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.

Expression profile of genes related to pregnancy maintenance in Dromedary Camel during the first trimester.

So far, few signals involved in embryo-maternal dialogue have been identified in pregnant she-camel. Our objective was to investigate expression profiles of genes relevant to uterine extracellular matrix remodeling (ITGB4, SLCO2A1, FOS, and JUN), uterine tissue vascularization, and placental formation (VEGFA, PGF, and PDGFA), embryonic growth and development (IGF1 and PTEN), plus cell death of uterine tissue (BCL2) in early pregnant versus non-pregnant she-camels. Forty genital tracts (20 pregnant and 20 non-pregnant) and blood samples were collected from abattoirs. Total RNA was extracted from uterine tissues and qRT-PCR was conducted for candidate genes. Serum concentrations of progesterone (P4) and estradiol17-ß (E2) were measured. Expression of ITGB4, FOS, and PGF genes increased (P < 0.001) in the right uterine horn of pregnant versus non-pregnant she-camels. Moreover, JUN, SLCO2A1, VEGFA, and PTEN mRNAs were up-regulated (P < 0.001) in various segments of uterine tissues in pregnant groups. The PDGFA transcript was over-expressed (P < 0.001) in both uterine horns of pregnant groups. Additionally, IGF1 was higher (P < 0.001) in the right horn and the uterine body of pregnant groups, and expression of BCL2 was increased (P < 0.001) in the pregnant uterine body. Moreover, serum concentrations of P4 were higher (P < 0.001) and E2 lower (P < 0.05) in pregnant she-camels. Taken together, the fine-tuning of genes related to implantation, matrix formation, vascularization, and placental formation is highly required for successful pregnancy in she-camels.

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.

Caffeine supplementation during IVM improves frequencies of nuclear maturation and preimplantation development of dromedary camel oocytes following IVF.

Caffeine supplementation during oocyte IVM has been reported to improve preimplantation embryo development and the quality of in vitro-produced blastocysts in a range of species; but no studies have been done in camels. The present study investigated the effect of caffeine supplementation during dromedary camel oocyte IVM on nuclear maturation rates, IVF events, and subsequent preimplantation development. Cumulus-oocyte complexes obtained at slaughter were matured in vitro in caffeine supplemented medium either for 30 hours (caffeine 30 hours) or in the medium without caffeine supplement for 24 hours and then transferred to freshly prepared IVM medium supplemented with 10 mM caffeine for another 6 hours (caffeine 6 hours). Cumulus-oocyte complexes matured for 30 hours in the medium without caffeine supplement were used as a control. Matured oocytes were fertilized in vitro by epididymal spermatozoa of mature male camels collected from a local slaughterhouse. Eighteen hours after insemination, presumptive zygotes were cultured in modified KSOMaa medium for 7 days. Maturation and fertilization rates were significantly higher in the caffeine 6-hour group compared with the control group (P < 0.05), whereas IVM of oocytes in caffeine-supplemented medium for 30 hours did not affect these parameters (P > 0.05). Interestingly, IVM of oocytes in caffeine supplemented medium for 6 hours significantly (P < 0.05) increased the frequencies of blastocyst development by more than two-fold when compared with control (27.78% vs. 11.76%). In conclusion, culturing dromedary camel oocytes in maturation medium without caffeine for 24 hours and then in the medium supplemented with 10 mM caffeine for 6 hours during 30-hour IVM can significantly improve frequencies of nuclear maturation, fertilization rate, and subsequent preimplantation development.