Determination of an extender protocol which will enhance the viability of frozenthawed bovine spermatozoa will allow producers to obtain higher conception rates due to the increased survival rate of the spermatozoa. Ejaculates of six Brangus bulls (age=18 months) were evaluated for spermatozoal motility, acrosomal integrity, and morphological characteristics (collectively called spermatozoal viability) in two experiments to test our hypotheses that (1) the treatment combination of a 4 hr cooling duration and a 2 hr equilibration with glycerol will result in optimum spermatozoal characteristics after freezing and thawing and (2) rank of three selected extenders relative to their effects on spermatozoal viability after freezing and thawing will be egg yolk-citrate (EC), egg yolk-tris (IMV), and skim milk (milk). In experiment 1, an ejaculate from each bull was partially extended and cooled to 4 ºC for either 2 or 4 hr and then allowed to equilibrate with the glycerolated extender for 2, 4, or 6 hr. Spermatozoal viability was assessed at 0, 3, 6, and 9 hr after thawing. In experiment 1, 4 hr of cooling resulted in a higher percentage of motile spermatozoa than did 2 hr of cooling. The 2 hr equilibration with glycerol yielded lower percentages of motile spermatozoa, acrosomal integrity, and morphologically normal spermatozoa than 4 and 6 hr equilibration durations with glycerol. In experiment 2, we observed a decrease in spermatozoal viability for all three extenders upon freezing and thawing. Viability of frozen-thawed spermatozoa extended in the milk was reduced for all incubation durations, and the IMV extender had a higher percentage of motile spermatozoa than the EC extender at 6 hr of incubation. A higher percentage of intact acrosomes was observed with the IMV extender; however, the EC extender had a higher percentage of morphologically normal spermatozoa than the IMV extender. Our results indicate that at cooling duration of 4 hr and a 4 hr equilibration with glycerol provide the highest level of spermatozoal viability post-thaw of the treatments evaluated and that the IMV extender enhances the percentage of spermatozoa with an intact acrosome for frozenthawed spermatozoa over the EC and skim milk extenders.

The effects of miticides on the reproductive physiology of queens and drones were examined. The first study examined the effects of Apistan (fluvalinate), Check Mite+ (coumaphos), and Apilife VAR (74% thymol) on sperm production and viability in drones. Drones from colonies treated with each miticide were collected at sexual maturity. Sperm production was determined by counting the number of sperm in the seminal vesicles. Sperm for viability assays was analyzed by dual fluorescent staining. Apilife VAR and coumaphos significantly lowered (P<0.0001) sperm production and coumaphos treatments caused a significant decrease (P<0.0001) in the sperm viability. The effects of miticides on queens was examined by treating queen-rearing colonies and examining the number and viability of sperm in the spermathecae of newly mated queens. Queens from each treatment group were collected after mating and the spermathecae were removed and analyzed. Colonies treated with coumaphos failed to provide viable queens and were excluded. Apilife VAR was found to significantly decrease (P<0.0016) sperm viability. No significant differences in sperm numbers were found between treatments. The effect of miticides on sperm viability over time was also examined. Drones were reared as described, but the spermatozoa were collected as pooled samples from groups of drones. The pooled samples from each treatment were subdivided and analyzed periods of up to 6 weeks. Random samples were taken from each treatment (n = 6 pools) over a period of 6 weeks. The exposure of drones to coumaphos during development significantly reduced sperm viability for all 6 weeks, and caused a large decline in week 6. The potential impacts of these results on queen performance and failure are discussed. / Master of Science in Life Sciences