Sperm Cryopreservation Affects Postthaw Motility, but Not Embryogenesis Or Larval Growth in the Brazilian ﬁsh Brycon Insignis (Characiformes) A.T.M

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Theriogenology 78 (2012) 803–810 www.theriojournal.com

Sperm cryopreservation affects postthaw motility, but not embryogenesis or larval growth in the Brazilian ﬁsh Brycon insignis (Characiformes) A.T.M. Viveirosa,*, Z.A. Isaúa,1, D. Caneppeleb, M.C. Leala a Department of Animal Science, Federal University of Lavras (UFLA), Lavras, Minas Gerais, Brazil b Hydrobiology and Aquaculture Station of the Hydroelectric Company of São Paulo (CESP), Paraibuna, São Paulo, Brazil Received 2 October 2011; received in revised form 5 March 2012; accepted 17 March 2012

Abstract Sperm cryopreservation is an important method for preserving genetic information and facilitating artificial reproduction. The objective was to investigate whether the cryopreservation process affects postthaw sperm motility, embryogenesis, and larval growth in the fish Brycon insignis. Sperm was diluted in methyl glycol and Beltsville Thawing solution, frozen in a nitrogen vapor vessel (dry shipper) and stored in liquid nitrogen. Half of the samples were evaluated both subjectively (% of motile sperm and motility quality score—arbitrary grading system from 0 [no movement] to 5 [rapidly swimming sperm]) and in a computer- assisted sperm analyzer (CASA; percentage of motile sperm and velocity). The other half was used for fertilization and the evaluation of embryogenesis (cleavage and gastrula stages), hatching rate, percentage of larvae with normal development and larval growth up to 112 days posthatching (dph). Fresh sperm was analyzed subjectively (percentage of motile sperm and motility quality score) and used as the control. In the subjective analysis, sperm motility significantly decreased from 100% motile sperm and quality score of 5 in fresh sperm to 54% motile sperm and quality score of 3 after thawing. Under computer-assisted sperm analyzer evaluation, postthaw sperm had 67% motile sperm, 122 ␮m/sec of curvilinear velocity, 87 ␮m/sec of straight-line velocity and 103 ␮m/sec of average path velocity. There were no significant differences between progenies (pooled data) for the percentage of viable embryos in cleavage (62%) or gastrula stages (24%) or in the hatching rate (24%), percentage of normal hatched larvae (93%), larval body weight (39.8 g), or standard length (12.7 cm) at 112 days posthatching. Based on these findings, cryopreserved sperm can be used as a tool to restore the population of endangered species, such as B. insignis, as well as for aquaculture purposes, without any concern regarding quality of the offspring. © 2012 Elsevier Inc. Open access under the Elsevier OA license.

Keywords: Sperm quality; CASA; Freezing; Semen; Teleost

1. Introduction

The genus Brycon belongs to the order Characi- formes, family Characidae, subfamily Bryconinae, and comprises more than 70 ﬁsh species distributed 1 This study is part of Z.A. Isaú’s PhD project. in Central and South America [1]. Brycon insignis ϩ ϩ * Corresponding author. Tel.: 55 35 38291223; fax: 55 35 (Steindachner, 1877), known as tiete tetra in English 38291231. E-mail address: [email protected]ﬂa.br; anatmviveiros@ and piabanha in Portuguese, is native to the Paraiba hotmail.com (A.T.M. Viveiros). do Sul River Basin [2] in the states of São Paulo,

Minas Gerais and Rio de Janeiro (southeastern Bra- The aim of the present study was to investigate zil). In the past, B. insignis was the fourth most whether the sperm cryopreservation process affects captured species in commercial fishing in this basin, postthaw sperm motility, embryogenesis and larval playing an important role in the regional economy growth in Brycon insignis. and sport fishing [3,4]. Many Brazilian fish species, including B. insignis, migrate upstream to spawn. 2. Materials and methods However, the reproductive cycle of some migratory species has been disrupted due to overfishing, pollu- 2.1. Fish handling, gamete collection, and initial tion, and changes to the course of rivers caused by evaluation the construction of hydroelectric dams. The genus All fish were handled in compliance with published Brycon is highly affected by these environmental guidelines for animal experimentation [23]. The B. in- changes and many species are on the red list of signis broodfish were selected from circular tanks at the Brazilian threatened fauna (e.g., B. insignis, B. nat- Hydrobiology and Aquaculture Station of the Energy tererii, B. opalinus, and B. orbignyanus [5]). Company of São Paulo (CESP) in Paraibuna, São The use of frozen or cryopreserved sperm is one Paulo, Brazil (23°23=10” S; 45°39=44” W) during the method for preserving genetic information across gen- spawning season (January and February). erations and facilitating artificial reproduction proce- Males at 4–5 yr of age and 245–335 g of body dures. Cryopreserved sperm kept in germ plasm banks weight (BW) with detectable running sperm under soft for an indefinite interval may help ensure genetic di- abdominal pressure were given a single intramuscular versity and allows reproductive success for population dose of carp pituitary extract (Argent Chemical Labo- management strategies, with cost reductions in stock ratory, Redmond, WA, USA) at 3 mg/kg BW and maintenance and potentially greater efficiency in selec- maintained at approximately 25 °C. Eight h later, the tive breeding [6]. Our research team recently developed urogenital papilla was carefully dried and sperm was a suitable protocol for B. insignis sperm cryopreserva- hand-stripped directly into test tubes. Sperm collection tion using methyl glycol as the cryoprotectant, com- was carried out at room temperature (approximately 25 bined with commercial extenders, such as Beltsville °C). Soon after collection, tubes containing sperm were Thawing solution (BTS) [7]. The same freezing me- placed in a polystyrene box containing crushed ice (5 Ϯ dium has successfully been employed with two other 2 °C). Contamination of sperm with water, urine, or species from the same genus: B. orbignyanus [8] and B. feces was carefully avoided. Immediately after collec- nattererii [9]. Although the application of cryopre- tion, 5 ␮L of each sample were placed on a glass slide served sperm has been encouraged [10], the perfor- and observed under a light microscope (Model L1000; mance of the offspring has not been fully explored [11] Bioval, Jiangbei, China) at magnification ϫ 400. As and a cautionary approach should therefore be taken fish sperm in seminal plasma should be immotile, any before this technique is widely used in conservation sperm motility observed was attributed to urine or wa- programs. ter contamination and the sample was discarded. In Sperm cryopreservation impairs the physical cell immotile samples (N ϭ 15 males), sperm motility was structure and DNA and exerts a negative effect on both ␮ triggered with 25 L of 1% NaHCO3 (Labsynth, Dia- sperm physiology and fertility in fish [12–16].Itisnot dema, SP, Brazil) as the activating agent and subjec- clear, however, whether this damage affects embryo tively estimated under a light microscope following the development and progeny viability [17]. Recent evalu- methodology established for species of the genus Bry- ations have been carried out on the survival and devel- con [7–9,24]. Only samples with at least 90% motile opment of embryos and juveniles of the rainbow/steel- sperm were used in the subsequent analyses. Motility head trout Oncorhynchus mykiss [17–19], as well as qualitative scores were assigned using an arbitrary grading larval malformations and ploidy of the common carp system from 0 (no movement) to 5 (rapidly swimming Cyprinus carpio [20,21] and African catfish Clarias sperm) [7]. Sperm volume was determined. The sperm gariepinus [22], which originated from cryopreserved (approximately 2 mL) from each male was centrifuged at sperm, with no significant differences between these 4500 ϫ g for 30 min at room temperature and osmolality and those which originated from fresh (control) sperm. of the seminal plasma was measured cryoscopically However, there have been no similar studies involving (Semi-Micro Osmometer K-7400, Knauer, Berlin, Ger- neotropical fish. many). A.T.M. Viveiros et al. / Theriogenology 78 (2012) 803–810 805

To harvest oocytes, three females at approximately 8 Lavras (UFLA), Lavras, Minas Gerais, Brazil, as de- yr of age (967 Ϯ 153 g BW) with detectable running scribed below. oocytes under soft abdominal pressure received two 2.3. Postthaw sperm motility and velocity doses of carp pituitary extract (0.5 and 4.5 mg/kg BW im) at a 12-h interval. Along with the second dose, the Half of the replicate straws (N ϭ 3 replicate straws ϫ females received a dose of human chorionic gonado- 6 males) were transferred back to the nitrogen vapor tropin (Pregnyl Schering-Plough, Kenilworth, NJ, vessel and transported by car from the CESP Aquacul- USA) at 1500 IU hCG/kg BW im and were hand- ture Station to the UFLA Laboratory of Semen Tech- stripped 8 h later at approximately 25 °C. Spawning nology (approximately 450 km). Upon arrival, the weight, spawning index (spawning weight ϫ 100/BW), straws were stored in liquid nitrogen. Approximately 1 number of oocytes/g spawned and number of oocytes yr later, the straws were thawed in a water bath at 60 °C per female were calculated. The oocytes were used for for 8 sec [7]. Postthaw sperm motility was estimated the fertilization trial (described below). immediately both subjectively, as described for fresh All analyses described above using fresh sperm and sperm, and objectively, using the CASA system. Mo- the fertilization trial were carried out at the facilities of tility was first triggered in Eppendorf tubes and the the CESP Aquaculture Station. sample was then transferred to a Leja counting chamber slide placed under a phase-contrast microscope (Nikon 2.2. Sperm cryopreservation Eclipse E200, Tokyo, Japan) at magnification ϫ 100 Sperm was cryopreserved following the methodol- with a green filter and pH 1 position. The microscope ogy previously described for B. insignis [7]. A freezing was connected to a video camera (Basler Vision Tech- medium comprised of a combination of methyl glycol nologies A602FC, Ahrensburg, Germany) generating

[CH3O(CH2)2OH, also known as ethylene glycol 100 images/sec; video recording started 10 sec postac- monomethyl ether; Vetec Química Fina Ltda, Duque de tivation. Each image was analyzed using the standard Caxias, RJ, Brazil] and BTS (Minitüb, Tiefenbach/ settings for ﬁsh by Sperm Class Analyzer software Landshut, Germany) was used. The BTS is a boar (SCA 2010, Microptics, S.L. Version 5.1, Barcelona, sperm extender containing 79.9% glucose, 12.7% so- Spain). Sperm was considered immotile when velocity Ͻ ␮ dium citrate, 2.7% EDTA, 2.7% NaHCO3, 1.6% KCl, was 20 m/sec. Although the SCA (Microptics) si- and 0.4% gentamycin sulfate; 318 mOsm were pre- multaneously assesses more than 15 sperm motility pared at 5% with deionized water (5 g BTS in 95 g endpoints, for brevity, only curvilinear velocity (VCL), deionized water) and adjusted to a pH of 7.6. Sperm straight line velocity (VSL) and average path velocity samples (N ϭ 6 males) were diluted to a ﬁnal propor- (VAP) were considered for analysis. To determine tion of 10% sperm, 10% methyl glycol, and 80% BTS. these velocities, each individual spermatozoon (N ϭ The samples were then loaded into 0.5-mL straws (N ϭ approximately 300 sperm per straw) was followed 6 replicate straws per male) and frozen in a nitrogen throughout the images and sperm trajectory was calcu- vapor vessel (dry vapor shipper; YDH-8, Chengdu lated. Golden Phoenix Liquid Nitrogen Container, Co., Ltd., 2.4. Fertilization process Chengdu, Sichuan, China) at approximately Ϫ170 °C. Using this methodology, straws reach Ϫ170 °C after One replicate straw from each male was thawed and approximately 329 sec at a freezing rate of approxi- the contents were pooled (N ϭ 3 pools of thawed sperm mately Ϫ35.6 °C/min between 21 and Ϫ170 °C [24]. from 6 males). Next, 2.3 mL of each pool of sperm was The straws were transferred to liquid nitrogen (cryo- mixed with 50 g of oocytes from each female (N ϭ 3). genic tank YDS-20, Chengdu Golden Phoenix Liquid To control oocyte quality, three pools of freshly col- Nitrogen Container, Co., Ltd., Chengdu, Sichuan, lected sperm from three other males (one pool contain- China) at Ϫ196 °C within 20 to 24 h for storage. ing fresh sperm of three males) were diluted 1:25 in Two days after freezing, half of the frozen replicate BTS and 2.3 mL were used to fertilize three aliquots of straws were thawed at 60 °C for 8 sec and used for the 50 g of oocytes from the same females. Fresh sperm fertilization trial at the facilities of the Aquaculture was challenged for its fertilization ability at a higher Station. The remaining straws were used for sperm dilution ratio due to its higher quality in comparison motility and velocity evaluations using a computer- with thawed sperm, as suggested in our previous study assisted sperm analyzer (CASA) at the Laboratory of [8]. Fertilization was initiated with the addition of 46 Semen Technology of the Universidade Federal de mL of tank water and mixed for 1 min. Subsequently, 806 A.T.M. Viveiros et al. / Theriogenology 78 (2012) 803–810

Table 1 progeny) were weighed and measured using an ichthyo- ϭ Ϯ Male body weight and fresh sperm features (N 15 males; mean meter. SD; minimum-maximum values) of Brycon insignis after carp pituitary treatment. 2.7. Statistical analysis Features Mean Ϯ SD Range Values are expressed as mean Ϯ SD. Statistical Body weight (g) 277 Ϯ 33 245–335 Subjective motility (%) 98 Ϯ 4 90–100 analyses were conducted with the R software program, Motility quality score (0–5)* 4.7 Ϯ 0.5 4–5 version 2.12.0 [26]. All data were tested for normal Sperm volume (mL) 4.4 Ϯ 2.5 1–8.8 distribution using the univariate procedure and for sig- Ϯ Seminal plasma osmolality (mOsm/kg) 340 20 306–390 niﬁcant differences using ANOVA. The level of signif- * A motility qualitative score was assigned using an arbitrary grad- icance for all statistical tests was set at 5% (P Ͻ 0.05). ing system from 0 (no movement) to 5 (rapidly swimming spermatozoa). 3. Results

184 mL of tank water was added and the samples were 3.1. Initial gamete evaluation mixed for another 2 min. Finally, the eggs were trans- The following mean sperm values were found for B. ferred to six 250-L funnel-type incubation units (three insignis males (N ϭ 15): 98% motile sperm, motility batches of oocytes fertilized with cryopreserved sperm quality score of 4.7, volume of 4.4 mL and seminal and three batches fertilized with fresh-control sperm) plasma osmolality of 340 mOsm/kg (Table 1). For fe- and incubated in a flow-through system at approxi- males, recently stripped oocytes had a mean spawning mately 23 °C. weight of 164 Ϯ 13 g, the spawning index was 16.9 Ϯ 2.5. Embryogenesis 0.1%, with 537 Ϯ 12 oocytes per g spawned and there were 87,803 Ϯ 6,367 oocytes per female. Approximately 80 embryos from each incubation unit (N ϭ 3 incubation units per progeny) were ran- 3.2. Postthaw sperm motility and velocity domly evaluated 1.5 and 10 h after fertilization, when In fresh sperm, subjective motility was 100% and embryos should, respectively be at the cleavage and quality score was 5, whereas these figures were 54% gastrula stages, with approximately 90% epiboly, based Ͻ on our previous study [25]. The embryos were placed in and 3 in postthaw sperm, respectively (P 0.05). In the Petri dishes, examined under a stereomicroscope (TIM CASA evaluation, postthaw sperm had 67% motile ␮ ␮ 2T Opton, Anatomic Microscopes and Anatomic Mod- sperm, a VCL of 122 m/sec, VSL of 87 m/sec, and ␮ els, Cotia, SP, Brazil) and placed back in the incubation VAP of 103 m/sec (Table 2). unit. The hatching rate and percentage of larvae with normal morphologic development (expressed as per- cent of normal larvae) were calculated. Recently hatched larvae were examined under the stereomicroscope and photographed with a digital camera (DSC- Table 2 W35, Sony Electronic, Inc., New York, NY, USA). Motility features (N ϭ 6 males; mean Ϯ SD) of fresh (subjective evaluation) and cryopreserved (subjective and CASA evaluations) 2.6. Larval growth sperm from Brycon insignis. Features Fresh Cryopreserved At 7 days posthatching (dph), all larvae were trans- Ϯ a Ϯ b ferred to two concrete tanks (one tank for each progeny) Motility quality score (0–5)* 5 0 3 1 Subjective motility (%) 100 Ϯ 0a 54 Ϯ 13b and 18 larvae per progeny type were fixed with 70% CASA motility (%) ND 67 Ϯ 9 ethanol and kept under refrigeration. Fixed larvae were Curvilinear velocity (␮m/sec) ND 122 Ϯ 26 individually weighed (Semi-Analytic Scale JA 3003n Straight-line velocity (␮m/sec) ND 87 Ϯ 22 Bioprecisa, Equipar, Ltda, Curitiba, PR, Brazil), photo- Average path velocity (␮m/sec) ND 103 Ϯ 26 graphed and measured (standard length; SL) using the Within a line, means without a common superscript letter differed (P Ͻ Adobe Photoshop CS4 Extend software (Adobe Systems 0.05). ϭ CASA, computer-assisted sperm analyzer; ND, not determined. Inc., San Jose, CA, USA). At 30 dph, fries (N 16 per * A motility qualitative score was assigned using an arbitrary grad- progeny) were weighed and measured using a common ing system from 0 (no movement) to 5 (rapidly swimming ruler. At 60 and 112 dph, fingerlings (N ϭ 35 per sperm). A.T.M. Viveiros et al. / Theriogenology 78 (2012) 803–810 807

3.3. Embryogenesis raises concerns regarding harm to the sperm during freezing and thawing. Some studies report altered cell Embryos originated from cryopreserved sperm de- membrane integrity and damage to cellular organelles veloped similar to those which originated from fresh [30,31]. sperm. Thus, the data from both progenies were pooled. Similar to other fish species, studies carried out on Ϯ At 1.5 h postfertilization, 62 20% of embryos were Characiformes have assessed the effects of cryopreser- viable and all were in the cleavage stage. At 10 to 11 h vation on sperm motility (subjectively or using CASA), Ϯ postfertilization, 24 10% of embryos were viable and vitality, mitochondrial function, and fertilization rates all were in the gastrula stage, with approximately 90% and most report a moderate reduction in at least one of epiboly. Hatching occurred 30 h postfertilization at a these parameters in comparison with fresh sperm Ϯ rate of 24 11% for both progenies. The percentage of [6,8,9,24,32,33]. In the present study, subjectively as- Ϯ larvae with normal morphologic development was 93 sessed B. insignis sperm motility decreased from 100% Ͼ 7%, with no difference (P 0.05) between progenies. motile sperm and a quality score of 5 in fresh sperm to 3.4. Larval growth 54% motile sperm and a quality score of 3 in postthaw sperm. Progenies originated from cryopreserved and fresh As fresh sperm was collected approximately 450 km Ͼ sperm developed similarly (P 0.05) during the 112- from the laboratory at which the CASA system is lo- day experiment. Thus, data from both progenies were cated and the transportation of sperm in the liquid phase Ϯ pooled. The larvae increased from a mean of 1.46 leads to an enormous decrease in quality (data not Ϯ Ϯ 0.27 cm SL and 0.04 0.01 g BW at 7 dph to 12.7 shown), fresh sperm was analyzed only with regard to Ϯ 1.8 cm SL and 39.8 16.3 g BW at 112 dph. subjective motility. The use of CASA for the evaluation of sperm motility is important, as the percentage of 4. Discussion motile sperm and sperm velocity are associated with reproductive success in fish [20,34–36]. However, Following development of a protocol for cryopre- there is a great necessity for the standardization of serving sperm from B. insignis in a previous investiga- CASA settings to allow comparisons among studies tion carried out by our research group [7], the present and fish species. The following text presents data pub- study investigated the effects of cryopreservation on lished on the sperm of species of Characiformes eval- postthaw sperm motility, embryogenesis (cleavage and uated using CASA with different numbers of frames gastrula stages), hatching rate and larval growth. Fresh per sec. In the present study, B. insignis sperm cryo- gamete quality was also evaluated. Fresh sperm motil- preserved in methyl glycol and BTS yielded 67% mo- ity (98% motile sperm and quality score of 4.7), volume tile sperm, a VCL of 122 ␮m/sec, VSL of 87 ␮m/sec, (4.4 mL) and seminal plasma osmolality (350 mOsmol/ and VAP of 103 ␮m/sec when analyzed using CASA at kg) from the 15 males used in this study were all within 100 frames per sec. In a previous study, sperm from the the range previously reported for this species [7,25] and same species was frozen in DMSO, glucose, and egg others from the subfamily Bryconinae [27]. Recently yolk in six dilution ratios and evaluated using CASA at stripped oocytes and spawning characteristics were all 50 frames per sec and the highest values were observed similar to previous observations for this species at a dilution ratio of 1:6 (sperm:extender): 86% motile [25,28]. Better knowledge regarding the characteristics sperm, a VCL of 82 ␮m/sec, VSL of 25 ␮m/sec, and of fresh sperm and oocytes is necessary to evaluate VAP of 68 ␮m/sec; however, when the dilution ratio gamete quality in commercial hatcheries before artifi- was increased to 1:10, postthaw quality decreased sig- cial reproduction in laboratories and experiments. nificantly to 22% motile sperm, a VCL of 10 ␮m/sec, ␮ ␮ 4.1. Postthaw sperm motility and velocity VSL of 1 m/sec, and VAP of 9 m/sec [4].Intwo other studies, postthaw sperm was evaluated using The cryopreservation process involves temperature CASA at 25 frames per sec. Pirapitinga (Piaractus reduction, cellular dehydration, freezing, and thawing. brachypomus) sperm frozen in methyl glycol and glu- Cell damage in varying degrees is induced by distinct cose yielded 81% motile sperm, a VCL of 60 ␮m/sec, mechanisms in each cryopreservation phase and the VSL of 33 ␮m/sec, and VAP of 46 ␮m/sec [33] and functional state of the frozen-thawed sperm is the result streaked prochilod (Prochilodus lineatus) sperm frozen of damage accumulated throughout the process [29]. in methyl glycol and powdered coconut water (ACP) The use of sperm cryopreservation in aquatic species yielded 85% motile sperm, a VCL of 54 ␮m/sec, VSL 808 A.T.M. Viveiros et al. / Theriogenology 78 (2012) 803–810 of 24 ␮m/sec, and VAP of 37 ␮m/sec [34]. It has been observed, this technology should be considered a safe suggested that the maximal number of frames per sec and effective reproductive tool. should be captured by a video camera so the CASA In the present study, there were no significant dif- system can evaluate motility more precisely [37]. ferences in the percentage of larvae with normal development between progenies originated from fresh and 4.2. Embryogenesis cryopreserved sperm. In fact, the highest value (97% Due to the difficulty in obtaining high quality normal larvae) was observed for the progeny originated oocytes in the subfamily Bryconinae, few studies have from cryopreserved sperm. Therefore, the presence of tested postthaw sperm for fertilization. In B. insignis, malformed larvae cannot be attributed to the use of artificial reproduction using fresh sperm yielded hatch- cryopreserved sperm. Similar results were reported in ing rates ranging from 0 to 85% [28]. A recent study by C. carpio [20,21] and C. gariepinus [22], for which the our research group [25] had a fertilization rate of 26%, use of cryopreserved sperm did not affect the number of whereas rates Ͻ30% are frequent at the CESP Aqua- malformed larvae. Furthermore, malformed C. gariepi- culture Station. nus larvae were examined by chromosome preparations B. insignis embryos originated from fresh and cryo- and most of these larvae were diploid, indicating that preserved sperm were observed in two stages. At 1.5 h larval malformation did not result from genetic prob- postfertilization, all viable embryos of both progeny lems [22]. The presence of malformed larvae is more types (62% of the total number of embryos) were in the likely attributable to inadequacies in the incubation cleavage stage. At 10 to 11 h postfertilization, all viable process [21], egg quality, or rearing conditions of the embryos (24% of total number of embryos) were at the fish [22]. end of the gastrula stage (approximately 90% epiboly; near blastopore closure). The end of the gastrula stage 4.3. Larval growth (blastopore closure) is considered the most appropriate Larval growth for B. insignis (evaluated in terms of moment for determination of the fertilization rate, as standard length and body weight) was similar between nonfertilized eggs develop parthenogenetically before progenies originated from fresh and cryopreserved blastopore closure [38]. Accordingly, there was a sig- sperm throughout the 112-day evaluation period. This nificant decrease in viable embryos between the cleav- is the first report on larval survival for a species of age and gastrula stages in both progenies. The hatching Characiformes originated from cryopreserved sperm. rate was 24% and the percentage of larvae with normal Similarly, there was no difference in O. mykiss growth development was 93%, with no significant differences between progenies originated from fresh and cryopre- between progenies. Therefore, embryogenesis was not served sperm during a 313-day experimental period in affected by sperm cryopreservation, as embryos origi- a previous study [17]. Moreover, progenies of the chan- nated from both progenies developed similarly. In con- nel catfish Ictalurus punctatus originated from cryopre- trast, recently published studies on O. mykiss found that served and control groups developed similarly during 1 sperm cryopreservation affected embryonic develop- yr of rearing [39]. Converesely, striped bass Morone ment and developmental stages and reduced survival from fertilization to eyed-egg as well as from fertiliza- saxatilis larvae originated from cryopreserved sperm tion to larvae [17–19]. There was no difference in mean were significantly larger than the control after a rearing survival from eyed-egg to larvae; therefore, the authors period of 45 or 47 days [40]. concluded that the survival differences between the two 4.4. Conclusions progenies in prior stages resulted from decreased survival from fertilization to eyed-egg followed by similar The percentage of motile sperm and motility quality survival from eyed-egg to larvae [18]. Sperm cryo- score decreased significantly after sperm cryopreserva- preservation can entail DNA damage and cryopre- tion. However, development of embryos and larvae served DNA-damaged sperm has reduced viability, fer- which originated from cryopreserved sperm was similar tilization and hatching rates in comparison with fresh to that of progeny which originated from fresh sperm. sperm [19]. Perhaps B. insignis sperm is more resistant Based on these findings, cryopreserved sperm can be to the cryopreservation procedures used in the present used as a tool to restore the population of endangered study in comparison with O. mykiss sperm. Further- species, such as B. insignis, as well as for aquaculture more, until definitive results demonstrating serious purposes, without any concerns regarding offspring harmful consequences from cryotechnology in fish are quality. A.T.M. Viveiros et al. / Theriogenology 78 (2012) 803–810 809

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