Factors affecting number of surface ovarian follicles and oocytes yield and quality in egyptian buffaloes

Three experiments were conducted to evaluate factors affecting number of surface ovarian follicles and oocytes yield and quality in buffalo. In Experiment 1, ovaries ($n = 126$) were collected in pairs from slaughtered anoestrus, early pregnant and cyclic buffaloes. Ovarian follicles (1-3, 4-9 and $\geq$ 10 mm diameter) were counted, aspirated and oocytes were recovered and evaluated. In Experiment 2, ovaries were divided into 2 groups. Group 1, ovaries bearing a CL ($n = 74$) and Group 2 non-bearing CL ($n = 74$), ovarian follicles (2-8 mm) were counted, aspirated and oocytes evaluated. In Experiment 3, oocytes were recovered using aspiration or slicing methods. In all experiments, oocytes were classified into good, fair, poor and denuded. Results showed that the development of small and total ovarian follicles are continuous and independent in early pregnant or cyclic buffalo cows, however, it significantly decreased ($P < 0.01$) in the ovaries of anoestrus buffaloes. Number of medium and large size follicles was significantly increased ($P < 0.01$) in cyclic buffaloes on Days 10-16 and 17-22 of oestrous cycle, while large follicles was significantly decreased ($P < 0.01$) in the ovaries of pregnant buffaloes. A significantly higher ($P < 0.01$) percentage of poor and denuded oocytes were recovered from ovaries of anoestrus and pregnant buffalo. While, the highest ($P < 0.01$) percentage of good quality oocytes were recovered from ovaries of cyclic buffaloes on Days 1-3 and 10-16 of oestrous cycle, eliciting that the stage of oestrous cycle is affecting the quality of buffalo oocytes. In addition, the presence of a CL stimulates the development of a significantly higher ($P < 0.01$) number ovarian follicles which produced a significantly higher ($P < 0.05$) number of good quality oocytes. Slicing of buffalo ovaries produced a significantly higher number of fair, poor and denuded oocytes. In conclusion, number of ovarian follicles and yield and quality of oocytes were affected by the reproductive status, stage of the oestrous cycle, presence of a CL and the method of oocytes retrieval

Impact of Nano-Bromocriptine on Egg Production Performance and Prolactin Expression in Layers

The current study aimed to investigate the potential use of nano-bromocriptine in improving the laying performance of late laying hens by modulating the prolactin gene expression. A total of 150 NOVOgen brown laying hens aged 70 weeks were randomly allocated into three groups of 50 birds each. The first group was kept as a control, while the second and the third groups were treated with bromocriptine and nano-bromocriptine, respectively, at a dose of 100 µg/kg body weight per week. The pause days, egg production, feed per dozen egg, and Haugh unit were determined on a monthly basis. Also, the relative prolactin gene expression in the pituitary gland was quantified using qPCR and the number of the ovarian follicles was determined after slaughtering at the 84th week of age. It was found that nano-bromocriptine and bromocriptine improved egg laying performance with minimal pause days, reduced feed per dozen egg, and depressed the relative prolactin gene expression; however, nano-bromocriptine treatment was significantly effective compared to bromocriptine. In conclusion, nano-bromocriptine might be beneficial for elongating sequences and reducing pauses

Intraovarian Injection of Reconstituted Lyophilized Growth-Promoting Factor Extracted from Horse Blood Platelets (L-GFequina) Increases Oocytes Recovery and In Vitro Embryo Production in Holstein Cows

The purpose of this study was to determine the impact of intraovarian injections of a reconstituted lyophilized growth-promoting factor extracted from horse blood platelets (L-GFequina) on the number of ovarian follicles, the recovery of cumulus&ndash;oocyte complexes (COCs), and embryo development to the blastocyst stage in Holstein cows. Thus, 12 Holstein cows were assigned to three protocols. According to the number of punctured follicles in protocol 1, ovum pick-up (OPU) was conducted on days 6 and 14 of the cycle (day 0 = estrus). In protocol 2, every large follicle (more than 7 mm) was removed, and 1 mL of L-GFequina was intraovarian injected (day 0). Two days later, equine chorionic gonadotropin (eCG) was administered, and OPU sessions were conducted on days 6, 10, and 14. The same ovarian stimulation procedure as that in protocol 2 was performed in protocol 3, except that equine L-GFequina was not supplied. OPU was carried out on days 6 and 10 of the cycle. The results indicate that the intraovarian injection of L-GFequina significantly (p &lt; 0.05) increased the number of OPU sessions per cycle, the recovery of cumulus&ndash;oocyte complexes (COCs), and the production of blastocysts. In conclusion, an intraovarian injection of L-GFequina can improves OPU-IVEP results in Holstein cows

Effect of Zinc and Nano Zinc on Developmental Competence of Buffalo Oocytes

This study aimed to investigate the impact of zinc sulfate and nano zinc oxide on the In-vitro maturation (IVM) of oocytes and on the In-vitro embryo developmental competence of buffaloes. Ovaries were obtained from the abattoir. Good quality oocytes (excellent &amp; good) were matured in tissue culture medium -199 (TCM-199) vs. TCM-199 +10-6M zinc sulfate vs. TCM-199 +10-6 M nano zinc oxide enriched by fetal calf serum 10% (FCS) + 10 μg/ml follicle-stimulating hormone + 50 μg/ml gentamicin. The oocyte maturation was done in the incubator for 22 h in a humidified environment with CO2 5% and 38.5°C. Frozen-thawed semen was used to fertilize Mature oocytes, which were then incubated for 18 hours, before being cultured by synthetic oviduct fluid (SOF) for 7 days. The obtained results showed that supplementing maturation medium with 10-6 M zinc sulfate and 10-6 M nano zinc oxide resulted in a significant (P˂0.05) rise in GIII cumulus cell expansion of buffalo oocytes by 52.93 %, and 59.75%, respectively, as compared to oocytes cultured in free medium (36.8%). G0 cumulus cell expansion showed a significant (P˂0.05) decrease in zinc sulfate and nano zinc oxide groups (7.85, 3.29 %, respectively) when compared with oocytes cultured in free medium (14.73 %). The rate of maturation of oocytes with polar bodies was significantly greater in the zinc sulfate and nano zinc oxide groups (86.98, and 92.43%, respectively) when compared with those matured in free medium (80.11%). The hatching (cleavage) rate was significantly greater (P˂0.05) in the zinc sulfate and nano zinc oxide groups (83.17, 87.66 respectively %) when compared with the TCM-199 (free medium) group (78.60%). The transferable embryos (blastocyst &amp; morula) rates significantly raised (P˂0.05) in the zinc sulfate (17.28 &amp; 19.87 %, respectively) and nano zinc oxide groups (21.23 &amp;26.21%, respectively) when compared with TCM-199 group (11.19 &amp;13.75 %, respectively). In conclusion, in vitro maturation rate and transferable embryo rates in buffaloes improve by adding zinc sulfate and nano zinc oxide to the medium of maturation

Effect of oxygen tension and antioxidants on the developmental competence of buffalo oocytes cultured in vitro

Aim: Oxidative stress (OS) is one of the major disruptors of oocyte developmental competence, which appears due to the imbalance between the production and neutralization of reactive oxygen species (ROS). Materials and Methods: In Experiment 1, buffalo oocytes were in vitro matured, fertilized, and cultured at 38.5°C under 5% CO2 + 20% O2 in standard CO2 incubator (OS) or under 5% O2 + 5% CO2 + 90% N2 (Multi-gas incubator, low O2). In Experiment 2, buffalo cumulus oocytes complexes (COCs) were matured in Basic maturation medium (BMM) composed of TCM199+ 10% FCS+ 10 μg/ml FSH+ 50 μg/ml gentamicin (control group) or in BMM supplemented with 50 μM ascorbic acid (ascorbic acid group) or 3.0 mM glutathione (glutathione group) or 10-5 M melatonin (melatonin group) and cultured at 38.5°C under 20% O2 for 24 h. Matured buffalo oocytes in control, ascorbic acid, or melatonin groups were fertilized and zygotes were cultured for 8 days under the same conditions. Results: In both experiments, maturation, cleavage, and blastocyst rates were recorded. Results showed that culture of buffalo oocytes under low O2 (5% O2) significantly increased maturation, cleavage, and blastocyst rates (p<0.05). Meanwhile, under 20% O2, addition of 10-5 M melatonin or 50 μM ascorbic acid to in vitro maturation (IVM) medium significantly improved cumulus cell expansion, nuclear maturation rates of buffalo oocytes (p<0.05), and increased cleavage and blastocyst rates (p<0.05). Conclusion: About 5% O2 is the optimum condition for in vitro production of buffalo embryos, and addition of 10-5 M melatonin to IVM medium for oocytes cultured under 20% O2 could alleviate the adverse effect of high oxygen tension and increased embryo yield

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

Abstract Background TGF-β signaling pathways regulate several crucial processes in female reproduction. AKT is a non-SMAD signaling pathway regulated by TGF-β 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-β 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

Effect of Melatonin-loaded Chitosan Nanoparticles (CMN) on Gene Expression of In-vitro Matured Buffalo Oocyte

This study aimed to evaluate the effect of melatonin and melatonin-loaded chitosan nanoparticle (CMN) supplementation to maturation media on buffalo oocyte maturation rate and relative expression of genes: growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), B Cell Lymphoma 2 (BCL2), Associated X protein (BAX) and superoxide dismutase 1 (SOD1). Buffalo ovaries were heaved from Al-Mounib slaughterhouse, cumulus-oocyte complexes (COCs) were in vitro matured in three different media, TCM-199 medium (control), TCM-199 with melatonin 10-9M, and TCM-199 with CMN 10-9M. The assessment of the nuclear maturation rate was carried out through the presence of the first polar body. In addition, the mature buffalo oocytes were stored on RNA later for genetic analysis of GDF9, BMP15, SOD1, BCL2, and BAX genes using quantitative real-time PCR (qRT-PCR). The results were reported that buffalo oocytes supplemented with melatonin-loaded chitosan nanoparticle (CMN) or melatonin have a significant effect on nuclear maturation rate 94.04±0.65 and 88.74±0.77 respectively when compared with buffalo oocytes matured with basic media (control) 79.67±1.35. Furthermore, buffalo oocytes supplemented with melatonin-loaded chitosan nanoparticle (CMN) or melatonin showed significant upregulation of GDF9, BMP15, SOD1, and BCL2 genes and significant downregulation of BAX gene when compared with oocyte matured with basic media (control). In conclusion, the results of nuclear maturation rate and relative expression pattern of GDF9, BMP15, SOD1, BCL2, and BAX reflect that melatonin-loaded chitosan nanoparticle (CMN) and melatonin` may play an important role in the buffalo oocytes developmental competence