ...... 213 Sperm Physiology and Activation ...... 213 Testicular Harvest ...... 215
xi
Cryopreservation ...... 216 Broader impacts ...... 218 References ...... 220 Literature Cited ...... 221 Appendix A. Testicular collections as a technique to increase milt availability in Sauger (Sander canadensis) ...... 261 Abstract ...... 261 Introduction ...... 262 Materials and methods ...... 265 Results ...... 274 Discussion ...... 276 Conclusions ...... 280 References ...... 282 Appendix B. Sauger (Sander canadensis) Milt Best Management Practices for Saugeye (S. vitreus x S. canadensis) Production ...... 289 Section 1: Milt Collection Techniques ...... 289 Section 2: Sauger Sperm Quality Control ...... 305 Section 3. Calculating Sperm Fertilization Doses ...... 311 Section 4: Long-term Milt Storage via Cryopreservation ...... 313 References ...... 318
xii
List of Tables
Table 1. Summary of cellular assessment characteristics, tools used for those assessments, and the target variable measured utilized in this dissertation...... 43
Table 2. Comparison of sperm motility characteristics and cell concentration among stripped sperm and sperm harvested from different regions of the testis (proximal, middle, or distal to the urogenital pore) in the Sauger (Sander canadensis). Means with differing superscripts within columns denote a significant Tukey’s HSD post hoc test mean comparisons (p < 0.05)...... 70
Table 3. Comparison of the proportion of highly fluorescent sperm cells (%) and viability (%) among Sauger sperm types and activation statuses for both wheat germ agglutinin (WGA) and concanavalin A (ConA) lectin probes (n = 12, 12, 7). Means with common Tukey’s HSD post hoc test superscripts do not differ. Data are reported as mean ± 1 standard error...... 109
Table 4. Comparison of Sauger sperm motility parameters among sperm pool types (n = 12). Means (± 1 standard error) with contrasting Tukey’s HSD post hoc test superscripts within a column indicated statistically significant differences among means (p < 0.05)...... 110
Table 5. Quantitative assessment of membrane fluidity among sperm types and activation statuses using the fluorescent probe merocyanine 540 (MC540). MC540 fluorescence on the y-axis was measured using flow cytometry and reported as arbitrary units (a.u.). Data are represented as mean ± 1SEM. Superscripts denote Tukey’s HSD post hoc test mean compressions (α = 0.05)...... 157
Table 6. Comparison of relative abundances for lipid metabolites derived from PLS-DA analysis among sperm types in Sauger sperm (Sander canadensis). Data are recorded as the mean ± 1 standard error of the mean in log transformed data. Means within rows with contrasting Tukey’s HSD superscripts are significantly different from one another (p < 0.05)...... 163
Table 7. Motility parameters and viability of Sauger (Sander canadensis) sperm pools used for plasma membrane analysis (n = 7). Data are reported as mean ± 1 standard error of the mean (SEM)...... 164
xiii
Table 8. Motility characteristics derived by the Hamilton Thorne Computer Assisted Sperm Analysis system (n = 11,339, (Kanuga et al., 2012b; Keel & Webster, 1990; Martínez-Pastor et al., 2011b))...... 205
Table 9. Comparison of the proportion (%) of Sauger spermatozoa exhibiting high quantities of intracellular Ca2+ and mean fluorescent intensity (a.u.) of all cells among sperm type and activation status (n = 6). Data are reported as the mean ± 1 SEM. Tukey’s HSD superscripts denote significant differences among group means (p < 0.05). Means within a row with contrasting Tukey’s HSD superscripts are significantly different (p < 0.05)...... 206
Table 10. Comparison of the proportion (%) of Sauger spermatozoa exhibiting high quantities of protein phosphorylation among sperm type and activation status (n = 6). Data are reported as the mean ± 1 SEM. Tukey’s HSD superscripts denote significant differences among group means (p < 0.05). Superscripts a,b,c, within a row denote differences among sperm types (testicular, stripped, and cryopreserved) while x,y within rows denote statistically significant differences between activation statuses (inactive vs. activated)...... 207
Table 11. Mean +/- 1 SD of the 8 CASA derived motility parameters for each of the three sperm subpopulations delineated by two-step clustering analysis...... 210
Table 12. Comparison of values for Sauger milt production and quality variables (mean 1 SE) in sperm collected using the stripping (n = 30) and testicular collection-sperm extraction (n = 17) procedure; Data were pooled per collection method regardless of collection iteration or preceding stripping regimen for sperm collection...... 287
Table 13. Sperm quality parameters (total motility, progressive motility, velocity and viability) for both stripped and testicular sperm during a 4-d storage period at 5°C...... 327
Table 14. Sauger milt characteristics (mean ± 1 SE) before and after treatment with hCG or saline (hCG n=16, control=17). Gain scores (Δ) were calculated by subtracting the initial measurement from the final measurement. Gain scores within the same row with different superscripts (a, b) differ (p < 0.05), based on repeated measures ANOVA. ... 328
Table 15. Motility characteristics of Sauger (Sander canadensis) spermatozoa cryopreserved using 7.5% methanol in modified ringers lactate compared to an unfrozen control. Superscripts indicate Tukey’s HSD post hoc mean comparison groups (p < 0.05, n = 4)...... 329
Table 16. Equine 590b Densimeter convertsion chart for Sauger (Sander canadensis) milt concentration assessment...... 330
Table 17. Item list and vendor information for equipment used during reproductive assessment and management of Sauger (Sander canadensis)...... 332 xiv
List of Figures
Figure 1. Ohio Department of Natural Resources – Division of Wildlife Saugeye production (2006-2017) in terms of millions of fry and fingerlings stocked. Red bars indicate the number of fry and grey bars represent the number of fingerlings produced. 40
Figure 2. Sauger sperm cryopreservation results from 2014-2018. Total motility (black), progressive motility (red), velocity (grey), and viability (white) are all compared among the 4 treatment groups: a fresh control and sperm frozen using base extenders at 350 mOsm/kg, 500 mOsm/kg, and 750 mOsm/kg. Extender 350 and 500 resulted in sufficiently high TM, PM, VAP, and viability compared to control, while 750 mOsm.kg severely reduced all measures or quality (n = 10)...... 41
Figure 3. Sauger sperm cryopreservation fertilization results 2018. Fertilization in a control sample (fresh sperm at 300,000 MSE, white) was compared to sperm cryopreserved using 350 mOsm/kg (red) and 500 mOsm/kg (grey) base extenders at both 300,000 and 600,000 MSE. Fresh sperm had significantly higher fertilization compared to frozen sperm (p < 0.05). Extender 350 mOsm/kg had higher fertilization than 500 mOsm/kg at both MSE. Super scripts denote statistically significant means among the two cryopreserved sperm base extenders and MSE ratios (n = 5)...... 42
Figure 4. Anatomical location of the Sauger reproductive tract (A) and experimental design (B) of histological analysis of the testes...... 71
Figure 5. Histological micrograph of a mature male Sauger testis during the A) prebreeding season (40×), B) breeding season (40×), and C) testis of immature individuals (100 ×). The bolded arrow indicates the main sperm duct...... 72
Figure 6. Testis cellular comparison between seasons (pre-breeding and breeding) and among testis sections (proximal, middle, and distal to the urogenital pore). (A) Cumulative proportions of each cell type for each treatment represented graphically. (B) Representative micrographs of (from left to right) spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, mature spermatozoa, interstitial cells, and lumen)...... 73
Figure 7. Main sperm duct epithelium comparison among proximal, middle, and distal portions of the Sauger testis. (A) Epithelium thickness (height µm) among different sections and between seasons (white: pre-breeding, black: breeding). (B-C) Representative micrographs of epithelium lateral folds in the proximal duct (B) and
xv simple squamous epithelium seen more commonly in the distal duct (C) and between epithelial fold regions in other sections...... 75
Figure 8. Electron micrographs of Sauger spermatozoa ultrastructure. (A) TEM image of the spermatozoa head and initial portion of the flagellum. (B) SEM image of the exterior of the spermatozoa head. (C) TEM image of the flagellum, flagellar ribbon, and axonemal organization (9 × 2 + 2 microtubule organization). Ax, axoneme; Cc, cytoplasmic channel; Dc, distal centriole; F, flagellum; Fr, flagellar ribbon; Mi, mitochondria; Nn; nuclear notch; Nu, nucleus; Pc, proximal centriole...... 76
Figure 9. Electron micrographs of sperm cell abnormalities and somatic cell fragments in the collection of testicular sperm in Sauger. (A) Incomplete chromatin condensation (*) of the sperm cell nucleus and Sertoli cell fragments (black arrow). (B) Commonly observed profile of a red blood cell (white arrow) seen in all testicular sperm samples. . 77
Figure 10. Alexa Fluor 488 – conjugated lectin staining distributions observed in Sauger sperm using 1000 × oil immersion fluorescent microscopy. Fluorescent staining distributions were defined as (1) apical, (2) homogenous, and (3) heterogeneous. Differential interference contract (DIC, a) images and corresponding fluorescent (b) images are adjacent (original image 400 × magnification)...... 105
Figure 11. Wheat germ agglutinin (WGA) fluorescent staining distributions (A) and fluorescent intensity (B) of testicular, stripped, and cryopreserved Sauger sperm in both the inactive and active states. (A) Mean proportions for each fluorescent staining distribution in each sperm type and activation status are given. Patten codes are as follows: homogenous [white], heterogeneous [grey], or apical staining [black]. (B) WGA fluorescent intensity (in relative fluorescent units) is expressed at mean ± 1 standard error (n = 10). Means with a common Tukey’s HSD superscript do not differ...... 106
Figure 12. Representative image of western blot for N-acetyl-glucosamine (GlcNAc) moieties on glycoproteins extracted from the Sauger sperm plasma membrane. Sperm were either testicular (lanes 1-2), stripped (lanes 3-4), or cryopreserved (lanes 5-6) in origin and in either the inactive (lanes 1, 3, and 5) or activated state (lanes 2, 4, and 6). Sperm glycoproteins containing GlNAc residues were visualized using 0.6 µg/mL HRP conjugated wheat germ agglutinin (WGA). A molecular weight ladder is included (leftmost column)...... 107
Figure 13. Concanavalin A (ConA) fluorescent staining distributions (A) and fluorescent intensity (B) of testicular, stripped, and cryopreserved Sauger sperm each in both the inactive and active states. Mean proportions for each fluorescent staining distribution in each sperm type and activation status are reported. Patten codes are as follows: homogenous [white] or heterogeneous [grey]. (B) ConA fluorescent intensity (in relative fluorescent units) is expressed at mean ± 1 standard error (n = 12). Means with a common Tukey’s HSD superscript do not differ...... 108
xvi
Figure 14. Sauger sperm cholesterol and protein content in ultracentrifugation separated lipid raft fractions. Fractions range from 1 (top of tube) through 12 (bottom of tube). Cholesterol content is on left y-axis and bars are separated by sperm types [testicular (red), grey (stripped), and cryopreserved (white)]. Different superscripts (Tukey’s HSD, p < 0.05) denote differences among sperm types based on cholesterol content differences in fraction 4, the proposed lipid rafts (p < 0.05). Protein content is on the right y-axis and lines connect protein content mean values from the same sperm type types [testicular (▲), grey (•), and cryopreserved (■)]. No differences were observed among protein contents between different sperm types. Data are represented as mean ± 1 SEM for cholesterol, and mea values alone for protein (n = 4 ...... 158
Figure 15. Distribution of flotillin-2, a lipid raft marker, in sucrose gradient ultracentrifugation separated fractions using western blot. Each type of Sauger sperm (testicular, stripped, and cryopreserved) was assessed for the flotillin-2 raft marker and β- actin was used to assess the validity of the separation technique (β-actin is a cytoskeletal protein and should only be localized to the pellet) using flotilin-2 (sc-28320) and beta- actin (ab8227), signal was generated using West Dura Super Signal (enhance chemiluminescence substrate), and imaged using a digital Amersham Imager 680 (Cytiva, MA, USA)...... 159
Figure 16. Lipid raft localization in Sauger sperm using flotilin-2 immunofluorescence. (Left) Differential interference contrast microscopy image (× 1,000). (Middle) Hoechst DNA stain localizing the nucleus. (Right) Flotilin-2 distribution showing the location of lipid rafts...... 161
Figure 17. Partial Least Squares – Discriminant Analysis (Left) plot of individual data points per treatment group and associated variables of importance (VIP) plot (Right) of LC-MS data obtained from cryopreserved (Group C), stripped (Group S), and testicular (Group T) Sauger spermatozoa. The top 15 metabolite variables important to group discrimination names are listedn on the left axis of the VIP plot...... 162
Figure 18. Electron micrographs of cryopreserved Sauger spermatozoa. (A) Scanning electron micrograph of spermatozoa showing widespread cellular damage. (B-D) Transmission electron micrographs demonstrating disrupted plasma membranes (B), absent nuclear membranes (C), and cytoplasmic blebs along the flagellum (D)...... 165
Figure 19. Localization of phosphorylated tyrosine (A-C), threonine (C-E), and serine (F-G) residues in Sauger sperm using immunofluorescence techniques. DIC micrographs of the sperm at × 1,000 magnification (A, C, F). Localization of the nucleus using Hoechst 3342 (B, D). Indirect immunofluorescent localization of phosphorylated tyrosine (C.), threonine (E.), and serine (G.) residues...... 208
Figure 20. Qualitative assessment of phosphoprotein diversity [threonine (A.), tyrosine (B.), and serine (C.)] among Sauger sperm types and activation statuses. Lanes correspond to the following treatments: 1, testicular, inactive; 2, testicular activated; 3, xvii stripped inactive; 4, stripped activated; 5, cryopreserved, inactive; 6, cryopreserved activated. Protein bands are measured on the y-axis as molecular weight (kDa)...... 209
Figure 21. A) Hierarchical clustering dendrogram of Sauger sperm trajectories pre- clustered K-means groupings, cut into three classification trees (k = 3) and B) graphical representation of the three clusters on a scatterplot displayed on principal components axes to maximize the amount of variance explained. Points on the plot represent invidual sperm trajectories ( n = 24,993)...... 211
Figure 22. Proportion of each sperm subpopulation [SP1, fast -linear (white); SP2, fast- nonlinear (grey); and SP3, slow-linear (black)] in each of the three sperm subtypes (Testicular, Stripped, and Cryopreserved). Results of the generalized linear model indicate that the proportion of sperm in each category differs in each case among all three sperm types (p’s < 0.05, Tukey’s HSD post hoc test)...... 212
Figure 23. Comparison of milt collection regimens [Stripped Twice (n = 10), Stripped Once + Testicular Collection and Semen Extraction (Group 1, n = 10), and Stripped Twice + Testicular Collections and Semen Extraction (Group 2, n = 7)] in terms of sperm type [total number of sperm (grey), total number of motile sperm (black), and number of total motile, morphologically normal sperm (white)]; Tukey’s HSD superscripts denote differences among group means within a sperm type (P < 0.05) ...... 288
Figure 24. Design of extended Sauger milt transport boxes. (A.) Ice packs are layered for ~ 2/3 of the depth of the box with 2 hand towels/ surgical towels to prevent contact between milt and ice. (B.) Extended semen vials are placed in a water-proof bag and allowed to float in an ice water slurry during transport. Cardboard or plastic is used to create a sloped surface allowing an ice-free pool of water for extended milt to rest in/on...... 319
Figure 25. Testicular harvest and sperm extraction from Sauger (Sander canadensis). (A) Sauger broodstock with the testis removed from the body cavity but still attached at the urogenital pore. (B) Sauger testis has been removed and transferred to a petri dish, lacerated along the lobe long-axis, and extender added to homogenize sperm. (C) End product of testicular harvest, a homogenized sample of testicular spermatozoa. Note the pink color due to blood contamination...... 320
Figure 26. Comparison of fertilization among stripped sperm (control) and testicular sperm at 20,000 MSE and 100,000 MSE in large-scale fertilization experiments (2019)...... 321
Figure 27. Percent of fertilized Saugeye eggs (mean ± 1 SE) at 8 d post fertilization in the three sperm-to-egg ratio treatments (20,000:1, 50,000:1, and 100,000:1). An egg was considered fertilized if it had reached the eyed-egg embryonic stage. Each treatment was replicated 3 times...... 322
xviii
Figure 28. Sperm quality analysis tools for use during Sauger milt analysis. (A) Model 569A Video Microscope (Animal Reproduction Systems, Chino, CA, USA). (B) 590B Equine Densimeter (ARS)...... 323
Figure 29. Examples of a range of Sauger sperm total motilities seen during milt analysis [(A) 0%, (B) 50%, (C) 95%)...... 324
Figure 30. Least-squares linear regression results of natural log transformed hemocytometer counts on natural log transformed 590b equine densimeter counts (n = 29) including a 95% confidence interval (Blawut et al., 2018b)...... 325
Figure 31. Images of the straw filling, sealing, equilibration, and cryopreservation processes. (A) Fill straws until the appropriately sized air bubble (a few mm) is present. (B) Remove straws from the sperm sample and continue to move semen through straw until the plug is wet. (C -D) Press the ends of the straw into PVC powder to form a few mm thick plug. (E) Dip the powder plug into water to for a seal. (F) “Crack the whip” move the air bubble into the center of the straw and wipe exterior of the plug end with a tissue/ lab wipe. (G) Place filled and sealed straws onto the floating rack and equilibrate for 10 min at 5C. (H) After equilibration, place floating rack into liquid nitrogen contained and cool for 10 min before plunging...... 326
xix
Chapter 1. Introduction
Assisted reproduction technologies (ARTs) are commonly used in the production of economically valuable fish species. The goal of these techniques is to maximize individual fertility by either assisting in reaching breeding status (e.g., through hormonal stimulation) or enhancing certain characteristics (e.g., sperm and egg quality and quantity). In aquaculture, the majority of these techniques are more developed for sperm than for eggs because (1) large quantities of sperm are usually available throughout the breeding season, and (2) oocytes are harder to manipulate because they are fragile, single-celled, and quality generally declines more rapidly over short periods of time
(Beirão et al., 2019). Examples of commonly used ARTs in aquaculture include sperm cryopreservation, short-term milt storage, sperm collection techniques to maximize milt availability, and maximum fertility insemination doses. In theory, ARTs should result in more effective use of sperm, maximized fertility, simplified broodstock management, and allows easy transfer of valuable genetic material among facilities (Cabrita et al., 2010).
However, these benefits are not always achieved.
Often, the unintended side effect of ARTs is a reduction in fertilization that precludes its practical application. The exterior surface, cell composition, viability, and motility of sperm can all be negatively impacted by components of ARTs (Leahy and
Gadella, 2011). Cryopreservation, in particular, has the potential to cause widespread
1 damage in gametes that can hinder post-thaw fertilization, rendering the efficacy of this technique as being very low (Hammerstedt et al., 1990; Holt, 2000). As a consequence, the large amount of sperm needed to achieve reasonable fertilization precludes the process from being viable at large-scale production (Watson, 2000). Additionally, some techniques, such as testicular harvest often result in a greater number of sperm collected, however, the fertilization potential may be much lower in a significant proportion of gametes collected (e.g., immature cells , Alavi et al., 2008; Blawut et al., 2020b; Mansour et al., 2004). Despite their limitations, these two techniques (cryopreservation and testicular harvest) see widespread use in the field of aquaculture as tools to address stripped milt shortages. However, the practical efficacy of these tools is often questionable due to negative effects on fertilization potential.
Our poor understanding of fish sperm physiology with respect to how ARTs affect fertilization potential is a major impediment to wide applicability of these techniques. Gaining a deeper understanding of the effects of sperm maturation and cryopreservation has broad applicability to the field of fish culture. With this knowledge,
ARTs can be optimized to maintain sperm fertilization potential faithfully through these damaging processes, ultimately increasing the efficacy of these techniques to the point where large-scale application is possible. This would be a great benefit to aquaculture, particularly for sport and food fish production.
Assisted reproduction technologies have been used extensively during the production of Saugeye (Sander vitreus × Sander canadensis) by the Ohio Department of
Natural Resource – Division of Wildlife (ODNR-DOW). The Saugeye (Sander vitreus ×
2
Sander canadensis) is a hybrid cross between a female Walleye (S. vitreus) and a male
Sauger (S. canadensis). This hybrid shows comparable rates of growth to Walleye
(Barton, 2011), comparable angling rates to Walleye (Quist et al., 2010), and high adaptability for pond culture (JR et al., 1982) making it well-suited for stocking in Ohio lakes and reservoirs.
Annually, angler harvest of these fish contributes more than $100,000,000 to the local and state economies, making Saugeye one of the most important fisheries in the state. Each year, the ODNR-DOW aims to produce enough fry and fingerlings to maintain this man-made fishery using in vitro fertilization during the short overlap in the two species’ breeding seasons. However, a combination of factors threatens to limit the number of Saugeye that can be produced annually, which can negatively affect stocking goals (Figure 1).
One of the limiting factors in the production of Saugeye is the availability of male
Sauger. Male Sauger are collected from the Ohio River by the ODNR-DOW staff immediately prior to the Walleye breeding season (mid-February – April) and held in human-care for the remainder of the breeding season in the London Isolation Facility
(London, OH). Sauger milt is collected from male Sauger via abdominal massage and taken to Berlin Reservoir, Mosquito Reservoir, or the Maumee River where fertilization of Walleye eggs occurs. However, Saugeye production can be extremely variable among years (Figure 1). The primary factors limiting Saugeye production are: 1) The availability of sufficient breeding age male Sauger collected from the source populations
(i.e., Ohio River), 2) Spring and winter precipitation patterns further exacerbating
3 collection efforts, and 3) Small volumes of sperm produced per fish. Frequently, insufficient numbers of male Sauger broodstock combined with the low volume of milt produced by each individual result in a limited supply of milt. This lack of sufficient milt stresses hatchery production by limiting Saugeye production despite increased efforts to collect adequate broodstock.
To combat poor broodstock/milt availability, current ODNR-DOW standard operating procedures utilize 2-3 milt collections per fish to reach these goals, often to the detriment of fish health and longevity. Additionally, effort by biologists to collect sufficient male broodstock for Saugeye production can be prohibitively expensive. Thus, maximizing the reproductive output of each male Sauger and providing long-term milt storage options to ameliorate broodstock and milt shortages using ARTs has been the primary goal of the ODNR-DOW and the Ohio State University Theriogenology lab. Our research has sought to minimize the impact of milt shortages as a production bottleneck for Saugeye. Our solutions to milt shortages are: 1) implementing the use of ARTs to allow for sustainable use of milt (e.g., preventing overuse and maximizing yield on an individual basis) and 2) storing excess milt long-term (≥ 1 year).
We investigated and validated commonly used ARTs in aquaculture including: establishing a sperm-to-egg ratio (10,000 MSE) that reduces the number of sperm applied by 66%, providing a quantity control tool in the densimeter, and also demonstrating testicular harvest as a superior method to strip spawning (7-10 fold increase in sperm yield per individual (Blawut et al., 2018, 2020b)). Collectively, these methods led to an excess sperm that can be utilized for long-term storage to safeguard variability among
4 years when male availability is extremely limited or completely absent. However, use of testicular harvest has raised questions regarding the quality of the resulting milt sample.
Variable fertilization at limited sperm-to-egg ratios, gross morphological defects, and concerns regarding potential ductal maturation’s effects on sperm physiological competence warrant further analysis (Appendix A).
To address the long-term storage potential for Sauger milt, we investigated the use of cryopreservation to provide supply of frozen milt for when broodstock are in short supply (Blawut et al., 2020a). Collectively, our results provided frozen Sauger sperm with high post-thaw quality (Figure 2) but a relatively low potential for fertilization
(Figure 3). Despite the progress made in Sauger sperm cryopreservation, several hurdles still remain before large-scale application can be achieved. At present, fertilization using frozen sperm from our current protocol (5.0 × 10 8 sperm/mL, Rathbun extender, 10%
DMSO, 4 mg/mL BSA) was unexpectedly low (< 20%) even when excess sperm (30-fold increase, 300,000 motile sperm/egg) were used during fertilization compared to fresh sperm. These results indicate that sperm physiology is altered in some way that is negatively impacting fertilization potential of frozen-thawed sperm but cannot be discerned using traditional quality assessments (Cabrita et al., 2010; Figueroa et al., 2016, p. 2; Pini et al., 2018a). Cryopreservation’s efficacy will remain low for the production of
Saugeye hybrids at larger scales until the source of cryodamage is identified and corrected. Without more meaningful biomarkers to assess the post-thaw fertilization potential of sperm, optimizing a cryopreservation protocol suitable for large-scale production will be expensive, time-consuming, and inefficient.
5
For many species, Sauger included, there is not a wealth of information available regarding sperm physiology and reproductive biology. Without this information, detailing the negative effects of ARTs on sperm function and the precise mechanisms responsible for reduced fertilization potential is impossible. Moreover, developing techniques and media to better preserve fertilization potential is a time-consuming series of exploratory trial and error experiments rather than a more targeted approach using sperm physiology biomarkers. For these reasons, a thorough investigation of reproductive biology and sperm physiology is required to achieve these goals.
The two most well studied aspects of fish reproductive biology are the seasonal cyclicity associated with spermatogenesis and the process of sperm activation in the external environment (Schulz et al., 2010; Torres et al., 2016), both of which are discussed below.
Seasonal Reproductive Development
Seasonal reproductive development is one of the most well studied aspects of fish sperm biology. Spermatogenesis and spermatozoa maturation are important first steps toward the development of fully fertile spermatozoa (Schulz et al., 2010). Temperate fishes show a strong seasonal cyclicity, often coinciding with spawning during the favorable conditions of spring or fall (Migaud et al., 2013). Spermatogenesis often begins months in advance of the spawning season. In a close relative of Sauger, the Walleye
(Sander vitreus), spermatogenesis began in August, spermatozoa were present as early as
September-October, and by the beginning of the breeding season in January 95% of cells
6 were spermatozoa (Malison et al., 1994; Malison and Held, 1996). Upon completion of spermatogenesis, sperm are released from their spermatocysts and migrate from the tubular lumen (i.e., seminiferous tubules) to the lobular lumen (i.e., main sperm duct) to undergo final sperm maturation (Billard, 1986). Sperm are often present in the main duct months prior to spawning, indicating that this structure may play a role in maintaining cell viability and contributing to overall sperm maturation processes.
During residency in the main duct, sperm maturity is further developed by contact with the seminal plasma. Sperm harvested from the testis periphery in Rainbow Trout
(Oncorhynchus mykiss) and Chum Salmon (Oncorhynchus keta) were immotile, whereas sperm harvested from the main sperm duct could achieve motility in K+ - free media
(Morisawa and Morisawa, 1986). The same is true for the European Eel (Anguilla anguilla ( Ohta et al., 1997; Ohta et al., 1997). A later study by Morisawa et al demonstrated a mechanism for maturation of testicular Masu Salmon sperm
(Oncorhynchus masou, Morisawa and Morisawa, 1988) within the main sperm duct.
Testicular plasma was found to contain less HCO3- and thus be more acidic than the main sperm duct. In turn, incubating testicular sperm in a high pH, high HCO3- media allowed sperm to attain motility within one hour. Furthermore, this pH-dependent maturation of testicular sperm was demonstrated to be mediated by the final maturation inducing progestogen, 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) in Rainbow
Trout and the European Eel (Miura et al., 1992; Miura and Miura, 2001). This hormonally driven rise in pH was associated with an increase in cellular cAMP required for motility initiation ability by cAMP imparting a maturational effect on the axoneme
7 competency via protein phosphorylation (Morisawa and Okuno, 1982). This information exists for salmonids and the European Eel, but it unavailable in a majority of fish species.
In summary, the fish testis and the spermatozoa generated by it are subject to both seasonal and spatial maturational forces. From a practical perspective, both the seasonal and spatially explicit origin of sperm from the testes could significantly impact the maturation status and quality of sperm harvested using ARTs. Thus, it is important to understand these two sources of variability and how they affect sperm harvested.
Sperm Activation
The most significant event in the life of a fish spermatozoa toward fertilization is motility activation (Billard, 1995). In externally fertilizing fish, activation is achieved through exposure to the external environment (e.g., hypo-osmotic shock for freshwater fish or hyper-osmotic shock for marine fish, Billard and Cosson, 1992). The osmotic and ionic (K+) differences between the cell and the external environment trigger activation the initiation of motility (Alavi and Cosson, 2006). Fish sperm cells are motile in these harsh environments for 30s in freshwater fish to 60 min in marine species. During this short time, sperm must make contact with the egg surface, navigate towards the single opening in the chorion (the micropyle), and fuse with the egg plasma membrane to achieve fertilization (Yanagimachi et al., 2017).
Despite the importance of this period of time to fertilization, there is a shocking paucity of information regarding additional Sauger sperm structures and how they are affected by activation. A number of other structures with known to play a role in sperm-
8 to-egg interactions as well as have implications for fertilization have been identified in sperm (Table 1). Unfortunately, these structures are poorly defined in fish spermatozoa and their relationship with sperm quality and fertilization potential remain unknow.
Below, we review the current knowledge for fish spermatozoa regarding a subset of these structures, e.g., the glycocalyx, the plasma membrane, and intracellular signaling involved in motility activation.
The Glycocalyx
The glycocalyx is composed of the extracellular glycoproteins and glycolipids in the plasma membrane of the cell (Tecle and Gagneux, 2015). These proteins and lipids are heavily glycosylated, and their sugars are often used as proxies to describe and monitor the glycoprotein movement or quantity within the glycocalyx. Extracellular glycoproteins have a wide variety of functions in the cell, however, chief among them is cell-cell communication. Examples of essential glycocalyx proteins and include fertilins, acidic epididymal glycoprotein-related protein (ARP), Izumo, and many others (Schröter et al., 1999). They are known to play essential roles in oviductal as well as oocyte complex interactions in mammalian species (Tecle and Gagneux, 2015). However, our understanding of the glycocalyx remains limited in fish spermatozoa.
The limited information that exists in internally fertilizing fish spermatozoa indicates that the glycocalyx plays a role in sperm chemotaxis toward the micropyle and/or sperm – egg membrane interactions. Early evidence of the glycocalyx in fish sperm suggested that lectins (carbohydrate binding proteins) bind to the acrosome region
9 of the Red spotted Shark, Schroederichthys chilensis, and Swordtail, Schroederichthys chilensis, and that their distribution changed as a result of development during spermatogenesis (Jonas‐Davies et al., 1983; Rojas and Esponda, 2001a). In externally fertilizing species, three heavily glycosylated proteins were identified and their contribution to fertilization and motility was assessed. First, a membrane protein, syndecan, rich in heparin sulfate (N-acetylglucosamine residues) was localized to the head of the Big-scaled Redfin, Triblodon hakonensis, spermatozoa (Kudo, 1998).
Additionally, blocking this protein using antibodies and other inhibitors reduced fertilization to 0 -17%. Second, Yu et al (2002a) identified a membrane ganglioside
((KDN)GM3 [KDNα2 3Galβ4Glcβ1Cer]) in the head of Rainbow Trout sperm that demonstrated strong binding to the micropyle region of oocytes. Finally, a sperm motility inhibiting glycoprotein in the sperm of Nile Tilapia (Oreochromis niloticus) was identified using lectins (e.g., concanavalin A, Lens culinarian agglutinin).
Similar to spermatozoa, oocytes of the Steelhead (Oncorhynchus mykiss), Black
Flounder (Pleuronectes obscurus), and Japanese Rice Fish (Oryzias latipes) displayed a specific localization of proteins and glycoproteins in circular pattern around the micropyle. Eggs treated with trypsin to eliminate these chorionic glycoproteins were less likely to experience sperm entry into the micropyle (Iwamatsu et al., 1997; Yanagimachi et al., 2017). Collectively, the results of these studies in fish suggest that the sperm glycocalyx plays an essential role in fertilization, whether through chemotaxis toward the micropyle or during sperm-egg fusion. Therefore, glycocalyx analysis may improve our understanding of fish sperm physiology and effects of ARTs on fertility.
10
The Plasma Membrane
The plasma membrane is the most important organelle to proper sperm cell function. Membranes are responsible for maintaining ionic composition, osmotic balance, transducing signals, and interacting with the oocyte (Flesch and Gadella, 2000). This structure is initially formed during spermatogenesis and further modified by a number of processes including ductal maturation and capacitation (Jones, 2002) as well as interaction with the oocyte (Gadella et al., 2001). What makes the sperm plasma membrane unique is its highly unsaturated composition, bilayer asymmetry, and lipid ordered domains essential to sperm-to-egg interactions (Tapia et al., 2018). Several of these plasma membrane characteristics have been studied in fish, but the existence of specific lipid domains, changes to membrane composition, and how these parameters are affected by ARTs remains unclear in a majority of fish species.
The composition of fish sperm plasma membranes is typical of many other taxa, with minor exceptions. Membrane composition can be affected by many factors, including: temperature (Engel et al., 2019), spatial location on the cell (e.g., head vs. tail
,J. Beirão et al., 2012), nutritional status (Nandi et al., 2007), and the period within the season (Martínez-Páramo et al., 2012). Sperm membranes are primarily composed of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids
(Lahnsteiner et al., 2009). However, they are unique because of an uncharacteristically high proportion of polyunsaturated fatty acids (e.g., docosahexaenoic acid ,DHA, in particular ) in the tails of glycerophospholipids (Bell et al., 1997). To a lesser degree,
11 sphingolipids and gangliosides show a species-specific quantity and diversity (Engel et al., 2020). Membrane cholesterol, another important effector of membrane physiology, represents a larger interchangeable pool in the membrane of Rainbow Trout. For example, motility and fertility could be maintained despite a 30 – 50% reduction in plasma membrane cholesterol (Müller et al., 2008). Synergistically, fatty acid chain length, degree of unsaturation, and cholesterol content contribute to the fluidity of the membrane. While an important characteristics to mammalian sperm, membrane fluidity has yet to have a demonstrated role in fish sperm beyond physiological adaptation to thermal conditions (Chakrabarty et al., 2007; Engel et al., 2019) and its link to membrane stability.
Membrane components do not occur randomly, and this structured arrangement imposes certain cellular characteristics. The fluid mosaic model is no longer the accepted model of membrane structure. Instead distinct lipid ordered (i.e., lipid raft) and lipid disordered regions exist within the membrane (Brown, 2006; Kawano et al., 2011; Nixon and Aitken, 2009). Lipid rafts are lipid ordered domains in the external leaflet of the plasma membrane that are enriched in cholesterol, sphingolipids, and signaling molecules
(Nixon and Aitken, 2009). These rafts play an important function in cell-cell communication during fertilization in mammalian sperm, and potentially in many other taxa (Kawano et al., 2011). Therefore, these structures, if present in fish, should be investigated thoroughly as they may play an essential role in sperm-oocyte interactions during fertilization could, in theory, be a source of sub-fertility following ART intervention.
12
Lipid raft composition render them insoluble at low temperatures in the presence of detergents. Researches use these “detergent resistant membranes” (DRMs) as equivalents to in vivo lipid rafts. Lipid rafts are an essential structure to mammalian sperm fertility. These structures undergo modifications during epididymal transit and seminal plasma exposure (Girouard et al., 2008), during capacitation (Khalil et al., 2006;
Shadan et al., 2004; Sleight et al., 2005; Thaler et al., 2006; van Gestel et al., 2005), and acrosome reaction (Miranda et al., 2009). Despite this knowledge in mammalian sperm, very few studies have investigated lipid rafts in fish. The majority of available studies in fish were conducted in somatic cells (Brogden et al., 2014; Zehmer and Hazel, 2003), with a single study dedicated to spermatozoa (Bai et al., 2019). The potential for lipid rafts to not only impact fertilization, but to also be potential targets of sub-lethal damage by cryopreservation warrants a more thorough analysis in fish sperm as potential biomarkers.
To summarize, plasma membrane analysis of Sauger sperm should be considered a necessary component of quality analysis to assess fertilization potential. In addition to traditional metrics of membrane function (i.e., viability) established methods such as metabolic fingerprinting using liquid chromatography – mass spectroscopy and membrane fluidity (i.e., fluidity) should be conducted to thoroughly assess membrane fitness. Though not yet fully established as essential to fish sperm, the importance of lipid rafts to fertilization in other taxa warrants further analysis in fish with regard to sperm quality assessments. This information is lacking in a majority of fish species subjected to
ARTs and is completely absent in the Sauger.
13
Intracellular Signaling
Sperm motility is considered to be universally important to fertilization potential
(Alavi, 2008; Tiersch and Green, 2011). Following osmotic shock, fish sperm must navigate to the micropyle of the egg during a 30 s to 60 min motile period to achieve fertilization (Alavi et al., 2019). Combined with the ease of assessing these parameters, motility analysis serves as the baseline quality assessment tool for many studies in fish sperm (Billard, 1995). While motility parameters can be correlated with fertilization potential in fresh sperm (Casselman et al., 2006), this metric of sperm quality falls short for sperm negatively impacted by ARTs (Boryshpolets et al., 2020). Because motility is a multifaceted process that includes multiple phases (signal transduction, protein phosphorylation, activation of the axoneme), any of these phases could be affected in a way that alters the ability of sperm motility to accurately portray fertilization potential.
Activation of the intracellular signaling pathway resulting from osmotic shock and leading to motility initiation have been described in several major fish models (Alavi et al., 2019; Zilli et al., 2017). Regardless of species-specific differences, a rise in intracellular Ca2+ is universally observed and thought to be a secondary messenger.
Calcium plays many roles in the cell, including propagation of activation stimuli, controlling flagella beating pattern, modifying sperm velocity (Dumorné et al., 2018;
Dzyuba & Cosson, 2014). Studies utilizing activation media, stimulants, and channel blockers have all but assured the importance of this ion to fish sperm motility (Alavi et al., 2011; Boitano and Omoto, 1992; Chen et al., 2020; Lissabet et al., 2020; Pérez et al.,
14
2016; Yanagimachi et al., 2017). Despite calcium’s importance to this process, surprisingly few studies have explored the effects of ARTs on cellular calcium. Altering cellular calcium homeostasis were associated with reduced motility and fertilization ability, suggesting calcium homeostasis could be a factor affecting cryopreserved Sauger sperm fertilization potential.
The final step in motility activation is activation of the sperm axoneme to achieve flagellar beating (Dumorné et al., 2018). Two major mechanisms are associated with protein phosphorylation in the axoneme and mitochondria, both of which act on protein kinases and phosphatases: 1) Ca2+ - calmodulin complexes, or 2) cAMP (Zilli et al.,
2017). Protein phosphorylation targets include structural proteins in the flagella, signaling proteins such as kinases and phosphatases, as well as metabolic proteins (Zilli et al.,
2017). Sperm require the ability for motility activation, which is conferred by phosphorylation during progestin induced sperm maturation (see Seasonal reproductive development above).
While motility is often an unreliable assessment of fertilization ability in frozen- thawed sperm (Tiersch and Green, 2011), more sophisticated analyses of motility characteristics have proven to provide a more valuable assessment of sperm quality
(Martínez-Pastor et al., 2011). Multivariate analyses, and clustering methods in particular, have become increasingly common in the field of animal andrology (Martínez-
Pastor et al., 2011). These methods seek to group similar observations into discrete clusters which can then be used to interpret effects of a wide array of experimental treatments. Clustering analysis has been used to classify and compare motile sperm
15 subpopulations in a number of fish species, including Gilthead Seabream (Sparaus auratus, Beirão et al., 2011), Atlantic Salmon (Salmo salar, Caldeira et al., 2018),
Tambaqui (Colossoma macropomum, Gallego et al., 2017a), European Eel (Anguilla
Anguilla, Gallego et al., 2015), steelhead (Kanuga et al., 2012), and Senegalese Sole
(Solea senegalensis, Martínez-Pastor et al., 2008). The subpopulations revealed in the studies mentioned above provide information that is not immediately evident from simple motility analysis and can be positively correlated with fertilization potential. However, fewer studies have determined the effect of various ARTs on motile sperm subpopulations in fish.
Assisted Reproduction Techniques
Assisted reproduction techniques are likely to impact these structures in fish spermatozoa, but the extent of their influence has yet to be described. Two of the main
ARTs used in aquaculture to increase milt availability and provide long-term milt storage have proven to impact sperm’s fertilization potential. This loss of fertility is similar to those in mammalian species, where ARTs effects on the glycocalyx, plasma membrane, and intracellular signaling have been described more thoroughly (Pini et al., 2018a;
Yeste, 2016). Thus, it stands to reason that ARTs may have similar effects on fertilization potential in both fish and mammal sperm. Below, we detail the known impacts of testicular harvest and cryopreservation on sperm physiology, with an emphasis on aquatic species.
16
Testicular Harvest
Testicular harvest is a widely used technique in the field of aquaculture as a milt collection alternative when a species produces small quantities of milt in response to strip spawning (Cabrita et al., 2008). However, this technique has not proven to be a panacea.
Some species respond well to this method – produce motile sperm with little effect on fertilization potential (Rurangwa et al., 2004; Kowalski et al., 2006)- while in other species the resulting sample is immotile and displays poor fertility (Alavi et al., 2008).
The disparity in fertility of the collected sperm may result from the collection of both mature (lobular lumen) and immature cells (tubular lumen) simultaneously (Mansour et al., 2004). At present, the definition of a mature sperm and the ability to discriminate them from immature cells is lacking in fish sperm. However, differences in certain sperm characteristics, those mentioned above, may offer an insight into sperm maturity.
There is a paucity of information regarding the physiology of sperm during the period of ductal maturation. In the limited reports available in fish, intra-testicular glycocalyx monitoring in Xiphorus helleri and Xiphorus maculatus during spermatogenesis (Jonas‐Davies et al., 1983; Rojas and Esponda, 2001b) revealed slight changes to the localization and relative quantities of sugar moieties in the sperm head.
No further information is available of regarding the glycocalyx and testicular development. Similarly, low total motility has been linked both to testicular harvest
(Morisawa and Morisawa, 1988, 1986; Hiromi Ohta et al., 1997; H. Ohta et al., 1997) and cryopreservation (Boryshpolets et al., 2020), comparatively few studies have addressed signal transduction (i.e., intracellular calcium and protein phosphorylation) or motile 17 sperm subpopulations. Lastly, membrane composition is also poorly described with regard to comparing testicular sperm and stripped sperm, with much more information available detailing the effects of cryopreservation (Díaz et al., 2019; He et al., 2011). This lack of information provides many opportunities for original research that would benefit sperm quality assessment and cell maturity discrimination in testicular harvest sperm.
More information is needed to determine the effect of testicular harvest in sperm physiology and to fully determine its practical efficacy. It is likely that testicular harvest results in the collection of immature sperm (tubular lumen) simultaneously with the more desirable mature sperm (lobular lumen). These tubular lumen sperm may be less fertile in addition to being hypothetically less motile as well and could have serious implications for practical efficacy of this technique. However, the specific metrics of sperm quality contributing to these differences in fertilization potential have not been adequately described in fish. This gap in knowledge would be beneficial to a species where testicular harvest is utilized and more generally toward assessing sperm maturity and quality.
Cryopreservation
Cryopreservation is a valuable tool for long-term storage of spermatozoa
(Martínez-Páramo et al., 2017); This technique, however, often results in significant damage to one or more essential sperm structures (Pini et al., 2018b; Tiersch and Green,
2011). These damages ultimately result in diminished fertilization capacity at post-thaw.
There are many potential sources of cryo-damage (e.g., reactive oxygen species, osmotic shock, cold shock, and physical damage) that have been well described (Tiersch and
18
Green, 2011). However, the effects of these sources of cryo-damage on the glycocalyx, plasma membrane, and intracellular environment in fish spermatozoa is lacking.
The sperm glycocalyx remains a poorly studies structure in fish. To the authors’ knowledge, there are no studies detailing the effect of cryopreservation on the fish sperm glycocalyx. Given the likely role of the glycocalyx in interaction with the oocyte (Kudo,
1998; Yu et al., 2002), it is possible that this structure may at least be partially responsible for poor fertilization rates in frozen sperm. This realization would certainly be a novel discovery and should provide new methods for assess sperm’s post-thaw fertilization potential.
Cryopreservation has been demonstrated to induce changes to the plasma membrane of fish spermatozoa. Sperm membranes are particularly susceptible to cryo- damage due to the high amounts of unsaturated fatty acids and sterols in the bilayer (Díaz et al., 2019; Horokhovatskyi et al., 2016; Klaiwattana et al., 2016). Lipid peroxidation has been identified as a potent source of membrane damage during freeze-thaw processes
(Klaiwattana et al., 2016; Martínez-Páramo et al., 2012; Sandoval‐Vargas et al., 2020).
The end result is decimated antioxidant scavenging systems leading to reduced motility, damaged membranes, DNA, and axoneme all to the detriment of fertilization potential.
More recently, lipid rafts have been suggested to suffer cryopreservation induced damage as well. Deterioration of the rafts as evidenced by loss of marker proteins to the seminal plasma, degradation of sphingomyelins, and spatial reorganization of rafts have been reported in fish (Bai et al., 2019; Dai et al., 2012; Dietrich et al., 2015; He et al., 2011). In agreement with these characteristics of membrane damage, membrane fluidity (associated
19 with compromised membrane stability) is often elevated in cryopreserved sperm as a result of bilayer destabilization (Pereira et al., 2019; Perry et al., 2019). Consequently, plasma membrane damage is likely a contributing factor to loss of fertility in cryopreserved sperm and should be further investigated.
Assisted reproduction techniques have been shown to affect all stages of motility in fish spermatozoa. Available evidence in the Striped Bass (Morone saxatillis) and
Atlantic Salmon spermatozoa (Figueroa et al., 2019; Guthrie et al., 2014) indicates that calcium homeostasis (either an increase or decrease relative to fresh) is associated with short-term storage and freeze-thaw. Likewise, cryopreservation may cause a change to the phosphorylation status of the axoneme and metabolism-related proteins, rendering them less able to achieve motility comparable to fresh sperm (Gazo et al., 2017; Li et al.,
2013; Zilli et al., 2017). And lastly, motile sperm subpopulation analysis has shown in
Colossoma macropmum and Sparus auratus the freeze-thaw process differentially affects sperm motility, and a small proportion of sperm exhibiting trajectories similar to those in fresh sperm that are highly correlated with fertilization potential (Beirão et al., 2011;
Gallego et al., 2015). While motility has been described for stripped, testicular, and cryopreserved sperm in the Sauger, a more detailed analysis of the activation pathway from initial signaling through the motility patterns produced has not been completed. At present, this information does not exist for Sauger despite its potential promise as a valuable biomarker of sperm quality.
20
Synthesis
The consensus from my review of relevant literature is that a number of physiological traits with a high probability of connection to fertilization success are poorly understood in most fish sperm, and completely absent in the Sauger. My dissertation serves to fill this gap in understanding by studying natural and artificially induced aspects of fish sperm physiology and their implications for sperm function and fertilization potential. The questions central to this work were: 1) Do sauger sperm demonstrate seasonal and main sperm duct maturation? 2) How does sperm physiology change as a result of motility activation? 3) Does testicular harvest result in the collection of immature cells? and 4) What parameters are most impacted by the freeze-thaw process and can these markers be related to fertilization potential? We hypothesized that: 1)
Sauger sperm and testes will show a seasonal and intra-ductal maturation potential as evidenced by testis morphology and changes to sperm characteristics, 2) Hypo-osmotic shock will result in changes to sauger sperm physiology, and 3) Testicular harvest and cryopreservation will alter inactive and activated sperm characteristics.
To achieve this goal, my collaborators and I used a combination of observational experiments to describe reproductive tract and spermatozoa structure, as well as controlled laboratory experiments to assess and compare spermatozoa physiological traits in inactive and activated sperm resulting from the most commonly used ARTs in Sauger
(i.e., strip-spawning, testicular harvest, and cryopreservation). In chapter 2, our objective was to describe the structure of the spermatozoa and the Sauger testis with the aim of illuminating maturational forces imparted on sperm as a result of seasonal changes and
21 intra-testicular structures. The focus of the remainder of the chapters in this dissertation was to determine the effects of hypo-osmotic shock, testicular harvest, and cryopreservation on the glycocalyx (Chapter 3), the plasma membrane (Chapter 4), and intracellular signaling (Chapter 5) in Sauger sperm. And lastly, we discuss the meaning of this research in the context of the practical application and implications for the field of aquaculture.
22
Literature Cited
Alavi, S.H., Rodina, M., Policar, T., Cosson, J., Kozak, P., Psenicka, M., Linhart, O.,
2008. Physiology and behavior of stripped and testicular sperm in Perca fluviatilis
L. 1758. Cybium 32, 162–163.
Alavi, S.M.H., 2008. Fish spermatology. Alpha Science International Ltd.
Alavi, S.M.H., Cosson, J., 2006. Sperm motility in fishes. (II) Effects of ions and
osmolality: A review. Cell Biol. Int. 30, 1–14.
https://doi.org/10.1016/j.cellbi.2005.06.004
Alavi, S.M.H., Cosson, J., Bondarenko, O., Linhart, O., 2019. Sperm motility in fishes:
(III) diversity of regulatory signals from membrane to the axoneme.
Theriogenology 136, 143–165.
https://doi.org/10.1016/j.theriogenology.2019.06.038
Alavi, S.M.H., Gela, D., Rodina, M., Linhart, O., 2011. Roles of osmolality, calcium —
Potassium antagonist and calcium in activation and flagellar beating pattern of
sturgeon sperm. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 160, 166–174.
https://doi.org/10.1016/j.cbpa.2011.05.026
Bai, C., Kang, N., Zhao, J., Dai, J., Gao, H., Chen, Y., Dong, H., Huang, C., Dong, Q.,
2019. Cryopreservation disrupts lipid rafts and heat shock proteins in yellow
catfish sperm. Cryobiology 87, 32–39.
https://doi.org/10.1016/j.cryobiol.2019.03.004
Barton, B.A., 2011. Biology, management, and culture of walleye and sauger. American
Fisheries Society Bethesda, Maryland. 23
Beirão, J., Boulais, M., Gallego, V., O’Brien, J.K., Peixoto, S., Robeck, T.R., Cabrita, E.,
2019. Sperm handling in aquatic animals for artificial reproduction.
Theriogenology 133, 161–178.
https://doi.org/10.1016/j.theriogenology.2019.05.004
Beirão, J., Cabrita, E., Pérez-Cerezales, S., Martínez-Páramo, S., Herráez, M.P., 2011.
Effect of cryopreservation on fish sperm subpopulations. Cryobiology 62, 22–31.
https://doi.org/10.1016/j.cryobiol.2010.11.005
Beirão, J., Zilli, L., Vilella, S., Cabrita, E., Fernández‐Díez, C., Schiavone, R., Herráez,
M.P., 2012. Fatty acid composition of the head membrane and flagella affects
Sparus aurata sperm quality. J. Appl. Ichthyol. 28, 1017–1019.
https://doi.org/10.1111/jai.12085
Bell, M.V., Dick, J.R., Buda, Cs., 1997. Molecular speciation of fish sperm
phospholipids: Large amounts of dipolyunsaturated phosphatidylserine. Lipids 32,
1085–1091. https://doi.org/10.1007/s11745-997-0140-y
Billard, R., 1995. Sperm physiology and quality. Broodstock Manag. Egg Larval Qual.
25–52.
Billard, R., 1986. Spermatogenesis and spermatology of some teleost fish species.
Reprod. Nutr. Dév. 26, 877–920. https://doi.org/10.1051/rnd:19860601
Billard, R., Cosson, M.P., 1992. Some problems related to the assessment of sperm
motility in freshwater fish. J. Exp. Zool. 261, 122–131.
https://doi.org/10.1002/jez.1402610203
24
Blawut, B., Wolfe, B., Moraes, C.R., Ludsin, S.A., Coutinho da Silva, M.A., 2018.
Increasing saugeye (S. vitreus × S. canadensis) production efficiency in a hatchery
setting using assisted reproduction technologies. Aquaculture 495, 21–26.
https://doi.org/10.1016/j.aquaculture.2018.05.027
Blawut, B., Wolfe, B., Moraes, C.R., Ludsin, S.A., Silva, M.A.C. da, 2020a. Use of
Hypertonic Media to Cryopreserve Sauger Spermatozoa. North Am. J. Aquac. 82,
84–91. https://doi.org/10.1002/naaq.10125
Blawut, B., Wolfe, B., Moraes, C.R., Sweet, D., Ludsin, S.A., Coutinho da Silva, M.A.,
2020b. Testicular collections as a technique to increase milt availability in sauger
(sander canadensis). Anim. Reprod. Sci. 212, 106240.
https://doi.org/10.1016/j.anireprosci.2019.106240
Boitano, S., Omoto, C.K., 1992. Trout sperm swimming patterns and role of intracellular
Ca++. Cell Motil. Cytoskeleton 21, 74–82. https://doi.org/10.1002/cm.970210109
Boryshpolets, S., Kholodnyy, V., Cosson, J., Dzyuba, B., 2020. Fish Sperm Quality
Evaluation After Cryopreservation, in: Betsy, J., Kumar, S. (Eds.),
Cryopreservation of Fish Gametes. Springer, Singapore, pp. 117–133.
https://doi.org/10.1007/978-981-15-4025-7_5
Brogden, G., Propsting, M., Adamek, M., Naim, H.Y., Steinhagen, D., 2014. Isolation
and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio
L.). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 169, 9–15.
https://doi.org/10.1016/j.cbpb.2013.12.001
25
Brown, D.A., 2006. Lipid Rafts, Detergent-Resistant Membranes, and Raft Targeting
Signals. Physiology 21, 430–439. https://doi.org/10.1152/physiol.00032.2006
Cabrita, E., Robles, V., Herráez, P., 2008. Methods in reproductive aquaculture: marine
and freshwater species. CRC Press.
Cabrita, E., Sarasquete, C., Martínez-Páramo, S., Robles, V., Beirão, J., Pérez-Cerezales,
S., Herráez, M. p., 2010. Cryopreservation of fish sperm: applications and
perspectives. J. Appl. Ichthyol. 26, 623–635. https://doi.org/10.1111/j.1439-
0426.2010.01556.x
Caldeira, C., García-Molina, A., Valverde, A., Bompart, D., Hassane, M., Martin, P.,
Soler, C., 2018. Comparison of sperm motility subpopulation structure among
wild anadromous and farmed male Atlantic salmon (Salmo salar) parr using a
CASA system. Reprod. Fertil. Dev.
Casselman, S.J., Schulte-Hostedde, A.I., Montgomerie, R., 2006. Sperm quality
influences male fertilization success in walleye (Sander vitreus). Can. J. Fish.
Aquat. Sci. 63, 2119–2125.
Chakrabarty, J., Banerjee, D., Pal, D., De, J., Ghosh, A., Majumder, G.C., 2007.
Shedding off specific lipid constituents from sperm cell membrane during
cryopreservation. Cryobiology 54, 27–35.
https://doi.org/10.1016/j.cryobiol.2006.10.191
Chen, Y., Wang, H., Wang, F., Chen, C., Zhang, P., Song, D., Luo, T., Xu, H., Zeng, X.,
2020. Sperm motility modulated by Trpv1 regulates zebrafish fertilization.
Theriogenology 151, 41–51. https://doi.org/10.1016/j.theriogenology.2020.03.032
26
Dai, T., Zhao, E., Lu, G., Che, K., He, Q., Lu, Y., Fang, Q., Wang, H., Zheng, L., Li, S.,
Huang, C., Dong, Q., 2012. Sperm cryopreservation of yellow drum Nibea
albiflora: A special emphasis on post-thaw sperm quality. Aquaculture 368–369,
82–88. https://doi.org/10.1016/j.aquaculture.2012.09.017
Díaz, R., Lee-Estevez, M., Quiñones, J., Dumorné, K., Short, S., Ulloa-Rodríguez, P.,
Valdebenito, I., Sepúlveda, N., Farías, J.G., 2019. Changes in Atlantic salmon
(Salmo salar) sperm morphology and membrane lipid composition related to cold
storage and cryopreservation. Anim. Reprod. Sci. 204, 50–59.
https://doi.org/10.1016/j.anireprosci.2019.03.004
Dietrich, M.A., Arnold, G.J., Fröhlich, T., Otte, K.A., Dietrich, G.J., Ciereszko, A., 2015.
Proteomic analysis of extracellular medium of cryopreserved carp (Cyprinus
carpio L.) semen. Comp. Biochem. Physiol. Part D Genomics Proteomics 15, 49–
57. https://doi.org/10.1016/j.cbd.2015.05.003
Dumorné, K., Figueroa, E., Cosson, J., Lee‐Estevez, M., Ulloa‐Rodríguez, P.,
Valdebenito, I., Farías, J.G., 2018. Protein phosphorylation and ions effects on
salmonid sperm motility activation. Rev. Aquac. 10, 727–737.
https://doi.org/10.1111/raq.12198
Engel, K.M., Dzyuba, V., Borys, D., Schiller, J., 2020. Different glycolipids in sperm
from different freshwater fishes – A high performance thin-layer
chromatography/electrospray ionization-mass spectrometry study. Rapid
Commun. Mass Spectrom. 34, 9. https://doi.org/10.1002/rcm.8875
27
Engel, K.M., Sampels, S., Dzyuba, B., Podhorec, P., Policar, T., Dannenberger, D.,
Schiller, J., 2019. Swimming at different temperatures: The lipid composition of
sperm from three freshwater fish species determined by mass spectrometry and
nuclear magnetic resonance spectroscopy. Chem. Phys. Lipids 221, 65–72.
https://doi.org/10.1016/j.chemphyslip.2019.03.014
Figueroa, E., Lee-Estevez, M., Valdebenito, I., Watanabe, I., Oliveira, R.P.S., Romero, J.,
Castillo, R.L., Farías, J.G., 2019. Effects of cryopreservation on mitochondrial
function and sperm quality in fish. Aquaculture 511, 634190.
https://doi.org/10.1016/j.aquaculture.2019.06.004
Figueroa, E., Valdebenito, I., Farias, J.G., 2016. Technologies used in the study of sperm
function in cryopreserved fish spermatozoa. Aquac. Res. 47, 1691–1705.
https://doi.org/10.1111/are.12630
Flesch, F.M., Gadella, B.M., 2000. Dynamics of the mammalian sperm plasma
membrane in the process of fertilization. Biochim. Biophys. Acta BBA - Rev.
Biomembr. 1469, 197–235. https://doi.org/10.1016/S0304-4157(00)00018-6
Gadella, B.M., Rathi, R., Brouwers, J.F.H.M., Stout, T.A.E., Colenbrander, B., 2001.
Capacitation and the acrosome reaction in equine sperm. Anim. Reprod. Sci.,
Symposium on Stallion Reproduction 68, 249–265.
https://doi.org/10.1016/S0378-4320(01)00161-0
Gallego, V., Cavalcante, S.S., Fujimoto, R.Y., Carneiro, P.C.F., Azevedo, H.C., Maria,
A.N., 2017. Fish sperm subpopulations: Changes after cryopreservation process
and relationship with fertilization success in tambaqui (Colossoma
28
macropomum). Theriogenology 87, 16–24.
https://doi.org/10.1016/j.theriogenology.2016.08.001
Gallego, V., Vílchez, M.C., Peñaranda, D.S., Pérez, L., Herráez, M.P., Asturiano, J.F.,
Martínez-Pastor, F., 2015. Subpopulation pattern of eel spermatozoa is affected
by post-activation time, hormonal treatment and the thermal regimen. Reprod.
Fertil. Dev. 27, 529–543. https://doi.org/10.1071/RD13198
Gazo, I., Dietrich, M.A., Prulière, G., Shaliutina-Kolešová, A., Shaliutina, O., Cosson, J.,
Chenevert, J., 2017. Protein phosphorylation in spermatozoa motility of
Acipenser ruthenus and Cyprinus carpio. Reproduction 154, 653–673.
https://doi.org/10.1530/REP-16-0662
Girouard, J., Frenette, G., Sullivan, R., 2008. Seminal Plasma Proteins Regulate the
Association of Lipids and Proteins Within Detergent-Resistant Membrane
Domains of Bovine Spermatozoa. Biol. Reprod. 78, 921–931.
https://doi.org/10.1095/biolreprod.107.066514
Guthrie, H.D., Welch, G.R., Woods, L.C., 2014. Effects of frozen and liquid hypothermic
storage and extender type on calcium homeostasis in relation to viability and ATP
content in striped bass (Morone saxatilis) sperm. Theriogenology 81, 1085–1091.
https://doi.org/10.1016/j.theriogenology.2014.01.035
Hammerstedt, R., Graham, J., Nolan, J., 1990. Cryopreservation of Mammalian Sperm -
What We Ask Them to Survive. J. Androl. 11, 73–88.
He, Q., Lu, G., Che, K., Zhao, E., Fang, Q., Wang, H., Liu, J., Huang, C., Dong, Q.,
2011. Sperm cryopreservation of the endangered red spotted grouper, Epinephelus
29
akaara, with a special emphasis on membrane lipids. Aquaculture 318, 185–190.
https://doi.org/10.1016/j.aquaculture.2011.05.025
Holt, W.V., 2000. Basic aspects of frozen storage of semen. Anim. Reprod. Sci. 62, 3–22.
https://doi.org/10.1016/S0378-4320(00)00152-4
Horokhovatskyi, Y., Sampels, S., Cosson, J., Linhart, O., Rodina, M., Fedorov, P.,
Blecha, M., Dzyuba, B., 2016. Lipid composition in common carp (Cyprinus
carpio) sperm possessing different cryoresistance. Cryobiology 73, 282–285.
https://doi.org/10.1016/j.cryobiol.2016.08.005
Iwamatsu, T., Yoshizaki, N., Shibata, Y., 1997. Changes in the chorion and sperm entry
into the micropyle during fertilization in the teleostean fish, Oryzias latipes. Dev.
Growth Differ. 39, 33–41. https://doi.org/10.1046/j.1440-169X.1997.00005.x
Jonas‐Davies, J. a. C., Winfrey, V., Olson, G.E., 1983. Plasma membrane structure in
spermatogenic cells of the swordtail (teleostei, Xiphophorus helleri). Gamete Res.
7, 309–324. https://doi.org/10.1002/mrd.1120070403
Jones, R., 2002. Plasma Membrane Composition and Organisation During Maturation of
Spermatozoa in the Epididymis. Epididymis Mol. Clin. Pract. 405–416.
https://doi.org/10.1007/978-1-4615-0679-9_23
JR, W.E.L., Johnson, D.L., Schell, S.A., 1982. Survival, Growth, and Food Habits of
Walleye X Sauger Hybrids (Saugeye) in Ponds. North Am. J. Fish. Manag. 2,
381–387. https://doi.org/10.1577/1548-8659(1982)2<381:SGAFHO>2.0.CO;2
Kanuga, M.K., Drew, R.E., Wilson-Leedy, J.G., Ingermann, R.L., 2012. Subpopulation
distribution of motile sperm relative to activation medium in steelhead
30
(Oncorhynchus mykiss). Theriogenology 77, 916–925.
https://doi.org/10.1016/j.theriogenology.2011.09.020
Kawano, N., Yoshida, K., Miyado, K., Yoshida, M., 2011. Lipid Rafts: Keys to Sperm
Maturation, Fertilization, and Early Embryogenesis [WWW Document]. J. Lipids.
https://doi.org/10.1155/2011/264706
Khalil, M.B., Chakrabandhu, K., Xu, H., Weerachatyanukul, W., Buhr, M., Berger, T.,
Carmona, E., Vuong, N., Kumarathasan, P., Wong, P.T.T., Carrier, D.,
Tanphaichitr, N., 2006. Sperm capacitation induces an increase in lipid rafts
having zona pellucida binding ability and containing sulfogalactosylglycerolipid.
Dev. Biol. 290, 220–235. https://doi.org/10.1016/j.ydbio.2005.11.030
Klaiwattana, P., Srisook, K., Srisook, E., Vuthiphandchai, V., Neumvonk, J., 2016. Effect
of cryopreservation on lipid composition and antioxidant enzyme activity of
seabass (Lates calcarifer) sperm. Iran. J. Fish. Sci. 15, 157–169.
Kudo, S., 1998. Role of sperm head syndecan at fertilization in fish. J. Exp. Zool. 281,
620–625. https://doi.org/10.1002/(SICI)1097-010X(19980815)281:6<620::AID-
JEZ10>3.0.CO;2-6
Lahnsteiner, F., Mansour, N., McNiven, M.A., Richardson, G.F., 2009. Fatty acids of
rainbow trout (Oncorhynchus mykiss) semen: Composition and effects on sperm
functionality. Aquaculture 298, 118–124.
https://doi.org/10.1016/j.aquaculture.2009.08.034
31
Leahy, T., Gadella, B.M., 2011. Sperm surface changes and physiological consequences
induced by sperm handling and storage. Reproduction 142, 759–778.
https://doi.org/10.1530/REP-11-0310
Li, P., Hulak, M., Li, Z.H., Sulc, M., Psenicka, M., Rodina, M., Gela, D., Linhart, O.,
2013. Cryopreservation of common carp (Cyprinus carpio L.) sperm induces
protein phosphorylation in tyrosine and threonine residues. Theriogenology 80,
84–89. https://doi.org/10.1016/j.theriogenology.2013.03.021
Lissabet, J.F.B., Belén, L.H., Lee-Estevez, M., Farias, J.G., 2020. Role of voltage-gated
L-type calcium channel in the spermatozoa motility of Atlantic salmon (Salmo
salar). Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 241, 110633.
https://doi.org/10.1016/j.cbpa.2019.110633
Malison, J.A., Held, J.A., 1996. Reproduction and spawning in walleye (Stizostedion
vitreum). J. Appl. Ichthyol. 12, 153–156. https://doi.org/10.1111/j.1439-
0426.1996.tb00081.x
Malison, J.A., Procarione, L.S., Barry, T.P., Kapuscinski, A.R., Kayes, T.B., 1994.
Endocrine and gonadal changes during the annual reproductive cycle of the
freshwater teleost,
Fish Physiol. Biochem. 13, 473–484. https://doi.org/10.1007/BF00004330
Mansour, N., Lahnsteiner, F., Berger, B., 2004. Characterization of the testicular semen
of the African catfish, Clarias gariepinus (Burchell, 1822), and its short-term
storage. Aquac. Res. 35, 232–244. https://doi.org/10.1111/j.1365-
2109.2004.00993.x
32
Martínez-Páramo, S., Diogo, P., Beirão, J., Dinis, M.T., Cabrita, E., 2012. Sperm lipid
peroxidation is correlated with differences in sperm quality during the
reproductive season in precocious European sea bass (Dicentrarchus labrax)
males. Aquaculture 358–359, 246–252.
https://doi.org/10.1016/j.aquaculture.2012.06.010
Martínez-Páramo, S., Horváth, Á., Labbé, C., Zhang, T., Robles, V., Herráez, P., Suquet,
M., Adams, S., Viveiros, A., Tiersch, T.R., Cabrita, E., 2017. Cryobanking of
aquatic species. Aquaculture 472, 156–177.
https://doi.org/10.1016/j.aquaculture.2016.05.042
Martínez-Pastor, F., Cabrita, E., Soares, F., Anel-Lopez, L., Dinis, M.T., 2008.
Multivariate cluster analysis to study motility activation of Solea senegalensis
spermatozoa: a model for marine teleosts. Reproduction 135, 449–459.
https://doi.org/10.1530/REP-07-0376
Martínez-Pastor, F., Tizado, E.J., Garde, J.J., Anel, L., de Paz, P., 2011. Statistical Series:
Opportunities and challenges of sperm motility subpopulation analysis11This
article is part of the Statistical Series guest-edited by Szabolcs Nagy.
Theriogenology 75, 783–795.
https://doi.org/10.1016/j.theriogenology.2010.11.034
Migaud, H., Bell, G., Cabrita, E., McAndrew, B., Davie, A., Bobe, J., Herráez, M.P.,
Carrillo, M., 2013. Gamete quality and broodstock management in temperate fish.
Rev. Aquac. 5, S194–S223. https://doi.org/10.1111/raq.12025
33
Miranda, P.V., Allaire, A., Sosnik, J., Visconti, P.E., 2009. Localization of Low-Density
Detergent-Resistant Membrane Proteins in Intact and Acrosome-Reacted Mouse
Sperm. Biol. Reprod. 80, 897–904. https://doi.org/10.1095/biolreprod.108.075242
Miura, T., Miura, C., 2001. Japanese Eel: A Model for Analysis of Spermatogenesis.
Zoolog. Sci. 18, 1055–1063. https://doi.org/10.2108/zsj.18.1055
Miura, T., Yamauchi, K., Takahashi, H., Nagahama, Y., 1992. The role of hormones in
the acquisition of sperm motility in salmonid fish. J. Exp. Zool. 261, 359–363.
https://doi.org/10.1002/jez.1402610316
Morisawa, M., Okuno, M., 1982. Cyclic AMP induces maturation of trout sperm
axoneme to initiate motility. Nature 295, 703–704.
https://doi.org/10.1038/295703a0
Morisawa, S., Morisawa, M., 1988. Induction of potential for sperm motility by
bicarbonate and pH in rainbow trout and chum salmon. J. Exp. Biol. 136, 13–22.
Morisawa, S., Morisawa, M., 1986. Acquisition of potential for sperm motility in
rainbow trout and chum salmon. J. Exp. Biol. 126, 89–96.
Müller, K., Müller, P., Pincemy, G., Kurz, A., Labbe, C., 2008. Characterization of sperm
plasma membrane properties after cholesterol modification: consequences for
cryopreservation of rainbow trout spermatozoa. Biol. Reprod. 78, 390–399.
Nandi, S., Routray, P., Gupta, S.D., Rath, S.C., Dasgupta, S., Meher, P.K.,
Mukhopadhyay, P.K., 2007. Reproductive performance of carp, Catla catla
(Ham.), reared on a formulated diet with PUFA supplementation. J. Appl.
Ichthyol. 23, 684–691. https://doi.org/10.1111/j.1439-0426.2007.00874.x
34
Nixon, B., Aitken, R.J., 2009. The biological significance of detergent-resistant
membranes in spermatozoa. J. Reprod. Immunol., Bio-immunoregulatory
Mechanisms Associated with Reproductive Organs: Relevance in Fertility and in
Sexually Transmitted Infections 83, 8–13.
https://doi.org/10.1016/j.jri.2009.06.258
Ohta, Hiromi, Ikeda, K., Izawa, T., 1997. Increases in concentrations of potassium and
bicarbonate ions promote acquisition of motility in vitro by Japanese eel
spermatozoa. J. Exp. Zool. 277, 171–180. https://doi.org/10.1002/(SICI)1097-
010X(19970201)277:2<171::AID-JEZ9>3.0.CO;2-M
Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., Iinuma, N., Hirose, K., 1997. Artificial
induction of maturation and fertilization in the Japanese eel, Anguilla japonica.
Fish Physiol. Biochem. 17, 163–169. https://doi.org/10.1023/A:1007720600588
Pereira, J.R., Pereira, F.A., Perry, C.T., Pires, D.M., Muelbert, J.R.E., Garcia, J.R.E.,
Corcini, C.D., Varela Junior, A.S., 2019. Dimethylsulfoxide, methanol and
methylglycol in the seminal cryopreservation of Suruvi, Steindachneridion
scriptum. Anim. Reprod. Sci. 200, 7–13.
https://doi.org/10.1016/j.anireprosci.2018.09.024
Pérez, L., Vílchez, M.C., Gallego, V., Morini, M., Peñaranda, D.S., Asturiano, J.F., 2016.
Role of calcium on the initiation of sperm motility in the European eel. Comp.
Biochem. Physiol. A. Mol. Integr. Physiol. 191, 98–106.
https://doi.org/10.1016/j.cbpa.2015.10.009
35
Perry, C.T., Corcini, C.D., Anciuti, A.N., Otte, M.V., Soares, S.L., Garcia, J.R.E.,
Muelbet, J.R.E., Varela, A.S., 2019. Amides as cryoprotectants for the freezing of
Brycon orbignyanus sperm. Aquaculture 508, 90–97.
https://doi.org/10.1016/j.aquaculture.2019.03.015
Pini, T., Leahy, T., de Graaf, S.P., 2018a. Sublethal sperm freezing damage:
Manifestations and solutions. Theriogenology 118, 172–181.
https://doi.org/10.1016/j.theriogenology.2018.06.006
Pini, T., Leahy, T., de Graaf, S.P., 2018b. Sublethal sperm freezing damage:
Manifestations and solutions. Theriogenology 118, 172–181.
https://doi.org/10.1016/j.theriogenology.2018.06.006
Quist, M.C., Stephen, J.L., Lynott, S.T., Goeckler, J.M., Schultz, R.D., 2010. An
Evaluation of Angler Harvest of Walleye and Saugeye in a Kansas Reservoir. J.
Freshw. Ecol. 25, 1–7. https://doi.org/10.1080/02705060.2010.9664351
Rojas, M.V., Esponda, P., 2001a. Plasma membrane glycoproteins during
spermatogenesis and in spermatozoa of some fishes. J. Submicrosc. Cytol. Pathol.
33, 133–140.
Rojas, M.V., Esponda, P., 2001b. Plasma membrane glycoproteins during
spermatogenesis and in spermatozoa of some fishes. J. Submicrosc. Cytol. Pathol.
33, 133–140.
Sandoval‐Vargas, L., Jiménez, M.S., González, J.R., Villalobos, E.F., Cabrita, E., Isler,
I.V., 2020. Oxidative stress and use of antioxidants in fish semen
cryopreservation. Rev. Aquac. n/a, 1–23. https://doi.org/10.1111/raq.12479
36
Schröter, S., Osterhoff, C., McArdle, W., Ivell, R., 1999. The glycocalyx of the sperm
surface. Hum. Reprod. Update 5, 302–313.
https://doi.org/10.1093/humupd/5.4.302
Schulz, R.W., de França, L.R., Lareyre, J.-J., LeGac, F., Chiarini-Garcia, H., Nobrega,
R.H., Miura, T., 2010. Spermatogenesis in fish. Gen. Comp. Endocrinol., Fish
Reproduction 165, 390–411. https://doi.org/10.1016/j.ygcen.2009.02.013
Shadan, S., James, P.S., Howes, E.A., Jones, R., 2004. Cholesterol Efflux Alters Lipid
Raft Stability and Distribution During Capacitation of Boar Spermatozoa. Biol.
Reprod. 71, 253–265. https://doi.org/10.1095/biolreprod.103.026435
Sleight, S.B., Miranda, P.V., Plaskett, N.-W., Maier, B., Lysiak, J., Scrable, H., Herr,
J.C., Visconti, P.E., 2005. Isolation and Proteomic Analysis of Mouse Sperm
Detergent-Resistant Membrane Fractions: Evidence for Dissociation of Lipid
Rafts During Capacitation. Biol. Reprod. 73, 721–729.
https://doi.org/10.1095/biolreprod.105.041533
Tapia, J.A., Macias‐Garcia, B., Miro‐Moran, A., Ortega‐Ferrusola, C., Salido, G.M.,
Peña, F.J., Aparicio, I.M., 2018. The Membrane of the Mammalian Spermatozoa:
Much More Than an Inert Envelope. Reprod. Domest. Anim. 47, 65–75.
https://doi.org/10.1111/j.1439-0531.2012.02046.x
Tecle, E., Gagneux, P., 2015. Sugar‐coated sperm: Unraveling the functions of the
mammalian sperm glycocalyx. Mol. Reprod. Dev. 82, 635–650.
https://doi.org/10.1002/mrd.22500
37
Thaler, C.D., Thomas, M., Ramalie, J.R., 2006. Reorganization of mouse sperm lipid
rafts by capacitation. Mol. Reprod. Dev. 73, 1541–1549.
https://doi.org/10.1002/mrd.20540
Tiersch, T.R., 2011. Process Pathways for Cryopreservation Research, Application and
Commercialization., in: Cryopreservation in Aquatic Species, 2nd Edition. World
Aquaculture Society, Baton Rouge, Louisiana, pp. 646–671.
Tiersch, T.R., Green, C.C., 2011. Cryopreservation in aquatic species, 2nd ed. World
aquaculture society Baton Rouge, LA.
Torres, L., Hu, E., Tiersch, T.R., 2016. Cryopreservation in fish: current status and
pathways to quality assurance and quality control in repository development.
Reprod. Fertil. Dev. 28, 1105. https://doi.org/10.1071/RD15388 van Gestel, R.A., Brewis, I.A., Ashton, P.R., Helms, J.B., Brouwers, J.F., Gadella, B.M.,
2005. Capacitation-dependent concentration of lipid rafts in the apical ridge head
area of porcine sperm cells. MHR Basic Sci. Reprod. Med. 11, 583–590.
https://doi.org/10.1093/molehr/gah200
Watson, P.F., 2000. The causes of reduced fertility with cryopreserved semen. Anim.
Reprod. Sci. 60–61, 481–492. https://doi.org/10.1016/S0378-4320(00)00099-3
Yanagimachi, R., Harumi, T., Matsubara, H., Yan, W., Yuan, S., Hirohashi, N., Iida, T.,
Yamaha, E., Arai, K., Matsubara, T., Andoh, T., Vines, C., Cherr, G.N., 2017.
Chemical and physical guidance of fish spermatozoa into the egg through the
micropyle,. Biol. Reprod. 96, 780–799. https://doi.org/10.1093/biolre/iox015
38
Yeste, M., 2016. Sperm cryopreservation update: Cryodamage, markers, and factors
affecting the sperm freezability in pigs. Theriogenology, Swine Reproduction 85,
47–64. https://doi.org/10.1016/j.theriogenology.2015.09.047
Yu, S., Kojima, N., Hakomori, S., Kudo, S., Inoue, S., Inoue, Y., 2002. Binding of
rainbow trout sperm to egg is mediated by strong carbohydrate-to-carbohydrate
interaction between (KDN)GM3 (deaminated neuraminyl ganglioside) and Gg3-
like epitope. Proc. Natl. Acad. Sci. U. S. A. 99, 2854–2859.
https://doi.org/10.1073/pnas.052707599
Zehmer, J.K., Hazel, J.R., 2003. Plasma membrane rafts of rainbow trout are subject to
thermal acclimation. J. Exp. Biol. 206, 1657–1667.
https://doi.org/10.1242/jeb.00346
Zilli, L., Schiavone, R., Vilella, S., 2017. Role of protein
phosphorylation/dephosphorylation in fish sperm motility activation: State of the
art and perspectives. Aquaculture, Recent advances in fish gametes and embryo
research 472, 73–80. https://doi.org/10.1016/j.aquaculture.2016.03.043
39
2017 2016 2015 2014 2013 2012 2011 2010 2009 2008 2007 2006
- 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Fish Produced ( x 10 6)
Figure 1. Ohio Department of Natural Resources – Division of Wildlife Saugeye production (2006-2017) in terms of millions of fry and fingerlings stocked. Red bars indicate the number of fry and grey bars represent the number of fingerlings produced.
40
140 120 100 80 60
40 and VAP(µm/s) and 20
TM (%), PM (%), Viability (%),(%),PM (%)Viability TM 0 Fresh 350 mOsm/kg 500 mOsm/kg 750 mOsm/kg Base Extender
Figure 2. Sauger sperm cryopreservation results from 2014-2018. Total motility (black), progressive motility (red), velocity (grey), and viability (white) are all compared among the 4 treatment groups: a fresh control and sperm frozen using base extenders at 350 mOsm/kg, 500 mOsm/kg, and 750 mOsm/kg. Extender 350 and 500 resulted in sufficiently high TM, PM, VAP, and viability compared to control, while 750 mOsm.kg severely reduced all measures or quality (n = 10).
41
***
60
)
%
(
n o
i 40
t
a
z
i
l
i
t
r e F a a 20 b b
0 Control 300,000 300,000 600,000 Motile Sperm−to−Egg Ratio
Figure 3. Sauger sperm cryopreservation fertilization results 2018. Fertilization in a control sample (fresh sperm at 300,000 MSE, white) was compared to sperm cryopreserved using 350 mOsm/kg (red) and 500 mOsm/kg (grey) base extenders at both
300,000 and 600,000 MSE. Fresh sperm had significantly higher fertilization compared to frozen sperm (p < 0.05). Extender 350 mOsm/kg had higher fertilization than 500 mOsm/kg at both MSE. Super scripts denote statistically significant means among the two cryopreserved sperm base extenders and MSE ratios (n = 5).
42
Table 1. Summary of cellular assessment characteristics, tools used for those assessments, and the target variable measured utilized in this dissertation.
Physiology Characteristics Analysis Tool Target References Electron Cell Ultrastructure (Figueroa et al., 2016) Microscopy Ultrastructure
Computer Motility Assisted Sperm (Billard and Cosson, 1992a) parameters Analysis Motility Multivariate Motile Sperm Clustering (Martínez-Pastor et al., 2011a) Subpopulations Analysis
Wheat germ N – acetyl- agglutinin (WGA) glucosamine Concanavalin A ⍺-mannose (Koehler, 1981) Glycocalyx (Con A) Peanut agglutinin β - galactose (PNA)
Membrane Filipin III (Flesch et al., 2001) Cholesterol Merocyanine 540 Membrane (Purdy et al., 2016) (MC540) Fluidity Cholera Toxin Ganglioside (Bai et al., 2019) Subunit β (CTB) GM1 Liquid Plasma Membrane Chromatography - Membrane Mass Lipid (Dietrich et al., 2019a) Spectroscopy Composition (LC-MS) Discontinuous Lipid Raft (Brogden et al., 2014a) ultracentrifugation Propidium Iodine Viability (Rurangwa et al., 2004) / Yo-Pro-1
Intracellular Fluo 4 AM (Guthrie et al., 2014) Calcium Protein Intracellular Signaling Anti- tyrosine,
Phosphoprotein threonine, and (Zilli et al., 2017) Antibodies serine phosphorylation
43
Chapter 2: Spatial and Temporal Changes in Testis Morphology and Sperm Ultrastructure of the Sportfish Sauger (Sander canadensis)
Abstract
Basic information pertaining to spermatogenesis in the economically important sportfish Sauger (Sander canadensis) is lacking. The objective of this study was to assess testicular morphology and spermatozoal structure spatially within the reproductive tract and temporally among seasons in the Sauger using light and electron microscopy. The testis exists as two separate lobes joined at the urogenital pore and is characterized as unrestricted lobular with seminiferous tubules terminating at the ventral periphery and coalescing dorsally on the main sperm duct. Differences were observed between the pre- breeding season (November) and breeding season (March), with every stage of spermatogenesis occurring in spermatocysts during pre-breeding season. By contrast, only spermatozoa were present in the tubules and main duct during the breeding season.
Longitudinal folds in the main duct epithelium increased in number with increasing proximity to the urogenital pore, greatly increasing epithelial height regardless of season.
Sauger spermatozoa consisted of an ovoid head, a midpiece containing 2-4 mitochondria incorporated into the head, and a single flagellum containing an asymmetrical lateral ribbon. Motile spermatozoa were found throughout the testis during the breeding season. 44
A decrease in sperm concentration was quantified moving toward the urogenital pore, suggesting a hydration effect by the main duct epithelium during the breeding season.
These observations fill an important knowledge gap regarding reproductive biology, including the sperm maturation process in the main duct of this impactful recreational fish species.
Introduction
As a taxonomic group, fish manifest wide variation in reproductive physiology compared to any other group of vertebrates, and these characteristics can vary even among closely related species (Sloman, 2011). Thus it is important to identify the reproductive characteristics of each species to help manage broodstock for assisted reproduction techniques (Alavi, 2008). Fish are known to display distinct seasonality in sperm production and quality. Following spermiation, sperm are not yet capable of fertilization as has been demonstrated in the Masu Salmon (Oncorhynchus masou,
Morisawa and Morisawa, 1988b) and European Eel (Anguilla anguilla, Hiromi Ohta et al., 1997; H. Ohta et al., 1997). Fish sperm are known to mature in the main sperm duct in response to a hormonally driven process of hydration (via progestins, e.g. 17α,20β – dihydroxy-4-pregmem-3-one (DHP, Schulz et al., 2010). This final maturation, necessary for sperm to acquire the ability to achieve motility via hypo-osmotic shock, is associated with an increase in sperm duct pH and intracellular cAMP (Miura et al., 1992a). While these changes have been described in relation to breeding and nonbreeding individuals, few investigations have described changes within the breeding season to testicular
45 histology, sperm cells, and the structure of the main sperm duct in relation to the proximity of the urogenital pore. The main sperm duct acts as a storage site for spermatozoa preceding spawning and thus could substantially impact sperm cell physiology and maturity. Analysis of testis histology and the sperm duct and also how they change within and among seasons is important in furthering our knowledge of reproductive physiology in freshwater fish.
No information regarding sperm and testis structure or maturational processes is available in the Sauger (Sander canadensis), an important sportfish species used in the production of hybrid Saugeye (S. vitreus x S. canadensis) that are commonly stocked through the midwestern United States. As Saugeye production is highly variable due to limited sperm production per individual Sauger compounded by fluctuations in populations and weather conditions, assisted production strategies are necessary for sustaining the multimillion-dollar recreational fishery industry in Ohio reservoirs.
Previous research has focused on managing sperm use (Blawut et al., 2018b), long-term storage of excess sperm using cryopreservation (Blawut et al., 2020a), and utilizing testicular harvest to enhance sperm yield (Appendix A). However, sperm quality (i.e., morphology, maturation, and motility) can vary as a result of the unintended consequences of these assisted reproduction techniques and thus could adversely impact
Saugeye production.
Systematic descriptions of the structure, organization, and seasonal changes in the
Sauger testis and spermatozoa are unavailable. Therefore, a fuller understanding of sperm maturation within the testis, as well as ultrastructural features of spermatozoa, could
46 substantially benefit our efforts to use assisted reproduction techniques in Saugeye production.
The objectives of this study were (i) to describe the morphological features of testicular structures and spermatozoa in the Sauger along the length of the lobules
(proximal, middle, and distal regions from the urogenital pore) during the pre-breeding and breeding seasons and (ii) to determine any potential seasonal differences in sperm quality. We hypothesized that maturational changes in both the testicular environment and spermatozoa are a result of seasonal changes associated with spawning and occur spatially along the sperm duct as sperm approach the common sperm duct prior to spawning.
Materials and methods
Experimental design
Temporal and spatial changes in testicular structure and maturation of Sauger spermatozoa were assessed by collecting and comparing histological data between the pre-breeding and breeding season and among testicular sections located proximal, middle, or distal to the urogenital pore. Testes from Sauger broodstock were harvested from six individuals prior to the beginning of the breeding season (November 2019) and seven during the reproductive season (March 2019). All testes were fixed and sectioned for histological analysis. Spatial and seasonal effects were examined by comparing spermatozoa cell types (e.g., spermatozoa, spermatids, primary spermatocytes etc.) and efferent duct epithelium thickness between the breeding and pre-breeding season.
47
Additionally, electron microscopy and traditional sperm assessments (motility parameters and spermatozoa concentration) were used to compare sperm ultrastructure and quality between stripped and testicular from different sections of the testis across the breeding season.
Broodstock Collection and Testicular Harvest
Ohio Department of Natural Resources – Division of Wildlife (ODNR-DOW) personnel used electrofishing to collect mature, male Sauger from the Ohio River near the
Greenup, KY, USA dam prior to the breeding season (November 2020) and again during the breeding season (late February to mid-March of 2019). Sauger broodstock were either euthanized on site using cervical transection (November) or transported by truck for ~2 hours in an aerated live-well in 0.5% NaCl solution to the London State Fish Hatchery isolation facility (London, OH). In the hatchery, broodstock were maintained in a ~2840
L indoor recirculating system at 5 to 6 °C with a flow rate of 45 to 57 L/hour. Fish were maintained in tanks with natural photoperiod exposure. Body mass (g) and total length
(mm) were recorded to the nearest 0.1 for each individual used in this study. The Ohio
State University (Columbus, OH) Institutional Animal Care and Use Committee approved all procedures and animal use prior to the beginning of the study (Protocol
#2015A00000008).
48
Histology
Testes used for histological analysis were prepared using standard methodology and then processed by The Ohio State University College of Veterinary Medicine
Comparative Mouse Phenotyping Shared Resource (Columbus, OH). Testes were removed from the body cavity and immersed in 10% neutral buffered formalin for 24 hours. After initial fixation, the testes were sectioned into three sections (total lobe length divided by three) corresponding to proximity to the urogenital pore: proximal, middle, and distal (Figure 1.B.). Three separate cuts were taken from the middle of each testicular section and allowed to fix for an additional 24 hours to ensure penetration of the fixative to the central portion of the tissues. Tissue samples were processed, embedded in paraffin wax, sectioned at 5µm thickness, and stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS).
Multiple measures were taken for each tissue section using the image analysis software, Image J (National Institutes of Health, Bethesda, MD). A microscope at 2,000 x magnification was used to take three random images from each histological slide after which images were exported to Image J for analysis. The approximate area of each cell type was estimated using methods similar to those used by Tomkiwicz et al (2011). A grid consisting of 80 points was overlain on the image and individual points were classified as spermatozoa (Spz), spermatids (Spt), secondary spermatocytes (Ssc), primary spermatocytes (Psc), spermatagonia (Spg), somatic cells (Int), or luminal space
(L). The area encompassed by each sperm cell type was estimated using the following formula:
49
푁푢푚푏푒푟 표푓 푝표𝑖푛푡푠 푚푎푡푐ℎ𝑖푛푔 푐푒푙푙 푡푦푝푒 푥 ( ) × 100 푇표푡푎푙 푁푢푚푏푒푟 표푓 푝표𝑖푛푡푠
Additionally, the height of the efferent duct epithelium was measured (to closest 1 µm) for each tissue section using Nikon Elements Advanced Research software (Nikon Inc.,
NY, USA). Measurements (n = 90) spanning proximal, middle, and distal regions of the main duct were recorded for each individual. Ten measurements were made for each of three serial sections per region to minimize effect of heterogeneity within a single section profile. The main duct epithelium thickness for an individual was determined by averaging all measurements made within a testicular region.
Scanning and Transmission Electron Microscopy
Scanning and transmission electron microscopy were used to assess stripped and testicular sperm ultrastructure. For scanning electron microscopy, samples of stripped and testicular sperm (from the whole tract) were fixed in 3% glutaraldehyde in Rathbun extender and stored at 4°C for approximately 3 months. Fixed sperm were adhered to a poly-L-lysine-coated coverslip for 10 min at room temperature. Coverslips were dehydrated using graded ethanol solutions (50%, 70%, 80%, 95%, and 100%) followed by graded HMDS solutions (25%, 50%, 75%, 100%). Coverslips were then allowed to dry in a fume hood overnight, mounted on aluminum stubs, and coated (~25 nm thickness) with gold/palladium (Cressington 108; Ted Pella Inc., Redding, CA). The samples were then observed and photographed using a Nova Nano scanning electron microscope (Nova NanoSEM 400; FEI, Hillsboro, OR).
50
For transmission electron microscopy, sperm samples from stripped individuals, as well as milt harvested from the proximal, middle, and distal portion of the testis, were pooled from three individuals during the breeding season and fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH = 7.4) with 0.15 M sucrose. After approximately 60 min of fixation, the fixative was removed, and cells were re-suspended in 0.1 M cacodylate buffer. Samples were shipped at room temperature overnight to the Colorado State
University Animal Reproduction and Biotechnology Laboratory where samples were further processed and evaluated using procedures developed to evaluate frog spermatogenesis (Lee and Veeramachaneni, 2005). Briefly, upon arrival, samples were post-fixed for 90 min in 1.0% osmium tetroxide (in 0.1 M cacodylate buffer), rinsed in buffer, and dehydrated through a graded series of ethanol solutions followed by rinsing in propylene oxide. Sample preparations were embedded in poly/Bed 812 (Polysciences
Inc., Warrington, PA) and sectioned using a Ultracut microtome (Richert-Jung, Vienna,
Austria). One-µm-thick sections were cut and mounted on glass slides, stained with toluidine blue for light microscopy to ensure optimal cut surface and to facilitate initial evaluation of cells. Then, ~80-nm-thin sections were cut and mounted on 300-mesh nickel grids and stained with uranyl acetate and lead citrate for characterization of ultrastructural features using a JOEL-1200EX transmission electron microscope (JEOL
USA, Inc., Peabody, Massachusetts).
51
Sperm Quality Assessment
Sperm motility potential and cell concentration were assessed in stripped sperm and testicular sperm from each segment of the testis. Sperm were collected using both strip-spawning on site and testicular harvest in the laboratory from three individual
Sauger during the breeding season. Strip-spawned sperm sampels were diluted with 1.0 mL Rathbun extender (Moore, 1987a) and stored at 5°C until further analysis. Each testis was divided into three equal parts according to length (proximal, middle, and distal) as described above. Each section was macerated in 1.0 mL Rathbun extender to release spermatozoa and then thoroughly mixed. Samples were then assessed using computer- assisted sperm analysis (CASA) and densimetry to determine motility, sperm velocity, and sperm concentration according to previously published methods (Blawut et al.,
2020a, 2018b).
Statistical Analysis
All statistical analyses were completed using R (R Development Core Team,
2009) using an alpha value of 0.05 to determine significance. Normality and homogeneity of variance of each predictor and dependent variable was confirmed using normal quantile and residual plots, respectively. Sauger body mass, total length, and gonadosomatic index (GSI: [testis mass/body mass] *100) were compared between the pre-breeding and breeding season using t-tests. Testicular cell types, main duct epithelium height, and sperm parameters (motility and concentration) were compared among testicular sections and between seasons using linear mixed models and Tukey’s
52
HSD post hoc mean comparison. A random effect for individual was added to the model to account for within-subject variation. All results are reported as mean ± 1 standard error of the mean (SEM).
Results
Testicular Structure
The Sauger testis exists as a paired organ suspended from the dorsal body cavity by a thin ligament (Figure 4.A). The point of origin is the urogenital pore, beginning as two lobes fused to form the common sperm duct. These lobes extend separately cranially where they terminate at the site of the main testicular artery in the vicinity of the pectoral girdle. The length of the testis from the urogenital pore to the tip of the longest lobe was approximately 90.6 ± 15.2mm (mean ± standard deviation). No accessory sex glands were observed. Immature testes were thin and transparent, whereas mature testes ranged from appearing pale white during the pre-breeding season to milky white at the time of spawning.
The Sauger testis can be classified as the lobular unrestricted type described by
Grier (1981), with spermatogonia distributed throughout the entire organ (Figure 5 A).
Seminiferous tubules (i.e., seminiferous epithelium containing Sertoli cells and developing germ cells) and tubular lumen, blindly terminate ventrally at the periphery of the testis under the thin tunica albuginea and extend dorsally toward the main sperm duct
(i.e., lobular lumen). Each testicular lobe contained a dorsally located main sperm duct that stretches along the entire length of the testis. The two main sperm ducts fuse to form the common duct immediately prior to the urogenital papilla. The main duct epithelium 53 consisted of simple squamous cells distally and transitions to cuboidal cells covering thick, branched longitudinal epithelial folds from middle to proximal segments. This duct was both empty and flattened during pre-breeding season or contained sperm cells and were fully expanded during the breeding season. No germ cells other than mature spermatozoa, were observed within the main or common ducts at any time. Germ cells in earlier stages of differentiation (spermatocytes – spermatids) were enclosed within spermatocysts. Seminiferous tubule walls were composed of myoid cells and connective tissue fibers surrounded by interstitial tissue containing Leydig cells and blood vessels.
Temporal changes to testicular morphology
Sauger body mass (268.5 ± 45.4 g) or total length (325.9 ± 33.2 mm) did not differ among seasons (p = 0.09 and p = 0.33). Gonadosomatic index, however, was significantly higher during the pre-breeding season (1.74 ± 0.15 %) compared to during the breeding season (1.06 ± 0.10%, p = 0.0017).
Histology revealed that Sauger testes displayed seasonal cyclicity. During the pre- breeding season, 6 of 9 male Sauger showed signs of testicular development (milk white, large testis), whereas the remaining three individuals were determined to be immature
(e.g., thin, transparent testis) based on gross testicular structure. At this time, spermatocysts containing all germ cell types from spermatogonia through spermatids were present (Error! Reference source not found. B). As early as November, s permatozoa were present in the lumen of the seminiferous tubules (Error! Reference source not found.A, 35 – 40% of cells), and several individuals had spermatozoa in the
54 main sperm duct. Spermatids as well as both secondary and primary spermatocytes were only observed during the pre-breeding season. Spermatids were more prevalent in the distal portion (29.6 ± 1.5 %) compared to proximal portions (21.9 ± 1.5 %), with the intermediate values in the middle portion.
Juvenile testes consisted primarily of spermatogonia dispersed throughout the lobular lumen without discernible seminiferous tubule structures evident (Figure 5 C).
All individuals sampled during March were in spawning state (readily released sperm with gentle abdominal pressure). Near all differentiating germ cells during the breeding season were spermatozoa (Error! Reference source not found. A). The r emainder of cells types counted were empty lumen space between spermatozoa and the interstitium (5 – 8%) or the interstitium itself (11 - 12%) regardless of proximity to the urogenital pore.
Spatial changes to testicular morphology
Differences in sperm cell composition and epithelial height were observed among the testicular sections. Main duct epithelial thickness increased in each testicular section moving from distal (35.5 ± 6.8 µm) to middle (62.5 ± 6.8 µm) to proximal (88.0 ± 6.8
µm, p < 0.001, Figure 7 A). No differences in epithelial thickness existed between seasons (p = 0.50). The increase in main duct epithelial height was a result of the increased frequency of longitudinal folds observed moving proximally. These folds were characterized by vacuolated, branching structure of the interstitium and cuboidal epithelial cell linings (Figure 7 B). Simple squamous epithelium was observed between
55 folds (Figure 7 C). Lack of PAS-positive staining in the Sauger testis and main sperm ductal epithelium indicated little to no glycogen content or polysaccharide secretion, respectively.
Sperm structure
Sauger spermatozoal structure is consistent with the highly conserved structure of most teleosts including closely related Percids (Figure 8 A). The head was approximately
1.81 ± 0.15 µm in height and 1.24 ± 0.12 µm deep, resulting in a more ovoid shape. A single flagellum averaging 26.45 ± 2.39 µm in length was inserted asymmetrically into one side of the head. An acellular cytoplasmic channel within the head surrounded the initial insertion point of the flagellum. The cytoplasm around this channel had approximately 2-4 irregularly shaped mitochondria. These characteristics give the sperm a mid-piece that was poorly differentiated from the sperm head. Upon exiting the head, the flagellum exhibited an asymmetric ribbon like protrusion of cytoplasm, referred to as the flagellar ribbon (Figure 8 B), along the length of the axoneme to its terminus. The axoneme itself consisted of a 9 × 2 + 2 microtubule arrangement with radial spokes and dynein arms (Figure 8 C). The nucleus of the sperm consisted of condensed chromatin material with a recognizable “nuclear notch” similar to those noted in other percids.
Adjacent to the nucleus was the centriolar complex. The proximal centriole rested above an electron-dense plate and was separated from the distal centriole by cytoplasm. The distal centriole possessed two electron-dense regions parallel to the orientation to the
56 flagellum, with an extension to the nucleus for attachment. The two centrioles were oriented at a 90° angle from one another.
Spatial changes in spermatozoa structure
Electron microscopy revealed subtle differences among sperm extracted from each of the sections of the testis and stripped sperm. These subtle differences largely reflect the state of chromatin condensation and perhaps the stripping process might have dislodged a few incompletely condensed late-stage spermatids. No apparent damage to cellular or nuclear membranes was noted in any samples observed during this study.
Fragments of seminiferous tubules including Sertoli cell nuclei, as well as spermatocysts, were observed; more cystic profiles were seen in the portion of the duct farthest from the urogenital pore (Figure 9 A). The distal section also showed some evidence of spermatids being present, confined to spermatocyst fragments. Interestingly, the middle section showed heterogenous sperm cell sizes and chromatin condensation within cysts unlike either the proximal or distal section, potentially indicating a maturational transition zone between fully mature spermatozoa in the proximal section and less advanced forms in the distal region. Red blood cells were found in each of the three testicular sections, most likely a result of the collection process (Figure 9 B).
Scanning electron microscopy revealed that testicular sperm were more likely to contain sperm heads lacking a flagellum than stripped sperm. Whether this difference is natural or was the result of decapitation during the process of testicular sperm collection and processing could not be determined from the current analysis.
Spermatozoa motility potential and concentration
57
Spermatozoa collected from the testis prior to the breeding season had little to no potential for motility (< 10% motile) and no milt could be manually strip-spawned from these individuals. Spermatozoa collected by strip-spawning and testicular harvest from each segment during the breeding season did possess the potential for motility. Sperm concentrations were highest in the distal portion of the testis, intermediate in the middle section, and lowest in the proximal portion of the testis and strip-spawned sperm (Table
2). Motility was significantly reduced in stripped sperm compared to testicular sperm, but is most likely a result of urine contamination as these values are not consistent with previous values for this species (Blawut et al., 2020a, 2018b).
Discussion
Our results provide a first look at testicular and spermatozoal ultrastructure of
Sauger as well as detail the effects of spatial and temporal maturational processes within the testis in this recreationally important sport fish species.
The structure of the Sauger spermatozoa and testis are similar to those seen in most other teleosts and other percids, specifically the Eurasian perch, Perca fluviatilis
(Hatef et al., 2011; Lahnsteiner et al., 1995), and pikeperch, Sander lucioperca (Křišťan et al., 2014; Lahnsteiner and Mansour, 2004). Common to nearly all teleosts, these three species all consisted of an ovoid head lacking an acrosome, a poorly differentiated midpiece, and a single flagellum possessing a flagellar ribbon typical of “aquasperm”.
However, the structure of Sauger sperm shared characteristics of both pikeperch and
Eurasian perch. The poorly defined midpiece in Sauger was more similar to that of the
58
Eurasian perch, in that 1-2 mitochondria were similarly arranged at the base of the nucleus and cytoplasmic channel (Lahnsteiner et al., 1995). However, the midpiece in the
Sauger was less conspicuous than in Eurasian perch as it retains a less distinct profile relative to the head. A nuclear notch was visible in Sauger spermatozoa, similar to the pikeperch and in contrast to the Eurasian perch. Its functional role has yet to be determined. Finally, the flagella of the three species differed in length and organization.
The Sauger flagellum was shorter than in pikeperch and Eurasian perch (26.5 µm vs. 30-
35 µm, respectively) and its flagellar ribbon was more asymmetrical with the longer extension on the nuclear side of the sperm than in either the pikeperch or Eurasian perch.
Sauger testis histology was also consistent with the results of other percid species. The more advanced unrestricted lobular type testis seen in Walleye (Malison et al., 1994) and
Eurasian perch (Perca fluviatilis, KROL et al. 2006) were also observed in Sauger.
All teleosts show cyclical reproductive development, and the Sauger is no exception. Seasonal progression of spermatogenesis in Sauger was consistent with the results seen in other percids. Specifically, the zingel (Zingel asper) and Eurasian perch both displayed proliferation and development of sperm cells as early as fall (e.g.,
October) proceeding the spring breeding season. In contrast to what was seen in Walleye
(Sander vitreus, (Malison et al., 1994), Sauger exhibited a significant proportion of sperm cells in earlier stages of spermatogenesis ( i.e. spermatocytes and spermatids) during
November (Chevalier et al., 2011; Krol et al., 2006).
Other differences were observed in the testis aside from sperm cell development among seasons. The gonadosomatic index (GSI) in Sauger was highest at a point
59 coinciding with active spermatogenesis and sperm cell proliferation. GSI has previously been shown to be highest prior to the breeding season in Eurasian perch (Krol et al.,
2006). However, as the GSI was decreasing leading up to spawning, the interstitium of the seminiferous tubules increased in thickness. Similar observations have been reported in Parasilurus aristotelis (Iliadou and Fishelson, 1995) and Perca fluviatilis (Krol et al.,
2006). We speculate spermatocyst remnants following spermiation may account for this increase in tubule wall thickness. Collectively, these results indicate that Sauger testis maturation is similar to what is noted in other percids. Moreover, the presence of spermatozoa within the testis of Sauger well-ahead of the breeding season strengthens the assertions that the main sperm duct may have an effect on sperm physiology given its role as a temporary sperm storage site.
Evidence of ductal maturation potential is widespread in mammalian species but has only recently been described in fish. The epididymis in mammals displays distinct regions based on cell types, lumen diameters, and epithelial height (Goyal, 1985). For instance, epididymis epithelial layer thickness decreases from the initial segment to the vas deferens (Dacheux et al., 2005; Goyal and Williams, 1991; Ibrahim and Singh, 2014;
Jones et al., 1984; Scott et al., 1963), presumably to facilitate sperm movement for ejaculation at the cauda section. In fish, sperm maturation has been shown to occurs in the spermatic duct of the smelt (Osmerus eperlanus L. ) and Masu Salmon
(Oncorhynchus masou) under the influence of milt hydration, a process driven by the
MIS hormone 17alpha,20beta-DP (Kowalski et al., 2012; Miura et al., 1992a; Morisawa and Morisawa, 1988b), potentially mediated by the ductal epithelium. In contrast,
60 sturgeon spermatozoa post-testicular maturation occurs as a result of urine contamination
(Boccaletto et al., 2018). In our study, we found that spermatozoa from each section of the testis were equally motile but also observed a decrease in sperm concentration in moving from distal to proximal portions. Similarly, spermatozoa of the whitefish
(Coregonus lavaretus L.) throughout the testis had the ability for motility activation
(Hliwa et al., 2010) which is in stark contrast to Morisawa and Morisawa (1988) who found distally located sperm were immotile until incubated with bicarbonate (i.e., a high pH environment). Both studies showed increased sperm concentrations in the distal potion compared to the proximal but contrasting motility potentials in the more distal spermatozoa.
Longitudinal folds in the epithelium of the main sperm duct were present in Sauger and substantially impacted histological structure. Extension of the interstitium of the main duct covered by a single layer of epithelium were common in the Sauger, but an increasingly high density of these folds directly contributed to the increase in epithelium thickness/height. Unlike other studies, we found no seasonal effect on the frequency or thickness of these structures (Muñoz et al., 2011; Walter et al., 2005). These folds were covered by cuboidal epithelium whereas the surrounding epithelium was simple squamous, and this relationship did not differ among seasons. Other studies have shown approaching spawning season contributed to a change in the epithelium cell type and function along the duct as seen in Leporinus microcephalus (Muñoz et al., 2011),
Leuciscus cephalus (Walter et al., 2005), and Gymnotus carapo (Souza et al., 2015).
More cuboidal epithelium in the duct proximal to the urogenital pore was associated with
61 decreased sperm concentrations in this section, potentially as a result of seminal fluid excretion similar to what is seen in the blue swimmer crab (Portunus pelagicus) (Ravi et al., 2014). However, a lack of PAS staining in the Sauger testis indicated the absence of polysaccharide secretory ability or glycogen content. Fish seminal plasma is often lacking detectable levels of polysaccharides (Lahnsteiner et al., 1993), leaving the possibility open for different secretory products being produced by the epithelium. Seminal plasma analysis from each section of the testis could provide more information regarding contributions from the ductal epithelium and is a potential area of interest for future studies.
A more comprehensive view of sperm maturation in Sauger could be achieved by overcoming some of the limitations in this study. A more complete sampling protocol to include multiple checkpoints per year (e.g., monthly) would be sure to shed more light on the transitional periods of recrudescence, initiation of spermatogenesis, and spermiation.
This sampling schedule would also be informative in assessing temporal and spatial acquisition of motility by Sauger sperm. And lastly, fertilization data from each of the testicular segments during the breeding season could provide more information on the physiological competence of sperm harvested from the testis with respect to spatial origin. Further investigation to address these limitations would be helpful to fully understand the reproductive physiology of sperm maturation in the Sauger.
62
Conclusions
In conclusion, our results were able to confirm testis and spermatozoa structures consistent with other members of Percidae. Most importantly, the data support a temporal shift in spawning status as well as the potential for spatial maturation of the spermatozoa within the testis like epididymal maturation in mammalian species. Specifically, changes in the main sperm duct epithelium (height and epithelial cell types) attributed to the size and frequency of longitudinal folds and heterogeneity of sperm ultrastructure moving distally support a role of the main sperm duct in potential post-testicular maturation.
63
References
Alavi, S.M.H., 2008. Fish spermatology. Alpha Science International Ltd.
Blawut, B., Wolfe, B., Moraes, C.R., Ludsin, S.A., Coutinho da Silva, M.A., 2018.
Increasing Saugeye (S. vitreus × S. canadensis) production efficiency in a
hatchery setting using assisted reproduction technologies. Aquaculture 495, 21–
26. https://doi.org/10.1016/j.aquaculture.2018.05.027
Blawut, B., Wolfe, B., Moraes, C.R., Ludsin, S.A., Silva, M.A.C. da, 2020a. Use of
Hypertonic Media to Cryopreserve Sauger Spermatozoa. North Am. J. Aquac. 82,
84–91. https://doi.org/10.1002/naaq.10125
Blawut, B., Wolfe, B., Moraes, C.R., Sweet, D., Ludsin, S.A., Coutinho da Silva, M.A.,
2020b. Testicular collections as a technique to increase milt availability in Sauger
(sander canadensis). Anim. Reprod. Sci. 212, 106240.
https://doi.org/10.1016/j.anireprosci.2019.106240
Boccaletto, P., Siddique, M.A.M., Cosson, J., 2018. Proteomics: A valuable approach to
elucidate spermatozoa post –testicular maturation in the endangered
Acipenseridae family. Anim. Reprod. Sci. 192, 18–27.
https://doi.org/10.1016/j.anireprosci.2018.03.033
Chevalier, C., de Conto, C., Exbrayat, J.-M., 2011. Reproductive biology of the
endangered percid Zingel asper in captivity: a histological description of the male
reproductive cycle. Folia Histochem. Cytobiol. 49, 486–496.
Dacheux, J.-L., Castella, S., Gatti, J.L., Dacheux, F., 2005. Epididymal cell secretory
activities and the role of proteins in boar sperm maturation. Theriogenology,
64
Proceedings of the V International Conference on Boar Semen Preservation 63,
319–341. https://doi.org/10.1016/j.theriogenology.2004.09.015
Goyal, H.O., 1985. Morphology of the bovine epididymis. Am. J. Anat. 172, 155–172.
https://doi.org/10.1002/aja.1001720205
Goyal, H.O., Williams, C.S., 1991. Regional differences in the morphology of the goat
epididymis: A light microscopic and ultrastructural study. Am. J. Anat. 190, 349–
369. https://doi.org/10.1002/aja.1001900404
Grier, H.J., 1981. Cellular Organization of the Testis and Spermatogenesis in Fishes. Am.
Zool. 21, 345–357. https://doi.org/10.1093/icb/21.2.345
Hatef, A., Alavi, S.M.H., Butts, I.A.E., Policar, T., Linhart, O., 2011. Mechanism of
action of mercury on sperm morphology, adenosine triphosphate content, and
motility in Perca fluviatilis (Percidae; Teleostei). Environ. Toxicol. Chem. 30,
905–914. https://doi.org/10.1002/etc.461
Hliwa, P., Dietrich, G.J., Dietrich, M., Nynca, J., Martyniak, A., Sobocki, M., Stabinski,
R., Ciereszko, A., 2010. Morphology and histology of cranial and caudal lobes of
whitefish (Coregonus lavaretus L.) testes. Fundam. Appl. Limnol. Arch. Fr
Hydrobiol. 176, 83–88. https://doi.org/10.1127/1863-9135/2010/0176-0083
Ibrahim, Z.H., Singh, S.K., 2014. Histological and morphometric studies on the
dromedary camel epididymis in relation to reproductive activity. Anim. Reprod.
Sci. 149, 212–217. https://doi.org/10.1016/j.anireprosci.2014.07.012
65
Iliadou, K., Fishelson, L., 1995. Histology and Cytology of Testes of the Catfish
Parasilurus aristotelis (Siluridae, Teleostei) from Greece. Jpn. J. Ichthyol.
41, 447–454. https://doi.org/10.11369/jji1950.41.447
Jones, R.C., Hinds, L.A., Tyndale-Biscoe, C.H., 1984. Ultrastructure of the epididymis of
the tammar, Macropus eugenii, and its relationship to sperm maturation. Cell
Tissue Res. 237, 525–535. https://doi.org/10.1007/BF00228437
Kowalski, R.K., Hliwa, P., Cejko, B.I., Krol, J., Stabinski, R., Ciereszko, A., 2012.
Quality and quantity of smelt (Osmerus eperlanus L.) sperm in relation to time
after hormonal stimulation. Reprod. Biol. 12, 231–246.
Křišťan, J., Hatef, A., Alavi, S.M.H., Policar, T., 2014. Sperm fine structure of the
pikeperch, Sander lucioperca (Percidae, Teleostei). Czech J. Anim. Sci. 59, 1–10.
Krol, J. (University of W. and M., Glogowski, J. (Polish A. of S., Demska-Zakes, K.
(University of W. and M., Hliwa, P. (University of W. and M., 2006. Quality of
semen and histological analysis of testes in Eurasian perch Perca fluviatilis L.
during a spawning period. Czech J. Anim. Sci. - UZPI Czech Repub.
Lahnsteiner, F., Berger, B., Weismann, T., Patzner, R., 1995. Fine structure and motility
of spermatozoa and composition of the seminal plasma in the perch. J. Fish Biol.
47, 492–508. https://doi.org/10.1111/j.1095-8649.1995.tb01917.x
Lahnsteiner, F., Mansour, N., 2004. Sperm fine structure of the pikeperch, Sander
lucioperca (Percidae, Teleostei). J. Submicrosc. Cytol. Pathol. 36, 309–312.
Lahnsteiner, F., Patzner, R.A., Welsmann, T., 1993. The spermatic ducts of salmonid
fishes (Salmonidae, Teleostei). Morphology, histochemistry and composition of
66
the secretion. J. Fish Biol. 42, 79–93. https://doi.org/10.1111/j.1095-
8649.1993.tb00307.x
Lee, S.K., Veeramachaneni, D.N.R., 2005. Subchronic Exposure to Low Concentrations
of Di-n-Butyl Phthalate Disrupts Spermatogenesis in Xenopus laevis Frogs.
Toxicol. Sci. 84, 394–407. https://doi.org/10.1093/toxsci/kfi087
Malison, J.A., Procarione, L.S., Barry, T.P., Kapuscinski, A.R., Kayes, T.B., 1994.
Endocrine and gonadal changes during the annual reproductive cycle of the
freshwater teleost,
Fish Physiol. Biochem. 13, 473–484. https://doi.org/10.1007/BF00004330
Miura, T., Yamauchi, K., Takahashi, H., Nagahama, Y., 1992. The role of hormones in
the acquisition of sperm motility in salmonid fish. J. Exp. Zool. 261, 359–363.
https://doi.org/10.1002/jez.1402610316
Moore, A., 1987. Short-Term Storage and Cryopreservation of Walleye Semen. Progress.
Fish-Cult. 49, 40–43. https://doi.org/10.1577/1548-
8640(1987)49<40:SSACOW>2.0.CO;2
Morisawa, S., Morisawa, M., 1988. Induction of potential for sperm motility by
bicarbonate and pH in Rainbow Trout and chum salmon. J. Exp. Biol. 136, 13–
22.
Muñoz, M.E., Batlouni, S.R., Vicentini, I.B.F., Vicentini, C.A., 2011. Testicular structure
and description of the seminal pathway in Leporinus macrocephalus
(Anostomidae, Teleostei). Micron 42, 892–897.
https://doi.org/10.1016/j.micron.2011.06.008
67
Ohta, Hiromi, Ikeda, K., Izawa, T., 1997. Increases in concentrations of potassium and
bicarbonate ions promote acquisition of motility in vitro by Japanese eel
spermatozoa. J. Exp. Zool. 277, 171–180. https://doi.org/10.1002/(SICI)1097-
010X(19970201)277:2<171::AID-JEZ9>3.0.CO;2-M
Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., Iinuma, N., Hirose, K., 1997. Artificial
induction of maturation and fertilization in the Japanese eel, Anguilla japonica.
Fish Physiol. Biochem. 17, 163–169. https://doi.org/10.1023/A:1007720600588
R Development Core Team, 2009. R: A language and environment for statistical
computing.
Ravi, R., Manisseri, M.K., Sanil, N.K., 2014. Structure of the male reproductive system
of the blue swimmer crab Portunus pelagicus (Decapoda: Portunidae). Acta Zool.
95, 176–185. https://doi.org/10.1111/azo.12017
Schulz, R.W., de França, L.R., Lareyre, J.-J., LeGac, F., Chiarini-Garcia, H., Nobrega,
R.H., Miura, T., 2010. Spermatogenesis in fish. Gen. Comp. Endocrinol., Fish
Reproduction 165, 390–411. https://doi.org/10.1016/j.ygcen.2009.02.013
Scott, T.W., Wales, R.G., Wallace, J.C., White, I.G., 1963. COMPOSITION OF RAM
EPIDIDYMAL AND TESTICULAR FLUID AND THE BIOSYNTHESIS OF
GLYCERYLPHOSPHORYLCHOLINE BY THE RABBIT EPIDIDYMIS.
Reproduction 6, 49–59. https://doi.org/10.1530/jrf.0.0060049
Sloman, K.A., 2011. The Diversity of Fish Reproduction: An Introduction, Encyclopedia
of Fish Physiology: From Genome to Environment, Vols 1-3. Elsevier Academic
Press Inc, San Diego.
68
Souza, L.C.M.D., Retamal, C.A., Rocha, G.M., Lopez, M.L., 2015. Morphological
evidence for a permeability barrier in the testis and spermatic duct of Gymnotus
carapo (Teleostei: Gymnotidae). Mol. Reprod. Dev. 82, 663–678.
https://doi.org/10.1002/mrd.22503
Walter, I., Tschulenk, W., Schabuss, M., Miller, I., Grillitsch, B., 2005. Structure of the
seminal pathway in the European chub, Leuciscus cephalus (Cyprinidae);
Teleostei. J. Morphol. 263, 375–391. https://doi.org/10.1002/jmor.10312
69
Table 2. Comparison of sperm motility characteristics and cell concentration among stripped sperm and sperm harvested from
different regions of the testis (proximal, middle, or distal to the urogenital pore) in the Sauger (Sander canadensis). a,b Mean values (±
SEM) with different superscript letters within a row differ significantly at P < 0.05.
Sperm Type Sperm Characteristics Distal Middle Proximal Stripped Total Motility (%) 43.7 ± 6.0 a 53.9 ± 6.0 a 55.7 ± 6.0 a 12.3 ± 6.0 b
Curvilinear Velocity (µm/s) 156.1 ± 12.9 154.0 ± 12.9 164.1 ± 12.9 121.0± 12.9 Sperm Concentration (×10 9 61.1 ± 7.1 a 45.2 ± 7.1 ab 26.9 ± 7.1 b 18.9 ± 7.1 b sperm/mL)
7
0
70
A.) Cranial Testicular Lobes Caudal
Urogenital Pore
30 mm
B.) Dital Middle Proximal
Urogenital Pore
30 mm
Figure 4. Anatomical location of the Sauger reproductive tract (A) and experimental design (B) of histological analysis of the testes.
71
A .
B .
C .
Figure 5. Histological micrograph of a mature male Sauger testis during the A) prebreeding season (40×), B) breeding season (40×), and C) testis of immature individuals (100 ×). The bolded arrow indicates the main sperm duct.
72
Figure 6. Testis cellular comparison between seasons (pre-breeding and breeding) and among testis sections (proximal, middle, and
distal to the urogenital pore). (A) Cumulative proportions of each cell type for each treatment represented graphically. (B)
Representative micrographs of (from left to right) spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids,
mature spermatozoa, interstitial cells, and lumen).
73
73
A. 100% 90%
80%
70%
60%
50%
40%
74 30%
Cumulative Proportion Cumulative Proportion (%)
20%
10%
0% Prebreeding Distal Prebreeding Median Prebreeding Proximal Breeding Distal Breeding Median Breeding Proximal Season / Proximity to Urogenital Pore Interstitial Cells Lumen Primary Spermatocytes Secondary Spermatocytes Spermatids Spermatogonia Spermatozoa B.
2* sp
Figure 6. 74
Figure 7. Main sperm duct epithelium comparison among proximal, middle, and distal portions of the Sauger testis. (A) Epithelium thickness (height µm) among different sections and between seasons (white: pre-breeding, black: breeding). (B-C)
Representative micrographs of epithelium lateral folds in the proximal duct (B) and simple squamous epithelium seen more commonly in the distal duct (C) and between epithelial fold regions in other sections.
75
A. B.)
Nu Pc Nu
Nu Mi FF Dc Nn
FrFr Mi Cc C.)C.) Mi Mi Fr Ax F
Figure 8. Electron micrographs of Sauger spermatozoa ultrastructure. (A) TEM image of the spermatozoa head and initial portion of the flagellum. (B) SEM image of the exterior of the spermatozoa head. (C) TEM image of the flagellum, flagellar ribbon, and axonemal organization (9 × 2 + 2 microtubule organization). Ax, axoneme; Cc, cytoplasmic channel; Dc, distal centriole; F, flagellum; Fr, flagellar ribbon; Mi, mitochondria; Nn; nuclear notch; Nu, nucleus; Pc, proximal centriole.
76
A B. .
*
Figure 9. Electron micrographs of sperm cell abnormalities and somatic cell fragments in the collection of testicular sperm in Sauger. (A) Incomplete chromatin condensation (*) of the sperm cell nucleus and Sertoli cell fragments (black arrow). (B) Commonly observed profile of a red blood cell (white arrow) seen in all testicular sperm samples.
77
Chapter 3: Changes to The Spermatozoa Glycocalyx and Its Role in Fertilization in
Sauger (Sander canadensis)
Abstract
The spermatozoa glycocalyx is a dynamic coating of extracellular glycoproteins known to facilitate the acrosome reaction and fertilization in mammals. Most fish sperm, however, contain no acrosome and are subjected to a different set of stimuli (osmotic shock in the external environment) prior to fertilization. At present, little is known about the composition and spatial distribution of sugar moieties nor the functional role of the glycocalyx in fish sperm. Moreover, the impact of assisted reproduction techniques (e.g., testicular harvest and cryopreservation) commonly used in aquaculture settings on this structure in fish sperm is unknown. The objective of this study was to describe and compare the composition and characteristics of the glycocalyx among sperm types
(stripped, testicular, and cryopreserved) and between activation statuses (inactive vs. activated) using Sauger as a model (Sander canadensis). Additionally, the importance of certain moieties (e.g., N-acetyl-glucosamine) to fertilization was investigated. Staining distributions (i.e., patterns, %), mean fluorescent intensity (a.u), and cell populations exhibiting high fluorescence (%) were measured for each treatment using fluorescent 78 microscopy and flow cytometry, respectively. Three lectins commonly used in mammalian sperm (Wheat germ agglutinin, WGA [N-acetyl-glucosamine]; ConA, concanavalin A [α-mannose]; and peanut agglutinin, PNA [β- galactose]) were used to monitor these variables in fish sperm. The Sauger glycocalyx contained GlcNAc and α- mannose but lacked β- galactose moieties. Testicular sperm exhibited fewer cells (20-
40% fewer) with high GlcNAc and α – mannose content than stripped and frozen sperm and was positively correlated with motility (r = 0.8 and 0.95), suggesting the glycocalyx is impacted by post-testicular maturation. Motility activation via hypo-osmotic shock caused a redistribution of GlcNAc to the apical region of the head of 40 - 50% of stripped and testicular sperm, respectively. Cryopreserved sperm showed significantly reduced apical staining following activation (< 5%) as well as a 2-3-fold increase in α-mannose availability. Additionally, fertilization was reduced by ~ 80% compared to a fresh control in both stripped sperm pre-treated with WGA to block GlcNAc during insemination and cryopreserved sperm. These results support a dynamic Sauger sperm glycocalyx which is modified during post-testicular maturation, motility activation, and plays a pivotal role in fertilization. Cryopreservation largely negated these changes, which may partially explain the reduced fertility observed in frozen sperm. Thus, analysis of the glycocalyx using lectins, e.g., WGA and ConA, as biomarkers provides insight into the maturational status and fertilization potential of Sauger sperm, and potentially other freshwater fish species.
79
Introduction
In mammals, the glycocalyx of sperm cells is a layer of glycoproteins and glycolipids that have significant implications for fertilization (Tecle and Gagneux,
2015b). Proteins essential to gamete-fusion (i.e. fertilins, ARP, Izumo etc) are critical components of the sperm glycocalyx and recent evidence suggests cryopreservation can alter the glycocalyx structure and potentially its function (Pini et al., 2018b; Schröter et al., 1999b). In contrast to mammalian sperm where the structure and function of the glycocalyx is well established, little is known about the glycocalyx of freshwater fish spermatozoa. The limited amount of evidence we do have is for an internally fertilizing fish species Xiphophorus maculatus and Schroederichthys chilensis (Rojas and Esponda,
2001b), and a small number of studies in externally fertilizing Rainbow Trout
(Oncorhynchus mykiss) (Kudo, 1998b; Yu et al., 2002b). Some changes in the plasma membrane glycocalyx of internally fertilizing fish species following motility activation resemble the changes observed in mammalian sperm during capacitation (Rojas and
Esponda, 2001b). Moreover, syndecan, a protein family rich in heparin sulfate (rich in N- acetyl-glucosamine/scialic acid), was found to be an essential sperm surface component to fertilization in Rainbow Trout (Oncorhynchus mykiss) (Kudo, 1998b) and interactions between these carbohydrates in sperm head gangliosides and carbohydrates in the micropyle region were important to fertilization (Yu et al., 2002b). Aside from the above references, not much is known regarding the glycocalyx components, spatial distribution, its dynamic nature, or which tools can be used to monitor this structure in externally fertilizing freshwater fish sperm. Given the important contribution of glycocalyx
80 components to sperm physiology in mammals, this structure in externally fertilizing freshwater fish could have a direct role in oocyte recognition during fertilization and could be adversely affected by manipulation using assisted reproduction technologies.
To address this knowledge gap, we investigated the presence, distribution, relative quantity of glycocalyx, and their importance to fertilization of freshwater fish sperm using Sauger (Sander canadensis) as a model. Sauger are the paternal contributor of the recreationally important Saugeye (S. vitreus × S. canadensis) and there is considerable interest in the large scale application and optimization of stripped, testicular, and frozen sperm to enhance annual production of these sport fish (Blawut et al., 2020a, 2020b,
2018b). However, limited and variable fertilization success using cryopreserved (Blawut et al., 2020a) and testicular sperm (Blawut et al., 2020b), respectively, suggests that certain aspects of the sperm cell’s physiology are adversely affected by these assisted reproduction techniques and warrant further investigation . In this study, differences in the cell glycocalyx among fresh testicular, stripped, and frozen-thawed sperm in both the inactive and activated state (after hypo-osmotic shock) were assessed to describe and compare the effects of hypo-osmotic shock, testicular harvest, and cryopreservation on this structure. We hypothesized differences exist in the glycocalyx of testicular sperm and stripped sperm due primarily to the lack of residency in the main testicular duct, which may act similarly to the mammalian epididymis to enhance maturation. Second, we hypothesized that cryopreservation would alter the distribution and decrease the quantity of glycoproteins in response to the harsh cooling/thawing environment. Third, we hypothesize motility activation by hypo-osmotic shock will affect the distribution and
81 quantity of these glycoproteins in the plasma membrane in freshwater fish. And lastly, we hypothesized that fertilization would be negatively impacted by blocking the availability of certain glycoproteins prior to insemination.
Materials and Methods
Experimental Design
A within-subjects experiment was conducted using a 3 x 2 factorial structure to describe and analyze the composition, distribution, quantity, and importance of glycocalyx components to fertilization utilizing lectins specific to certain sugar moieties
(GlcNAc, α – mannose, and β-galactose). The two factors tested were sperm type
(testicular, stripped, and cryopreserved) and activation status (inactive and activated).
Sperm from three individuals was collected by strip-spawning and testicular harvest as described previously (Blawut et al., 2020b) and pooled based on the method of collection
(n = 12 pools total per sperm type). Stripped sperm was cryopreserved according to a previously published protocol for Sauger (Blawut et al., 2020a). Each of these three sperm types (testicular, stripped, and cryopreserved) were analyzed in both the inactive and activated states for the presence, distribution, and relative amount of sugar moieties.
Additionally, the relevance of GlcNAc to fertilization was assessed using samples pretreated with lectins to block glycoprotein GlcNAc availability.
82
Broodstock, Milt Collection, Pooling, and Analysis
Sauger broodstock were collected and maintained using methods reported previously (Blawut et al., 2020a, 2020b, 2018b). In summary, Ohio Department of
Natural Resources – Division of Wildlife (ODNR-DOW) personnel collected mature, male Sauger from the Ohio River near the Greenup, KY, USA dam via electrofishing during the 2018 and 2019 breeding seasons. Sauger were transported by truck for ~2 hours in an aerated live-well 0.5% NaCl solution to the London State Fish Hatchery isolation facility (London, OH, USA) where they were maintained in a ~2840 L indoor recirculating system at 5 to 6 °C with a flow rate of 45 to 57 L/hour. Fish were maintained in tanks with natural photoperiod exposure. The Ohio State University
(Columbus, OH, USA) Institutional Animal Care and Use Committee approved all procedures and animal use prior to the beginning of the study (Protocol
#2015A00000008).
Milt was collected from Sauger using both strip-spawning and testicular harvest.
Individuals were strip-spawned by placing them in dorsal recumbency, drying the urogenital pore thoroughly to avoid premature activation (Billard and Cosson, 1992b), and stripped to collect milt using abdominal massage and a 1.0 mL rubberless syringe
(S7510-1, Thermo Fisher Scientific, MA, USA). Immediately after collection, the total volume of the stripped milt was determined (nearest 0.01 mL) and the sample was extended 1:3 with 350 mOsm/kg Rathbun extender ((g/l): CaCl2·2H2O, 0.117;
MgCl2·6H2O, 0.134; Na2HPO4, 0.236; KCl, 1.872; NaCl, 6.578; glucose, 10.000; citric
83 acid, 0.100; NaOH, 0.254; and bicine, 1.06; pH 8.5 (Bergeron et al., 2002a; Moore,
1987b)). Extended milt was maintained at 5° C in a Styrofoam box until further use.
The same individuals were then euthanized for testicular harvest with an overdose of tricaine sulfate (MS-222, Western Chemical, Ferndale, WA, USA) at a dose of ~ 1 g/L in 2018 or cervical transection followed by decapitation in 2019. Euthanized individuals were stored on ice and transported to the laboratory (< 1 h transport). Once in the lab, testicular sperm was harvested from each euthanized individual using methodology described by Blawut et al (Blawut et al., 2020b). Briefly, the testes of euthanized fish were removed from the body cavity. Then each lobe of the testes was cut along its length with a scalpel and pressure was applied to the lacerated lobes from the urogenital pore to the tip of the lobe to expel sperm. Testicular milt was then diluted using 3.0 mL of
Rathbun extender, homogenized, and transferred to a test tube for storage at 5°C as described above.
Sample motility was assessed objectively using computer assisted sperm analysis
(CASA, Hamilton Thorne, MA, USA). Stripped and testicular sperm were diluted to 0.5
× 10 9 sperm/mL prior to analysis. Cryopreserved sperm was analyzed undiluted. Two µL of sperm was activated in 38µL of 0.22µM filtered hatchery water containing 1% BSA at a pH of 8.3. Activated sperm were loaded into a Microtool chamber (#B7, Cytonix LLC,
MD, USA) and analyzed at 10s post-activation using CASA software using acquisition parameters described previously (Blawut et al., 2020a).
Milt collected using strip spawning and testicular harvest were pooled by sperm type to prevent individual biases. Stripped sperm samples from three Sauger with motility
84
> 70% were combined (i.e., pooled) and maintained at 5°C prior to cryopreservation. The corresponding testicular milt samples from the same cohort of three Sauger were also pooled and maintained at 5°C until analysis.
Sperm Cryopreservation and Thawing
A subsample of pooled stripped sperm was cryopreserved using an established protocol. (Blawut et al., 2020a). To summarize, stripped milt was diluted to 1.0 × 10 9 sperm/mL using Rathbun extender supplemented with 4 mg/mL bovine serum albumin
(BSA). After a 10 min equilibration period, sperm were further diluted (1:1, v: v) with
Rathbun + BSA supplemented with dimethyl sulfoxide (DMSO). The resulting diluent consisted of 5.0 × 10 8 sperm/mL in Rathbun plus 4 mg/mL BSA and 10% DMSO as cryoprotectants. During the 10-min equilibration with the cryoprotectant, samples were loaded into 0.5 mL straws and then cooled in vapor at 3 cm above liquid nitrogen for 10 min. Samples were then plunged into liquid nitrogen and stored at - 196C prior to thawing at 37°C for 10 s in a water bath. Thawed samples were maintained at 5°C until further analysis.
Fluorescent Microscopy
To assess the distribution of specific sugar moieties on the plasma membrane glycocalyx, Sauger sperm were stained with fluorescent conjugated lectins using previously published protocols (Pelaez et al., 2011; Rojas and Esponda, 2001b; Pini et al., 2018c) with some modifications. Inactive sperm stock solutions were prepared by
85 diluting sperm to 5.0 × 10 8 sperm/mL using Rathbun extender containing either no
DMSO (for stripped and testicular sperm) or 10% DMSO (cryopreserved sperm) to maintain media osmolality, and thus inactive status. Activated sperm stock solutions were prepared by diluting sperm 20-fold in 0.22 µm filtered hatchery well water (pH 8.3, 35 mOsm.kg) for 30 s, and then diluted 1:1 with 2× Rathbun to prevent further osmotically driven changes to sperm structure and physiology (i.e., loss of viability). Separate aliquots of inactive and activated sperm stock solutions were incubated for 30 min with
10 μg/mL wheat germ agglutinin (WGA, specific to N-acetyl-glucosamine [GlcNAc]), 7
μg/mL peanut agglutinin (PNA, specific to β-galactose [β-gal]), or 100 μg/mL concanavalin (ConA, specific to α-mannose) conjugated to Alexa Fluor 488 (Thermo
Scientific) at 5°C (previously optimized to give adequate fluorescence without strong background). Propidium iodine (PI, 1.2 μM) was added to each sample for the last 10 min of incubation to stain non-viable cells. Viable sperm cells (n = 100) from each sample were analyzed using a Nikon Eclipse Ti and Intensilight C-HGFIE lamp with a 1000 × oil objective (Nikon Inc., NY, USA). The distribution of fluorescence on individual sperm membrane was classified as heterogeneous, homogenous, or apical (Fig 1). An unstained control was used to assess auto fluorescence to prevent biases during distribution assessment.
Flow Cytometry
Flow cytometry analysis was used to quantify lectin binding to the plasma membrane of the different sperm types and status. Similar to the methods described in
86 section 2.4, sperm were diluted to approximately 5.0 10 7 sperm/ mL from inactivate and activated stock solutions and then incubated for 30 min with either 10 μg/mL WGA,
μg/mL PNA, or 100 μg/mL ConA. Samples were stained using 1.2 µM PI for the last 10 min of incubation to discriminate viable and non-viable cells. Approximately 20,000 viable sperm cells per sample were analyzed using an NXT Attune Acoustic Flow
Cytometer (Thermo Fisher Scientific). Forward and side scatterplots were used to exclude debris, and forward area and height scatterplots were used to remove doublets from the analysis. Alexa Fluor 488 was detected using the 530/40 nm band pass filter at
300 V and PI was detected using the 613/20 nm band pass filter at 460 V. No compensation was required due to little spectral overlap. Quantitative endpoints included sample mean fluorescent intensity (M.F.I., arbitrary units) and the proportion of cells exhibiting high fluorescence (%).
Lectin Western Blot
Qualitative changes to glycocalyx GlcNAc sugar moieties were visualized using lectin blotting. Sperm cell membranes were extracted from samples (stripped, testicular, and cryopreserved sperm in both the inactive and activated state) using Mem-PER Plus membrane extraction buffer (Thermo Scientific) according to manufacturer instructions.
Total protein concentrations were measured using a bicinchoninic acid assay (Smith et al., 1985a). Membrane lysates were stored at - 80°C until further analysis. Protein content
(10 µg) was normalized per lane and separated by electrophoresis for 1 hour at 180 V in a
4-15% sodium dodecyl sulfite page gel (Criterion Cell, Bio-Rad, CA, USA) and blotted
87 onto PVDF membrane for 1.25 h at 100V (Criterion Blotter, Bio-Rad). Membranes were blocked for 1 h at room temperature using Carbo-Free Blocking Solution (Vector Labs,
CA, USA) and then labeled for 15 min at room temperature using 1 µg/mL horseradish peroxidase conjugated wheat germ agglutinin (Vector Labs). Membranes were washed three times (5 min each) in PBS and imaged with Amersham 600 digital imager (GE
Healthcare, IL, USA) using ECL Plus substrate (Thermo Fisher Scientific).
Fertilization
The importance of GlcNAc moieties to fertilization was assessed by comparing fertility among stripped sperm (control), stripped sperm treated with the WGA lectin
(specific to GlcNAc), and cryopreserved sperm. Prior to insemination, stripped sperm was diluted to 5.0 × 10 8 sperm/mL. A subsample of diluted stripped sperm was treated with 10 μg/mL WGA for at least 30 min prior to insemination to bind to sperm GlcNAc moieties. Cryopreserved sperm was thawed as described above (section 2.3) immediately prior to insemination. Approximately 1,500 eggs collected from 2-3 female Walleye
(Sander vitreus) captured from the Maumee River (Toledo, OH) were inseminated using
300,000 motile sperm/egg from each of the three treatment groups (i.e., stripped, stripped
+ WGA, cryopreserved). This ratio is known to exceed the minimum number of stripped sperm required to reach maximum fertilization during the production of Saugeye (Blawut et al., 2018b). Activation was accomplished using a 10-fold dilution in hatchery well water (pH 8.3, ~ 35 mOsm/kg). After insemination was complete, fertilized oocytes were treated with a 400-ppm tannic acid solution (3 min), and a 50-ppm ovadine solution for
88
30 min (Western Chemical) prior to packaging and transport to the London State Fish
Hatchery (< 3h, London, OH, USA). Upon arrival, a second 100 ppm ovadine treatment was applied to fertilized eggs for 10 min. Treated eggs were incubated in a Heath tray incubator (Marisource Inc., WA, USA) using well water with a flow rate of 22 L/ min at
12 °C for approximately 12 d, at which time they reached the eyed-stage of development.
The proportion of eyed-stage Saugeye embryos was determined in 100 eggs for each replicate using a dissection scope at 40 × magnification. The experiment was replicated twice (n = 2).
Data Analysis
All analyses were completed using R statistical software (R Development Core
Team, 2009) with an alpha value of 0.05. Mean fluorescent intensity of WGA (n = 10) and ConA (n =12) staining was compared using linear mixed models to quantify the effect of sperm type (fixed factor: testicular, stripped, or cryopreserved) and status (fixed factor: inactive or active). A random effect for pool of origin was included in the model.
Staining pattern distribution (%), proportion of cells expressing high fluorescence (%), and viability (%) were analyzed using logistic regression to compare the staining patterns seen based on sperm type (fixed factor: testicular, stripped, or cryopreserved) and status
(fixed factor: inactive or active). A random effect for pool of origin was included in each model. Motility variables were compared among sperm types using a linear mixed model including a fixed effect for sperm type and random effect of pool of origin. Total motility’s relationship with lectin staining patterns and proportion of highly fluorescent
89 cells were assessed using Pearson correlations and linear regression (sperm type as a fixed factor and motility as a covariate). One data point was removed from the preceding analyses, thought to be the result of poor testicular harvest technique (excessively low motility rate after extended interval between euthanasia and harvest, < 20% motility). For each model, least square means, SE, and confidence intervals for the probabilities were calculated and Tukey’s HSD post-hoc tests were used to compare means among treatment combinations. Fertilization and percent change in fertilization after treatment with WGA are reported. All data are expressed as mean ± 1 SEM.
Results
Sauger spermatozoa from each group in this study stained strongly with WGA and ConA, suggesting the glycocalyx contained both GlcNAc / sialic acid and α– mannose moieties. Staining was seen throughout the head and tail in three possible distributions: apical, homogenous, and heterogenous (Figure 10). However, no visible staining was observed using fluorescent microscopy or flow cytometry for PNA on the surface of Sauger sperm indicating that the Sauger sperm glycocalyx lacks terminal β- galactose.
Wheat germ agglutinin staining distribution, fluorescent intensity, and highly fluorescent cell populations were affected by sperm type, activation status, and their interactive effects (Figure 11, Table 3). First, a significant interaction was found between sperm type and activation status for homogenous, heterogeneous, and apical staining (all p < 0.0001). In inactive sperm, the predominant WGA staining patterns
90 observed were homogenous and heterogeneous distributions of fluorescence throughout both the head and tail (Figure 11 A). Frozen sperm contained fewer homogenously stained sperm (~ 40% lower) and more heterogeneously stained sperm (30-50% greater) than stripped or testicular sperm in the inactive state. Only a small proportion of inactive sperm, regardless of sperm type, exhibited an apical staining pattern (1.6 – 4.6 %).
However, after activation, the proportion of sperm with apical staining increased 3 to 12- fold in all three sperm types. Activated testicular and stripped sperm showed greater proportions of apical staining (39.1 ± 2.3 and 51.1 ± 2.4 %), which was highest in stripped sperm (p < 0.05). By contrast, cryopreserved sperm in the active state had the lowest proportion of apical stained sperm (4.6 ± 0.7 %).
Second, the results of WGA fluorescent intensity analysis revealed a trend toward an interaction between sperm type and its activation status (p = 0.06, Figure 11 B), with cryopreserved sperm showing the seemingly highest fluorescence after activation.
Statistically, mean fluorescence differed among sperm types (p < 0.001). Testicular sperm exhibited consistently lower levels of WGA compared to fresh and frozen sperm.
Third, an interaction between sperm type and activation status was found in regard to the proportion of highly fluorescent cells stained with WGA (Table 3, p =
0.01). The results were similar to the weak trend identified in mean fluorescent intensity.
Testicular sperm contained the lowest proportion of cells with high fluorescence (49-
65%), with stripped and cryopreserved sperm showing a significantly larger proportion
(83 – 93%). Cryopreserved sperm after activation contained the largest proportion of highly fluorescent cells (93.1 ± 2.5 %).
91
The sugar moiety GlcNAc was prominent in one membrane protein as determined using western blot. Wheat germ agglutinin blotting indicated a single band of strong staining at approximately 35 kDa with no changes associated with sperm type or activation status (Figure 12).
Concanavalin A staining distribution, fluorescent intensity, and high fluorescent cell populations also differed among sperm types, activation status, and their combined interactive effects (Figure 13, Table 3). Firstly, no apical staining was observed using
ConA. Significant interactions between sperm type and activation status were found for both heterogeneous (p = 0.002) and homogenous staining (p = 0.003). The most prominent staining distribution observed in Sauger sperm, independent of sperm type or activation status, was homogenously stained cells (> 90 % in all samples, Figure 13 A).
The amount of homogenously stained sperm in cryopreserved samples increased ~4% following motility activation. By contrast, testicular and stripped sperm remained at a constant level after activation compared to the inactive state.
Secondly, ConA mean fluorescent intensity was affected by sperm type (p <
0.001) but not activation status (p = 0.50). ConA fluorescence was highest in cryopreserved sperm (Figure 13 B), approximately 2-3 times higher compared to stripped and testicular sperm. No difference in ConA fluorescence was found between stripped and testicular sperm.
Lastly, sperm populations exhibiting high ConA fluorescence were affected by an interaction of sperm type and motility activation (p < 0.0001, Table 3). Testicular samples again contained fewer sperm displaying high fluorescence than stripped or
92 cryopreserved samples, and it was affected by motility activation (increase by 20%).
Stripped and cryopreserved sperm populations did not change as a result of motility activation. A small difference in the proportion of highly fluorescent sperm existed between inactive stripped sperm (83.9 ± 2.4%) and activated cryopreserved sperm (91.8
± 1.4%).
Motility characteristics and viability differed among sperm types and activation statuses. Results of motility comparison among sperm types are listed in Table 4. Total motility was 20% higher in stripped sperm compared to both testicular and cryopreserved. However, testicular sperm were more similar to stripped sperm in regard to sperm trajectories. The main differences between testicular and stripped sperm were reduced curvilinear velocity and amplitude of lateral head displacement. Every measure of sperm motility was affected by cryopreservation with the exception of straight-line velocity, which did not differ among treatments. Sperm viability was affected by an interactive effect of sperm type and activation status (p < 0.001, Table 4). Testicular sperm experienced a reduction in viability (17 %) following motility activation while stripped and cryopreserved did not.
Motility’s relationship with glycocalyx parameters (mean fluorescent intensity and proportion of highly fluorescent cells) depended on the type of sperm. Moderately strong, positive correlations were found between total motility and apical WGA staining and the proportion of cells containing high proportions of GlcNAc and α-mannose (r =
0.54, 0.42, and 0.42, respectively). Further assessment using analysis of covariance indicated that apical WGA staining’s correlation was driven by differences among sperm
93 types, and total motility was not a useful predictor in the model (β = 0.09, p = 0.51). By contrast, an interaction between motility and sperm type on the proportion of highly fluorescent cells was found using WGA or ConA (both p < 0.01). Motility in testicular sperm (range 36.9 – 72.9 %) showed a positive relationship with the proportion of highly fluorescent cells for ConA (β = 1.97(SE = 0.33), R2= 0.81, p < 0.001) and WGA (β =
1.21(0.40), R2= 0.76, p = 0.005). This relationship was not observed in stripped or cryopreserved sperm, however. Finally, a strong, positive relationship was found between the proportions of highly fluorescent cells stained using ConA and WGA (r = 0.95, p <
0.001). These results suggest that both lectins and their respective moieties are qualitatively proportional.
Sugar moieties contained within the glycocalyx were found to play a role during fertilization. Blocking GlcNAc /sialic acid by pre-incubating samples with WGA prior to insemination had a negative effect on fertilization (n = 2, mean SD). Despite high fertilization in stripped sperm, treating the same sample with WGA caused an ~ 80% reduction in fertilization relative to the stripped control (fresh: 79.3 4.2%, WGA- treated: 14.0 1.4%). The reduction in fertilization success in WGA-treated sperm was similar to the decrease in fertilization observed in between stripped and cryopreserved sperm (17.4 3.7%).
Discussion
The purpose of this study was to describe the composition and dynamics of the sperm glycocalyx in Sauger, an externally fertilizing freshwater fish. The glycocalyx of
94
Sauger sperm was found to contain two of the three components, GlcNAc and α- mannose, which are known to be major components of the mammalian and internally fertilizing fishes’ sperm glycocalyx. While β-galactose was not observed in the membrane of Sauger sperm, GlcNAc and α-mannose showed different distributions, relative abundances, and high fluorescence populations among the three sperm types and after motility activation by hypo-osmotic shock. These results provide the first observations into the dynamic nature of freshwater fish glycocalyx with implications for fertility.
The importance of glycocalyx components (most notably GlcNAc /sialic acid) to fertilization in freshwater fish has been studied but not in great detail. The dynamic nature of these components and their response to assisted reproductive technologies has not been reported before (Kudo, 1998b; Yanagimachi et al., 2017a; Yu et al., 2002b). The increase in apical staining following hypo-osmotic shock during this study demonstrates an important change in sperm physiology as a result of motility activation. It is interesting to note the lack of correlation between motility and apical staining. This suggests that while the two processes share a common stimulus (i.e., hypo-osmotic shock), they are independent processes. Moreover, these changes resulting from motility activation suggest that the GlcNAc components may be involved in fertilization/cell-cell adhesion in the micropyle region. Redistribution of these components to the apical portion of the head, presumably the structure to first contact the egg, suggests these components may interact with the chorion, micropyle, and oocyte plasma membrane.
Interestingly, the funnel-like micropyle leading to the oolemma in Rainbow Trout eggs
95 are also rich in sugar moieties (e.g. GlcNAc), indicating that glycocalyx components are concentrated in areas relevant to fertilization in both the male and female gametes
(Yanagimachi et al., 2013). Furthermore, treating stripped sperm with the WGA lectin to block GlcNAc moieties reduced fertilization approximately 80%, solidifying the role of the glycocalyx in fertilization. Consistent with our findings regarding GlcNAc, heparin sulfate ( a structure rich in GlcNAc moieties) seemed to be an important glycocalyx component to Rainbow Trout sperm, and blocking it with antibodies reduced fertilization to approximately 15% (Kudo, 1998b). Collectively, our findings show that the glycocalyx of fish sperm could be involved in chemical guidance of the sperm toward and into the micropyle or interaction with the oolemma.
Lectins such as WGA, PNA, and ConA are often used in mammalian sperm to indicate the capacitation status or maturity of sperm (Jiménez et al., 2003; Liu, 2016;
Sakaguchi et al., 2009). Our results suggest that WGA and ConA could be used for similar applications in fish sperm. Using both WGA and ConA, we found that a larger proportion of cells in testicular sperm were classified as low fluorescent cells when compared to stripped sperm. Moreover, in testicular sperm alone, the motility potential of each sample was strongly correlated with the proportion of cells with high glycoprotein content. This comparative increase in cell glycoprotein content may indicate a similar phenomenon seen during epididymal maturation in mammal sperm where certain components (e.g., sialic acid) of the sperm plasma membrane increase in abundance during post-testicular transport (Schröter et al., 1999b). However, a loss of membrane sialic acid / GlcNAc in addition to a redistribution of sugar moieties is associated with
96 capacitation leading to the acrosome reaction, hyperactive motility, and fertilization in the female tract in a majority of mammalian species (Liu, 2016). By contrast, we did not observe loss of glycoproteins during any process (maturation, motility activation, or cryopreservation) in Sauger sperm. Regardless, in the Sauger, WGA and ConA have proven useful as an indicator of sperm’s maturational status during testicular residency.
Despite having a higher proportion of immature sperm, successful fertilization can still be achieved with testicular sperm using practical sperm-to-egg ratios (Blawut et al., 2020b).
Cryopreservation induced the most significant alterations in the fish sperm glycocalyx. Available -mannose increased 2-3-fold in frozen sperm compared to stripped and testicular sperm. Ram (Ovus domesticus) sperm showed similar trends to our study as evidenced by an increase in the α-mannose content of the glycocalyx after cryopreservation, which was attributed to oxidative damage (Pini et al., 2018c). Apart from increased α-mannose availability, few meaningful differences were found in the proportion of cells exhibiting high fluorescence in both WGA and ConA between stripped and frozen sperm, suggesting sugar moieties are not being lost as a result of the freeze-thaw process. These results suggest ConA staining can be useful as an indicator of glycocalyx degradation in fish sperm. For example, the glycocalyx of somatic cells in early stages of apoptosis show an increase in α-mannose and β-galactose availability at the cell surface (Bilyy and Stoika, 2003; Bilyy et al., 2004; Taatjes et al., 2008).
Together, these results suggest that the increase in α-mannose content seen in Sauger sperm indicates early stages of cellular apoptosis. Regarding glycocalyx function, cryopreserved sperm did not demonstrate the ability for hypo-osmotic shock to induce
97 apical staining of GlcNAc to the same degree seen in stripped and testicular sperm, despite becoming motile. Again, the relative quantity of GlcNAc was not affect by cryopreservation, suggesting that the failure to demonstrate apical staining was due to a lack of membrane redistribution rather than loss of these moieties. Furthermore, the our previous findings of reduced fertilization ability in frozen Sauger sperm (Blawut et al.,
2020a) combined with reduced fertilization when blocking GlcNAc moieties and lost moiety mobility in frozen sperm demonstrated here indicates that the loss of GlcNAc redistribution is at least partially responsible for reduced fertility seen in cryopreserved sperm. Collectively, lectin analysis of the frozen Sauger sperm glycocalyx indicates reduced fertilization can be explained by a lack of sugar moiety mobility potentially resulting from membrane changes associated with early stages of cellular apoptosis.
Our results highlight a number of avenues for future research. The limited number of sugars visualized in this study likely does not capture the full spectrum of complexity in these cells. A more complete view of the changes to the quantity of moieties could be taken using lectin microarrays (Xin et al., 2018), which could delineate good and poor quality sperm samples and post-thaw quality assessment. Fluorescent conjugated lectins have proven to be an important analysis tool for sperm quality, and our results suggest that WGA and ConA may be a valuable sperm quality assessment tool in freshwater fish.
Additionally, cryopreservation’s implications for fertility concerning GlcNAc could help to explain, at least in part, why superficially competent frozen sperm lacks the level fertilization as seen in fresh sperm. Future studies should focus on different
98 cryopreservation protocols (e.g., cryoprotectants, base medias, additives) effects on the sperm glycocalyx and further relate these changes to fertilization.
Conclusion
The results presented here describe the dynamic nature of the spermatozoa glycocalyx in freshwater fish exhibiting an external fertilization reproductive pattern.
Herein, we demonstrated that β – galactose is absent in Sauger sperm while GlcNAc and
α – mannose were major components throughout both sperm head and tail. Testicular sperm exhibited fewer cells with high GlcNAc and α – mannose content (20- 40% lower), and these parameters were strongly correlated with motility potential. Combined, these results indicate that changes to the glycocalyx are associated with final sperm maturation in the main sperm duct. Motility activation via hypo-osmotic shock caused a redistribution of GlcNAc to the apical region of the head of 40-50% of stripped and testicular sperm. Cryopreservation significantly impacted both α-mannose content (2 to
3-fold increase) and prevented GlcNAc redistribution (<5% apically stained cells) following hypo-osmotic shock. Further, we were able to show that treating Sauger sperm with WGA prior to insemination reduced fertilization rates by ~ 80 %, implicating an essential role of GlcNAc in fertilization. The implications of this research indicate that freshwater fish sperm glycocalyx goes through a natural series of structural and physiological changes in response to post-testicular maturation and external stimuli, such as motility activation in a hypo-osmotic environment. However, cryopreservation largely negated the activation effect on sugar moiety redistribution and caused apoptosis-like
99 changes to the glycocalyx, which at least partially explained the significant reduction in fertilization ability observed in frozen sperm. Thus, lectins specific to N-acetyl- glucosamine and α-mannose may have value as biomarkers to quantify maturational status and fertilization potential in freshwater fish sperm.
100
References
Bergeron, A., Vandenberg, G., Proulx, D., Bailey, J.L., 2002. Comparison of extenders,
dilution ratios and theophylline addition on the function of cryopreserved Walleye
semen. Theriogenology 57, 1061–1071. https://doi.org/10.1016/S0093-
691X(01)00707-5
Billard, R., Cosson, M.P., 1992. Some problems related to the assessment of sperm
motility in freshwater fish. J. Exp. Zool. Part Ecol. Genet. Physiol. 261, 122–131.
Bilyy, R.O., Antonyuk, V.O., Stoika, R.S., 2004. Cytochemical study of role of alpha-d-
mannose- and beta-d-galactose-containing glycoproteins in apoptosis. J. Mol.
Histol. 35, 829–838. https://doi.org/10.1007/s10735-004-1674-z
Bilyy, R.O., Stoika, R.S., 2003. Lectinocytochemical detection of apoptotic murine
leukemia L1210 cells. Cytometry A 56A, 89–95.
https://doi.org/10.1002/cyto.a.10089
Blawut, B., Wolfe, B., Moraes, C.R., Ludsin, S.A., Coutinho da Silva, M.A., 2018.
Increasing Saugeye (S. vitreus × S. canadensis) production efficiency in a
hatchery setting using assisted reproduction technologies. Aquaculture 495, 21–
26. https://doi.org/10.1016/j.aquaculture.2018.05.027
Blawut, B., Wolfe, B., Moraes, C.R., Ludsin, S.A., Silva, M.A.C. da, 2020a. Use of
Hypertonic Media to Cryopreserve Sauger Spermatozoa. North Am. J. Aquac. 82,
84–91. https://doi.org/10.1002/naaq.10125
101
Blawut, B., Wolfe, B., Moraes, C.R., Sweet, D., Ludsin, S.A., Coutinho da Silva, M.A.,
2020b. Testicular collections as a technique to increase milt availability in Sauger
(sander canadensis). Anim. Reprod. Sci. 212, 106240.
https://doi.org/10.1016/j.anireprosci.2019.106240
Fàbrega, A., Puigmulé, M., Dacheux, J.-L., Bonet, S., Pinart, E., 2012. Glycocalyx
characterisation and glycoprotein expression of Sus domesticus epididymal sperm
surface samples. Reprod. Fertil. Dev. 24, 619–630.
https://doi.org/10.1071/RD11064
Jiménez, I., González-Márquez, H., Ortiz, R., Herrera, J.A., Garcı́a, A., Betancourt, M.,
Fierro, R., 2003. Changes in the distribution of lectin receptors during
capacitation and acrosome reaction in boar spermatozoa. Theriogenology 59,
1171–1180. https://doi.org/10.1016/S0093-691X(02)01175-5
Koehler, J.K., 1981. Lectins as Probes of the Spermatozoon Surface. Arch. Androl. 6,
197–217. https://doi.org/10.3109/01485018108987531
Kudo, S., 1998. Role of sperm head syndecan at fertilization in fish. J. Exp. Zool. 281,
620–625. https://doi.org/10.1002/(SICI)1097-010X(19980815)281:6<620::AID-
JEZ10>3.0.CO;2-6
Liu, M., 2016. Capacitation-Associated Glycocomponents of Mammalian Sperm.
Reprod. Sci. 23, 572–594. https://doi.org/10.1177/1933719115602760
Moore, A., 1987. Short-Term Storage and Cryopreservation of Walleye Semen. Progress.
Fish-Cult. 49, 40–43. https://doi.org/10.1577/1548-
8640(1987)49<40:SSACOW>2.0.CO;2
102
Pelaez, J., Bongalhardo, D.C., Long, J.A., 2011. Characterizing the glycocalyx of poultry
spermatozoa: III. Semen cryopreservation methods alter the carbohydrate
component of rooster sperm membrane glycoconjugates. Poult. Sci. 90, 435–443.
https://doi.org/10.3382/ps.2010-00998
Pini, T., Leahy, T., Graaf, S.P. de, 2018. Seminal plasma and cryopreservation alter ram
sperm surface carbohydrates and interactions with neutrophils. Reprod. Fertil.
Dev. 30, 689–702. https://doi.org/10.1071/RD17251
R Development Core Team, 2009. R: A language and environment for statistical
computing.
Rojas, M.V., Esponda, P., 2001. Plasma membrane glycoproteins during spermatogenesis
and in spermatozoa of some fishes. J. Submicrosc. Cytol. Pathol. 33, 133–140.
Sakaguchi, Y., Iwata, H., Kuwayama, T., Monji, Y., 2009. Effect of N-acetyl-D-
glucosamine on bovine sperm-oocyte interactions. J. Reprod. Dev. 55, 676–684.
Schröter, S., Osterhoff, C., McArdle, W., Ivell, R., 1999. The glycocalyx of the sperm
surface. Hum. Reprod. Update 5, 302–313.
https://doi.org/10.1093/humupd/5.4.302
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano,
M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985.
Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85.
https://doi.org/10.1016/0003-2697(85)90442-7
Taatjes, D.J., Sobel, B.E., Budd, R.C., 2008. Morphological and cytochemical
determination of cell death by apoptosis. Histochem. Cell Biol. 129, 33–43.
103
Tecle, E., Gagneux, P., 2015. Sugar‐coated sperm: Unraveling the functions of the
mammalian sperm glycocalyx. Mol. Reprod. Dev. 82, 635–650.
https://doi.org/10.1002/mrd.22500
Xin, A., Wu, Y., Lu, H., Cheng, L., Gu, Y., Diao, H., Chen, G., Wu, B., Li, Z., Tao, S.,
Sun, X., Shi, H., 2018. Comparative analysis of human sperm glycocalyx from
different freezability ejaculates by lectin microarray and identification of ABA as
sperm freezability biomarker. Clin. Proteomics 15.
https://doi.org/10.1186/s12014-018-9195-z
Yanagimachi, R., Cherr, G., Matsubara, T., Andoh, T., Harumi, T., Vines, C., Pillai, M.,
Griffin, F., Matsubara, H., Weatherby, T., Kaneshiro, K., 2013. Sperm Attractant
in the Micropyle Region of Fish and Insect Eggs. Biol. Reprod. 88.
https://doi.org/10.1095/biolreprod.112.105072
Yanagimachi, R., Harumi, T., Matsubara, H., Yan, W., Yuan, S., Hirohashi, N., Iida, T.,
Yamaha, E., Arai, K., Matsubara, T., Andoh, T., Vines, C., Cherr, G.N., 2017.
Chemical and physical guidance of fish spermatozoa into the egg through the
micropyle,. Biol. Reprod. 96, 780–799. https://doi.org/10.1093/biolre/iox015
Yu, S., Kojima, N., Hakomori, S., Kudo, S., Inoue, S., Inoue, Y., 2002. Binding of
Rainbow Trout sperm to egg is mediated by strong carbohydrate-to-carbohydrate
interaction between (KDN)GM3 (deaminated neuraminyl ganglioside) and Gg3-
like epitope. Proc. Natl. Acad. Sci. U. S. A. 99, 2854–2859.
https://doi.org/10.1073/pnas.052707599
104
Figure 10. Alexa Fluor 488 – conjugated lectin staining distributions observed in Sauger sperm using 1000 × oil immersion fluorescent microscopy. Fluorescent staining distributions were defined as (1) apical, (2) homogenous, and (3) heterogeneous.
Differential interference contract (DIC, a) images and corresponding fluorescent (b) images are adjacent (original image 400 × magnification).
105
Figure 11. Wheat germ agglutinin (WGA) fluorescent staining distributions (A) and fluorescent intensity (B) of testicular, stripped, and cryopreserved Sauger sperm in both the inactive and active states. (A) Mean proportions for each fluorescent staining distribution in each sperm type and activation status are given. Patten codes are as follows: homogenous [white], heterogeneous [grey], or apical staining [black]. (B) WGA fluorescent intensity (in relative fluorescent units) is expressed at mean ± 1 standard error
(n = 10). Means with a common Tukey’s HSD superscript do not differ.
106
Figure 12. Representative image of western blot for N-acetyl-glucosamine (GlcNAc) moieties on glycoproteins extracted from the Sauger sperm plasma membrane. Sperm were either testicular (lanes 1-2), stripped (lanes 3-4), or cryopreserved (lanes 5-6) in origin and in either the inactive (lanes 1, 3, and 5) or activated state (lanes 2, 4, and 6).
Sperm glycoproteins containing GlNAc residues were visualized using 0.6 µg/mL HRP conjugated wheat germ agglutinin (WGA). A molecular weight ladder is included
(leftmost column).
107
Figure 13. Concanavalin A (ConA) fluorescent staining distributions (A) and fluorescent intensity (B) of testicular, stripped, and cryopreserved Sauger sperm each in both the inactive and active states. Mean proportions for each fluorescent staining distribution in each sperm type and activation status are reported. Patten codes are as follows: homogenous [white] or heterogeneous [grey]. (B) ConA fluorescent intensity (in relative fluorescent units) is expressed at mean ± 1 standard error (n = 12). Means with a common
Tukey’s HSD superscript do not differ.
108
Table 3. Comparison of the proportion of highly fluorescent sperm cells (%) and viability (%) among Sauger sperm types and
activation statuses for both wheat germ agglutinin (WGA) and concanavalin A (ConA) lectin probes (n = 12, 12, 7). Data are reported
as mean ± 1 standard error.
Sperm Type / Activation Status
Lectin Probe Testicular Stripped Cryopreserved
Inactive Activated Inactive Activated Inactive Activated
WGA 49.5 ± 9.5 d 65.8 ± 8.6 c 83.3 ± 5.3 b 86.8 ± 4.4 b 87.2 ± 4.3 b 93.1 ± 2.5 a
ConA 49.1 ± 4.2 e 68.6 ± 3.7 d 83.9 ± 2.4 c 86.6 ± 2.1 bc 90.0 ± 1.6 ab 91.8 ± 1.4 a
Viability 94.2 ± 1.7 a 77.1 ± 1.7 d 91.5 ± 1.7 ab 85.2 ± 1.7 bc 85.2 ± 1.7 bc 79.9 ± 1.7 cd
1
09 a,b Mean values (± SEM) with different superscript letters within a row differ significantly at P < 0.05.
109
Table 4. Comparison of Sauger sperm motility parameters among sperm pool types (n =
12).
Sperm Type Characteristics Testicular Stripped Cryopreserved
Total Motility (%) 53.9 ± 2.5 b 76.7 ± 2.5 a 56.6 ± 2.5 b Curvilinear Velocity (µm/s) 156 ± 3.6 b 178 ± 3.6 a 100 ± 3.6 c Straight Line Velocity (µm/s) 47.2 ± 4.4 43.9 ± 4.4 50.5 ± 4.4 Average Path Velocity (µm/s) 107.7 ± 4.2 a 115.1 ± 4.2 a 78.6 ± 4.2 b
b a Straightness (%) 43.4 ± 3.0 b 36.3 ± 3.0 66.4 ± 3.0 Linearity (%) 32.9 ± 3.3 b 25.8 ± 3.3 b 52.6 ± 3.3 a Beat Cross Frequency (Hz) 15.6 ± 0.7 b 13.1 ± 0.7 c 21.8 ± 0.7 a Wobble (%) 70.0 ± 2.3 ab 65.5 ± 2.3 b 76.9 ± 2.3 a Head Amplitude (µm) 10.7 ± 0.4 b 13.0 ± .04 a 5.4 ± 0.4 c a,b Mean values (± SEM) with different superscript letters within a row differ significantly at P < 0.05.
110
Chapter 4: Effect of Assisted Reproduction Techniques on Sauger (Sander
canadensis) Spermatozoa Plasma Membrane Physiology
Abstract
The sperm plasma membrane is a multifunctional organelle essential to fertilization. However, assisted reproduction techniques often negatively affect this structure, resulting in reduced fertility. These reductions have been attributed to plasma membrane damage in a wide array of species, including fish. Considerable research has been conducted on the fish sperm membrane, but few have examined the effect of cryopreservation and other assisted reproduction techniques (ARTs) on not only membrane composition, but also specific characteristics (e.g., fluidity) and organization
(e.g., lipid rafts). Herein, we determined the effect of three ARTs (testicular harvest, strip spawning, and cryopreservation) on the composition of the sperm membrane, using
Sauger (Sander canadensis) sperm as a model. To this end, a combination of fluorescent dyes (e.g., merocyanine 540, filipin III, cholera toxin subunit β), LC-MS analysis of membrane lipids, and membrane ultracentrifugation coupled with plate assays and immunofluorescence to describe and compare sperm fluidity, membrane composition, and lipid raft composition and distribution among sperm types. Stripped Sauger sperm became more fluid following motility activation (40% increase in highly fluid cells and
2× increase in fluorescence) and contained typical lipid rafts restricted to the midpiece.
Testicular harvest yielded sperm with characteristics similar to stripped sperm. By
111 contrast, cryopreservation impacted every aspect of membrane physiology. Two cell populations, one highly fluid and the other rigid, resulted from the freeze-thaw process.
Cryopreservation reduced lipid raft cholesterol content by 44% and flotilin-2 (a lipid raft marker) was partially displaced owing to a decreased in buoyancy. Unlike stripped and testicular sperm, LC-MS analysis revealed increases in oxidative damage markers, membrane destabilization, and apoptotic signaling in cryopreserved sperm.
Ultrastructural analysis also revealed widespread physical damage to the membrane following freeze-thaw. Sperm motility, however, was unrelated to any measure of membrane physiology used in this study. Our results demonstrate that ARTs have the potential to significantly affect sperm plasma membrane, but not always detrimentally.
These results provide multiple potential biomarkers of sperm quality as well as insight into the likely sources of sub-fertility resulting from use of ARTs.
Introduction
The plasma membrane is one of the most important components for the proper structure and function of a mature spermatozoa (Cabrita et al., 2008). Sperm membranes are unique in that they are enriched in polyunsaturated fatty acids, contain lipid-ordered signaling domains (i.e., lipid rafts), and demonstrate high membrane fluidity, which collectively allow for membrane fusion with the oolema during fertilization (Tapia et al.,
2018). Unfortunately, these same properties make the sperm plasma membrane susceptible to a variety of natural and artificially induced damages (Holt, 2000; Xin et al.,
2019). For example, assisted reproduction techniques (ARTs) often result in reduced fertilization as a consequence of plasma membrane damage (Hammerstedt et al., 1990;
Parks and Graham, 1992). Processes such as lipid peroxidation, lipid phase transition, and 112 osmotic stress associated with ARTs are commonly referenced sources of membrane damage (Tapia et al., 2018). Consequently, use of these techniques has serious implications for fertilization ability and thus practical large-scale application in aquaculture. In particular, fish sperm fertilization has been shown to be negatively impacted by ARTs commonly used in aquaculture (e.g., testicular harvest and cryopreservation). Yet, our knowledge of which aspects of sperm physiology are being impaired remains largely speculative. Given the importance of the plasma membrane to fertilization success, this structure should be a primary target for sperm quality assessment.
Assisted reproduction techniques are used in the production of Saugeye (Sander vitreus × S. canadensis) to augment recreational fishing opportunities in Ohio reservoirs.
Recently, sperm cryopreservation and testicular harvest in Sauger (S. canadensis) have been introduced as tools to help stabilize annual Saugeye production by maximizing reproductive potential in the limited pool of male broodstock (Blawut et al., 2020a,
2020b). However, poor or variable fertilization resulting from cryopreservation and testicular harvest, respectively, suggest that the physiology of sperm resulting from these techniques is being negatively affected. Because maturation of the plasma membrane during sperm storage in the main sperm duct is associated with changes to membrane composition, organization, and characteristics, harvesting the sperm prematurely (i.e., testicular harvest) could cause variable fertilization due to the size of the mature sperm population (Blawut et al., 2020b). Additionally, cryopreservation is known to cause substantial damage to the plasma membrane, which can partially reduce post-thaw fertilization (Cabrita et al., 2014, 2010; Parks and Graham, 1992). Beyond these few
113 studies of sperm viability, all of which here use traditional assessment measures, little information is available regarding Sauger sperm membrane physiology and how ARTs affect those parameters. This gap in knowledge is important because preventing substantial damage to the membrane associated with these ARTs will lead to higher fertility and higher large-scale application efficacy.
To uncover the effects on these ARTs on the plasma membrane in Sauger spermatozoa, we sought describe and compare membrane physiology among three sperm types (stripped, testicular, and cryopreserved) and activation statuses (inactive and activated via hyposmotic shock). Currently, a multitude of metrics have been used to asses aspects of fish sperm physiology (Cabrita et al., 2014). While phospholipids, fatty acids, and cholesterol content have proven to be indicative of overall sperm quality, information is limited regarding membrane ultrastructure, fluidity, or lipid raft characteristics and their relation to sperm quality in the Sauger. Further investigation of these membrane physiological variables beyond composition alone would provide more detail into the effects of ARTs on spermatozoa to the benefit of future protocol optimization.
Membrane lipid composition is an important factor affecting sperm quality and its physiological properties. Sperm cells are composed primarily of saturated fatty acids
(SFA) but are also enriched in mono and polyunsaturated fatty acids (MUFA and PUFA), particularly in polyunsaturated FA c22:6 (docosahexaenoic acid) (Bell et al., 1997, 1996).
Generally, sperm containing higher amounts of unsaturated fatty acids are considered to be of higher quality (Pustowka et al., 2000). Fish sperm lipid composition varies widely among species (Engel et al., 2020), among individuals of the same species (Díaz et al.,
114
2018), among seasons (Martínez-Páramo et al., 2012), as a result of nutritional background (Bell et al., 1996), and even spatially within a single sperm cell (J. Beirão et al., 2012). Interestingly, motility activation via hypo-osmotic shock does not affect membrane composition (Dadras et al., 2017). By contrast, membrane fluidity, an attribute dictated by membrane packing order and SFA/USFA ratio, was elevated in Cyprinus carpio sperm following hypostatic shock (Krasznai et al., 2003a). These results suggest that, while membrane composition is unaffected by activation, elevated fluidity may result from a change in membrane organization. A clearer understanding of membrane composition, the effects of both hypo-osmotic shock on membrane fluidity, and the effects of ARTs on both measures would benefit our understanding of fish membrane physiology and its relation to fertility.
Fish sperm membrane properties can also be dictated by the order imposed from its structural organization (Müller et al., 2008). It is generally accepted that the fluid mosaic model does not adequately describe the structure of the cellular plasma membrane
(Parks and Graham, 1992). The external leaflet of the membrane contains lipid micro domains (i.e., lipid-ordered domains or lipid rafts) that are enriched in cholesterol, sphingolipids, and saturated fatty acids whose hydrogen bonds and hydrophobic interactions result in high stability (Nixon and Aitken, 2009). Lipid rafts are resistant to low concentrations of detergents at approximately 4°C, and thus these “detergent- resistant membranes” are often extracted and analyzed as approximations of in vivo lipid rafts (Brown, 2006).These structures have been studied extensively in the sperm of mammalian species and only recently in fish. Lipid rafts play an important role in sperm- egg interactions (both with the zona pellucida and the oocyte itself) and their composition
115 and distribution are a result of physiological adaptation to maturation and capacitation in mammalian species (Khalil et al., 2006b; Shadan et al., 2004b; Thaler et al., 2006; Tsai et al., 2007; van Gestel et al., 2005b). Recent studies in fish have confirmed the presence of lipid rafts in somatic cells of Cyprinus carpio (Brogden et al., 2014b), Gadus morhua
(Gylfason et al., 2010), Oncorhynchus mykiss (Zehmer and Hazel, 2004, 2003), Ictalurus punctatus, and Carassius auratus L. (Garcia-Garcia et al., 2012). In fish sperm, lipid raft integrity was documented to be negatively affected by cryopreservation in Pelteobagrus fulvidraco (Bai et al., 2019) and Cyprinus carpio (Dietrich et al., 2015). Ganglioside
GM1 and flotilin (lipid raft components) were either reduced following cryopreservation or found to be elevated in the post-thaw media, suggesting a loss of these components.
Additionally, a loss of sphingomyelin, another lipid raft component, in both the Red
Spotted Grouper (Epinephelus akaara) and the Yellow Drum (Nibea albiflora, Dai et al.,
2012; He et al., 2011b) following cryopreservation suggests potential negative effects on lipid raft stability. At present, a thorough analysis of lipid raft composition and distribution has not been completed in fish sperm nor have the effects of different ARTs been tested using detergent-resistant membrane methodology. Filling these gaps is important in advancing our understanding of membrane damage and its relationship with sub-fertility resulting from ARTs.
To address these knowledge gaps, we conducted a laboratory experiment to determine the effect of ARTs on the fish sperm plasma membrane using Sauger as a model. Our primary objectives were to (1) characterize and compare the sperm membrane’s overall lipid composition, lipid raft composition, and ultrastructure from sperm acquired through testicular harvest, stripping, and after cryopreservation and (2)
116 assess and compare sperm membrane fluidity and lipid raft distributions within sperm among sperm types and between activation statuses (inactive vs. activated). We hypothesized that lipid composition would differ among sperm types owing to a lack of exposure to seminal plasma and cryopreservation-induced damage relative to a strip- spawned control. We hypothesized that detergent-resistant membranes (i.e., lipid rafts) would be present in fish sperm, have a distribution dependent on activation status, and their cholesterol content would depend on the type of sperm assayed. Lastly, we hypothesized that membrane fluidity would increase as a result of both motility activation via hypo-osmotic shock and following cryopreservation.
Materials and Methods
Media and Chemicals
All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise specified. Primary antibodies specific to flotilin-2 and β-actin were purchased from Santa Cruz Biotechnology (#sc-28320, CA, USA) and Abcam (ab8227, Cambridge,
UK). Amplex Red Cholesterol Assay Kits (A12216), BCA protein quantitation kits
(23227), Yo-Pro-1 (Y3603), fluorescent secondary antibodies (A28175), Slow-fade gold
(S36940), and ECL substrates (West Dura SuperSignal, #34075) were purchased from
ThermoFisher Scientific (Waltham, MA, USA). The fluorescent cholesterol dye (Filipin
III) was purchased from Caymen Chemical (70440, Ann Arbor, MI, USA). All electrophoresis and western blotting equipment, gels, and membranes were purchased from Bio-Rad (Hercules, CA, USA). Horseradish peroxidase conjugated secondary antibodies were purchased from Cell Signaling (#7076, #7074, Danvers, MA, USA).
117
Experimental design
A three-factor treatment structure was used to compare the sperm plasma membrane composition and lipid raft composition among sperm types (testicular, stripped, and cryopreserved). A second 3 × 2 factorial experiment was used to compare the membrane fluidity, cholesterol, and lipid raft marker quantity and distributions among sperm types (testicular, stripped, and cryopreserved) and between activation statuses
(inactive and activated via hypo-osmotic shock). Sperm were collected from each Sauger using both strip-spawning and testicular harvest. High-quality samples were pooled based on sperm type to prevent individual biases. Stripped sperm were cryopreserved according to a previously used protocol (Blawut et al., 2020a). For each of these three sperm types
(testicular, stripped, and cryopreserved), the following metrics were analyzed in inactive sperm only: lipid composition (n = 5 individuals), detergent resistant membranes protein and cholesterol content (n = 4 pools), and membrane structure using electron microscopy
(n = 1 pool). Membrane fluidity, ganglioside GM1, flotilin-2, and cholesterol content were assessed using flow cytometry and fluorescent microscopy in inactive as well activated sperm from all three sperm types (n = 1 – 7 pools).
Broodstock Acquisition, Milt Collection, and Motility Analysis
Sauger broodstock were acquired and maintained in human care using methods describe previously (Blawut et al., 2018b). In summary, Ohio Department of Natural
Resources – Division of Wildlife (ODNR-DOW) personnel collected mature, male
Sauger from the Ohio River near the Greenup, KY, USA dam via electrofishing during the breeding season (March 2020). Sauger broodstock were transported by truck for ~2
118 hours in an aerated live-well containing 0.5% NaCl solution to the London State Fish
Hatchery isolation facility (London, OH, USA). The specimens were maintained in a
~2840 L indoor recirculating system at 5 to 6 °C with a flow rate of 45 to 57 L/hour. Fish were maintained in tanks with natural photoperiod exposure. The Ohio State University
(Columbus, OH, USA) Institutional Animal Care and Use Committee approved all procedures and animal use prior to the beginning of the study (Protocol
#2015A00000008).
Milt used for this study was collected using two different techniques, strip- spawning and testicular harvest using previously published methodology (Blawut et al.,
2020b). Individuals were strip-spawned by placing them in dorsal recumbancy, drying the cloaca thoroughly to avoid premature activation (Billard and Cosson, 1992), and stripped to collect milt using abdominal massage and a 1.0 mL rubberless syringe that was placed at the opening of the cloaca. Total milt volume was determined (nearest 0.01 mL) and the milt was immediately extended milt 1:2 with 350 mOsm/kg Rathbun extender ((g/l): CaCl2·2H2O, 0.117; MgCl2·6H2O, 0.134; Na2HPO4, 0.236; KCl, 1.872;
NaCl, 6.578; glucose, 10.000; citric acid, 0.100; NaOH, 0.254; and bicine, 1.06; pH 8.5
(Bergeron et al., 2002b; Moore, 1987b). Extended milt was maintained in centrifuge tubes at 5° C in a Styrofoam box until further use.
The same individuals were euthanized for testicular sperm harvest using cervical transection followed by decapitation. Euthanized individuals were stored on ice and transported to the Theriogenology laboratory (< 1 h, Columbus, OH). Once in the lab, the testis of euthanized fish was removed from the body cavity then cleaned of extraneous fluids and debris. Each lobe of the testis was cut along its length with a scalpel and
119 pressure was applied to the lacerated lobes from the cloaca to the tip of the lobe to expel sperm. Testicular milt was then diluted using 3.0 mL of Rathbun extender, gently homogenized, and transferred to a test tube for storage at 5° C.
Sperm motility was assessed objectively using computer-assisted sperm analysis
(CASA, Hamilton Thorne, MA, USA). Stripped and testicular sperm were diluted to 0.5
× 10 9 sperm/mL prior to analysis. Cryopreserved sperm samplers were analyzed undiluted. Two µL of sperm was activated in 38 µL of 0.22 µM filtered hatchery water containing 1% BSA at a pH of 8.3. Activated sperm were loaded into a Microtool chamber (#B7, Cytonix LLC, MD) and analyzed at 10 s post-activation using CASA software with acquisition parameters described previously (Blawut et al., 2020a).
To prevent potential individual biases, milt samples were pooled among three individuals based on sperm type. Stripped sperm samples from three Sauger with motility
> 70% were combined (i.e., pooled) and maintained at 5°C prior to cryopreservation (n =
7). Likewise, testicular harvest milt samples from same cohort of three Sauger were also pooled at 5°C. By contrast, samples submitted for LC-MS were analyzed based on individual samples rather than pooling.
Sperm Cryopreservation and Thawing
Sperm samples were cryopreserved using a protocol developed by our laboratory described previously (Blawut et al., 2020a). In brief, fresh, stripped milt was diluted to
1.0 × 10 9 sperm/mL using Rathbun extender supplemented with 4 mg/mL bovine serum albumin (BSA). After 10 min of equilibration, sperm were further diluted (1:1, v: v) with
Rathbun + BSA supplemented containing 20% dimethyl sulfoxide (DMSO). The final
120 diluent consisted of 0.5 × 10 9 sperm/mL in Rathbun plus 4 mg/mL BSA and 10% DMSO as cryoprotectants. During the 10 min cryoprotectant equilibration, samples were loaded into 0.5 mL straws and sealed before cooling at 3 cm above liquid nitrogen for 10 min.
Samples were plunged and stored in liquid nitrogen for approximately 1 month prior to thawing at 37°C for 10 s in a water bath and maintenance at 5°C prior to analysis.
Flow Cytometry
Flow cytometry analysis was conducted using methods described in Blawut et al
(in review). Inactive (1:40 dilution in isotonic extender) and activated (1:20 dilution in hatchery water for 30 s followed by an additional 1:2 dilution in 2× Rathbun extender) sperm stock solutions were prepared and maintained at 4°C prior to the staining procedures outlined below.
Merocyanine 540- Membrane fluidity was assessed using the lipophilic dye
Merocyanine 540 (MC540). MC540 is more easily incorporated into fluid membranes with a lower packing order (Williamson et al., 1983). Approximately 2.0 × 10 6 inactive sperm of each type were stained with 1.2 M Yo-Pro-1 viability counterstain for 5 min at
4C. Sperm were then diluted (1:10) with either isotonic extender (inactive) or hatchery water (activated) containing 2.7 mM MC540 for approximately 10 min at 4C. Samples were then analyzed using fluorescent microscopy for qualitative descriptions of staining or flow cytometry for quantitative assessment of binding.
Flow cytometric analysis was completed for MC540 using an NXT Attune
Acoustic Flow Cytometer (ThermoFisher Scientific, Waltham, MA, USA). Merocyanine
540 was detected using the yellow laser (561 nm) and 585/16 (YL2) emission filter at
121
350mV. Yo-Pro-1 was detected using the blue laser (488 nm) and 530/30 (BL1) emission filter at 200V. Data were recorded using the following instrument settings: flow rate =
12.5 µl/minute; cell capture = 20,000 cells; positive gates = 1.0 ± 103. Forward and side scatterplots were used to exclude debris, whereas forward area and height scatterplots were used to remove doublets from the analysis. Endpoints included mean MC540 fluorescent intensity (M.F.I., arbitrary units) and the proportion of cells exhibiting high fluorescence (i.e., high fluidity, %) for viable cells only.
Filipin III - Membrane cholesterol content was assessed using the naturally fluorescent, cholesterol-specific membrane probe filipin III. Approximately 2.0 × 10 6 sperm from each stock solution (inactive and activated) for each type were stained for approximately 30 min using 30 mM filipin III at 4C. Propidium iodine (PI) was used as a viability counterstain by staining for the last 10 min of incubation using 1.2 mM dye. A
BD LSR Fortessa flow cytometer (Beckman, Dickson and Company, USA) was used for assessing filipin III fluorescence using the ultraviolet laser (355 nm) and a 450/30 bandpass filter at 350 mV. PI was detected using the 613/20 nm band pass filter at 460 V.
No compensation was required. Mean filipin III fluorescent intensity (M.F.I., arbitrary units) was recorded for each sample.
Cholera Toxin Subunit β (GM1) - Ganglioside GM1, a known lipid raft component in other species, was assessed in Sauger sperm using cholera toxin subunit β.
Approximately 2.0 × 10 6 sperm from each stock solution (inactive and activated) per sperm type were stained using 0 – 20 µg/mL of the fluorescent conjugated CTβ probe for
122
30 min, counterstained for 10 min using 1.2 mM PI, then assessed using flow cytometry to describe GM1 distribution and quantity. Propidium iodine was detected using the yellow laser (561 nm) and 695/40 (BL3) emission filter at 400 mV. CTβ was detected using the blue laser (488 nm) and 530/30 (BL1) emission filter at 350 mV. No compensation was required due to little spectral overlap. Endpoints included mean GM1 fluorescent intensity (M.F.I., arbitrary units) and the proportion of cells exhibiting high fluorescence (i.e., high GM1 content, %) for viable cells only.
Fluorescent Microscopy
Sperm for fluorescent microscopy analysis were stained as described in the section above. After incubation with fluorescent dyes, sperm were adhered to poly-L- lysine treated microscope slides for 5 min at 4°C in a humid chamber. Samples were assessed using a Nikon Eclipse Ti and Intensilight C-HGFIE lamp with a 1000 × oil objective (Nikon Inc., NY, USA) to capture representative images of staining patterns observed.
Detergent Resistant Membrane (DRM) Analysis
DRM Extraction - Detergent resistant membranes, approximately equivalent to lipid rafts, were extracted using a low concentration of detergent at 4°C and separated utilizing discontinuous sucrose gradient floatation (Brown, 2006). One billion sperm per sperm type (testicular, stripped, or cryopreserved) were aliquoted into each tube and centrifuged for 5 min at 1,200 × g (4°C) to remove the original media. Sperm were suspended in isotonic extender and then lysed using a 1:10 dilution of concentrated lysis
123 buffer to yield 0.1% Triton X-100 in TBS containing 1 × Halt Protease and Phosphatase inhibitors and 1mM PMSF. Sperm were lysed on ice for 30 min with occasional vortexing. Cells were also mechanically lysed using 30 passes through a 25×G needle.
Lysates were then mixed 1:1 with 80% sucrose-TBS to yield a 40% sucrose lysate. These lysates were then overlaid with 4 mL of 30% sucrose-TBS and 3 mL of a 5% sucrose-
TBS solution in a 12 mL polyallomer ultracentrifugation tube (Seton Scientific, CA,
USA). These discontinuous gradients were centrifuged for 18 hours at 4°C at 32,400
RPM with no brake (~ 180,000 × G, L-80 Ultracentrifuge, Beckman Coulter, USA).
Fractions (1 mL) were taken from the top to the bottom (11 total aliquots) with the last aliquot containing the cell pellet. Fractioned lysates were stored at -80C until further analysis. Samples were thawed at 37°C for ~ 1 min and then maintained at 4°C prior to analysis.
Protein quantification - Protein content in each sample was determined using bicinchoninic acid assay (Smith et al., 1985b) according to manufacturer instructions
(Pierce BCA assay kit, ThermoFisher Scientific). Ultracentrifugation fractions (1 - 11) and accompanying BSA standards were loaded into a 96-well plate and maintained at
4°C. Samples were diluted with working reagent, sealed with parafilm, and incubated at
37°C for 30 min. Absorbance at 562 nm was recorded for each sample using a Cytation 5 microplate reader (BioTek, VT, USA). Protein content (nearest µg) was calculated using the linear regression equation generated from the standard curve (R2 = 0.99).
124
Cholesterol quantification - Cholesterol content (nearest µM) in each fraction was assessed using the commercially available Amplex Red Cholesterol assay according to manufacturer specifications (ThermoFisher Scientific). Ultracentrifugation fraction samples (50 L) and standard (0 µM - 25µM) aliquots were arranged in a 96-well plate and maintained at 4°C. A working reagent consisting of 300 µM Aplex Red reagent, 2
U/mL HRP, 2 U/mL cholesterol oxidase, and 0.2 U/mL cholesterol oxidase was added
(50µL total) to initiate the reaction. Plates were sealed with parafilm and incubated for 30 min at 37°C. Fluorescent intensity of each well was recorded (ex: 560/10, em: 590/10 nm) and used to calculate cholesterol content using a polynomial regression equation derived from the standard curve (R2 = 0.99).
Western blot - The validity of the ultracentrifugation separation of raft and non- raft fractions was confirmed by assessing the distribution of a detergent resistant membrane marker (flotilin -2) and cytoskeletal protein (β-actin) in lysate fractions using western blot. Equal volumes of each ultracentrifugation fractions were diluted with 4 ×
Laemmli loading buffer (Laemmli, 1970) containing 2-mercaptoethanol, boiled for 5 min at 100°C, and loaded into the wells of a 4-15% Criterion XTG polyacrylamide gel.
Protein was separated using electrophoresis for 1 hour at 175 V and transferred to a
0.22µm PVDF membrane for 75 min at 100 V at 4°C. Membranes were blocked for 1 hour using 3% milk in TBST (0.1% Tween20) and probed with primary antibody
(flotilin-2: 1:1,000 or β-actin: 1:5,000) in 3% milk-TBST overnight at 4°C. Blots were then incubated for 1 hour at room temperature with horse radish peroxidase- conjugated secondary antibodies (flotilin-2: #7076 - 1:10,000 ; β-actin: #7074 -1:10,000). Lastly,
125 blots were imaged using enhance chemiluminescent substrate (West Dura SuperSignal) and an Amersham Imager 600 (General Electric, Boston, MA, USA) digital blot imager.
Immunofluorescence - Flotilin-2 distribution in sperm was assessed using a modified immunofluorescence protocol for fish described by Koubek et al. (2008). Sperm from each sperm type and activation status (1:20 dilution in either isotonic or hypo- osmotic media) were attached to poly-L-lysine treated 8-well chambered slides (ibidi
GmbH, Gräfelfing, DEU) for 10 min at 4C in a humid chamber. Sperm were fixed for
20 min at 4C in 3.6% paraformaldehyde and then washed thoroughly with TBS.
Nonspecific binding was blocked with 3% BSA in TBS for 60 min at 4C. Sperm were then incubated probed with the flotilin-2 monoclonal antibody (sc-28320, 1:500) in 3%
BSA-TBST overnight at 4C. An Alexa-fluor488 conjugated polyclonal secondary antibody (A28175, 1µg/mL) was applied for 30 min at 37C. Slides were washed in triplicate with TBS, mounted using Slow-Fade Gold anti-fade mounting media, and assessed using fluorescent microscopy. Staining distribution (e.g., apical, homogenous, heterogeneous, and other) was assessed in 100 sperm per sample.
Untargeted metabolomics analysis
Liquid chromatography – mass spectroscopy (LC-MS) was used to analyze the lipid composition of the Sauger sperm plasma membrane. Membrane lipids were extracted from sperm using a slight modification of the Blithe and Dyer method (1959) as described by Reis et al (2013). All reagents used for this experiment were sufficient LC-
MS grade. Sperm of each sperm type (500 million) were aliquoted to tubes, centrifuged at
126
1,200 x G for 5 min at 4C, and re-suspended in 160 L of either isotonic extender.
Lipids from each sample were then extracted using chloroform: methanol (1:2) for 10 min at 4°C in a Bioruptor water bath (Diagenode, Denville, NJ, USA) to fully lyse the cells. Water was added to a final ratio of 1:2:1 of chloroform, methanol and H2O, vigorously homogenized, and then centrifuged for 5 min at 2,000 × g. The upper aqueous layer was transferred to a separate tube and subjected to a second round of chloroform extraction with the addition CHCl3 and 12M HCl. Following centrifugation at 2,000 × g for 5 min, the resulting organic layers were combined, washed with LC-MS-grade H2O, and centrifuged. This final organic layer was dried for 60 min using an Eppendorf
Vacufuge Concentrator (Eppendorf, GER) and then stored at -80°C prior to analysis.
Samples were re-suspended in 100µL of CHCl3: MeOH (1:1 ratio), sonicated, and then further diluted with 100 µL of MeOH prior to injection.
The samples were injected at 5 µL onto a Thermo Scientific UltiMate 3000 HPLC using an Agilent Poroshell 120 SB-C18 column (2x100 mm, 2.7 µm particle size) with a solvent system of 60:40 water: acetonitrile with 13 mM ammonium formate for solvent A and 90:10 IPA: ACN with 13 mM ammonium formate for solvent B. A flow rate of 200
µL/min was run throughout with an initial gradient of 32 % solvent B until 1.5 minutes then a linear increase to 45 % B ending at minute 4, another increase to 52 % ending at minute 5, up to 55 % by minute 8, up to 60 % by minute 11, up to 70 % by minute 14, up to 75 % by minute 18, and up to 97% by minute 21, holding 97% until minute 25, back down to 32 % by minute 27, and ending the run at 32 % at minute 30. Ionization of samples following LC separation was done with a Heated Electrospray ionization (HESI) source with runs performed in positive and negative modes with ESI voltages of 4 and 3
127 kV, respectively, with a sheath gas of 15, an auxiliary gas of 5, and capillary temperature of 320 ⁰C. MS data collection was performed using Data-Dependent Analysis with a mass range of 106-1600 m/z, an MS resolution of 70000, an MSMS resolution of 35000, and 30 second dynamic exclusion with top 10 ion selection each MS cycle.
All feature detection was performed using Progenesis QI with each run imported using the RAW QE Plus Orbitrap MS data. Runs were aligned to the pooled quality control samples (a combined set of all 18 samples), which were run every 10 samples.
Sample alignment of runs matched to pooled references below 85 % were removed from statistical analysis and features of p-values less than 0.05 were considered statistically significant. Additionally, only those features which had MS/MS fragmentation analysis were considered as valid features. Each possible metabolite was identified using the
Human Metabolome Database sdf file uploaded into Progenesis MetaScope with a precursor tolerance of 10 ppm and a theoretical fragmentation tolerance of 100 ppm along with a retention time window of 6 seconds. Adducts included for positive mode identification included M+H, M+Na, M+K, M+2H, and 2M+H, whereas negative mode included M-H, M+Cl, 2M-H, and M-2H. In total, 154 metabolic features were tentatively identified with Lipid Maps codes and a 0.01 p-value cut-off. The list of features from the
Progenesis QI alignment and detection were submitted to MetabolAnalyst for normalization by sum, auto scaling, and subsequent statistical analysis.
Scanning and Transmission Electron Microscopy
Scanning and transmission electron microscopy were used to assess stripped and testicular sperm ultrastructure. For scanning electron microscopy (SEM), samples of
128 stripped and testicular sperm (from the whole tract) were fixed in 3% glutaraldehyde in
Rathbun extender and stored at 4°C for approximately 3 months. Fixed sperm were adhered to a poly-L-lysine coated coverslip for 10 min at room temperature. Coverslips were dehydrated using graded ethanol solutions (50%, 70%, 80%, 95%, and 100%) and followed by graded HMDS solutions (25%, 50%, 75%, 100%). Coverslips were allowed to dry in a fume hood overnight, mounted on aluminum stubs, and sputter coated with a thin layer gold/palladium coating approximately 25 nm thick (Cressington 108; Ted Pella
Inc., Redding, CA). The samples were then observed and photographed using a Nova
Nano scanning electron microscope (Nova NanoSEM 400; FEI, Hillsboro, OR).
For transmission electron microscopy (TEM), stripped, testicular, and cryopreserved sperm samples were preserved in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH = 7.4). After approximately 60 min of fixation, the fixative was removed, and cells were re-suspended in 0.1 M cacodylate buffer. Samples were shipped at room temperature to the Colorado State University Animal Reproduction and Biotechnology
Laboratory where samples were further processed and evaluated using procedures used for evaluating frog spermatogenesis (Lee and Veeramachaneni, 2005). Briefly, upon arrival, samples were post-fixed for 90 min in 1.0% osmium tetroxide (in 0.1 M cacodylate buffer), rinsed in buffer, and dehydrated through a graded series of ethanol solutions followed by rinsing in propylene oxide. Sample preparations were embedded in poly/Bed 812 (Polysciences Inc., Warrington, PA) and sectioned using an Ultracut microtome (Richert-Jung, Vienna, Austria). One-µm-thick sections were cut and mounted on glass slides, stained with toluidine blue for light microscopy to ensure optimal cut surface and to facilitate initial evaluation of cells. Then, ~80-nm-thin sections
129 were cut and mounted on 300-mesh nickel grids and stained with uranyl acetate and lead citrate for characterization of ultrastructural features using a JOEL-1200EX transmission electron microscope (JEOL USA, Inc., Peabody, Massachusetts).
Statistical Analysis
A variety of statistical models were used to compare sperm physiology traits among sperm types and between activation statuses. All analyses were completed in R (R
Development Core Team, 2019) with an alpha value of 0.05. All values are expressed as mean ± 1 standard error of the mean (SEM) unless otherwise stated.
Membrane Characteristics - Mean fluorescent intensity of cell populations was compared among groups using linear mixed models with two fixed factors: (1) sperm type: testicular, stripped, and cryopreserved; (2) activation status: inactive and activated.
A random factor was included to account for within-pool variance. Highly fluorescent sperm populations (%) were compared among groups using logistic regression with the same predictors as for the above treatment structure. Tukey’s honestly significant difference (HSD) was used for all post hoc comparisons.
Detergent-Resistant Membranes - Lipid raft cholesterol content (µM) was also compared among membrane fractions (e.g., lipid rafts at fraction 4) as well as among sperm types using a linear mixed model. A random effect of pool of origin was included to account for within pool variance. A one-way ANOVA was used to compare total
130 cholesterol content (all fractions summed) among sperm types. Tukey’s HSD was used for all post hoc comparisons.
Untargeted Metabolomics - Membrane lipid metabolite quantities generated from
LC-MS was compared among sperm types (testicular, stripped, and cryopreserved) using multivariate statistical analysis. Partial least squares discriminant analysis (PLS-DA) was used to separate the metabolome among sperm types and determine the contribution of specific variables to the model (determined by VIP score). The number of variables included was determined by a VIP cutoff value of 1.0. Univariate ANOVA with post-hoc
Tukey’s HSD tests were used to compare metabolite abundances for model variables among sperm types.
Motility and Viability - Motility parameters and viability were compared among sperm types using linear mixed models with a fixed factor of sperm type and a random factor of pool of origin. Post-hoc mean comparisons were made using Tukey’s HSD. And lastly, Pearson r correlation was used to assess the relationships among sperm motility, viability, and membrane fluidity.
Results
Membrane characteristics
Membrane fluidity, as assessed using the lipophilic dye MC540, was affected by an interaction between sperm type and activation status (p < 0.001, Table 5). Testicular and stripped sperm membrane fluidity increased approximately 2-fold following
131 activation. By contrast, membrane fluidity was elevated approximately 4-fold in cryopreserved sperm compared to stripped and testicular sperm and did not change as a result of hypo-osmotic shock. After activation, cryopreserved sperm membrane fluidity was 1.6-fold higher than in activated testicular and stripped sperm in the same status.
Further analysis of sperm subpopulations exhibiting high MC540 fluorescence within samples revealed a slightly different pattern (Table 5). The proportion of highly fluorescent cells depended on sperm type (p < 0.001) and activation status (p < 0.001).
Highly fluorescent cells (%) were lowest in inactive stripped and testicular sperm (< 7%) but increased to approximately 40% after motility activation. Inactive cryopreserved sperm were more likely to be highly fluorescent compared to stripped and testicular sperm (29.53 ± 2.2%), but also increased to 75.15 ± 2.2 % following motility activation.
These results indicate that the sperm plasma membrane was destabilized by the freeze- thaw process.
Cryopreserved sperm membrane fluidity displayed abnormal patterns that were exclusive to this group. The intensity of low fluorescent frozen sperm (~ 70%) of sperm in the inactive state was 37 % and 45 % lower than in testicular or stripped sperm, respectfully, indicating a higher level of membrane rigidity. This low fluidity population experienced a 2-fold increase in fluorescent intensity following motility activation, while those of testicular and stripped sperm remained unchanged. The excessively fluid cells in the inactive cryopreserved sperm mentioned above (~30% of cells) seemed to disappear following motility activation, likely becoming nonviable following hypo-osmotic shock.
Moreover, fluorescent intensity of activated frozen sperm was 1.6-fold higher than
132 activated sperm from other two treatments, indicating a higher level of fluidity than other treatments following activation.
Fluorescent microscopy of membrane fluidity revealed a single staining pattern in sperm that was unaffected by sperm type or activation status. Sperm were stained homogenously with a subjectively brighter head and midpiece compared to the tail.
No differences were observed in the quantity of membrane cholesterol among sperm type or activation statuses using filipin III (p > 0.838,), nor were any discernable staining patterns existent other than the homogenous distribution observed using fluorescent microscopy. Sperm were stained homogenously throughout the head and tail with no indication of regional staining intensity differences.
Cholera toxin subunit β did not bind to the plasma membrane of sperm at any concentration tested [0 – 20 µg/mL], contrary to Yellow Catfish (Pelteobagrus fulvidraco) where this probe was shown to bind successfully to sperm (Bai et al., 2019).
Detergent-Resistant Membranes
Detergent-resistant membranes (DMRs) were successfully extracted using the reported discontinuous sucrose gradient ultracentrifugation (i.e., floatation) protocol
(Figure 14). An opaque band was observed at the interface of the 5% and 30% sucrose layers (i.e., fraction 4, Figure 14 insert), consistent with the hypothetical buoyancy of
DRMs. Fraction 4 contained a disproportionate amount of cholesterol relative to the remained of the fractions regardless of sperm type (19 – 34% of cholesterol). The amount of cholesterol in the lipid raft fraction (fraction 4) depended on the type of sperm assayed
(p = 0.03, Figure 14). Testicular and stripped sperm had a similar cholesterol content (3.
133
23 ± 0.27 µM and 3.23 ± 0.27 µM, respectively). By contrast, cryopreserved sperm showed a 44% reduction in DRM cholesterol content (1.82 ± 0.27 µM) compared to stripped sperm. Collectively, the total content of cholesterol from all fractions summed together was highest in testicular sperm (13.46 ± 1.52 µM), and lowest in cryopreserved sperm (8.66 ± 1.52 µM) with stripped sperm showing intermediate values (10.93 ± 1.52
µM, p = 0.02). Protein concentrations in each fraction spiked at fraction 4 before receding through the rest of the 30% sucrose fractions (4-6), and from there markedly increasing in concentration throughout the pelleted cell debris (fraction 7-11).
Western blot analysis revealed that the distribution of the lipid raft marker flotilin-2 was affected by the type of sperm being assayed (
Figure 15). Testicular sperm showed a wider range of fractions with detectable levels of the flotilin-2 protein (4 - 11). Stripped sperm contained flotilin-2 primarily in fraction 4 and 5, with some detected in fraction 6-7. Cryopreserved sperm exhibited what appeared to be a disruption of the lipid raft. Flotilin-2 was distributed from fraction 4 through fraction 8, crossing the 30-40% sucrose barrier, indicating a change to raft buoyancy as a result of cholesterol loss.
The lipid raft marker, flotilin-2, was localized to the insertion point of the flagellum into the sperm head (Figure 16). The pattern appeared as 2-3 distinct patches located above the midpiece containing the mitochondria and centriolar complex. The remainder of sperm head and tail were lightly stained as well. Flotilin-2 distribution did not differ among different types of sperm activation statuses.
134
Membrane Lipid Composition
Untargeted metabolomics analysis of the Sauger sperm membrane revealed substantial impacts of cryopreservation on membrane composition, but little effect from testicular harvest (Figure 17). Multivariate analysis using PLS-DA identified 15 metabolites with a VIP score greater than 1.0, suggesting these were responsible for discrimination amongst treatments. Univariate analysis revealed no differences between stripped and testicular sperm in any of the 15 metabolites (one-way ANOVA, p < 0.05,
Table 6). However, cryopreserved sperm differed from stripped and testicular sperm for all measured variables. Specifically, metabolites such as hydroxylated cholesterol, cholesterol esters, lactones, hydroxylated fatty acids, lyso-phosphatidylcholine, and certain ceramides (2 of 4) were more prevalent in cryopreserved sperm. By contrast, only certain ceramides (2 of 4) and diacylglycerol were higher in stripped and testicular sperm.
Motility and Viability
Traditional sperm quality assessments revealed differences among sperm types (p
< 0.001, Table 7Table 7). Cryopreservation reduced all measured variables relative to stripped sperm, resulting in sperm that moved slowly and linearly. Testicular harvest resulted in similar motility and viability to cryopreserved sperm. However testicular sperm were more circular and swam at higher velocities compared to cryopreserved sperm. No correlation was found between sperm motility and the proportion of cells with high MC540 fluorescence following activation (p = 0.17). Viability, on the other hand, was negatively correlated with the proportion of highly MC540 fluorescent cells (r = -
135
0.48, p = 0.045). However, this relationship was driven by the differences among sperm types. When analyzed separately for each sperm types, no correlations were found between viability and fluidity within a given sperm type. These results confirm that motility and viability assessment cannot be relied upon as assessments of membrane quality and function.
Sperm Ultrastructure
Electron microscopy revealed that Sauger sperm plasma and nuclear membranes were severely damaged as a result of cryopreservation (Figure 18Figure 18). The most common abnormalities were swollen, whorled, and ruptured plasma and nuclear membranes (A). The nuclear material did not show any visible de-condensation (B), but the head membranes, flagellar apparatus, and nuclear membranes were often damaged (B
& C). Cytoplasmic blebs were also observed along the flagella on the axoneme of within the flagellar ribbon (D). Nearly all sperm were afflicted by one or multiple forms of damage. Interestingly, all of the samples used for both scanning and transmission electron microscopy displayed reasonable total motility (50-60%), further highlighting the possibility of widespread damage in the spermatozoa that is not evident from motility and viability assessment alone.
Discussion
The overall goal of this study was to describe and quantify sperm plasma membrane physiology in Sauger as a result of motility activation and how the membrane composition and structure differs among sperm yielded from several ARTs. We found the
Sauger sperm membrane to be a dynamic structure with a complex composition and 136 structure similar to other reports in spermatozoa. Testicular harvest and cryopreservation, two widely used assisted reproduction techniques, were found to have varying effects on the sperm plasma membrane’s composition, structure, and in some cases motility activation’s impact on membrane fluidity. We also found testicular harvest to mildly impact membrane physiology, whereas cryopreservation was found to be detrimental to all sperm characteristics assayed. These results offer insight to the physiological response of sperm to these different ARTs, provide further evidence for main sperm duct sperm maturation in fish, expand our knowledge of fish sperm cryobiology, and provide future avenues for protocol optimization to maintain membrane competence.
Detergent-resistant membranes were successfully isolated, characterized, and their distribution described for the first time in the spermatozoa of fish. DRMs were characterized by their buoyancy during ultracentrifugation separation, which is attributed to a high cholesterol content (Brown, 2006; Kawano et al., 2011). Moreover, flotilin-2, a common component of DRMs in other tissues and species, was most prevalent in the buoyant fractions, while the cytoskeleton (assessed using β-actin) was restricted to the pellet. In contrast to lipid raft analysis in Yellow Catfish (Pelteobagrus fulvidraco, Bai et al., 2019), we were unable to reliably confirm ganglioside GM1, another raft component, staining in Sauger sperm. Cholera toxin has been known to show poor affinity for fish
GM1 compared to commercial antibodies (Garcia-Garcia et al., 2012), such as those used by Bai et al (2019), potentially explaining the discrepancy between our studies. While the exact function of fish sperm lipid rafts was not tested herein, and remains unknown at present, their restricted distribution to the midpiece suggests a potential role in membrane fusion or cellular respiration. For example, flotilin was found to be transiently
137 phosphorylated following hypo-osmotic shock in the common carp suggesting a role in the activation response (Gazo et al., 2017). Additionally, studies in the rose bitterling
(Rhodeus ocellats) found that up to 70% of sperm were oriented with the flagella insertion and midpiece in contact with the oocyte plasma membrane (Ohta, 1991; Ohta and Iwamatsu, 1983). It is also interesting to note that lipid raft distribution was unaffected by hypo-osmotic shock, unlike certain glycoproteins of the Sauger glycocalyx with a demonstrated role in fertilization (Chapter 3). It is possible that multiple lipid raft subclasses exist within the sperm cell, as demonstrated in murine sperm (Asano et al.,
2009). Thus, the possibility exists that another type of lipid raft exists with a connection to the glycocalyx. By contrast, lipid raft redistribution in mammalian species typically corresponds to preparation for and execution of the acrosome reaction (Tsai et al., 2007).
Since Sauger sperm contain no acrosome (Chapter 2), a lack of redistribution is consistent with known fish sperm cell morphology.
Membrane fluidity, an important characteristic to sperm of other taxa, was elevated by hypo-osmotic shock in our study. Based on mean fluorescent intensity alone, our results were in agreement with earlier accounts of elevated membrane fluidity in common carp following activation (Krasznai et al., 2003a, 2003b). However, a more detailed assessment of individual sperm revealed a different pattern. Similar to capacitation in mammalian sperm, only a small proportion of sperm exhibited enhanced fluidity (< 40%) in response to activation. Experiments by Krasznai et al. (2003 a, b) implicated increased membrane fluidity during the activation response of common carp sperm but also that this increase could both be inhibited using a stretch activated channel blocker, gadolinium that also blocked motility. By contrast, we found a lack of
138 correlation between motility and fluidity, suggesting that the two processes are not codependent, other than perhaps through a common stimulus. Likewise, Purdy et al (
2016) also found no correlation among fertilization and membrane fluidity in
Oncorhynchus mykiss in fresh sperm. Given these contrasting findings, the role of membrane fluidity in fish sperm remains unclear. However, we speculate that elevated fluidity is a sign of membrane instability, as it was highest in cryopreserved sperm.
The effect of testicular harvest’s effect on sperm was less compared to the effect of cryopreservation. Aside from lower motility, the largest differences between stripped and testicular sperm were in the composition and the distribution of flotilin-2 in lipid rafts. These results are resonant of differences seen in between early epididymal and ejaculated mammalian sperm (Haidl and Opper, 1997; Jones, 2002; Parks and
Hammerstedt, 1985; Rejraji et al., 2006). Exposure to seminal plasma during epididymal maturation in mammalian species (e.g., bovine) is often associated with changes to membrane composition, favoring a more fluid and organized state (Rejraji et al., 2006).
This remodeling process in bovine sperm includes loss of cholesterol and GM1 from the cell and rafts, as well as changes to the raft protein composition (Girouard et al., 2008b).
The results of our study support this model. The lipid raft marker flotilin-2 was visibly more condensed in fractions 4-5 (i.e., lipid rafts) in stripped sperm compared to testicular sperm. However, the differences seen in our study between testicular and stripped sperm seemed to have a relatively small impact on fertilization based on previously published evidence describing similar fertility among sperm types (Appendix A, Blawut et al.,
2020b). Thus, our results corroborate that during testicular harvest, a portion of immature
139 cells are collected alongside mature spermatozoa. However, in Sauger, immature cell presence cells do not appear to inhibit the practical applicability of this technique.
Cryopreservation altered every aspect of the sperm plasma membrane composition, structure, and functionality in Sauger. These changes are likely at least partially responsible for the low post-thaw fertility observed previously in this species
(Blawut et al., 2020a). Findings from our study are consistent with widespread damage to the plasma membrane composition, likely as a result of oxidative stress (Ball, 2008;
Ertmer et al., 2017). Significant increases in hydroxylated cholesterol, hydroxylated fatty acids, lactones, and lyso-phospholipids, as well as increases in certain ceramides, have been associated with oxidative damage that alters membrane characteristics and intracellular apoptotic signaling (Flesch and Gadella, 2000; Schiller et al., 2000).
Ceramides are products of sphingolipid signaling pathways and are generally regarded to be an effector of cellular apoptosis (Kalo and Roth, 2011; Peña et al., 1997).
Interestingly, 2 of 4 ceramides in this analysis were elevated in cryopreserved sperm with the others elevated in stripped and testicular sperm. Lactones, most notable for quorum sensing in bacteria, have also been shown to induce the acrosome reaction and cellular apoptosis in human sperm (Rennemeier et al., 2009) and could be part of apoptosis signaling. However, little information exists regarding the function of ceramides and lactones in fish spermatozoa. The evidence presented above supports apoptotic-like changes in the plasma membrane of cryopreserved sperm resulting from lipid peroxidation damage.
Fluidity closely mirrored these changes in membrane composition for cryopreserved sperm. The majority (75%) of inactive sperm exhibited low fluorescence,
140 with a magnitude of approximately half that of stripped and testicular sperm. These results suggest a more rigid membrane in frozen sperm. This rigidifying process has also been reported in the sperm of humans, chickens, and other fish species (Blesbois et al.,
2005; Giraud et al., 2000). While high post-thaw fluidity in avian sperm is an indicator of high fertility (Blesbois et al., 2008), the lowest fertility was seen in frozen-thawed sperm samples despite their high membrane fluidity. We suspect that an inability to control membrane asymmetry during the activation process in the harsh hypo-osmotic environment is the likely the cause of this elevated fluidity.
Lipid rafts have long been known to be prime targets of cryopreservation induced damage. Lipid raft components, e.g. cholesterol and sphingomyelin, are commonly reduced membrane components in fish and avian sperm following cryopreservation (Dai et al., 2012; Díaz et al., 2019; He et al., 2011a; Ushiyama et al., 2016, 2017; Dietrich et al., 2015). Correspondingly, Sauger lipid raft were affected by cryopreservation as evidenced by the nearly 45% reduction in cholesterol and buoyance changes seen in wider distribution of the DRM marker. Because lipid rafts are known to contain proteins important for oocyte complex and oocyte-sperm interaction (Nixon et al., 2009; Nixon and Aitken, 2009a), disturbances in these rafts is thought to negatively affect fertilization.
Further investigation is needed to determine if the observed dissociation of lipid rafts may be a contributing factor to low fertilization potential of cryopreserved sperm observed in Sauger and other species (Bai et al., 2019; He et al., 2011a)
Interestingly, these changes observed in the spermatozoa of Sauger following cryopreservation did not inhibit motility in frozen sperm. Total motility was similar between testicular (67.7 ± 2.6 %) and cryopreserved sperm (65.3 ± 2.6 %) and was
141 unrelated to membrane fluidity, lipid raft integrity, or lipid composition. Fish sperm has previously been shown to have high resiliency to membrane perturbations. Müller et al (
2008), for example, found Oncorhynchus mykiss sperm motility to be unaffected by up to a 30-50% reduction in membrane cholesterol. In fact, cholesterol affected both fluidity and phosphatidylserine translocation ability, both of which increased in the presence of decreasing cholesterol without any effect on fertilization capacity. However, fertilization testing by Müller et al (2008) used a high sperm-to-egg ratio, potentially masking reductions in quality with excessive amounts of sperm. Additionally, lipid rafts were not investigated. Our results echo the notion that traditional measures of sperm quality (e.g., motility) are insufficient biomarkers of fertility (Asturiano et al., 2017; Cabrita et al.,
2014) and suggests that the membrane plays more than just one role in sperm activation
While we feel that our findings are robust, our study is limited in several ways.
First, lipid raft analysis has been widely adopted in the field of reproduction, but the methodology used here does have its drawbacks. Detergent-resistant membrane analysis is sometimes discredited by some researchers as an artifact of the extraction process
(Brown, 2006; Sonnino and Prinetti, 2013). While it is true that the DRMs and lipid raft are not completely synonymous, the research conducted thus far in spermatozoa has been consistent and results suggest the findings do have in vivo implications. Second, several probes used in this study were unsuccessful regarding binding (CTβ) or inconsistent with other methods measuring similar characteristics (filipin III). Low homology of GM1 between fish and mammalian species (Bai et al., 2019) or suboptimal staining procedures
(filipin III may be more effective with fixation prior to analysis) may explain their lack of staining efficacy.
142
Conclusions
In summary, this research was able to describe Sauger sperm membrane physiology and compare the effects of commonly used assisted reproduction techniques impacts on membrane composition, structure, and fluidity. First, we determined that hypo-osmotic shock caused an increase in membrane fluidity for a proportion of cells and was unrelated to sperm motility. Thus, motility cannot be used as a proxy for membrane quality in Sauger sperm. Second, we provided a detailed isolation protocol and characterization of lipid raft composition and distribution in the sperm cell membrane.
These results are novel in that lipid rafts have not been adequately studied to date in fish sperm, despite their role in sperm-egg interactions in other species. Third, we supported main sperm duct maturation of Sauger sperm through comparative differences in lipid raft structure between testicular and stripped sperm. This finding reinforces our previous assertion that sperm mature physiologically within the main sperm duct (Chapter 2 and
3), which may have implications for the practical application of testicular harvest. And lastly, our results indicated that cryopreservation negatively affected membrane composition, lipid raft cholesterol content, and membrane fluidity as a result of oxidative damage during the freeze-thaw process. These results indicate potential sources of sub- fertility resulting from the freeze-thaw process, most of which are structures that are not routinely evaluated during quality assessment. Collectively, these results deepen our understanding of natural reproductive physiology in the Sauger, enhance our knowledge of fish sperm cryobiology, and provide multiple membrane biomarkers for sperm quality.
In turn, these results have implications as future biomarkers of membrane quality in any aquatic species where ARTs substantially impact sperm fertilization potential. 143
References
Asano, Atsushi, Vimal Selvaraj, Danielle E. Buttke, Jacquelyn L. Nelson, Karin M.
Green, James E. Evans, and Alexander J. Travis. 2009. “Biochemical
Characterization of Membrane Fractions in Murine Sperm: Identification of Three
Distinct Sub-Types of Membrane Rafts.” Journal of Cellular Physiology
218(3):537–48. doi: 10.1002/jcp.21623.
Asturiano, Juan F., Elsa Cabrita, and Ákos Horváth. 2017. “Progress, Challenges and
Perspectives on Fish Gamete Cryopreservation: A Mini-Review.” General and
Comparative Endocrinology 245:69–76. doi: 10.1016/j.ygcen.2016.06.019.
Bai, Chenglian, Ning Kang, Junping Zhao, Jun Dai, Hui Gao, Yuanhong Chen, Haojia
Dong, Changjiang Huang, and Qiaoxiang Dong. 2019. “Cryopreservation
Disrupts Lipid Rafts and Heat Shock Proteins in Yellow Catfish Sperm.”
Cryobiology 87:32–39. doi: 10.1016/j.cryobiol.2019.03.004.
Ball, Barry A. 2008. “Oxidative Stress, Osmotic Stress and Apoptosis: Impacts on Sperm
Function and Preservation in the Horse.” Animal Reproduction Science
107(3):257–67. doi: 10.1016/j.anireprosci.2008.04.014.
Beirão, J., L. Zilli, S. Vilella, E. Cabrita, C. Fernández‐Díez, R. Schiavone, and M. P.
Herráez. 2012. “Fatty Acid Composition of the Head Membrane and Flagella
Affects Sparus Aurata Sperm Quality.” Journal of Applied Ichthyology
28(6):1017–19. doi: 10.1111/jai.12085.
Bell, M. V., J. R. Dick, and Cs. Buda. 1997. “Molecular Speciation of Fish Sperm
Phospholipids: Large Amounts of Dipolyunsaturated Phosphatidylserine.” Lipids
32(10):1085–91. doi: 10.1007/s11745-997-0140-y.
144
Bell, Michael V., James R. Dick, Mark Thrush, and Juan Carlos Navarro. 1996.
“Decreased 20:4n − 620:5n − 3 Ratio in Sperm from Cultured Sea Bass,
Dicentrarchus Labrax, Broodstock Compared with Wild Fish.” Aquaculture
144(1):189–99. doi: 10.1016/S0044-8486(96)01311-7.
Bergeron, A., G. Vandenberg, D. Proulx, and J. L. Bailey. 2002. “Comparison of
Extenders, Dilution Ratios and Theophylline Addition on the Function of
Cryopreserved Walleye Semen.” Theriogenology 57(3):1061–71. doi:
10.1016/S0093-691X(01)00707-5.
Blawut, Bryan, Barbara Wolfe, Christa R. Moraes, Stuart A. Ludsin, and Marco A.
Coutinho da Silva. 2018. “Increasing Saugeye (S. Vitreus × S. Canadensis)
Production Efficiency in a Hatchery Setting Using Assisted Reproduction
Technologies.” Aquaculture 495:21–26. doi: 10.1016/j.aquaculture.2018.05.027.
Blawut, Bryan, Barbara Wolfe, Christa R. Moraes, Stuart A. Ludsin, and Marco A.
Coutinho da Silva. 2020. “Use of Hypertonic Media to Cryopreserve Sauger
Spermatozoa.” North American Journal of Aquaculture 82(1):84–91. doi:
10.1002/naaq.10125.
Blawut, Bryan, Barbara Wolfe, Christa R. Moraes, Douglas Sweet, Stuart A. Ludsin, and
Marco A. Coutinho da Silva. 2020. “Testicular Collections as a Technique to
Increase Milt Availability in Sauger (Sander Canadensis).” Animal Reproduction
Science 212:106240. doi: 10.1016/j.anireprosci.2019.106240.
Blesbois, E., I. Grasseau, and F. Seigneurin. 2005. “Membrane Fluidity and the Ability of
Domestic Bird Spermatozoa to Survive Cryopreservation.” Reproduction
129(3):371–78. doi: 10.1530/rep.1.00454.
145
Blesbois, E., I. Grasseau, F. Seigneurin, S. Mignon-Grasteau, M. Saint Jalme, and M. M.
Mialon-Richard. 2008. “Predictors of Success of Semen Cryopreservation in
Chickens.” Theriogenology 69(2):252–61. doi:
10.1016/j.theriogenology.2007.09.019.
Bligh, E. G., and W. J. Dyer. 1959. “A Rapid Method of Total Lipid Extraction and
Purification.” Canadian Journal of Biochemistry and Physiology 37(8):911–17.
doi: 10.1139/o59-099.
Brogden, Graham, Marcus Propsting, Mikolaj Adamek, Hassan Y. Naim, and Dieter
Steinhagen. 2014. “Isolation and Analysis of Membrane Lipids and Lipid Rafts in
Common Carp (Cyprinus Carpio L.).” Comparative Biochemistry and Physiology
Part B: Biochemistry and Molecular Biology 169:9–15. doi:
10.1016/j.cbpb.2013.12.001.
Brown, Deborah A. 2006. “Lipid Rafts, Detergent-Resistant Membranes, and Raft
Targeting Signals.” Physiology 21(6):430–39. doi: 10.1152/physiol.00032.2006.
Cabrita, E., S. Martínez-Páramo, P. J. Gavaia, M. F. Riesco, D. G. Valcarce, C.
Sarasquete, M. P. Herráez, and V. Robles. 2014. “Factors Enhancing Fish Sperm
Quality and Emerging Tools for Sperm Analysis.” Aquaculture 432:389–401. doi:
10.1016/j.aquaculture.2014.04.034.
Cabrita, E., C. Sarasquete, S. Martínez-Páramo, V. Robles, J. Beirão, S. Pérez-Cerezales,
and M. p. Herráez. 2010. “Cryopreservation of Fish Sperm: Applications and
Perspectives.” Journal of Applied Ichthyology 26(5):623–35. doi: 10.1111/j.1439-
0426.2010.01556.x.
146
Cabrita, Elsa, Vanesa Robles, and Paz Herráez. 2008. Methods in Reproductive
Aquaculture: Marine and Freshwater Species. CRC Press.
Dadras, Hadiseh, Sabine Sampels, Amin Golpour, Viktoriya Dzyuba, Jacky Cosson, and
Borys Dzyuba. 2017. “Analysis of Common Carp Cyprinus Carpio Sperm
Motility and Lipid Composition Using Different in Vitro Temperatures.” Animal
Reproduction Science 180:37–43. doi: 10.1016/j.anireprosci.2017.02.011.
Dai, Tongren, Enhui Zhao, Gang Lu, Kai Che, Qiutao He, Yonglin Lu, Qiongshan Fang,
Hansheng Wang, Leyun Zheng, Suping Li, Changjiang Huang, and Qiaoxiang
Dong. 2012. “Sperm Cryopreservation of Yellow Drum Nibea Albiflora: A
Special Emphasis on Post-Thaw Sperm Quality.” Aquaculture 368–369:82–88.
doi: 10.1016/j.aquaculture.2012.09.017.
Díaz, Rommy, Manuel Lee-Estévez, Elías Figueroa, Patricio Ulloa-Rodríguez, Néstor
Sepúlveda, and Jorge G. Farias. 2018. “Study of the Membrane Lipid
Composition of Atlantic Salmon (Salmo Salar) Spermatozoa and Its Relation with
Semen Quality.” Aquaculture Research 49(7):2603–2607.
Díaz, Rommy, Manuel Lee-Estevez, John Quiñones, Kelly Dumorné, Stefania Short,
Patricio Ulloa-Rodríguez, Ivan Valdebenito, Néstor Sepúlveda, and Jorge G.
Farías. 2019. “Changes in Atlantic Salmon (Salmo Salar) Sperm Morphology and
Membrane Lipid Composition Related to Cold Storage and Cryopreservation.”
Animal Reproduction Science 204:50–59. doi: 10.1016/j.anireprosci.2019.03.004.
Dietrich, Mariola A., Georg J. Arnold, Thomas Fröhlich, Kathrin A. Otte, Grzegorz J.
Dietrich, and Andrzej Ciereszko. 2015. “Proteomic Analysis of Extracellular
Medium of Cryopreserved Carp (Cyprinus Carpio L.) Semen.” Comparative
147
Biochemistry and Physiology Part D: Genomics and Proteomics 15:49–57. doi:
10.1016/j.cbd.2015.05.003.
Engel, Kathrin M., Viktoriya Dzyuba, Dzyuba Borys, and Jürgen Schiller. 2020.
“Different Glycolipids in Sperm from Different Freshwater Fishes – A High
Performance Thin-Layer Chromatography/Electrospray Ionization-Mass
Spectrometry Study.” Rapid Communications in Mass Spectrometry 34(19):9.
doi: 10.1002/rcm.8875.
Ertmer, Franziska, Harriëtte Oldenhof, Saskia Schütze, Karl Rohn, Willem F. Wolkers,
and Harald Sieme. 2017. “Induced Sub-Lethal Oxidative Damage Affects
Osmotic Tolerance and Cryosurvival of Spermatozoa.” Reproduction, Fertility
and Development 29(9):1739–50. doi: 10.1071/RD16183.
Flesch, Frits M., and Barend M. Gadella. 2000. “Dynamics of the Mammalian Sperm
Plasma Membrane in the Process of Fertilization.” Biochimica et Biophysica Acta
(BBA) - Reviews on Biomembranes 1469(3):197–235. doi: 10.1016/S0304-
4157(00)00018-6.
Garcia-Garcia, Erick, Leon Grayfer, James L. Stafford, and Miodrag Belosevic. 2012.
“Evidence for the Presence of Functional Lipid Rafts in Immune Cells of
Ectothermic Organisms.” Developmental & Comparative Immunology
37(2):257–69. doi: 10.1016/j.dci.2012.03.009.
Gazo, Ievgeniia, Mariola A. Dietrich, Gérard Prulière, Anna Shaliutina-Kolešová, Olena
Shaliutina, Jacky Cosson, and Janet Chenevert. 2017. “Protein Phosphorylation in
Spermatozoa Motility of Acipenser Ruthenus and Cyprinus Carpio.”
Reproduction 154(5):653–73. doi: 10.1530/REP-16-0662.
148 van Gestel, R. A., I. A. Brewis, P. R. Ashton, J. B. Helms, J. F. Brouwers, and B. M.
Gadella. 2005. “Capacitation-Dependent Concentration of Lipid Rafts in the
Apical Ridge Head Area of Porcine Sperm Cells.” MHR: Basic Science of
Reproductive Medicine 11(8):583–90. doi: 10.1093/molehr/gah200.
Giraud, M. N., C. Motta, D. Boucher, and G. Grizard. 2000. “Membrane Fluidity Predicts
the Outcome of Cryopreservation of Human Spermatozoa.” Human Reproduction
15(10):2160–64. doi: 10.1093/humrep/15.10.2160.
Girouard, Julie, Gilles Frenette, and Robert Sullivan. 2008. “Seminal Plasma Proteins
Regulate the Association of Lipids and Proteins Within Detergent-Resistant
Membrane Domains of Bovine Spermatozoa.” Biology of Reproduction
78(5):921–31. doi: 10.1095/biolreprod.107.066514.
Gylfason, Gudjón Andri, Erna Knútsdóttir, and Bjarni Ásgeirsson. 2010. “Isolation and
Biochemical Characterisation of Lipid Rafts from Atlantic Cod (Gadus Morhua)
Intestinal Enterocytes.” Comparative Biochemistry and Physiology Part B:
Biochemistry and Molecular Biology 155(1):86–95. doi:
10.1016/j.cbpb.2009.10.006.
Haidl, G., and C. Opper. 1997. “Changes in Lipids and Membrane Anisotropy in Human
Spermatozoa during Epididymal Maturation.” Human Reproduction 12(12):2720–
23. doi: 10.1093/humrep/12.12.2720.
Hammerstedt, Roy H., James K. Graham, and John P. Nolan. 1990. “Cryopreservation of
Mammalian Sperm: What We Ask Them to Survive.” Journal of Andrology
11(1):73–88. doi: 10.1002/j.1939-4640.1990.tb01583.x.
149
He, Qiutao, Gang Lu, Kai Che, Enhui Zhao, Qiongshan Fang, Hansheng Wang, Jing Liu,
Changjiang Huang, and Qiaoxiang Dong. 2011a. “Sperm Cryopreservation of the
Endangered Red Spotted Grouper, Epinephelus Akaara, with a Special Emphasis
on Membrane Lipids.” Aquaculture 318(1):185–90. doi:
10.1016/j.aquaculture.2011.05.025.
He, Qiutao, Gang Lu, Kai Che, Enhui Zhao, Qiongshan Fang, Hansheng Wang, Jing Liu,
Changjiang Huang, and Qiaoxiang Dong. 2011b. “Sperm Cryopreservation of the
Endangered Red Spotted Grouper, Epinephelus Akaara, with a Special Emphasis
on Membrane Lipids.” Aquaculture 318(1):185–90. doi:
10.1016/j.aquaculture.2011.05.025.
Holt, W. V. 2000. “Basic Aspects of Frozen Storage of Semen.” Animal Reproduction
Science 62(1):3–22. doi: 10.1016/S0378-4320(00)00152-4.
Jones, Roy. 2002. “Plasma Membrane Composition and Organisation During Maturation
of Spermatozoa in the Epididymis.” The Epididymis: From Molecules to Clinical
Practice 405–16. doi: 10.1007/978-1-4615-0679-9_23.
Kalo, Dorit, and Zvi Roth. 2011. “Involvement of the Sphingolipid Ceramide in Heat-
Shock-Induced Apoptosis of Bovine Oocytes.” Reproduction, Fertility and
Development 23(7):876–88. doi: 10.1071/RD10330.
Kawano, Natsuko, Kaoru Yoshida, Kenji Miyado, and Manabu Yoshida. 2011. “Lipid
Rafts: Keys to Sperm Maturation, Fertilization, and Early Embryogenesis.”
Journal of Lipids. Retrieved October 19, 2017
(https://www.hindawi.com/journals/jl/2011/264706/abs/).
150
Khalil, Maroun Bou, Krittalak Chakrabandhu, Hongbin Xu, Wattana Weerachatyanukul,
Mary Buhr, Trish Berger, Euridice Carmona, Ngoc Vuong, Premkumari
Kumarathasan, Patrick T. T. Wong, Danielle Carrier, and Nongnuj Tanphaichitr.
2006. “Sperm Capacitation Induces an Increase in Lipid Rafts Having Zona
Pellucida Binding Ability and Containing Sulfogalactosylglycerolipid.”
Developmental Biology 290(1):220–35. doi: 10.1016/j.ydbio.2005.11.030.
Koubek, P., A. Kralova, M. Psenicka, and J. Peknicova. 2008. “The Optimization of the
Protocol for Immunofluorescence on Fish Spermatozoa.” Theriogenology
70(5):852–58. doi: 10.1016/j.theriogenology.2008.05.050.
Krasznai, Zoltán, Masaaki Morisawa, Zoárd Tibor Krasznai, Sachiko Morisawa, Kazuo
Inaba, Zsuzsa Kassai Bazsáné, Bálint Rubovszky, Béla Bodnár, Antal Borsos, and
Teréz Márián. 2003. “Gadolinium, a Mechano-Sensitive Channel Blocker,
Inhibits Osmosis-Initiated Motility of Sea- and Freshwater Fish Sperm, but Does
Not Affect Human or Ascidian Sperm Motility.” Cell Motility 55(4):232–43. doi:
10.1002/cm.10125.
Krasznai, Zoltán, Masaaki Morisawa, Sachiko Morisawa, Zoárd Tíbor Krasznai, Lajos
Trón, Rezső Gáspár, and Teréz Márián. 2003. “Role of Ion Channels and
Membrane Potential in the Initiation of Carp Sperm Motility.” Aquatic Living
Resources 16(5):445–49. doi: 10.1016/S0990-7440(03)00054-8.
Laemmli, U. K. 1970. “Cleavage of Structural Proteins during the Assembly of the Head
of Bacteriophage T4.” Nature 227(5259):680–85. doi: 10.1038/227680a0.
Martínez-Páramo, S., P. Diogo, J. Beirão, M. T. Dinis, and E. Cabrita. 2012. “Sperm
Lipid Peroxidation Is Correlated with Differences in Sperm Quality during the
151
Reproductive Season in Precocious European Sea Bass (Dicentrarchus Labrax)
Males.” Aquaculture 358–359:246–52. doi: 10.1016/j.aquaculture.2012.06.010.
Moore, A. 1987. “Short-Term Storage and Cryopreservation of Walleye Semen.”
Progressive Fish-Culturist 49(1):40–43. doi: 10.1577/1548-
8640(1987)49<40:SSACOW>2.0.CO;2.
Müller, Karin, Peter Müller, Gwenaëlle Pincemy, Anke Kurz, and Catherine Labbe.
2008. “Characterization of Sperm Plasma Membrane Properties after Cholesterol
Modification: Consequences for Cryopreservation of Rainbow Trout
Spermatozoa.” Biology of Reproduction 78(3):390–399.
Nixon, Brett, and R. John Aitken. 2009a. “The Biological Significance of Detergent-
Resistant Membranes in Spermatozoa.” Journal of Reproductive Immunology
83(1):8–13. doi: 10.1016/j.jri.2009.06.258.
Nixon, Brett, and R. John Aitken. 2009b. “The Biological Significance of Detergent-
Resistant Membranes in Spermatozoa.” Journal of Reproductive Immunology
83(1):8–13. doi: 10.1016/j.jri.2009.06.258.
Nixon, Brett, Amanda Bielanowicz, Eileen A. Mclaughlin, Nongnuj Tanphaichitr,
Michael A. Ensslin, and R. John Aitken. 2009. “Composition and Significance of
Detergent Resistant Membranes in Mouse Spermatozoa.” Journal of Cellular
Physiology 218(1):122–34. doi: 10.1002/jcp.21575.
Ohta, Tadayuki. 1991. “Initial Stages of Sperm-Egg Fusion in the Freshwater Teleost,
Rhodeus Ocellatus Ocellatus.” The Anatomical Record 229(2):195–202. doi:
10.1002/ar.1092290206.
152
Ohta, Tadayuki, and Takashi Iwamatsu. 1983. “Electron Microscopic Observations on
Sperm Entry into Eggs of the Rose Bitterling, Rhodeus Ocellatus.” Journal of
Experimental Zoology 227(1):109–19. doi: 10.1002/jez.1402270115.
Parks, J. E., and J. K. Graham. 1992. “Effects of Cryopreservation Procedures on Sperm
Membranes.” Theriogenology 38(2):209–22. doi: 10.1016/0093-691X(92)90231-
F.
Parks, John E., and Roy H. Hammerstedt. 1985. “Developmental Changes Occurring in
the Lipids of Ram Epididymal Spermatozoa Plasma Membrane.” Biology of
Reproduction 32(3):653–68. doi: 10.1095/biolreprod32.3.653.
Peña, Louis A., Zvi Fuks, and Richard Koksnick. 1997. “Stress-Induced Apoptosis and
the Sphingomyelin Pathway.” Biochemical Pharmacology 53(5):615–21. doi:
10.1016/S0006-2952(96)00834-9.
Purdy, P. H., E. A. Barbosa, C. J. Praamsma, and G. J. Schisler. 2016. “Modification of
Trout Sperm Membranes Associated with Activation and Cryopreservation.
Implications for Fertilizing Potential.” Cryobiology 73(1):73–79. doi:
10.1016/j.cryobiol.2016.05.008.
Pustowka, C., M. A. McNiven, G. F. Richardson, and S. P. Lall. 2000. “Source of Dietary
Lipid Affects Sperm Plasma Membrane Integrity and Fertility in Rainbow Trout
Oncorhynchus Mykiss (Walbaum) after Cryopreservation.” Aquaculture Research
31(3):297–305. doi: 10.1046/j.1365-2109.2000.00416.x.
R Development Core Team. 2019. R: A Language and Environment for Statistical
Computing. R Foundation for Statistical Computing, Vienna, Austria. URL
http://R-project.org.
153
Reis, Ana, Alisa Rudnitskaya, Gavin J. Blackburn, Norsyahida Mohd Fauzi, Andrew R.
Pitt, and Corinne M. Spickett. 2013. “A Comparison of Five Lipid Extraction
Solvent Systems for Lipidomic Studies of Human LDL.” Journal of Lipid
Research 54(7):1812–24. doi: 10.1194/jlr.M034330.
Rejraji, Hanae, Benoit Sion, Gerard Prensier, Martine Carreras, Claude Motta, Jean-
Marie Frenoux, Evelyne Vericel, Genevieve Grizard, Patrick Vernet, and Joël R.
Drevet. 2006. “Lipid Remodeling of Murine Epididymosomes and Spermatozoa
During Epididymal Maturation.” Biology of Reproduction 74(6):1104–13. doi:
10.1095/biolreprod.105.049304.
Rennemeier, Claudia, Torsten Frambach, Florian Hennicke, Johannes Dietl, and Peter
Staib. 2009. “Microbial Quorum-Sensing Molecules Induce Acrosome Loss and
Cell Death in Human Spermatozoa.” Infection and Immunity 77(11):4990–97.
doi: 10.1128/IAI.00586-09.
Schiller, Jürgen, Jürgen Arnhold, Hans-Jürgen Glander, and Klaus Arnold. 2000. “Lipid
Analysis of Human Spermatozoa and Seminal Plasma by MALDI-TOF Mass
Spectrometry and
NMR Spectroscopy — Effects of Freezing and Thawing.” Chemistry and Physics of
Lipids 106(2):145–56. doi: 10.1016/S0009-3084(00)00148-1.
Shadan, Sadaf, Peter S. James, Elizabeth A. Howes, and Roy Jones. 2004. “Cholesterol
Efflux Alters Lipid Raft Stability and Distribution During Capacitation of Boar
Spermatozoa.” Biology of Reproduction 71(1):253–65. doi:
10.1095/biolreprod.103.026435.
154
Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D.
Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk. 1985.
“Measurement of Protein Using Bicinchoninic Acid.” Analytical Biochemistry
150(1):76–85. doi: 10.1016/0003-2697(85)90442-7.
Sonnino, S., and A. Prinetti. 2013. “Membrane Domains and the ‘Lipid Raft’ Concept.”
Current Medicinal Chemistry 20(1):4–21.
Tapia, J. A., B. Macias‐Garcia, A. Miro‐Moran, C. Ortega‐Ferrusola, G. M. Salido, F. J.
Peña, and I. M. Aparicio. 2018. “The Membrane of the Mammalian Spermatozoa:
Much More Than an Inert Envelope.” Reproduction in Domestic Animals
47(s3):65–75. doi: 10.1111/j.1439-0531.2012.02046.x.
Thaler, Catherine D., Monzy Thomas, and Jenniffer R. Ramalie. 2006. “Reorganization
of Mouse Sperm Lipid Rafts by Capacitation.” Molecular Reproduction and
Development 73(12):1541–49. doi: 10.1002/mrd.20540.
Tsai, Pei-Shiue, Pei-Shiue Tsai, Klaas J. De Vries, Pei-Shiue Tsai, Klaas J. De Vries,
Mieke De Boer-Brouwer, Nuria Garcia-Gil, Renske A. Van Gestel, Pei-Shiue
Tsai, Klaas J. De Vries, Mieke De Boer-Brouwer, Nuria Garcia-Gil, Renske A.
Van Gestel, Ben Colenbrander, Bart M. Gadella, and Theo Van Haeften. 2007.
“Syntaxin and VAMP Association with Lipid Rafts Depends on Cholesterol
Depletion in Capacitating Sperm Cells.” Molecular Membrane Biology
24(4):313–24. doi: 10.1080/09687860701228692.
Ushiyama, Ai, Naoto Ishikawa, Atsushi Tajima, and Atsushi Asano. 2016. “Comparison
of Membrane Characteristics between Freshly Ejaculated and Cryopreserved
155
Sperm in the Chicken.” The Journal of Poultry Science advpub. doi:
10.2141/jpsa.0160043.
Ushiyama, Ai, Atsushi Tajima, Naoto Ishikawa, and Atsushi Asano. 2017.
“Characterization of the Functions and Proteomes Associated with Membrane
Rafts in Chicken Sperm.” PLOS ONE 12(11):e0186482. doi:
10.1371/journal.pone.0186482.
Williamson, P., K. Mattocks, and R. A. Schlegel. 1983. “Merocyanine 540, a Fluorescent
Probe Sensitive to Lipid Packing.” Biochimica Et Biophysica Acta 732(2):387–
93. doi: 10.1016/0005-2736(83)90055-x.
Xin, Miaomiao, Hamid Niksirat, Anna Shaliutina‐Kolešová, Mohammad Abdul Momin
Siddique, Jan Sterba, Sergii Boryshpolets, and Otomar Linhart. 2019. “Molecular
and Subcellular Cryoinjury of Fish Spermatozoa and Approaches to Improve
Cryopreservation.” Reviews in Aquaculture n/a(n/a). doi: 10.1111/raq.12355.
Zehmer, John K., and Jeffrey R. Hazel. 2003. “Plasma Membrane Rafts of Rainbow
Trout Are Subject to Thermal Acclimation.” Journal of Experimental Biology
206(10):1657–67. doi: 10.1242/jeb.00346.
Zehmer, John K., and Jeffrey R. Hazel. 2004. “Membrane Order Conservation in Raft
and Non-Raft Regions of Hepatocyte Plasma Membranes from Thermally
Acclimated Rainbow Trout .” Biochimica et Biophysica Acta (BBA) -
Biomembranes 1664(1):108–16. doi: 10.1016/j.bbamem.2004.04.008.
156
Table 5. Quantitative assessment of Sauger spermmembrane fluidity among sperm types and activation statuses using the fluorescent
probe merocyanine 540 (MC540, n = 7). Data are represented as mean ± 1SEM. Different superscript letters denote Tukey’s HSD post
hoc test mean compressions (α = 0.05).
Sperm Type / Activation Status
15 Characteristic Testicular Stripped Cryopreserved
7
Inactive Activated Inactive Activated Inactive Activated
MC540 Populations 4.1 ± 0.9 b X 39.1 ± 2.7 b Y 6.3 ± 1.1 b X 44.5 ± 2.7 b Y 29.5 ± 2.2 a X 75.6 ± 2.2 a Y (%)
MC540 MFI (a.u.) 663 ± 191 III 1,541 ± 191 II 731 ± 191 III 1,494 ± 191 II 2,995 ± 191 I 2,504 ± 191 I
a,b Mean values (± SEM) with different superscript letters within a row differ significantly at P < 0.05.
X,Y Mean values (± SEM) with different superscript letters within a row differ significantly at P < 0.05.
I,II, III Mean values (± SEM) with different superscript letters within a row differ significantly at P < 0.05.
157
a a 600
3 ( Content Protein
M) b 400 2
200 g) Cholesterol ( 1
0 0 1 2 3 4 5 6 7 8 9 10 11 Fraction Sample
Figure 14. Sauger sperm cholesterol and protein content in ultracentrifugation separated lipid raft fractions. Fractions range from 1 (top of tube) through 12 (bottom of tube).
Cholesterol content is on left y-axis and bars are separated by sperm types [testicular
(red), grey (stripped), and cryopreserved (white)]. Different superscripts (Tukey’s HSD, p
< 0.05) denote differences among sperm types based on cholesterol content differences in fraction 4, the proposed lipid rafts (p < 0.05). Protein content is on the right y-axis and lines connect protein content mean values from the same sperm type types [testicular
(▲), grey (•), and cryopreserved (■)]. No differences were observed among protein contents between different sperm types. Data are represented as mean ± 1 SEM for cholesterol, and mea values alone for protein (n = 4
158
Ultracentrifugation Fraction 1 2 3 4 5 6 7 8 9 10 11 Testicular 53 kDa 38 kDa Flotilin-2
53 kDa
38 kDa Β - Actin
Stripped 1
59 53 kDa
38 kDa Flotilin-2 53 kDa
38 kDa Β - Actin
Cryopreserved 53 kDa
38 kDa Flotilin-2 53 kDa
38 kDa Β - Actin
Figure 15. Distribution of flotillin-2, a lipid raft marker, in sucrose gradient ultracentrifugation separated fractions using western blot.
Each type of Sauger sperm (testicular, stripped, and cryopreserved) was assessed for the flotillin-2 raft marker and β-actin was used to
assess the validity of the separation technique (β-actin is a cytoskeletal protein and should only be localized to the pellet)…………….
159
Figure 15. Continued. Blots were probed using flotilin-2 (sc-28320) and β-actin (ab8227), signal was generated using West Dura
Super Signal (enhance chemiluminescence substrate),and imaged using a digital Amersham Imager 680 (Cytiva, MA, USA).
16
0
160
Figure 16. Lipid raft localization in Sauger sperm using flotilin-2 immunofluorescence.
(Left) Differential interference contrast microscopy image (× 1,000). (Middle) Hoechst
DNA stain localizing the nucleus. (Right) Flotilin-2 distribution showing the location of lipid rafts.
161
Figure 17. Partial Least Squares – Discriminant Analysis (Left) plot of individual data points per treatment group and associated variables of importance (VIP) plot (Right) of
LC-MS data obtained from cryopreserved (Group C), stripped (Group S), and testicular
(Group T) Sauger spermatozoa. The top 15 metabolite variables important to group discrimination names are listedn on the left axis of the VIP plot.
162
Table 6. Comparison of relative abundances for lipid metabolites derived from PLS-DA analysis among sperm types in Sauger sperm. Data are reported as the mean 1 standard error of the mean (SEM) in log transformed data.
Sperm Type Lipid Common Name Testicular Stripped Cryopreserved
N-tetradecanoyl-homoserine lactone 0.00 ± 0.43 b 0.74 ± 0.43 b 11.57 ± 0.43 a
N-octanoyl-homoserine lactone 1.01 ± 0.59 b 0.00 ± 0.59 b 14.19 ± 0.59 a
17:0 Cholesterol ester 10.00 ± 0.11 b 10.20 ± 0.11 b 11.60 ± 0.11 a
15:0 Cholesterol ester 11.40 ± 0.05 b 11.40 ± 0.05 b 12.70 ± 0.05 a
Cer(d18:0/20:0) 12.60 ± 0.26 a 12.10 ± 0.26 a 10.70 ± 0.26 b
PC(18:3(9Z,12Z,15Z)/0:0) 6.29 ± 0.81 b 4.46 ± 0.81 b 11.50 ± 0.81 a
(25S)-5α-cholestan-3β,4β,6α,7α,8β,15β,16β,26- b b a octol 9.59 ± 0.45 8.40 ± 0.45 13.11 ± 0.45
Cer(t16:0(15Me)/23:0(2OH[R])) 8.78 ± 0.22 b 8.87 ± 0.22 b 10.89 ± 0.22 a
DG(18:2(9Z,12Z)/20:3(8Z,11Z,14Z)/0:0)[iso2] 10.54 ± 1.02 a 9.95 ± 1.03 a 1.67 ± 1.04 b
N-decanoyl-homoserine lactone 0.65 ± 0.39 b 0.00 ± 0.39 b 12.40 ± 0.39 a
3-hydroxyisovalerylcarnitine 1.87 ± 0.92 b 1.77 ± 0.92 b 8.17 ± 0.92 a
Cer(d14:1/24:0) 13.00 ± 0.24 a 12.50 ± 0.24 a 11.10 ± 0.24 b
Cer(d20:0/22:0) 12.30 ± 0.07 b 12.10 ± 0.07 b 13.50 ± 0.07 a
Hydroxypropionylcarnitine 1.17 ± 0.94 b 1.81 ± 0.94 b 8.72 ± 0.94 a
Arachidyl palmitate 11.40 ± 0.08 b 11.50 ± 0.08 b 13.00 ± 0.08 a a,b Means within rows with contrasting Tukey’s HSD superscripts are significantly different from one another (p < 0.05).
163
Table 7. Motility parameters and viability of Sauger sperm pools used for plasma membrane analysis (n = 7). Data are reported ae the mean 1 standard error of the mean.
Sperm Type Characteristics Testicular Stripped Cryopreserved
Total Motility (%) 67.7 ± 2.6 b 81.7 ± 2.6 a 65.3 ± 2.6 b
Curvilinear Velocity (µm/s) 168.6 ± 4.0 b 184.5 ± 4.0 a 98.1 ± 4.0 c
Straight Line Velocity (µm/s) 77.2 ± 2.3 a 77.1 ± 2.3 a 51.5 ± 2.3 b
Average Path Velocity (µm/s) 142.0 ± 3.6 a 152.7 ± 3.6 a 77.5 ± 3.6 b
Straightness (%) 55.8 ± 1.8 b 50.9 ± 1.8 b 69.5 ± 1.8 a
Linearity (%) 47.4 ± 1.5 b 43.2 ± 1.8 b 55.7 ± 1.8 a
Beat Cross Frequency (Hz) 12.9 ± 1.0 b 10.3 ± 1.0 b 22.3 ± 1.0 a
Wobble (%) 83.6 ± 0.5 a 82.7 ± 0.5 a 78.7 ± 0.5 b
Head Amplitude (µm) 12.09 ± 0.5 b 14.33 ± 0.5 a 5.09 ± 0.5 c
Viability (%) 77.0 ± 2.0 b 89.8 ± 2.0 a 73.6 ± 2.0 b a,b Mean values (± SEM) with different superscript letters within a row differ significantly at P < 0.05.
164
A. B.
C. D.
Figure 18. Electron micrographs of cryopreserved Sauger spermatozoa. (A) Scanning electron micrograph of spermatozoa showing widespread cellular damage. (B-D)
Transmission electron micrographs demonstrating disrupted plasma membranes (B), absent nuclear membranes (C), and cytoplasmic blebs along the flagellum (D).
165
Chapter 5. Assisted Reproduction Techniques’ Effects on Spermatozoa Calcium
Homeostasis, Protein Phosphorylation, and Motile Subpopulations
Abstract
Sperm motility analysis is a poor indicator of fertilization success in sperm produced by assisted reproduction techniques (ARTs), pointing to the need for other ways to identify how these ARTs negatively affect fertilization ability. To this end, we assessed and compared different aspects of motility (intracellular Ca2+, protein phosphorylation, and motile sperm subpopulations) in sperm resulting from three ARTs
(strip-spawning, testicular harvest, and cryopreservation). A combination of microscopy, flow cytometry, western blot, and multivariate analysis were used to achieve these goals.
Our results indicated a significant effect of testicular harvest and cryopreservation on sperm intracellular signaling and motility variables. Motility activation in stripped sperm was associated with a 2-fold increase in intracellular Ca2+, threonine phosphorylation of a
35kDa protein, and a majority of motile cells classified as fast-nonlinear (66.70 ± 6.48%).
Testicular sperm, while displaying unaltered Ca2+ influx ability, exhibited fewer sperm with protein phosphorylation levels equal to those seen in stripped sperm and contained
13% fewer fast, nonlinear sperm. Cryopreservation altered all measures of sperm signal transduction and motility. Calcium was artificially elevated and unresponsive to hypo- osmotic shock induced influx, threonine phosphorylation in 35kDa protein was present in
166 inactive sperm, tyrosine and serine phosphorylation were reduced in ~ 10% of cells, and fast, nonlinear sperm were reduced by 53% following freeze-thaw. Our results indicate the substantial effects ARTs can have on the physiology of sperm motility as well as added potential biomarkers as tools for describing sperm quality.
Introduction
Assisted reproduction techniques (ARTs) are commonly used in aquaculture to maximize the reproductive potential of broodstock. These techniques are most numerous in male reproductive management because sperm are more easily manipulated and stored than eggs (Beirão et al., 2019). Unfortunately, some ARTs have unintended negative effects on sperm fertility. For example, ARTs such as testicular harvest and cryopreservation used with Sauger (Sander canadensis) during the production of recreationally valuable Saugeye (S. vitreus × S. canadensis) result in highly variable or poor fertilization despite only slightly reduced sperm motility (Appendix A, Blawut et al.,
2020a, 2018b). While sperm motility is used by most researchers as a primary measure of sperm quality, this parameter often comes up short in predicting sperm fertilization potential. Moreover, motility is a multifaceted process consisting of activation of signal transduction via specific ions (e.g., Ca2+ and K+) and protein phosphorylation, as well as initiation and modulation of flagella beating. Thus, assessing these parameters instead of total motility alone could help explain the effects of ARTs on sperm intracellular signaling and its relationship to fertilization potential.
While several motility activation pathways have been described in well- documented aquatic species (Alavi et al., 2019; Alavi and Cosson, 2006, 2005), nearly all pathways include an initial rise in calcium. Calcium is a ubiquitous second messenger in 167 most cells that encodes information through temporal and spatial patterns of concentration (Zilli et al., 2008). Upon release into the external environment, differences in osmolality and ionic concentrations (low K+, high Ca2+) across the cell membrane are responsible for initiating the intracellular pathway that results in motility (Dzyuba and
Cosson, 2014). While its exact role may differ among most fishes, most species require elevated intracellular calcium for motility activation mediated by voltage activated channels (Alavi and Cosson, 2006; Butts et al., 2013; Pérez et al., 2016). Calcium has many roles including : 1.) propagation of activation stimulus, 2.) controlling flagella beating pattern, and 3.) modifying sperm velocity (Dumorné et al., 2018; Dzyuba and
Cosson, 2014). An increase in cellular calcium is essential to propagating this signal
(Krasznai et al., 2000). Calcium aids in cAMP synthesis in salmonids or acts in concert with calmodulin to transform external stimuli to cellular action via protein phosphorylation. Once in the motile state, calcium concentrations also affect motility patterns. Cells responding to elevated calcium via Catsper channels in mammalian species exhibit hyperactivation characterized by erratic, non-linear swimming patterns
(Brenker et al., 2012). Similarly, the calcium content of fish sperm, which is modified via similar channels (Chen et al., 2020; Lissabet et al., 2020), affects motility patterns (Alavi et al., 2011b; Boitano and Omoto, 1992; Pérez et al., 2016) and participates in processes such as chemotaxis toward the egg’s micropyle (Seifert et al., 2015; Yanagimachi et al.,
2017b). As a result, calcium is a primary candidate for assessing the initial stages of motility activation, as well as motility characteristics in fish sperm.
Phosphorylation and dephosphorylation of proteins in the axoneme of spermatozoa is the final step toward initiating motility. Secondary messengers (cAMP
168 and Ca2+) activate protein kinases and phosphatases usually in the form of calmodulin, protein kinase A (PKA), and protein kinase C (PKC), which then act on motility initiation and regulatory proteins (Zilli et al., 2017). Phosphorylation of tyrosine, threonine, and serine residues in several low molecular weight proteins have been associated with motility activation in the sea bream (Sparus aurata) and salmonids (Inaba et al., 1998;
Zilli et al., 2008). Along with the axoneme, proteins involved in energy production, signaling platforms, and heat shock proteins are also phosphorylated during activation
(Gazo et al., 2017). This evidence suggests that other important pathways, not just motility, are activated via hypo-osmotic shock (i.e., activation). However, ARTs, primarily cryopreservation, have been shown to increase protein phosphorylation in mammalian sperm (“cryo-capacitation”, Naresh and Atreja, 2015) and alter protein phosphorylation patterns in inactive (i.e., immotile in isotonic extender) and activated
(i.e., after exposure to osmotic shock) sea bream sperm (Zilli et al., 2008). At present, little information regarding motility activation or the effects of ARTs on sperm protein phosphorylation is available in fish.
Once motility is initiated, the duration of swimming ranges from 1 min in freshwater fish to more than 10 min in marine fish, during which time sperm must also navigate to the micropyle of the egg to achieve fertilization using a combination of chemical and physical cues (Browne et al., 2015; Yanagimachi et al., 2017b). Motile sperm are a heterogeneous group of cells, with some individuals exhibiting certain either desirable trajectories and others undesirable ones (Amann and Waberski, 2014).
Classifying sperm motility patterns using multivariate techniques offers a way to describe motility patterns that can inform sperm quality and fertility (Martínez-Pastor et al.,
169
2011b). Motile sperm subpopulation analysis has proven to be a valuable technique in assessing sperm quality in a variety of taxa. In fish, sperm subpopulations have been used to reveal differences in the motility of sperm from both captive and wild Shortnose
Sturgeon (Acipenser brevirostrum) and Atlantic Salmon (Salmo salar), cryopreserved sperm in Tambaqui (Colossoma macropomum) and Seabream (Sparus aurata), and sperm activated in various activation medias in both steelhead (Oncorhynchus mykiss) and Senegalese Sole (Solea senegalensis) (Beirão et al., 2011; Caldeira et al., 2018;
Gallego et al., 2017a; Gilroy and Litvak, 2019; Kanuga et al., 2012a). A majority of these studies have resulted in 3-4 distinct sperm subpopulations (Martínez-Pastor et al., 2011a), strongly defined by sperm velocity and trajectory shape (circular vs. linear). These sperm subpopulations were demonstrated to be susceptible to effects of motility duration, activation media, and ARTs such as cryopreservation. However, few studies have attempted to relate these populations with fertilization, and in the few examples that exist, definitive conclusions could not be reached relating subpopulations to fertility (Beirão et al., 2011). Thus, this type of analysis could provide a more detailed link between sperm motility and fertilization that is applicable to all aquatic species.
To address these knowledge gaps, we conducted an experiment to determine the effects of ARTs on intracellular signaling and sperm motility subpopulations after motility activation in the spermatozoa of Sauger (Sander canadensis). Specifically, we sought to: (1) characterize the effect of motility activation on intracellular Ca2+ and protein phosphorylation, (2) determine how testicular harvest and cryopreservation affect the relationship between activation status and intracellular signaling, and (3) compare motile Sauger sperm subpopulations among sperm collected using 3 different ARTs (i.e.,
170 strip-spawning, testicular harvest, and cryopreservation). We hypothesized that (1) Ca2+ and protein phosphorylation would increase following motility activation in stripped and testicular sperm (2) sperm harvested via testicular harvest would be similar to stripped sperm, (3) cryopreservation would interfere with Ca2+ and protein phosphorylation in both the inactive and activated state, and (4) sperm motility trajectories would differ among sperm types.
Materials and Methods
Reagents and Antibodies
All extender reagents used in this study were purchased from Millipore Sigma
(Sigma Aldrich, St. Louis, MO, USA). Intracellular calcium probes (Fluo-4 AM,
#F14201) and associated diluent (20% pluronic acid in DMSO, #P3000MP) were purchased from ThermoFisher Scientific (Waltham, MA, USA). Primary antibodies for phosphor-tyrosine (#8954, CST), phosphor-threonine (#9386, CST), and phosphor-serine
(#05-1000X, MS) were purchased from Cell Signaling Technology (CST, Dancers, MA,
USA) and Millipore Sigma, respectively. Beta-actin primary antibodies were purchased from Abcam (#ab8227, Cambridge, MA, USA). Anti-mouse and anti-rabbit secondary antibodies conjugated to horseradish-peroxidase (#7076 and #7074, respectively) and
Alexa Fluor 488 antibodies (#4408 and #4412, respectively) were purchased through Cell
Signaling Technology. Viability counter stains for flow cytometry (Propidium iodide
(PI), # P3566) was purchased through Thermo Fisher Scientific.
171
Experimental Design
A 3 × 2 factorial experimental design was used to compare intracellular signaling
(e.g., calcium and protein phosphorylation) in sperm from 3 ARTs (stripped, testicular, and cryopreserved sperm) and activation statuses (inactive vs. activated). Sperm was collected from Sauger using strip-spawning and testicular harvest (Appendix A).
Multiple sperm samples were pooled per sperm type to reduce bias resulting from individual variation. An aliquot of pooled stripped sperm was cryopreserved according to a published protocol for Sauger (Blawut et al., 2020a). For each of these three sperm types (testicular, stripped, and cryopreserved), the following metrics were analyzed in inactive (i.e., immotile in isotonic extender) and activated (i.e., after dilution in hyposmotic media, hatchery H2O) spermatozoa: intracellular calcium content using the fluorescent indicator Fluo 4AM as well as phosphoprotein abundance, distribution, and diversity using a combination of flow cytometry, immunofluorescence, and western blot.
Additionally, motile sperm subpopulations were delineated using multivariate statistical techniques (a two-step clustering technique) and subpopulation relative proportions were compared among sperm types. Computer-assisted sperm analysis data were compiled from 2018, 2019, and 2020 sampling seasons subjected to clustering analysis. Relative proportions of each sperm subpopulation were compared among sperm type and correlations were used to relate subpopulations to fertilization.
Broodstock, Milt Collection, and Pooling
Sauger broodstock were acquired and maintained in human care using methods describe previously (Blawut et al., 2018b). In summary, Ohio Department of Natural
172
Resources – Division of Wildlife (ODNR-DOW) personnel collected mature, male
Sauger from the Ohio River near the Greenup, KY, USA dam via electrofishing during the breeding season (March 2020). Sauger broodstock were transported by truck for ~2 hours in an aerated live-well containing 0.5% NaCl solution to the London State Fish
Hatchery isolation facility (London, OH, USA). The specimens were maintained in a
~2840 L indoor recirculating system at 5 to 6 °C with a flow rate of 45 to 57 L/hour. Fish were maintained in tanks with natural photoperiod exposure. The Ohio State University
(Columbus, OH, USA) Institutional Animal Care and Use Committee approved all procedures and animal use prior to the beginning of the study (Protocol
#2015A00000008).
Milt used for this study was collected from a cohort of male Sauger (3 individuals each) using two different techniques, strip-spawning and testicular harvest or dissection during the 2019 and 2020 breeding seasons. This process was replicated 9 times (n = 9) on a total of 27 fish. Individuals were strip-spawned by placing them in dorsal recumbence, drying the cloaca thoroughly to avoid premature activation (Billard and
Cosson, 1992a), and stripped to collect milt using abdominal massage and a 1.0 mL rubberless syringe (S7510-1, Thermo Fisher Scientific, MA, USA) that was placed at the opening of the cloaca. Immediately after collection, total volume of milt produced was determined to the nearest 0.01 mL and immediately extended milt 1:2 with 350 mOsm/kg Rathbun extender ((g/l): CaCl2·2H2O, 0.117; MgCl2·6H2O, 0.134;
Na2HPO4, 0.236; KCl, 1.872; NaCl, 6.578; glucose, 10.000; citric acid, 0.100; NaOH,
0.254; and bicine, 1.06; pH 8.5 (Moore, 1987a; Bergeron et al., 2002b). Extended milt was maintained at 5° C in a Styrofoam box until further use.
173
The same individuals were euthanized using cervical transection followed by decapitation. Euthanized individuals were stored on ice and transported to the
Theriogenology laboratory (< 1 h, Columbus, OH). Once in the lab, testicular sperm was harvested from the testes using previously published methods (Appendix A, Blawut et al.,
2020b). In brief, each lobe of the testes was cut along its length with a scalpel and applied pressure to the lacerated lobes from the cloaca to the tip of the lobe to expel sperm.
Testicular milt was then diluted using 3.0 mL of Rathbun extender, gently homogenized, and transferred to a test tube for storage at 5°C.
Milt collected using strip spawning was pooled before cryopreservation and subsequent plasma membrane analyses to prevent individual bias. Stripped sperm samples from three Sauger with motility > 70% were combined (i.e., pooled) and maintained at 5°C prior to cryopreservation. Corresponding testicular sperm samples from the same individuals were also pooled.
Sperm Cryopreservation and Thawing
Sperm was cryopreserved using the protocol derived by our laboratory (Blawut et al., 2020a). In brief, fresh, stripped milt was diluted to 1.0 × 10 9 sperm/mL using
Rathbun extender supplemented with 4 mg/mL bovine serum albumin (BSA). After a 10 min equilibration, sperm was further diluted (1:1, v: v) with Rathbun plus BSA supplemented with dimethyl sulfoxide (DMSO). The resulting diluent consisted of 0.5 ×
10 9 sperm/mL in Rathbun plus 4 mg/mL BSA and 10% DMSO as cryoprotectants.
During the 10 min equilibration with the cryoprotectant, samples were loaded into 0.5 mL straws and then cooled at 3 cm above liquid nitrogen for 10 min. Samples were then
174 plunged and stored in liquid nitrogen for approximately 1 month prior to thawing at 37°C for 10 s in a water bath and maintenance at 5°C.
Motility Analysis
Sperm motility was assessed using computer-assisted sperm analysis (CASA) less than 3 hours after collection in fresh sperm, and immediately after thawing in cryopreserved sperm using methods previously described (Blawut et al., 2020a). Stripped and testicular sperm samples were diluted with Rathbun extender (1:50) and 2 µL of diluted milt was activated in 18 µL of hatchery water containing 1% BSA to prevent sticking (Billard and Cosson, 1992b). Cryopreserved samples were activated directly in hatchery water and analyzed. Motility was assessed objectively with a Ceros II CASA system (Hamilton Thorne, Beverly, MA, USA) at 10 s post-activation in a 2 µL sample of activated sperm in a Cytonix microchamber (20 µm depth, Cytonix LLC, Beltsville,
MD, USA). Images were captured at a rate of 60 Hz for 30 frames. Progressively motile sperm were considered to have an average path velocity (VAP) of >50 µm/s and a straightness of > 80.0%. A minimum VAP of 20 µm/s was used as a criterion for motile cells to reduce the effects of drift (Park et al., 2012). Other CASA settings included: exposure = 80 ms; gain = 300; minimum head brightness = 152; min head size = 1 µm2; max head size = 19 µm2; capillary correction = 1.3; max photometer = 70; min photometer = 60; and minimum total cell count = 200. Endpoints for data collected on each sperm track are listed in Table 8.
175
Intracellular Calcium
The fluorescent probed Fluo-4 AM was used to assess intracellular Ca2+ concentration and distribution among treatments. Testicular, stripped, and cryopreserved sperm at an initial concentration of 0.5 × 10 9 sperm/mL were first diluted 40-fold using isotonic media (i.e., Rathbun or Rathbun ± 10% DMSO). Aliquots 1.0 × 10 6 sperm were then loaded with 10 µM of the fluorescent intracellular calcium indicator, Fluo-4 AM containing 0.5% pluronic acid and 2% DMSO to prevent compartmentalization, for 30 min at 4°C. After 20 min, cells were then counterstained with propidium iodide (PI, 6
µM) for 10 min at 4°C to assess viability. Prior to analysis, samples were diluted 1:20 in either isotonic holding media (Rathbun or Rathbun ± 10% DMSO) or in activating media
(hatchery water) to assess cellular calcium content in the inactive and activated state, respectively.
Flow cytometry analysis was conducted using an Attune Acoustic flow cytometer
(Thermo Fisher, MA, USA). Cells were gated to exclude debris and doublets. Fluo-4 was detected using the 488 nm excitation laser and 530/30 emission bandpass filter (BL1). PI was detected using the 488 nm excitation laser and 695/40 nm bandpass filter (BL3).
Compensation was calculated and applied to minimize signal spillover among channels.
Mean fluorescent intensity (MFU, arbitrary units, a.u.) and the percentage of cells exhibiting high fluorescence was determined from ≥ 10,000 viable cells (- PI, viable) per sample.
Fluorescent microscopy was used to assess the distribution of calcium in the sperm cell. Sperm were stained and activated as described above and then viewed using a
Nikon Eclipse Ti microscope and Intensilight C-HGFIE lamp with a 1,000 × oil objective
176
(Nikon Inc., NY, USA). Representative images of viable cells were taken using Nikon
Advanced Research Software.
Protein Phosphorylation
A combination of immunofluorescence, flow cytometry, and western blot were used to assess protein phosphorylation in Sauger sperm cells among sperm types and activation statuses. The description of each method is reported below.
Immunofluorescence - Fluorescent microscopy was used to qualitatively describe the distribution of phosphoproteins labeled with fluorescent antibodies using methodology described by Koubek et al. (2008) with slight modification. Inactive sperm from each sperm type at approximately 1.25 × 10 7 sperm/mL were attached to poly-L- lysine pre-treated 8 well chamber slides (#80841, ibidi, DE) for 10 min at 4°C. Sperm were then treated with isotonic extender (inactive) or hatchery water (activated). The wells were then drained, and sperm were fixed using 3.6 % paraformaldehyde for 30 min at 4°C. Cells were permeabilized with 100% methanol for 20 min at - 20°C and washed using TBS. Sperm were then blocked for 1 hour at 4°C in 3% BSA-TBST and incubated overnight in primary antibody solution (1:250 dilution in BSA-TBST). After washing, cells were incubated for 1hour at 37°C in appropriate secondary antibody solution
(1:1,000 dilution in BSA-TBST). Slides were mounted using Pro-long gold anti-fade mounting media (ThemoFisher Scientific). Sealed slides were stored protected from light at 4°C until analyzed. Sperm cells (n = 100) from each sample were assessed using a
177
Nikon Eclipse Ti and Intensilight C-HGFIE lamp with a 1,000 × oil objective to describe the localization of staining.
Flow cytometry- Phosphoprotein abundance was assessed using flow cytometry to quantitatively describe phosphorylation events. Aliquots of sperm (50 million) were centrifuged and treated with isotonic extender (inactive) or hatchery water (activated) for
30 s and permeabilized using 90% methanol. Samples were stored at -80°C until analysis
(5 months). Aliquots of preserved sperm were centrifuged and rehydrated in flow cytometry buffer (TBS, 1.0 % BSA, 5 mM EDTA) sperm prior to downstream processing. Sperm were blocked for 1 hour at room temperature in 3% BSA (IgG-Free,
Protease-Free; Jackson ImmunoResearch Laboratories, PA, USA) TBS and then further diluted 1:1 in blocking buffer containing primary antibodies for anti-phosphotyrosine
(1:250), anti-phosphothreonine (1:250), or anti-phosphoserine (1:250). Samples were incubated with primary antibody overnight at 4°C. After centrifugation and washing in
TBS, samples were incubated for 1 hour at 37°C in Alexa-Fluor 488 conjugated secondary antibody solution (1:500, 1:1,000, and 1:500 in 3% BSA-TBST, respectively).
Prior to analysis, sperm samples were washed and re-suspended in TBS containing 6 µM
PI at approximately 1.0 × 10 9 sperm/mL.
Flow cytometry analysis was conducted using an Attune Acoustic flow cytometer
(Thermo Fisher, MA, USA). Cells were gated to exclude debris and doublets. Alexa
Fluor 488 was detected using the 488 nm excitation laser and 530/30 emission bandpass filter (BL1). PI was detected using the 488 nm excitation laser and 695/40 nm bandpass filter (BL3) to exclude non-cellular debris. Compensation was not necessary owing to
178 low spectral overlap. Mean fluorescent intensity (MFU, a.u.) and the percentage of cells exhibiting high fluorescence was determined from ≥ 10,000 cells in each sample (n = 6).
Phosphoprotein Western Blot - Protein Extraction - Protein was extracted from
Sauger sperm using a similar technique used by Zilli et al (2008). One billion sperm from each sperm type and activation status were centrifuged and then held inactive or activated using hatchery water. Sperm were again centrifuged and then re-suspended in isotonic lysis buffer (1.0% Triton X-100, 1 mM PMSF, 1× Halt Protease and Phosphatase
Inhibitor, and 1 mM EDTA in TBS ± 10% DMSO). Lysates were incubated for 30 min on ice, vortexed periodically (10s each), and passed through a 25 × gauge for mechanical disruption. Cellular debris was pelleted at 18,000 × G for 10 min at 4°C. Protein content was determined using a commercially available Bicinchoninic acid assay kit (Thermo
Fisher) and a Citation 5 (BioTek, USA) microplate reader according to manufacturer instructions. Lysates were stored at -80°C for approximately 5 months prior to use.
Sample lysates were thawed at 37°c for 30 s and then maintained on ice
Western Blot- Extracted protein (30 p per sample) was diluted in 4× Laemmli buffer containing 10% 2-β-mercaptoethanol and denatured by boiling for 5 min at 100°C
(Laemmli, 1970). Samples were loaded into the wells of a 4-15% gradient polyacrylamide Criterion TGX gel (Bio-Rad; Hercules, California) along with a
Amersham™ ECL™ Rainbow™ Marker - Full range (Millipore Sigma). Proteins were separated using electrophoresis for 60 min at 175V and transferred to polyvinylidene difluoride membrane (PVDF, Bio-Rad) for 75 min at 100V at 4°C. Membranes were
179 immediately washed using TBST (0.1% Tween20) and then blocked in 3% BSA-TBST for 60 minutes at room temperature. Membranes were washed and then incubated overnight at 4 °C with primary antibodies for phosphotyrosine (P-Tyr, 1:80,000), phosphothreonine (P-Thr, 1:80,000), or phosphoserine (P-Ser, 1:20,000) in 3% BSA-
TBST. Membranes were then washed and incubated in a secondary antibody solution (P-
Tyr - 1:50,000; P-Thr - 1:50,000; P-Ser – 1:40,000) for 60 min at room temperature.
Blots were washed and then imaged using Pierce ECL Plus chemiluminescent substrate
(ThermoFisher Scientific) with an Amersham 600 digital imager (GE Healthcare, IL,).
Statistical Analysis
A combination of linear mixed models and multivariate statistical analyses were used to assess the impact of sperm type and activation status on Sauger sperm signaling and motility. All analyses were completed using Program R (R Development Core Team,
2019) with a significance set at p < 0.05. All values are expressed as mean ± 1 standard error of the mean (SE), unless otherwise stated.
Intracellular calcium and Protein Phosphorylation- Comparisons of intracellular
Ca2+ and phosphorylation fluorescent intensity (MFI) among sperm types (testicular, stripped, and cryopreserved) in the inactive and activated states were made using linear mixed effect models. Comparisons of cell populations exhibiting high fluorescence (%) for intracellular calcium and protein phosphorylation were made among sperm types and activation statuses using a generalized linear mixed model fitted with a binomial distribution. Fixed effects for both models included sperm type and activation status as
180 well as their interaction. Random terms included the pool of origin and year of collection when the experiment spawned more than one spawning season (Ca2+ only). Tukey’s honestly significant difference (HSD) post-hoc test was used to compare means. Model assumptions were confirmed using normal quantile plots (normality) and residual plots
(homogeneity of variance).
Motility Analysis - Sperm motility characteristics (CASA data) were compared among sperm types. Traditional motility variables (Table 8) were compared among the three sperm types (fixed factor with 3 levels: testicular, striped, and cryopreserved) using linear mixed models (including pool and year as random factors) with Tukey’s HSD post hoc tests conducted on the least square means probabilities.
Clustering Analysis - The main objective of this portion of the analysis was to describe and compare motile sperm subpopulations among sperm types. All motility variables included in the analysis are listed in Table 8. To ensure the accuracy of sperm measurements, sperm tracks with fewer than 30 frames captured (i.e., late-tracked, and border crossers) were removed from analysis. Only motile sperm were considered for this analysis. All variables were scaled to mean = 0 and SD = 1 to minimize the effects that variables with large values (i.e. curvilinear velocity) would have over smaller values (i.e. amplitude of lateral head displacement, Martínez-Pastor et al., 2011b).
We then used a two – step clustering analysis was used to define motile sperm subpopulations (José Beirão et al., 2012; Dorado et al., 2010; Gallego et al., 2017b;
Kanuga et al., 2012b; Martínez-Pastor et al., 2011b) using the cluster, factoextra, and
181
FactoMineR packages in R (R Development Core Team, 2009). First, principal component analysis was used to generate a subset of uncorrelated axes that accounted for a majority of the overall variation inherent in the data. The first two PCA axes were extracted and used in subsequent analyses because their Kaiser criteria exceeded 1.0. The first two PCA axes were used and accounted for 80.2% of the original variation (PCA1 =
46.3, PCA2 = 33.9). Next, K-means agglomerative clustering with a Euclidean distance metric was used to partition the 24,993 sperm trajectories based on their PCA dimensions
(PCA1 and PCA2) into 15 pre-clusters. Lastly, a Euclidean distance matrix was calculated between pre-clusters, and a hierarchical clustering algorithm using Ward’s linage method further reduced the number of resulting clusters. The resulting clusters were described using the 8 measured CASA variables (mean ± standard deviations). The percentage of sperm in each subpopulation category was compared among the three sperm types (testicular, striped, and cryopreserved) using linear mixed models (including pool and year as random factors) with Tukey’s HSD post hoc tests conducted on the least square means probabilities.
Results
Intracellular calcium
An interactive effect of sperm type and activation status on intracellular calcium content was observed (Table 9, p = 0.016). Both testicular and stripped sperm had an approximately 3-fold increase in intracellular calcium as a result of activation. In contrast, cryopreserved sperm did not increase intracellular calcium content as a result of motility activation. In fact, intracellular calcium was already 2-fold higher in inactivated frozen sperm than either testicular or stripped sperm in the activated state. 182
Analysis of highly fluorescent sperm populations revealed similar results as mean fluorescent intensity (Table 9). The proportion of sperm exhibiting high fluorescence was affected by an interaction between sperm type and activation status (p = 0.016). Stripped and testicular sperm both had a relatively small population (< 15%) of sperm with high amounts of intracellular calcium prior to activation, but that population increased to 74 and 84%, respectively, following motility activation. Cryopreserved sperm, however, always contained high proportion of sperm characterized by high intracellular calcium
(91-93%) and that did not change as a result of motility activation.
Fluorescent staining using Fluo 4 AM did not identify specific staining patterns.
All sperm were stained homogenously regardless of sperm type or activation status, indicating calcium was present throughout the cell rather than being localized to a particular structure.
Protein Phosphorylation
Sperm subpopulation analysis of protein phosphorylation using immuno-fluorescent flow cytometry is reported in Table 10. Least squared mean and SEM are reported for each group along with Tukey’s HSD post hoc groupings.
Phosphotyrosine- Tyrosine phosphorylation staining intensity was affected by both sperm type (p < 0.0001) and activation status (p = 0.049), with no interactive effect observed (Table 10). The largest population of sperm exhibiting high fluorescence was seen in stripped sperm (80.4 ± 1.69 %), somewhat less in cryopreserved sperm (74.6 ±
1.95%), and least in testicular sperm (69.2 ± 2.15%). Highly fluorescent sperm
183 populations increased by approximately 3% following motility activation in all sperm types (73.5 ± 1.86 and 76.4 ± 1.75%).
In tyrosine, a ~ 100kDa protein was phosphorylated in all samples, but seemed subjectively, to be more prevalent in stripped and cryopreserved sperm (Figure 20 A).
Additionally, a possible decrease in phosphorylation of that protein due to motility activation was evident. In support of this notion, phosphotyrosine staining was prominent in a triangular patch of staining located directly over the location of the mitochondria and flagella insertion point (Figure 19). The remainder of the cell was also stained, but at a lesser intensity
Phosphothreonine- Threonine phosphorylation staining intensity was affected by sperm type (p < 0.0001) and activation status (p = 0.028), but no interaction was observed (Table 10). Stripped and cryopreserved sperm had a larger percentage high fluorescence cells (76.4 ± 2.44% and 67.8 ± 2.57, respectively) than in testicular sperm
(60.1 ± 2.79 %). Motility activation caused a 4% increase in highly fluorescent sperm
(inactive 64.6 ± 2.55 and activated 68.2 ± 2.44%) collectively among sperm types. While not statistically significant, a trend was observed in the interactive effect of motility activation and sperm type (p = 0.066). In this scenario, cryopreserved sperm were the only to increase in fluorescence (+ 8.5% following activation), while testicular and stripped sperm did not respond to activation. Testicular sperm fluorescence (59.8-60.3%) was significantly lower than stripped (70.3-71.6%).
Phosphothreonine exhibited the largest changes resulting from motility activation
(Figure 20 B). In activated sperm, a ~ 35kDa protein was phosphorylated in all sperm
184 types, but testicular and cryopreserved sperm showed some evidence of pre-activation phosphorylation of those proteins. Phosphothreonine displayed no discernable staining distribution (Figure 19). All cells were stained homogenously throughout the head and flagellum.
Phosphoserine- Serine phosphoserine staining intensity was affected by only sperm type (p < 0.0001, Table 10). Stripped sperm displayed the largest population of high fluorescence (80.1 ± 3.1%), followed by testicular sperm (74.0 ± 3.7%), and fewest in cryopreserved sperm (67.3 ± 4.2%). No differences in phosphoserine content were observed between inactive vs activated sperm (74.7 ± 3.54% and 73.6 ± 3.64%, respectively; p = 0.5103).
In serine phosphorylation, the most notable difference among sperm types is the loss of a ~55 kDa protein serine phosphorylation in cryopreserved sperm (Figure 20 C).
However, little evidence exists for motility activation causing changes to the phosphorylation of serine residues (e.g., specific proteins phosphorylated or dephosphorylated). Additionally, no differences were seen in the distribution of phosphoserine. Its localization, like phosphotyrosine, included a prominent triangular patch of staining located directly over the location of the mitochondria and flagella insertion point with weaker staining throughout the rest of the head and flagellum
(Figure 19).
185
Sperm Subpopulations
Two PCA axes were chosen from principal component analysis to use for further clustering based on the Kaiser criteria. Dimension 1, which accounted for 46.33% of the variation, was positively associated with the straightness of the sperm tracks (high STR,
LIN, VSL). By contrast, dimension 2 (33.91%) was positively associated with the velocity of sperm (high VAP, VSL, VCL, WOB).
Three sperm subpopulations were generated by two-step clustering process
(Figure 21, Table 11). The coefforetic distance of the hierarchical clustering data to the original data was 0.63, indicating good maintenance of the original data structure.
Subpopulation 3 (S3) consisted of “slow-linear sperm” (34.8% of sperm). Subpopulation
2 (S2) was indicated to be sperm with a high curvilinear velocity and low straightness and was the most common type (45.1%, “fast-nonlinear”). Subpopulation 1 (S1) was characterized by sperm with a fast velocity (VAP), high straightness, and linearity
(20.1% of sperm, “fast-linear”).
Motile sperm subpopulations were not distributed evenly among sperm types
(Figure 22). Cryopreserved samples were characterized by a majority of sperm in SP1
(64.9 ± 1.7%), 26.4 ± 1.7 % SP2, and 8.7 ± 1.0% SP3. By contrast, strip-spawned sperm had the highest proportion of SP2 sperm (83.2 ± 1.9%), and low proportions of SP1 and
S3 cells (8.2 ± 1.3% and 8.6 ±1.4 %, respectively. Testicular sperm was intermediate between stripped and frozen in terms of SP1 (20.12 ± 2.0%) and SP2 (75.00 ± 2.16%) but was composed of a similar proportion of cells in SP3 (4.84 ± 1.07%) compared to strip- spawned sperm.
186
Discussion
To date, little information exists regarding intracellular signaling in the sperm of
Sauger in relation to motility activation or the effect of assisted reproduction techniques on this relationship. In this study, we found Sauger spermatozoa to share certain cellular responses to hypo-osmotic shock with other fish species. Intracellular Ca2+increased and protein phosphorylation was altered. Additionally, several motile sperm subpopulations were identified with fast- nonlinear sperm being most numerous. However, these variables were affected by ARTs. Testicular sperm contained fewer sperm with levels of protein phosphorylation comparable to strip-spawned sperm. Otherwise, calcium homeostasis and circular motility were the same or slightly reduced, respectively, compared to stripped sperm. By contrast, cryopreservation affected every variable measured including cellular calcium content, abolishing Ca2+ increase following hypo- osmotic shock, lowering protein phosphorylation, and altering motile sperm subpopulations. Our collective findings demonstrate the negative effects of cryopreservation on normal sperm physiology associated with motility activation but also offer insights as to which changes contribute to reduced fertilization potential.
Hypo-osmotic shock accompanying motility activation is associated with changes to intracellular signaling (Alavi et al., 2019). Consistent with a majority of other fish species studied (Gallego et al., 2014; Krasznai et al., 2003b), hypo-osmotic shock was associated with a nearly 2-fold increase in Sauger sperm intracellular Ca2+ content relative to inactive sperm. This rise in intracellular Ca2+ depended on external calcium in the activation media, as evidenced by a lack of calcium increase after activation in distilled water (data not shown). Unlike many other studies conducted in fish spermatozoa, we were only partially able to replicate increases in protein phosphorylation 187 during motility activation using western blot. Studies in seabream, common carp, and starlet have shown transient phosphorylation of low molecular weight proteins following activation (Gazo et al., 2017; Zilli et al., 2008), although only a single 35kDa protein was differentially phosphorylated. In agreement with this lack of protein phosphorylation using western blot, flow cytometry revealed no quantitative differences in protein phosphorylation among inactive and activated sperm. Thus, the possibility exists that our methodology needs to be optimized to capture these transient protein phosphorylation events to draw more definitive conclusions (Gazo et al., 2017). These results provide a detailed description of the early events in in Sauger sperm motility initiation.
Spermatozoa resulting from testicular harvest displayed characteristics of sperm in the process of final sperm maturation. Protein phosphorylation was approximately 20% lower in tyrosine, serine, and threonine residues. Maturation within the testes via
17α,20β-dihydroxy-4-pregnen-3-one in Rainbow Trout (Oncorhynchus mykiss) has been associated with elevated main duct pH, increases in sperm cAMP, phosphorylation of the axoneme, and enhanced motility potential (Miura et al., 1992b; Morisawa and Okuno,
1982). Sperm Ca2+ homeostasis, however, did not differ between stripped and testicular sperm results in our study. This is in contrast to other reports that testicular sperm of fish
(e.g., sterlet, Acipenser ruthenus) require 10 6 times higher external Ca2+ to initiate motility than stripped sperm. Moreover, Sauger testicular sperm motility did not show differences in Ca2+ homeostasis related to external calcium concentrations (e.g., capable of motility in distilled water containing no added calcium). It is likely that the lower percentage of cells with high protein phosphorylation results from lack of residency within the main sperm duct. These immature cells may be sperm that reside in the tubular
188 lumen as opposed to the main sperm duct (lobular lumen). Regardless, high fertilization potential observed in testicular sperm (Appendix A, Blawut et al., 2020b) suggests a sufficiently large proportion of functioning sperm are collected using this ART and thus reinforces its high practical application efficacy.
Unlike testicular harvest, cryopreservation resulted in significant changes in sperm physiology. Surprisingly, few studies have been conducted on the effect of cryopreservation on intracellular calcium despite its importance to motility. Previous studies in Rainbow Trout (Purdy et al., 2016) and Atlantic Salmon (Salmo salar,
Figueroa et al., 2019) reported a stable or diminished Ca2+ content following freeze- thaw. However, in our results and those of Guthrie et al in Striped Bass (Morone saxatilis, Guthrie et al., 2014)), intracellular Ca2+ was elevated in cryopreserved sperm.
Moreover, intracellular Ca2+ did not increase in frozen Sauger sperm following hypo- osmotic shock. Interestingly, motility was still initiated in frozen sperm despite the lack of Ca2+ increase. In Rainbow Trout sperm, glycerol treated sperm displayed elevated intracellular Ca2+ but were still able to achieve motility activation through an alternative pathway, instead utilizing Ca2+ efflux (Takei et al., 2012). At present, the exact mechanism responsible for elevated Ca2+ are unknown. We speculate cryoprotectant addition or leaky membranes associated with lipid phase transition (Drobnis et al., 1993) during freeze-thaw could be affecting intracellular Ca2+ accumulation. Optimizing cryoprotectant choice, method of addition, and freeze-thaw regiments may lead to more typical Ca2+ homeostasis post-thaw, and potentially enhanced fertility.
Contrary to reports in other fish species and mammalian taxa, cryopreservation caused a reduction in protein phosphorylation in a portion of cells. Cryo-capacitation,
189 associated with elevated Ca2+ and tyrosine phosphorylation in sperm from mammalian species is a prevalent source of reduced fertilization (Thomas et al., 2006). Similarly,
Gilthead Seabream (Sparus aurata) spermatozoa have also been demonstrated to have increased threonine phosphorylation (both before and after activation) compared to a stripped control (Zilli et al., 2008). In our study, however, threonine phosphorylation did not change following cryopreservation, although inactivated sperm did show signs of phosphorylation of certain proteins altered by motility activation (~ 35kDa). By contrast, tyrosine and serine phosphorylation decreased in a proportion of cells relative to the strip- spawned control. At present, the identity of the proteins altered by motility activation or cryopreservation is unknown and thus physiological consequences of these changes cannot be speculated.
Finally, motile sperm subpopulation analysis identified distinct subpopulations that are negatively impacted by testicular harvest and cryopreservation. Consistent with most other studies (Beirão et al., 2011; Caldeira et al., 2018; Gallego et al., 2017b, 2015;
Kanuga et al., 2012b), three sperm populations were identified using our two-step clustering analysis. In contrast to what has been reported in Senegalese Sole (Solea snegalensis) and Atlantic Salmon (Kanuga et al., 2012b; Martínez-Pastor et al., 2008), the dominant motility pattern observed in Sauger sperm was associated with high VCL and ALH but also low STR and LIN. This subpopulation was smaller in testicular sperm
(13% lower) and cryopreserved sperm (53% lower) compared to the stripped control.
While we did not examine their relationship with fertilization has not be studied in
Sauger, previous reports in Gilthead Seabream showed a positive correlation between fast-nonlinear sperm and fertilization potential. Moreover, our analysis was able to
190 provide more detail regarding motility than traditional univariate comparisons. While slow-linear sperm were most prominent in cryopreserved sperm, clustering also was able to show a small proportion of sperm maintained fast-nonlinear motility patterns and would be more likely to retain fertility. As demonstrated in Sparus aurata (Beirão et al.,
2011), this technique could be used to screen different cryopreservation protocol combinations that retain the higher proportion of fast-nonlinear sperm more likely to be fertile. A biomarker such as this could greatly enhance cryopreservation protocol development in Sauger, and many other fish species.
Several limitations exist regarding methodology used in our study that should be considered. First, the protocol use to extract proteins for phosphoprotein western blot could have affected our ability to assess the transient phosphorylation events (Gazo et al.,
2017). Late addition of the lysis buffer compared to imminent phosphorylation events associated with activity could me we were unable retain transiently phosphorylated proteins for our analysis. However, our methods should still be able to reliably identify phosphorylation of proteins associated with motility activation in inactivated cryopreserved sperm as a result of altered signaling, of which we found some evidence.
Secondly, we found a slightly different trend in motility subpopulation analysis compared to other studies using multivariate analysis. Instead of most sperm exhibiting high velocities and high straightness, Sauger sperm that were fast-nonlinear were far more numerous. These results may be affected by the environment in which motility was assessed. The depth of the chamber (20 µm) may have resulted in sperm being imaged in close proximity to the cover glass. In this case, sperm have been shown to have a high affinity for following the surface (Elgeti et al., 2010). Stripped and testicular sperm
191 seemed to have a high affinity for these surfaces, exhibiting a “drilling” like motion of concentric rings. Interestingly, fewer frozen sperm were capable of thigmotactic movement (swimming along a surface), which is hypothesized to be an important factor facilitating the search for and entry into the egg micropyle (Ishimoto et al., 2016;
Kholodnyy et al., 2020; Yanagimachi et al., 2017b). Our results suggest this ability is lost in cryopreserved sperm and could be implicated in sperm’s substantially lower ability to achieve fertilization despite exceedingly high sperm-to-egg ratios.
Conclusions
Our results provide a description of basic Sauger sperm motility, including calcium signaling, protein phosphorylation, and motile sperm trajectories. We also quantified and described the potential negative effects of ARTs routinely used in the production of Saugeye. We found that hypo-osmotic shock induced a 2-fold increase in intracellular Ca2+, alteration of the phosphoproteome – particularly for threonine, and a dominant motility pattern consisting of fast-nonlinear motion. We further supported the model of spermatozoa maturation within the main sperm duct as evidenced by lower percentages of testicular sperm with high phosphoprotein content and changes to motile sperm subpopulations. Finally, cryopreservation induced major changes to all three sperm motility measures. Elevated cellular Ca2+, absent Ca2+ influx resulting from hypo-osmotic shock, reduced or altered protein phosphorylation, and slow-linear sperm trajectories all resulted from the cryopreservation process. All of these suggest a negative effect on fertilization but verification of their effect on fertilization is needed. Collectively, our results point toward sperm collection methods and cryopreservation’s ability to negatively impact motility by impacting aspects of signal initiation (Ca2+ influx) and 192 axoneme maturity (protein phosphorylation) that motility analysis alone does not capture.
Thus, this work provides a deeper understanding of Sauger sperm motility, negative effects of commonly used ARTs, and potential biomarkers for assessing sperm’s physiological changes and fertilization potential. These findings will be of value to further optimization of ARTs to more effectively retain sperm’s ability to respond to activation, ultimately leading to higher practical efficacy of these techniques.
193
References
Alavi, S. M. H., Gela, D., Rodina, M., & Linhart, O. (2011). Roles of osmolality,
calcium—Potassium antagonist and calcium in activation and flagellar beating
pattern of sturgeon sperm. Comparative Biochemistry and Physiology Part A:
Molecular & Integrative Physiology, 160(2), 166–174.
https://doi.org/10.1016/j.cbpa.2011.05.026
Alavi, Sayyed Mohammad Hadi, & Cosson, J. (2005). Sperm motility in fishes. I. Effects
of temperature and pH: A review. Cell Biology International, 29(2), 101–110.
https://doi.org/10.1016/j.cellbi.2004.11.021
Alavi, Sayyed Mohammad Hadi, & Cosson, J. (2006). Sperm motility in fishes. (II)
Effects of ions and osmolality: A review. Cell Biology International, 30(1), 1–14.
https://doi.org/10.1016/j.cellbi.2005.06.004
Alavi, Sayyed Mohammad Hadi, Cosson, J., Bondarenko, O., & Linhart, O. (2019).
Sperm motility in fishes: (III) diversity of regulatory signals from membrane to
the axoneme. Theriogenology, 136, 143–165.
https://doi.org/10.1016/j.theriogenology.2019.06.038
Amann, R. P., & Waberski, D. (2014). Computer-assisted sperm analysis (CASA):
Capabilities and potential developments. Theriogenology, 81(1), 5-17.e3.
https://doi.org/10.1016/j.theriogenology.2013.09.004
Beirão, J., Cabrita, E., Pérez-Cerezales, S., Martínez-Páramo, S., & Herráez, M. P.
(2011). Effect of cryopreservation on fish sperm subpopulations. Cryobiology,
62(1), 22–31. https://doi.org/10.1016/j.cryobiol.2010.11.005
194
Beirão, José, Boulais, M., Gallego, V., O’Brien, J. K., Peixoto, S., Robeck, T. R., &
Cabrita, E. (2019). Sperm handling in aquatic animals for artificial reproduction.
Theriogenology, 133, 161–178.
https://doi.org/10.1016/j.theriogenology.2019.05.004
Beirão, José, Zilli, L., Vilella, S., Cabrita, E., Schiavone, R., & Herráez, M. P. (2012).
Improving Sperm Cryopreservation with Antifreeze Proteins: Effect on Gilthead
Seabream (Sparus aurata) Plasma Membrane Lipids. Biology of Reproduction,
86(2). https://doi.org/10.1095/biolreprod.111.093401
Bergeron, A., Vandenberg, G., Proulx, D., & Bailey, J. L. (2002). Comparison of
extenders, dilution ratios and theophylline addition on the function of
cryopreserved Walleye semen. Theriogenology, 57(3), 1061–1071.
https://doi.org/10.1016/S0093-691X(01)00707-5
Billard, R., & Cosson, M. P. (1992a). Some problems related to the assessment of sperm
motility in freshwater fish. Journal of Experimental Zoology Part A: Ecological
Genetics and Physiology, 261(2), 122–131.
Billard, R., & Cosson, M. P. (1992b). Some problems related to the assessment of sperm
motility in freshwater fish. Journal of Experimental Zoology Part A: Ecological
Genetics and Physiology, 261(2), 122–131.
Blawut, B., Wolfe, B., Moraes, C. R., Ludsin, S. A., & Coutinho da Silva, M. A. (2018).
Increasing Saugeye (S. vitreus × S. canadensis) production efficiency in a
hatchery setting using assisted reproduction technologies. Aquaculture, 495, 21–
26. https://doi.org/10.1016/j.aquaculture.2018.05.027
195
Blawut, B., Wolfe, B., Moraes, C. R., Ludsin, S. A., & Silva, M. A. C. da. (2020). Use of
Hypertonic Media to Cryopreserve Sauger Spermatozoa. North American Journal
of Aquaculture, 82(1), 84–91. https://doi.org/10.1002/naaq.10125
Blawut, B., Wolfe, B., Moraes, C. R., Sweet, D., Ludsin, S. A., & Coutinho da Silva, M.
A. (2020). Testicular collections as a technique to increase milt availability in
Sauger (sander canadensis). Animal Reproduction Science, 212, 106240.
https://doi.org/10.1016/j.anireprosci.2019.106240
Boitano, S., & Omoto, C. K. (1992). Trout sperm swimming patterns and role of
intracellular Ca++. Cell Motility and the Cytoskeleton, 21(1), 74–82.
https://doi.org/10.1002/cm.970210109
Brenker, C., Goodwin, N., Weyand, I., Kashikar, N. D., Naruse, M., Krähling, M.,
Müller, A., Kaupp, U. B., & Strünker, T. (2012). The CatSper channel: A
polymodal chemosensor in human sperm. The EMBO Journal, 31(7), 1654–1665.
https://doi.org/10.1038/emboj.2012.30
Browne, R. K., Kaurova, S. A., Uteshev, V. K., Shishova, N. V., McGinnity, D., Figiel,
C. R., Mansour, N., Agnew, D., Wu, M., Gakhova, E. N., Dzyuba, B., & Cosson,
J. (2015). Sperm motility of externally fertilizing fish and amphibians.
Theriogenology, 83(1), 1-13.e8.
https://doi.org/10.1016/j.theriogenology.2014.09.018
Butts, I. A. E., Alavi, S. M. H., Mokdad, A., & Pitcher, T. E. (2013). Physiological
functions of osmolality and calcium ions on the initiation of sperm motility and
swimming performance in redside dace, Clinostomus elongatus. Comparative
196
Biochemistry and Physiology Part A: Molecular & Integrative Physiology,
166(1), 147–157. https://doi.org/10.1016/j.cbpa.2013.05.011
Caldeira, C., García-Molina, A., Valverde, A., Bompart, D., Hassane, M., Martin, P., &
Soler, C. (2018). Comparison of sperm motility subpopulation structure among
wild anadromous and farmed male Atlantic salmon (Salmo salar) parr using a
CASA system. Reproduction, Fertility and Development.
Chen, Y., Wang, H., Wang, F., Chen, C., Zhang, P., Song, D., Luo, T., Xu, H., & Zeng,
X. (2020). Sperm motility modulated by Trpv1 regulates zebrafish fertilization.
Theriogenology, 151, 41–51.
https://doi.org/10.1016/j.theriogenology.2020.03.032
Dorado, J., Molina, I., Muñoz-Serrano, A., & Hidalgo, M. (2010). Identification of sperm
subpopulations with defined motility characteristics in ejaculates from Florida
goats. Theriogenology, 74(5), 795–804.
Drobnis, E. Z., Crowe, L. M., Berger, T., Anchordoguy, T. J., Overstreet, J. W., &
Crowe, J. H. (1993). Cold shock damage is due to lipid phase transitions in cell
membranes: A demonstration using sperm as a model. Journal of Experimental
Zoology Part A: Ecological Genetics and Physiology, 265(4), 432–437.
Dumorné, K., Figueroa, E., Cosson, J., Lee‐Estevez, M., Ulloa‐Rodríguez, P.,
Valdebenito, I., & Farías, J. G. (2018). Protein phosphorylation and ions effects
on salmonid sperm motility activation. Reviews in Aquaculture, 10(3), 727–737.
https://doi.org/10.1111/raq.12198
197
Dzyuba, V., & Cosson, J. (2014). Motility of fish spermatozoa: From external signaling
to flagella response. Reproductive Biology, 14(3), 165–175.
https://doi.org/10.1016/j.repbio.2013.12.005
Elgeti, J., Kaupp, U. B., & Gompper, G. (2010). Hydrodynamics of Sperm Cells near
Surfaces. Biophysical Journal, 99(4), 1018–1026.
https://doi.org/10.1016/j.bpj.2010.05.015
Figueroa, E., Lee-Estevez, M., Valdebenito, I., Watanabe, I., Oliveira, R. P. S., Romero,
J., Castillo, R. L., & Farías, J. G. (2019). Effects of cryopreservation on
mitochondrial function and sperm quality in fish. Aquaculture, 511, 634190.
https://doi.org/10.1016/j.aquaculture.2019.06.004
Gallego, V., Cavalcante, S. S., Fujimoto, R. Y., Carneiro, P. C. F., Azevedo, H. C., &
Maria, A. N. (2017a). Fish sperm subpopulations: Changes after cryopreservation
process and relationship with fertilization success in tambaqui (Colossoma
macropomum). Theriogenology, 87(Supplement C), 16–24.
https://doi.org/10.1016/j.theriogenology.2016.08.001
Gallego, V., Cavalcante, S. S., Fujimoto, R. Y., Carneiro, P. C. F., Azevedo, H. C., &
Maria, A. N. (2017b). Fish sperm subpopulations: Changes after cryopreservation
process and relationship with fertilization success in tambaqui (Colossoma
macropomum). Theriogenology, 87(Supplement C), 16–24.
https://doi.org/10.1016/j.theriogenology.2016.08.001
Gallego, V., Martínez-Pastor, F., Mazzeo, I., Peñaranda, D. S., Herráez, M. P., Asturiano,
J. F., & Pérez, L. (2014). Intracellular changes in Ca2+, K+ and pH after sperm
motility activation in the European eel (Anguilla anguilla): Preliminary results.
198
Aquaculture, 418–419, 155–158.
https://doi.org/10.1016/j.aquaculture.2013.10.022
Gallego, V., Vílchez, M. C., Peñaranda, D. S., Pérez, L., Herráez, M. P., Asturiano, J. F.,
& Martínez-Pastor, F. (2015). Subpopulation pattern of eel spermatozoa is
affected by post-activation time, hormonal treatment and the thermal regimen.
Reproduction, Fertility and Development, 27(3), 529–543.
https://doi.org/10.1071/RD13198
Gazo, I., Dietrich, M. A., Prulière, G., Shaliutina-Kolešová, A., Shaliutina, O., Cosson, J.,
& Chenevert, J. (2017). Protein phosphorylation in spermatozoa motility of
Acipenser ruthenus and Cyprinus carpio. Reproduction, 154(5), 653–673.
https://doi.org/10.1530/REP-16-0662
Gilroy, C. E., & Litvak, M. K. (2019). Swimming kinematics and temperature effects on
spermatozoa from wild and captive shortnose sturgeon (Acipenser brevirostrum).
Animal Reproduction Science, 204, 171–182.
https://doi.org/10.1016/j.anireprosci.2019.03.022
Guthrie, H. D., Welch, G. R., & Woods, L. C. (2014). Effects of frozen and liquid
hypothermic storage and extender type on calcium homeostasis in relation to
viability and ATP content in striped bass (Morone saxatilis) sperm.
Theriogenology, 81(8), 1085–1091.
https://doi.org/10.1016/j.theriogenology.2014.01.035
Inaba, K., Morisawa, S., & Morisawa, M. (1998). Proteasomes regulate the motility of
salmonid fish sperm through modulation of cAMP-dependent phosphorylation of
an outer arm dynein light chain. Journal of Cell Science, 111(8), 1105–1115.
199
Ishimoto, K., Cosson, J., & Gaffney, E. A. (2016). A simulation study of sperm motility
hydrodynamics near fish eggs and spheres. Journal of Theoretical Biology, 389,
187–197. https://doi.org/10.1016/j.jtbi.2015.10.013
Kanuga, M. K., Drew, R. E., Wilson-Leedy, J. G., & Ingermann, R. L. (2012a).
Subpopulation distribution of motile sperm relative to activation medium in
steelhead (Oncorhynchus mykiss). Theriogenology, 77(5), 916–925.
https://doi.org/10.1016/j.theriogenology.2011.09.020
Kanuga, M. K., Drew, R. E., Wilson-Leedy, J. G., & Ingermann, R. L. (2012b).
Subpopulation distribution of motile sperm relative to activation medium in
steelhead (Oncorhynchus mykiss). Theriogenology, 77(5), 916–925.
https://doi.org/10.1016/j.theriogenology.2011.09.020
Keel, B. A., & Webster, B. W. (1990). Handbook of the Laboratory Diagnosis and
Treatment of Infertility. CRC Press.
Kholodnyy, V., Gadêlha, H., Cosson, J., & Boryshpolets, S. (2020). How do freshwater
fish sperm find the egg? The physicochemical factors guiding the gamete
encounters of externally fertilizing freshwater fish. Reviews in Aquaculture,
12(2), 1165–1192. https://doi.org/10.1111/raq.12378
Koubek, P., Kralova, A., Psenicka, M., & Peknicova, J. (2008). The optimization of the
protocol for immunofluorescence on fish spermatozoa. Theriogenology, 70(5),
852–858. https://doi.org/10.1016/j.theriogenology.2008.05.050
Krasznai, Z., Márián, T., Izumi, H., Damjanovich, S., Balkay, L., Trón, L., & Morisawa,
M. (2000). Membrane hyperpolarization removes inactivation of Ca2+ channels,
leading to Ca2+ influx and subsequent initiation of sperm motility in the common
200
carp. Proceedings of the National Academy of Sciences of the United States of
America, 97(5), 2052–2057.
Krasznai, Z., Morisawa, M., Morisawa, S., Krasznai, Z. T., Trón, L., Gáspár, R., &
Márián, T. (2003). Role of ion channels and membrane potential in the initiation
of carp sperm motility. Aquatic Living Resources, 16(5), 445–449.
https://doi.org/10.1016/S0990-7440(03)00054-8
Laemmli, U. K. (1970). Cleavage of Structural Proteins during the Assembly of the Head
of Bacteriophage T4. Nature, 227(5259), 680–685.
https://doi.org/10.1038/227680a0
Lissabet, J. F. B., Herrera Belén, L., Lee-Estevez, M., Risopatrón, J., Valdebenito, I.,
Figueroa, E., & Farías, J. G. (2020). The CatSper channel is present and plays a
key role in sperm motility of the Atlantic salmon (Salmo salar). Comparative
Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 241,
110634. https://doi.org/10.1016/j.cbpa.2019.110634
Martínez-Pastor, F., Cabrita, E., Soares, F., Anel-Lopez, L., & Dinis, M. T. (2008).
Multivariate cluster analysis to study motility activation of Solea senegalensis
spermatozoa: A model for marine teleosts. Reproduction, 135(4), 449–459.
https://doi.org/10.1530/REP-07-0376
Martínez-Pastor, F., Tizado, E. J., Garde, J. J., Anel, L., & de Paz, P. (2011a). Statistical
Series: Opportunities and challenges of sperm motility subpopulation
analysis11This article is part of the Statistical Series guest-edited by Szabolcs
Nagy. Theriogenology, 75(5), 783–795.
https://doi.org/10.1016/j.theriogenology.2010.11.034
201
Martínez-Pastor, F., Tizado, E. J., Garde, J. J., Anel, L., & de Paz, P. (2011b). Statistical
Series: Opportunities and challenges of sperm motility subpopulation
analysis11This article is part of the Statistical Series guest-edited by Szabolcs
Nagy. Theriogenology, 75(5), 783–795.
https://doi.org/10.1016/j.theriogenology.2010.11.034
Miura, T., Yamauchi, K., Takahashi, H., & Nagahama, Y. (1992). The role of hormones
in the acquisition of sperm motility in salmonid fish. Journal of Experimental
Zoology, 261(3), 359–363. https://doi.org/10.1002/jez.1402610316
Moore, A. (1987). Short-Term Storage and Cryopreservation of Walleye Semen.
Progressive Fish-Culturist, 49(1), 40–43. https://doi.org/10.1577/1548-
8640(1987)49<40:SSACOW>2.0.CO;2
Morisawa, M., & Okuno, M. (1982). Cyclic AMP induces maturation of trout sperm
axoneme to initiate motility. Nature, 295(5851), 703–704.
https://doi.org/10.1038/295703a0
Naresh, S., & Atreja, S. K. (2015). The protein tyrosine phosphorylation during in vitro
capacitation and cryopreservation of mammalian spermatozoa. Cryobiology,
70(3), 211–216. https://doi.org/10.1016/j.cryobiol.2015.03.008
Park, D. S., Egnatchik, R. A., Bordelon, H., Tiersch, T. R., & Monroe, W. T. (2012).
Microfluidic mixing for sperm activation and motility analysis of pearl Danio
zebrafish. Theriogenology, 78(2), 334–344.
https://doi.org/10.1016/j.theriogenology.2012.02.008
Pérez, L., Vílchez, M. C., Gallego, V., Morini, M., Peñaranda, D. S., & Asturiano, J. F.
(2016). Role of calcium on the initiation of sperm motility in the European eel.
202
Comparative Biochemistry and Physiology Part A: Molecular & Integrative
Physiology, 191, 98–106. https://doi.org/10.1016/j.cbpa.2015.10.009
Purdy, P. H., Barbosa, E. A., Praamsma, C. J., & Schisler, G. J. (2016). Modification of
trout sperm membranes associated with activation and cryopreservation.
Implications for fertilizing potential. Cryobiology, 73(1), 73–79.
https://doi.org/10.1016/j.cryobiol.2016.05.008
R Development Core Team. (2009). R: A language and environment for statistical
computing. R Foundation for Statistical Computing, Vienna, Austria. http://R-
project.org
R Development Core Team. (2019). R: A language and environment for statistical
computing. R Foundation for Statistical Computing, Vienna, Austria. URL
http://R-project.org. http://R-project.org
Seifert, R., Flick, M., Bönigk, W., Alvarez, L., Trötschel, C., Poetsch, A., Müller, A.,
Goodwin, N., Pelzer, P., Kashikar, N. D., Kremmer, E., Jikeli, J., Timmermann,
B., Kuhl, H., Fridman, D., Windler, F., Kaupp, U. B., & Strünker, T. (2015). The
CatSper channel controls chemosensation in sea urchin sperm. The EMBO
Journal, 34(3), 379–392. https://doi.org/10.15252/embj.201489376
Takei, G. L., Mukai, C., & Okuno, M. (2012). Transient Ca2+ mobilization caused by
osmotic shock initiates salmonid fish sperm motility. Journal of Experimental
Biology, 215(4), 630–641. https://doi.org/10.1242/jeb.063628
Thomas, A. D., Meyers, S. A., & Ball, B. A. (2006). Capacitation-like changes in equine
spermatozoa following cryopreservation. Theriogenology, 65(8), 1531–1550.
https://doi.org/10.1016/j.theriogenology.2005.08.022
203
Yanagimachi, R., Harumi, T., Matsubara, H., Yan, W., Yuan, S., Hirohashi, N., Iida, T.,
Yamaha, E., Arai, K., Matsubara, T., Andoh, T., Vines, C., & Cherr, G. N.
(2017). Chemical and physical guidance of fish spermatozoa into the egg through
the micropyle,. Biology of Reproduction, 96(4), 780–799.
https://doi.org/10.1093/biolre/iox015
Zilli, L., Schiavone, R., Storelli, C., & Vilella, S. (2008). Effect of cryopreservation on
phosphorylation state of proteins involved in sperm motility initiation in sea
bream. Cryobiology, 57(2), 150–155.
https://doi.org/10.1016/j.cryobiol.2008.07.006
Zilli, Loredana, Schiavone, R., & Vilella, S. (2017). Role of protein
phosphorylation/dephosphorylation in fish sperm motility activation: State of the
art and perspectives. Aquaculture, 472, 73–80.
https://doi.org/10.1016/j.aquaculture.2016.03.043
204
Table 8. Sauger sperm motility characteristics derived by the Hamilton Thorne Computer
Assisted Sperm Analysis system (n = 24,993, Kanuga et al., 2012b; Keel and Webster,
1990; Martínez-Pastor et al., 2011b).
Motility Unit Range Derivation/ Interpretation Parameter
Average Path Distance over and average path, averaged μm/s 20.00 – 200.87 Velocity (VAP) over 1/6th of all frames per unit time.
Straight Line Straight-line distance from 1st and last μm/s 5.00-195.00 Velocity (VSL) recorded point per unit time.
Curvilinear Actual distance traveled along path per unit μm/s 21.96 – 307 Velocity (VCL) time.
Amplitude of
Lateral Head Amplitude of variations of the actual sperm μm 0.50 – 32.94 Displacement head trajectory along average path.
(ALH)
Beat Cross The time – average rate that the curvilinear HZ 1.00 – 59.99 Frequency (BCF) path crosses the average path.
Straightness % 2.90 – 99.95 VSL/VAP, straightness of the average path. (STR)
Linearity (LIN) % 2.11 - 99.40 VSL/VCL, linearity of the curvilinear path.
Wobble (WOB) % 22.44 – 103.69 VAP/VCL, efficiency of forward progression.
205
Table 9. Comparison of the percentage of Sauger spermatozoa exhibiting high quantities of intracellular Ca2+ and mean fluorescent
intensity (a.u.) of all cells among sperm types (testicular, stripped, and cryopreserved) and activation status (inactive and activated).
Data are reported as the mean 1 standard error of the mean (n = 6). Tukey’s HSD superscripts denote significant differences among
group means (p < 0.05).
Sperm Type / Activation Status
Characteristic Testicular Stripped Cryopreserved
Inactive Activated Inactive Activated Inactive Activated
High Calcium (%) 13.24 ± 86.76 c 74.52 ± 4.00 b 14.67 ± 2.74 c 84.22 ± 2.87 b 93.25 ± 1.47 a 91.48 ± 1.77 a
Mean Fluorescent 6,091 ± 2,466 c 17,547 ± 2,466 b 7,003 ± 2,466 c 19,509 ± 2,466 b 30,471 ± 2,466 a 32,823 ± 2,466 a
20 Intensity (a.u.)
6
a,b,c Mean values (± SEM) with different superscript letters within a row differ significantly at P < 0.05.
206
Table 10. Comparison of the proportion (%) of Sauger spermatozoa exhibiting high quantities of protein phosphorylation among
sperm type and activation status (n = 6). Data are reported as the mean ± 1 SEM. Tukey’s HSD superscripts denote significant
differences among group means (p < 0.05).
Sperm Type / Activation Status
Characteristic Testicular Stripped Cryopreserved
Inactive Activated Inactive Activated Inactive Activated
Phosphotyrosine 67.6 ± 2.58 cx 70.7 ± 2.48 cy 79.0 ± 2.12 ax 81.7 ± 1.97 ay 73.2 ± 2.38 bx 76.0 ± 2.26 by
Phosphothreonine 59.8 ± 3.13 bx 60.3 ± 3.12 by 70.3 ± 2.80 ax 71.6 ± 2.75 ay 63.4 ± 3.05 ax 71.9 ± 2.73 ay
b b a a c c 20 Phosphoserine 74.6 ± 3.84 73.4 ± 3.95 81.1 ± 3.19 79.2 ± 3.39 67.2 ± 4.44 67.4 ± 4.39
7
Superscripts a,b,c, within a row denote differences among sperm types (testicular, stripped, and cryopreserved) while x,y within rows
denote statistically significant differences between activation statuses (inactive vs. activated).
207
Figure 19. Localization of phosphorylated tyrosine (A-C), threonine (C-E), and serine
(F-G) residues in Sauger sperm using immunofluorescence techniques. DIC micrographs of the sperm at × 1,000 magnification (A, C, F). Localization of the nucleus using
Hoechst 3342 (B, D). Indirect immunofluorescent localization of phosphorylated tyrosine
(C.), threonine (E.), and serine (G.) residues.
208
Figure 20. Qualitative assessment of phosphoprotein diversity [threonine (A.), tyrosine
(B.), and serine (C.)] among Sauger sperm types and activation statuses. Lanes correspond to the following treatments: 1, testicular, inactive; 2, testicular activated; 3, stripped inactive; 4, stripped activated; 5, cryopreserved, inactive; 6, cryopreserved activated. Protein bands are measured on the y-axis as molecular weight (kDa).
209
Table 11. Mean +/- 1 standard deviation of the 8 CASA-derived Sauger sperm motility variables for each of the three sperm subpopulations delineated by two-step clustering analysis.
Sperm Subpopulation Parameter 1 2 3
Number of Sperm (%) 5024 (20.10 %) 11,269 (45.09 %) 8,700 (34.81 %)
VAP (μm/s) 153.03 ± 29.00 108.46 ± 32.16 62.05 ± 30.63
VSL (μm/s) 92.08 ± 34.95 24.79 ± 13.89 46.62 ± 28.06
VCL (μm/s) 176.01 ± 29.90 175.14 ± 34.57 81.87 ± 32.96
ALH (μ) 12.13 ± 5.97 14.62 ± 5.77 3.88 ± 2.33
BCF (Hz) 9.56 ± 6.77 10.58 ± 7.99 26.38 ± 12.76
STR (%) 59.92 ± 17.78 23.23 ± 11.30 73.94 ± 20.94
LIN (%) 52.48 ± 17.95 14.22 ± 7.29 57.02 ± 23.33
WOB (%) 87.12 ± 8.89 62.44 ± 15.13 75.31 ± 17.67
210
Figure 21. A) Hierarchical clustering dendrogram of Sauger sperm trajectories pre- clustered K-means groupings, cut into three classification trees (k = 3) and B) graphical representation of the three clusters on a scatterplot displayed on principal components axes to maximize the amount of variance explained. Points on the plot represent invidual sperm trajectories ( n = 24,993).
211
1.00
0.75
0.50
0.25 Cumulative Proportion (%) Proportion Cumulative
0.00
Testicular Stripped Cryopreserved Sperm Type
Sperm Subpopulations SP1 SP2 SP3
Figure 22. Proportion of each sperm subpopulation [SP1, fast -linear (white); SP2, fast- nonlinear (grey); and SP3, slow-linear (black)] in each of the three sperm subtypes
(Testicular, Stripped, and Cryopreserved). Results of the generalized linear model indicate that the proportion of sperm in each category differs in each case among all three sperm types (p’s < 0.05, Tukey’s HSD post hoc test).
212
Chapter 6. Conclusion
The overall goal of this dissertation was to provide a thorough investigation of reproductive biology of Sauger, Sauger sperm physiology, and the effects of assisted reproduction techniques on sperm physiology. First, we were able to describe seasonal reproductive development of Sauger and characterize the main sperm duct as potential structure responsible for final sperm maturation prior to spawning. Second, we provided a detailed description of sperm physiology in the Sauger but, more importantly, how it changed in response to activation in the hypo-osmotic environment. And lastly, through this research we were able to determine the extent to which testicular harvest and cryopreservation altered sperm physiology and how they were able to respond to activation. These results have substantial implications for the practical application of these techniques in not only Sauger, but likely many other fish species. While we were able to identify many differences and similarities among sperm types, some changes in sperm physiology were more likely to be associated with fertilization potential than others.
Sperm Physiology and Activation
213
Our results provide the first details description of sperm structure and physiology in the Sauger. The ultrastructure of the Sauger testes and spermatozoa were consistent with those of other percid and teleost species. Additionally, our results also provided a description of the glycocalyx, plasma membrane, and intracellular signaling pathways found in Sauger sperm. These characteristics are poorly described in a majority of other fish species and represent novel advances in fish sperm biology. Novel parameters, such as these, offer new possibilities to the field of aquaculture for the sperm quality assessment.
Most importantly, we were able to provide a detailed account of the changes associated with activation, a ubiquitous event in fish spermatozoa. Many aspects of cell physiology were altered when diluted in hypo-osmotic hatchery water. Specifically, those changes were increased intracellular Ca2+, protein threonine phosphorylation, and motility patterns characterized by fast-nonlinear motility. Fast-nonlinear motility may be helpful in helping sperm swim along the surface of the egg (Elgeti et al., 2010; Ishimoto et al., 2016; Kholodnyy et al., 2020). Also, during this short period glucosamine moieties in the glycocalyx were redistributed to the apical portion of the sperm head. It is likely these moieties are involved in chemotaxis/ mechanosensory detection and interaction with the egg chorion and micropyle (Yanagimachi et al., 2017). Sperm cells were able to accomplish all of these changes while also maintaining membrane stability (e.g., fluidity) upon exposure to the harsh hypo-osmotic environment. Therefore, it is likely that these characteristics are essential to the proper function and fertilization capacity of fish sperm.
214
Even more important that simply understanding sperm physiology is our ability to discern the effects of ARTs on these parameters and overall sperm quality. In this dissertation we found testicular harvest and cryopreservation, both common ARTs used during Saugeye production, to have differing effects on sperm physiology and fertilization potential; one of which, testicular harvest, was not overtly detrimental while the other, cryopreservation, which was overwhelmingly detrimental to sperm physiology.
Testicular Harvest
Testicular harvest proved to be a valuable tool for hatchery staff during the production of Saugeye. Using this technique, a similar level of fertilization capacity could be achieved while also increasing sperm yield per fish by approximately 7-fold
(Appendix A). However, fertilization rates were somewhat variable at more limiting sperm-to-egg ratios. One goal of the research completed during this study was aimed at explaining characteristics of testicular sperm responsible for this variability.
Cells harvested from the testes of Sauger were composed of a heterogenous population of spermatozoa believed to be the result of colleting mature and immature cells simultaneously. A combination of evidence regarding seasonal reproductive development, main sperm duct morphology, and the heterogeneity in resulting sperm physiologies supports the concept that testicular harvest results in the collection of both immature and mature cells. Further, we support the concept that sperm maturation within the testes is likely a result of residency in the main sperm duct, characterized by the presence of epithelial folds. Poorer sperm maturity, characterized by fewer membrane glycocalyx moieties, lower amounts of protein phosphorylation, and differential lipid raft
215 structure, is likely to result from sperm within the tubular ducts (i.e., seminiferous tubules) compared to the main sperm duct.
Testicular harvest, despite the collection of immature cells, displayed high practical efficacy. The results reported in this dissertation indicate that despite a high number of sperm demonstrating what appears to be less developed traits, the population of mature, fertile sperm is sufficiently large to achieve fertilization high enough for routine application. It is essential, however, for sperm-to-egg ratios to be managed to prevent unexpectedly low fertilization during the production of Saugeye. We conclude that the testicular harvest procedure can be used in male Sauger broodstock as a way to increase total milt yield per individual.
Cryopreservation
Cryopreservation altered nearly every aspect of sperm physiology measured in this study to the detriment of fertilization potential. In fact, the results of our work indicated that frozen sperm were in the early stages of cellular apoptosis – cell death.
Moreover, in this state of disarray frozen sperm were unable to respond to the activation stimulus with one exception – motility. The fact that sperm in early stages of apoptosis are capable of motility only reaffirms the notion that motility is not a good predictor of fertilization capacity.
Cryopreservation was not only detrimental to all measures of sperm physiology but also affected how sperm responded to the motility stimulus. The evidence provided here indicates cryopreservation rendered a vast majority of spermatozoa in early stages or
216 cellular apoptosis. Enhanced fluidity indicative of membrane disruption, elevated intracellular Ca2+, increased -mannose availability, and loss of lipid raft stability were all associated with cryopreserved sperm, and thus low fertility. Moreover, spermatozoa were refractory to activation stimuli with regards to certain physiological changes, N- acetyl glucosamine redistribution and Ca2+ influx. Given the importance of the plasma membrane to cell function, we attributed the vast majority of altered physiology to changes in the plasma membrane. Evidence of lipid peroxidation observed during our investigation points to oxidative damage as a likely source damage during freezing.
Among the many differences observed between fresh and frozen sperm, the glycocalyx alone was related to contrasting fertilization potential. As demonstrated by our fertilization experiments, the loss of glycocalyx moiety mobility observed in frozen sperm was associated with an 82% reduction in fertilization. This same reduction was observed in fresh sperm when lectins were used to block glucosamine availability. Thus, it is our assertion that the loss of glucosamine activity observed in frozen sperm is at least partially responsible for poor fertilization at post-thaw. Widespread plasma membrane damage resulting from lipid peroxidation, phospholipase activity, and osmotic stress may render the plasma membrane unable to facilitate the glycocalyx redistribution observed in fertile sperm.
Presently, cryopreservation is not yet practical for large-scale application during the production of Saugeye hybrids. A 30 to 60-fold increase in sperm required to achieve only 20% fertilization is not sustainable at the level of Saugeye production by the ODNR-
DOW. The limited amount of milt collected from each individual combined with the low
217 concentration of sperm per cryopreservation unit makes the ability freeze, thaw, and handle large quantities of sperm the next hurdle toward large-scale application of this technique. However, increasing fertilization potential of frozen sperm (e.g., decreasing the number of sperm required to reach sufficient fertilization) will help to alleviate the need for massive quantities of sperm needed for large-scale application. New biomarkers proposed by our work should be used in future investigation to formulate cryopreservation media more protective toward sperm physiology and thus fertilization potential at post-thaw. Through the work reported here, practical efficacy of cryopreservation should be more easily attained using physiological biomarkers rather than ineffective and misleading measures, such as motility, when optimizing this particular technique.
Broader impacts
The work presented in this dissertation has broad impacts for use of ARTs in the field of aquaculture. This exploration of basic sperm biology and how a wide variety of traits, some of which are newly described for fish sperm, provides an abundance of research probabilities toward making ARTs less harmful to fertilization potential.
Biomarkers for sperm fertilization competence developed during this research (e.g., glucosamine redistribution) could replace motility assessment as a measure of post-thaw quality. This transition will allow researchers to screen a large number of candidate cryopreservation protocols (i.e., extenders, cryoprotectants, protein sources, antioxidants, etc.) in a short amount of time. Screening using this method can narrow down a large
218 number of protocols to a select few for further optimization. Protocol optimization aimed at maintaining sperm physiology, not just motility potential, should lead to more effective cryopreservation media with a better prognosis for large-sale application. We expect these results to have a substantial impact in the development of ARTs in not only the
Sauger, but all relevant aquatic species.
219
References
Elgeti, J., Kaupp, U.B., Gompper, G., 2010. Hydrodynamics of Sperm Cells near
Surfaces. Biophys J 99, 1018–1026. https://doi.org/10.1016/j.bpj.2010.05.015
Ishimoto, K., Cosson, J., Gaffney, E.A., 2016. A simulation study of sperm motility
hydrodynamics near fish eggs and spheres. Journal of Theoretical Biology 389,
187–197. https://doi.org/10.1016/j.jtbi.2015.10.013
Kholodnyy, V., Gadêlha, H., Cosson, J., Boryshpolets, S., 2020. How do freshwater fish
sperm find the egg? The physicochemical factors guiding the gamete encounters
of externally fertilizing freshwater fish. Reviews in Aquaculture 12, 1165–1192.
https://doi.org/10.1111/raq.12378
Yanagimachi, R., Harumi, T., Matsubara, H., Yan, W., Yuan, S., Hirohashi, N., Iida, T.,
Yamaha, E., Arai, K., Matsubara, T., Andoh, T., Vines, C., Cherr, G.N., 2017.
Chemical and physical guidance of fish spermatozoa into the egg through the
micropyle,. Biol Reprod 96, 780–799. https://doi.org/10.1093/biolre/iox015
220
Literature Cited
Alavi, S.H., Rodina, M., Policar, T., Cosson, J., Kozak, P., Psenicka, M., Linhart, O.,
2008. Physiology and behavior of stripped and testicular sperm in Perca fluviatilis
L. 1758. Cybium 32, 162–163.
Alavi, S.M.H., 2008. Fish spermatology. Alpha Science International Ltd.
Alavi, S.M.H., Cosson, J., 2006. Sperm motility in fishes. (II) Effects of ions and
osmolality: A review. Cell Biology International 30, 1–14.
https://doi.org/10.1016/j.cellbi.2005.06.004
Alavi, S.M.H., Cosson, J., 2005. Sperm motility in fishes. I. Effects of temperature and
pH: a review. Cell Biology International 29, 101–110.
https://doi.org/10.1016/j.cellbi.2004.11.021
Alavi, S.M.H., Cosson, J., Bondarenko, O., Linhart, O., 2019. Sperm motility in fishes:
(III) diversity of regulatory signals from membrane to the axoneme.
Theriogenology 136, 143–165.
https://doi.org/10.1016/j.theriogenology.2019.06.038
Alavi, S.M.H., Gela, D., Rodina, M., Linhart, O., 2011a. Roles of osmolality, calcium —
Potassium antagonist and calcium in activation and flagellar beating pattern of
221
sturgeon sperm. Comparative Biochemistry and Physiology Part A: Molecular &
Integrative Physiology 160, 166–174. https://doi.org/10.1016/j.cbpa.2011.05.026
Alavi, S.M.H., Gela, D., Rodina, M., Linhart, O., 2011b. Roles of osmolality, calcium —
Potassium antagonist and calcium in activation and flagellar beating pattern of
sturgeon sperm. Comparative Biochemistry and Physiology Part A: Molecular &
Integrative Physiology 160, 166–174. https://doi.org/10.1016/j.cbpa.2011.05.026
Amann, R.P., Waberski, D., 2014. Computer-assisted sperm analysis (CASA):
Capabilities and potential developments. Theriogenology 81, 5-17.e3.
https://doi.org/10.1016/j.theriogenology.2013.09.004
Asano, A., Selvaraj, V., Buttke, D.E., Nelson, J.L., Green, K.M., Evans, J.E., Travis,
A.J., 2009. Biochemical characterization of membrane fractions in murine sperm:
Identification of three distinct sub-types of membrane rafts. Journal of Cellular
Physiology 218, 537–548. https://doi.org/10.1002/jcp.21623
Asturiano, J.F., Cabrita, E., Horváth, Á., 2017. Progress, challenges and perspectives on
fish gamete cryopreservation: A mini-review. General and Comparative
Endocrinology, Proceedings of the Fifth International Workshop on the Biology
of Fish Gametes 245, 69–76. https://doi.org/10.1016/j.ygcen.2016.06.019
Bai, C., Kang, N., Zhao, J., Dai, J., Gao, H., Chen, Y., Dong, H., Huang, C., Dong, Q.,
2019. Cryopreservation disrupts lipid rafts and heat shock proteins in yellow
catfish sperm. Cryobiology 87, 32–39.
https://doi.org/10.1016/j.cryobiol.2019.03.004
222
Ball, B.A., 2008. Oxidative stress, osmotic stress and apoptosis: Impacts on sperm
function and preservation in the horse. Animal Reproduction Science, Special
Issue: Proceedings of the 5th International Symposium on Stallion Reproduction
107, 257–267. https://doi.org/10.1016/j.anireprosci.2008.04.014
Barton, B.A., 2011. Biology, management, and culture of Walleye and Sauger. American
Fisheries Society Bethesda, Maryland.
Beirão, J., Boulais, M., Gallego, V., O’Brien, J.K., Peixoto, S., Robeck, T.R., Cabrita, E.,
2019. Sperm handling in aquatic animals for artificial reproduction.
Theriogenology 133, 161–178.
https://doi.org/10.1016/j.theriogenology.2019.05.004
Beirão, J., Cabrita, E., Pérez-Cerezales, S., Martínez-Páramo, S., Herráez, M.P., 2011.
Effect of cryopreservation on fish sperm subpopulations. Cryobiology 62, 22–31.
https://doi.org/10.1016/j.cryobiol.2010.11.005
Beirão, J., Zilli, L., Vilella, S., Cabrita, E., Fernández‐Díez, C., Schiavone, R., Herráez,
M.P., 2012. Fatty acid composition of the head membrane and flagella affects
Sparus aurata sperm quality. Journal of Applied Ichthyology 28, 1017–1019.
https://doi.org/10.1111/jai.12085
Beirão, José, Zilli, L., Vilella, S., Cabrita, E., Schiavone, R., Herráez, M.P., 2012.
Improving Sperm Cryopreservation with Antifreeze Proteins: Effect on Gilthead
Seabream (Sparus aurata) Plasma Membrane Lipids. Biol Reprod 86.
https://doi.org/10.1095/biolreprod.111.093401
223
Bell, M.V., Dick, J.R., Buda, Cs., 1997. Molecular speciation of fish sperm
phospholipids: Large amounts of dipolyunsaturated phosphatidylserine. Lipids 32,
1085–1091. https://doi.org/10.1007/s11745-997-0140-y
Bell, M.V., Dick, J.R., Thrush, M., Navarro, J.C., 1996. Decreased 20:4n − 620:5n − 3
ratio in sperm from cultured sea bass, Dicentrarchus labrax, broodstock compared
with wild fish. Aquaculture 144, 189–199. https://doi.org/10.1016/S0044-
8486(96)01311-7
Bergeron, A., Vandenberg, G., Proulx, D., Bailey, J.L., 2002a. Comparison of extenders,
dilution ratios and theophylline addition on the function of cryopreserved Walleye
semen. Theriogenology 57, 1061–1071. https://doi.org/10.1016/S0093-
691X(01)00707-5
Bergeron, A., Vandenberg, G., Proulx, D., Bailey, J.L., 2002b. Comparison of extenders,
dilution ratios and theophylline addition on the function of cryopreserved Walleye
semen. Theriogenology 57, 1061–1071. https://doi.org/10.1016/S0093-
691X(01)00707-5
Billard, R., 1986. Spermatogenesis and spermatology of some teleost fish species.
Reprod. Nutr. Dévelop. 26, 877–920. https://doi.org/10.1051/rnd:19860601
Billard, R., Cosson, M.P., 1992a. Some problems related to the assessment of sperm
motility in freshwater fish. Journal of Experimental Zoology Part A: Ecological
Genetics and Physiology 261, 122–131.
224
Billard, R., Cosson, M.P., 1992b. Some problems related to the assessment of sperm
motility in freshwater fish. Journal of Experimental Zoology Part A: Ecological
Genetics and Physiology 261, 122–131.
Bilyy, R.O., Antonyuk, V.O., Stoika, R.S., 2004. Cytochemical study of role of alpha-d-
mannose- and beta-d-galactose-containing glycoproteins in apoptosis. Journal of
Molecular Histology 35, 829–838. https://doi.org/10.1007/s10735-004-1674-z
Bilyy, R.O., Stoika, R.S., 2003. Lectinocytochemical detection of apoptotic murine
leukemia L1210 cells. Cytometry Part A 56A, 89–95.
https://doi.org/10.1002/cyto.a.10089
Blawut, B., Wolfe, B., Moraes, C.R., Ludsin, S.A., Coutinho da Silva, M.A., 2018a.
Increasing Saugeye (S. vitreus × S. canadensis) production efficiency in a
hatchery setting using assisted reproduction technologies. Aquaculture 495, 21–
26. https://doi.org/10.1016/j.aquaculture.2018.05.027
Blawut, B., Wolfe, B., Moraes, C.R., Ludsin, S.A., Coutinho da Silva, M.A., 2018b.
Increasing Saugeye (S. vitreus × S. canadensis) production efficiency in a
hatchery setting using assisted reproduction technologies. Aquaculture 495, 21–
26. https://doi.org/10.1016/j.aquaculture.2018.05.027
Blawut, B., Wolfe, B., Moraes, C.R., Ludsin, S.A., Silva, M.A.C. da, 2020a. Use of
Hypertonic Media to Cryopreserve Sauger Spermatozoa. North American Journal
of Aquaculture 82, 84–91. https://doi.org/10.1002/naaq.10125
Blawut, B., Wolfe, B., Moraes, C.R., Sweet, D., Ludsin, S.A., Coutinho da Silva, M.A.,
2020b. Testicular collections as a technique to increase milt availability in Sauger
225
(sander canadensis). Animal Reproduction Science 212, 106240.
https://doi.org/10.1016/j.anireprosci.2019.106240
Blesbois, E., Grasseau, I., Seigneurin, F., 2005. Membrane fluidity and the ability of
domestic bird spermatozoa to survive cryopreservation. Reproduction 129, 371–
378. https://doi.org/10.1530/rep.1.00454
Blesbois, E., Grasseau, I., Seigneurin, F., Mignon-Grasteau, S., Saint Jalme, M., Mialon-
Richard, M.M., 2008. Predictors of success of semen cryopreservation in
chickens. Theriogenology 69, 252–261.
https://doi.org/10.1016/j.theriogenology.2007.09.019
Bligh, E.G., Dyer, W.J., 1959. A Rapid Method of Total Lipid Extraction and
Purification. Can. J. Biochem. Physiol. 37, 911–917. https://doi.org/10.1139/o59-
099
Boccaletto, P., Siddique, M.A.M., Cosson, J., 2018. Proteomics: A valuable approach to
elucidate spermatozoa post –testicular maturation in the endangered
Acipenseridae family. Animal Reproduction Science 192, 18–27.
https://doi.org/10.1016/j.anireprosci.2018.03.033
Boitano, S., Omoto, C.K., 1992. Trout sperm swimming patterns and role of intracellular
Ca++. Cell Motil. Cytoskeleton 21, 74–82. https://doi.org/10.1002/cm.970210109
Boryshpolets, S., Kholodnyy, V., Cosson, J., Dzyuba, B., 2020. Fish Sperm Quality
Evaluation After Cryopreservation, in: Betsy, J., Kumar, S. (Eds.),
Cryopreservation of Fish Gametes. Springer, Singapore, pp. 117–133.
https://doi.org/10.1007/978-981-15-4025-7_5
226
Brenker, C., Goodwin, N., Weyand, I., Kashikar, N.D., Naruse, M., Krähling, M., Müller,
A., Kaupp, U.B., Strünker, T., 2012. The CatSper channel: a polymodal
chemosensor in human sperm. The EMBO Journal 31, 1654–1665.
https://doi.org/10.1038/emboj.2012.30
Brogden, G., Propsting, M., Adamek, M., Naim, H.Y., Steinhagen, D., 2014a. Isolation
and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio
L.). Comparative Biochemistry and Physiology Part B: Biochemistry and
Molecular Biology 169, 9–15. https://doi.org/10.1016/j.cbpb.2013.12.001
Brogden, G., Propsting, M., Adamek, M., Naim, H.Y., Steinhagen, D., 2014b. Isolation
and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio
L.). Comparative Biochemistry and Physiology Part B: Biochemistry and
Molecular Biology 169, 9–15. https://doi.org/10.1016/j.cbpb.2013.12.001
Brown, D.A., 2006. Lipid Rafts, Detergent-Resistant Membranes, and Raft Targeting
Signals. Physiology 21, 430–439. https://doi.org/10.1152/physiol.00032.2006
Browne, R.K., Kaurova, S.A., Uteshev, V.K., Shishova, N.V., McGinnity, D., Figiel,
C.R., Mansour, N., Agnew, D., Wu, M., Gakhova, E.N., Dzyuba, B., Cosson, J.,
2015. Sperm motility of externally fertilizing fish and amphibians.
Theriogenology 83, 1-13.e8. https://doi.org/10.1016/j.theriogenology.2014.09.018
Butts, I.A.E., Alavi, S.M.H., Mokdad, A., Pitcher, T.E., 2013. Physiological functions of
osmolality and calcium ions on the initiation of sperm motility and swimming
performance in redside dace, Clinostomus elongatus. Comparative Biochemistry
227
and Physiology Part A: Molecular & Integrative Physiology 166, 147–157.
https://doi.org/10.1016/j.cbpa.2013.05.011
Cabrita, E., Martínez-Páramo, S., Gavaia, P.J., Riesco, M.F., Valcarce, D.G., Sarasquete,
C., Herráez, M.P., Robles, V., 2014. Factors enhancing fish sperm quality and
emerging tools for sperm analysis. Aquaculture 432, 389–401.
https://doi.org/10.1016/j.aquaculture.2014.04.034
Cabrita, E., Robles, V., Herráez, P., 2008. Methods in reproductive aquaculture: marine
and freshwater species. CRC Press.
Cabrita, E., Sarasquete, C., Martínez-Páramo, S., Robles, V., Beirão, J., Pérez-Cerezales,
S., Herráez, M. p., 2010. Cryopreservation of fish sperm: applications and
perspectives. Journal of Applied Ichthyology 26, 623–635.
https://doi.org/10.1111/j.1439-0426.2010.01556.x
Caldeira, C., García-Molina, A., Valverde, A., Bompart, D., Hassane, M., Martin, P.,
Soler, C., 2018. Comparison of sperm motility subpopulation structure among
wild anadromous and farmed male Atlantic salmon (Salmo salar) parr using a
CASA system. Reproduction, Fertility and Development.
Casselman, S.J., Schulte-Hostedde, A.I., Montgomerie, R., 2006. Sperm quality
influences male fertilization success in Walleye (Sander vitreus). Canadian
Journal of Fisheries and Aquatic Sciences 63, 2119–2125.
Chakrabarty, J., Banerjee, D., Pal, D., De, J., Ghosh, A., Majumder, G.C., 2007.
Shedding off specific lipid constituents from sperm cell membrane during
228
cryopreservation. Cryobiology 54, 27–35.
https://doi.org/10.1016/j.cryobiol.2006.10.191
Chen, Y., Wang, H., Wang, F., Chen, C., Zhang, P., Song, D., Luo, T., Xu, H., Zeng, X.,
2020. Sperm motility modulated by Trpv1 regulates zebrafish fertilization.
Theriogenology 151, 41–51. https://doi.org/10.1016/j.theriogenology.2020.03.032
Chevalier, C., de Conto, C., Exbrayat, J.-M., 2011. Reproductive biology of the
endangered percid Zingel asper in captivity: a histological description of the male
reproductive cycle. Folia histochemica et cytobiologica 49, 486–496.
Dacheux, J.-L., Castella, S., Gatti, J.L., Dacheux, F., 2005. Epididymal cell secretory
activities and the role of proteins in boar sperm maturation. Theriogenology,
Proceedings of the V International Conference on Boar Semen Preservation 63,
319–341. https://doi.org/10.1016/j.theriogenology.2004.09.015
Dadras, H., Sampels, S., Golpour, A., Dzyuba, V., Cosson, J., Dzyuba, B., 2017. Analysis
of common carp Cyprinus carpio sperm motility and lipid composition using
different in vitro temperatures. Animal Reproduction Science 180, 37–43.
https://doi.org/10.1016/j.anireprosci.2017.02.011
Dai, T., Zhao, E., Lu, G., Che, K., He, Q., Lu, Y., Fang, Q., Wang, H., Zheng, L., Li, S.,
Huang, C., Dong, Q., 2012. Sperm cryopreservation of yellow drum Nibea
albiflora: A special emphasis on post-thaw sperm quality. Aquaculture 368–369,
82–88. https://doi.org/10.1016/j.aquaculture.2012.09.017
Díaz, R., Lee-Estévez, M., Figueroa, E., Ulloa-Rodríguez, P., Sepúlveda, N., Farias, J.G.,
2018. Study of the membrane lipid composition of Atlantic salmon (Salmo salar)
229
spermatozoa and its relation with semen quality. Aquaculture Research 49, 2603–
2607.
Díaz, R., Lee-Estevez, M., Quiñones, J., Dumorné, K., Short, S., Ulloa-Rodríguez, P.,
Valdebenito, I., Sepúlveda, N., Farías, J.G., 2019. Changes in Atlantic salmon
(Salmo salar) sperm morphology and membrane lipid composition related to cold
storage and cryopreservation. Animal Reproduction Science 204, 50–59.
https://doi.org/10.1016/j.anireprosci.2019.03.004
Dietrich, M.A., Arnold, G.J., Fröhlich, T., Otte, K.A., Dietrich, G.J., Ciereszko, A., 2015.
Proteomic analysis of extracellular medium of cryopreserved carp (Cyprinus
carpio L.) semen. Comparative Biochemistry and Physiology Part D: Genomics
and Proteomics 15, 49–57. https://doi.org/10.1016/j.cbd.2015.05.003
Dietrich, M.A., Nynca, J., Ciereszko, A., 2019a. Proteomic and metabolomic insights into
the functions of the male reproductive system in fishes. Theriogenology 132,
182–200. https://doi.org/10.1016/j.theriogenology.2019.04.018
Dietrich, M.A., Samczuk, P., Ciborowski, M., Nynca, J., Parfieniuk, E., Pietrowska, K.,
Kowalczyk, T., Kretowski, A., Ciereszko, A., 2019b. Metabolic fingerprinting of
carp and Rainbow Trout seminal plasma. Aquaculture 501, 178–190.
https://doi.org/10.1016/j.aquaculture.2018.11.017
Dorado, J., Molina, I., Muñoz-Serrano, A., Hidalgo, M., 2010. Identification of sperm
subpopulations with defined motility characteristics in ejaculates from Florida
goats. Theriogenology 74, 795–804.
230
Drobnis, E.Z., Crowe, L.M., Berger, T., Anchordoguy, T.J., Overstreet, J.W., Crowe,
J.H., 1993. Cold shock damage is due to lipid phase transitions in cell
membranes: a demonstration using sperm as a model. Journal of Experimental
Zoology Part A: Ecological Genetics and Physiology 265, 432–437.
Dumorné, K., Figueroa, E., Cosson, J., Lee‐Estevez, M., Ulloa‐Rodríguez, P.,
Valdebenito, I., Farías, J.G., 2018. Protein phosphorylation and ions effects on
salmonid sperm motility activation. Reviews in Aquaculture 10, 727–737.
https://doi.org/10.1111/raq.12198
Dzyuba, V., Cosson, J., 2014. Motility of fish spermatozoa: from external signaling to
flagella response. Reproductive Biology 14, 165–175.
https://doi.org/10.1016/j.repbio.2013.12.005
Elgeti, J., Kaupp, U.B., Gompper, G., 2010. Hydrodynamics of Sperm Cells near
Surfaces. Biophys J 99, 1018–1026. https://doi.org/10.1016/j.bpj.2010.05.015
Engel, K.M., Dzyuba, V., Borys, D., Schiller, J., 2020. Different glycolipids in sperm
from different freshwater fishes – A high performance thin-layer
chromatography/electrospray ionization-mass spectrometry study. Rapid
Communications in Mass Spectrometry 34, 9. https://doi.org/10.1002/rcm.8875
Engel, K.M., Sampels, S., Dzyuba, B., Podhorec, P., Policar, T., Dannenberger, D.,
Schiller, J., 2019. Swimming at different temperatures: The lipid composition of
sperm from three freshwater fish species determined by mass spectrometry and
nuclear magnetic resonance spectroscopy. Chemistry and Physics of Lipids 221,
65–72. https://doi.org/10.1016/j.chemphyslip.2019.03.014
231
Ertmer, F., Oldenhof, H., Schütze, S., Rohn, K., Wolkers, W.F., Sieme, H., 2017.
Induced sub-lethal oxidative damage affects osmotic tolerance and cryosurvival
of spermatozoa. Reprod. Fertil. Dev. 29, 1739–1750.
https://doi.org/10.1071/RD16183
Figueroa, E., Lee-Estevez, M., Valdebenito, I., Watanabe, I., Oliveira, R.P.S., Romero, J.,
Castillo, R.L., Farías, J.G., 2019. Effects of cryopreservation on mitochondrial
function and sperm quality in fish. Aquaculture 511, 634190.
https://doi.org/10.1016/j.aquaculture.2019.06.004
Figueroa, E., Valdebenito, I., Farias, J.G., 2016. Technologies used in the study of sperm
function in cryopreserved fish spermatozoa. Aquaculture Research 47, 1691–
1705. https://doi.org/10.1111/are.12630
Flesch, F.M., Brouwers, J.F.H.M., Nievelstein, P.F.E.M., Verkleij, A.J., Golde, L.M.G.
van, Colenbrander, B., Gadella, B.M., 2001. Bicarbonate stimulated phospholipid
scrambling induces cholesterol redistribution and enables cholesterol depletion in
the sperm plasma membrane. Journal of Cell Science 114, 3543–3555.
Flesch, F.M., Gadella, B.M., 2000. Dynamics of the mammalian sperm plasma
membrane in the process of fertilization. Biochimica et Biophysica Acta (BBA) -
Reviews on Biomembranes 1469, 197–235. https://doi.org/10.1016/S0304-
4157(00)00018-6
Gadella, B.M., Rathi, R., Brouwers, J.F.H.M., Stout, T.A.E., Colenbrander, B., 2001.
Capacitation and the acrosome reaction in equine sperm. Animal Reproduction
232
Science, Symposium on Stallion Reproduction 68, 249–265.
https://doi.org/10.1016/S0378-4320(01)00161-0
Gallego, V., Cavalcante, S.S., Fujimoto, R.Y., Carneiro, P.C.F., Azevedo, H.C., Maria,
A.N., 2017a. Fish sperm subpopulations: Changes after cryopreservation process
and relationship with fertilization success in tambaqui (Colossoma
macropomum). Theriogenology 87, 16–24.
https://doi.org/10.1016/j.theriogenology.2016.08.001
Gallego, V., Cavalcante, S.S., Fujimoto, R.Y., Carneiro, P.C.F., Azevedo, H.C., Maria,
A.N., 2017b. Fish sperm subpopulations: Changes after cryopreservation process
and relationship with fertilization success in tambaqui (Colossoma
macropomum). Theriogenology 87, 16–24.
https://doi.org/10.1016/j.theriogenology.2016.08.001
Gallego, V., Martínez-Pastor, F., Mazzeo, I., Peñaranda, D.S., Herráez, M.P., Asturiano,
J.F., Pérez, L., 2014. Intracellular changes in Ca2+, K+ and pH after sperm
motility activation in the European eel (Anguilla anguilla): Preliminary results.
Aquaculture 418–419, 155–158.
https://doi.org/10.1016/j.aquaculture.2013.10.022
Gallego, V., Vílchez, M.C., Peñaranda, D.S., Pérez, L., Herráez, M.P., Asturiano, J.F.,
Martínez-Pastor, F., 2015. Subpopulation pattern of eel spermatozoa is affected
by post-activation time, hormonal treatment and the thermal regimen. Reprod.
Fertil. Dev. 27, 529–543. https://doi.org/10.1071/RD13198
233
Garcia-Garcia, E., Grayfer, L., Stafford, J.L., Belosevic, M., 2012. Evidence for the
presence of functional lipid rafts in immune cells of ectothermic organisms.
Developmental & Comparative Immunology 37, 257–269.
https://doi.org/10.1016/j.dci.2012.03.009
Gazo, I., Dietrich, M.A., Prulière, G., Shaliutina-Kolešová, A., Shaliutina, O., Cosson, J.,
Chenevert, J., 2017. Protein phosphorylation in spermatozoa motility of
Acipenser ruthenus and Cyprinus carpio. Reproduction 154, 653–673.
https://doi.org/10.1530/REP-16-0662
Gilroy, C.E., Litvak, M.K., 2019. Swimming kinematics and temperature effects on
spermatozoa from wild and captive shortnose sturgeon (Acipenser brevirostrum).
Animal Reproduction Science 204, 171–182.
https://doi.org/10.1016/j.anireprosci.2019.03.022
Giraud, M.N., Motta, C., Boucher, D., Grizard, G., 2000. Membrane fluidity predicts the
outcome of cryopreservation of human spermatozoa. Hum Reprod 15, 2160–
2164. https://doi.org/10.1093/humrep/15.10.2160
Girouard, J., Frenette, G., Sullivan, R., 2008a. Seminal Plasma Proteins Regulate the
Association of Lipids and Proteins Within Detergent-Resistant Membrane
Domains of Bovine Spermatozoa. Biol Reprod 78, 921–931.
https://doi.org/10.1095/biolreprod.107.066514
Girouard, J., Frenette, G., Sullivan, R., 2008b. Seminal Plasma Proteins Regulate the
Association of Lipids and Proteins Within Detergent-Resistant Membrane
234
Domains of Bovine Spermatozoa. Biol Reprod 78, 921–931.
https://doi.org/10.1095/biolreprod.107.066514
Goyal, H.O., 1985. Morphology of the bovine epididymis. American Journal of Anatomy
172, 155–172. https://doi.org/10.1002/aja.1001720205
Goyal, H.O., Williams, C.S., 1991. Regional differences in the morphology of the goat
epididymis: A light microscopic and ultrastructural study. American Journal of
Anatomy 190, 349–369. https://doi.org/10.1002/aja.1001900404
Grier, H.J., 1981. Cellular Organization of the Testis and Spermatogenesis in Fishes.
American Zoologist 21, 345–357. https://doi.org/10.1093/icb/21.2.345
Guthrie, H.D., Welch, G.R., Woods, L.C., 2014. Effects of frozen and liquid hypothermic
storage and extender type on calcium homeostasis in relation to viability and ATP
content in striped bass (Morone saxatilis) sperm. Theriogenology 81, 1085–1091.
https://doi.org/10.1016/j.theriogenology.2014.01.035
Gylfason, G.A., Knútsdóttir, E., Ásgeirsson, B., 2010. Isolation and biochemical
characterisation of lipid rafts from Atlantic cod (Gadus morhua) intestinal
enterocytes. Comparative Biochemistry and Physiology Part B: Biochemistry and
Molecular Biology 155, 86–95. https://doi.org/10.1016/j.cbpb.2009.10.006
Haidl, G., Opper, C., 1997. Changes in lipids and membrane anisotropy in human
spermatozoa during epididymal maturation. Hum Reprod 12, 2720–2723.
https://doi.org/10.1093/humrep/12.12.2720
Hammerstedt, R., Graham, J., Nolan, J., 1990. Cryopreservation of Mammalian Sperm -
What We Ask Them to Survive. Journal of Andrology 11, 73–88.
235
Hammerstedt, R.H., Graham, J.K., Nolan, J.P., 1990. Cryopreservation of Mammalian
Sperm: What We Ask Them to Survive. Journal of Andrology 11, 73–88.
https://doi.org/10.1002/j.1939-4640.1990.tb01583.x
Hatef, A., Alavi, S.M.H., Butts, I.A.E., Policar, T., Linhart, O., 2011. Mechanism of
action of mercury on sperm morphology, adenosine triphosphate content, and
motility in Perca fluviatilis (Percidae; Teleostei). Environmental Toxicology and
Chemistry 30, 905–914. https://doi.org/10.1002/etc.461
He, Q., Lu, G., Che, K., Zhao, E., Fang, Q., Wang, H., Liu, J., Huang, C., Dong, Q.,
2011a. Sperm cryopreservation of the endangered red spotted grouper,
Epinephelus akaara, with a special emphasis on membrane lipids. Aquaculture
318, 185–190. https://doi.org/10.1016/j.aquaculture.2011.05.025
He, Q., Lu, G., Che, K., Zhao, E., Fang, Q., Wang, H., Liu, J., Huang, C., Dong, Q.,
2011b. Sperm cryopreservation of the endangered red spotted grouper,
Epinephelus akaara, with a special emphasis on membrane lipids. Aquaculture
318, 185–190. https://doi.org/10.1016/j.aquaculture.2011.05.025
Hliwa, P., Dietrich, G.J., Dietrich, M., Nynca, J., Martyniak, A., Sobocki, M., Stabinski,
R., Ciereszko, A., 2010. Morphology and histology of cranial and caudal lobes of
whitefish (Coregonus lavaretus L.) testes. Fundamental and Applied Limnology /
Archiv f??r Hydrobiologie 176, 83–88. https://doi.org/10.1127/1863-
9135/2010/0176-0083
Holt, W.V., 2000. Basic aspects of frozen storage of semen. Animal Reproduction
Science 62, 3–22. https://doi.org/10.1016/S0378-4320(00)00152-4
236
Horokhovatskyi, Y., Sampels, S., Cosson, J., Linhart, O., Rodina, M., Fedorov, P.,
Blecha, M., Dzyuba, B., 2016. Lipid composition in common carp (Cyprinus
carpio) sperm possessing different cryoresistance. Cryobiology 73, 282–285.
https://doi.org/10.1016/j.cryobiol.2016.08.005
Ibrahim, Z.H., Singh, S.K., 2014. Histological and morphometric studies on the
dromedary camel epididymis in relation to reproductive activity. Animal
Reproduction Science 149, 212–217.
https://doi.org/10.1016/j.anireprosci.2014.07.012
Iliadou, K., Fishelson, L., 1995. Histology and Cytology of Testes of the Catfish
Parasilurus aristotelis (Siluridae, Teleostei) from Greece. Japanese Journal
of Ichthyology 41, 447–454. https://doi.org/10.11369/jji1950.41.447
Inaba, K., Morisawa, S., Morisawa, M., 1998. Proteasomes regulate the motility of
salmonid fish sperm through modulation of cAMP-dependent phosphorylation of
an outer arm dynein light chain. Journal of Cell Science 111, 1105–1115.
Ishimoto, K., Cosson, J., Gaffney, E.A., 2016. A simulation study of sperm motility
hydrodynamics near fish eggs and spheres. Journal of Theoretical Biology 389,
187–197. https://doi.org/10.1016/j.jtbi.2015.10.013
Iwamatsu, T., Yoshizaki, N., Shibata, Y., 1997. Changes in the chorion and sperm entry
into the micropyle during fertilization in the teleostean fish, Oryzias latipes.
Development, Growth & Differentiation 39, 33–41.
https://doi.org/10.1046/j.1440-169X.1997.00005.x
237
Jiménez, I., González-Márquez, H., Ortiz, R., Herrera, J.A., Garcı́a, A., Betancourt, M.,
Fierro, R., 2003. Changes in the distribution of lectin receptors during
capacitation and acrosome reaction in boar spermatozoa. Theriogenology 59,
1171–1180. https://doi.org/10.1016/S0093-691X(02)01175-5
Jonas‐Davies, J. a. C., Winfrey, V., Olson, G.E., 1983. Plasma membrane structure in
spermatogenic cells of the swordtail (teleostei, Xiphophorus helleri). Gamete
Research 7, 309–324. https://doi.org/10.1002/mrd.1120070403
Jones, R., 2002. Plasma Membrane Composition and Organisation During Maturation of
Spermatozoa in the Epididymis. The Epididymis: From Molecules to Clinical
Practice 405–416. https://doi.org/10.1007/978-1-4615-0679-9_23
Jones, R.C., Hinds, L.A., Tyndale-Biscoe, C.H., 1984. Ultrastructure of the epididymis of
the tammar, Macropus eugenii, and its relationship to sperm maturation. Cell
Tissue Res. 237, 525–535. https://doi.org/10.1007/BF00228437
Jr, J.R.S., Flickinger, S.A., 1995. Field Collection and Short-Term Storage of Walleye
Semen. The Progressive Fish-Culturist 57, 182–187.
https://doi.org/10.1577/1548-8640(1995)057<0182:FCASTS>2.3.CO;2
JR, W.E.L., Johnson, D.L., Schell, S.A., 1982. Survival, Growth, and Food Habits of
Walleye X Sauger Hybrids (Saugeye) in Ponds. North American Journal of
Fisheries Management 2, 381–387. https://doi.org/10.1577/1548-
8659(1982)2<381:SGAFHO>2.0.CO;2
238
Kalo, D., Roth, Z., 2011. Involvement of the sphingolipid ceramide in heat-shock-
induced apoptosis of bovine oocytes. Reprod. Fertil. Dev. 23, 876–888.
https://doi.org/10.1071/RD10330
Kanuga, M.K., Drew, R.E., Wilson-Leedy, J.G., Ingermann, R.L., 2012a. Subpopulation
distribution of motile sperm relative to activation medium in steelhead
(Oncorhynchus mykiss). Theriogenology 77, 916–925.
https://doi.org/10.1016/j.theriogenology.2011.09.020
Kanuga, M.K., Drew, R.E., Wilson-Leedy, J.G., Ingermann, R.L., 2012b. Subpopulation
distribution of motile sperm relative to activation medium in steelhead
(Oncorhynchus mykiss). Theriogenology 77, 916–925.
https://doi.org/10.1016/j.theriogenology.2011.09.020
Kawano, N., Yoshida, K., Miyado, K., Yoshida, M., 2011. Lipid Rafts: Keys to Sperm
Maturation, Fertilization, and Early Embryogenesis [WWW Document]. Journal
of Lipids. https://doi.org/10.1155/2011/264706
Keel, B.A., Webster, B.W., 1990. Handbook of the Laboratory Diagnosis and Treatment
of Infertility. CRC Press.
Khalil, M.B., Chakrabandhu, K., Xu, H., Weerachatyanukul, W., Buhr, M., Berger, T.,
Carmona, E., Vuong, N., Kumarathasan, P., Wong, P.T.T., Carrier, D.,
Tanphaichitr, N., 2006a. Sperm capacitation induces an increase in lipid rafts
having zona pellucida binding ability and containing sulfogalactosylglycerolipid.
Developmental Biology 290, 220–235.
https://doi.org/10.1016/j.ydbio.2005.11.030
239
Khalil, M.B., Chakrabandhu, K., Xu, H., Weerachatyanukul, W., Buhr, M., Berger, T.,
Carmona, E., Vuong, N., Kumarathasan, P., Wong, P.T.T., Carrier, D.,
Tanphaichitr, N., 2006b. Sperm capacitation induces an increase in lipid rafts
having zona pellucida binding ability and containing sulfogalactosylglycerolipid.
Developmental Biology 290, 220–235.
https://doi.org/10.1016/j.ydbio.2005.11.030
Kholodnyy, V., Gadêlha, H., Cosson, J., Boryshpolets, S., 2020. How do freshwater fish
sperm find the egg? The physicochemical factors guiding the gamete encounters
of externally fertilizing freshwater fish. Reviews in Aquaculture 12, 1165–1192.
https://doi.org/10.1111/raq.12378
Klaiwattana, P., Srisook, K., Srisook, E., Vuthiphandchai, V., Neumvonk, J., 2016. Effect
of cryopreservation on lipid composition and antioxidant enzyme activity of
seabass (Lates calcarifer) sperm. Iranian Journal of Fisheries Sciences 15, 157–
169.
Koehler, J.K., 1981. Lectins as Probes of the Spermatozoon Surface. Archives of
Andrology 6, 197–217. https://doi.org/10.3109/01485018108987531
Koubek, P., Kralova, A., Psenicka, M., Peknicova, J., 2008. The optimization of the
protocol for immunofluorescence on fish spermatozoa. Theriogenology 70, 852–
858. https://doi.org/10.1016/j.theriogenology.2008.05.050
Kowalski, R.K., Hliwa, P., Cejko, B.I., Krol, J., Stabinski, R., Ciereszko, A., 2012.
Quality and quantity of smelt (Osmerus eperlanus L.) sperm in relation to time
after hormonal stimulation. Reprod. Biol. 12, 231–246.
240
Krasznai, Z., Márián, T., Izumi, H., Damjanovich, S., Balkay, L., Trón, L., Morisawa,
M., 2000. Membrane hyperpolarization removes inactivation of Ca2+ channels,
leading to Ca2+ influx and subsequent initiation of sperm motility in the common
carp. Proc Natl Acad Sci U S A 97, 2052–2057.
Krasznai, Z., Morisawa, M., Krasznai, Z.T., Morisawa, S., Inaba, K., Bazsáné, Z.K.,
Rubovszky, B., Bodnár, B., Borsos, A., Márián, T., 2003a. Gadolinium, a
mechano-sensitive channel blocker, inhibits osmosis-initiated motility of sea- and
freshwater fish sperm, but does not affect human or ascidian sperm motility. Cell
Motility 55, 232–243. https://doi.org/10.1002/cm.10125
Krasznai, Z., Morisawa, M., Morisawa, S., Krasznai, Z.T., Trón, L., Gáspár, R., Márián,
T., 2003b. Role of ion channels and membrane potential in the initiation of carp
sperm motility. Aquatic Living Resources 16, 445–449.
https://doi.org/10.1016/S0990-7440(03)00054-8
Křišťan, J., Hatef, A., Alavi, S.M.H., Policar, T., 2014. Sperm fine structure of the
pikeperch, Sander lucioperca (Percidae, Teleostei). Czech J. Anim. Sci 59, 1–10.
Krol, J. (University of W. and M., Glogowski, J. (Polish A. of S., Demska-Zakes, K.
(University of W. and M., Hliwa, P. (University of W. and M., 2006. Quality of
semen and histological analysis of testes in Eurasian perch Perca fluviatilis L.
during a spawning period. Czech Journal of Animal Science - UZPI (Czech
Republic).
241
Kudo, S., 1998a. Role of sperm head syndecan at fertilization in fish. J. Exp. Zool. 281,
620–625. https://doi.org/10.1002/(SICI)1097-010X(19980815)281:6<620::AID-
JEZ10>3.0.CO;2-6
Kudo, S., 1998b. Role of sperm head syndecan at fertilization in fish. J. Exp. Zool. 281,
620–625. https://doi.org/10.1002/(SICI)1097-010X(19980815)281:6<620::AID-
JEZ10>3.0.CO;2-6
Laemmli, U.K., 1970. Cleavage of Structural Proteins during the Assembly of the Head
of Bacteriophage T4. Nature 227, 680–685. https://doi.org/10.1038/227680a0
Lahnsteiner, F., Berger, B., Weismann, T., Patzner, R., 1995. Fine structure and motility
of spermatozoa and composition of the seminal plasma in the perch. Journal of
Fish Biology 47, 492–508. https://doi.org/10.1111/j.1095-8649.1995.tb01917.x
Lahnsteiner, F., Mansour, N., 2004. Sperm fine structure of the pikeperch, Sander
lucioperca (Percidae, Teleostei). J Submicrosc Cytol Pathol 36, 309–312.
Lahnsteiner, F., Mansour, N., McNiven, M.A., Richardson, G.F., 2009. Fatty acids of
Rainbow Trout (Oncorhynchus mykiss) semen: Composition and effects on
sperm functionality. Aquaculture 298, 118–124.
https://doi.org/10.1016/j.aquaculture.2009.08.034
Lahnsteiner, F., Patzner, R.A., Welsmann, T., 1993. The spermatic ducts of salmonid
fishes (Salmonidae, Teleostei). Morphology, histochemistry and composition of
the secretion. Journal of Fish Biology 42, 79–93. https://doi.org/10.1111/j.1095-
8649.1993.tb00307.x
242
Leahy, T., Gadella, B.M., 2011. Sperm surface changes and physiological consequences
induced by sperm handling and storage. Reproduction 142, 759–778.
https://doi.org/10.1530/REP-11-0310
Lee, S.K., Veeramachaneni, D.N.R., 2005. Subchronic Exposure to Low Concentrations
of Di-n-Butyl Phthalate Disrupts Spermatogenesis in Xenopus laevis Frogs.
Toxicol Sci 84, 394–407. https://doi.org/10.1093/toxsci/kfi087
Li, P., Hulak, M., Li, Z.H., Sulc, M., Psenicka, M., Rodina, M., Gela, D., Linhart, O.,
2013. Cryopreservation of common carp (Cyprinus carpio L.) sperm induces
protein phosphorylation in tyrosine and threonine residues. Theriogenology 80,
84–89. https://doi.org/10.1016/j.theriogenology.2013.03.021
Lissabet, J.F.B., Belén, L.H., Lee-Estevez, M., Farias, J.G., 2020a. Role of voltage-gated
L-type calcium channel in the spermatozoa motility of Atlantic salmon (Salmo
salar). Comparative Biochemistry and Physiology Part A: Molecular &
Integrative Physiology 241, 110633. https://doi.org/10.1016/j.cbpa.2019.110633
Lissabet, J.F.B., Herrera Belén, L., Lee-Estevez, M., Risopatrón, J., Valdebenito, I.,
Figueroa, E., Farías, J.G., 2020b. The CatSper channel is present and plays a key
role in sperm motility of the Atlantic salmon (Salmo salar). Comparative
Biochemistry and Physiology Part A: Molecular & Integrative Physiology 241,
110634. https://doi.org/10.1016/j.cbpa.2019.110634
Liu, M., 2016. Capacitation-Associated Glycocomponents of Mammalian Sperm. Reprod
Sci 23, 572–594. https://doi.org/10.1177/1933719115602760
243
Malison, J.A., Held, J.A., 1996. Reproduction and spawning in Walleye (Stizostedion
vitreum). Journal of Applied Ichthyology 12, 153–156.
https://doi.org/10.1111/j.1439-0426.1996.tb00081.x
Malison, J.A., Procarione, L.S., Barry, T.P., Kapuscinski, A.R., Kayes, T.B., 1994.
Endocrine and gonadal changes during the annual reproductive cycle of the
freshwater teleost,
Fish Physiol Biochem 13, 473–484. https://doi.org/10.1007/BF00004330
Mansour, N., Lahnsteiner, F., Berger, B., 2004. Characterization of the testicular semen
of the African catfish, Clarias gariepinus (Burchell, 1822), and its short-term
storage. Aquaculture Research 35, 232–244. https://doi.org/10.1111/j.1365-
2109.2004.00993.x
Martínez-Páramo, S., Diogo, P., Beirão, J., Dinis, M.T., Cabrita, E., 2012. Sperm lipid
peroxidation is correlated with differences in sperm quality during the
reproductive season in precocious European sea bass (Dicentrarchus labrax)
males. Aquaculture 358–359, 246–252.
https://doi.org/10.1016/j.aquaculture.2012.06.010
Martínez-Páramo, S., Horváth, Á., Labbé, C., Zhang, T., Robles, V., Herráez, P., Suquet,
M., Adams, S., Viveiros, A., Tiersch, T.R., Cabrita, E., 2017. Cryobanking of
aquatic species. Aquaculture 472, 156–177.
https://doi.org/10.1016/j.aquaculture.2016.05.042
Martínez-Pastor, F., Cabrita, E., Soares, F., Anel-Lopez, L., Dinis, M.T., 2008.
Multivariate cluster analysis to study motility activation of Solea senegalensis
244
spermatozoa: a model for marine teleosts. Reproduction 135, 449–459.
https://doi.org/10.1530/REP-07-0376
Martínez-Pastor, F., Tizado, E.J., Garde, J.J., Anel, L., de Paz, P., 2011a. Statistical
Series: Opportunities and challenges of sperm motility subpopulation
analysis11This article is part of the Statistical Series guest-edited by Szabolcs
Nagy. Theriogenology 75, 783–795.
https://doi.org/10.1016/j.theriogenology.2010.11.034
Martínez-Pastor, F., Tizado, E.J., Garde, J.J., Anel, L., de Paz, P., 2011b. Statistical
Series: Opportunities and challenges of sperm motility subpopulation
analysis11This article is part of the Statistical Series guest-edited by Szabolcs
Nagy. Theriogenology 75, 783–795.
https://doi.org/10.1016/j.theriogenology.2010.11.034
Migaud, H., Bell, G., Cabrita, E., McAndrew, B., Davie, A., Bobe, J., Herráez, M.P.,
Carrillo, M., 2013. Gamete quality and broodstock management in temperate fish.
Reviews in Aquaculture 5, S194–S223. https://doi.org/10.1111/raq.12025
Miranda, P.V., Allaire, A., Sosnik, J., Visconti, P.E., 2009. Localization of Low-Density
Detergent-Resistant Membrane Proteins in Intact and Acrosome-Reacted Mouse
Sperm. Biol Reprod 80, 897–904. https://doi.org/10.1095/biolreprod.108.075242
Miura, T., Miura, C., 2001. Japanese Eel: A Model for Analysis of Spermatogenesis. jzoo
18, 1055–1063. https://doi.org/10.2108/zsj.18.1055
245
Miura, T., Yamauchi, K., Takahashi, H., Nagahama, Y., 1992a. The role of hormones in
the acquisition of sperm motility in salmonid fish. Journal of Experimental
Zoology 261, 359–363. https://doi.org/10.1002/jez.1402610316
Miura, T., Yamauchi, K., Takahashi, H., Nagahama, Y., 1992b. The role of hormones in
the acquisition of sperm motility in salmonid fish. Journal of Experimental
Zoology 261, 359–363. https://doi.org/10.1002/jez.1402610316
Moore, A., 1987a. Short-Term Storage and Cryopreservation of Walleye Semen.
Progress. Fish-Cult. 49, 40–43. https://doi.org/10.1577/1548-
8640(1987)49<40:SSACOW>2.0.CO;2
Moore, A., 1987b. Short-Term Storage and Cryopreservation of Walleye Semen.
Progress. Fish-Cult. 49, 40–43. https://doi.org/10.1577/1548-
8640(1987)49<40:SSACOW>2.0.CO;2
Morisawa, M., Okuno, M., 1982. Cyclic AMP induces maturation of trout sperm
axoneme to initiate motility. Nature 295, 703–704.
https://doi.org/10.1038/295703a0
Morisawa, S., Morisawa, M., 1988a. Induction of potential for sperm motility by
bicarbonate and pH in Rainbow Trout and chum salmon. Journal of Experimental
Biology 136, 13–22.
Morisawa, S., Morisawa, M., 1988b. Induction of potential for sperm motility by
bicarbonate and pH in Rainbow Trout and chum salmon. J. Exp. Biol. 136, 13–
22.
246
Morisawa, S., Morisawa, M., 1986. Acquisition of potential for sperm motility in
Rainbow Trout and chum salmon. J. Exp. Biol. 126, 89–96.
Müller, K., Müller, P., Pincemy, G., Kurz, A., Labbe, C., 2008. Characterization of sperm
plasma membrane properties after cholesterol modification: consequences for
cryopreservation of Rainbow Trout spermatozoa. Biology of Reproduction 78,
390–399.
Muñoz, M.E., Batlouni, S.R., Vicentini, I.B.F., Vicentini, C.A., 2011. Testicular structure
and description of the seminal pathway in Leporinus macrocephalus
(Anostomidae, Teleostei). Micron 42, 892–897.
https://doi.org/10.1016/j.micron.2011.06.008
Nandi, S., Routray, P., Gupta, S.D., Rath, S.C., Dasgupta, S., Meher, P.K.,
Mukhopadhyay, P.K., 2007. Reproductive performance of carp, Catla catla
(Ham.), reared on a formulated diet with PUFA supplementation. Journal of
Applied Ichthyology 23, 684–691. https://doi.org/10.1111/j.1439-
0426.2007.00874.x
Naresh, S., Atreja, S.K., 2015. The protein tyrosine phosphorylation during in vitro
capacitation and cryopreservation of mammalian spermatozoa. Cryobiology 70,
211–216. https://doi.org/10.1016/j.cryobiol.2015.03.008
Nixon, B., Aitken, R.J., 2009a. The biological significance of detergent-resistant
membranes in spermatozoa. Journal of Reproductive Immunology, Bio-
immunoregulatory Mechanisms Associated with Reproductive Organs: Relevance
247
in Fertility and in Sexually Transmitted Infections 83, 8–13.
https://doi.org/10.1016/j.jri.2009.06.258
Nixon, B., Aitken, R.J., 2009b. The biological significance of detergent-resistant
membranes in spermatozoa. Journal of Reproductive Immunology, Bio-
immunoregulatory Mechanisms Associated with Reproductive Organs: Relevance
in Fertility and in Sexually Transmitted Infections 83, 8–13.
https://doi.org/10.1016/j.jri.2009.06.258
Nixon, B., Bielanowicz, A., Mclaughlin, E.A., Tanphaichitr, N., Ensslin, M.A., Aitken,
R.J., 2009. Composition and significance of detergent resistant membranes in
mouse spermatozoa. Journal of Cellular Physiology 218, 122–134.
https://doi.org/10.1002/jcp.21575
Ohta, Hiromi, Ikeda, K., Izawa, T., 1997. Increases in concentrations of potassium and
bicarbonate ions promote acquisition of motility in vitro by Japanese eel
spermatozoa. Journal of Experimental Zoology 277, 171–180.
https://doi.org/10.1002/(SICI)1097-010X(19970201)277:2<171::AID-
JEZ9>3.0.CO;2-M
Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., Iinuma, N., Hirose, K., 1997. Artificial
induction of maturation and fertilization in the Japanese eel, Anguilla japonica.
Fish Physiology and Biochemistry 17, 163–169.
https://doi.org/10.1023/A:1007720600588
248
Ohta, T., 1991. Initial stages of sperm-egg fusion in the freshwater teleost, Rhodeus
ocellatus ocellatus. The Anatomical Record 229, 195–202.
https://doi.org/10.1002/ar.1092290206
Ohta, T., Iwamatsu, T., 1983. Electron microscopic observations on sperm entry into
eggs of the rose bitterling, Rhodeus ocellatus. Journal of Experimental Zoology
227, 109–119. https://doi.org/10.1002/jez.1402270115
Park, D.S., Egnatchik, R.A., Bordelon, H., Tiersch, T.R., Monroe, W.T., 2012.
Microfluidic mixing for sperm activation and motility analysis of pearl Danio
zebrafish. Theriogenology 78, 334–344.
https://doi.org/10.1016/j.theriogenology.2012.02.008
Parks, J.E., Graham, J.K., 1992. Effects of cryopreservation procedures on sperm
membranes. Theriogenology 38, 209–222. https://doi.org/10.1016/0093-
691X(92)90231-F
Parks, J.E., Hammerstedt, R.H., 1985. Developmental Changes Occurring in the Lipids of
Ram Epididymal Spermatozoa Plasma Membrane. Biol Reprod 32, 653–668.
https://doi.org/10.1095/biolreprod32.3.653
Pelaez, J., Bongalhardo, D.C., Long, J.A., 2011. Characterizing the glycocalyx of poultry
spermatozoa: III. Semen cryopreservation methods alter the carbohydrate
component of rooster sperm membrane glycoconjugates. Poultry Science 90,
435–443. https://doi.org/10.3382/ps.2010-00998
249
Peña, L.A., Fuks, Z., Koksnick, R., 1997. Stress-induced apoptosis and the
sphingomyelin pathway. Biochemical Pharmacology 53, 615–621.
https://doi.org/10.1016/S0006-2952(96)00834-9
Pereira, J.R., Pereira, F.A., Perry, C.T., Pires, D.M., Muelbert, J.R.E., Garcia, J.R.E.,
Corcini, C.D., Varela Junior, A.S., 2019. Dimethylsulfoxide, methanol and
methylglycol in the seminal cryopreservation of Suruvi, Steindachneridion
scriptum. Animal Reproduction Science 200, 7–13.
https://doi.org/10.1016/j.anireprosci.2018.09.024
Pérez, L., Vílchez, M.C., Gallego, V., Morini, M., Peñaranda, D.S., Asturiano, J.F., 2016.
Role of calcium on the initiation of sperm motility in the European eel.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative
Physiology 191, 98–106. https://doi.org/10.1016/j.cbpa.2015.10.009
Perry, C.T., Corcini, C.D., Anciuti, A.N., Otte, M.V., Soares, S.L., Garcia, J.R.E.,
Muelbet, J.R.E., Varela, A.S., 2019. Amides as cryoprotectants for the freezing of
Brycon orbignyanus sperm. Aquaculture 508, 90–97.
https://doi.org/10.1016/j.aquaculture.2019.03.015
Pini, T., Leahy, T., de Graaf, S.P., 2018a. Sublethal sperm freezing damage:
Manifestations and solutions. Theriogenology 118, 172–181.
https://doi.org/10.1016/j.theriogenology.2018.06.006
Pini, T., Leahy, T., Graaf, S.P. de, 2018b. Seminal plasma and cryopreservation alter ram
sperm surface carbohydrates and interactions with neutrophils. Reprod. Fertil.
Dev. 30, 689–702. https://doi.org/10.1071/RD17251
250
Pini, T., Leahy, T., Graaf, S.P. de, 2018c. Seminal plasma and cryopreservation alter ram
sperm surface carbohydrates and interactions with neutrophils. Reprod. Fertil.
Dev. 30, 689–702. https://doi.org/10.1071/RD17251
Purdy, P.H., Barbosa, E.A., Praamsma, C.J., Schisler, G.J., 2016. Modification of trout
sperm membranes associated with activation and cryopreservation. Implications
for fertilizing potential. Cryobiology 73, 73–79.
https://doi.org/10.1016/j.cryobiol.2016.05.008
Pustowka, C., McNiven, M.A., Richardson, G.F., Lall, S.P., 2000. Source of dietary lipid
affects sperm plasma membrane integrity and fertility in Rainbow Trout
Oncorhynchus mykiss (Walbaum) after cryopreservation. Aquaculture Research
31, 297–305. https://doi.org/10.1046/j.1365-2109.2000.00416.x
Quist, M.C., Stephen, J.L., Lynott, S.T., Goeckler, J.M., Schultz, R.D., 2010. An
Evaluation of Angler Harvest of Walleye and Saugeye in a Kansas Reservoir.
Journal of Freshwater Ecology 25, 1–7.
https://doi.org/10.1080/02705060.2010.9664351
R Development Core Team, 2019. R: A language and environment for statistical
computing. R Foundation for Statistical Computing, Vienna, Austria. URL
http://R-project.org.
R Development Core Team, 2009. R: A language and environment for statistical
computing.
251
Ravi, R., Manisseri, M.K., Sanil, N.K., 2014. Structure of the male reproductive system
of the blue swimmer crab Portunus pelagicus (Decapoda: Portunidae). Acta
Zoologica 95, 176–185. https://doi.org/10.1111/azo.12017
Reis, A., Rudnitskaya, A., Blackburn, G.J., Mohd Fauzi, N., Pitt, A.R., Spickett, C.M.,
2013. A comparison of five lipid extraction solvent systems for lipidomic studies
of human LDL. J. Lipid Res. 54, 1812–1824. https://doi.org/10.1194/jlr.M034330
Rejraji, H., Sion, B., Prensier, G., Carreras, M., Motta, C., Frenoux, J.-M., Vericel, E.,
Grizard, G., Vernet, P., Drevet, J.R., 2006. Lipid Remodeling of Murine
Epididymosomes and Spermatozoa During Epididymal Maturation. Biol Reprod
74, 1104–1113. https://doi.org/10.1095/biolreprod.105.049304
Rennemeier, C., Frambach, T., Hennicke, F., Dietl, J., Staib, P., 2009. Microbial
Quorum-Sensing Molecules Induce Acrosome Loss and Cell Death in Human
Spermatozoa. Infection and Immunity 77, 4990–4997.
https://doi.org/10.1128/IAI.00586-09
Rojas, M.V., Esponda, P., 2001a. Plasma membrane glycoproteins during
spermatogenesis and in spermatozoa of some fishes. Journal of submicroscopic
cytology and pathology 33, 133–140.
Rojas, M.V., Esponda, P., 2001b. Plasma membrane glycoproteins during
spermatogenesis and in spermatozoa of some fishes. Journal of submicroscopic
cytology and pathology 33, 133–140.
252
Rurangwa, E., Kime, D.E., Ollevier, F., Nash, J.P., 2004. The measurement of sperm
motility and factors affecting sperm quality in cultured fish. Aquaculture 234, 1–
28.
Sakaguchi, Y., Iwata, H., Kuwayama, T., Monji, Y., 2009. Effect of N-acetyl-D-
glucosamine on bovine sperm-oocyte interactions. J. Reprod. Dev. 55, 676–684.
Sandoval‐Vargas, L., Jiménez, M.S., González, J.R., Villalobos, E.F., Cabrita, E., Isler,
I.V., 2020. Oxidative stress and use of antioxidants in fish semen
cryopreservation. Reviews in Aquaculture n/a, 1–23.
https://doi.org/10.1111/raq.12479
Schiller, J., Arnhold, J., Glander, H.-J., Arnold, K., 2000. Lipid analysis of human
spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR
spectroscopy — effects of freezing and thawing. Chemistry and Physics of Lipids
106, 145–156. https://doi.org/10.1016/S0009-3084(00)00148-1
Schröter, S., Osterhoff, C., McArdle, W., Ivell, R., 1999a. The glycocalyx of the sperm
surface. Hum Reprod Update 5, 302–313. https://doi.org/10.1093/humupd/5.4.302
Schröter, S., Osterhoff, C., McArdle, W., Ivell, R., 1999b. The glycocalyx of the sperm
surface. Hum Reprod Update 5, 302–313. https://doi.org/10.1093/humupd/5.4.302
Schulz, R.W., de França, L.R., Lareyre, J.-J., LeGac, F., Chiarini-Garcia, H., Nobrega,
R.H., Miura, T., 2010. Spermatogenesis in fish. General and Comparative
Endocrinology, Fish Reproduction 165, 390–411.
https://doi.org/10.1016/j.ygcen.2009.02.013
253
Scott, T.W., Wales, R.G., Wallace, J.C., White, I.G., 1963. COMPOSITION OF RAM
EPIDIDYMAL AND TESTICULAR FLUID AND THE BIOSYNTHESIS OF
GLYCERYLPHOSPHORYLCHOLINE BY THE RABBIT EPIDIDYMIS.
Reproduction 6, 49–59. https://doi.org/10.1530/jrf.0.0060049
Seifert, R., Flick, M., Bönigk, W., Alvarez, L., Trötschel, C., Poetsch, A., Müller, A.,
Goodwin, N., Pelzer, P., Kashikar, N.D., Kremmer, E., Jikeli, J., Timmermann,
B., Kuhl, H., Fridman, D., Windler, F., Kaupp, U.B., Strünker, T., 2015. The
CatSper channel controls chemosensation in sea urchin sperm. The EMBO
Journal 34, 379–392. https://doi.org/10.15252/embj.201489376
Shadan, S., James, P.S., Howes, E.A., Jones, R., 2004a. Cholesterol Efflux Alters Lipid
Raft Stability and Distribution During Capacitation of Boar Spermatozoa. Biol
Reprod 71, 253–265. https://doi.org/10.1095/biolreprod.103.026435
Shadan, S., James, P.S., Howes, E.A., Jones, R., 2004b. Cholesterol Efflux Alters Lipid
Raft Stability and Distribution During Capacitation of Boar Spermatozoa. Biol
Reprod 71, 253–265. https://doi.org/10.1095/biolreprod.103.026435
Sleight, S.B., Miranda, P.V., Plaskett, N.-W., Maier, B., Lysiak, J., Scrable, H., Herr,
J.C., Visconti, P.E., 2005. Isolation and Proteomic Analysis of Mouse Sperm
Detergent-Resistant Membrane Fractions: Evidence for Dissociation of Lipid
Rafts During Capacitation. Biol Reprod 73, 721–729.
https://doi.org/10.1095/biolreprod.105.041533
254
Sloman, K.A., 2011. The Diversity of Fish Reproduction: An Introduction, Encyclopedia
of Fish Physiology: From Genome to Environment, Vols 1-3. Elsevier Academic
Press Inc, San Diego.
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano,
M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985a.
Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150,
76–85. https://doi.org/10.1016/0003-2697(85)90442-7
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano,
M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985b.
Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150,
76–85. https://doi.org/10.1016/0003-2697(85)90442-7
Sonnino, S., Prinetti, A., 2013. Membrane Domains and the “Lipid Raft” Concept.
Current Medicinal Chemistry 20, 4–21.
Souza, L.C.M.D., Retamal, C.A., Rocha, G.M., Lopez, M.L., 2015. Morphological
evidence for a permeability barrier in the testis and spermatic duct of Gymnotus
carapo (Teleostei: Gymnotidae). Molecular Reproduction and Development 82,
663–678. https://doi.org/10.1002/mrd.22503
Taatjes, D.J., Sobel, B.E., Budd, R.C., 2008. Morphological and cytochemical
determination of cell death by apoptosis. Histochemistry and cell biology 129,
33–43.
255
Takei, G.L., Mukai, C., Okuno, M., 2012. Transient Ca2+ mobilization caused by
osmotic shock initiates salmonid fish sperm motility. Journal of Experimental
Biology 215, 630–641. https://doi.org/10.1242/jeb.063628
Tapia, J.A., Macias‐Garcia, B., Miro‐Moran, A., Ortega‐Ferrusola, C., Salido, G.M.,
Peña, F.J., Aparicio, I.M., 2018. The Membrane of the Mammalian Spermatozoa:
Much More Than an Inert Envelope. Reproduction in Domestic Animals 47, 65–
75. https://doi.org/10.1111/j.1439-0531.2012.02046.x
Tecle, E., Gagneux, P., 2015a. Sugar‐coated sperm: Unraveling the functions of the
mammalian sperm glycocalyx. Mol Reprod Dev 82, 635–650.
https://doi.org/10.1002/mrd.22500
Tecle, E., Gagneux, P., 2015b. Sugar‐coated sperm: Unraveling the functions of the
mammalian sperm glycocalyx. Mol Reprod Dev 82, 635–650.
https://doi.org/10.1002/mrd.22500
Thaler, C.D., Thomas, M., Ramalie, J.R., 2006. Reorganization of mouse sperm lipid
rafts by capacitation. Molecular Reproduction and Development 73, 1541–1549.
https://doi.org/10.1002/mrd.20540
Thomas, A.D., Meyers, S.A., Ball, B.A., 2006. Capacitation-like changes in equine
spermatozoa following cryopreservation. Theriogenology 65, 1531–1550.
https://doi.org/10.1016/j.theriogenology.2005.08.022
Tiersch, T.R., 2011. Process Pathways for Cryopreservation Research, Application and
Commercialization., in: Cryopreservation in Aquatic Species, 2nd Edition. World
Aquaculture Society, Baton Rouge, Louisiana, pp. 646–671.
256
Tiersch, T.R., Green, C.C., 2011. Cryopreservation in aquatic species, 2nd ed. World
aquaculture society Baton Rouge, LA.
Torres, L., Hu, E., Tiersch, T.R., 2016. Cryopreservation in fish: current status and
pathways to quality assurance and quality control in repository development.
Reproduction, Fertility and Development 28, 1105.
https://doi.org/10.1071/RD15388
Tsai, P.-S., Tsai, P.-S., Vries, K.J.D., Tsai, P.-S., Vries, K.J.D., Boer-Brouwer, M.D.,
Garcia-Gil, N., Gestel, R.A.V., Tsai, P.-S., Vries, K.J.D., Boer-Brouwer, M.D.,
Garcia-Gil, N., Gestel, R.A.V., Colenbrander, B., Gadella, B.M., Haeften, T.V.,
2007. Syntaxin and VAMP association with lipid rafts depends on cholesterol
depletion in capacitating sperm cells. Molecular Membrane Biology 24, 313–324.
https://doi.org/10.1080/09687860701228692
Ushiyama, A., Ishikawa, N., Tajima, A., Asano, A., 2016. Comparison of Membrane
Characteristics between Freshly Ejaculated and Cryopreserved Sperm in the
Chicken. The Journal of Poultry Science advpub.
https://doi.org/10.2141/jpsa.0160043
Ushiyama, A., Tajima, A., Ishikawa, N., Asano, A., 2017. Characterization of the
functions and proteomes associated with membrane rafts in chicken sperm. PLOS
ONE 12, e0186482. https://doi.org/10.1371/journal.pone.0186482 van Gestel, R.A., Brewis, I.A., Ashton, P.R., Helms, J.B., Brouwers, J.F., Gadella, B.M.,
2005a. Capacitation-dependent concentration of lipid rafts in the apical ridge head
257
area of porcine sperm cells. Mol Hum Reprod 11, 583–590.
https://doi.org/10.1093/molehr/gah200 van Gestel, R.A., Brewis, I.A., Ashton, P.R., Helms, J.B., Brouwers, J.F., Gadella, B.M.,
2005b. Capacitation-dependent concentration of lipid rafts in the apical ridge head
area of porcine sperm cells. Mol Hum Reprod 11, 583–590.
https://doi.org/10.1093/molehr/gah200
Walter, I., Tschulenk, W., Schabuss, M., Miller, I., Grillitsch, B., 2005. Structure of the
seminal pathway in the European chub, Leuciscus cephalus (Cyprinidae);
Teleostei. Journal of Morphology 263, 375–391.
https://doi.org/10.1002/jmor.10312
Watson, P.F., 2000. The causes of reduced fertility with cryopreserved semen. Animal
Reproduction Science 60–61, 481–492. https://doi.org/10.1016/S0378-
4320(00)00099-3
Williamson, P., Mattocks, K., Schlegel, R.A., 1983. Merocyanine 540, a fluorescent
probe sensitive to lipid packing. Biochim. Biophys. Acta 732, 387–393.
https://doi.org/10.1016/0005-2736(83)90055-x
Xin, A., Wu, Y., Lu, H., Cheng, L., Gu, Y., Diao, H., Chen, G., Wu, B., Li, Z., Tao, S.,
Sun, X., Shi, H., 2018. Comparative analysis of human sperm glycocalyx from
different freezability ejaculates by lectin microarray and identification of ABA as
sperm freezability biomarker. Clin Proteomics 15. https://doi.org/10.1186/s12014-
018-9195-z
258
Xin, M., Niksirat, H., Shaliutina‐Kolešová, A., Siddique, M.A.M., Sterba, J.,
Boryshpolets, S., Linhart, O., 2019. Molecular and subcellular cryoinjury of fish
spermatozoa and approaches to improve cryopreservation. Reviews in
Aquaculture n/a. https://doi.org/10.1111/raq.12355
Yanagimachi, R., Cherr, G., Matsubara, T., Andoh, T., Harumi, T., Vines, C., Pillai, M.,
Griffin, F., Matsubara, H., Weatherby, T., Kaneshiro, K., 2013. Sperm Attractant
in the Micropyle Region of Fish and Insect Eggs. Biol Reprod 88.
https://doi.org/10.1095/biolreprod.112.105072
Yanagimachi, R., Harumi, T., Matsubara, H., Yan, W., Yuan, S., Hirohashi, N., Iida, T.,
Yamaha, E., Arai, K., Matsubara, T., Andoh, T., Vines, C., Cherr, G.N., 2017a.
Chemical and physical guidance of fish spermatozoa into the egg through the
micropyle,. Biol Reprod 96, 780–799. https://doi.org/10.1093/biolre/iox015
Yanagimachi, R., Harumi, T., Matsubara, H., Yan, W., Yuan, S., Hirohashi, N., Iida, T.,
Yamaha, E., Arai, K., Matsubara, T., Andoh, T., Vines, C., Cherr, G.N., 2017b.
Chemical and physical guidance of fish spermatozoa into the egg through the
micropyle,. Biol Reprod 96, 780–799. https://doi.org/10.1093/biolre/iox015
Yu, S., Kojima, N., Hakomori, S., Kudo, S., Inoue, S., Inoue, Y., 2002a. Binding of
Rainbow Trout sperm to egg is mediated by strong carbohydrate-to-carbohydrate
interaction between (KDN)GM3 (deaminated neuraminyl ganglioside) and Gg3-
like epitope. Proc. Natl. Acad. Sci. U.S.A. 99, 2854–2859.
https://doi.org/10.1073/pnas.052707599
259
Yu, S., Kojima, N., Hakomori, S., Kudo, S., Inoue, S., Inoue, Y., 2002b. Binding of
Rainbow Trout sperm to egg is mediated by strong carbohydrate-to-carbohydrate
interaction between (KDN)GM3 (deaminated neuraminyl ganglioside) and Gg3-
like epitope. Proc. Natl. Acad. Sci. U.S.A. 99, 2854–2859.
https://doi.org/10.1073/pnas.052707599
Zehmer, J.K., Hazel, J.R., 2004. Membrane order conservation in raft and non-raft
regions of hepatocyte plasma membranes from thermally acclimated Rainbow
Trout . Biochimica et Biophysica Acta (BBA) - Biomembranes 1664, 108–116.
https://doi.org/10.1016/j.bbamem.2004.04.008
Zehmer, J.K., Hazel, J.R., 2003. Plasma membrane rafts of Rainbow Trout are subject to
thermal acclimation. Journal of Experimental Biology 206, 1657–1667.
https://doi.org/10.1242/jeb.00346
Zilli, L., Schiavone, R., Storelli, C., Vilella, S., 2008. Effect of cryopreservation on
phosphorylation state of proteins involved in sperm motility initiation in sea
bream. Cryobiology 57, 150–155. https://doi.org/10.1016/j.cryobiol.2008.07.006
Zilli, L., Schiavone, R., Vilella, S., 2017. Role of protein
phosphorylation/dephosphorylation in fish sperm motility activation: State of the
art and perspectives. Aquaculture, Recent advances in fish gametes and embryo
research 472, 73–80. https://doi.org/10.1016/j.aquaculture.2016.03.043
260
Appendix A. Testicular collections as a technique to increase milt availability in
Sauger (Sander canadensis)1
Abstract
This study was conducted to compare quality and quantity of sperm collected from Sauger (S. canadensis) using two collection methods: stripping alone and testicular tissue collection combined with stripping. Sperm were collected from Sauger broodstock
(n = 20) during the breeding season. Fish were randomly assigned to two sperm collection groups: (1) stripping once or (2) stripping twice before testicular tissue collection for obtaining additional sperm. Sperm motility variables, morphology, total number produced, and fertilization (%) were compared using the two collection methods.
Testicular sperm had greater total motility (70.1 ± 2.1% compared with 44.3 ± 5.7%) but there were fewer morphologically normal cells (76.4 ± 1.3% compared with 92.8 ± 1.0%) compared to sperm collected using the stripping procedure. Sperm collection regimen utilizing testicular collections and sperm extractions in combination with stripping
1 Blawut B, Wolfe B, Moraes CR, Sweet D, Ludsin SA, Coutinho da Silva MA. Testicular collections as a technique to increase milt availability in Sauger (Sander canadensis). Animal Reproduction Science 2020;212:106240. https://doi.org/10.1016/j.anireprosci.2019.106240.
261 resulted in a ~tenfold increase in total number of motile and morphologically normal sperm (39.5 ± 4.1 x 10 9) compared with the currently utilized two sequential sperm stripping collection procedures alone (3.6 ± 4.1 x 10 9 sperm). In large-scale studies
(150,000 eggs), fertilization, using sperm collected from testicular tissues (1.0 x 105 motile sperm/egg), was similar to sperm collected with only the stripping procedure (71.2
± 5.5 %, 81.2 ± 5.5 %, P = 0.265). The results of this study indicate testicular collection combined with sperm extractions allows for collection of sperm of a quantity and quality to maximize fry production and reduce the problems with lack of broodstock availability for sperm collection.
Introduction
The lack of success in production of Saugeye, a hybrid cross between a female
Walleye (S. vitreus) and a male Sauger (S. canadensis), exemplifies some of the difficulties of using traditional sperm collection techniques in fish propagation programs.
Annual Saugeye production is necessary for sustaining the multimillion-dollar recreational fishery sector in Ohio reservoirs. This production is highly variable due to the relatively greater winter and spring precipitation patterns that prevent the collection of sufficient Sauger broodstock to meet production demand for sperm. Additionally, Sauger produce a small volume of milt (< 0.5 mL), thus a small number of sperm are obtained when collection procedures are used, which further limits the supply of sperm during the production of Saugeye hybrids. To compensate for these limitations, the personnel of the
Ohio Department of Natural Resources, Division of Wildlife, with oversight of Saugeye
262 stocking in Ohio, collect sperm using the stripping procedure from each male Sauger broodstock two to three times during the course of the breeding season. While a previous study has attempted to maximize efficiency of sperm use during fertilization, attempts to increase the amount of sperm collected from each individual using hormonal treatments were unsuccessful (Blawut et al., 2018). To mitigate the effects of the male and the lack of an adequate supply of sperm, alternative milt collection techniques to increase the quantity and quality of sperm collected from each Sauger should be investigated.
While collection of milt using the stripping procedure can result in a sufficient quality and quantity of sperm for many fish species [e.g., trout (Hajirezaee et al., 2009)], testicular sperm collection utilizing euthanization and testicular tissue collection is a viable alternative for species where sperm are in short supply and/or of less-than- desirable quality (Rurangwa et al., 2004; Kowalski et al., 2006). Importantly, testicular collection is superior to sperm collection using the stripping procedure in a large number of commercially valuable species [common snook (Centropomus undecimalis), African catfish (Clarias gariepinus), Japanese eel (Anguilla japonica), freshwater gobies
(Rhinogobius sp), captive Red Snapper (Lutjanus cempechanus)] (Ohta et al., 1997;
Viveiros et al., 2002; Tiersch et al., 2004; Yokoi et al., 2008; Bardon-Albaret et al.,
2015). The results from these previous studies indicate that a large number of spermatozoa can be obtained from the testis that are not procured using the stripping procedure for sperm collection. Testicular tissue harvest may not be a sustainable procedure, however, because the quality of sperm collected using this procedure may be less because some gametes may not be mature at the time of collection as compared with
263 sperm collected using the stripping procedure (Mansour et al., 2004). In many cases, sperm collected directly from the testicles have a lesser motility and there is a lesser fertility when these sperm are used to fertilize eggs because the larger number of immature cells and mechanical damage to the sperm that is associated with use of the technique of direct sperm collection from the testis (Alavi et al., 2008). For Sauger broodstock, it is unknown whether the collection of sperm directly from testicular tissues after tissue collection is an effective method for increasing the quantity of viable sperm that could be effectively used for fertilizing eggs as compared with collection of sperm using the stripping procedure.
Only a small number of techniques are used to collect sperm from other closely related Sander and Perca species. Milt is typically collected from Sander sp. using either the stripping procedure [Walleye, Sander vitreus (Moore, 1987; Rinchard et al., 2005;
Casselman et al., 2006)] or using a catheter [pikeperch Sander luciperca (Sarosiek et al.,
2016)]. Sperm collection using the stripping procedure is also used in other percids such as the yellow perch [Perca flavescens,(Ciereszko and Dabrowski, 1993)] while catheterization was found to be superior for sperm collection in Eurasian perch [(Perca fluviatiles (Żarski et al., 2017)]. While the use of the catheterization procedure resulted in an increase in values for sperm quality variables compared to when there was use of the stripping procedure for sperm collection in pikeperch and Eurasian perch (Sarosiek et al.,
2016; Król et al., 2018), the results were considered to confounded by avoiding urine contamination which can have detrimental effects on sperm of these species. Testicular tissue was used for sperm collection in Perca fluviatilis, however, few details were
264 provided comparing sperm viability variables for sperm collected from testicular tissues and those procured using the stripping procedure in terms of total milt yield (Alavi et al.,
2008). Testicular collections may provide a method to enhance the amount of sperm collected per individual while also avoiding urine contamination.
To evaluate the potential of using testicular collections to help personnel of agencies overcome current limitations in Saugeye production, a controlled laboratory approach was used. To determine the effect of different collection methods, sperm quantity and quality was compared when sperm were collected using the stripping procedure and testicular tissue procurement followed with use of sperm extraction techniques. The hypothesis was that sperm procured using testicular collection and sperm extraction methods would be of greater quantity, but have lesser quality than sperm collected using conventional stripping procedures.
Materials and methods
Broodstock collection and care
The ODNR-DOW personnel collected mature, male Sauger from the Ohio River near the Greenup, KY, USA dam via electrofishing during late February to mid-March.
Sauger broodstock were transported by truck for ~2 hours in an aerated live-well 0.5%
NaCl solution to the London State Fish Hatchery isolation facility (London, OH, USA) where the specimens were maintained in a ~2840 L indoor recirculating system at 5 to 6
°C with a flow rate of 45 to 57 L/hour. Fish were maintained in tanks with natural photoperiod exposure. The Ohio State University (Columbus, OH, USA) Institutional
265
Animal Care and Use Committee approved all procedures and animal use prior to the beginning of the study (Protocol #2015A00000008).
Study design
Milt from twenty male Sauger was collected using both the stripping and testicular tissue-sperm extraction collection procedures for sperm collection to compare the quantity and quality of sperm. Fish were assigned to two groups: Group 1) Sperm were collected using the stripping procedure once followed by testicular tissue collection- sperm extraction 5 d later, and Group 2) Sperm were collected using the stripping procedure twice followed by testicular tissue collections-sperm extractions, each at 5 d intervals. Sperm of each milt sample were assessed for: motility, cell concentration of milt, total sperm number, and cell morphology. These metrics were compared between samples collected using the sequential strip-spawning procedures in the same fish (n =
10), and all the collection information for each of strip spawning (n = 30) and testicular collections (n = 17) were pooled by sperm type and compared between the two collection types. Procedures used were the current sperm collection regimen (twice using stripping procedure only, n = 10) that was compared with use of the sperm stripping procedure once followed by testicular tissue collection and sperm extraction from the tissue (Group
1, n = 10) and strip spawning twice followed by testicular collections and sperm extractions (Group 2, n = 7, three were euthanized before the end of the study). There were comparisons among the procedures in terms of the total number of sperm cells, motile sperm cells, morphologically normal sperm cells, and total number of motile cells
266 collected using each procedure. Additional male Sauger were collected using both techniques later and pooled to assess fertilization potential in small batches (~1,000, n = 4
-5) and hatchery-relevant large batches (~150,000, n = 3) of Walleye eggs, respectively.
Milt collection
Male Sauger used in this study were approximately 313.9 ± 5.0 mm in total length and 233.5 ± 14.6 g in mass (mean ± 1 SD). At the beginning of the study, sperm were collected from all 20 fish using the stripping procedure. No hormonal treatments were used to induce spermiation. Unanaesthetized individuals were placed in dorsal recumbancy, and the urogenital pore was thoroughly dried to prevent premature milt release. Milt was collected using abdominal massage and a 1.0 mL rubber-less syringe that was placed at the opening of the urogenital pore. Milt samples contaminated with urine or feces were discarded. Immediately after collection, the total volume of milt produced was determined to the nearest 0.01 mL and milt was immediately extended with
350 mOsm/kg Rathbun extender (1:2, (g/l): CaCl2·2H2O, 0.117; MgCl2·6H2O, 0.134;
Na2HPO4, 0.236; KCl, 1.872; NaCl, 6.578; glucose, 10.0; citric acid, 0.10; NaOH, 0.254; and bicine, 1.06; pH 8.5 (Moore, 1987)). Milt samples were maintained at 5 °C until further analysis (< 1.5 h). All individuals were marked using an intramuscular passive integrated transponder (PIT) tag (Biomark, Boise, Idaho, USA) with attachment to the first dorsal fin for future identification before returning the specimens to the holding tank.
Milt samples were transported at 5 C to the OSU Theriogenology laboratory (Columbus,
OH, USA) in a sealed styrofoam box.
267
After the initial milt collection using the stripping procedure, sperm were collected from the same fish 5 d later using two different techniques: with use of the stripping procedure and testicular collection-extraction procedure. Broodstock were randomly assigned to one of two groups: Group 1) Milt collection using the stripping procedure a second time or Group 2) Euthanization, testicular tissue collection, and sperm extraction from the testis. Randomly selected individuals from Group 2 were euthanized with an overdose of tricaine sulfate (MS222, Western Chemical, Ferndale, WA, USA) at a dose of ~ 1 g/L. Milt was collected using the stripping procedure from the remaining ten individuals (Group 1) which were later euthanized 5 days after the time of the second collection by stripping (10 d total after initial collection) and for testicular tissue collection-sperm extraction. The carcasses of euthanized specimens were stored and transported to the Ohio State Theriogenology Laboratory on crushed ice (< 3 h). In the laboratory, the testes were removed from the euthanized fish body cavity and extraneous fluids and debris were discarded. Testicular weight and length were recorded to the nearest 0.001 g and 0.01 mm, respectfully (XS105 Dual Range, Mettler Toledo,
Columbus, OH, USA). There was an incision of each lobe of the testis along its length with a scalpel and there was hand pressure applied from the urogenital pore to the tip of each incised lobe to extract testicular sperm. Seminal pH was measured using test strips
(Fisher Scientific, Pittsburgh, PA, USA) and then each sample was diluted with 3.0 mL of Rathbun extender followed by gentle homogenization of sperm in this solution and subsequent transfer to a test tube occurred for storage at 5 °C.
268
CASA motility, viability, and concentration assessment
Sperm quality (motility characteristics and cell viability) was assessed immediately upon returning to the laboratory from the hatchery (< 2 h). Each sample was diluted with
Rathbun extender (1:50) and the sperm in 2 µL of diluted milt was activated in 18 µL of distilled water containing 1% bovine serum albumin (BSA). Sperm motility was assessed using a Ceros II computer assisted sperm analysis (CASA) system (Hamilton Thorne,
Beverly, MA, USA) with approximately 12 seconds of activation occurring in the sperm contained in 2 µL sample of milt with activation occurring in a Cytonix microchamber
(20 µm depth, Cytonix LLC, Beltsville, MD, USA). Images were obtained at a rate of 60
Hz for 30 frames. Progressively motile sperm were considered to have an average path velocity (VAP) of >50 µm/s and a straightness of >80.0%. A minimum VAP of 20 µm/s was used as a criterion for motile cells to reduce the effects of drift (Park et al., 2012).
Other CASA settings included: exposure = 80 ms; gain = 300; minimum head brightness
= 152; min head size = 1 µm2; max head size = 19 µm2; capillary correction = 1.3; max photometer = 70; min photometer = 60; and minimum total cell count = 200.
Sperm plasma membrane integrity (i.e., viability) was assessed using the Live/Dead®
Sperm Viability kit according to manufacturer instructions (Molecular Probes, Eugene,
OR, USA). Briefly, milt aliquots were diluted to 1.0 mL using Rathbun extender (1:15, v: v), supplemented with 10% BSA and 10 mM HEPES, and then there was addition of 2
µL (0.2 µM) and 5 µL (1.2 µM) of SYBR 14 and propidium iodide (PI) dye, respectively.
Samples were incubated at 5 °C for 10 min and there was subsequent assessment of the sperm using an Accu-Scope 3025 (Accu-Scope Commack, NY, USA) fluorescent
269 microscope at × 1,000. Sperm (n = 100) from each sample were evaluated to determine the percentage of cells with intact plasma membranes (SYBR positive, PI negative).
To assess sperm concentration in each sample, cells were manually counted in diluted sperm samples using a hemocytometer (Fisher Scientific, Hampton, NH, USA, Cat
#0267110). Diluted testicular sperm (20 µl) were preserved by applying a 40-fold dilution in a 3% glutaraldehyde solution. Sperm concentration was determined manually, using a previously reported method (Blawut et al., 2018), with a hemocytometer and the total number of sperm collected using both the stripping and testicular dissection-sperm extraction procedures was determined based on the hemocytometer estimate (sperm/mL) and sample volume (mL).
Spermatozoa morphology
Spermatozoa structure was determined by assessing morphology of sperm cells in each sample using oil immersion microscopy. Sperm in 20 µl of each milt sample were preserved in 980 µL of 3% glutaraldehyde solution. Sperm (n = 100) from each sample were analyzed at a 1000 × magnification (Accu-Scope 3025, Commack, NY, USA) using oil emersion to determine the percentage of sperm with normal morphology.
Spermatozoa abnormalities included: detached heads, broken tails, distal and proximal reflexes, macrocephaly, coiled tails and cytoplasmic droplets.
270
Fertilization
During the breeding season of 2018, milt was collected from 20 additional male
Sauger using stripping procedure for collection and testicular tissues were collected to compare fertilization capacity using previously described methods (Blawut et al., 2018).
Samples collected using the same method were pooled (two-or three specimens per pool, n = four pools for stripped and n = five pools for testicular tissue) and stored at 5 ºC overnight. Walleye (Sander vitreus) eggs for fertilization were obtained from mature specimens captured from their natural habitat in the Berlin Reservoir (Deerfield, OH,
USA) in the spawning area of the reservoir using trap nets set by ODNR-DOW staff.
Oocytes were collected from unanesthetized Walleye using abdominal massage, after which females were returned to the reservoir. Oocytes contaminated with water, urine, feces, or blood were discarded. Approximately 1,500 eggs (~ 4.2 g) from two or three females were fertilized using either sperm collected using the stripping procedure at
300,000 motile sperm/egg or testicular sperm at 300,000 and 600,000 motile sperm/egg
(MSE) collected from males (n = 14) and pooled the day prior to fertilization and storage at 4 °C overnight. Total motility of sperm in pools collected using the stripping and testicular collection-sperm extraction procedures used in this experiment were 74.5 ±
11.3% and 63.6 ± 3.1% (mean ± SD), respectively. Fertilized eggs were then treated with a 400-ppm tannic acid solution for 3 min, and a 50-ppm ovadine (Western Chemical, OR,
USA) prior to transport to the London Isolation Facility (London, OH, USA).
Immediately after the eggs were transferred into the isolation facility, eggs were disinfected with an additional 10 min, 100-ppm ovadine solution before there was
271 incubation of eggs in each batch in a 7.62 cm diameter PVC disc (2.5 cm depth) sealed on both ends with 200 μm nylon mesh held in place by a 10.16 cm stainless steel clamp.
Discs were incubated at 12 °C with a 22.7 liters/minute flow rate until embryos developed eyes at 11 d post fertilization with this embryo phenotype being indicative of subsequent hatching success (Latif et al., 1999). Oocytes (n = 100) of each replicate were collected, preserved (in 3% glutaraldehyde), and analyzed to determine the percentage of viable embryos using a dissection scope (Rocky Mountain Microscope Company, Ft.
Collins, CO, USA).
During the 2019 breeding season, there were collections of sperm using the testicular collection-sperm extraction and specimen stripping procedures from nine male
Sauger to compare fertilization capacity of sperm at a lesser, more practical sperm-to-egg ratio (100,000 MSE) using hatchery-relevant quantities of eggs (~150,000 eggs).
Percentage sperm motility was approximately 61.7 ± 2.9% and 37.8 ± 26.2% (mean ±
SD) for sperm collected using the stripping and testicular collection-sperm extraction procedures, respectively. A total of 150,000 eggs (~ 617 g) were combined from two or three females in a large plastic bowl and immediately fertilized using sperm collected using the stripping procedure as a positive control for egg quality at 300,000 MSE or sperm collected using the testicular collection-sperm extraction procedure at 100,000
MSE. Fertilized oocytes were treated as previously described in this manuscript. Each replicate was placed in a plastic bag, filled with compressed oxygen, and sealed prior to a
2 hr period of transport to the Hebron State Fish Hatchery (Hebron, OH, USA). After transfer of eggs into the hatchery, eggs were subjected to a second ovadine treatment
272 using procedures described previously in this manuscript and were subsequently incubated in McDonald style hatching jars in a well-water fed flow-through system at 12
°C. The proportion of eyed eggs per sample was assessed using procedures that were described previously in this manuscript at 9 d of incubation (eyed stage).
Statistical analysis
All statistical analyses were completed using R (R Development Core Team,
2009) Alpha was set at 0.05. Normality and homogeneity of variance of each predictor and dependent variable was confirmed using normal quantile and residual plots, respectively. Sperm quality [total motility (%), progressive motility (%), sperm velocity
(µm/s), and cell viability (%)] and quantity [total sperm produced, sperm concentration, and milt volume (mL)] differences between first and second milt collections using the stripping procedure within the same individual were compared using a repeated measures
ANOVA and then the data from all milt collections using the stripping procedure and all testicular harvest-sperm extraction procedures for collections were pooled by sperm type and compared using an independent one-way ANOVA. Milt yield using the three collection regimens [1) Stripping twice (the current ODNR-DOW protocol) compared with 2) Stripping once plus testicular collection-sperm extraction compared with 3)
Stripping twice plus testicular collection-sperm extraction] in terms of total sperm, total motile sperm, and total motile, morphologically normal sperm was compared using independent one-way ANOVAs. Correlations were used to determine the nature of the associations among body and testis measurements with sperm production values. General
273 linear mixed models were used to compare fertilization (%) among insemination treatments [1) 300,000 MSE stripped sperm (positive control compared with 2) Testicular sperm at 300,000 MSE compared with 3) Testicular sperm at 600,000 MSE], including sperm pool origin as a random factor, and post-hoc mean comparisons using the Tukey’s
HSD test. The same model was again used for large-scale fertilization experiments using the two insemination treatments: 1) 300,000 MSE stripped sperm (positive control) compared with testicular sperm at 100,000 MSE. All results are represented as least square means ± 1 SEM.
Results
Repeated sperm collections using the stripping procedure resulted in a lesser, as compared with use of the testicular collection-sperm extraction procedure, sperm volume and total number of sperm collected, but did not have an effect on values for other sperm variables. Total volume of milt collected by imposing the first collection procedure was
~two-fold greater than the volume collected from the same specimen 5 d later (0.22 ±
0.03 mL and 0.11 ± 0.03 mL, respectively, P < 0.001). Sperm concentration did not differ between samples collected sequentially but total sperm collected was ~two-fold less at the second compared with the first collection (4.8 × 109 ± 1.8 × 109 sperm compared with
10.6 × 109 ± 1.6 × 109, respectively, P < 0.01).
Testicular mass was approximately 2.5 ± 0.3 g with a gonadosomatic index of 1.0
± 0.3%. The lengths of the left and right testicle were 79.6 ± 0.2 and 79.3 ± 0.4 mm,
274 respectively. Testicular sperm production was correlated with body mass (r = 0.81), total length (r = 0.83), and testicular weight (r = 0.83).
Sperm production and quality differed with use of the different collection techniques (Table 12). With use of the testicular sperm collection-sperm extraction procedure, there was a 25% greater total motility, 10% greater progressive motility, and a seven-fold increase in sperm collected compared with use of the stripping procedure for sperm collection (all P < 0.001). Sperm velocity and viability did not differ when there was use of the different procedures for sperm collection (P = 0.74 and P = 0.80, respectively). The number of morphologically normal sperm cells with use of the testicular collection-sperm extraction procedure was less by about 16% compared with collections using the stripping procedure (P < 0.001).
The regimen used to collect sperm affected the number and quality of sperm from each individual (Figure 23). Total sperm collected of all types (total, motile, morphologically normal motile) was ~seven to ten-fold less using the regimens that did not include testicular collection. Sperm yield (total, motile, motile morphologically normal) did not differ (all P > 0.05) as a result of conducting one and two milt collections prior to testicular collection.
Egg fertilization rates did not differ when there were sperm used that were collected using the different procedures or when there were the different sperm-to-egg ratios evaluated in this study. The results from the preliminary small-scale fertilization assessment using sperm collected utilizing the stripping procedure and testicular collection-sperm extraction procedure were similar at 11-days of the fertilization period
275
(P = 0.48, Table 12). Additionally, increasing the number of sperm that were collected using the testicular collection-sperm extraction procedure for fertilization from 300,000 to 600,000 motile sperm per egg did not affect fertilization rate (70.0 ± 5.3% compared with 68.2 ± 4.8%, P = 0.81). One observation was removed from the analysis as an outlier to meet the assumptions of the test (testicular 300,000 MSE, ~15% fertilization).
In the large-scale fertilization experiment (150,000 eggs per insemination), there was no difference in fertilization rate (P = 0.265) when there was use of sperm collected using the stripping procedure as a positive control (81.2 ± 5.5%) and sperm collected using the testicular collection-sperm extraction procedure at 100,000 MSE (71.2 ± 5.5%).
Discussion
With an overarching goal being the helping of agencies such as the ODNR-DOW overcome sperm number limitations in the production of Saugeye, experiments were conducted to compare sperm numbers collected, fertilization, and protocol suitability using different sperm collection procedures. Specifically, there was use of the commercially used stripping procedure and there was comparison of outcomes using this procedure to one where there is testicular collections and sperm extractions combined with the use of the stripping procedure for sperm collection. It was confirmed that repeated use of the stripping procedure for sperm collection had diminishing outcomes with regard to sperm number obtained for use in egg fertilization procedures, further supporting the notion that the use of the traditional method for sperm collection is not optimal for obtaining sperm for fertilization. By contrast, testicular collection combined with sperm extraction was found to be an advantageous method in terms of number of 276 sperm collected, and sperm motility, although there were more morphological defects of sperm collected directly from the testes compared to those collected using the milt stripping procedure. Even though there were these differences in sperm morphology, fertilization was similar when there was use of sperm for fertilization that were obtained using the two procedures for milt collection. Collectively, these results indicate the use of the testicular collection-sperm extraction procedure in the hatchery setting is a viable alternative to using the stripping procedure for sperm collection from the limited numbers of broodstock that are available for this purpose.
Results of the present study highlight the need for alternative milt collection techniques. According to current protocols, the stripping procedure is imposed for sperm collections from male Saugers two or three times before the end of the breeding season.
Consistent with findings in the present study, results from previous studies with Walleye
(Sander vitreus) and Caspian brown trout (Salmo trutta caspius) indicated milt production and quality decreased as a result of repeatedly imposing sperm collection procedures (Satterfield Jr and Flickinger, 1995; Hajirezaee et al., 2009). Combined with the fact that Sauger produce <1.0 mL of milt per collection, the reduction in sperm quantity as a consequence of sequential collections in the present study further emphasizes the need for alternative collection techniques, such as the testicular collection-semen extraction procedure evaluated in the present study for increasing the number of sperm collected.
Similar to results from other studies, in the present study there was found to be an untapped reservoir of sperm within the testis that is apparently not released when using
277 the stripping procedure for milt collection. Inconsistent with results in other studies with smelt (Osmerus eperlanus L.) and Eurasian perch (Perca fluviatilis) (Kowalski et al.,
2006; Alavi et al., 2008), results from the present study indicate sperm collected from the testicular tissue were of similar quality to those in milt collected using the stripping procedure, except for the greater progressive motility and fewer cells with normal morphology of sperm collected from the testicular tissues. Similar to northern pike (Esox lucius), coho salmon (Oncorhynchus kisutch), and zebrafish (Danio rerio) (Morisawa and
Morisawa, 1988; Hulak et al., 2008; Jing et al., 2009), in the present study there was approximately seven-fold more sperm in milt collected from the testis as compared with that collected using the stripping procedure. Interestingly, collecting sperm using the stripping procedure once or twice before testicular collections for sperm procurement did not affect the total amount of sperm collected with use of the testicular collection-sperm extraction procedure. Morphologically, there were more abnormal cells in the sperm collected from the testicular tissues with the two most common defects being decapitations and broken tails. These deformities may be the result of sperm damage that occurred as a result of the procedures applied to the testis for milt collection or the presence of immature sperm cells in the testicular milt (Mansour et al., 2004; Rodina et al., 2008). Results from the present study indicate testicular collections for procuring milt may be more beneficial than the use of the traditional stripping procedure for sperm collection currently used by a majority of aquaculture enterprises in which there is Sauger production, such as the ODNR-DOW. Furthermore, testicular collections in combination with use of the stripping procedure for sperm procurement proved to be a way to greatly
278 increase sperm numbers available for fertilization procedures; an advancement that will likely lead to lesser problems as a result of sperm limitations in Sauger hatcheries.
Even though there were a considerable number of sperm cells in testicular milt that are immature or damaged, there were no differences in fertility between when there was use of sperm for fertilizations that were collected using the stripping and testicular collection-sperm extraction procedures. Initially in the small-scale fertilization experiment in which there was evaluation of fertilization rate with a 300,000 motile sperm-per-egg ratio, it was found that there were similar fertilization rates when sperm were used that were collected using the two procedures. In the subsequent large- fertilization experiment, there were no differences when there was use of sperm for fertilization that were collected using the two procedures at sperm ratios of 100,000
MSE, a practical insemination dose for hatchery enterprises. The scalability of testicular sperm usage from small to large-scale fertilization in the production of Saugeye further indicates the suitability of the testicular collection-sperm extraction method for hatchery use. It, therefore, is recommended that a 100,000 MSE ratio should be used during fertilization at both small and large scales of Saugeye production.
The main limitation of collecting sperm from testicular tissues are that broodstock need to be sacrificed during collection and the presence of morphologically immature cells in the sample. Sauger broodstock are collected from the Ohio River annually and are maintained with human care throughout the breeding season, after which these specimens are euthanized. As a result, loss of broodstock during sperm harvest should not be considered a factor limiting production in subsequent years. In species where broodstock
279 are obtained by capturing in their natural habitat that are maintained for several years, loss of individuals through euthanization would reduce the availability of males, such as in species that are rare in nature and with the practice of euthanization there could be a decrease in overall genetic diversity (for example species at risk of threatened or endangered status). Future directions for this line of research includes examining the testicular sperm physiology and fertility at lesser sperm-to-egg ratios than used in the present study to further quantify the overall impact of procedural advancements in gamete management on Saugeye production relative to use of traditional procedures of stripping for sperm collections.
Conclusions
Collectively, findings in the present study indicate milt collection with use of testicular collection-semen extraction procedures is a viable alternative to use of the stripping procedure for sperm collection. Contrary to the original hypothesis for the present study, results indicate milt collection using the stripping procedure results in collection of a relatively small number of sperm with lesser motility whereas sperm collection using the testicular collection-sperm extraction procedure results in a ~seven- fold increase in sperm collected that are of comparable quality. Additionally, even though there were differences in values for sperm variables between sperm collected using the stripping and testicular collection-semen extraction procedures, there were highly acceptable fertilization rates with use of sperm collected using both procedures.
Collection regimens need to be developed for utilizing the single stripping procedure for
280 sperm collection followed by use of the testicular collection-semen extraction procedure to maximize the amount of sperm collected while also minimizing labor requirements.
Ultimately results of the present study provide stakeholders with captive breeding and hatchery programs a means to overcome the sperm number limitations in Sauger and likely other species in which there are limited sperm volumes for aquaculture purposes or in which males are in short supply.
281
References
Alavi, S.H., Rodina, M., Policar, T., Cosson, J., Kozak, P., Psenicka, M., Linhart, O.,
2008. Physiology and behavior of stripped and testicular sperm in Perca fluviatilis
L. 1758. Cybium 32, 162–163.
Bardon-Albaret, A., Brown-Peterson, N.J., Lemus, J.T., Apeitos, A., Saillant, E.A., 2015.
A histological study of gametogenesis in captive red snapper Lutjanus
campechanus. Aquac. Res. 46, 901–908. https://doi.org/10.1111/are.12244
Blawut, B., Wolfe, B., Moraes, C.R., Ludsin, S.A., Coutinho da Silva, M.A., 2018.
Increasing Saugeye (S. vitreus × S. canadensis) production efficiency in a
hatchery setting using assisted reproduction technologies. Aquaculture 495, 21–
26. https://doi.org/10.1016/j.aquaculture.2018.05.027
Casselman, S.J., Schulte-Hostedde, A.I., Montgomerie, R., 2006. Sperm quality
influences male fertilization success in Walleye (Sander vitreus). Can. J. Fish.
Aquat. Sci. 63, 2119–2125.
Ciereszko, A., Dabrowski, K., 1993. Estimation of sperm concentration of Rainbow
Trout , whitefish and yellow perch using a spectrophotometric technique.
Aquaculture 109, 367–373.
Hajirezaee, S., Mojazi Amiri, B., Mirvaghefi, A.R., 2009. Effects of stripping frequency
on semen quality of endangered Caspian brown trout, Salmo trutta caspius. Am. J.
Anim. Vet. Sci. 4, 65–71.
282
Hulak, M., Rodina, M., Linhart, O., 2008. Characteristics of stripped and testicular
Northern pike (Esox lucius) sperm: spermatozoa motility and velocity. Aquat.
Living Resour. 21, 207–212. https://doi.org/10.1051/alr:2008022
Jing, R., Huang, C., Bai, C., Tanguay, R., Dong, Q., 2009. Optimization of activation,
collection, dilution, and storage methods for zebrafish sperm. Aquaculture 290,
165–171. https://doi.org/10.1016/j.aquaculture.2009.02.027
Kowalski, R.K., Hliwa, P., Andronowska, A., Krol, J., Dietrich, G.J., Wojtczak, M.,
Stabinski, R., Ciereszko, A., 2006. Semen biology and stimulation of milt
production in the European smelt (Osmerus eperlanus L.). Aquaculture 261, 760–
770. https://doi.org/10.1016/j.aquaculture.2006.08.038
Król, J., Żarski, D., Bernáth, G., Palińska-Żarska, K., Krejszeff, S., Długoński, A.,
Horváth, Á., 2018. Effect of urine contamination on semen quality variables in
Eurasian perch Perca fluviatilis L. Anim. Reprod. Sci. 197, 240–246.
https://doi.org/10.1016/j.anireprosci.2018.08.034
Latif, M.A., Bodaly, R.A., Johnston, T.A., Fudge, R.J.P., 1999. Critical Stage in
Developing Walleye Eggs. N. Am. J. Aquac. 61, 34–37.
https://doi.org/10.1577/1548-8454(1999)061<0034:CSIDWE>2.0.CO;2
Mansour, N., Lahnsteiner, F., Berger, B., 2004. Characterization of the testicular semen
of the African catfish, Clarias gariepinus (Burchell, 1822), and its short-term
storage. Aquac. Res. 35, 232–244. https://doi.org/10.1111/j.1365-
2109.2004.00993.x
283
Moore, A., 1987. Short-Term Storage and Cryopreservation of Walleye Semen. Prog.
Fish-Cult. 49, 40–43. https://doi.org/10.1577/1548-
8640(1987)49<40:SSACOW>2.0.CO;2
Morisawa, S., Morisawa, M., 1988. Induction of potential for sperm motility by
bicarbonate and pH in Rainbow Trout and chum salmon. J. Exp. Biol. 136, 13–
22.
Ohta, H., Tanaka, H., Kagawa, H., Okuzawa, K., Iinuma, N., 1997. Artificial fertilization
using testicular spermatozoa in the Japanese eel Anguilla japonica. Fish. Sci. 63,
393–396.
Park, D.S., Egnatchik, R.A., Bordelon, H., Tiersch, T.R., Monroe, W.T., 2012.
Microfluidic mixing for sperm activation and motility analysis of pearl Danio
zebrafish. Theriogenology 78, 334–344.
https://doi.org/10.1016/j.theriogenology.2012.02.008
R Development Core Team, 2009. R: A language and environment for statistical
computing.
Rinchard, J., Dabrowski, K., Van Tassell, J.J., Stein, R.A., 2005. Optimization of
fertilization success in Sander vitreus is influenced by the sperm : egg ratio and
ova storage. J. Fish Biol. 67, 1157–1161. https://doi.org/10.1111/j.0022-
1112.2005.00800.x
Rodina, M., Policar, T., Linhart, O., Rougeot, C., 2008. Sperm motility and fertilizing
ability of frozen spermatozoa of males (XY) and neomales (XX) of perch (Perca
284
fluviatilis). J. App. Ichthyol. 24, 438–442. https://doi.org/10.1111/j.1439-
0426.2008.01137.x
Rurangwa, E., Kime, D.E., Ollevier, F., Nash, J.P., 2004. The measurement of sperm
motility and factors affecting sperm quality in cultured fish. Aquaculture 234, 1–
28. https://doi.org/10.1016/j.aquaculture.2003.12.006
Sarosiek, B., Dryl, K., Krejszeff, S., Żarski, D., 2016. Characterization of pikeperch
(Sander lucioperca) milt collected with a syringe and a catheter. Aquaculture 450,
14–16. https://doi.org/10.1016/j.aquaculture.2015.06.040
Satterfield Jr, J.R., Flickinger, S.A., 1995. Field collection and short-term storage of
Walleye semen. Prog. Fish-Cult. 57, 182–187.
Tiersch, T.R., Wayman, W.R., Skapura, D.P., Neidig, C.L., Grier, H.J., 2004. Transport
and cryopreservation of sperm of the common snook, Centropomus undecimalis
(Bloch). Aquac. Res. 35, 278–288. https://doi.org/10.1111/j.1365-
2109.2004.01013.x
Viveiros, A.T.M., Fessehaye, Y., ter Veld, M., Schulz, R.W., Komen, J., 2002. Hand-
stripping of semen and semen quality after maturational hormone treatments, in
African catfish Clarias gariepinus. Aquaculture 213, 373–386.
https://doi.org/10.1016/S0044-8486(02)00036-4
Yokoi, K., Ohta, H., Hosoya, K., 2008. Sperm motility and cryopreservation of
spermatozoa in freshwater gobies. J. Fish Biol. 72, 534–544.
https://doi.org/10.1111/j.1095-8649.2007.01719.x
285
Żarski, D., Bernáth, G., Król, J., Cejko, B.I., Bokor, Z., Palińska-Żarska, K., Milla, S.,
Fontaine, P., Krejszeff, S., 2017. Effects of hCG and salmon gonadoliberine
analogue on spermiation in the Eurasian perch ( Perca fluviatilis ).
Theriogenology 104, 179–185.
https://doi.org/10.1016/j.theriogenology.2017.08.022
286
Table 12. Comparison of values for Sauger milt production and quality variables (mean
1 SE) in sperm collected using the stripping (n = 30) and testicular collection-sperm extraction (n = 17) procedure.
Milt Characteristic Stripped Testicular
Total Motility (%) 44.27 ± 5.7 b 70.11 ± 2.1 a
Progressive Motility (%) 8.05 ± 1.4 b 18.49 ± 1.6 a
Velocity (μm/s) NS 111.84 ± 3.3 113.45 ± 2.5
Viability (%) NS 94.30 ± 1.5 95.82 ± 0.7
Morphology (%)* 92.83 ± 0.7 a 76.41 ± 1.6 b
Total Sperm (× 109) 7.71 ± 1.4 b 55.4 ± 5.0 a
Fertilization (%) NS† 59.90 ± 5.3 70.0 ± 5.3
Data were pooled per collection method regardless of collection iteration or preceding
stripping regimen for sperm collection
NS Denotes non-significant differences among groups (P > 0.05) a,b Means with a similar superscript within a row do not differ (one-way ANOVA,
Tukey’s HSD)
Morphology is measured as the percentage of morphologically normal spermatozoa
†Sample sizes for small-scale fertilization experiments (300,000 MSE) were n = 4 and n =
5 for stripped and testicular sperm, respectively
287
Figure 23. Comparison of milt collection regimens [Stripped Twice (n = 10), Stripped
Once + Testicular Collection and Semen Extraction (Group 1, n = 10), and Stripped
Twice + Testicular Collections and Semen Extraction (Group 2, n = 7)] in terms of sperm type [total number of sperm (grey), total number of motile sperm (black), and number of total motile, morphologically normal sperm (white)]; Tukey’s HSD superscripts denote differences among group means within a sperm type (P < 0.05)
288
Appendix B. Sauger (Sander canadensis) Milt Best Management Practices for Saugeye (S. vitreus x S. canadensis) Production
Section 1: Milt Collection Techniques
Introduction
Milt collection from broodstock is the first potential obstacle faced at the hatchery in terms of maximizing individual sperm yield. Sperm yield can vary widely according to season (early, mid, late breeding season) and among individuals based on age, size, and relative condition. The goal of the collection process is to gather as much milt from each individual as possible.
One factor affecting individual sperm yield if the method of sperm collection. In some cases, traditional milt collection techniques are not effective. Small sperm volumes and contamination issues can severely hamper the production process. Sauger exemplify these problems perfectly, and many of these problems are related to the collection methodology. Sauger sperm is currently collected from broodstock using two techniques: strip-spawning and testicular harvest. Both have distinct advantages and disadvantages relative to the ultimate goal of maximizing sperm collection and effectively managing available broodstock. Additionally, administering human chorionic gonadotropin (hCG) prior to collection has been shown to have positive effects on milt handling characteristics, and could be a practical tool during Sauger milt collection. 289
This chapter details the techniques of strip spawning, testicular harvest, short-term milt storage, and hCG administration as well as common problems associated with these techniques and how best to avoid them.
Materials and Methods
The materials needed, vendors, prices, and unit sizes for items in this section can be found in Table 17.
Strip-Spawning
Strip spawning is currently the most commonly used method of milt collection from male Sauger broodstock. Individuals are captured from the holding tank, placed in dorsal recumbency, and thoroughly dried to prevent premature activation of milt with tank water. Strip spawning is accomplished by applying pressure using the thumb and fingers along the body wall and abdomen starting from the pectoral fins and ending at the urogenital pore. Milt is collected from the urogenital pore using a serological pipette or a
1.0 mL all-plastic syringe.
Milt can be contaminated with the following bodily fluids:
Urine/Feces – contamination with either will significantly lower sperm motility
potential. Avoid contamination and discard potentially contaminated samples.
• Dabbing the end of the syringe with contaminated sperm at the end on
paper towels can draw the contamination out and preserve the rest of the
sample.
290
• Slight urine contamination can be reversed by diluting with extender as
soon as possible.
Blood – blood contamination is not detrimental to sperm (Jr and Flickinger,
1995), but severe contamination with blood will negatively affect sperm
concentration assessment.
Loose seminal plasma – loose seminal plasma released along with sperm usually
presents as a clear liquid expressed with or without a viscous “noodle” of
unmixed sperm. This is a common occurrence and there is no evidence that this
negatively affects sperm quality.
Following collection, milt should be diluted approximately 1-part semen to 3-parts extender, homogenized, and stored at 4 - 5C (39 – 41F) prior to use.
Multiple milt samples can be pooled together and stored in a 4.5 mL cryovial.
Reactive oxygen species production can result from excessive exposure to air (air space in the vessel) and should be avoided by sealing the container. Milt should be homogenized 1 – 2 times per day by inverting the container multiple times to prevent sperm from accumulating in the bottom of the tube, resulting in cell death and quality degradation. The cap should also be opened to allow new oxygen into the tube, and then recapped.
291
Short-Term Storage and Milt Handling
Proper dilution and handling of extended Sauger sperm are essential for successful short-term storage. We have found a 1:4 extension (1-part sperm to 3-parts extender) to be more appropriate than a 1:3 extension during short-term storage of sperm.
However, extended milt with high cell concentrations (> 1.5 × 10 10 sperm/mL) are prone to a faster, more precipitous decline in sperm quality during cooled storage (see results).
In these instances, particularly for - but not limited to - testicular sperm, we have found that adding an additional 0.5-1.0 mL extender per 3.0 mL of extended milt can have a positive effect on sperm motility.
Sperm should be stored and transported at 4 - 5°C and the vessel temperature should be maintained as consistently as possible. A common refrigerator at 4 - 5°C is sufficient when sperm is not in transit. Samples should NOT be placed on the uppermost shelf near the back because this can freeze the vial, effectively killing sperm. The refrigerator should be opened as infrequently as possible to maintain a stable internal temperature.
Aside from natural quality loss over time, improper milt transportation to and from the site of fertilization is the most likely cause of loss of fertility in extended semen pools. Caution should be used when handling extended milt as well. Extended milt should always remain as close as 4°C as possible.
Possible sources responsible for decreased fertilization during routine hatchery procedures include: (1) sub-optimal transport box set-up, (2) variable temperature during transport and use, and (3) over-handling of milt pools.
292
1. Box preparation is essential to proper milt storage during transportation.
Preparation of the box, insulation, and cooling element prior to placing milt into
the cooler is vital. Depending on the size of the box and the amount of ice/cooling
blocks used, the air temperature could take as long as 2 hours to reach 4°C before
adding the sperm vials. Therefore, we recommend preparing the transport box as
early as possible, even the night before if fertilization occurs early in the morning.
2. Placing the Styrofoam box in the direct line of a heater vent, exposed to the sun,
or opening the box more frequent than is necessary will all contribute to a higher
than optimal temperature within the box. For this reason, we recommend
transporting the box in a safe location away heat sources and direct exposure to
sun. Additionally, the box should not be opened until milt needs to be aliquotted
for fertilization. Larger containers are less amenable to temperature fluctuations
when the box is opened. We recommend a larger box for milt transport. The box
we have used to transport extended milt in the past are approximately 30mm (L) ×
24mm (W) × 24mm (D).
3. Poor handing of extended milt can also contribute to quality degradation.
Exposure to sunlight, extended contact with warm hands, as well as using wet/
unclean instruments can negatively affect sperm as well. For this reason, we
recommend using disposable pipettes to aliquot milt no more than once or allow
to dry thoroughly overnight before re-use.
293
While individually these actions may seem mild, several days of repeated occurrences combined with natural degradation of sperm quality over time can significantly impact fertilization and milt shelf-life.
Transportation of Sauger milt has been accomplished using two different methods
(Figure 24) in the OSU Theriogenology department: samples stored (1) dry above ice/icepacks and (2) partially submerged in an ice water slurry. Both have been used successfully by our lab to transport milt. Ice water has advantages and disadvantages over cooled air alone. The primary advantage is that water temperature is more stable than the air and will be less affected by opening the box. Disadvantages include possible milt activation with water and contact with ice can cause the sperm to freeze. Accidental water introduction into extended milt samples and freezing should be avoided at all costs. We avoid these potential pitfalls but placing the sperm vials in a Ziploc back to prevent water contamination as well as keeping vials and ice separate using a cardboard/ plastic divided under a hand towel/surgical towel.
We recommend that newer sperm vials (e.g. collected the day prior) and old vials are transported in different containers. This will allow hatchery staff to use the old milt first without disturbing the newer samples by repeatedly opening the box. This is especially important on days where fresh milt was prepared and transported, but not used that day.
Sperm pool fertilization potential cannot be assessed without the use of microscopic examination. Continued observation of sperm motility is the only reliable method to assess sperm fertilization potential. Extended Walleye milt has been
294 successfully stored for 10-14d at 4°C by Moore (1987), but no such studies have investigated the full short-term storage potential of Sauger milt. Our recommendations for short-term storage are approximately 4-5d given the results of comparative studies
(see below). If motility of certain pools decreases suddenly, fertilization may still be able to be preserved by adjusting the insemination dose (Section 3).
Testicular Harvest
Testicular harvest in Sauger has recently been proven by our lab to maximize individual milt yield and eliminate the possibility of urine and feces contamination all while delivering a consistently high-quality sperm sample (Blawut et al. 2020a).
Broodstock Euthanasia
Sauger broodstock have been euthanized using two methods for testicular harvest:
MS-222 and cervical transection. Euthanasia using MS-222 has the potential to deprive the testes and the sperm of oxygen prior to harvest which can negatively affect sperm quality. Cervical transection is the current and preferred method of euthanasia because it avoids oxygen depletion, is rapid, is cost effective, and does not require handling chemicals.
1. Make a deep cut with a filet knife, approximately one inch behind the eye to sever
the spinal cord.
2. Decapitate the fish to guarantee complete and humane euthanasia, being careful
not to cut the tips of the testes that extend cranially beyond the pectoral region.
295
Testicular Harvest Procedure
• Make a T-shaped incision on the ventral surface using a scalpel (Figure 25):
o Across the body just below the pectoral fins
o From the middle of the first incision down to the cloaca (using scissors
will give more control).
o Either pin back the belly folds, or you can just cut them off to leave the
cavity exposed.
o Use care not to cut too deep or you will cut the testes and
lose/contaminate sperm.
• Clamp the testes as close to the cloaca as possible.
• Use scissors to break the pectoral bones.
• Sever the ligament and blood vessel under the pectoral girdle connected to the
testis lobes (Figure 25 A)
• Break/cut the ligament holding the testes to the ventral body cavity wall along the
entire testis length
• Carefully cut the liberated testes above the clamp at the urogenital pore and
remove them from the body. Place in petri dish.
• Try to remove any blood vessels on the lobes before making any further incisions.
o This will minimize blood contamination. While not detrimental, it can
affect longevity and the ability of the densimeter to measure sperm
concentration.
296
• Use the scalpel to cut from the cloaca to the tip of each of the lobes.
• Roll a pipette up and down over the lobes, from the cloaca to the tips, to liberate
sperm.
• Add 3.0 mL of Rathbun Extender to the petri dish (Figure 25 B)
• Wash the testes with extender, use the side of the pipette squeeze sperm from the
lobes again, and then dispose of the tissue.
• Use the pipette to gently homogenize the sample (Figure 25 C).
o Pipetting too fast and making air bubbles will kill sperm.
o You can scour patches of thick sperm from the dish surface by using the
force of liquid leaving the pipette to wash sperm from the surface.
• Transfer the homogenized sample to a tissue culture flask/test tube/cryovial
• Store at 4 - 5 C.
Comparison of Fertilization and Short-Term Storage of Stripped and Testicular Milt
Fertilization was compared among stripped sperm and testicular sperm at several different sperm to egg ratios (20,000, 100,000, 300,000 and 600,000 motile sperm per egg (MSE)) at using small (1,000 oocytes) and large scales (150,000 oocytes).
Fertilization was compared between the two sperm types using the proportion of eggs to reach the eyed stage of development (9-11d).
We compared short-term storage potential at 5° C for 3 -6 days in stripped and testicular milt. We pooled high-quality sperm demonstrating motility of > 50% collected from multiple fish by stripping (1-part milt to 3-parts extender) or testicular dissection
297
(3.0 mL added during homogenizing) to a final volume of ~ 2.0 mL in a 4.0 mL cryovial and stored the samples at 5°C over a period of 3 -6d. Four and eight pools of sperm were created using stripped sperm and testicular sperm, respectively. Pools were mixed twice daily by inverting the tube multiple times. Vials were placed horizontally on racks instead of vertically to lessen sperm sedimentation intensity. We determined total motility (%), progressive motility (%), sperm velocity (µm/s), and cell viability (%) using previously described methods at the time of collection and then once every 24 hours for the remained of the storage period.
Human Chorionic Gonadotropin (hCG) Treatments
Fish can be treated with hCG according to these guidelines: 550 IU/kg hCG
(Chorulon, Merck Animal Health Management, NJ, USA) intramuscularly (ventral to the first dorsal fin) or intraperitoneal. A five-day waiting period prior to sperm collection was used in the 2015 study. The ability of hCG to enhance sperm production in fish that had been strip spawned previously was assessed.
Results and Discussion
Sauger milt volume, concentration, and total sperm yield was highly variable among individual broodstock using the strip spawning technique (mean ± SD). Milt volume was approximately 0.18 ± 0.15 mL (CV= 84.3, n = 243). Sperm concentration and total sperm yields were 36.42 x 109 ± 30.79 × 109 sperm per mL (CV= 84.5%, n=
298
117) and 14.87 x 109 ± 13.18 × 109 sperm total per individual (CV= 88.7%, n= 123), respectively.
Repeated sperm collections using the stripping procedure resulted in a lesser sperm volume and total number of sperm collected but did not have an effect on values for other sperm variables. Total volume of milt collected by imposing the first collection procedure was about two-fold greater than the volume collected from the same specimen
5 d later (0.22 ± 0.03 mL and 0.11 ± 0.03 mL, respectively, P < 0.001). Sperm concentration did not differ between samples collected sequentially, but total sperm collected was around two-fold less at the second collection compared with the first collection (4.8 × 109 ± 1.8 × 109 sperm compared with 10.6 × 109 ± 1.6 × 109, respectively, P < 0.01; Blawut et al. 2020a).
Strip Spawning vs. Testicular Harvest
Sperm production and quality differed with use of the different collection techniques (Figure 23, Appendix A). With use of the testicular sperm collection-sperm extraction procedure, there was a 25% greater total motility, 10% greater progressive motility, and a seven-fold increase in sperm collected compared with use of the stripping procedure for sperm collection (all P < 0.001). Sperm velocity and viability did not differ when there was use of the different procedures for sperm collection (P = 0.74 and P =
0.80, respectively). The number of morphologically normal sperm cells with use of the testicular collection-sperm extraction procedure was less by about 16% compared with collections using the stripping procedure (P < 0.001; Blawut et al. 2020a).
299
Collection Regiment
The regimen used to collect sperm affected the number and quality of sperm from each individual (Table 12, Appendix A). Sperm collected of all types (total number of sperm, total number of motile sperm, and total number of motile and morphologically normal sperm) were about seven -ten-fold less using the regimens that did not include testicular collection. Sperm yield (total number of sperm, total number of motile sperm, and total number of motile and morphologically normal sperm) did not differ (all P >
0.05) as a result of conducting one and two milt collections prior to testicular collection
(Blawut et al. 2020a)
Short-Term Storage
No differences were seen between stripped and testicular sperm in terms of short- term storage at 5C over the course of 3 days (Table 13). However, sperm velocity in both treatments did decrease during the 3d study period. At 6d of storage, we observed a significant decrease in motility (~ 20% and ~ 8% total motility in stripped and testicular sperm, respectively). Motility on day 6 of storage was found to be negative correlated with sperm concentration (r = - 0.69), with samples at concentrations of > 15 billion sperm/mL averaging 0.53% motility compared to 16.9% at less than 15 billion cells / mL.
Therefore, careful consideration must be given to sperm concentration in pooled milt samples during extended short-term storage. Milt pools exceeding 15 billion sperm/mL should be diluted to ≤ 10 billion sperm/mL.
300
Fertilization
The results from the preliminary small-scale fertilization assessment using sperm collected utilizing the stripping procedure and testicular collection-sperm extraction procedure were similar at 11-days of the fertilization period (P = 0.48, Table 12,
Appendix A). Additionally, increasing the number of sperm that were collected using the testicular collection-sperm extraction procedure for fertilization from 300,000–600,000 motile sperm per egg (MSE) did not affect fertilization rate (70.0 ± 5.3% compared with
68.2 ± 4.8%, P = 0.81). One observation was removed from the analysis as an outlier to meet the assumptions of the test (testicular, at 300,000 MSE, ∼15% fertilization).
In the large-scale fertilization experiment (150,000 eggs per insemination), there was no difference in fertilization rate (P = 0.26) when there was use of sperm collected using the stripping procedure as a positive control (81.2 ± 5.5%) and sperm collected using the testicular collection-sperm extraction procedure at 100,000 MSE (71.2 ± 5.5%).
However, fertilization at 20,000 MSE was highly variable (Figure 26). Total motility
(TM) was low in one pool (~ 7% TM) and resulted in approximately 2% fertilization at
20,000 MSE, but statistically no differences were seen because of the high variability due to this observation. However, increasing MSE addition up to 100,000 (a five-fold increase) resulted in fertilization equivalent to the fresh control (~ 65%).
Treating Sauger with hCG had variable effects on sperm characteristics (Table
14). Sauger treated with hCG produced a higher volume of milt at the post-treatment sampling than fish treated with the saline control (p = 0.01). By contrast, hCG had no
301 obvious effects sperm cell concentration (p = 0.08) or total sperm production (p = 0.18).
No tank temperature effect (p = 0.27–0.75) or temperature × treatment interaction effects were found on any of the three dependent variables (p = 0.47–0.97). Thus, hCG was effective in inducing milt hydration (increasing milt volume through LH endocrine action), and can be a tool to enhance handling characteristics, but was not effective in this context in increasing total sperm yield.
Management Implications
Strip spawning is a viable tool for milt collection, but significant variation in the amount and quality of milt collected using this method leave room for improvement.
Using testicular sperm harvest has multiple advantages when compared to strip spawning:
1. Little to no possibility of milt contamination by urine or feces
2. Higher total sperm yield compared to strip spawning (7-10x)
However, testicular sperm harvest cannot be relied upon when strip spawning fails to yield any sperm. If Sauger suddenly are no longer producing sperm upon strip spawning (e.g., 2018 when fish stopped producing sperm due to a sudden temperature shift), testicular sperm will be of low quality (< 10% motility) as the testes are transitioning to a state of regression.
Despite the superiority of testicular harvest as a collection technique, strip spawning fish still has a place in the hatchery production of Saugeye. Milt can be collected from Sauger in the initial stages of Saugeye production using strip spawning to
302 prevent excessive depletion of broodstock via euthanasia. Then testicular harvest can be used later in the season to remove broodstock from the system while also maximizing sperm yield per individual. A larger volume of sperm can be obtained from testicular harvest than strip spawning, meaning that fewer fish and less labor will be required to achieve larger yields of sperm.
Short-term storage of extended Sauger sperm is an essential technique during the production of Saugeye. Our results demonstrate that sperm quality is maintained through
3-4 days of storage at 4°C, with only a small decrease in sperm velocity. However, by day 6 motility had decreased to approximately 18.75% in stripped sperm and 5.6 % in testicular sperm. Fertility was not assessed but was expected to be poor at that point due to low motility. In contrast, Moore (1987) was able to store Walleye semen at 1:2 at 1°C for 10-14 days in extender containing 180 µg/mL ampicillin (fertilization ~ 90.8%). Our results combined with the fact that Walleye semen generally is less concentrated than is found in the Sauger, and our past experiences suggests the concentration of sperm in the sample may ultimately affects short-term storage potential. Steps should be taken to ensure above average concentrations of cells in sperm pools are reduced to further enhance short-term storage capability (~ 10 × 10 9 sperm/mL).
Lastly, hCG administration can be a useful tool in certain situations given its ability to increase milt volume. Seasonal or low water temperature effects can limit the amount of sperm collected due to low volume or highly viscous milt samples. Treating fish with hCG could be useful in that it will enhance volume and fluidity, making collection more efficient and practical. However, total sperm production is not affected
303 by this treatment. Thus, hCG is simply a tool to enhance milt hydration and facilitate milt collection, not increase reproductive output in Sauger.
304
Section 2: Sauger Sperm Quality Control
Introduction
Sperm pools and individual samples have been shown to vary considerably in both sperm cell quality and sperm cell density. These characteristics are two primary factors controlling sample fertility. High variability among samples can account for variable fertilization rates and hatching rates, which ultimately interfere with fry production. Thus, it is important to quantify (total sperm) and qualify (percentage motility) samples used for fertilization to modify insemination doses appropriately.
A standard insemination dose of 0.5 mL per quart of eggs may achieve fertilization, but variation among pools in terms of motility and concentration can and will lead to variable fertilization, as well as under- or over-application of the limited milt available. Results of the research we’ve conducted have shown that only a small number of motile sperm are needed to achieve high fertilization (Figure 27). Reduced return on investment at higher doses is not advised, as it is the most prevalent form of wasting sperm. Sperm limitation caused by poor utilization of extended milt can tax hatchery staff and fisheries biologists responsible for broodstock collection. Thus, it is imperative that the sperm pool be assessed adequately to ensure efficient usage to alleviate the stress on all facets of hatchery production of Saugeye.
As an example of the importance of sperm quality assessment, we refer to a specific instance during the 2018 breeding season. A testicular sperm pool with approximately 7.80% motility was used for fertilization experiments. Fertilization was low (2%) when 20,000 motile sperm per egg were applied to the 1-quart egg mass. 305
However, a 5-fold increase in motile sperm applied to each egg (100,000 vs. 20,000) restored fertility to the same levels seen in higher quality pools (~65%). Knowing the concentration and motility of each samples is essential to correctly identifying and correcting insemination dosages.
Materials and Methods.
The materials needed, vendors, prices, and unit sizes for items in this section can be found in Table 17.
Setup
• Make activation solution: 1% bovine serum albumin in hatchery water (pH to
8.5). Store at 5o C.
o This solution prevents the sperm from sticking to the glass.
• Dilution tubes: aliquot 490 µL of Rathbun extender into a 1.50 mL Eppendorf
tube, cap, and store at 5o C.
o Without this step, the sperm are too concentration to estimate motility
accurately
• Turn on microscope and monitor (Figure 28 A).
• Plug in the densimeter and turn it on (Figure 28 B).
• Purge the formalin pump of air by dispensing formalin into a waste container
o The pump is calibrated to give exactly 3320 µL of formalin. Air bubbles in
the tip of the tubing will decrease the amount of formalin, resulting in an
inaccurate estimate.
306
Sample Preparation
• Make sure the pool is thoroughly mixed and maintained at 4-5 o C.
• Aliquot 10 µL of milt from the pool with a micropipette and mix with the
premade dilution tube (490 µL Rathbun extender). Cap and invert 5-10 times and
store at 5° C.
Motility Assessment
• Get a slide and coverslip and place on the microscope stage
• Add 18 µL of activation solution (1% BSA in hatchery water, pH 8.5) to the
center of the slide.
• From the diluted milt sample, take 2 µL of sperm and release into the water
droplet.
• QUICKLY: Use pipette tip to mix the sample. When removing the tip from the
slide, go back to the center of the drop and lift up.
o This will help to reduce drift in the sample. Motility estimation in a sample
that drifts under the scope is difficult and inaccurate
• QUICKLY: put the coverslip over the mixture and put it under the microscope
objective
o Careful to avoid air bubbles. Large bubbles will make analysis more
difficult.
307
The whole process of activation should take no more than 5-10 seconds. Sauger sperm are only motile for 30 seconds, and motility estimates at 10-15 seconds after activation are most related to fertilization potential.
• From the monitor, you can estimate the percent of motile cells in the sperm
sample (Figure 29 A-C).
o It is the percentage of sperm showing forward, arching movement
o Sperm that vibrate in place are not considered motile
Sperm Cell Concentration Assessment
• Fill a cuvette with 3320 µL of formalin from the pump (1 full pump)
o Cuvettes have 2 smooth and 2-ridged surfaces. Only touch the ridge sides.
The beam of light passes through the smooth surfaces. Smudges will result
in inaccurate readings.
• Place the cuvette in the sample port on the machine, close the door, press ZERO
o Ridges surfaces should face the machine you. The beam passes
horizontally through the sample.
• Thoroughly mix the diluted sperm sample by inverting gently 5-10 times.
• Use the ARS pipette and tips to collect 180 µL of sperm from the diluted sperm.
o Be sure the tip is filled completely, with no bubbles. It often takes
multiple passes with the pipette to clear all air bubbles.
308
o Wipe the sides of the pipette off with a kimwipe before adding sperm to
the cuvette. Excess sperm on the outside of the tip will result in
inaccurate measurements.
• After the machine reads “Add 180 µL of sperm”, remove the cuvette and
thoroughly mix the sperm sample with formalin. Cap and invert the sample 10
times.
• Put the cuvette in the machine and press COUNT.
The densimeter will give you a reading in millions of cells/ml (M/mL). This measure will be used to convert the horse semen estimate to fish sperm estimate using the equation below.
Converting 590b Measurements to Fish Sperm Cells
Look up the densimeter estimate in the attached table (Table 16). Follow the line across to the column labeled “Sauger Sperm Concentration.” This value is the estimate of sperm in the sample from the horse sperm estimate, converted using a linear regression equation we validated (Figure 30; R2 = 0.96). This estimate takes into account the 50- fold dilution used to make the sample that analyses were performed on, giving you the sperm concentration in the original sample.
309
Management Implications
At the end of this process, pooled milt motility and sperm concentration will have been accurately assessed. The next step is to use the two measurements to calculate the amount of milt needed to apply per quart of Walleye eggs to meet insemination standards.
310
Section 3. Calculating Sperm Fertilization Doses
This chapter focuses on the calculations needed to convert the pooled milt assessment characteristics derived from Section 2 into a standardized insemination dose for Saugeye production.
Calculating the Concentration of Motile Sperm in the Sample
The equation below will give you the concentration of motile sperm (i.e. fertile sperm) per milliliter of sample. We will use this equation in the next step to determine the amount of sperm needed for fertilization.
푀표푡𝑖푙푒 푆푝푒푟푚 푆푝푒푟푚 = 푇표푡푎푙 푀표푡𝑖푙𝑖푡푦 (%) × 푆푝푒푟푚 퐶표푛푐푒푛푡푟푎푡𝑖표푛 ( ) 푚퐿 푚퐿
• Total Motility = Motility Estimate (%) / 100 (e.g. 75% is 0.75)
• Sperm Concentration = x × 109 sperm/mL or x × 1010 sperm/mL
Calculating Fertilization Doses
This equation below will tell you the volume of sperm per quart of eggs needed to reach the 20,000 motile sperm per egg standard. This value should be calculated for each pool prior to fertilization.
푒푔푔푠 푠푝푒푟푚 [150,000 푞푢푎푟푡 × 20,000 푚표푡𝑖푙푒 푒푔푔 ] 퐹푒푟푡𝑖푙𝑖푧푎푡𝑖표푛 퐷표푠푒 (푚퐿 ) = ⁄푞푢푎푟푡 푐표푛푐푒푛푡푟푎푡𝑖표푛 푀표푡𝑖푙푒 푆푝푒푟푚 푚퐿 푚𝑖푙푡 311
• The numerator is the standardized number of motile sperm required per egg for
efficient fertilization. It is a fixed factor.
• The denominator is the concentration of motile sperm per milliliter of milt,
calculated using the previous equation.
Management Implications
This chapter provides users with the equations and standards necessary to use
Sauger milt most effectively. By calculating the volume of sperm need to obtain the optimal insemination dose, sperm pools can be used more efficiently during the fertilization process. This will help to prevent over-application of sperm that would otherwise contribute to sperm availability shortages as well as ensure more consistent fertilization from variable quality sperm pools.
312
Section 4: Long-term Milt Storage via Cryopreservation
Introduction
Sperm cryopreservation offers the ability to store excess sperm at subzero temperatures (-196°C) for extended periods of time. This tool is commonly used in the biomedical, agricultural, and more recently in the aquaculture fields. By using a published protocol for Sauger (Blawut et al., 2020a) with some more recently optimized media variants, hatchery staff can use the protocol listed in this chapter to cryopreserve unused Sauger sperm for use during years when broodstock at in short supply. While the current protocol is not well suited for large scale application, it has merit in laboratory settings, preservation of valuable genetic lines, or production of limited number of fry to establish regional strain cross breeding.
Methodology
The materials needed, vendors, prices, and unit sizes for items in this section can be found in Table 17.
Setup
1) Dilute spermatozoa to 1.0 × 10 9 sperm/mL using Modified Ringers lactate
(MRL) containing 4mg/mL BSA.
a. Modifications to Ringers lactate include increasing osmolality to 300
mOsm/lk and pH to 8.5 (originally, 245 mOsm/kg, pH 6.5)
2) Dilute sperm with equal part 15% methanol in MRL + 4mg/mL BSA. Mix
thoroughly. Incubate at 10 minutes, at 4°C on ice. 313
3) During this incubation load the diluent into 0.5 mL semen straws (A-F)
a. Aspirate diluted semen until only 5-10 millimeters of empty space exists
between the liquid front and the cotton plug (Figure 31 A). Remove from
liquid and continue to pull up until the cardboard ring wets (completing
the top seal (Figure 31 B)).
b. This also adds an air bubble to the straw. Without this bubble, straws will
burst / explode during the freezing process at the frozen liquid expands.
4) Seal the end of the straws using sealing powder, and for a plug by briefly dipping
into water (Figure 31 C-F)
a. Wipe off extra powder before and after dipping into water. The straws will
stick to one another and any surface they come into contact with if you
don’t remove the excess powder.
b. Move the air bubble into the middle of the straw by “cracking the whip”
so expanding liquid does not force the sealant out during freezing.
5) After the 10-minute equilibration, place straws on the ARS freezing rack, and
transfer the floating rack to the freezing chamber (Figure 31 G-H)
a. The freezing chamber should be filled with LN2 more than 20 minutes
prior to floating straws.
6) Let the straws cool for 10 minutes, and then plunge the straws into LN2 in the
bottom of the cooling chest and allow them to rest in LN2 for 10 minutes.
a. This is the final freezing step after which samples are stable.
314
7) Use pre-cooled tongs/forceps to put straws into the goblets pre-attached to labeled
canes.
a. Do not bring straws above LN2 liquid surface if possible – will begin to
warm and could have negative consequences on quality.
8) Transfer canes to liquid nitrogen dewar.
a. Maintain LN2 level in sampler storage dewar.
i. We recommend keeping the canes submerged in LN2.
ii. Maintain a minimum of 7 cm of liquid depth in the tank; less will
allow samples to thaw. Prematurely thawed samples are unusable
and should be disposed.
9) One straw should be thawed and assessed for motility.
10) Thaw straws by RAPIDLY moving the straw from LN2 to a 37°C water bath for
10 seconds.
11) Cut the straw into a 0.6 mL centrifuge tube and assess motility as describe before
– no dilution in extender prior to activation needed.
Motility duration is much shorter in cryopreserved sperm, so thoroughly mix
as quickly as possible.
12) Maintain thawed semen at 4-5°C prior to analysis. Samples should not be held for
more than 5 minutes before use.
315
Results
Cryopreservation altered a number of motility parameters compared to stripped sperm (Table 15). Motility was reduced by 28%, all measures of velocity were reduced by between 22.5% (VCL) and upwards of 50% (45 and 49% for VAP and VSL), track linearity decreased by 32.4%, and wobble was reduced by 30% in cryopreserved sperm compared to an unfrozen control. The only parameter to increase following freezing was
BCF. In general, after freezing, motile sperm were active for a shorter period of time (~
15 sec compared to 30 sec in fresh sperm) and swam less vigorously during that time.
Fertility trials using small batches of oocytes (~ 1,000 oocytes per replicate) revealed a diminished capacity for fertilization using cryopreserved sperm (Figure 6, p =
0.002). Stripped sperm fertilized oocytes at approximately 73.4 ± 6.06 % using an overdose of 300,000 motile sperm per egg. By contrast, cryopreserved sperm was only able to reach a mean fertilization rate of 29.8 ± 6.06%, approximately 40.6% of the unfrozen control.
Management Implications
At present, cryopreservation of Sauger milt has limited efficacy at large scales, such as in the production of Saugeye using frozen Sauger sperm. Excessively high sperm- to-egg ratios needed for fertilization combined with the lower maximum fertility reached using frozen sperm precludes use of this technology in the hatchery system. For example, at the sperm concentration used for freezing, and the approximately 300,000 motile
316 sperm/mL needed for fertilization, about 450 mL of sperm per 450,000 eggs is required - which is approximately 900 straws.
At this time, sperm cryopreservation is not feasible during the production of
Saugeye. Further development of the cryopreservation process should focus on improving the fertility of frozen-thawed sperm. We have shown that frozen-thawed
Sauger sperm, while motile to an acceptable degree, had significantly damaged plasma membranes and intracellular signaling pathways that do not respond to motility activation the way fresh sperm does. By addressing these problems and potentially making fertility equivalent to fresh sperm, a more reasonable amount of sperm applied per quart of eggs would make cryopreservation’s large-scale application efficacy more likely.
317
References
Bergeron, A., G. Vandenberg, D. Proulx, and J. L. Bailey. 2002. Comparison of
extenders, dilution ratios and theophylline addition on the function of
cryopreserved Walleye semen. Theriogenology 57(3):1061–1071.
Blawut, B., B. Wolfe, C. R. Moraes, S. A. Ludsin, and M. A. Coutinho da Silva. 2018.
Increasing Saugeye (S. vitreus × S. canadensis) production efficiency in a
hatchery setting using assisted reproduction technologies. Aquaculture 495:21–
26.
Blawut, B., B. Wolfe, C. R. Moraes, D. Sweet, S. A. Ludsin, and M. A. Coutinho da
Silva. 2020a. Testicular collections as a technique to increase milt availability in
Sauger (sander canadensis). Animal Reproduction Science 212:106240.
Blawut, B., B. Wolfe, C. R. Moraes, S. A. Ludsin, and M. A. C. da Silva. 2020b. Use of
Hypertonic Media to Cryopreserve Sauger Spermatozoa. North American Journal
of Aquaculture 82(1):84–91.
Jr, J. R. S., and S. A. Flickinger. 1995. Field Collection and Short-Term Storage of
Walleye Semen. The Progressive Fish-Culturist 57(3):182–187.
Moore, A. 1987. Short-Term Storage and Cryopreservation of Walleye Semen.
Progressive Fish-Culturist 49(1):40–43.
318
A.) B.)
Sperm Pools / Air Divider Space
Layered Ice Ice Water Packs
Figure 24. Design of extended Sauger milt transport boxes. (A.) Ice packs are layered for
~ 2/3 of the depth of the box with 2 hand towels/ surgical towels to prevent contact between milt and ice. (B.) Extended semen vials are placed in a water-proof bag and allowed to float in an ice water slurry during transport. Cardboard or plastic is used to create a sloped surface allowing an ice-free pool of water for extended milt to rest in/on.
319
A.
B. C.
Figure 25. Testicular harvest and sperm extraction from Sauger (Sander canadensis).
(A) Sauger broodstock with the testis removed from the body cavity but still attached at the urogenital pore. (B) Sauger testis has been removed and transferred to a petri dish, lacerated along the lobe long-axis, and extender added to homogenize sperm. (C) End product of testicular harvest, a homogenized sample of testicular spermatozoa. Note the pink color due to blood contamination.
320
100 a
a
)
%
(
n 75
o
i t
a a
z
i
l
i
t
r e
F 50
d
e
y
E
y a
D 25
9
0
Fresh − Control Testicular 20,000 Testicular 100,000 Fertilization Treatment
Figure 26. Comparison of fertilization among stripped sperm (control) and testicular sperm at 20,000 MSE and 100,000 MSE in large-scale fertilization experiments (2019).
321
Figure 27. Percent of fertilized Saugeye eggs (mean ± 1 SE) at 8 d post fertilization in the three sperm-to-egg ratio treatments (20,000:1, 50,000:1, and 100,000:1). An egg was considered fertilized if it had reached the eyed-egg embryonic stage. Each treatment was replicated 3 times.
322
A. B.
Figure 28. Sperm quality analysis tools for use during Sauger milt analysis. (A) Model
569A Video Microscope (Animal Reproduction Systems, Chino, CA, USA). (B) 590B
Equine Densimeter (ARS).
323
A. B. C.
Figure 29. Examples of a range of Sauger sperm total motilities seen during milt analysis
[(A) 0%, (B) 50%, (C) 95%).
324
Figure 30. Least-squares linear regression results of natural log transformed hemocytometer counts on natural log transformed 590b equine densimeter counts (n =
29) including a 95% confidence interval (Blawut et al., 2018b).
325
A. B. C. D. E. F.
G. H.
Figure 31. Images of the straw filling, sealing, equilibration, and cryopreservation processes. (A) Fill straws until the appropriately sized air bubble (a few mm) is present.
(B) Remove straws from the sperm sample and continue to move semen through straw until the plug is wet. (C -D) Press the ends of the straw into PVC powder to form a few mm thick plug. (E) Dip the powder plug into water to for a seal. (F) “Crack the whip” move the air bubble into the center of the straw and wipe exterior of the plug end with a tissue/ lab wipe. (G) Place filled and sealed straws onto the floating rack and equilibrate for 10 min at 5C. (H) After equilibration, place floating rack into liquid nitrogen contained and cool for 10 min before plunging.
326
Table 13. Sperm quality parameters (total motility, progressive motility, velocity and viability) for both stripped and testicular sperm
during a 4-d storage period at 5°C.
Storage Time Parameter Sperm Type Day 0 Day 1 Day 2 Day 3
Total Motility (%) Testicular 71.36 ± 2.64 69.56 ± 2.64 72.03 ± 2.64 66.34 ± 2.64
NS Stripped 68.49 ± 3.74 70.45 ± 3.74 63.70 ± 3.74 63.50 ± 3.74
Testicular 119.50 ± 4.99 a 121.29 ± 4.99 a 105.80 ± 4.99 ab 95.60 ± 4.99 b Velocity (µm/s) a ab ab ab
32 Stripped 122.05 ± 7.05 111.58 ± 7.05 110.50 ± 7.05 96.66 ± 7.05
7
Testicular 95.75 ± 0.79 96.00 ± 0.79 94.88 ± 0.79 95.00 ± 0.79 Viability (%) NS Stripped 94.50 ± 1.12 95.50 ± 1.12 96.50 ± 1.12 94.75 ± 1.12
a,b Means with a similar superscript within a block do not differ (repeated measure ANOVA, Tukey’s HSD).
NS No statistical differences among treatments.
327
Table 14. Sauger milt characteristics (mean ± 1 SE) before and after treatment with hCG or saline (hCG n=16, control=17).
Treatment hCG Control
Quality Parameter Pre Post ∆ Pre Post ∆
Milt Volume (mL/kg) 0.64 ± 0.15 0.93 ± 0.19 0.28 ± 0.27a 0.94 ± 0.14 0.31 ± 0.05 -0.63 ± 0.12b
Sperm Concentration 5.42 × 1010 ± 3.95 × 1010 ± -4.54 × 1010 ± 4.99 × 1010 ± 5.66 × 1010 ± 1.20 × 10 10 ±
(Sperm/mL) 6.15 × 109 3.66× 109 1.89 × 109 a 6.00 × 109 6.27 × 109 3.31 × 1010 a
Total Sperm Production 4.11 × 1010 ± 3.11 × 1010 ± -1.00 × 1010 ± 5.21 × 10 10 ± 1.68 × 1010 ± -3.53 × 1010 ±
32
8 (Sperm/kg) 1.23 × 1010 4.67 × 109 1.50 × 1010 a 1.15 × 10 10 3.44 × 109 1.28 × 1010 a
Gain scores (Δ) were calculated by subtracting the initial measurement from the final measurement. Gain scores within the same row
with different superscripts (a, b) differ (p < 0.05), based on repeated measures ANOVA.
a,b Mean values (± SEM) with different superscript letters within a row differ significantly at P < 0.05.
328
Table 15. Motility characteristics of Sauger (Sander canadensis) spermatozoa cryopreserved using 7.5% methanol in modified ringers lactate compared to an unfrozen control (n = 4).
Sperm Type Motility Characteristic Fresh Cryopreserved
Total Motility 62.1 ± 2.78 a 44.2 ± 2.78 b
Amplitude of Head Displacement (µm) 10.89 ± 1.45 6.92 ± 1.46
Linearity (%) 37.00 ± 3.34 a 25.00 ± 3.34 b
Straightness (%) 46.5 ± 4.69 48.1 ± 4.69
Velocity Average Path (µm/s) 122.60 ± 5.21 a 66.80 ± 5.21b
Straight Line Velocity (µm/s) 59.10 ± 3.38 a 30.00 ± 3.38 b
Curvilinear Velocity (µm/s) 169.00 ± 8.54 a 131.00 ± 8.54 b
Beat Cross Frequency (Hz) 16.40 ± 2.53 b 28.20 ± 2.53 b
Wobble (%) 73.30 ± 1.07 a 51.30 ± 1.07 b
a,b Mean values (± SEM) with different superscript letters within a row differ significantly at P < 0.05.
329
Table 16. Equine 590b Densimeter convertsion chart for Sauger (Sander canadensis) milt concentration assessment.
Densimeter Densimeter Densimeter Sperm Pool Densimeter Sperm Pool (Millions per (Millions Reading Concentration Reading Concentration mL) per mL) 53 5.30E+07 7.218E+09 125 1.25E+08 7.218E+09 54 5.40E+07 7.411E+09 126 1.26E+08 7.411E+09 55 5.50E+07 7.606E+09 127 1.27E+08 7.606E+09 56 5.60E+07 7.803E+09 128 1.28E+08 7.803E+09 57 5.70E+07 8.001E+09 129 1.29E+08 8.001E+09 58 5.80E+07 8.201E+09 130 1.30E+08 8.201E+09 59 5.90E+07 8.402E+09 131 1.31E+08 8.402E+09 60 6.00E+07 8.604E+09 132 1.32E+08 8.604E+09 61 6.10E+07 8.808E+09 133 1.33E+08 8.808E+09 62 6.20E+07 9.013E+09 134 1.34E+08 9.013E+09 63 6.30E+07 9.220E+09 135 1.35E+08 9.220E+09 64 6.40E+07 9.428E+09 136 1.36E+08 9.428E+09 65 6.50E+07 9.637E+09 137 1.37E+08 9.637E+09 66 6.60E+07 9.848E+09 138 1.38E+08 9.848E+09 67 6.70E+07 1.006E+10 139 1.39E+08 1.006E+10 68 6.80E+07 1.027E+10 140 1.40E+08 1.027E+10 69 6.90E+07 1.049E+10 141 1.41E+08 1.049E+10 70 7.00E+07 1.070E+10 142 1.42E+08 1.070E+10 71 7.10E+07 1.092E+10 143 1.43E+08 1.092E+10 72 7.20E+07 1.114E+10 144 1.44E+08 1.114E+10 73 7.30E+07 1.136E+10 145 1.45E+08 1.136E+10 74 7.40E+07 1.158E+10 146 1.46E+08 1.158E+10 75 7.50E+07 1.180E+10 147 1.47E+08 1.180E+10 76 7.60E+07 1.203E+10 148 1.48E+08 1.203E+10 77 7.70E+07 1.225E+10 149 1.49E+08 1.225E+10 78 7.80E+07 1.248E+10 150 1.50E+08 1.248E+10 79 7.90E+07 1.270E+10 151 1.51E+08 1.270E+10 80 8.00E+07 1.293E+10 152 1.52E+08 1.293E+10 81 8.10E+07 1.316E+10 153 1.53E+08 1.316E+10 82 8.20E+07 1.339E+10 154 1.54E+08 1.339E+10 83 8.30E+07 1.363E+10 155 1.55E+08 1.363E+10 84 8.40E+07 1.386E+10 156 1.56E+08 1.386E+10 85 8.50E+07 1.409E+10 157 1.57E+08 1.409E+10 330
Table 16. Continued Densimeter Densimeter Densimeter Sperm Pool Densimeter Sperm Pool (Millions per (Millions Reading Concentration Reading Concentration mL) per mL) 86 8.60E+07 1.433E+10 158 1.58E+08 1.433E+10 87 8.70E+07 1.456E+10 159 1.59E+08 1.456E+10 88 8.80E+07 1.480E+10 160 1.60E+08 1.480E+10 89 8.90E+07 1.504E+10 161 1.61E+08 1.504E+10 90 9.00E+07 1.528E+10 162 1.62E+08 1.528E+10 92 9.20E+07 1.576E+10 164 1.64E+08 1.576E+10 93 9.30E+07 1.601E+10 165 1.65E+08 1.601E+10 94 9.40E+07 1.625E+10 166 1.66E+08 1.625E+10 95 9.50E+07 1.650E+10 167 1.67E+08 1.650E+10 96 9.60E+07 1.674E+10 168 1.68E+08 1.674E+10 97 9.70E+07 1.699E+10 169 1.69E+08 1.699E+10 98 9.80E+07 1.724E+10 170 1.70E+08 1.724E+10 99 9.90E+07 1.749E+10 171 1.71E+08 1.749E+10 100 1.00E+08 1.774E+10 172 1.72E+08 1.774E+10 101 1.01E+08 1.799E+10 173 1.73E+08 1.799E+10 102 1.02E+08 1.825E+10 174 1.74E+08 1.825E+10 103 1.03E+08 1.850E+10 175 1.75E+08 1.850E+10 104 1.04E+08 1.875E+10 176 1.76E+08 1.875E+10 105 1.05E+08 1.901E+10 177 1.77E+08 1.901E+10 106 1.06E+08 1.927E+10 178 1.78E+08 1.927E+10 107 1.07E+08 1.953E+10 179 1.79E+08 1.953E+10 108 1.08E+08 1.978E+10 180 1.80E+08 1.978E+10 109 1.09E+08 2.004E+10 181 1.81E+08 2.004E+10 110 1.10E+08 2.031E+10 182 1.82E+08 2.031E+10 111 1.11E+08 2.057E+10 183 1.83E+08 2.057E+10 112 1.12E+08 2.083E+10 184 1.84E+08 2.083E+10 113 1.13E+08 2.109E+10 185 1.85E+08 2.109E+10 114 1.14E+08 2.136E+10 186 1.86E+08 2.136E+10 115 1.15E+08 2.163E+10 187 1.87E+08 2.163E+10 116 1.16E+08 2.189E+10 188 1.88E+08 2.189E+10 117 1.17E+08 2.216E+10 189 1.89E+08 2.216E+10 118 1.18E+08 2.243E+10 190 1.90E+08 2.243E+10 119 1.19E+08 2.270E+10 191 1.91E+08 2.270E+10 120 1.20E+08 2.297E+10 192 1.92E+08 2.297E+10 121 1.21E+08 2.324E+10 193 1.93E+08 2.324E+10 122 1.22E+08 2.351E+10 194 1.94E+08 2.351E+10
331
Table 17. Item list and vendor information for equipment used during reproductive assessment and management of Sauger (Sander canadensis).
Item Purpose Vendor 1.0 mL syringe Milt collection Fisher Scientific 14-817-25 2.0 mL Eppendorf Milt storage Fisher Scientific 05-402-24C tubes 5.0 mL cryovial Pooled milt storage Fisher Scientific 03-337-7H 3 inch Petri Dish Testicular harvest Fisher Scientific S33580A Scalpel + #10 Testicular harvest Fisher Scientific 08-927-5A blade Forceps Testicular harvest Fisher Scientific 13-812-10 Chorulon hCG, spawning aide Merck Animal Health Equine 590b Animal Reproduction Systems Concentration assessment Densimeter Kit 590B-MOD1 Animal Reproduction Systems 537- Cuvettes and Caps Concentration assessment 204-205C/100 ARS Pipette Tips Concentration assessment Animal Reproduction Systems ARS Microscope Animal Reproduction Systems Motility assessment and Monitor VMK-304 1.5 mL Eppendorf Milt dilution for Fisher Scientific 05-402 tubes assessment Microscope slides Motility assessment Fisher Scientific 12-550-A4 Microscope Cover Motility assessment Fisher Scientific 115-183-89 Slips Bovine serum Prevents sperm from Sigma Aldrich A6003 albumin sticking to the glass NaOH (1N)/ HCl Fisher Scientific SS266-1 / Fisher pH adjustments (1N) Scientific SA48-500 Pipettes Sperm dilution Fisher Scientific 14-388-100 Pipette Tips 300-1000 µL Fisher Scientific 02-707-480 30-300 µL Fisher Scientific 02-707-479 0.1 – 10 µL Fisher Scientific 02-707-474 0.1 – 20 µL Fisher Scientific 02-707-475 15 and 50 mL Extending semen for Fisher Scientific 14-959-49A Falcon tubes freezing
332
Table 17. Continued Item Purpose Vendor
Modified Ringers Freezing media Fisher Scientific AAJ67572AP Lactate Bovine serum Extracellular Sigma Aldrich A6003 albumin cryoprotectant Methanol Internal cryoprotectant Sigma Aldrich 34860-1L-R 0.5 mL sealing Animal Reproduction Systems 537- Sperm freezing unit straws 670-CLE/2500 Straw sealing Animal Reproduction Systems 537- Sealing straws powder 671 Fills multiple straws at a Animal Reproduction Systems FSF- Straw filler time 101-KIT Liquid nitrogen Cooling straws in Animal Reproduction Systems FSR- floating rack nitrogen vapor 101 Liquid Nitrogen Freezing and storage - Animal Reproduction Systems 537- Transfer Dewar Transporting LN2 721 Animal Reproduction Science 537- Storage Dewar Long-term straw storage 711 Handling and retrieving Animal Reproduction Systems SFF- Tongs/Forceps straws 101-12 Nitrogen canes and Animal Reproduction Systems 537- Straw storage goblets 734/75 & 537-733/150 Animal Reproduction Systems FCT- Cane labels Sample identification WHT/100
333