Browsing by Author "Walters, Anneke H."
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- Analysis of early lactation reproductive characteristics in Holstein cowsWalters, Anneke H. (Virginia Tech, 2000-09-12)Ultrasound-guided transvaginal follicular aspiration was used to obtain oocytes from cows to study follicular development and oocyte morphology. Follicular aspiration was conducted once during wk 1 to 12 postpartum on 120 lactating cows with 6 groups, separated by biweekly intervals. Approximately one half of the aspirated cows at each session were from the early groups (wk 1-2, 3-4, or 5-6) and the other half from the later groups (wk 7-8, 9-10, or 11-12). On the day of aspiration the number of follicles on each ovary, and their sizes, small (2-5 mm), medium (6-10 mm) and large (≥ 11 mm), were recorded. The collected oocytes were morphologically classified into 4 grades, with 4 = excellent, 3 = good, 2 = fair, and 1 = poor. Blood samples from the jugular vein and follicular fluid samples from the largest follicle were collected in order to perform hormone and metabolite assays. Environmental data were obtained from the local airport. There was a significant (P < .01) quadratic days pre- and postpartum by parity interaction for BCS. Body condition score for older cattle was the lowest at 90 d prior to calving and changed the least amount over time, while youngest cattle had the highest initial BCS at d 90 prior to calving and had the greatest change in BCS over time. Body condition score was the highest during summer calving season (3.3 ± .06) compared to BCS during winter calving season (2.6 ± .06). But the loss in BCS was greater for cows that calved in summer (-0.53 ± .06) compared to cows that calved in winter (-0.07 ± .08). Increased serum NEFA concentrations with simultaneous decreases in serum insulin concentrations for younger cattle implied a more negative EB status than for older cattle. The total number of follicles and total number of oocytes retrieved was significantly (P < .001) affected by a linear days postpartum by parity interaction with younger cattle having linear increases compared to decreases in the total number of follicles for older cattle. Oocyte quality score was affected by the quadratic days postpartum by parity interaction (P < .01) and calving season (P < .01). Younger cattle had higher initial quality scores compared to older cattle, but older cattle had higher quality oocytes towards the end of the 12 wk period compared with younger cattle. Younger cattle had higher E2 and IGF-I concentrations in follicular fluid associated with a higher number of total follicles and number of oocytes, compared to older cattle. However, oocyte quality of younger cattle seemed to be reduced and oocytes were less competent than for older cattle. Cattle in 3rd and greater lactation showed very little change in BCS and hormone and metabolite measures during early lactation, with no apparent decrease in oocyte quality, despite the aging effect on follicle numbers. This study demonstrated that conditions related to early lactation have a negative effect on oocyte quality and endocrine measures of dairy cattle and that animals of different ages are differentially affected.
- In vitro assessment of fertilization and embryo development with Bovine spermatozoa after scrotal insulationWalters, Anneke H. (Virginia Tech, 2004-11-12)Fertilization and cleavage of bovine embryos depend not only on maternal involvement, but also on the paternal contributions that involve more than just providing the haploid male genome. Therefore, the overall objective of this project was to determine the impact of morphologically abnormal spermatozoa on fertilization, subsequent embryonic development, and embryo quality at the cellular level. Four experiments used morphologically abnormal semen samples collected and cryopreserved from four Holstein bulls before (Pre) and after a scrotal insulation (PI) period of 48 h. Zygotes were cultured for 8 d when a developmental score was assigned to each embryo; subpopulations were subjected to either the TUNEL or caspase assays to determine apoptosis. In the final experiment pronuclear decondensation for presumptive zygotes was evaluated by differential interference contrast microscopy at 3 h time intervals from 6 to 18 h post in vitro insemination (hpi). Morphological evaluation of semen samples revealed a decrease (P < 0.01) in the percentages of normal spermatozoa in the PI samples in comparison with the Pre samples for Bulls I and Bull III (74 to 22.3% and 67.7 to 0.5 %, respectively) and the scrotal insulation effects persisted from the time of cleavage through blastocyst formation for Bulls I and III and corresponded with a similar decrease in blastocyst development for PI samples in experiment 1 regardless of which semen separation method was used. Likewise, the overall pronuclear decondensation rate for the PI zygotes of Bull I and III showed no increase over time and remained predominantly at PN1 stage (1.5 ± 0.17; 1.8 ± 0.22, respectively). In contrast, the development for Bull II and Bull IV were unaffected. The embryo quality assessment revealed that the caspase intensity increased significantly for both Bull I (217 ± 147) and Bull III (229 ± 98) for the PI embryo groups compared to those of Bull II (98 ± 115) and Bull IV (90 ± 111). In conclusion, the tested separation methods used seemed inadequate in their ability to provide potentially competent sperm for IVF. The decrease in embryonic development appears to be multifaceted and related to the changes in head shape morphology and we suggest the failure in normal pronuclear formation is associated with an absence of normal decondensation of the penetrating spermatozoon. The inability to consistently measure apoptosis in early stage embryos complicates the assessment of differences in embryo quality. These observations support the hypothesis of uncompensable seminal traits in IVF with abnormal spermatozoa and provide compelling evidence that the effect of morphologically abnormal spermatozoa occurred prior to cleavage, thus is manifested during the early stages of fertilization.