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Effect of human leukemia inhibitory factor on in vitro development of IVF-derived bovine morulae
231
Theriogenology
EFFECT OF HUMAN LEUKEMIA INHIBITORY FACTOR ON IN VITRO DEVELOPMENT OF IVF-DERIVED BOVINE MORULAE Y.M. Han, ES. Lee’, T. Mogoe’, K.K. Lee and Y. Fukui’ Genetic Engineering Research Institute, KIST, Taejon 305-333 Korea ‘Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080 Japan This study was conducted to investigate whether human leukemia inhibitory factor (KIF) would improve the subsequent development of IVF-derived bovine morulae and blastocysts. To obtain IVF-derived bovine morulae, oocytes matured and fertilized in vitro were cultured in 0.5 ml of synthetic oviduct fluid (SOF) medium supplemented with 10% human serum (HS) for 5 d at 39C under the gas atmosphere of 5% Con, 5% 02, 90% N2. Morulae were cultured in 0.5 ml of SOF medium with or without 5000 unit/ml recombinant hLlF (AMRAD Co. Ltd.. Kew Victoria, Australia) for 2 or 3 d (2 groups). To investigate effect of hLlF addition on the subsequent development of motulae, SOF medium was supplemented with 8 mg/ml BSA instead of HS. To test whether hLlF affects the subsequent development of IVF-derived bovine blastocysts, blastocysts that developed from SOF medium with or without hLlF at Day 7 and 8 of culture were frozen and again cultured in SOF medium with or without the addition of hLlF for 3 days after thawing (4 groups). Excellent and good blastocysts were frozen by a conventional slow freezing method using 10% glycerol in PBS. Survival of frozen-thawed bovine embryos was evaluated for re-expansion and hatching of blastocysts during 3 d of culture. Table 1. Effect of hLlF addition on the subsequent development of IVF-derived bovine blastocysts after freezing and thawing Before freezing (+I
(-1
After thawing
No.embryos cultured
Survival rate(%)
Hatching rate(%)
(+)
42
61.9
28.6a
(-1
40
67.5
25.0a
(+I
46
58.7
6.5b
(-1
45
62.2
6.7b
(+),(-I : With or without hLIF. respectively. ; a vs b : PcO.05 Developmental rates of IVF-derived bovine morulae and early blastocysts to blastocysts in the groups with or without hLlF addition before freezing were 41.9% (127/303) and 81.3% (43/53), and 40.7% (12X02) and 868% (46/53), respectively. There was no significant difference in the developmental rates to blastocysts between with and without hLlF addition. However, the addition of hLlF before freezing significantly improved the hatching rate of IVF-derived bovine morulae (PiO.05). whereas the addition of hLlF after thawing did not increase the subsequent development of the blastocysts (Table 1). These results indicate that hLlF added at the Day 5 morula stage has contributed to bovine embryonic development through hatching process after freezing and thawing.
Theriogenology
EFFECT OF HUMAN LEUKEMIA INHIBITORY FACTOR ON IN VITRO DEVELOPMENT OF IVF-DERIVED BOVINE MORULAE Y.M. Han, ES. Lee’, T. Mogoe’, K.K. Lee and Y. Fukui’ Genetic Engineering Research Institute, KIST, Taejon 305-333 Korea ‘Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080 Japan This study was conducted to investigate whether human leukemia inhibitory factor (KIF) would improve the subsequent development of IVF-derived bovine morulae and blastocysts. To obtain IVF-derived bovine morulae, oocytes matured and fertilized in vitro were cultured in 0.5 ml of synthetic oviduct fluid (SOF) medium supplemented with 10% human serum (HS) for 5 d at 39C under the gas atmosphere of 5% Con, 5% 02, 90% N2. Morulae were cultured in 0.5 ml of SOF medium with or without 5000 unit/ml recombinant hLlF (AMRAD Co. Ltd.. Kew Victoria, Australia) for 2 or 3 d (2 groups). To investigate effect of hLlF addition on the subsequent development of motulae, SOF medium was supplemented with 8 mg/ml BSA instead of HS. To test whether hLlF affects the subsequent development of IVF-derived bovine blastocysts, blastocysts that developed from SOF medium with or without hLlF at Day 7 and 8 of culture were frozen and again cultured in SOF medium with or without the addition of hLlF for 3 days after thawing (4 groups). Excellent and good blastocysts were frozen by a conventional slow freezing method using 10% glycerol in PBS. Survival of frozen-thawed bovine embryos was evaluated for re-expansion and hatching of blastocysts during 3 d of culture. Table 1. Effect of hLlF addition on the subsequent development of IVF-derived bovine blastocysts after freezing and thawing Before freezing (+I
(-1
After thawing
No.embryos cultured
Survival rate(%)
Hatching rate(%)
(+)
42
61.9
28.6a
(-1
40
67.5
25.0a
(+I
46
58.7
6.5b
(-1
45
62.2
6.7b
(+),(-I : With or without hLIF. respectively. ; a vs b : PcO.05 Developmental rates of IVF-derived bovine morulae and early blastocysts to blastocysts in the groups with or without hLlF addition before freezing were 41.9% (127/303) and 81.3% (43/53), and 40.7% (12X02) and 868% (46/53), respectively. There was no significant difference in the developmental rates to blastocysts between with and without hLlF addition. However, the addition of hLlF before freezing significantly improved the hatching rate of IVF-derived bovine morulae (PiO.05). whereas the addition of hLlF after thawing did not increase the subsequent development of the blastocysts (Table 1). These results indicate that hLlF added at the Day 5 morula stage has contributed to bovine embryonic development through hatching process after freezing and thawing.