Effect of bovine oocyte vitrification with EGTA and post-warming recovery with resveratrol on meiotic spindle, mitochondrial function, reactive oxygen species, and developmental competence.

The present study aimed to determine the effects of the addition of EGTA to vitrification solutions and a post-warming recovery period supplemented with 1 µM resveratrol on meiotic spindle integrity, mitochondrial activity, ATP content, reactive oxygen species (ROS) levels, and developmental potential of partially denuded, vitrified-warmed bovine oocytes. Results of microtubule distribution and chromosomal arrangement indicated that resveratrol supplementation, irrespective to EGTA addition, reduced the incidence of abnormal meiotic spindles to similar levels of the control group. Mitochondrial membrane potential was similar in all groups, but ATP content was negatively affected by the vitrification-warming procedure and failed to recover after 4 h of post-warming culture. Resveratrol caused the reduction of ROS to lower levels of the control group, and showed the lowest ROS levels when combined with EGTA treatment. Oocytes in all vitrification groups presented lower developmental potential when compared to fresh oocytes. However, oocytes that underwent vitrification supplemented with EGTA and post-warming culture along with resveratrol showed higher developmental competence compared with vitrified-warmed oocytes not supplemented with resveratrol. The results of our study indicate that submitting vitrified-warmed, partially denuded bovine oocytes to a post-warming recovery period supplemented with 1 µM resveratrol improves vitrification outcomes. However, the benefits of EGTA on vitrification and warming of bovine oocytes need to be further investigated.

Recovery of spindle morphology and mitochondrial function through extended culture after vitrification-warming of bovine oocytes.

The present study aimed to determine the effects of vitrification on the meiotic spindle and mitochondrial function of bovine oocytes submitted to different times of post-warming culture. Partially denuded cumulus-oocyte complexes were vitrified at different maturation times (18-, 20-, and 24-h) using a two-step cryoprotectant addition protocol and submitted to 6-, 4-, or 0-h of post-warming extended culture in maturation medium. Microtubule configuration and chromosomal arrangement were analyzed after 0- and 6-h of extended culture, whereas mitochondrial membrane potential and ATP content were measured at 0-, 4-, and 6-h of post-warming recovery. Results of meiotic spindle integrity revealed that vitrified-warmed oocytes that underwent 6-h of culture had similar incidence of normal microtubule configuration and chromosomal arrangement as compared to fresh oocytes, but higher than oocytes in the vitrification control group (no culture). Mitochondrial membrane potential was not different in all the vitrification groups, but the oocytes that were cultured for 4-h after warming had similar levels compared to fresh oocytes. ATP concentration in all vitrification groups was lower than the control group. However, oocytes cultured for 6-h had the lowest rate of ATP depleted oocytes among the vitrification groups. The results of this study indicate that extended culture after warming promotes the recovery of the meiotic spindle and, to some extent, mitochondrial function of vitrified-warmed metaphase II bovine oocytes.

Evaluation of Seasonal Heat Stress on Transcriptomic Profiles and Global DNA Methylation of Bovine Oocytes.

Heat stress affects oocyte developmental competence and is a major cause of reduced fertility in heat stressed cattle. Negative effects of heat stress on the oocyte have been observed at morphological, biochemical and developmental levels. However, the mechanisms by which heat stress affects the oocyte at the transcriptional and epigenetic levels remain to be further elucidated. Here we aimed to investigate the effect of heat stress on oocyte quality, transcriptomic profiles and DNA methylation of oocytes collected through the transition from spring to summer under Louisiana conditions. Summer season resulted in a lower number of high quality oocytes obtained compared to the spring season. There was no difference in in vitro maturation rates of oocytes collected during spring as compared to summer. RNA sequencing analysis showed that a total of 211 and 92 genes were differentially expressed as a result of heat stress in GV and MII oocytes, respectively. Five common genes (E2F8, GATAD2B, BHLHE41, FBXO44, and RAB39B) were significantly affected by heat in both GV and MII oocytes. A number of pathways were also influenced by heat stress including glucocorticoid biosynthesis, apoptosis signaling, and HIPPO signaling in GV oocytes, and Oct4 pluripotency, Wnt/beta-catenin signaling, and melatonin degradation I in MII oocytes. In addition, fluorescent immunocytochemistry analysis showed no difference in global levels of DNA methylation and DNA hydroxymethylation at either the GV or MII stage between spring and summer oocytes. The results of this study contribute to a better understanding of the effect of heat stress on the molecular mechanisms altered in bovine oocytes.

Pregnancy Rates Following Low-Temperature Storage of Large Equine Embryos Before Vitrification.

Satisfactory pregnancy rates can now be achieved following the cryopreservation of large equine embryos. Nonetheless, its wide application might be limited by the fact that the cryopreservation of large equine embryos requires a specialized micromanipulation equipment and micromanipulation/vitrification skills. Alternatives should be developed to increase its utilization and widespread application in the commercial equine industry. To determine if large equine embryos are able to remain viable during transport from farms to specialized centers for embryo cryopreservation, we evaluated pregnancy rates following the low-temperature storage of large equine embryos before vitrification. Grade 1 embryos (n = 37) were randomly assigned to six treatments consisting of day of collection (Day 7 or 8 after ovulation) and cooling for 0, 12, or 24 hours before vitrification in a factorial design. Pregnancy rates of Day 7 embryos cooled for 12 and 24 hours were 55.5% and 75%, respectively. Pregnancy rates of Day 8 embryos cooled for 12 and 24 hours were 0 and 16.6%, respectively. Day 7 cooled embryos resulted in higher pregnancy rate compared with Day 8 cooled embryos (64.7% and 7.7%, respectively; P < .05). Pregnancy rate comparison of cooled embryos grouped by diameter showed that embryos <550 µm resulted in a higher pregnancy rate compared with embryos >550 µm (71.4% and 12.5% respectively; P < .05). In conclusion, Day 7 equine embryos up to 550 µm can be cooled to temperatures of 9-12°C for 12 or 24 hours before vitrification and result in satisfactory pregnancy rates.