132 POST-HATCHING BOVINE EMBRYO DEVELOPMENT IN VITRO: RELATIONSHIP BETWEEN SEX AND SIZE

Abstract

In vitro post-hatching embryos culture is a procedure that allows the establishment of more accurate tools for evaluating embryo developmental potential without the need of transferring them to recipient animals (Vejlsted et al. 2006 Theriogenology, 65, 153–165). It is well established that in the in vitro embryo production (IVP) technique, the sex ratio is imbalanced in favor of male embryos. The difference in sex ratio observed in the blastocyst stage at day 7 may be attributed to a variety of factors including developmental speed. However, whether or not this difference in sex ratio and speed of development continues after hatching is not known. The objective of this study was to evaluate post-hatching embryonic development until day 11 after in vitro fertilization (day 0) associating embryo size and gender. A total of 468 oocytes, obtained from abattoir-derived ovaries, were used. They were matured, fertilized, and cultured in vitro for 8 days in synthetic oviduct fluid medium (SOF Nutricell^®) and incubated at 39°C in 5% CO₂ in air. Degenerated embryos on day 8 and non-hatching embryos on day 9 were removed from culture droplets, and only hatched blastocysts were kept. Then, embryos were measured using a graduated ocular and post-hatching development (PHD) medium (Brandão et al. 2005 Biol. Reprod. 71, 2048–2055) was added in each well, being the final medium 1:1 of SOF:PHD. On day 11, the embryos were evaluated under stereomicroscope and only morphologically normal blastocysts were measured and frozen at –80°C, for gender diagnosis. The DNA from frozen samples was extracted with trizol reagent, sodium citrate solution (0.1 m), and ethanol. Sex embryos determination was performed by PCR and visualized in 2% agarose gel. Data were analyzed using the Mann-Whitney test. The results show that the majority (69%) of the embryos that reached blastocyst stage at day 7 developed in the PHD system until day 11. From the initial oocytes, 144 embryos (30.1%) and 146 (31.1%) embryos had reached the blastocyst stage at days 7 and 8, respectively. At day 9, 89 (19%) embryos were hatched and 65 embryos (13.9%) developed until day 11, of which only 48 embryos (73.8%) had a clear trophoblast. No difference (P > 0.05) in the percentage of male and female embryos was observed when embryos were evaluated at day 11 of culture. In addition the mean size was similar (P > 0.05) for female (467.24 ± µm, n = 19) and male (478.84 ± 190.21 µm, n = 29) embryos. The results suggest that after post-hatching culture the differences in sex ration and in gender development in IVP bovine embryos are not evident. The development until day 11 showed that post-hatching in vitro culture of bovine blastocyst can be used for embryo evaluation in later phase of development However, several questions still remain to be investigated regarding post-hatching culture of bovine blastocysts before it can be used as a tool to evaluate in vitro embryos.

Supported by Embrapa and UnB, Brazil.