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Pregnancy rates and briths after co-culture of cumulus cells with bovine embryos derived from in vitro fertilization of in-vitro matured follicular oocytes
PREGNANCY RATES AND BIRTHS AFTER CC-CULTURE OF CUMULUS CELLS WITH BOVINE EMBRYOS DERIVED FROM IN VITRO FERTILIZATION OF IN-VITRO MATURED FOLLICULAR OOCYTES Y. Kajiharal, N. Kometanil, S. Kobayashi’, Y. Shitanaka*, Y. Koshibal, K. Hishiyamal, K. Shiraiwal and K. Gotos 1 Technical Research Center, Marubeni Feed Co., Ltd. Otaru 1292, Shinbe-cho, Ono, Hyogo 675-13 2 ho-Ham Foods Co., Inc. Takahata 4-27, Nishinomiya, Hyogo 663 3 Department of Animal Science, Faculty of Agriculture, Kagoshima University, Kagoshima, 890 Japan
The objective of this study was to determine the optimum time of transfer for bovine blastocysts derived from in vitro fertilization of in-vitro matured follicular oocytes. The complete method for bovine in vitro fertilization has been described previously (Jpn.J.Anim.Reprod. 33:173,1987; J.Reprod.Fert. 83:753,1988). The following changes were made in the present experiment. Follicular oocytes were aspirated by puncturing follicles l-7 mm in diameter with a hypodermic needle. Oocytes were matured in vitro for 20 to 22 h in a 39°C incubator with 5% CO2 in air. The medium used for maturation was 25 mM HEPES-buffered TCM-199 supplemented with 5% calf serum and antibiotics. Frozen-th~ed spermatozoa (ejaculated) were washed three times with B.O. solution with 5 mM theophylline, 2.5 mg/ml BSA and 10 pgglml heparin immediately after thawing. The washed s~rmatozoa were then incubated (15x10s ceils/ml) for 2 to 3 h before incubation with matured oocytes. Fertilization was conducted by introducing 215 matured oocytes/lOO ul sperm microdrop for 5 to 6 h. Development medium used was TCM-199 supplemented with 1% calf serum. At 78 to 83 h after fertilization, cumulus cells attached to the ova were removed, and those attached to the bottom of the culture dish were maintained for co-culture. Ova were then cultured in dishes with cumulus cells (CCL) or with combinations of cumulus cells and monolayers of uterine endometrial cells (CUL) to evaluate further development. The embryos that developed into blastocysts were transferred after 7 to 9 days (fertilization = day 0). One to three embryos were non-surgically transferred to recipients on days 5.5 to 8.5 of the estrous cycle (estrus = day 0; single embryo, n = 1; two embryos, n = 70; three embryos, n =L26). Ultrasound was used to determine pregnancy = 55 days after estrus. Redpient/emkyo synchrony -3 -2.5 -2 -1.5 -1 Oto+l
No. of recipients 10 8 22 17 27 13
No. pregnant 3 5 18 10 16 6
30 b 63 ab 81.8 a 68.8 eb
e.b Means with different superscripts in the same colomn are significantly different (P
Maturation and fertilization rates of oocytes were 94.0% (1091116) and 62.9% (7311i6), respectively. Development rate of ova (to blastocyst stage) was higher (P.cO.01) in CUL at 39.4%(54/137) than in CCL at 25.9%(23~888). The relative synchrony between recipients and embryos had an effect on pregnancy rates. Pregnancy rates were higher (PeO.01) for -2 days than -3 days recipien~emb~o asynchrony. To date there have been 39 single births, 13 sets of twins and 2 sets of triplets. This work was supported by Livestock Embryo Research Cooperative of Japan.
264
JANUARY 1996 VOL. 33 NO. 1
The objective of this study was to determine the optimum time of transfer for bovine blastocysts derived from in vitro fertilization of in-vitro matured follicular oocytes. The complete method for bovine in vitro fertilization has been described previously (Jpn.J.Anim.Reprod. 33:173,1987; J.Reprod.Fert. 83:753,1988). The following changes were made in the present experiment. Follicular oocytes were aspirated by puncturing follicles l-7 mm in diameter with a hypodermic needle. Oocytes were matured in vitro for 20 to 22 h in a 39°C incubator with 5% CO2 in air. The medium used for maturation was 25 mM HEPES-buffered TCM-199 supplemented with 5% calf serum and antibiotics. Frozen-th~ed spermatozoa (ejaculated) were washed three times with B.O. solution with 5 mM theophylline, 2.5 mg/ml BSA and 10 pgglml heparin immediately after thawing. The washed s~rmatozoa were then incubated (15x10s ceils/ml) for 2 to 3 h before incubation with matured oocytes. Fertilization was conducted by introducing 215 matured oocytes/lOO ul sperm microdrop for 5 to 6 h. Development medium used was TCM-199 supplemented with 1% calf serum. At 78 to 83 h after fertilization, cumulus cells attached to the ova were removed, and those attached to the bottom of the culture dish were maintained for co-culture. Ova were then cultured in dishes with cumulus cells (CCL) or with combinations of cumulus cells and monolayers of uterine endometrial cells (CUL) to evaluate further development. The embryos that developed into blastocysts were transferred after 7 to 9 days (fertilization = day 0). One to three embryos were non-surgically transferred to recipients on days 5.5 to 8.5 of the estrous cycle (estrus = day 0; single embryo, n = 1; two embryos, n = 70; three embryos, n =L26). Ultrasound was used to determine pregnancy = 55 days after estrus. Redpient/emkyo synchrony -3 -2.5 -2 -1.5 -1 Oto+l
No. of recipients 10 8 22 17 27 13
No. pregnant 3 5 18 10 16 6
30 b 63 ab 81.8 a 68.8 eb
e.b Means with different superscripts in the same colomn are significantly different (P
Maturation and fertilization rates of oocytes were 94.0% (1091116) and 62.9% (7311i6), respectively. Development rate of ova (to blastocyst stage) was higher (P.cO.01) in CUL at 39.4%(54/137) than in CCL at 25.9%(23~888). The relative synchrony between recipients and embryos had an effect on pregnancy rates. Pregnancy rates were higher (PeO.01) for -2 days than -3 days recipien~emb~o asynchrony. To date there have been 39 single births, 13 sets of twins and 2 sets of triplets. This work was supported by Livestock Embryo Research Cooperative of Japan.
264
JANUARY 1996 VOL. 33 NO. 1