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Survival of porcine GV and M II-oocytes after exposure to cryoprotectants and cooling to 10 °C
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Theriogenology SURVIVAL OF PORCINE GV AND M II-OOCYTES AFTER EXPOSURE TO CRYOPROTECTANTS AND COOLING TO 10 “C Huang, W. -T. and Holtz, W. Institute of Animal Husbandry, University of Goettingen Albrecht-Thaer Weg 3, D-37075 Goettingen, Germany
The aim of the present investigation was to devise ways of cryopreserving porcine oocytes. As preliminary studies, we examined the toxicity of several candidate ctyoprotectants and the effect of cooling to 10°C. Pig ovaries were obtained from the local abattoir. Antral follicles of 2-6 mm diameter were nicked and cumulus-oocyte-complexes (COC) were scraped out into a phosphate buffered saline solution (Dulbecco’s PBS) containing 2% fetal calf serum (FCS). Some of the oocytes were matured to metaphase II (M II) by culture for 44 h in M-199 supplemented with 5 III/ml hCG, 2.5 IU/ml eCG and 10% porcine follicular fluid at 39°C in 5% CO2 in air. The success rate of this step was 80%. In the first experiment, oocytes at the germinal vesicle (GV; n=2077) and M II (n=755) stage were equilibrated in PBS+lS% FCS containing 1.5 M of glycerol (Gly), 1,Zpropanediol (PROH), dimethylsulfoxide (DMSO) or ethylene glycol (EG) in three steps (0.5 M, 1.0 M and 1.5 M for, respectively, 5, 5 and 10 min) at room temperature (2O’C). Cryoprotectant was diluted out in four steps at 20°C by serial transfer of oocytes into 0.3 M sucrose containing 0.5 M cryoprotectant, 0.3 M sucrose, 0.15 M sucrose and then in IVM/lVF medium for 5 min each. A control group was cultured without cryoprotectant (untreated oocytes). Survival was assessed by: 1) membrane integrity using FDA staining, and 2) cleavage following maturation (as above), fertilization and culture of GV oocytes (IVMFC) and IVFC of M II oocytes. Matured oocytes were co-cultured with spermatozoa for 6 h in M-199 supplemented with Na-pyruvate, Ca-lactate, glucose, caffeine and 12% FCS and then for 90 h in mBMOC-2 supplemented with 10% FCS and 0.4% BSA. Afterwards one half of each group was stained with FDA. FDA survival rates for Gly, PROH, DMSO, EG and untreated oocytes were, respectively, 95ti (SEM), 96i2,96+3,99i2 and 99fl% for M II oocytes and 82f4,94fl, 95f1, 92X2 and 94ti% for GV oocytes. As a next step, M II oocytes were in vitro-fertilized and cultured for 90 h (IVFC). GVoocytes were in vitro-matured before undergoing similar treatment (IVMFC). The corresponding cleavage rates were respectively, 21f5, 43+10, 34f8, 32*7 and 32+7% for M II oocytes and 39M, 38f5,44K!, 34s and 48&3% for GV oocytes. In a second experiment, GV (n=927) and M II (n=996) oocytes were equilibrated in the four cryoprotectants as described above and then cooled to 10°C at l”C/min. After 10 min at 10°C and rewarming to room temperature, one half of the oocytes was stained with FDA, the other half was in vitro-fertilized and cultured as described above. FDA survival rates for Gly, PROH, DMSO, EG and untreated oocytes, respectively, were 23+6, 26*4, 26f10, 40+14 and 98+1% for M II-oocytes and 34f12, 51+18, 48f17, 40f17 and 87+5% for GV-oocytes. The corresponding cleavage rates afler IVFC were 0, l+l, 2f2, 2+2 and 42+4% for M II-oocytes and all zeros for cooled GV-oocytes except 37510% for the untreated controls. It may be concluded that porcine GV- und M II-oocytes are not damaged by a range of cryoprotectants but will, nevertheless, succumb to a drop in temperature to 10°C
Theriogenology SURVIVAL OF PORCINE GV AND M II-OOCYTES AFTER EXPOSURE TO CRYOPROTECTANTS AND COOLING TO 10 “C Huang, W. -T. and Holtz, W. Institute of Animal Husbandry, University of Goettingen Albrecht-Thaer Weg 3, D-37075 Goettingen, Germany
The aim of the present investigation was to devise ways of cryopreserving porcine oocytes. As preliminary studies, we examined the toxicity of several candidate ctyoprotectants and the effect of cooling to 10°C. Pig ovaries were obtained from the local abattoir. Antral follicles of 2-6 mm diameter were nicked and cumulus-oocyte-complexes (COC) were scraped out into a phosphate buffered saline solution (Dulbecco’s PBS) containing 2% fetal calf serum (FCS). Some of the oocytes were matured to metaphase II (M II) by culture for 44 h in M-199 supplemented with 5 III/ml hCG, 2.5 IU/ml eCG and 10% porcine follicular fluid at 39°C in 5% CO2 in air. The success rate of this step was 80%. In the first experiment, oocytes at the germinal vesicle (GV; n=2077) and M II (n=755) stage were equilibrated in PBS+lS% FCS containing 1.5 M of glycerol (Gly), 1,Zpropanediol (PROH), dimethylsulfoxide (DMSO) or ethylene glycol (EG) in three steps (0.5 M, 1.0 M and 1.5 M for, respectively, 5, 5 and 10 min) at room temperature (2O’C). Cryoprotectant was diluted out in four steps at 20°C by serial transfer of oocytes into 0.3 M sucrose containing 0.5 M cryoprotectant, 0.3 M sucrose, 0.15 M sucrose and then in IVM/lVF medium for 5 min each. A control group was cultured without cryoprotectant (untreated oocytes). Survival was assessed by: 1) membrane integrity using FDA staining, and 2) cleavage following maturation (as above), fertilization and culture of GV oocytes (IVMFC) and IVFC of M II oocytes. Matured oocytes were co-cultured with spermatozoa for 6 h in M-199 supplemented with Na-pyruvate, Ca-lactate, glucose, caffeine and 12% FCS and then for 90 h in mBMOC-2 supplemented with 10% FCS and 0.4% BSA. Afterwards one half of each group was stained with FDA. FDA survival rates for Gly, PROH, DMSO, EG and untreated oocytes were, respectively, 95ti (SEM), 96i2,96+3,99i2 and 99fl% for M II oocytes and 82f4,94fl, 95f1, 92X2 and 94ti% for GV oocytes. As a next step, M II oocytes were in vitro-fertilized and cultured for 90 h (IVFC). GVoocytes were in vitro-matured before undergoing similar treatment (IVMFC). The corresponding cleavage rates were respectively, 21f5, 43+10, 34f8, 32*7 and 32+7% for M II oocytes and 39M, 38f5,44K!, 34s and 48&3% for GV oocytes. In a second experiment, GV (n=927) and M II (n=996) oocytes were equilibrated in the four cryoprotectants as described above and then cooled to 10°C at l”C/min. After 10 min at 10°C and rewarming to room temperature, one half of the oocytes was stained with FDA, the other half was in vitro-fertilized and cultured as described above. FDA survival rates for Gly, PROH, DMSO, EG and untreated oocytes, respectively, were 23+6, 26*4, 26f10, 40+14 and 98+1% for M II-oocytes and 34f12, 51+18, 48f17, 40f17 and 87+5% for GV-oocytes. The corresponding cleavage rates afler IVFC were 0, l+l, 2f2, 2+2 and 42+4% for M II-oocytes and all zeros for cooled GV-oocytes except 37510% for the untreated controls. It may be concluded that porcine GV- und M II-oocytes are not damaged by a range of cryoprotectants but will, nevertheless, succumb to a drop in temperature to 10°C