Optimizing The Culture And Cryopreservation Techniques Of IVF Embryo In Bovine

Bovine in vitro embryo production could play a central role in cattle production systems. However, it is an inefficient process at present. There are important technical limitations to their production that reduce the efficiency because of sub-optimal oocyte and embryo culture conditions and cryopreservation methods. These experiments were conducted to study the key techniques associated with in vitro embryo production in bovine so as to establish a more efficient and practical system.1 Effects of bovine oviductal or granulosa cells on developmental competence of bovine oocytes following maturation, fertilization and culture in vitroExperiments were conducted to study the effects of oviductal cell monolayer (OCM) and granulose cell monolayer (GCM) on developmental competence of bovine oocytes derived from abattoir ovaries following in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). In experiment 1, bovine cumulus oocyte complexes (COCs) were aspirated from follicles and classified into 3 morphological categories according to the surrounding cumulus cells:Grade1 (≥4 layers), Grade2 (2 to 3 layers) and Grade3 (0 to 1 layer). Oocytes were co-cultured with or without granulose cells (1×106个/ml) in IVM and IVC medium. The presence of granulosea cells during maturation and culture did not affect the cleavage rate,6～8 cells rate and blastocyst rates (P﹥0.05)of oocytes in grade 1. However, the cleavage rate,6～8 cells rate and blastocyst rates in grade 2 and grade 3 were higher (P<0.05) when COCs were cultured in the presence of GCM than when cultured in the absence of GCM. In experiment 2, oocytes (COCs and naked oocytes) were co-cultured with OCM, GCM or without both (control) in IVM and IVC medium. Following IVM, IVF and IVC, more oocytes in both OCM and GCM groups reached cleavage, 6～8 cells and blastocyst than no control group (P<0.05),although there was no significant difference(P﹥0.05)between OCM and GCM groups. In experiment 3, about 20 oocytes derived from every one cow were cultured and fertilized in different size of medium drops (30μL,50μL,100μL and 200μL). The blastocyst developmental rates of 30μL and 50μL groups were higher than 100μL and 200μL treatments (P<0.05).2 Effects of follicle size and bovine follicular fluid on developmental competence of bovine oocytes following maturation, fertilization and culture in vitroExperiments were conducted to study the effects of follicle size and bovine follicular fluid (BFF) on developmental competence of bovine oocytes derived from abattoir ovaries following in vitro maturation, fertilization and culture. In experiment 1, bovine COCs (cumulus oocyte complex) derived from follicle of different sizes were allocated to four treatments according to follicles size: >8mm; 2～8mm; preantral follicle(PF); and mix above COCs together (mix). The results showed that follicle size directly affected the development of oocytes and mixing all sizes of follicle oocytes together could stimulate development of immature oocytes from PF. In experiment 2, 10% of bovine follicular fluid (BFF) derived from competent follicles (>8 mm ), small follicles(2~8mm ) or all sizes of follicles (mix) were supplemented to the maturation medium. Following in vitro maturation, feitilization and culture, more COCs in three treatments reached cleavage and blastocyst than no BFF control group (P<0.05).However, 10% of BFF from competent follicles made the oocytes and embryos sticking together and may easily cause some of them lost. In experiment 3,different concentrations (10%, 20% and 40%) of BFF from all follicles (>2 mm) were supplemented to the maturation medium. The results showed that different concentrations of BFF could support in vitro maturation of COCs and subsequent development capacity, however, 20% and 40% of BFF could seriously cause oocytes and embryos adhered together. Therefore adding 10% of mixed BFF in maturation medium is the best choice.3 Effects of different culture systems on the in vitro production of bovine embryosThe objective was to study the development potential and quality of in vitro produced bovine embryos cultured individually or in groups. The well of the well system(WOW) were produced using a heated steel needle in the bottom of petri dish, creating a small well with a V-shape 1 cm apart, covered individual with a 20μL drop of medium. Methods for the modified"well of well"system(mWOW) were the same as the WOW, except for following differences: (1) the wells with a V-shape were created 1 mm apart in the culture dishes, (2) the wells were covered in groups with a 400μL drop of medium. Oocytes and presumptive zygotes were cultured in three culture systems: drop in group (control); WOW individually; mWOW in group. Cleavage, blastocyst rates and total cell number of embryos at days 6, 7, and 8 were similar (P>0.05) for drop and mWOW in groups treatments. However, cleavage, blastocyst rates and total cell number at days 6, 7, and 8 were lower (P<0.05) in embryos cultured individually in WOW than those cultured in groups in the drop and the mWOW. Lower hatching rates (P<0.05) were observed in the WOW treatment than the drop and the mWOW treatments. But, there was no difference between the drop and the mWOW treatments. In additional, total cell number at days 6, 7, and 8 for three tratments were all decline gradually. In conclusion, oocytes and embryos cultured in groups in the mWOW system had the better development potential and quality than embryos cultured individually in the WOW system in TCM199 medium for the in vitro production of bovine embryos. The faster the embryos developed in vitro cuture, the better the quality were.4 Study on the immature bovine oocyte vitrification methodsThe aim of this study was to compare the efficiency of immature bovine COCs (cumulus oocyte complex) vitrification in different concentrations of ethylene glycol (EG) and dimethylsulfoxide (DMSO), or by different vitrification methods of in straw or in open pulled straws (OPS). In the experiment 1, COCs were exposed to the cryoprotectant for either 30 or 60 s in final solutions of VS1 (10% EG + 10% DMSO) or VS2 (20% EG + 20% DMSO) or VS3 (25% EG + 25% DMSO). Maturation rates of 30 s and 60 s exposure groups both in VS1 and VS2 were not different from the control. Maturation rate of 30 s exposure group in VS3 was also not different from the control, but 60 s cryoprotectant exposure was toxic, decreasing maturation rate. In the experiment 2, the COCs were vitrified in OPS using 30 s exposure in final solutions of VS1 or VS2 or VS3. The vitrification with VS2 resulted in highest maturation rate (67.9%) and blastocyst rate (5.4%) as compared with VS1 (61.1%; 0) and VS3 (63.6%; 3.6%) treatments. Experiment 3 were conducted to study the effect of different vitrifications methods, straw and OPS, on survival, in vitro maturation, in vitro fertilization, and post-fertilization development of vitrified-thawed immature bovine COCs (cumulus oocyte complex). The results as follows: The recovery of post-thaw morphologically normal COCs in straw and OPS were 59.4%±4.3 and 77.9%±4.1 resp(P<0.01);The maturation rates were 48.2%±5.3 and 66.0%±5.8 resp(P<0.01);The cleavage rates were18.5%±2.0 and 32.8%±1.4 resp(P<0.01);The percentage of 8-cell embryo were 14.8%±2.5 and 24.8%±1.5 resp(P<0.01);The percentage of morula in straw, OPS and fresh oocytes were 0, 5.3%±1.1and 21.0%±3.8 resp(P<0.01);The percentage of blastocyst in straw , OPS and fresh oocytes were 0, 4.0%±1.0and 17.1%±2.7 resp(P<0.01). In conclusion, it is possible to cryopreserve immature bovine oocytes by exposure for 30 s in cryoprotectant solution with 20% EG and 20% DMSO and vitrification in OPS, although the blastocyst rate is low after IVF.5 Closed pulled straw vitrification of in vitro produced and in vivo produced bovine embryosThe objective was to evaluate the efficiency of the closed pulled straw (CPS) method for cryopreserving in vitro and in vivo produced bovine embryos. Based on the open pulled straw (OPS) protocol, the top end of CPS was closed by heated tweezers in a flame to prevent the cryoprotectant medium containing embryos from contacting the liquid nitrogen. Bovine in vitro or in vivo morulae and early blastocysts embryos were frozen by slow cryopreservation, OPS vitrification, or CPS vitrification. Morphology of post-thawed embryos was evaluated and normal embryos were used for successive culture for 72 h. There were no significant differences between OPS and CPS freezing groups in post-thawed in vitro produced embryos regarding rates of morphologically normal embryos (mean±SD, 87.9±5.2 vs 85.4±4.9), survival at 24 h (58.0±6.8 vs 56.3±4.4), and survival at 72 h (35.2±6.0 vs 34.9±6.7). However, both OPS and CPS vitrification resulted in higher post-thawed rates of morphologically normal embryo, and survival at 24 and 72 h than the slow freezing method (P<0.05). Similar results were obtained for in vivo derived embryos. We concluded that CPS vitrification was a feasible method to cryopreserve both in vitro and in vivo derived bovine embryos. This method not only eliminated the risk of embryo contamination by preventing contact with liquid nitrogen, it also retained the advantages of the OPS vitrification method.