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Impact of nano‑selenium on nuclear maturation and genes expression profile of buffalo oocytes matured in vitro [2020-10-20]

Abstract Supplementation of maturation media with antioxidant (bulk form) improves oocyte maturation. However, the influence of adding antioxidant (nano-particles) on oocyte maturation is not well known. We aimed to evaluate the effect of selenium nano-particles (SeNP) and bulk selenium (Se) on buffalo oocytes maturation, in terms nuclear maturation and molecular level. Oocytes were distributed into four groups; 1st group was control, 2nd group was supplied with Se (10 ng/ml), 3rd and 4th groups were supplied with 1 μg/ml SeNP (67 nm), and SeNP (40 nm), respectively. Matured oocytes were fixed and stained to determine nuclear maturation. Oocytes and COC after IVM were stored at − 80 °C, for RNA isolation and qRT-PCR for selected genes. The Se and seNP (40 nm) had a positive effect on oocytes nuclear maturation rates. Apoptosis-related cysteine peptidase (CASP3) was reduced in all supplemented groups. Anti-Mullerian hormone (AMH) was up-regulated in oocytes supplemented with SeNP (40 nm). In COC, AMH increased in group supplemented with SeNP (67 nm). In oocytes, phospholipase A2 group III (PLA2G3) decreased in all supplemented groups. While in COC, PLA2G3increased in group supplied with Se. In COC, luteinizing hormone/choriogonadotropin receptor (LHCGR) increased in groups supplied with Se or SeNP (40 nm).Glutathione peroxidase 4 (GPX4) increased in all supplemented groups, in oocytes and COC. In oocytes, superoxide dismutase (SOD) was up-regulated in supplemented groups {Se and SeNP (67 nm)}.The DNA methyltransferase (DNMT) in oocytes was reduced in supplemented groups. In oocytes, the POU class 5 homeobox 1 (OCT4) increased in all supplemented groups. In COC, the OCT4 was over-expressed in group supplemented with SeNP (40 nm). Selenium supplementation in bulk or nano-particle improved in vitro buffalo oocytes maturation, viaup-regulation of antioxidant defense and development competence genes. SeNP (smaller size, 40 nm) induced higher expression of antioxidant gene. download attachment

Influence of Epidermal Growth Factor with Cysteamine on in -Vitro Buffalo Embryo Development [2020-10-20]

R IMPROVING embryo development in buffalo, two experiments were conducted. The first one was carried out to evaluate the different concentrations (0, 5, 25, 50 ng/ml) of epidermal growth factors (EGF) on developmental competence of buffalo oocytes. The selected oocytes were cultured in the four concentrations of EGF. The embryo cleavage rate was significantly higher in oocytes exposed to 5, 25, 50 ng/ml EGF than control. There were no significant difference in four groups (0, 5, 25, 50 ng/ml EGF) in the rate of morula. But, better cleavage and blastocyst rates were observed at 5 ng/ml EGF. In the second experiment, the additive effect of 5 ng/ml EGF with 50 μM cysteamine on maturation and embryo development was studied. Oocytes were collected, matured and cultured in three groups. In the first group, media supplemented with 5 ng/ml EGF + 50 μM cysteamine combination. In the second group, media supplemented with EGF. The third group was supplemented with cysteamine. There was a significant increase in cleavage rate in combination group than EGF (P< 0.05) and cysteamine (P< 0.01) groups. But there was no significant difference in cleavage rate between EGF and cysteamine. The morula percentage was nearly similar in the three groups. But blastocyst rate was significantly (P

Effect of Cumulus Cells and Meiotic Stages on Survivability and Meiotic Competence in Vitrified Buffalo Oocytes [2020-10-20]

Abstract.- Cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. Buffalo oocytes are sensitive to chilling injury, making their cryopreservation very difficult. To improve the efficacy of vitrification in buffalo oocytes, two experiments were conducted in this study, the first one was to evaluate the effect of existence of cumulus cells on the viability and maturation of vitrified oocytes. Oocytes with complete cumulus oocyte complexes (COCs), partial (COCs) and denuded were matured in vitro for 22h. The oocytes were vitrified in a mixture of 3M DMSO + 3M EG by the straw method. After warming the oocytes were cultured for further 2h. There was significant increase in morphologically normal, survivability and maturation rate of COCs and partial COCs than denuded oocytes. The second experiment was to evaluate the effect of meiotic stage on the viability and maturation of vitrified oocytes. Mature (22h) and immature (0h) oocytes were vitrified by the straw method. After warming, the oocytes were evaluated morphologically, and then cultured in vitro to complete the maturation period to 24h. Survivability of vitrified oocytes directly after warming was higher significantly (P< 0.05) in mature than immature oocytes. Also, the nuclear maturation rate was significantly higher (p

Establishment a protocol for total RNA isolation from buffalo fresh and frozen semen for molecular applications [2020-10-20]

Abstract To date, there is no an established protocol for total RNA isolation in Egyptian buffalo spermatozoa. The present study aimed (I) to establish a defined protocol for total RNA isolation from fresh and frozen spermatozoa, (II) to evaluate RNA quality and quantity from different extraction methods and studying gene expression. Warm and standard room temperature modified QIAzol Lysis Reagents were used for total RNA extraction. The quality and quantity of extracted RNA were checked, and subsequently qRT-PCR was performed using androgen receptor-like and three reference gene primers (GAPDH, ACTB and 18S). The warm modified QIAzol Lysis Reagents resulted higher yield of good quality RNA from fresh (569.54 ± 18.83 ng/μl) and frozen spermatozoa (110.59 ± 4.43 ng/μl), compared to standard room temperature modified QIAzol (421.26 ± 7.18 ng/μl) and (29.07 ± 5.25 ng/μl), for fresh and frozen semen samples respectively. The 260/280 ratio was 1.90 and 1.89 for fresh and frozen isolated semen by warm method respectively. The integrity of RNA was good and appeared as a sharp band on 2% agarose gel. The most stable reference gene was 18S. Reliable extraction method of high quality RNA yield could be a step forward for understanding mechanisms of spermatogenesis for improving male fertility. K E Y W O R D S Egyptian buffalo, reference genes, RNA isolation, spermatozoa, warm QIAzol

Impact of Using Brilliant Cresyl Blue Stain on Oocyte and Embryo Selection [2018-01-02]

T HE SUCCESS of in vitro embryo production is ultimately dependent on the number and quality of cumulus-oocyte complexes harvested from ovaries. The morphological criteria are routinely used for oocytes selection in most laboratories. There is brilliant cresyl blue stain (BCB) test allowing the selection of developmentally competent oocytes which is a simple, quick, economic and feasible protocol. BCB is known to be a non-invasive methodology that allows the selection of immature oocytes in several species. BCB test determine the intra cellular activity of glucose-6-phosphate dehydrogenase (G6PDH) that gradually decreases its activity as oocytes reach their growth phase. The using of BCB test before IVM is still controversial, so this review describes the use of BCB staining test for oocytes and embryo selection to investigate the impact of application of this stain in in-vitro maturation and embryo production in animals. Key words: Oocytes quality, brilliant cresyl blue stain, embryo quality, selection, in vitro maturation.

Factors Affecting Buffalo Oocytes Maturation [2018-01-02]

Abstract: in vitro maturation, fertilization and culturing of buffalo oocytes were considered the most important application, to improve buffalo reproductivety and productivity. Oocyte maturation is the first and most critical step towards successful in vitro embryo production. There are many factors affect the maturation of buffalo oocytes as culture media, method of recovery, oocyte quality, season of collection and ovarian status and others. Proper oocytes selection in the laboratory is crucial for successful embryo production in vitro. Little information is available on in vitro maturation and fertilization of buffalo oocytes. in vitro fertilization results obtained by several workers were relatively poor when compared to cattle. So the present review aimed to throw some lights on the factors affecting oocytes in vitro maturation in buffalo. Key words: in vitro Maturation Buffalo Oocytes

Effect of Season of the year and Ovarian Structures on Oocytes Recovery Rate, Quality and Meiotic Competence in Egyptian Buffaloes Al-shimaa Al-H. H. El-1 Naby, 2Karima G.H.M. Mahmoud, 2Youssef F. Ahmed, 1Mahmoud E.A. Abouel-Roos and 1Alaa E. Abdel-Ghaffar [2018-01-02]

Abstract: The present study aimed to evaluate the effect of season of the year and ovarian structure (corpus luteum) on the recovery rate, quality and maturation of Egyptian buffalo oocytes. For this purpose, ovaries were collected from slaughtered buffaloes and oocytes were scored after recovery by aspiration. Oocytes were matured in vitro for 24 h and evaluated for nuclear maturation after staining by 1% orcein stain. Results showed a significantly (P

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