Expression profile of viability and stress response genes as a result of resveratrol supplementation in vitrified and in vitro produced cattle embryos.

BACKGROUND: Resveratrol, a potent antioxidant, is known to induce the up-regulation of the internal antioxidant system. Therefore, it holds promise as a method to mitigate cryopreservation-induced injuries in bovine oocytes and embryos. This study aimed to (i) assess the enhancement in the quality of in vitro produced bovine embryos following resveratrol supplementation and (ii) monitor changes in the expression of genes associated with oxidative stress (GPX4, SOD, CPT2, NFE2L2), mitochondrial function (ATP5ME), endoplasmic reticulum function (ATF6), and embryo quality (OCT4, DNMT1, CASP3, ELOVL5). METHODS AND RESULTS: Three groups of in vitro bovine embryos were cultured with varying concentrations of resveratrol (0.01, 0.001, and 0.0001 µM), with a fourth group serving as a control. Following the vitrification process, embryos were categorized as either good or poor quality. Blastocysts were then preserved at - 80 °C for RNA isolation, followed by qRT-PCR analysis of selected genes. The low concentrations of resveratrol (0.001 µM, P < 0.05 and 0.0001 µM, P < 0.01) significantly improved the blastocyst rate compared to the control group. Moreover, the proportion of good quality vitrified embryos increased significantly (P < 0.05) in the groups treated with 0.001 and 0.0001 µM resveratrol compared to the control group. Analysis of gene expression showed a significant increase in OCT4 and DNMT1 transcripts in both good and poor-quality embryos treated with resveratrol compared to untreated embryos. Additionally, CASP3 expression was decreased in treated good embryos compared to control embryos. Furthermore, ELOVL5 and ATF6 transcripts were down-regulated in treated good embryos compared to the control group. Regarding antioxidant-related genes, GPX4, SOD, and CPT2 transcripts increased in the treated embryos, while NFE2L2 mRNA decreased in treated good embryos compared to the control group. CONCLUSIONS: Resveratrol supplementation at low concentrations effectively mitigated oxidative stress and enhanced the cryotolerance of embryos by modulating the expression of genes involved in oxidative stress response.

Ovarian and uterine arteries blood flow velocities waveform, hormones and nitric oxide in relation to ovulation in cows superstimulated with equine chorionic gonadotropin and luteolysis induction 10 and 17 days after ovulation.

To investigate the ovarian responses, ovarian and uterine hemodynamics, circulating ovarian hormones, and nitric oxide (NO) with their relations in superstimulated cows. Eight Holstein Friesian dry cows previously synchronized with CIDR underwent rectal Doppler ultrasound scanning and blood sampling after administrating eCG (1500 I.U) on day 10 of the second ovulation (day -5). Cows were treated with 12.5 mg prostaglandin F2α (PGF2α) on days 10 and 17 after ovulation. Estradiol, progesterone, and NO were measured. Results showed that from ≥ 13 follicles, five follicles ovulated from both ovaries. The ovulated follicles increased antrum colored area and colored area % till day -1. The developed corpora lutea (CLs) attained similar diameter, area, colored area, and colored area % from day 2 till day 15. The peak point of velocity (PSV) of uterine arteries decreased while that of ovarian arteries increased from day -4 to day 0. Both ovarian arteries diameter, resistance index (RI), PSV, end velocity (EDV) and systolic/diastolic ratio (S/D) positively correlated (P < 0.0001), but their pulsatility index (PI) negatively correlated (P < 0.0001). The uterine arteries PI, RI, PSV, EDV, time average velocity (TAMV) and S/D negatively correlated (P < 0.0001) but their diameters positively correlated. Estradiol increased but progesterone decreased from day -5 till day 0. After ovulation, P4 reached maximum values on day 9 and started to decrease till day 19.NO showed one peak on day -3 and another one from day 3 to day 9. Conclusions: Blood flow of ovarian arteries is different from uterine arteries and depended on pre- or post-ovulation.

Dynamics of uterine and ovarian arteries flow velocity waveforms and their relation to follicular and luteal growth and blood flow vascularization during the estrous cycle in Friesian cows.

Doppler ultrasonography enabled understanding of the reproductive system hemodynamics in cyclic and pregnant cattle. To confirm the hypothesis that the ipsilateral ovarian and uterine arterial blood flows to the ovulating ovary are higher than the contralateral one along days and phases (follicular, early luteal, mid-luteal, late luteal) of the estrous cycle, eight cyclic spontaneously ovulating cows were scanned with Doppler ultrasound each other day along three oestrous cycles to monitor the follicular dynamics, the vascularization of the ovulatory follicle (OF), the corpus luteum (CL) developmental dynamics, the ipsilateral and the contralateral ovarian and uterine arterial diameters and their blood flow. Results proved the hypothesis. Both days and phases of the estrus cycle influenced (P = 0.0001) the follicular dynamic, the luteal hemodynamics, the ovarian and uterine hemodynamic. The ovulatory wave and the mid-luteal non-ovulatory wave had expanding numbers and the diameters of small, medium and large follicles. Though area, antral area, vascularization area of the OF ascended from Day -4 to the day of ovulation (Day 0), but the percent of its vascularization area and that of granulose layer increased till Day -3. The CL diameter increase till Day 15, and its vascularization area increased till Day 13, but its% of vascularization area ascended (P = 0.0001) from Days 1-4 and declined from Days 9-13. Both RI and PI of the ipsilateral ovarian artery were lower than the contralateral one; but, both obtained high values during the follicular phase. A linear increase (P = 0.0001) of uterine horns vascularization area and both ovarian and uterine arteries diameters, PSV and EDV from follicular to late luteal phases accompanied a linear decrease of their PI and RI. In conclusion, the ovarian and uterine blood flows vary according to the estrous day, estrous phase, the ovulating ovary, ovulatory follicle growth and corpus luteum developmental stage.

Functional role of AKT signaling in bovine early embryonic development: potential link to embryotrophic actions of follistatin.

BACKGROUND: TGF-ß signaling pathways regulate several crucial processes in female reproduction. AKT is a non-SMAD signaling pathway regulated by TGF-ß ligands essential for oocyte maturation and early embryonic development in the mouse, but its regulatory role in bovine early embryonic development is not well established. Previously, we demonstrated a stimulatory role for follistatin (a binding protein for specific members of TGF-ß superfamily) in early bovine embryonic development. The objectives of the present studies were to determine the functional role of AKT signaling in bovine early embryonic development and embryotrophic actions of follistatin. METHODS: We used AKT inhibitors III and IV as pharmacological inhibitors of AKT signaling pathway during the first 72 h of in vitro embryo culture. Effects of AKT inhibition on early embryonic development and AKT phosphorylation were investigated in the presence or absence of exogenous follistatin. RESULTS: Pharmacological inhibition of AKT signaling resulted in a significant reduction in early embryo cleavage, and development to the 8- to 16-cell and blastocyst stages (d7). Treatment with exogenous follistatin increased AKT phosphorylation and rescued the inhibitory effect of AKT inhibitors III and IV on AKT phosphorylation and early embryonic development. CONCLUSIONS: Collectively, results suggest a potential requirement of AKT for bovine early embryonic development, and suggest a potential role for follistatin in regulation of AKT signaling in early bovine embryos.