Carnosine supplementation in cryopreservation solution improved frozen-thawed bovine embryo viability.

Cryopreservation adversely affects embryo quality and viability in vitro.We investigated the effects of cryopreservation solutions supplemented with the antioxidant carnosine on frozen-thawed bovine embryo viability. Bovine blastocysts were produced in vitro and cryopreserved using slow freezing. The rates of re-expanded and hatched blastocysts in the 50 µg/ml carnosine-supplemented group at 4, 24, and 48 h after thawing were higher than those in the control (P< 0.05) group. In frozen-thawed embryos, cryopreservation solution supplemented with carnosine (50 µg/ml) significantly reduced reactive oxygen species (ROS) production(P < 0.05), decreased TUNEL-positive apoptotic cells (P< 0.05), and increased the mRNA expression of BCL2 (P< 0.05), an apoptosis suppressor gene. The expression of translocase of outer mitochondrial membrane 20 (TOMM20), which is involved in protein mitochondrial transport, in the carnosine (50 µg/ml)-treated embryos was significantly higher than that in the control group (P < 0.05). ATP production in frozen-thawed embryos in the 50 µg/ml carnosine-supplemented group was significantly higher than that in the control group (P< 0.05), however no significant difference in the total number of cells per embryo among the groups was observed. These results suggest that supplementing the cryopreservation solution with carnosine can improve the viability of frozen-thawed bovine embryos by reducing oxidative damage.

Effects of short-term dietary supplementation on the number of ovarian follicles, quantity and quality of oocytes, and in vitro embryo production in Japanese Black cows.

This study aimed to test the hypothesis that short-term supplementation with a high-energy diet promotes embryo production following ovum pick-up (OPU) in Japanese Black cows. After a period of adaptation to the maintenance diet, a 200% maintenance diet was fed to the high-energy diet group (HD group, n = 6) for four weeks, and a maintenance diet was fed to the other group (MD group, n = 6). OPU-in vitro fertilization (IVF) procedures were performed on days 14, 21, and 28; follicles and oocytes were counted and morphologically graded, and cultivable oocytes were cultured for in vitro maturation, fertilization, and culture. The mean plasma insulin concentrations on days 14 and 21 were significantly higher in the HD group than in the MD group (P < 0.05). The number of follicles observed at OPU, recovered oocytes, cultivable (Grades 1 to 4) oocytes, and the rate of degenerated (Grade 6) oocytes in the HD group were significantly higher than those in the MD group (P < 0.05). The proportion of cleaved oocytes was lower in the HD group than in the MD group (P < 0.05); consequently, there was no significant difference in the number of blastocysts obtained between the HD and MD groups. The present findings suggest that high-energy diets can promote follicular growth in parallel with an increase in plasma concentrations of insulin, but have a detrimental effect on the quality of oocytes with the OPU-IVF procedure in Japanese Black cows.

Mild hypothermia promotes the viability of in vitro-produced bovine blastocysts and their transcriptional expression of the cold-inducible transcription factor Rbm3 during in vitro culture.

In this study, we evaluated the effects of holding in vitro-produced bovine blastocysts under mild hypothermia (33°C or 35°C), by examining viability and hatching rates of day 7 blastocysts (day 0: in vitro fertilization) cultured for 6 days and transcriptional expression of cold-inducible transcription factors Cirp and Rbm3, implicated in mild hypothermia-induced cellular protection against various types of stress. In the normothermic control (38.5°C), viability of the embryos decreased rapidly after day 10, and most samples were degenerated on day 13. However, mild hypothermia, particularly at 33°C, resulted in maintenance of high embryonic survival rates until day 13 (77.1% on day 13) and significant increases in transcriptional expression of Rbm3 in day 11 embryos compared with those at 38.5°C. Thus, our results suggested that upregulation of Rbm3 may occur in response to mild hypothermia in many bovine embryos, providing insights into the effects of mild hypothermia on embryo quality.

Embryogenesis of vitrified mature bovine oocytes is improved in the presence of multi-layered cumulus cells.

This study was aimed at evaluating the effects of multi-layered cumulus cells (MCCs) during vitrification and in vitro fertilization (IVF) of mature bovine oocytes and embryogenesis after IVF. The rates of cleavage and blastocyst formation were higher in vitrified and fertilized oocytes with MCCs than in denuded oocytes (P < 0.05), but were comparable to the rates in fresh oocytes with MCCs or without (denuded). When the MCC-enclosed oocytes were denuded before IVF, blastocyst formation rate reduced compared with that in vitrified oocytes with MCCs (P < 0.05). This suggested that the MCCs surrounding the mature bovine oocytes play important roles during cryopreservation: protecting them against freezing and promoting their survival and development post IVF, thereby increasing the success rates of IVF and embryonic development. Herein, we showed for the first time that calves could be produced using only 14-19 vitrified mature oocytes with MCCs from the ovaries of individual cows post slaughter.