Differences in amniotic amino acid concentrations between pregnancies obtained with transfer of vitrified thawed in vitro-produced embryos and with natural mating in sheep.

In vitro embryo production (IVP) and cryopreservation are associated with a high incidence of pregnancy complications and fetal abnormalities that may be linked with alterations of placental development. The amniotic fluid is partly derived from the transport of water and solutes across the placenta and provides the fetus with amino acids (AAs), which are the building blocks for biomolecules involved in physiological growth and development. To better understand the anomalies associated with IVP pregnancies, the present study was conducted to test the hypothesis that amniotic concentrations of AAs differ in pregnancies derived from vitrified/thawed (V/T) IVP embryos compared with gestations obtained with natural mating (NM) in sheep. Amniotic fluid was sampled in ewes that were pregnant after transfer of V/T IVP embryos and that had conceived with NM between Days 60 and 65 (V/T, n = 6; NM, n = 11) and between Days 80 and 85 (V/T, n = 5; NM, n = 14) of gestation via ultrasound-guided amniocentesis. Concentrations of 16 AAs in the amniotic fluid were measured using high-performance liquid chromatography. From Days 60 to 65 of gestation, concentrations of cystine, phenylalanine, and isoleucine were lower in V/T compared with NM ewes. From Days 80 to 85 of pregnancy, the mean concentrations of cystine and lysine were lower in the V/T versus NM groups. The total AA concentration per ewe was similar between the groups from Days 60 to 65 and 80 to 85 of gestation and decreased by 55% from Days 60 to 65 and 80 to 85 of gestation in all ewes. The most abundant AA from Days 60 to 65 of gestation was alanine in both groups, whereas from Days 80 to 85, the most abundant AAs were alanine in NM and glycine in V/T ewes; cystine was the less abundant detectable AA in all ewes at both stages of gestation. Results report that V/T IVP embryos have decreased concentrations of individual AAs in the amniotic fluid during the second trimester of gestation possibly because of an impaired placental vasculogenesis or because of a reduced placental transport. These novel findings are relevant to unravel the mechanisms responsible for the issues of pregnancies achieved with the transfer of IVP and cryopreserved embryos.

Effect of caffeine treatment before vitrification on MPF and MAPK activity and spontaneous parthenogenetic activation of in vitro matured ovine oocytes.

BACKGROUND: Molecules that stabilize protein kinases may be useful in overcoming the deleterious effects of cryopreservation. OBJECTIVE: To evaluate the effect of caffeine treatment before vitrification of in vitro matured ovine oocytes on the activity of MPF and MAPK as well as the spontaneous parthenogenetic activation after 24 h culture. MATERIALS AND METHODS: Oocytes obtained from slaughterhouse sheep ovaries were in vitro matured for 21 h, incubated for 3 h with or without caffeine and then vitrified. After warming, oocytes were processed for the analysis of chromatin configuration and the evaluation of spontaneous parthenogenetic activation (24 h in vitro culture). Fresh in vitro matured oocytes were used as control. RESULTS: Caffeine treatment before vitrification maintained the MPF activity at a level similar to that of fresh oocytes, and reduced the spontaneous parthenogenetic activation in comparison with oocytes that were not-treated with caffeine. CONCLUSION: Caffeine treatment prolongs the meiotic arrest of vitrified MII oocytes, likely via its action of stabilizing the MPF level.