This study was designed to: 1. Determine the effects of sperm dose and retrograde sperm blockage on mean number accessory sperm/ova. 2. Evaluate the relationship between mean number accessory sperm/ova and fertilization status/embryo quality. 3. Determine if mean number accessory sperm/ova or embryo quality are affected by semen quality. 4. Compare the percentage of morphologically normal accessory sperm with the percentage of normal cells in the inseminate. Using excised reproductive tracts, a French insemination rod housed in a 24-gauge Foley catheter was determined to be effective in blocking retrograde flow of semen following insemination. In a preliminary study, blocked vs conventional inseminations (control) were made using average quality frozen semen at 20 x 10⁶ sperm/dose. Although not different (P > .1), the mean number accessory sperm/ovum was 20 ± 40 (n= 24) and 13 ± 28 (n= 26) for the blocked and control methods, respectively. In Expt. 1, the conventional (control) and blocked system were again compared in a 2x2 factorial using low quality semen at 20 and 40 x 10⁶ sperm/dose. Mean number accessory sperm/ova was not affected by dose, blocking, nor the interaction. Embryo quality was negatively affected by blocking (P < .1), and unaffected by sperm dose. In Expt. 2, embryo quality and accessory sperm numbers were unaffected by a 40 x 10⁶ sperm dose of either average or below average quality semen. However, embryo quality tended to be improved by the average quality semen. Accessory sperm were significantly enriched with morphologically normal cells when compared with those in the inseminate (P < .01). Viable quality embryos (poor thru excellent) had the highest mean number accessory sperm/ovum (16.2 ± 28.9), most unfertilized ova (UFO) contained zero accessory sperm (.27 ± .83) and degenerates embryos were intermediate in number (5.4 ± 8.7). The relationship between embryo quality and accessory sperm number appears to vary in response to semen quality. / Master of Science

Four experiments were conducted to evaluate the viability and fertility of bovine spermatozoa encapsulated in microcapsules and microgels. In Experiment I, one of two morphologically distinct sperm types i.e. marker or unmarked bull spermatozoa (100 x 10⁶ sperm/bull) were encapsulated in protamine sulfate microcapsules and simultaneously inseminated with the reciprocal sperm type unencapsulated. Insemination of both sperm types unencapsulated served as a control. Accessory sperm embedded in the zona pellucida were counted and morphologically classified 6 to 7 d post insemination. From microencapsulated inseminates, accessory sperm populations did not increase over the unencapsulated controls, but contributed 25.7% of the accessory sperm population. ln Experiment 2, an in vitro study was performed to evaluate the maintenance of viability for bovine spermatozoa encapsulated in PIPES, HEPES, or saline microgels. Neat semen was pooled from five bulls (50 x 10° sperm/bull), encapsulated in alginate microgels, and incubated at 37 C for 8 h. The unencapsulated control displayed greater maintenance of viability for percent intact acrosomes and motility when compared to all treatments. By 8 h incubation, PlPES and HEPES were not significantly different, but demonstrated greater maintenance of viability when compared to saline microgel treatments. In Experiment 3, PIPES microgels were heterospermically inseminated with equal numbers (20 x l0⁶ sperm/bull) of frozen-thawed marker bull and normal bull spermatozoa as explained in Experiment 1. Microencapsulated treatments contributed significantly lower numbers of accessory sperm when compared to unencapsulated controls. In Experiment 4, one of the two morphologically distinct sperm types (20 x l0⁶ frozen-thawed sperm/bull) were encapsulated in protamine sulfate microcapsules and the reciprocal sperm type was encapsulated in PIPES microgels. A total of 21 accessory sperm were recovered from 30 embryos which demonstrates the ability of microencapsulated spermatozoa to fertilize an oocyte. / Master of Science