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Elaine M Carnevale, Marco A Coutinho da Silva, Lisa J Maclellan, George E Seidel Jr, and Edward L Squires

Parentage identification was used to test the developmental competence of oocytes cultured under different conditions and fertilized in vivo after oocyte transfer. Oocytes were collected transvaginally from follicles of estrous mares approximately 22 h after administration of human chorionic gonadotropin. Oocytes were cultured for approximately 16 h in one of three media, with or without addition of hormones and growth factors. Groups of three or four oocytes, cultured in different media, were transferred into the oviduct contralateral to a recipient’s own ovulation. Recipients were inseminated with semen from two different stallions at 15 h before and 2.5 h after oocyte transfer. Sixteen days after transfer, embryos were recovered from uteri and submitted for parentage testing. The percentage of oocytes resulting in embryonic vesicles was nearly identical (P > 0.05) for transferred oocytes (32/44, 73%) versus ovulated oocytes of recipients (9/13, 69%). More (P < 0.01) oocytes were fertilized by sperm inseminated before (35/38, 92%) versus after (3/38, 8%) oocyte transfer. Tissue culture medium (TCM)-199 was superior to equine maturation medium I (EMMI; a SOF-based medium) for culturing oocytes (P < 0.05), although addition of hormones and growth factors during culture did not improve (P > 0.05) development of embryos.

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Marco A Coutinho da Silva, George E Seidel Jr, Edward L Squires, James K Graham, and Elaine M Carnevale

The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the sperm through Percoll did not increase sperm binding to the ZP but did remove any positive effect of EZM on sperm–ZP binding. In Experiment IV, exposure of either sperm or ZP to EZM before co-incubation did not increase sperm binding to ZP. In Experiment V, sperm diluted in TALP containing skim milk, EZM, or INRA96 bound more efficiently to the ZP than sperm diluted in TALP without milk proteins. In Experiment VI, sodium caseinate, native phosphocaseinate, and caseinoglycopeptide increased sperm binding to the ZP. In conclusion, diluents containing milk or milk proteins markedly enhanced the number of sperm bound to both equine and bovine ZP.

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Giovana D Catandi, Yusra M Obeidat, Corey D Broeckling, Thomas W Chen, Adam J Chicco, and Elaine M Carnevale

Advanced maternal age is associated with a decline in fertility and oocyte quality. We used novel metabolic microsensors to assess effects of mare age on single oocyte and embryo metabolic function, which has not yet been similarly investigated in mammalian species. We hypothesized that equine maternal aging affects the metabolic function of oocytes and in vitro-produced early embryos, oocyte mitochondrial DNA (mtDNA) copy number, and relative abundance of metabolites involved in energy metabolism in oocytes and cumulus cells. Samples were collected from preovulatory follicles from young (≤14 years) and old (≥20 years) mares. Relative abundance of metabolites in metaphase II oocytes (MII) and their respective cumulus cells, detected by liquid and gas chromatography coupled to mass spectrometry, revealed that free fatty acids were less abundant in oocytes and more abundant in cumulus cells from old vs young mares. Quantification of aerobic and anaerobic metabolism, respectively measured as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a microchamber containing oxygen and pH microsensors, demonstrated reduced metabolic function and capacity in oocytes and day-2 embryos originating from oocytes of old when compared to young mares. In mature oocytes, mtDNA was quantified by real-time PCR and was not different between the age groups and not indicative of mitochondrial function. Significantly more sperm-injected oocytes from young than old mares resulted in blastocysts. Our results demonstrate a decline in oocyte and embryo metabolic activity that potentially contributes to the impaired developmental competence and fertility in aged females.