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Improving the embryo developmental competence and success of in vitro produced calves during hot season in Buffalo
Kandil, O., Dohreig, R., Abdoon, A...
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Heat stress is a major problem for animal breeding in tropical and equatorial area as well as in the Mediterranean countries. This translates into several pathologies and high rates of reproductive failure through early embryonic loss in buffalo. The present work was conducted to investigate:1) Effect of cold and hot season in the oocyte quality and in vitro embryo development competence of buffalo cultured in buffalo oviduct epithelial cell (BOEC).2) Effect of heat stress (41◦ C) & IGF during in vitro oocyte maturation in oocyte maturation rate and embryo developmental competence.3)Transfer of in vitro produced embryo during hot season to Egyptian buffalo. Egyptian buffalo’s ovaries were collected during hot (June-August) and cold (December-February) season from abattoir. Oocytes were classified to Excellent (Ex), Good (G), Fair (F), Denuded (D). EX and G oocytes were matured in in-vitro maturation medium (IVM) (TCM-199+ 10% FCS + 10 µg/ml FSH+ 10 ng/ml EGF+ 50 µg/ml gentamicin (gn) for 22 h in 38.5C and 5% CO2 . The matured oocytes (1st pb) fertilized using Frozen – thawed buffalo semen, for 18 hours incubation. Zygotes cultured in SOFM + 10% FCS, 5 µg/ml insulin and 50 µg /ml gentamicin, with or without BOEC and incubated for 8 days. 2) Ex & G oocytes IVM in TCM-199 + 10% FCS+ 10 µg/ml FSH + 10 ng/ml IGF + 50 µg/ml, for 41◦ C /1hr- then 38.5◦ C vs. 38.5◦ C incubation for 22 hours. Then matured oocytes were fertilized and cultured as described before. Blastocyst were Fixed for cell counting to validate the quality of blastocyst. 3) Non-surgical embryo transfer during hot season in National Research Centre farm, Fresh Two in-vitro produced embryos (IVM,TCM+IGF, cultured in BOEC) were transferred to each buffaloes came in natural oestrus (buffalo number=5).Pregnancy diagnosis after 40 days from transfer. Results, Oocytes were collected from (349) buffalo ovaries giving an average of 2.4 oocytes/ovary (range 1.8 – 3.0 oocytes/ovary).There were highly significant (P<0.01) differences in the recovery rate of total oocytes between the two seasons, cold and hot season (3.02% ± 005 and 1.76% ± 0.05 respectively). Analysis of quality of buffalo oocytes revealed that, there were highly significant (P<0.01) differences in the mean % ± SE of excellent and good quality oocytes between the two seasons cold (38.63% ± 0.5, 51.35±0.50 resp.) and hot (11.29% ± 0.64, 21.37±0.74 resp.). While, fair and denuded oocytes increased with higher significant differences (P< 0.01) in summer (58.45% ± 0.86) when compared with the cold (9.86% ± 0.23). Maturation rate (85- 84 %), cleavage rate (75-71 %), blastocyst rate (33- 2%) were significantly higher (P<0.1, P<0.5) in cold temperature when compared with hot temperature season, in range 71-70%, 63-60 % and 22-17 respectively. In vitro culture of embryo using BOEC vs. without BOEC significantly increase the blastocyst rate either in cold (33 vs. 27 %) or hot temperature (22 vs. 17 %). The cell number of the blastocyst was significantly higher (P<0.1) in the cold temperature (mean= 106-90) when compared with hot temperature (80-60) and in vitro culturing in BOEC significantly increase (P<0.1) the blastocyst cell number either in cold or hot season. Effect of heat stress 41◦ C during in vitro oocyte maturation on in vitro embryo developmental competence in buffalo. Higher temperature 41◦ C for one hour during in vitro oocyte maturation in buffalo significantly (P<0.01) decreased the mean ± SD and rate of maturation (32±3.79, 51), cleavage (16.5±2.65, 26) and blastocyst (13.5±2.65, 22), when compared with in vitro oocyte maturation in 38.5◦ C (77.50±7.46, 87%, 42.67±2.52, 72% and 37.00±5.03, 41% respectively). Non- Surgical transfer of in vitro produced fresh buffalo embryos to five buffalos (day 6 of natural oestrus), pregnancy and calving of two calves (2/5, 40% Emy & Medo) with 42 kg body wright.
In conclusion
Heat stress either during hot season (summer) or through experimental rise temperature during in vitro maturation of buffalo oocytes significantly decrease the in vitro oocyte competence and blastocyst rate. Supplementation of IGF in in vitro maturation medium and co-culture of in vitro fertilized oocytes using BOEC decreased effect of heat stress and improved in vitro embryo production of buffalo. Success of calving of two calves (EMY& Medo) through transfer of IVP buffalo embryos matured in TCM+IGF during hot season (summer).
Keywords: Buffalo, season, BOEC, embryo, ET
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