- Email: [email protected]
In vitro maturation of caprine oocytes in different concentrations of estrous goat serum
Small Ruminant Research 64 (2006) 186–189
Technical note
In vitro maturation of caprine oocytes in different concentrations of estrous goat serum S.D. Kharche ∗ , A.K. Goel, S.K. Jindal, N.K. Sinha Female Reproduction Laboratory, PRSM Division, Central Institute for Research on Goats, Makhdoom, P.O. Farah 281122, Mathura, UP, India Received 19 August 2004; received in revised form 1 April 2005; accepted 11 April 2005 Available online 6 June 2005
Abstract The aim of the study was to evaluate the optimum concentration of the estrous goat serum for goat in vitro oocyte maturation. Goat ovaries were collected from a local abattoir and transported within 3 h to the laboratory in a warm saline solution (30–35 ◦ C), supplemented with 100 IU penicillin G and 100 g streptomycin sulfate/ml. Cumulus-oocyte complexes (COC’s) were obtained by puncture of 1–4 mm diameter follicles with a 18G needle attached to a disposable syringe, washed 10 times in TCM-199 hepes modification with Earl’s salts and supplemented with 50 g/ml gentamycin, 100 g/ml l-glutamine and 0.25 mM sodium pyruvate without any serum supplementation. The COC’s were randomly divided into four groups. Group 1 (n = 105) COC’s were fresh control and cultured in TCM-199 medium without serum supplementation. Group 2 (n = 105) COC’s were washed five times and cultured in TCM-199 medium supplemented with 10% EGS. Group 3 (n = 110) COC’s were washed five times and cultured in TCM-199 medium supplemented with 15% EGS. Group 4 (n = 112) COC’s were washed five times and cultured in TCM-199 medium supplemented with 20% EGS. After 24–27 h of IVM, oocytes were denuded with the aid of 0.1% hyaluronidase and passing them through a fine pipette, fixed for 24–48 h in a mixture of acetic acid and alcohol (1:3) at room temperature, stained for 10 min with 1% (w/v) orcein in 45% acetic acid and examined for the evidence of different stages of maturation. Significantly higher (p < 0.01) maturation rates of oocytes (61–78%) were observed in all concentrations of estrous goat serum compared to those without serum supplementation. However, no significant difference was observed between the 10 and 15% serum supplemented group. A significant difference in maturation percentage (p < 0.01) was observed between 10 and 20% serum supplementation in culture media. In conclusion, it can be said that the supplementation of 20% EGS in TCM-199 medium could be used to achieve optimal maturation rate. © 2005 Published by Elsevier B.V. Keywords: Caprine; Estrous goat serum; Follicle puncture; Oocyte maturation; Ovary
1. Introduction ∗ Corresponding author. Tel.: +91 565 2763260; fax: +91 565 2763246. E-mail addresses: [email protected], [email protected] (S.D. Kharche).
0921-4488/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.smallrumres.2005.04.005
In vitro maturation (IVM) is one of the essential steps in the in vitro fertilization (IVF) process. Several workers have studied different aspects of IVM in
S.D. Kharche et al. / Small Ruminant Research 64 (2006) 186–189
mammalian oocytes (Pawshe et al., 1996; Kharche et al., 2005). In most of the studies, the basic medium was supplemented with hormones and different concentrations of serum. The maturation medium and the selection of protein supplements and hormones for IVM play an important role in subsequent IVF and in vitro development (Pawshe et al., 1996). Caprine oocytes have also been studied for different aspects of maturation (Mogas et al., 1997a). In all the experiments maturation media were supplimented with fetal bovine serum (FBS) (Martin-Lunas et al., 1996), calf serum (Crozet et al., 1993), estrous goat serum (EGS) (Pawshe et al., 1996; Tajik and Shams Esfandabadi, 2003) and bovine serum albumin + EGS (Rajikin et al., 1994) as protein supplement. Hormone supplementation varied in all experiments. Tajik and Shams Esfandabadi (1998) reported no significant differences in IVM of caprine oocytes for different concentrations of estrous goat serum during January–June (non-breeding season). Tropical goats are generally classified as non-seasonal with major estrous activity being concentrated during certain periods of the year (Goel and Agrawal, 2000). There is, however, not much information regarding on the efficiency of different estrous goat serum concentration on in vitro maturation of caprine oocytes in tropical goats. The present experiment was planned during the natural peak breeding season for Indian goats (July–November) season to determine the optimum concentration of EGS to be used for maturation of theoocytes.
2. Materials and methods Twenty goats were observed for the occurence of estrous daily twice at 12 h intervals in the experiment herd at the Institute. Estrous serum from these goats was collected 12 h following the onset of estrous and filtered through Millipore filters (with a pore size of 0.22 m). The estrous goat serum was heat inactivated at 56 ◦ C for 30 min in a water bath, dispensed into 1 ml aliquots and stored at −20 ◦ C until used (Rao et al., 2002). Goat ovaries were collected from a local abattoir and transported within 3 h to the laboratory in a warm saline solution (30–35 ◦ C), supplemented with 100 IU penicillin G and 100 g streptomycin sulfate/ml. Cumulus-oocytes complexes (COC’s) were obtained by the puncturing of 1–4 mm diameter fol-
187
licles with a 18G needle, attached to a disposable syringe. The COC’s were washed 10 times in TCM-199 (Cat. No. M 7528, Sigma) hepes modification solution with Earl’s salts and supplemented with 50 g/ml gentamycin (Cat. No. G 1264, Sigma), 100 g/ml lglutamine (Cat. No. G 3126, Sigma) and 0.25 mM sodium pyruvate (Cat. No. P 4562, Sigma) without any serum supplementation. The COC’s were randomly divided into four groups. Group 1 (n = 105) COC’s were taken fresh control and cultured in TCM199 medium without serum supplementation. Group 2 (n = 105) COC’s were washed five times and cultured in TCM-199 medium supplemented with 10% EGS. Group 3 (n = 110) COC’s were washed five times and cultured in TCM-199 medium supplemented with 15% EGS. Group 4 (n = 112) the COC’s were washed five times and cultured in TCM-199 medium supplemented with 20% EGS. Ten to 15 cumulus intact caprine oocytes were transferred into a 50 l drop of each group of the culture media (control medium or serum supplemented media) under warm mineral oil (Cat. No. M 8410, Sigma) in a tissue culture dish (35 mm × 10 mm), equilibrated for 2 h in a CO2 incubator before the oocytes were added. These oocytes were cultured for 24–27 h at 38.5 ◦ C under humidified atmosphere of 5% CO2 in air. Oocytes were denuded after 27 h following culture by treating with TCM-199 containing 0.1% hyaluronidase (Cat. No. H 4272, Sigma) and passing them through a fine pipette. Oocytes were fixed for 24–48 h in a mixture of acetic acid and alcohol (1:3) at room temperature, stained for 10 min with 1% (w/v) orcein in 45% acetic acid and examined for evidence of different stages of maturation under a phase contrast microscope. The different stages of maturation examined based on chromosomal configuration were assigned to germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase-I (M-I) and metaphase-II (M-II) categories. A chromosome configuration was designated as GV, when having a single large nucleus with uniformly distributed filamentous chromatin subsequently condensing to form a ring of condensed chromatin around the compact nucleus. In the GVBD category, the nucleolus and nuclear membrane had disappeared and chromosomes appeared as condensed and coiled up filaments. The metaphaseI stage was recognized by the appearance of paired chromosomes and bivalents, while in the metaphase-II
188
S.D. Kharche et al. / Small Ruminant Research 64 (2006) 186–189
Table 1 Effect of different concentrations of estrous goat serum on the in vitro maturation rate of caprine oocytes Groups
EGS (%)
No. of oocytes
GV (%)
GVBD (%)
M-I (%)
M-II (%)
1 2 3 4
00.00 10.00 15.00 20.00
105 105 110 112
42 10 8 5
22 12 6 7
11 18 16 12
30 (28.6)a 65 (61.9)b,c 80 (72.7)b,c 88 (78.6)c,d
Values in columns with different superscripts (a, b, c, d) differ significantly (p < 0.01); GV, germinal vesicle; GVBD, germinal vesicle breakdown; M-I, metaphase-I; M-II, metaphase-II.
emission of first polar body, resulting in the formation of haploid set of chromosomes in the oocytes was observed. The percentages of M-II stage oocytes were calculated in different groups. The collected data were analyzed by Chi square analysis. The level of significance was observed at the 1% level (Snedecor and Cochran, 1989).
3. Results and discussion The results are presented in Table 1. The maturation rate of the caprine oocytes in media without protein supplemented was 28.57% as shown by the control group. However, the protein supplemented in the form of 10% EGS significantly (p < 0.01) increased the maturation rate to 61.9%. The maturation rate further increased with 15 and 20% EGS supplementation to 72.7 and 78.6%, respectively. A significant difference (p < 0.01) in the in vitro maturation rate was recorded between the serum supplemented and nonsupplemented medium. The maturation rate of oocytes also significantly differed with the addition of 20% EGS, compared to 10% EGS. However, the maturation rate between 15 and 20% EGS supplementation did not differ significantly (Table 1). Experiments on in vitro maturation of caprine oocytes using TCM-199 supplemented with EGS, but without hormones, revealed a maturation rate of 52.7% (Pawshe et al., 1996) and 57.6% (Mogas et al., 1995). Mogas et al. (1997a,b) reported the addition of FSH, LH and estradiol-17 as well as EGS in TCM-199 medium to increase the maturation rate to 72.4 and 64.1% in adult and pre pubertal goats, respectively. Similarly Pawshe et al. (1996) also reported a maturation rate of 62.6% following the supplementation of hormones and EGS. In the present study a 61–78%
maturation rate with estrous goat serum was recorded in caprine oocytes without hormone supplementation. Tajik and Shams Esfandabadi (2003) reported an even higher maturation rate of oocytes in culture medium containing EGS without hormones. Samake et al. (2000) reported higher maturation rate (100%) from immature oocytes aspirated by laparotomy or via ovariectomized ovaries. In these studies, donor goats were superovulated with synchromate ear implants, followed by an intramascular injection of FSH. In mammalian females, the primordial germ cells migrate to the genital ridges following the establishment of the gonadal rudiments. Here the cells are enclosed by somatic cells and become primordial follicles, in which meiosis is initiated within the embryo. The maturation of follicular oocytes is normally arrested at the prophase-I of the first meiotic division and the oocyte remain in the dormant stage, called a dictate nucleus. At this stage, nuclear material is enveloped and the resulting structure is called a germinal vesicle. The oocyte remain at this stage until the onset of puberty. Under the influence of gonadotropins and particularly in response to the LH surge, oocytes resume meiosis just before ovulation. This results in the disappearance of the nuclear membrane and germinal vesicle breakdown, followed by chromosome condensation with the occurrence of the M-I stage. Subsequently, upon extrusion of the first polar body, the oocytes reach the M-II stage and remain at this stage until penetration by the spermatozoa. Oocytes remained in the GV stage from the onset to 6–8 h of culture. The GVBD occurred between 7 and 9 h and the metaphase-I became established within 12–18 h. Finally most oocytes reach the metaphase-II stage after 27 h. Resumption of meiosis and subsequent sequential configuration are species specific (Le Gall et al., 1992). The configuration of meiotic chromosomes at the time of co-culture of oocytes with spermatozoa for in
S.D. Kharche et al. / Small Ruminant Research 64 (2006) 186–189
vitro fertilization has a direct influence on the final success of fertilization. The M-II stage which is also known as the second phase of meiotic arrest, is consider as having have completed nuclear maturation of the oocytes required for successful fertilization of oocytes. Obviously, nuclear maturation of oocytes along with cytoplasmic maturation is important at the completion of meiotic division for success of fertilization. Therefore, the in vitro maturation process is supposed to be completed when the highest percentage of M-II oocytes is observed.
4. Conclusion The results indicated that supplementation of 20% EGS in TCM-199 medium could be used to achieve optimal maturation rates.
Acknowledgement The authors wish to thanks to the Director C.I.R.G., Makhdoom, Farah, Mathura, UP, India for providing the facilities needed.
References Crozet, N., De Smedt, V., Ahmed-Ali, M., Sevellec, C., 1993. Normal development following in vitro oocytes maturation and fertilization in the goat. Theriogenology 39, 206. Goel, A.K., Agrawal, K.P., 2000. Distribution pattern of estrous in Jakhrana goats. Indian J. Anim. Sci. 70, 589–590. Kharche, S.D., Sharma, G.T., Majumdar, A.C., 2005. In vitro maturation and fertilization of goat oocytes vitrified at germinal vesicle stage. Small Rumin. Res. 57, 81–84.
189
Le Gall, F., Gall, I., De smedt, V., 1992. Changes in protein synthesis pattern during in vitro maturation of goat oocytes. Mol. Reprod. Dev. 32, 1–8. Martin-Lunas, M., Martino, A., Paramino, M.T., Palomo, M..J., Mogas, M.T., Bielsa, M.A., Andols, P., Martinez, P., 1996. Effect of oocytes sperm co incubation on acrosome reaction in the goat. Therigenology 46, 320–330. Mogas, T., Izquierdo, M.D., Palomo, M.J., Paramio, M.T., 1995. Effect of hormones, serum source and culture system on the IVM and IVF of prepubertal goat oocytes and subsequent embryo development. Theriogenology 43, 284. Mogas, T., Palomo, M.J., Izquierdo, M.D., Paramio, M.T., 1997a. Development capacity of in vitro matured and fertilized oocytes from prepubertal and adult goats. Theriogenology 47, 1189–1203. Mogas, T., Palomo, M.J., Izquierdo, M.D., Paramio, M.T., 1997b. Morphological events during in vitro fertilization of prepubertal goat oocytes matured in vitro. Theriogenology 48, 815– 829. Pawshe, C.H., Palanisamy, A., Taneja, M., Jain, S.K., Totey, S.M., 1996. Comparison of various maturation treatments on in vitro maturation of goat oocytes and their early embryonic development and cell numbers. Theriogenology 46, 971– 982. Rajikin, M.H., Yusoff, M., Abdullah, R.B., 1994. Ultrastructural studies of developing goat oocytes in vitro. Theriogenology 42, 1003–1016. Rao, B.S., Naidu, K.S., Amarnath, D., Vagdevi, R., Rao, A.S., Brahmaiah, K.V., Rao, V.H., 2002. In vitro maturation of sheep oocytes in different media during breeding and non breeding season. Small Rumin. Res. 43, 31–36. Samake, S., Amoah, E.A., Mobini, S., Gazal, O., Gelaye, S., 2000. In vitro fertilization of goat oocyts during the non-breeding season. Small Rumin. Res. 35, 49–54. Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods. Eighth Editrion Iowa State University Press, Ames, IA, USA. Tajik, P., Shams Esfandabadi, N., 1998. Effects of estrous goat serum on in vitro maturation of caprine oocytes in non breeding season. In: Proceedings of the XVII Nordic Veterinary Congress, 4–7 August, Helsinki, Finland, p. 348. Tajik, P., Shams Esfandabadi, N., 2003. In vitro maturation of caprine oocytes in different culture media. Small Rumin. Res. 47, 155–158.
Technical note
In vitro maturation of caprine oocytes in different concentrations of estrous goat serum S.D. Kharche ∗ , A.K. Goel, S.K. Jindal, N.K. Sinha Female Reproduction Laboratory, PRSM Division, Central Institute for Research on Goats, Makhdoom, P.O. Farah 281122, Mathura, UP, India Received 19 August 2004; received in revised form 1 April 2005; accepted 11 April 2005 Available online 6 June 2005
Abstract The aim of the study was to evaluate the optimum concentration of the estrous goat serum for goat in vitro oocyte maturation. Goat ovaries were collected from a local abattoir and transported within 3 h to the laboratory in a warm saline solution (30–35 ◦ C), supplemented with 100 IU penicillin G and 100 g streptomycin sulfate/ml. Cumulus-oocyte complexes (COC’s) were obtained by puncture of 1–4 mm diameter follicles with a 18G needle attached to a disposable syringe, washed 10 times in TCM-199 hepes modification with Earl’s salts and supplemented with 50 g/ml gentamycin, 100 g/ml l-glutamine and 0.25 mM sodium pyruvate without any serum supplementation. The COC’s were randomly divided into four groups. Group 1 (n = 105) COC’s were fresh control and cultured in TCM-199 medium without serum supplementation. Group 2 (n = 105) COC’s were washed five times and cultured in TCM-199 medium supplemented with 10% EGS. Group 3 (n = 110) COC’s were washed five times and cultured in TCM-199 medium supplemented with 15% EGS. Group 4 (n = 112) COC’s were washed five times and cultured in TCM-199 medium supplemented with 20% EGS. After 24–27 h of IVM, oocytes were denuded with the aid of 0.1% hyaluronidase and passing them through a fine pipette, fixed for 24–48 h in a mixture of acetic acid and alcohol (1:3) at room temperature, stained for 10 min with 1% (w/v) orcein in 45% acetic acid and examined for the evidence of different stages of maturation. Significantly higher (p < 0.01) maturation rates of oocytes (61–78%) were observed in all concentrations of estrous goat serum compared to those without serum supplementation. However, no significant difference was observed between the 10 and 15% serum supplemented group. A significant difference in maturation percentage (p < 0.01) was observed between 10 and 20% serum supplementation in culture media. In conclusion, it can be said that the supplementation of 20% EGS in TCM-199 medium could be used to achieve optimal maturation rate. © 2005 Published by Elsevier B.V. Keywords: Caprine; Estrous goat serum; Follicle puncture; Oocyte maturation; Ovary
1. Introduction ∗ Corresponding author. Tel.: +91 565 2763260; fax: +91 565 2763246. E-mail addresses: [email protected], [email protected] (S.D. Kharche).
0921-4488/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.smallrumres.2005.04.005
In vitro maturation (IVM) is one of the essential steps in the in vitro fertilization (IVF) process. Several workers have studied different aspects of IVM in
S.D. Kharche et al. / Small Ruminant Research 64 (2006) 186–189
mammalian oocytes (Pawshe et al., 1996; Kharche et al., 2005). In most of the studies, the basic medium was supplemented with hormones and different concentrations of serum. The maturation medium and the selection of protein supplements and hormones for IVM play an important role in subsequent IVF and in vitro development (Pawshe et al., 1996). Caprine oocytes have also been studied for different aspects of maturation (Mogas et al., 1997a). In all the experiments maturation media were supplimented with fetal bovine serum (FBS) (Martin-Lunas et al., 1996), calf serum (Crozet et al., 1993), estrous goat serum (EGS) (Pawshe et al., 1996; Tajik and Shams Esfandabadi, 2003) and bovine serum albumin + EGS (Rajikin et al., 1994) as protein supplement. Hormone supplementation varied in all experiments. Tajik and Shams Esfandabadi (1998) reported no significant differences in IVM of caprine oocytes for different concentrations of estrous goat serum during January–June (non-breeding season). Tropical goats are generally classified as non-seasonal with major estrous activity being concentrated during certain periods of the year (Goel and Agrawal, 2000). There is, however, not much information regarding on the efficiency of different estrous goat serum concentration on in vitro maturation of caprine oocytes in tropical goats. The present experiment was planned during the natural peak breeding season for Indian goats (July–November) season to determine the optimum concentration of EGS to be used for maturation of theoocytes.
2. Materials and methods Twenty goats were observed for the occurence of estrous daily twice at 12 h intervals in the experiment herd at the Institute. Estrous serum from these goats was collected 12 h following the onset of estrous and filtered through Millipore filters (with a pore size of 0.22 m). The estrous goat serum was heat inactivated at 56 ◦ C for 30 min in a water bath, dispensed into 1 ml aliquots and stored at −20 ◦ C until used (Rao et al., 2002). Goat ovaries were collected from a local abattoir and transported within 3 h to the laboratory in a warm saline solution (30–35 ◦ C), supplemented with 100 IU penicillin G and 100 g streptomycin sulfate/ml. Cumulus-oocytes complexes (COC’s) were obtained by the puncturing of 1–4 mm diameter fol-
187
licles with a 18G needle, attached to a disposable syringe. The COC’s were washed 10 times in TCM-199 (Cat. No. M 7528, Sigma) hepes modification solution with Earl’s salts and supplemented with 50 g/ml gentamycin (Cat. No. G 1264, Sigma), 100 g/ml lglutamine (Cat. No. G 3126, Sigma) and 0.25 mM sodium pyruvate (Cat. No. P 4562, Sigma) without any serum supplementation. The COC’s were randomly divided into four groups. Group 1 (n = 105) COC’s were taken fresh control and cultured in TCM199 medium without serum supplementation. Group 2 (n = 105) COC’s were washed five times and cultured in TCM-199 medium supplemented with 10% EGS. Group 3 (n = 110) COC’s were washed five times and cultured in TCM-199 medium supplemented with 15% EGS. Group 4 (n = 112) the COC’s were washed five times and cultured in TCM-199 medium supplemented with 20% EGS. Ten to 15 cumulus intact caprine oocytes were transferred into a 50 l drop of each group of the culture media (control medium or serum supplemented media) under warm mineral oil (Cat. No. M 8410, Sigma) in a tissue culture dish (35 mm × 10 mm), equilibrated for 2 h in a CO2 incubator before the oocytes were added. These oocytes were cultured for 24–27 h at 38.5 ◦ C under humidified atmosphere of 5% CO2 in air. Oocytes were denuded after 27 h following culture by treating with TCM-199 containing 0.1% hyaluronidase (Cat. No. H 4272, Sigma) and passing them through a fine pipette. Oocytes were fixed for 24–48 h in a mixture of acetic acid and alcohol (1:3) at room temperature, stained for 10 min with 1% (w/v) orcein in 45% acetic acid and examined for evidence of different stages of maturation under a phase contrast microscope. The different stages of maturation examined based on chromosomal configuration were assigned to germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase-I (M-I) and metaphase-II (M-II) categories. A chromosome configuration was designated as GV, when having a single large nucleus with uniformly distributed filamentous chromatin subsequently condensing to form a ring of condensed chromatin around the compact nucleus. In the GVBD category, the nucleolus and nuclear membrane had disappeared and chromosomes appeared as condensed and coiled up filaments. The metaphaseI stage was recognized by the appearance of paired chromosomes and bivalents, while in the metaphase-II
188
S.D. Kharche et al. / Small Ruminant Research 64 (2006) 186–189
Table 1 Effect of different concentrations of estrous goat serum on the in vitro maturation rate of caprine oocytes Groups
EGS (%)
No. of oocytes
GV (%)
GVBD (%)
M-I (%)
M-II (%)
1 2 3 4
00.00 10.00 15.00 20.00
105 105 110 112
42 10 8 5
22 12 6 7
11 18 16 12
30 (28.6)a 65 (61.9)b,c 80 (72.7)b,c 88 (78.6)c,d
Values in columns with different superscripts (a, b, c, d) differ significantly (p < 0.01); GV, germinal vesicle; GVBD, germinal vesicle breakdown; M-I, metaphase-I; M-II, metaphase-II.
emission of first polar body, resulting in the formation of haploid set of chromosomes in the oocytes was observed. The percentages of M-II stage oocytes were calculated in different groups. The collected data were analyzed by Chi square analysis. The level of significance was observed at the 1% level (Snedecor and Cochran, 1989).
3. Results and discussion The results are presented in Table 1. The maturation rate of the caprine oocytes in media without protein supplemented was 28.57% as shown by the control group. However, the protein supplemented in the form of 10% EGS significantly (p < 0.01) increased the maturation rate to 61.9%. The maturation rate further increased with 15 and 20% EGS supplementation to 72.7 and 78.6%, respectively. A significant difference (p < 0.01) in the in vitro maturation rate was recorded between the serum supplemented and nonsupplemented medium. The maturation rate of oocytes also significantly differed with the addition of 20% EGS, compared to 10% EGS. However, the maturation rate between 15 and 20% EGS supplementation did not differ significantly (Table 1). Experiments on in vitro maturation of caprine oocytes using TCM-199 supplemented with EGS, but without hormones, revealed a maturation rate of 52.7% (Pawshe et al., 1996) and 57.6% (Mogas et al., 1995). Mogas et al. (1997a,b) reported the addition of FSH, LH and estradiol-17 as well as EGS in TCM-199 medium to increase the maturation rate to 72.4 and 64.1% in adult and pre pubertal goats, respectively. Similarly Pawshe et al. (1996) also reported a maturation rate of 62.6% following the supplementation of hormones and EGS. In the present study a 61–78%
maturation rate with estrous goat serum was recorded in caprine oocytes without hormone supplementation. Tajik and Shams Esfandabadi (2003) reported an even higher maturation rate of oocytes in culture medium containing EGS without hormones. Samake et al. (2000) reported higher maturation rate (100%) from immature oocytes aspirated by laparotomy or via ovariectomized ovaries. In these studies, donor goats were superovulated with synchromate ear implants, followed by an intramascular injection of FSH. In mammalian females, the primordial germ cells migrate to the genital ridges following the establishment of the gonadal rudiments. Here the cells are enclosed by somatic cells and become primordial follicles, in which meiosis is initiated within the embryo. The maturation of follicular oocytes is normally arrested at the prophase-I of the first meiotic division and the oocyte remain in the dormant stage, called a dictate nucleus. At this stage, nuclear material is enveloped and the resulting structure is called a germinal vesicle. The oocyte remain at this stage until the onset of puberty. Under the influence of gonadotropins and particularly in response to the LH surge, oocytes resume meiosis just before ovulation. This results in the disappearance of the nuclear membrane and germinal vesicle breakdown, followed by chromosome condensation with the occurrence of the M-I stage. Subsequently, upon extrusion of the first polar body, the oocytes reach the M-II stage and remain at this stage until penetration by the spermatozoa. Oocytes remained in the GV stage from the onset to 6–8 h of culture. The GVBD occurred between 7 and 9 h and the metaphase-I became established within 12–18 h. Finally most oocytes reach the metaphase-II stage after 27 h. Resumption of meiosis and subsequent sequential configuration are species specific (Le Gall et al., 1992). The configuration of meiotic chromosomes at the time of co-culture of oocytes with spermatozoa for in
S.D. Kharche et al. / Small Ruminant Research 64 (2006) 186–189
vitro fertilization has a direct influence on the final success of fertilization. The M-II stage which is also known as the second phase of meiotic arrest, is consider as having have completed nuclear maturation of the oocytes required for successful fertilization of oocytes. Obviously, nuclear maturation of oocytes along with cytoplasmic maturation is important at the completion of meiotic division for success of fertilization. Therefore, the in vitro maturation process is supposed to be completed when the highest percentage of M-II oocytes is observed.
4. Conclusion The results indicated that supplementation of 20% EGS in TCM-199 medium could be used to achieve optimal maturation rates.
Acknowledgement The authors wish to thanks to the Director C.I.R.G., Makhdoom, Farah, Mathura, UP, India for providing the facilities needed.
References Crozet, N., De Smedt, V., Ahmed-Ali, M., Sevellec, C., 1993. Normal development following in vitro oocytes maturation and fertilization in the goat. Theriogenology 39, 206. Goel, A.K., Agrawal, K.P., 2000. Distribution pattern of estrous in Jakhrana goats. Indian J. Anim. Sci. 70, 589–590. Kharche, S.D., Sharma, G.T., Majumdar, A.C., 2005. In vitro maturation and fertilization of goat oocytes vitrified at germinal vesicle stage. Small Rumin. Res. 57, 81–84.
189
Le Gall, F., Gall, I., De smedt, V., 1992. Changes in protein synthesis pattern during in vitro maturation of goat oocytes. Mol. Reprod. Dev. 32, 1–8. Martin-Lunas, M., Martino, A., Paramino, M.T., Palomo, M..J., Mogas, M.T., Bielsa, M.A., Andols, P., Martinez, P., 1996. Effect of oocytes sperm co incubation on acrosome reaction in the goat. Therigenology 46, 320–330. Mogas, T., Izquierdo, M.D., Palomo, M.J., Paramio, M.T., 1995. Effect of hormones, serum source and culture system on the IVM and IVF of prepubertal goat oocytes and subsequent embryo development. Theriogenology 43, 284. Mogas, T., Palomo, M.J., Izquierdo, M.D., Paramio, M.T., 1997a. Development capacity of in vitro matured and fertilized oocytes from prepubertal and adult goats. Theriogenology 47, 1189–1203. Mogas, T., Palomo, M.J., Izquierdo, M.D., Paramio, M.T., 1997b. Morphological events during in vitro fertilization of prepubertal goat oocytes matured in vitro. Theriogenology 48, 815– 829. Pawshe, C.H., Palanisamy, A., Taneja, M., Jain, S.K., Totey, S.M., 1996. Comparison of various maturation treatments on in vitro maturation of goat oocytes and their early embryonic development and cell numbers. Theriogenology 46, 971– 982. Rajikin, M.H., Yusoff, M., Abdullah, R.B., 1994. Ultrastructural studies of developing goat oocytes in vitro. Theriogenology 42, 1003–1016. Rao, B.S., Naidu, K.S., Amarnath, D., Vagdevi, R., Rao, A.S., Brahmaiah, K.V., Rao, V.H., 2002. In vitro maturation of sheep oocytes in different media during breeding and non breeding season. Small Rumin. Res. 43, 31–36. Samake, S., Amoah, E.A., Mobini, S., Gazal, O., Gelaye, S., 2000. In vitro fertilization of goat oocyts during the non-breeding season. Small Rumin. Res. 35, 49–54. Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods. Eighth Editrion Iowa State University Press, Ames, IA, USA. Tajik, P., Shams Esfandabadi, N., 1998. Effects of estrous goat serum on in vitro maturation of caprine oocytes in non breeding season. In: Proceedings of the XVII Nordic Veterinary Congress, 4–7 August, Helsinki, Finland, p. 348. Tajik, P., Shams Esfandabadi, N., 2003. In vitro maturation of caprine oocytes in different culture media. Small Rumin. Res. 47, 155–158.