Date Available


Year of Publication


Degree Name

Master of Science (MS)

Document Type

Master's Thesis


Agriculture, Food and Environment


Veterinary Science

First Advisor

Dr. Edward L. Squires


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 oneway 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.

Digital Object Identifier (DOI)