In vitro Embryo Production

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Selection of Stallions for in vitro Embryo Production by ICSI in a Commercial Program

Herrera C.; Dufourq P.; Freije M.; Morikawa I.; Centeno J.E.; Aristi V.; Menghini L.; Sporleder C.

Genetec S.A., Ruta 28 km 6.5, Pilar, Buenos Aires, Argentina.


vum pick up (OPU) and in vitro embryo production by ICSI have become useful techniques to obtain offspring from high merit mares that fail to reproduce by AI and embryo transfer. Semen with proved in vivo fertility is used to fertilize in vitro the oocytes obtained by OPU, assuming these samples will show the same efficiency as in vivo. Additionally, very few oocytes are obtained from each mare and therefore, the best possible semen needs to be used to perform ICSI.  The aim of this study was to compare the efficiency of different stallions to produce embryos in vitro using oocytes obtained from slaughterhouse ovaries.

Mare ovaries were transported from an abbatoir located 60km from our laboratory. Oocytes were obtained by aspiration of follicles with a  19G needle connected to an aspiration pump. All oocytes were matured in vitro for 24h in TCM199 supplemented with 1mM glutamine, 0.19mM sodium pyruvate, 5g/ml FSH (Bioniche, Canada), 100ng/ml EGF, 100ng/ml IGF-I and 10% FCS (SIGMA, New Zealand) at 38º C, 5% CO2 in air. Sperm cells were obtained on the same day of ICSI from different stallions housed at our facility. ICSI was performed as previously described (Herrera et al. 2008), except no exogenous activation was used. All injected oocytes were incubated in SOFm with 19 mM D-glucose and 10% FBS at 38º C, in 7% O2 and 5% CO2 for 7 days. Some embryos were transferred to recipient mares. The rest were used on a different experiment.

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The number of cleaved oocytes, morulae or blastocysts and pregnancies obtained were compared between stallions and analyzed by ANOVA and Fisher’s Exact Test.

Only cleaved oocytes from stallion 5 differed significantly when compared to stallions 3 and 6. All other stallions did not differ on their ability to fertilize oocytes in vitro by ICSI.  The number of embryos that developed in vitro did not differ significantly between stallions. Only embryos from stallions 3,4,6 and 7 were transferred to recipient mares and the number of pregnancies obtained did not differ between these four stallions.

When possible, stallions used to fertilize oocytes obtained by OPU should be previously tested with abbatoir oocytes for their ability to produce embryos in vitro. In this study, we conclude that stallion 5 should not be used for ICSI of oocytes of high merit mares due to its low fertilizing ability.

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