Effects of ghrelin on in vitro nuclear maturation and subsequent embryo development of immature bovine oocytes

Development of efficient culture system to support embryonic development would be valuable when percentage of produced embryos reaching to the blastocyst stage is important. However, the rate of bovine embryo production in vitro is still lower than expected. Present study was performed to investigate the effect of ghrelin on nuclear maturation and subsequent bovine embryo development in vitro. Cumulus-oocyte-complexes were collected from slaughterhouse ovaries and randomly allocated in each treatment groups. Five different concentrations of ghrelin (0, 5, 50, 500 and 1000 ng mL-1) were added to the in vitro maturation medium (Hepes-buffered medium 199+fetal calf serum+gonadotrophins+insulin+antibiotics). The proportion of oocytes developed to metaphase II stage was significantly increased at 5 and 50 ng mL-1 ghrelin (86.32±3.38 and 89.77±2.92%, respectively). The result also indicated that adding high concentration of ghrelin adversely affect (p<0.05) the nuclear maturation rates of bovine oocytes. However, the subsequent embryo development was not significantly affected by addition of ghrelin to the IVM medium. This study showed that inclusion of 5-50 ng mL-1 ghrelin in maturation medium may have beneficial effects on nuclear maturation of bovine oocytes in vitro

The role of metalloproteinase and hypoxia conditions in endometrial cells and embryo implantation

In the process of implantation, metalloproteinase enzymes play a key role in basement membrane degradation and endometrial extracellular matrix. The activity of these enzymes is impeded by binding Tissue Inhibitors of Metalloproteinase (TIMP). The oxygen concentration in the mammalian uterus at the time of implantation is about 2-5%. It is seen that the imposition of hypoxia on cancer cells increases the expression of metalloproteinase enzymes and reduces the expression of metalloproteinase inhibitors, resulting in increased cell invasion. To know the effect of Hypoxia-Inducible Factor (HIF) and other related factors, we decided to evaluate hypoxic conditions on endometrial epithelial cells of the uterus and roll of matrix metalloproteinases (MMPs) on angiogenesis and invasion of the embryo during implantation. In this study, human and mouse endometrial epithelial cells were incubated for 24-48 hours in hypoxic conditions. Subsequently, the expression level of TIMP-1 was measured in mouse and human epithelial cells by Real-Time PCR technique. The cell viability in hypoxic conditions was evaluated by MTT assay. Our results demonstrated that hypoxia reduced the quantitative gene expression of TIMP-1 in the human and mouse endometrial epithelial cells compared to the control group. It can be concluded that applying hypoxic conditions by reducing the TIMP-1 expression and consequently increasing MMP expression, may improve the embryo implantation rate

Selection of immature bovine oocytes using Brilliant Cresyl Blue enhances nuclear maturity after vitrification

Beside cooling/warming rates and composition of vitrification solution, developmental stage of immature oocytes may also affect their vitrification outcome. The aim of the present study was to evaluate the selection effect of developmentally competent immature bovine oocytes by Brilliant Cresyl Blue (BCB) on maturity of oocytes after vitrification. Oocytes were obtained from slaughterhouse ovaries. Only oocytes with 4-5 layers of cumulus cells and homogenous cytoplasm were used. After exposure to BCB stain, immature oocytes were divided into colored (BCB+) and colorless (BCB-) cytoplasm groups. Immature oocytes were equilibrated in VS1 (7.5 Ethylene Glycol (EG)+7.5% DMSO) for 10-12 min and then exposed to VS2 (15% EG+ 15% DMSO+0.5M sucrose) for 1 min. Thereafter, oocytes were loaded on Cryotop and directly plunged into liquid nitrogen. After warming, oocytes were examined for presence of polar body and nuclear maturity. Higher number of oocytes in BCB+group extruded first polar body in comparison with other vitrified groups but not significantly (p>0.05). Compared to the BCB- oocytes, there was significantly lower percentage of degeneration for BCB+oocytes (p<0.05). Within vitrified groups, reaching to the MII stage was significantly higher in BCB+group (51.5%) compared with BCB and vitrified-control groups (27.9 and 40.3%, respectively). These results indicated that selection of potent immature bovine oocytes using brilliant cresyl blue improved the nuclear maturity of immature oocytes after vitrification. In addition, this selection can be a valuable tool to improve the vitrification outcome

Cryotop device enhances vitrification outcome of immature Bovine Oocytes.

The aim of this study was to evaluate the effectiveness of different cryodevices (Open Pulled Straw (OPS), Electron Microscopy Grid (EMG) and cryotop for vitrification of immature bovine oocytes. Polar body, MII stage, survivability and subsequent developmental rates were compared. Only oocytes with 4-5 layers of cumulus cells were used. Oocytes were equilibrated in the first vitrification solution (VS1; HS+10% DMSO+10% Ethylene Glycol (EG)) for 30-45 sec and then in the second vitrification solution (VS2; 20% DMSO+20% EG+0.5 M Sucrose) for 25 sec. Within 30 sec they were mounted on one of the cryodevices and directly plunged into Liquid Nitrogen (LN2) for 10 days. Immature oocytes vitrified using cryotop represented higher rate of polar body extrusion and nuclear maturity (p<0.05). The highest survivability resulted from cryotop and EMG groups and no significant difference found between them. Vitrified oocytes in cryotop group had highest cleavage and blastocyst rates. All of the mean measured rates for vitrified/warmed immature oocytes were significantly lower than that of control group (p<0.05). In conclusion, results of this study showed the superiority of cryotop device for vitrification of immature bovine oocytes which resulted in higher viability and subsequent embryo development

Effect of equilibration temperature on in vitro viability and subsequent embryo development of vitrified-warmed immature bovine oocytes

Problem statement: Vitrification is replacing conventional slow freezing to cryopreserve gametes and embryos especially for in vitro production of embryo in domestic animal species. However, the results are still not satisfactory. The aim of this experiment was to study the effect of different equilibration temperatures on in vitro viability of immature bovine oocytes after vitrification. Approach: Oocytes were obtained from slaughterhouse ovaries. Only grade one oocytes were used. Oocytes were equilibrated in three different temperatures: 32, 37, or 41°C. Immature oocytes were equilibrated in VS1 (7.5 Ethylene Glycol (EG) + 7.5% DMSO) for 10-12 min and then exposed to VS2 (15% EG + 15%DMSO + 0.5M sucrose) for one min. Thereafter oocytes were loaded on hand-made Cryotop and directly plunged into liquid nitrogen. After warming, oocytes were examined for viability, maturation, cleavage and blastocyst production. Results: Oocytes that were equilibrated at 37°C had significantly higher (p<0.05) viability than 41°C, but there were no significant difference between 37 and 41 with 32°C. Maturation rate in 37°C group was significantly higher compared with other groups. The highest percentage of degenerated and germinal vesicle stage oocytes were obtained from 41°C than 32 and 37°C. Cleavage rate of 37°C group (38.77%) was greater than other groups (30.84 and 28.95% for 32 and 41°C, respectively). The highest blastocyst rate was also produced when oocytes equilibrated at 37°C (6.45%). Conclusion: In conclusion, these results indicated that immature bovine oocytes can be equilibrated successfully at 37°C while higher or lower temperature can significantly decrease their subsequent viability and development

Effects of hormonal and oocyte activation treatments on in vitro embryo development and interspecies hybridization of bovidar embryos following intracytoplasmic sperm injection

In countries in the tropics such as Malaysia, local cattle are not suitable for the feedlot production system because of their poor meat production performance. Therefore, hybridization of domestic cattle with local wild bovids (Bos gaurus hubbacki and Bos javanicus) which are able to thrive very well in the tropics and at the same time develop a large muscular body and sturdy limbs may be an alternative way to improve livestock production system in Malaysia. In vitro embryo production (IVEP) technique plays a crucial role to manage hybridization and therefore may improve the desirable characteristics of livestock. Although IVEP techniques have received great attention in recent years, the rate of transferable blastocysts is quite low. In the present study, to improve the yield of in vitro produced bovine embryos, the effect of different concentrations of insulin and ghrelin hormones on in vitro development of bovine oocytes was evaluated. Insulin at 10 μg/ml significantly improved the proportion of immature bovine oocytes that reached the metaphase II stage. Furthermore, addition of 10 μg/ml insulin into the culture medium showed a positive effect on bovine embryo development until the morula stage. It was also found that supplementation with 50 ng/ml ghrelin in maturation and culture media enhanced nuclear maturation and blastocyst formation rates, respectively. Although inclusion of 50 ng/ml ghrelin in the culture medium strikingly increased the population of inner cell mass (ICM), trophoectoderm (TE) and total cell number cells of bovine blastocysts, no significant difference was detected in ratio of ICM: total cell number per blastocysts between the different treatment groups compared to the control. In order to improve the in vitro bovine embryo development following intracytoplasmic sperm injection (ICSI), a variety of single and combined artificial oocyte activation treatments were assessed. Data analysis showed that single artificial oocyte activation by strontium chloride (20 mM) and combination of strontium chloride followed by ethanol (7%) significantly increased the cleavage rate and in vitro bovine embryo development (p<0.05) following ICSI. However, no significant difference was detected between treatments regarding the quality of the blastocysts evaluated by differential staining of ICM and TE cells. Modified maturation and culture solutions and artificial oocyte activation (AOA) methods resulted from the present study were applied to assess the possibility and efficiency of advanced assisted reproductive techniques on in vitro production of hybrid bovine embryos. Furthermore, the effects of different diameters of the sperm injection needle on in vitro development of bovine embryos were also evaluated. The results indicated that frozen-thawed Bos gaurus hubbacki and Bos javanicus sperm were able to fertilize in vitro matured bovine oocytes and produced interspecies hybrid embryos following both in vitro fertilization (IVF) and ICSI methods at acceptable rates. In addition, the inner diameter of the injection pipette had significant effects on the in vitro production of hybrid embryos. In conclusion, the in vitro mass production of hybrid embryos that were successfully achieved in the present study can be considered as the first step to introduce a novel source of meat for the future

Nuclear maturation of immature bovine oocytes after vitrification using open pulled straw and cryotop methods

To date, at least two well known methods have been widely used for vitrification of oocytes and embryos at different stages in a variety of species. However, there is no reported data regarding the comparative effectiveness of these two methods for vitrification of immature bovine oocytes. The objective of this study is to compare the nuclear maturation of immature bovine oocytes vitrified using open pulled straw (OPS) and cryotop methods. Two experiments were conducted in this study. In the first experiment, cytotoxicity of vitrification solutions (VS) from both methods was studied. After removal of cryoprotectants, cumulus oocyte complexes (COCs) was cultured in vitro and cleavage rate was monitored on Day 2 post-insemination (pi), whereas, morulae and blastocyst yields on Days 5 and 8 pi, respectively. The VS solutions significantly reduced zygotic cleavage rate, morulae and blastocyst rates compared with the control group (P < 0.05). The lowest cleavage rate resulted from prolonged exposure time to OPS-VS solutions (35.1%; P < 0.05). However, the morulae and blastocyst rates were significantly higher (P < 0.05) for embryos derived from oocytes exposed to cryotop solutions (40.5 and 22.4%, respectively). In the second experiment, effectiveness of both vitrification methods was compared for cryopreservation of immature bovine oocytes. After warming, COCs were cultured in vitro for 24 h. The polar body (PB+) and metaphase-II (MII) stage rates differed significantly among treatment groups. Oocytes vitrified using cryotop solution and device showed higher percentages of PB+ (36%) and MII (51%) rates. In addition, the lowest percentage of degenerated oocytes resulted from cryotop solution. The highest degenerated oocytes obtained by equilibration in OPS solution and vitrified using OPS device (40%; P < 0.05). In conclusion, our data demonstrated that cryotop solution was less toxic to the immature bovine oocytes and vitrification with the cryotop method resulted in higher survival and nuclear maturation rates