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Parthenogenetic development of in vitro matured oocytes of buffalo (Bubalus bubalis)
Theriogenology41:312,
1994
PARTHENOGENETIC DEVELOPMENT OF IN VITRO MATURED OOCYTES OF BUFFALO (Buba/us buba//s) M. Taneja and G. Singh Embryo Biotechnology Laboratory, National Institute of Immunology, YNU Complex, Aruna Asaf Ali Marg, New Delhi 110 067 INDIA The objective of this study was to find out the optimal conditions for activation of in vitro matured (IVM) buffalo oocytes using electrical pulses. The effects of field strength (kV/cm), number of electrical pulses and the age of oocytes on activation and development of IVM oocytes were therefore investigated. Oocytes were aspirated from 1-8 mm follicles, matured in vitro in Ham's F10 + hormones (LH, FSH and E2) + 10 % FCS for 24 h. Cumulus cells were removed by repeated pipetting in the presence of 1 mg/ml hyaluronidase and oocytes were selected for the presence of first polar body (PB). The electrical DC pulses were applied to the PB positive oocytes at 28 hours post maturation (hpm) (unless otherwise indicated) in a 1.0 mm round wire chamber overlaid with 0.3 M Mannitol containing 100/~M CaC12 and 100/~M MgC12 using a Zimmerman Cell Fusion Apparatus (Model Z 1000, GCA/Precision Scientific Group, Chicago, IL). The control IVM oocytes were briefly incubated in mannitol without stimulation. Oocytes were either mounted 16 h after the pulse and assessed for formation of pronuclei, or co-cultured for 8 days with buffalo oviductal cells in CRlaa (Rosenkrans and First, 1991,Theriogenology,35:266) supplemented with 10% FCS. Each experiment was repeated 3-4 times and the data analyzed by Fisher's exact test. In Experiment 1, the IVM oocytes were stimulated with a single pulse of 0.2, 1.0 or 2.0 kV/cm for 40 #sec, resulting in activation rates of 7% (n=17), 43% (n=17) and 25% (n=16) respectively (P < 0.05),however, only 2.0kV/cm pulse led to development to blastocyst stage (6%,n=16). In Experiment 2, the oocytes were stimulated with one, two, three, four or five pulses (1 sec apart) of 2.0 kV/cm for 40 ~sec and the observed activation rates were 73 %, 45 %, 55 %, 55 % and 73% ( n = l l in all the groups) respectively (P > 0.05).Of control, 9% ( n = l l ) oocytes were activated which was lower (P < 0.05) than the pulsed groups (group with 2 pulses was an exception). A similar blastocyst development rate (5%, n=20) was observed with 3 and 4 pulses, while the other treatments as well as the control group did not result in development to blastocyst. In Experiment 3, the oocytes were pulsed at 24, 28 or 32 hpm with three pulses (1 sec apart) of 2.0 kV/cm for 40 /~sec. The activation rates increased with age. They were 18% ( n = l l ) at 24 hpm, 44% (n=9) at 28 hpm and further increased to 73% ( n = l l ) at 32 hpm (P > 0.05). Rates of development to blastocysts were similar at 24 and 28 hpm (4%, n=24) while it increased to 24% (n=24) at 32 hpm (P > 0.05). These data indicate that buffalo oocytes can be activated by a single electrical pulse, however, multiple pulses of high field strength apparently yield a better rate of parthenogenetic development. Furthermore, both activation and development appear to be dependent on oocyte age. This study was supported by the Dept. of Biotechnology, Govt. of India.
312
Copyright © 1994 Butterworth-Heinemann
1994
PARTHENOGENETIC DEVELOPMENT OF IN VITRO MATURED OOCYTES OF BUFFALO (Buba/us buba//s) M. Taneja and G. Singh Embryo Biotechnology Laboratory, National Institute of Immunology, YNU Complex, Aruna Asaf Ali Marg, New Delhi 110 067 INDIA The objective of this study was to find out the optimal conditions for activation of in vitro matured (IVM) buffalo oocytes using electrical pulses. The effects of field strength (kV/cm), number of electrical pulses and the age of oocytes on activation and development of IVM oocytes were therefore investigated. Oocytes were aspirated from 1-8 mm follicles, matured in vitro in Ham's F10 + hormones (LH, FSH and E2) + 10 % FCS for 24 h. Cumulus cells were removed by repeated pipetting in the presence of 1 mg/ml hyaluronidase and oocytes were selected for the presence of first polar body (PB). The electrical DC pulses were applied to the PB positive oocytes at 28 hours post maturation (hpm) (unless otherwise indicated) in a 1.0 mm round wire chamber overlaid with 0.3 M Mannitol containing 100/~M CaC12 and 100/~M MgC12 using a Zimmerman Cell Fusion Apparatus (Model Z 1000, GCA/Precision Scientific Group, Chicago, IL). The control IVM oocytes were briefly incubated in mannitol without stimulation. Oocytes were either mounted 16 h after the pulse and assessed for formation of pronuclei, or co-cultured for 8 days with buffalo oviductal cells in CRlaa (Rosenkrans and First, 1991,Theriogenology,35:266) supplemented with 10% FCS. Each experiment was repeated 3-4 times and the data analyzed by Fisher's exact test. In Experiment 1, the IVM oocytes were stimulated with a single pulse of 0.2, 1.0 or 2.0 kV/cm for 40 #sec, resulting in activation rates of 7% (n=17), 43% (n=17) and 25% (n=16) respectively (P < 0.05),however, only 2.0kV/cm pulse led to development to blastocyst stage (6%,n=16). In Experiment 2, the oocytes were stimulated with one, two, three, four or five pulses (1 sec apart) of 2.0 kV/cm for 40 ~sec and the observed activation rates were 73 %, 45 %, 55 %, 55 % and 73% ( n = l l in all the groups) respectively (P > 0.05).Of control, 9% ( n = l l ) oocytes were activated which was lower (P < 0.05) than the pulsed groups (group with 2 pulses was an exception). A similar blastocyst development rate (5%, n=20) was observed with 3 and 4 pulses, while the other treatments as well as the control group did not result in development to blastocyst. In Experiment 3, the oocytes were pulsed at 24, 28 or 32 hpm with three pulses (1 sec apart) of 2.0 kV/cm for 40 /~sec. The activation rates increased with age. They were 18% ( n = l l ) at 24 hpm, 44% (n=9) at 28 hpm and further increased to 73% ( n = l l ) at 32 hpm (P > 0.05). Rates of development to blastocysts were similar at 24 and 28 hpm (4%, n=24) while it increased to 24% (n=24) at 32 hpm (P > 0.05). These data indicate that buffalo oocytes can be activated by a single electrical pulse, however, multiple pulses of high field strength apparently yield a better rate of parthenogenetic development. Furthermore, both activation and development appear to be dependent on oocyte age. This study was supported by the Dept. of Biotechnology, Govt. of India.
312
Copyright © 1994 Butterworth-Heinemann