Browsing by Author "Carnevale, Elaine M., advisor"
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Item Open Access Characterization of selected gene expression patterns as potential markers for oocyte quality in young versus old mares(Colorado State University. Libraries, 2010) Rodrigues, Bernardo de Lima, author; Clay, Colin M., advisor; Carnevale, Elaine M., advisor; Tjalkens, Ronald B., committee member; Bouma, Gerrit J., committee member; Bruemmer, Jason E., committee memberAs a female ages a series of alterations in normal physiology take place, during this process fecundity decreases. The reproductive system starts to shut down as a consequence of hormonal, histological and anatomical changes, and in the middle of this process, playing a critical role, is the oocyte. As in women, fertility decreases with aging in mares. Recently the mare has been suggested as a promising model to study age-related infertility in women due to similarities in the reproductive cycle and similar age-associated reproductive changes. During the last decades the use of assisted reproductive techniques (ART) in human and veterinary medicine to treat infertility has increased. Unfortunately, ART can only partially compensate for declining fertility- particularly the age related decline in fertility. Therefore, we need to understand the mechanisms involved in age-associated infertility and improve both the diagnostic tools and the techniques currently used in ART. In that regard the identification of reliable oocyte quality markers is of great interest, specifically of extrinsic markers in follicular cells and follicular fluid (FF). Follistatin (FS1) and anti-müllerian hormone (AMH) have been suggested as potential oocyte quality markers. In addition, the rate of apoptosis in follicular cells has also been suggested to be a good indicator of oocyte quality In this study, our goal was to use the young and old mare model to obtain competent and incompetent oocytes, respectively, to try and elucidate the involvement of apoptosis of follicular cells and/or of the oocyte in the determination of oocyte quality. Oocytes, follicular fluid, granulosa and cumulus cells were collected through transvaginal follicular aspirations from young ( 4-10 years) and old (>20 years) mares. Preovulatory follicles were aspirated 30-36 h post induction of follicular maturation, which was performed by administration (i.v) of a combination of deslorelin and hCG when the biggest follicle reached 35 mm. We used real time PCR to examine expression of pro-apoptotic ( CASP2, CASP 3) and pro-survival (XIAP) genes, as well as of FST and AMH expression in these cell types. In addition we measured androgens and estrogens in FF and calculated the androgens to estrogens ratio to assess follicular atresia. We also sought to determine FF concentrations of FST and AMH, and relate it to oocyte quality. There was no difference in CASP3 expression levels in granulosa and cumulus cells between the two age groups. In addition, there was no difference in CASP2 and CASP3 mRNA expression in oocytes from young and old mares. XIAP mRNA levels were expressed 3.3 fold higher in oocytes from young when compared to old mares, and there was a tendency for XIAP to be more highly expressed in granulosa cells of young mares. In contrast, the levels of XIAP mRNA in cumulus cells were 1.46 fold higher in old when compared to young mares. There was no difference in the expression levels of AMH in granulosa cells between young and old mares, but in cumulus cells there was a tendency for AMH to be higher expressed in cells from old vs. young mares. Unfortunately we were not able to analyze AMH FF concentrations. FST mRNA levels in oocytes were similar between the age groups, but FST concentrations in FF of preovulatory follicles from young mares (197 ± 16.7 ng/mL) were higher (p=0.02) than in FF from old (153.3 ± 22.7 ng/mL) mares. In both age groups FST FF concentrations in preovulatory follicles significantly decreased when compared to mid-estrus and post-deviation follicles. In conclusion, we believe that our data suggest that FST follicular fluid levels can be a non-invasive marker to assess oocyte quality in the horse, and that FST levels decrease in preovulatory follicles of the horse. In addition, expression levels of caspase-3 in follicular cells, and caspases 3 and 2 in the oocyte, does not seem to be involved in the mechanism of fertility loss in the old mare. Finally, XIAP mRNA levels may be important for oocyte quality in the horse.Item Open Access Cryopreservation of cooled semen for equine intracytoplasmic sperm injection(Colorado State University. Libraries, 2010) Daigneault, Bradford W., author; Carnevale, Elaine M., advisor; Denniston, David J., committee member; Graham, James K., committee member; Bruemmer, Jason E., committee memberThe development of intracytoplasmic sperm injection (ICSI) for use in the horse has resulted in unique needs for semen. Modifications of ways to handle sperm for ICSI-related procedures were investigated through a series of three experiments. In Experiment 1, semen was cooled for 24 h and then frozen to provide a viable population of sperm for ISCI. The objectives of this study were to compare the efficacy of: (1) two extenders for cooling semen prior to freezing and (2) four extenders for freezing cooled semen. Stallion semen was extended in two commercial cooling extenders (CST and INRA96) and cooled for 24 h before freezing in four extenders. Freezing extenders were a modified- French (FR8), Lactose-EDT A (LAC 10), glycerol (G), and glycerol egg yolk (GEY). Extenders were added directly to cooled semen and diluted to a final concentration of 20 x 106 sperm/mL before freezing. After thawing, sperm were evaluated at 0, 45 and 75 min. Cooling extender did not affect sperm motility after freezing. Total motility and progressive motility was similar (p>0.05) for all freezing extenders at O and 45 min, but sperm frozen in glycerol egg yolk (GEY) yielded the highest total and progressively motile sperm at 75 min. Differences in motility over time were not detected except for sperm frozen in glycerol (G) in which total and progressive motility declined from Oto 75 min. For all extenders, some motile sperm were obtained from semen cooled for 24 h and then frozen. In Experiment 2, five media were evaluated for holding sperm to determine which best maintained the motility of stallion sperm over time. The experiment was designed to determine appropriate media in which to handle sperm for ICSI selection procedures. The media compared were: 1) GIVF, 2) FDCM, 3) TALP, 4) Emcare (EM) and 5) Mare Mojo (MM). The only media designed for holding sperm was T ALP; the remaining media were formulated as holding media for oocytes and embryos. Motility of sperm was analyzed every hour for 5 h. No differences in total or progressive motility were detected among groups until 5 h. At 5 h, sperm held in TALP had higher (p<0.05) total and progressive motility than all other groups. Sperm can be held in any of the five media evaluated and remain motile for ICSI. However, T ALP resulted in the best sperm motility over time. Experiment 3 was performed to evaluate a hyaluronic acid (HA) binding assay for the selection of sperm for ICSI. Sperm binding to hyaluronic acid has been characterized for many species. Sperm that bind to HA have been shown to exhibit more normal chromosomal structures and improve fertilization. The objective of Experiment 3 was to determine if fresh and frozen stallion sperm bind to HA when introduced to premanufactured hyaluronic acid binding kits (HBA, Biocoat Inc., Horsham, PA). Fresh semen pooled from two stallions exhibited approximately 15% of sperm bound to HA. Four frozen samples from Experiment 1 were thawed and pooled. Frozen sperm did not bind to the assays. For fresh and frozen samples, sperm displayed some hyperactivation, uncharacteristic circling, and tails bent at the mid-piece. Sperm were observed to agglutinate in congregations of fifty or more sperm. Hyaluronic acid seemed to have an effect on stallion sperm that has not been described for other species. Further investigation of HA binding for selection of sperm for ICSI is warranted.Item Open Access Oocyte metabolism – a potential link between mare conditions and impaired fertility(Colorado State University. Libraries, 2023) Di Donato Catandi, Giovana, author; Carnevale, Elaine M., advisor; Krisher, Rebecca L., advisor; Chicco, Adam J., committee member; Chen, Thomas W., committee memberMaternal advanced aging and obesity are known for negatively affecting reproductive outcomes by directly impacting the oocyte and the follicular environment, where the oocyte develops and matures. Success of early embryonic development relies on appropriate ability of the oocyte to produce energy. Whether maternal conditions of the mare impact oocyte metabolic function had not been previously determined. In the studies described throughout this dissertation, novel microsensors were utilized to quantify aerobic and anaerobic metabolism of single equine oocytes. Additional and complementing end points were obtained through high- resolution respirometry of granulosa cells and metabolomic profiling of oocytes and cumulus cells. The overarching hypothesis of this dissertation is that mare conditions known to impair fertility, namely advanced age and obesity, affect oocyte metabolism, ultimately impairing oocyte developmental potential. It was additionally hypothesized that dietary supplementation to old or obese mares would reach and affect the ovarian follicular environment and the oocyte, improving its metabolic function and quality. To test these hypotheses, a series of three projects were conducted to: 1) Investigate effects of mare advanced aging on oocyte metabolism; 2) Determine the potential of diet supplementation to old mares to improve oocyte metabolism; 3) Investigate effects of mare obesity on oocyte metabolism and the potential of diet supplementation on normalizing metabolic alterations. Findings from these projects revealed that mare advanced aging impairs oocyte aerobic and anaerobic metabolic function, contributing to limited embryonic metabolism and development after intracytoplasmic sperm injection (ICSI). Short-term dietary supplementation to old mares with feed additives, specifically formulated to improve mitochondrial metabolism and overall equine health, was able to improve mitochondrial metabolism of granulosa cells and oocytes, promoting greater embryonic rates after ICSI in comparison to a control grain supplementation. Additionally, the findings here reported demonstrate that mare obesity promotes several alterations in the ovarian follicle, including excess of reactive oxygen species production by granulosa cells, lipid accumulation in cumulus cells and oocytes, and excessive oocyte aerobic and anaerobic metabolism. Dietary supplementation to obese mares with similar feed components mitigated many of the obesity-associated follicular changes, likely contributing to oocyte quality. Collectively, these novel discoveries contribute to knowledge and understanding of the direct effects of maternal conditions of the mare on the ovarian follicle and oocyte, elucidating cellular mechanisms by which advanced aging and obesity disturb fertility. Furthermore, these findings reveal the benefits of dietary interventions in improving oocyte metabolism and quality. Dietary supplementation represents a non-invasive and feasible approach to tackle female subfertility. Assuredly the results presented throughout this dissertation will contribute to the equine reproduction industry, with potential to have a translational impact on the human fertility industry, by not only elucidating direct effects of maternal conditions on oocyte metabolism, but also by providing a practical method for rescuing it in vivo.