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Sex Reversal in Yellow Perch (Perca Flavescens) to Produce Functional Neomale Sperm Donors

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Sex Reversal in Yellow Perch (Perca Flavescens) to Produce Functional Neomale Sperm Donors Sex Reversal in Yellow Perch (Perca flavescens) to Produce Functional Neomale Sperm Donors THESIS Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Graduate School of The Ohio State University By Kristen Towne Graduate Program in Environment and Natural Resources The Ohio State University 2016 Master’s Examination Committee: Dr. Konrad Dabrowski, Advisor Dr. Robert Gates Dr. Suzanne Gray Dr. Roman Lanno Copyrighted by Kristen Marie Towne 2016 Abstract Yellow perch (Perca flavescens) are a popular game and food fish in the Great Lakes region. However, intensive culture of this species in the North Central Region is still in its infancy. In this species, females grow faster and to a larger size than males, making them more valuable in aquaculture. In fact, a population of 1,000 female yellow perch can gross approximately $555 more than the same number of fish exhibiting a 1:1 male:female ratio at the current fillet value of $14 per pound. Attempts have been made to produce monosex populations of yellow perch through sex reversal, but have resulted in the formation of nonfunctional, intersex fish due to the initiation of treatment after the beginning of gonadal differentiation. Yellow perch require more elaborate techniques to conduct sex reversal than many other species due to the beginning of sexual differentiation occurring before the transition to formulated feeds. This thesis consists of two sex reversal experiments carried out in 2015 and 2016. The 2015 studies evaluated the success of hormonal masculinization using the synthetic steroid hormone 17α- methyltestosterone (MT) by two treatment methods, diet enrichment and immersion, as well as two initiation times with respect to fish size, 12 mm and 14 mm total length. This study also evaluated the feminizing capacity of another steroid hormone, estradiol-17β, in yellow perch exposed via immersion treatments beginning at the same two size classes. All fish were checked for spermiation by manual stripping in late January/early February ii of 2016. Although the groups that had been exposed to the masculinizing hormone beginning at 12 mm total length displayed significantly more spermiating males than those exposed beginning at 14 mm (P < 0.01), the control groups of both size classes also displayed a sex ratio significantly deviated toward males (P < 0.01). In addition, the 12 mm feminizing hormone-treated group displayed a sex ratio significantly deviated toward males (P < 0.05). During the hormone exposure phase, fish were maintained at water temperatures that were much higher than those found in Lake Erie during this time of ontogenetic development to optimize growth. Therefore, it was hypothesized that these high water temperatures inadvertently induced masculinization. In the spring of 2016, two additional sex reversal experiments were conducted, this time directly evaluating the effect of temperature on gonadal differentiation. Three egg ribbons were obtained from Mill Creek Perch Farm, LLC, and divided into two groups each: one exposed to water temperatures of 15.6 ± 1.9o C, and the other to temperatures of 24.0 ± 1.3o C for a duration of 43 days. The sex ratio of each group was checked by dissection in September of 2016, with no group showing a sex ratio that significantly differed from the expected 1:1 sex ratio (P < 0.05). The other temperature sex reversal experiment was conducted on the progeny of a sample of the control males from the 2015 hormonal sex reversal experiment. Similar to the other temperature experiment, 10 day old larvae were separated into warm (23.1 ± 0.2o C) and cool (16.4 ± 1.0o C) water systems for a duration of 23 days. The sex ratio was checked by dissection in October of 2016. Of the five fathers evaluated, two produced progeny populations that significantly deviated toward females (P < 0.05), indicating that these two fish were likely masculinized females/neomales. The iii remaining three fathers produced progeny populations that did not significantly differ from the expected sex ratio. Additionally, the sex ratios of individual males of the warm groups did not significantly differ from the cool groups (P < 0.05). The results from these two temperatures used in these experiments indicate that high water temperatures do not affect gonadal differentiation in juvenile yellow perch. Therefore, the skewed sex ratios in the control groups of the 2015 hormonal sex reversal experiment may have been caused by some environmental or food-containing factor(s) yet to be determined. However, to our knowledge, this is the first work to produce all-female populations of yellow perch from functional neomale sperm donors. iv Acknowledgements First and foremost, I would like to thank my advisor, Dr. Konrad Dabrowski, for his endless hours of support and guidance. I would also like to thank the other members of my examination committee and the kind and helpful staff of the School of Environment and Natural Resources. I would like to thank my dear friends and family for their support and understanding during stressful spawning seasons. Lastly, this research would not have been possible without the patience, guidance, and assistance of Mohammad Alam, Thomas Delomas, Kevin Fisher, John Grayson, Megan Kemski, Mackenzie Miller, and Shib Nath Saha. v Vita June 2009……………….Brecksville-Broadview Heights High School, Broadview Heights, Ohio May 2013……………………………………………………..……….B.S. Animal Science, Cornell University January 2015 to present…...Graduate Teaching Associate, SENR, The Ohio State University Field of Study Major Field: Environment and Natural Resources Specialization: Fisheries and Wildlife Science vi Table of Contents Abstract………………………………………………………………………………….………………………………...…..ii Acknowledgements………………………………………………………………..……………………………..……….v Vita…………………………………………………………………………….……………………………………….………..vi Field of Study………………………………………………………………………..…………………………………….…vi Table of Contents……………………………………………………………….………………………………………..vii List of Tables…………………………………..………………………………………………………………………………x List of Figures………………………………………………….…………………………………………………………….xi Chapter 1: Introduction to Yellow Perch and Sex Reversal……………………………………..………1 Taxonomy and Physical Characteristics……………………………………………………………….1 Market Demand for Yellow Perch………………………………………………….……………………1 Sex Reversal………………………………………………………………………………………………………..3 Hormonal Sex Reversal……………………………………………………………………………4 Environmental Sex Reversal………………………………………………………………….11 Goals and Objectives……………………………………………..……………………………..………….13 Hypotheses……………………………………………………………………………………………………….14 Chapter 2: Determination of the Optimum Timing and Exposure Method for Hormonal Sex Reversal in Yellow Perch……………………………………..…………………………….……….16 Introduction…………………………………………………………………..…………………………………16 vii Methods…………………………………………………………….…………………………………………….20 Gamete Collection and Incubation……………………………………………..…………21 Larval Rearing…………………………………………….…………………………………………22 Enrichment of Artemia nauplii……………………………………………..……………….23 Sex Reversal…………………………………………….……………………………………………24 Growout………………………………………………………………………………..………………28 Analysis of F2 Generation……………………………………………………………..………29 Statistical Analysis……………………………………………………………..………………….31 Results…………………………………………………..………………………………………………………….32 Method of Exposure………………………………………………………..……………………32 Time of Initiation……………………………………………………..……………………………34 Evaluation of F2 Generation……………………………………..…………………………..35 Discussion…………………………..…………………………………………………………………………….35 Chapter 3: Exploration of the Potential for Temperature-Induced Sex Reversal in Yellow Perch…………………………………………..……………………………………………………………………42 Introduction………………………………………..……………………………………………………………42 Methods…………………………………………….…………………………………………………………….44 Experiment 1……………………………………………………………..………………………….44 Experiment 2………………………………………………………..……………………………….47 Statistical Analysis……………………………………..………………………………………….50 Results……………………………..……………………………………………………………………………….52 viii Experiment 1…………………………………………………..…………………………………….52 Experiment 2………………………………………………..……………………………………….53 Discussion………………………..……………………………………………………………………………….55 Future Research……………………………………….………………………………………………………………….58 Literature Cited (AFS)……………………………………………….………………………………………………….60 Appendix A: T Test and ANOVA Tables……………………………………………..………………………….74 ix List of Tables Table 1. Gonadosomatic indices of yellow perch fed diets containing different levels of methyltestosterone (MT) or cholesterol (C, control) for 84 days. Numbers are expressed as mean ± SE. Different letters next to the numbers within a column indicate a significant difference between groups (P < 0.01). Adapted from Malison et al. (1986)………………………………………………….18 Table 2. Summary of the hormone exposures for both Group 14 mm and Group 12 mm…………....27 Table 3. Water quality parameters during the hormone exposure phase of Yellow Perch larvae in Group 14 mm. Values are given as mean ± standard deviation. No values within a column differed significantly (ANOVA, P < 0.05)…………………………………………………………………………………………………..27 Table 4. Water quality parameters during the hormone exposure phase of Yellow Perch larvae in Group 12 mm. Values are given as mean ± standard deviation. No values within a column differed significantly (ANOVA, P < 0.05)…………………………………………………………………………………………………..28 Table 5. Total weight (TW), gonadosomatic index (GSI), and percent females of progenies (n = 5) produced by two control males from Group 14 mm (Chapter 2) that had been raised at 23.1 ± 0.2o C (Warm) or 16.4 ± 1.0o C (Cool). Values are given as mean ± standard
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