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Effect of different activators on development of activated in vitro matured caprine oocytes Sharma, J. R.1; Agarwal, S.2; Kharche, S. D.3*; Goel, A. K.3; Jindal, S. K.3 and Agarwal, S. K.3 1
MSc, Physiology Reproduction and Shelter Management Division, Central Institute for Research on Goats (CIRG), Makhdoom, Farah-281122, Mathura, Uttar Pradesh, India; 2Ph.D. Scholar, Physiology Reproduction and Shelter Management Division, Central Institute for Research on Goats (CIRG), Makhdoom, Farah-281122, Mathura, Uttar Pradesh, India; 3Physiology Reproduction and Shelter Management Division, Central Institute for Research on Goats (CIRG), Makhdoom, Farah-281122, Mathura, Uttar Pradesh, India *
Correspondence: S. D. Kharche, Physiology Reproduction and Shelter Management Division, Central Institute for Research on Goats (CIRG), Makhdoom, Farah-281122, Mathura, Uttar Pradesh, India. E-mail: [email protected] (Received 21 May 2014; revised version 5 Aug 2014; accepted 27 Oct 2014)
Summary This study was designed to compare the effectiveness of different activation treatments for activation of in vitro matured oocytes and their developmental potency in mCR2aa medium so as to obtain maximum number of embryos. A total of 1090 cumulus oocyte complexes (COC’s) were collected from 480 ovaries. In vitro matured oocytes were randomly divided into four groups. Group 1 in vitro matured oocytes (n=226) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. Group 2 in vitro matured oocytes (n=294) were exposed to 7% ethanol for 5 min followed by treatment with 10 µg/ml CHX for 4 h in mCR2aa medium. Group 3 in vitro matured oocytes (n=325) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP and 10 µg/ml CHX for 4 h in mCR2aa medium. Group 4 in vitro matured oocytes (n=108) were cultured for 4 h without any chemical treatment in mCR2aa medium (control). The cleavage rate in groups 1, 2, 3 and 4 was 54.42%, 44.55%, 51.69% and 0.00%, respectively. The percentage of morula and blastocyst production in group 1, group 2 and group 3 was 26.01%, 29.77% and 29.76% and 2.43%, 1.52% and 1.78%, respectively. These results suggest that the activation of in vitro matured oocytes by 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa is most favorable for parthenogenetic caprine embryos production. Key words: Parthenogenesis, Ethanol activation, Morula, Blastocyst, Caprine
Introduction Methods used for parthenogenetic activation of oocytes are electrical pulse method (Kono et al., 1989), ethanol (Kharche et al., 2013), chemical activation (Mishra et al., 2006) or by combined electrical-chemical parthenogenetic activation of oocytes (Hossenni et al., 2008). There are several other methods available for the induction of parthenogenetic activation that promote an increase in intracellular free calcium concentrations by the release of calcium from cytoplasmic stores such as: calcium ionophore (Funahashi et al., 1994), ethanol (Loi et al., 1998), A-23187, calcium ionophore and cycloheximide alone (Nussbaum and Prather, 1995) or combined with a protein phosphorylation inhibitor, 6dimethyl amino purine (6-DMAP) (Liu and Yang, 1999), electrical shock (Kim et al., 1996), CaCl2 (Machaty et al., 1996), Ca-EDTA (Zaeand Ryoo, 2007), G protein stimulation (Machaty et al., 1996), Ionomycin (Loi et al., 1998), ultrasound (Sato et al., 2005), strontium (Meo et al., 2004) and magnetic field (Max et al., 2007). Methods used for parthenogenetic activation could be divided into two groups according to the mechanism used to increase intracellular calcium. One method for obtaining free calcium is by exposure to strontium or ionomycin to release cytoplasmic calcium. A second IJVR, 2015, Vol. 16, No. 1, Ser. No. 50, Pages 42-46
method is to promote influx of calcium from the extracellular medium by using an electrical stimulus or ethanol exposure (Meo et al., 2004). Currently, combined treatment of two activation stimuli are commonly used to ensure better development of reconstructed oocytes. Limited literature on activation protocols for goat oocytes is available, creating the need for effective oocyte activation protocols that can be used during nuclear transfer in goats. Therefore, in the present study attempts were made to compare different activation protocol on the development competence of parthenogenetic goat embryos. Thus, the present study was designed with the objectives to study the effect of different chemical activation protocols on cleavage rate of in vitro matured goat oocytes and to compare the development of parthenogenetic embryos produced from different chemical activation protocols.
Materials and Methods Recovery of oocytes and in vitro maturation (IVM) During the spring season, ovaries from goats, regardless of the stage of estrous cycle, were collected from a local abattoir located at Agra. Goats in this area are not seasonal breeders. A total of 1090 oocytes (Fig.
Iranian Journal of Veterinary Research, Shiraz University
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1A) were recovered by using follicle puncture technique. The collected oocytes were finally graded under the inverted phase contrast as per the method of Kharche et al. (2008). The cumulus oocyte complexes (COCs) were matured in maturation media (TCM-199 (Sigma) containing L-glutamine (100 µg/ml), sodium pyruvate (0.25 mmol), gentamycin (50 µg/ml), FSH (5 µg/ml), LH (10 µg/ml), oestradiol-17β (1 µg/ml) supplemented with 10% FBS and 3 mg/ml BSA) for 27 h in humidified 5% CO2 at 38.5°C in CO2 incubator. Out of a total 1090 oocytes recovered, 1036 matured (95.04%).
Parthenogenetic activation of in vitro matured oocytes The cleavage rate of chemically activated in vitro matured goat oocytes in group 1 (activation with ethanol + DMAP), group 2 (activation with ethanol + CHX), group 3 (activation with ethanol + DMAP + CHX) and group 4 (no activation), were 54.42%, 44.55%, 51.69% and 0.00%, respectively. Statistically (Chi-square) no significant difference was observed in cleavage rate between group 2 and 3 and group 1 and 3. In group 1 significantly higher (P
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Effect of different activators on development of activated in vitro matured caprine oocytes Sharma, J. R.1; Agarwal, S.2; Kharche, S. D.3*; Goel, A. K.3; Jindal, S. K.3 and Agarwal, S. K.3 1
MSc, Physiology Reproduction and Shelter Management Division, Central Institute for Research on Goats (CIRG), Makhdoom, Farah-281122, Mathura, Uttar Pradesh, India; 2Ph.D. Scholar, Physiology Reproduction and Shelter Management Division, Central Institute for Research on Goats (CIRG), Makhdoom, Farah-281122, Mathura, Uttar Pradesh, India; 3Physiology Reproduction and Shelter Management Division, Central Institute for Research on Goats (CIRG), Makhdoom, Farah-281122, Mathura, Uttar Pradesh, India *
Correspondence: S. D. Kharche, Physiology Reproduction and Shelter Management Division, Central Institute for Research on Goats (CIRG), Makhdoom, Farah-281122, Mathura, Uttar Pradesh, India. E-mail: [email protected] (Received 21 May 2014; revised version 5 Aug 2014; accepted 27 Oct 2014)
Summary This study was designed to compare the effectiveness of different activation treatments for activation of in vitro matured oocytes and their developmental potency in mCR2aa medium so as to obtain maximum number of embryos. A total of 1090 cumulus oocyte complexes (COC’s) were collected from 480 ovaries. In vitro matured oocytes were randomly divided into four groups. Group 1 in vitro matured oocytes (n=226) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. Group 2 in vitro matured oocytes (n=294) were exposed to 7% ethanol for 5 min followed by treatment with 10 µg/ml CHX for 4 h in mCR2aa medium. Group 3 in vitro matured oocytes (n=325) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP and 10 µg/ml CHX for 4 h in mCR2aa medium. Group 4 in vitro matured oocytes (n=108) were cultured for 4 h without any chemical treatment in mCR2aa medium (control). The cleavage rate in groups 1, 2, 3 and 4 was 54.42%, 44.55%, 51.69% and 0.00%, respectively. The percentage of morula and blastocyst production in group 1, group 2 and group 3 was 26.01%, 29.77% and 29.76% and 2.43%, 1.52% and 1.78%, respectively. These results suggest that the activation of in vitro matured oocytes by 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa is most favorable for parthenogenetic caprine embryos production. Key words: Parthenogenesis, Ethanol activation, Morula, Blastocyst, Caprine
Introduction Methods used for parthenogenetic activation of oocytes are electrical pulse method (Kono et al., 1989), ethanol (Kharche et al., 2013), chemical activation (Mishra et al., 2006) or by combined electrical-chemical parthenogenetic activation of oocytes (Hossenni et al., 2008). There are several other methods available for the induction of parthenogenetic activation that promote an increase in intracellular free calcium concentrations by the release of calcium from cytoplasmic stores such as: calcium ionophore (Funahashi et al., 1994), ethanol (Loi et al., 1998), A-23187, calcium ionophore and cycloheximide alone (Nussbaum and Prather, 1995) or combined with a protein phosphorylation inhibitor, 6dimethyl amino purine (6-DMAP) (Liu and Yang, 1999), electrical shock (Kim et al., 1996), CaCl2 (Machaty et al., 1996), Ca-EDTA (Zaeand Ryoo, 2007), G protein stimulation (Machaty et al., 1996), Ionomycin (Loi et al., 1998), ultrasound (Sato et al., 2005), strontium (Meo et al., 2004) and magnetic field (Max et al., 2007). Methods used for parthenogenetic activation could be divided into two groups according to the mechanism used to increase intracellular calcium. One method for obtaining free calcium is by exposure to strontium or ionomycin to release cytoplasmic calcium. A second IJVR, 2015, Vol. 16, No. 1, Ser. No. 50, Pages 42-46
method is to promote influx of calcium from the extracellular medium by using an electrical stimulus or ethanol exposure (Meo et al., 2004). Currently, combined treatment of two activation stimuli are commonly used to ensure better development of reconstructed oocytes. Limited literature on activation protocols for goat oocytes is available, creating the need for effective oocyte activation protocols that can be used during nuclear transfer in goats. Therefore, in the present study attempts were made to compare different activation protocol on the development competence of parthenogenetic goat embryos. Thus, the present study was designed with the objectives to study the effect of different chemical activation protocols on cleavage rate of in vitro matured goat oocytes and to compare the development of parthenogenetic embryos produced from different chemical activation protocols.
Materials and Methods Recovery of oocytes and in vitro maturation (IVM) During the spring season, ovaries from goats, regardless of the stage of estrous cycle, were collected from a local abattoir located at Agra. Goats in this area are not seasonal breeders. A total of 1090 oocytes (Fig.
Iranian Journal of Veterinary Research, Shiraz University
43
1A) were recovered by using follicle puncture technique. The collected oocytes were finally graded under the inverted phase contrast as per the method of Kharche et al. (2008). The cumulus oocyte complexes (COCs) were matured in maturation media (TCM-199 (Sigma) containing L-glutamine (100 µg/ml), sodium pyruvate (0.25 mmol), gentamycin (50 µg/ml), FSH (5 µg/ml), LH (10 µg/ml), oestradiol-17β (1 µg/ml) supplemented with 10% FBS and 3 mg/ml BSA) for 27 h in humidified 5% CO2 at 38.5°C in CO2 incubator. Out of a total 1090 oocytes recovered, 1036 matured (95.04%).
Parthenogenetic activation of in vitro matured oocytes The cleavage rate of chemically activated in vitro matured goat oocytes in group 1 (activation with ethanol + DMAP), group 2 (activation with ethanol + CHX), group 3 (activation with ethanol + DMAP + CHX) and group 4 (no activation), were 54.42%, 44.55%, 51.69% and 0.00%, respectively. Statistically (Chi-square) no significant difference was observed in cleavage rate between group 2 and 3 and group 1 and 3. In group 1 significantly higher (P
