Developmental kinetics and viability of bovine embryos produced in vitro with sex-sorted semen.

The present study aimed to elucidate the developmental kinetics, growth potential, and viability of bovine embryos produced in vitro with sexed semen. Bovine oocytes were fertilized in vitro using unsorted and X-sorted semen from the same Holstein bulls, and the kinetics of in vitro development were continuously monitored for 10 d through time-lapse cinematography. The blastocyst formation rate was lower in the X-sorted group than in the unsorted group (P < 0.01), whereas the normal fertilization rate did not differ between groups. Morphokinetic evaluation revealed that the incidence of blastomere fusion during the first cleavage division, termed reverse cleavage, was higher in the X-sorted group (P < 0.01). Furthermore, embryos produced with X-sorted semen showed slower growth throughout the developmental period than embryos produced with unsorted semen (P < 0.01). The cell number of the trophectoderm and inner cell mass of blastocysts was reduced in the X-sorted group (P < 0.01). In embryos that developed to the blastocyst stage, the hatchability (P < 0.05), chromosomal normality (P < 0.01), and survivability after the conventional frozen-thawing process (P < 0.05) were reduced in the X-sorted group compared to that in the unsorted group, indicating a compromised viability of embryos derived from X-sorted semen. Taken together, the first cleavage dysmorphism, delayed embryo growth, and impaired viability of embryos developed to the blastocyst stage may explain the mechanism of reduced fertility in embryos derived from sexed semen. The kinetic evaluation of early embryo development and de-selection of embryos presenting the aberrant first cleavage would be valid for clinical application to produce sexed embryos with high implantation potential.

Glutathione ethyl ester improved the age-induced decline in the developmental competence of bovine oocytes.

The aberrant redox regulation and anti-oxidative defense is one of the main causes of age-induced decline in oocytes quality and embryo development in mammals. The present study aimed to elucidate the effect of glutathione ethyl ester (GSH-OEt), a cell-permeable glutathione (GSH) donor, on the developmental competence of oocytes in cows with advanced reproductive age. Oocytes were collected from cows aged 30-50 months or >120 months, which were defined as young or aged, respectively, and subjected to in vitro maturation (IVM) in the presence of 5 mM of GSH-OEt. In aged cows, the GSH level in follicular fluid was lower, and the intracellular levels of reactive oxygen species (ROS) in post-IVM oocytes was higher than those in young cows. GSH-OEt supplementation during IVM reduced the ROS contents of oocyte in aged cows but not in young cows. GSH-OEt treatment promoted the meiotic progression and increased the proportion of oocytes with mature cytoplasm containing evenly dispersed cortical granules in aged cows. After in vitro fertilization, the normal fertilization and development to the blastocyst stage were enhanced by GSH-OEt in aged cows to levels comparable to those in young cows. Further, oocyte maturation in the presence of GSH-OEt increased the proportion of diploid blastocyst in aged cows. In contrast, GSH-OEt failed to enhance the oocyte maturation, fertilization, and embryo development in young cows. Taken together, the exogenous supplementation of GSH-OEt during IVM modulated the age-related oxidative damage of bovine oocytes and improved the developmental competence of oocytes in aged cows. Oocytes presented a distinct response to GSH-OEt treatment depending on the donor age. GSH-OEt supplementation during IVM could be of practical value through the efficiency improvement of chromosomally normal embryo production in aged cows.

Prevention of retained fetal membranes and improvement in subsequent fertility with oxytocin administration in cows with assisted calving.

In dairy cows, the efficacy of oxytocin treatment for preventing retained fetal membranes (RFM) is controversial. The physiological condition of cows associated with the calving process may affect the action of oxytocin. This study aimed to elucidate the difference in the efficacy of exogenous oxytocin treatment immediately after calving among cows that received various obstetric interventions. The calving ease was recorded using a score of 1-5, and assisted birth was defined as a score of 2 or more. Cows that required calving assistance (assisted, n = 28) due to delayed calving progression had a prolonged time from calving to expulsion of the fetal membrane (P < 0.01), and impaired reproductive performance compared to cows that did not receive calving assistance (unassisted, n = 78). The effect of oxytocin treatment was determined using cows that did not expel their fetal membrane within 3 h after calving. Cows were randomly divided into the control (unassisted, n = 41; assisted, n = 22) or oxytocin group (unassisted, n = 33; assisted, n = 10). Oxytocin (50 IU) was administered intramuscularly to the cows in the oxytocin group between 3 and 6 h after calving, while no treatment was administered in the control group. In cows with assisted birth, oxytocin administration accelerated placental expulsion (P < 0.05) and improved several reproductive parameters, such as the number of services until conception (P < 0.05) and the calving to conception intervals (P < 0.05) compared to the control group. On the other hand, oxytocin administration slightly accelerated placental expulsion (P < 0.05), but failed to improve fertility in cows with unassisted birth. The results indicate that the action of oxytocin varies depending on the calving situation of the cows. Oxytocin administration during the early postpartum period could prevent RFM and improve the decline in reproductive performance associated with calving assistance.

Growth potential of bovine embryos presenting abnormal cleavage observed through time lapse cinematography.

Time-lapse monitoring (TLM) has emerged as a novel technology for the continuous and noninvasive evaluation of embryos. TLM has revealed the prevalence of specific dysmorphisms such as abnormal development during the early-cleavage stage of embryos. However, little information is available on the prevalence and consequences of abnormal cleavage in bovine embryos. Hence, this study aimed to investigate growth potential of bovine embryos presenting abnormal cleavage, such as reverse cleavage (RC), direct cleavage (DC), and irregular and unsmooth ruffling of the oolema membrane (ruffling). Bovine embryos derived through in vitro fertilization (IVF) were cultured in the microwell culture dishes, and the kinetics of in vitro development were observed through TLM at 20-min intervals for 10 d. Approximately 36% of embryos that developed into a blastocyst presented abnormal cleavage. Morphokinetic evaluations revealed that RC, DC, and ruffling embryos showed slower development compared to embryos with normal cleavage (P < 0.01). Embryos with RC and DC, but not ruffling, revealed impaired hatchability (P < 0.05) with increased collapses of the blastocyst cavity until hatching (P < 0.0001). Moreover, the RC and DC embryos presented increased chromosomal aneuploidy (P < 0.05). These results suggest a compromised viability of embryos with RC and DC. This is the first report that clarified the effect of abnormal cleavage on the morphokinetics and growth potential of bovine IVF embryos. Results indicate that the kinetic evaluation of bovine embryos using the time-lapse imaging system will be beneficial for selecting embryos with a high viability.

Peptidoglycan inhibits progesterone and androstenedione production in bovine ovarian theca cells.

Uterine bacterial infection perturbs uterine and ovarian functions in postpartum dairy cows. Peptidoglycan (PGN) produced by gram-positive bacteria has been shown to disrupt the ovarian function in ewes. The aim of this study was to determine the effect of PGN on steroid production in bovine theca cells at different stages of follicular development. Bovine theca cells isolated from pre- and post-selection ovarian follicles (<8.5mm and >8.5mm in diameter, respectively) were cultured in vitro and challenged with PGN. Steroid production was evaluated by measuring progesterone (P4) and androstenedione (A4) concentration in culture media after 48 h or 96 h of culture. Bovine theca cells expressed PGN receptors including Toll-like receptor 2 and nucleotide-binding oligomerization domain 1 and 2. Treatment with PGN (1, 10, or 50 µg/ml) led to a decrease in P4 and A4 production by theca cells in both pre- and post-selection follicles. The mRNA expression of steroidogenic enzymes were decreased by PGN treatment. Moreover, A4 production was further suppressed when theca cells of post-selection follicles were simultaneously treated by PGN and lipopolysaccharide (0.1, 1, or 10 µg/ml). These findings indicate that bacterial toxins may act locally on ovarian steroidogenic cells and compromise follicular development in postpartum dairy cows.

Effects of lipopolysaccharide (LPS) and peptidoglycan (PGN) on estradiol production in bovine granulosa cells from small and large follicles.

In cows, postpartum uterine infection due to bacteria that produce lipopolysaccharide (LPS) or peptidoglycan (PGN) leads to ovarian dysfunction. The aim of this study was to determine the effect of LPS and/or PGN on estradiol production from granulosa cells from small and large follicles in the bovine ovary. Granulosa cells from small and large ovarian follicles were exposed to LPS and/or PGN in vitro. LPS inhibited the expression of TLR4, CD14, MD2 and NOD1 genes in FSH-treated granulosa cells from small follicles. LPS suppressed estradiol (E2) production in granulosa cells from small and large follicles, while PGN inhibited E2 production in granulosa cells from large follicles. LPS or PGN did not affect granulosa cell survival. Although LPS alone suppressed E2 production in granulosa cells from small and large follicles, E2 production was not further suppressed when PGN was added to culture medium with LPS alone. Our data demonstrated that susceptibility to LPS or PGN in granulosa cells depends on the follicle developmental stage. The results of the present study suggest that ovarian dysfunction in cows with postpartum uterine infection may be caused by inhibitory effects of LPS and PGN on E2 production in granulosa cells.