The present study investigated the vitrification-induced deterioration of mitochondrial functions that may reduce the developmental ability of post-warming bovine embryos. In addition, the effect of supplementation of the culture medium with resveratrol on the mitochondrial functions and post-warming embryonic development was examined. Two days after in vitro fertilization, embryos with 8–12 cells (referred to hereafter as 8-cell embryos) were vitrified and warmed, followed by in vitro incubation for 5 days in a culture medium containing either the vehicle or 0.5 μM resveratrol. Vitrification reduced embryonic development until the blastocyst stage, reduced the ATP content of embryos, and impaired the mitochondrial genome integrity, as determined by real-time polymerase chain reaction. Although the total cell number and mitochondrial DNA copy number (Mt-number) of blastocysts were low in the vitrified embryos, the Mt-number per blastomere was similar among the blastocysts derived from fresh (non-vitrified) and vitrified-warmed embryos. Supplementation of the culture medium with resveratrol enhanced the post-warming embryonic development and reduced the Mt-number and reactive oxygen species level in blastocysts and blastomeres without affecting the ATP content. An increase in the content of cell-free mitochondrial DNA in the spent culture medium was observed following cultivation of embryos with resveratrol. These results suggested that vitrification induces mitochondrial damages and that resveratrol may enhance the development of post-warming embryos and activates the degeneration of damaged mitochondria, as indicated by the increase in the cell-free mitochondrial DNA content in the spent culture medium and the decrease in the Mt-number of blastocysts and blastomeres.

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One of the major difference between the in vivo and in vitro embryonic environments is the stiffness of the culture substrate. Xanthan gum (XG) and locust bean gum (LBG) are natural materials that are safe, inexpensive and easy to handle. In this study, we investigated the effects of using a polysaccharide culture substrate made from 1% XG and 1% LBG (XG‐LBG gel) on bovine embryonic development. Oocytes collected from bovine ovaries were subjected to maturation, and fertilization to generate embryos at an early developmental stage (>4 cell stage). Cleaved embryos were further cultured in a well of 96‐well cell culture plate coated with or without XG‐LBG gel for 5 days. While the developmental rate up to the blastocyst stage did not differ between the two culture systems (control, 38.0 vs. gel, 38.6%), blastocysts developed on the XG‐LBG gel produced significantly high cell numbers and ATP content. Embryos cultured on XG‐LBG gels for 24 hr had high expression levels of F‐actin and a highly even distribution of E‐cadherin. In addition, embryos developed on XG‐LBG gel demonstrated increased translocation of YAP to the nucleus and increased connective tissue growth factor (CTGF) protein levels (downstream of Hippo signalling). These findings suggest that soft culture substrates improve embryonic development by enhancing mechanotransduction, including YAP‐CTGF signalling.

The objective of this study was to analyze factors affecting the pregnancy rates after transfer of IVF-derived Japanese Black embryos. Holstein cows and heifers (n=7250) were selected as recipients, and embryo transfers were performed for 3yr (between 1998 and 2000). The IVM-IVF procedure was performed according to a method previously described (Hamano S and Kuwayama M 1993 Theriogenology 39, 703–712). IVF-derived embryos that developed into expanded blastocysts (grade 1, manual of IETS) after 7 to 8 days (insemination=Day 0) were used for this study. Some of these embryos were frozen in TCM-199 supplemented with 1.4M glycerol, 20% calf serum, and 0.25M sucrose. The embryos were seeded at −6°C, held at −6°C for 10min, and then cooled to −25°C at a rate of 0.33°Cmin−1. Frozen embryos were thawed in a 30 to 35°C water bath after 10s of air thawing. Fresh (n=3952) or frozen-thawed (n=3298) embryos were nonsurgically transferred to recipients on Days 6 to 9 of the estrous cycle. Data collected at the time of embryo transfer included recipient parity (cow or heifer), whether recipient estrus was natural or synchronized with PGF2α, cloprostenol or CIDR, methods of estrous confirmation (showing standing heat, rectal palpation of ovary without standing heat, or showing only mucous vulvular discharge), number of examinations of the CL by palpation per rectum (twice on the day before embryo transfer and the day of embryo transfer, or once on the day of embryo transfer), type of embryos (fresh or frozen), and day of the estrous cycle at the time of embryo transfer. CATMOD procedures of SAS were used to determine the factors affecting the pregnancy rate. Overall pregnancy rates were 37.3% (n=2704). Whether recipient estrus was natural or synchronized and the type of embryos did not influence the pregnancy rates. Heifers had significantly higher pregnancy rates than cows (44.0% v. 33.0%, respectively, P<0.05). Pregnancy rates among the subset of heifers and cows showing standing heat were significantly higher than those showing only mucous vulvular discharge (39.5% v. 33.5%, respectively, P<0.05). Examining the CL twive had a significantly higher pregnancy rate than did a single examination of the CL (41.1% v. 35.6%, respectively, P<0.05). Pregnancy rate on Day 8 (38.4%, 1358/3533) of the estrous cycle at the time of embryo transfer was significantly higher than on Days 6 (27.7%, 23/83) and 7 (36.2%, 1235/3408) (P<0.05), and the pregnancy rate on Day 6 of the estrous cycle at the time of embryo transfer tended to be lower than on Day 9 (38.9%, 88/226) (P<0.08). These results demonstrate that confirming standing heat, performing CL examination twice before embryo transfer, freezing high quality embryos, and performing embryo transfers on Day 8 resulted in an improved pregnancy rate for the transfer of IVF-derived embryos.