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Dha) from a Heterotrophically Grown Microalgae, Vitamin E, and Selenium on Stallion Sperm Motion Characteristics

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Dha) from a Heterotrophically Grown Microalgae, Vitamin E, and Selenium on Stallion Sperm Motion Characteristics University of Kentucky UKnowledge Theses and Dissertations--Veterinary Science Veterinary Science 2016 EFFECTS OF FEEDING A YEAST-BASED SUPPLEMENT CONTAINING DOCOSAHEXAENOIC ACID (DHA) FROM A HETEROTROPHICALLY GROWN MICROALGAE, VITAMIN E, AND SELENIUM ON STALLION SPERM MOTION CHARACTERISTICS Lauren D. Goedde University of Kentucky, [email protected] Digital Object Identifier: http://dx.doi.org/10.13023/ETD.2016.377 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Goedde, Lauren D., "EFFECTS OF FEEDING A YEAST-BASED SUPPLEMENT CONTAINING DOCOSAHEXAENOIC ACID (DHA) FROM A HETEROTROPHICALLY GROWN MICROALGAE, VITAMIN E, AND SELENIUM ON STALLION SPERM MOTION CHARACTERISTICS" (2016). Theses and Dissertations-- Veterinary Science. 26. https://uknowledge.uky.edu/gluck_etds/26 This Master's Thesis is brought to you for free and open access by the Veterinary Science at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Veterinary Science by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Lauren D. Goedde, Student Dr. Edward L. Squires, Major Professor Dr. Daniel K. Howe, Director of Graduate Studies EFFECTS OF FEEDING A YEAST-BASED SUPPLEMENT CONTAINING DOCOSAHEXAENOIC ACID (DHA) FROM A HETEROTROPHICALLY GROWN MICROALGAE, VITAMIN E, AND SELENIUM ON STALLION SPERM MOTION CHARACTERISTICS THESIS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the College of Agriculture, Food, and Environment at the University of Kentucky By: Lauren Dianne Goedde Lexington, Kentucky Director of Thesis: Dr. Edward L. Squires Lexington, Kentucky 2016 Copyright © Lauren Dianne Goedde, 2016 ABSTRACT OF THESIS EFFECTS OF FEEDING A YEAST-BASED SUPPLEMENT CONTAINING DOCOSAHEXAENOIC ACID (DHA) FROM A HETEROTROPHICALLY GROWN MICROALGAE, VITAMIN E, AND SELENIUM ON STALLION SPERM MOTION CHARACTERISTICS The use of cooled and frozen stallion semen has become quite popular. However, there are some stallions that have sperm that are quite susceptible to cold shock. Thus, there is a need for techniques that will alter sperm so that they can withstand the stresses of cooling and freezing and thus improve pregnancy rates achieved with cooled or frozen spermatozoa. Studies have shown that a diet high in omega-3 (n-3) fatty acids can improve the motility of cooled and frozen/thawed sperm. Many of the omega-3 fatty acid products for stallions have low levels of docosahexaenoic acid (DHA) and are based on fish oil, which may have reduced palatability. The objectives of this study were to determine if a yeast-based supplement containing 15g DHA from a heterotrophically grown microalgae, 1000 IU Vitamin E and 2mg selenium as selenized yeast would enhance the motility of fresh, cooled, and frozen-thawed stallion sperm. Twelve stallions, 3 to 12 y old were used. Semen was collected every other day for two weeks (July) and sperm motion parameters (total and progressive motility) were determined by computer assisted sperm analysis (CASA) on the last three ejaculates. These ejaculates were cooled to 5°C (Equitainer, Hamilton Thorne) and held for 48 hr. Stallions were then paired based on CASA values for fresh and cooled semen, age of stallion, sperm output and body condition and randomly assigned to either control or treatment. Stallions were fed one of two dietary treatments for 60 d: A basal diet, Control, 0.4% BW as concentrate and 1.8% BW as grass hay, and Treated, basal diet plus 160g of a yeast-based supplement containing 15g DHA from a heterotrophically grown microalgae, 1000 IU Vitamin E and 2mg selenium as selenized yeast (Alltech Inc., Nicholasville, KY). Consumption of the supplement was accepted within a few days of feeding. Beginning on day 46, stallions had semen collected every other day until day 60. Sperm motion parameters were assessed with CASA. For cryopreservation, sperm were loaded into 0.5-mL straws and frozen with a programmable freezer. Using flow cytometric analysis, sperm viability, mitochondrial membrane potential, lipid peroxidation, acrosomal status, and membrane fluidity were measured on frozen-thawed sperm. Sperm viability was measured using both a SYBR-14/PI stain as well as a Yo-Pro+/PI- stain. Sperm mitochondrial membrane potential was evaluated using JC-1, and lipid peroxidation measured using BODIPY 581/591 C11. Acrosomal status was analyzed using FITC-PNA/PI and membrane fluidity evaluated using Merocyanine 540/Yo-Pro. Data was analyzed using a mixed model and significance was set to p ≤ 0.05. Although a significant improvement in total and progressive motility was seen in both the fresh and the cooled spermatozoa in sperm from the DHA/Vit E/Sel treated stallions, there was no significant difference noted at any of the parameters measured in the frozen-thawed sperm. An oxidative challenge experiment was also conducted in which frozen-thawed sperm were deliberately stressed with FeCl2 and H2O2. To evaluate the effects of the oxidative challenge, a matched pairs analysis was used. To analyze differences in the way that sperm from control stallions and DHA/Vit E/Sel treated stallions handled the oxidative stress imposed upon them, a one- way ANOVA was used to compare the percent increase in positive (oxidative stressed) cells. Results for the oxidative challenge study proved that treatment with the FeCl2 and H2O2 oxidative challenge was effective at inducing oxidative stress to the sperm membrane in both control and DHA/Vit E/Sel treated stallions, however no significant difference was seen in the way that control versus treated stallion sperm withstood the oxidative challenge. KEYWORDS: DHA, Stallion, Sperm, Oxidative Stress Lauren Dianne Goedde 5-22-2016 EFFECTS OF FEEDING A YEAST-BASED SUPPLEMENT CONTAINING DOCOSAHEXAENOIC ACID (DHA) FROM A HETEROTROPHICALLY GROWN MICROALGAE, VITAMIN E, AND SELENIUM ON STALLION SPERM MOTION CHARACTERISTICS By Lauren Dianne Goedde Dr. Edward L. Squires Director of Thesis Dr. Daniel K. Howe Director of Graduate Studies 05/24/2016 Acknowledgements Words cannot possibly describe the debt of gratitude that I owe to my endlessly supportive boyfriend and best friend, Elliott Pyles. There are many difficulties I likely would not have overcome without him by my side, always encouraging and lifting me up. I also carry immense appreciation for my parents, Mark and Lisa Goedde, who strongly supported education and a strong work ethic for myself and my brothers throughout our childhood. Without them as perfect role models, I would likely not have had the discipline to follow my dreams. My advisors Dr. Ed Squires and Dr. Barry Ball gave me the guidance and direction that I needed in order to pursue this research and ultimately my Master’s Degree. I greatly appreciate their help and support throughout my two years with the University of Kentucky. I also owe it to Dr. Squires, whose kindness and willingness to meet with me in 2012 first sparked my interest in the UK Veterinary Science program. The inputs of Drs. Squires, Ball, Troedsson, and Lawrence in the planning and execution of this project were vital to its successful completion. I would also like to thank Michelle Wynn, who was my best buddy when it came to tackling difficult coursework—Studying together made it possible. I am very happy to consider her a close friend rather than just a co-worker. Dr. Alejandro Esteller-Vico was essential in completing the statistical analysis for this project, and I greatly appreciate his willingness to devote a significant amount of time into our data. Dr. Kirsten Scoggin also generously devoted her time to assisting with this project, including everything from re-calibrating the flow cytometer to giving much needed advice. I greatly appreciate the assistance of Gabriel Davolli, who taught me proper stallion handling techniques and introduced me to much of the stallion lab equipment. Drs. Fernando Peña and Juan Maria Gallardo were
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