The effects of pretreatment with Cyclosporin A and Docetaxel before vitrification of porcine immature oocytes on subsequent embryo development.

In the present study, we attempted to improve the developmental competence of vitrified immature porcine oocytes by the preservation of mitochondrial properties using Cyclosporin A (CsA, inhibitor of mitochondrial membrane permeability transition) and Docetaxel (stabilizer of microtubules, hence mitochondrial distribution). In Experiment 1, Mitotracker red staining revealed reduced mitochondrial activity (MA) in vitrified/warmed oocytes at 0 and 22 h of in vitro maturation (IVM) compared with fresh ones. However, by at 46 h of IVM, MA levels in vitrified oocytes were similar to those in fresh control. Treatment of oocytes with CsA or Docetaxel improved MA at 0 h and 22 h of IVM compared with non-treated vitrified oocytes. However, there were no significant differences among groups in percentages of survival, maturation and embryo development after subsequent IVM and parthenogenetic activation. Nevertheless, a pretreatment with a combination of 10 µg/mL CsA and 0.05 µM Docetaxel improved the blastocyst formation of vitrified oocytes compared with non-treatment counterparts (11.2 ± 1.6% vs 5.9 ± 1.6%, P < 0.05). In conclusion, vitrification reduced mitochondrial activity in GV-stage oocytes during 0-22 h of IVM; however, it was normalized by 46 h IVM. Docetaxel or CsA pretreatment alone did not improve development competence of vitrified oocytes. However, pretreatment with a combination of CsA and Docetaxel could improve blastocyst formation rates.

Assessment of pregnancy-associated glycoprotein (PAG) concentrations in swamp buffalo samples from fetal and maternal origins by using interspecies antisera.

Pregnancy-associated glycoproteins (PAG) constitute a large family of glycoproteins found in the outer placental epithelial cell layer of the placenta in Eutherian species. In ruminants, they are noted to be structurally closely related among the different species. This study was designed to determine PAG concentrations in maternal and fetal plasma, allantoic and amniotic fluids in buffalo species. Antisera (AS) generated in rabbits against distinct PAG molecules were used in three radioimmunoassay (RIA)-PAG systems: RIA-1 (antiserum raised against bovine PAG67kDa; AS#497), RIA-2 (antiserum raised against caprine PAG55 + 62 kDa; AS#706) or RIA-3 (antiserum raised against buffalo PAG; AS#859). Samples were collected at a slaughterhouse (n = 67). PAG concentrations determined by RIA-2 gave significantly higher results in both allantoic and amniotic fluids (12.7 ± 2.1 ng/mL and 24.0 ± 7.3 ng/mL, respectively). Regarding maternal and fetal plasma, PAG concentrations obtained by RIA-2 (21.8 ± 2.4 ng/mL and 20.2 ± 2.5 ng/mL, respectively) and RIA-3 (25.0 ± 2.2 ng/mL and 21.9 ± 3.2 ng/mL, respectively) were higher than those obtained by RIA-1 (15.5 ± 1.4 ng/mL and 16.1 ± 1.8 ng/mL, respectively). The correlation among the three systems was very high. The study clearly reveals the ability of different PAG-RIA systems to measure PAG concentration in swamp buffalo samples.

Direct freezing of cattle embryos after partial dehydration at room temperature.

A new and simple method for freezing of bovine morulae and blastocysts was developed. Embryos were predehydrated at room temperature, frozen at -30 degrees C (cooling rate = 12 degrees C/min), and plunged into liquid nitrogen. This method was compared in vitro and in vivo to the slow freezing method (0.3 degrees C/min to -30 degrees C). Predehydration of the embryos in 1.5M glycerol was achieved by sucrose solution that makes the cells osmotically shrink. After the predehydrated morulae and blastocysts were frozen and thawed, 6 .4% (33 52 ) were developed in vitro for 48h and 44.2% (23 52 ) were hatched. Development obtained with slowly frozen embryos were 70.8% (17 24 ) and 58.3% (14 24 ) respectively. After transfer to recipient heifers, 33.3% (7 21 ) of the embryos frozen according this new method developed normally into viable foetuses or calves. This was the case for 48.5% (16 33 ) of the slowly frozen embryos.