We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes

Capacitation is a complex and not well-understood process that encompasses all the molecular changes sperm must undergo to successfully fertilize an oocyte. In vitro fertilization has remained elusive in the horse, as evidenced by low in vitro fertilization (IVF) rates (0%–33%); moreover, only two foals have ever been produced using IVF. Incubation of stallion sperm in modified Whittens supplemented with bovine serum albumin and sodium bicarbonate yielded significant rates of time-dependent protein tyrosine phosphorylation and induced acrosomal exocytosis, consistent with capacitation. The objective of this study was to characterize stallion sperm hyperactivation and to test whether hyperactivation of capacitated sperm supported equine IVF. Treatment of sperm with procaine, an anesthetic shown to induce hyperactivation in other mammalian species, resulted in the decrease of three motility variables indicative of hyperactivation: straight line velocity (P = 0.029), straightness (P = 0.001), and linearity (P = 0.002). We demonstrated that procaine-induced hyperactivation was not regulated by changes in protein tyrosine phosphorylation and that it did not induce acrosomal exocytosis in capacitated sperm compared with calcium ionophore (P > 0.05), similar to findings in the bovine. Most notably, by coupling our capacitating conditions with the induction of hyperactivation using procaine, we have achieved the novel result of substantial and reproducible percentages of fertilized mare oocytes (60.7%) in our IVF experiments. Conversely, sperm incubated in capacitating conditions but not treated with procaine did not fertilize (0%). These results support the hypothesis that capacitation and hyperactivation are required for successful IVF in the equine.

The chromatin configuration of resting horse oocytes and the time course of in vitro oocyte maturation was characterized using a fluorescent, DNA-spec

Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P<0.05). In Experiment 3, blastocyst development rate was 6–11% after injection of sperm lyophilized from fresh or frozen–thawed semen, suspended in SE. In Experiment 4, sperm lyophilized 3.5 months or 1 week previously, suspended in SE, yielded similar blastocyst rates (6 and 3% respectively). Rates of normal pregnancy after transfer were 7/10 and 5/7 for embryos from control and lyophilized sperm treatments respectively. Three pregnancies from the lyophilized sperm treatments were not terminated, resulting in two healthy foals. Parentage testing determined that one foal originated from the lyophilized sperm; the other was the offspring of the stallion providing the sperm extract. Further testing indicated that two of five additional embryos in the lyophilized sperm treatment originated from the stallion providing the sperm extract. We conclude that both lyophilized stallion sperm and stallion sperm processed by multiple unprotected freeze–thaw cycles (as for sperm extract) can support production of viable foals. To the best of our knowledge, this is the first report on production of live offspring by fertilization with lyophilized sperm in a non-laboratory animal species. Abstract Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P<0.05). In Experiment 3, blastocyst development rate was 6–11% after injection of sperm lyophilized from fresh or frozen–thawed semen, suspended in SE. In Experiment 4, sperm lyophilized 3.5 months or 1 week previously, suspended in SE, yielded similar blastocyst rates (6 and 3% respectively). Rates of normal pregnancy after transfer were 7/10 and 5/7 for embryos from control and lyophilized sperm treatments respectively. Three pregnancies from the lyophilized sperm treatments were not terminated, resulting in two healthy foals. Parentage testing determined that one foal originated from the lyophilized sperm; the other was the offspring of the stallion providing the sperm extract. Further testing indicated that two of five additional embryos in the lyophilized sperm treatment originated from the stallion providing the sperm extract. We conclude that both lyophilized stallion sperm and stallion sperm processed by multiple unprotected freeze–thaw cycles (as for sperm extract) can support production of viable foals. To the best of our knowledge, this is the first report on production of live offspring by fertilization with lyophilized sperm in a non-laboratory animal species.

Horse oocytes were collected from an abattoir over a 15-mo period. After classification of follicle size and cumulus morphology, oocytes were either f

Horse oocytes with expanded (EX) cumuli appear to have greater meiotic competence than do horse oocytes with compact (CP) cumuli but are thought to co