Skip to main content Accessibility help

Production of buffalo embryos using oocytes from in vitro grown preantral follicles

  • P.S.P. Gupta (a1), H.S. Ramesh (a1), B.M. Manjunatha (a1), S. Nandi and J.P. Ravindra (a1)...


The present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80–100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.


Corresponding author

1All correspondence to: P.S.P. Gupta, National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore-560 030, India. E-mail:;


Hide All
Aboul-Ela, M.B.E. (2000). Superovulation in the buffaloes: constraints and manipulation. Buffalo J. 16, 120.
Andrade, E.R., Amorim, C.A., Matos, M.H.T., Rodrigues, A.P.R., Silva, J.R.V., Dode, M.A.N. & Figueiredo, J.R. (2002). Evaluation of saline and coconut water solutions in the preservation of sheep preantral follicles in situ. Small Rumin. Res. 43, 235–43.
Andrade, E.R., Seneda, M.M., Alfieri, A.A., Oliveira, J.R., Figueiredo, R. & Toniolli, I. (2005). Effect of indol acetic acid concentration in the activation and growth of ovine preantral follicles. Arq. Bras. Med. Vet. Zootec. 57, 334–39.
Blume, H., Vale-Filho, V.R., Marques, A.P. Jr & Saturnino, H.M. (1988). Evaluation of coconut water for bovine embryo culture. Arq. Bras. Med. Vet. Zootec. 50, 395–9.
Cecconi, S., Barboni, B., Coccia, M. & Mattioli, M. (1999). In vitro development of sheep preantral follicles. Biol. Reprod. 60, 594601.
Combarnous, Y. & Nunes, F. J. (1995). Sperm extender including indole derivative. International patent no. 2725342: PCT/FR95/01322(FR 94-12122).
Costa, S.H.F., Santos, R.R., Ferreira, M.A.L., Machado, V.P., Rodrigues, A.P.R., Ohashi, O.M. & Figueiredo, J.R. (2002) Preservation of goat preantral follicles in saline or coconut water solution. Brazilian J. Vet. Res. Anim. Sci. 39, 324–30.
Danell, B. (1987). Oestrus behaviour, ovarian morphology and cyclical variation in the follicular system and endocrine pattern in water buffalo heifers. Ph.D Dissertation, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Eppig, J.J. (1991). Intercommunication between mammalian oocytes and companion somatic cells. Mol. Reprod. Dev. 13, 569–74.
Eppig, J.J. & Schroeder, A.C. (1989) Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilisation in vitro. Biol. Reprod. 41, 268–76.
Guoliang, X., Byskov, A.G. & Andersen, C.Y. (1994). Cumulus cells secrete a meiosis-inducing substance by stimulation with forskolin and dibutyric cyclic adenosine monophosphate. Mol. Reprod. Dev. 39, 1724.
Gupta, P.S.P., Nandi, S., Ravindranatha, B.M & Sarma, P.V. (2001a). Isolation of preantral follicles from buffalo ovaries. Vet. Rec. 148, 543–4.
Gupta, P.S.P., Nandi, S., Ravindranatha, B.M. & Sarma, P.V. (2001b). Effect of commercially available PMSG on maturation, fertilization and embryo development of buffalo oocytes in vitro. Reprod. Fertil. Dev. 13, 355–60.
Gupta, P.S.P., Nandi, S., Ravindranatha, B.M. & Sarma, P.V. (2002a). In vitro culture of buffalo (Bubalus bubalis) preantral follicles. Theriogenology 57, 1839–54.
Gupta, P.S.P., Nandi, S., Ravindranatha, B.M. & Sarma, P.V. (2002b). Trypan blue staining to differentiate live and dead buffalo oocytes and its effect on embryo development in vitro. Buffalo J. 18, 321–30.
Gupta, P.S.P., Ramesh, H.S., Nandi, S. & Ravindra, J.P. (2006) Recovery of large preantral follicles from buffalo ovary: effect of season and corpus luteum. Anim. Reprod. Sci. (in press). doi: 10.1016/j.anireprosci.2006.11.011
Gutierrez, C.G., Ralph, J.H., Telfer, E.E., Wilmut, I. & Webb, R. (2000). Growth and antrum formation of bovine preantral follicles in long term culture in vitro. Biol. Reprod. 62, 1322–28.
Huanmin, Z. & Yong, Z. (2000). In vitro development of caprine ovarian preantral follicles. Theriogenology 54, 641–50.
Itoh, T., Kacchi, M., Abe, H., Sendai, Y. & Hoshi, H. (2002). Growth, antrum formation and estradiol production of bovine preantral follicles cultured in a serum free medium. Biol. Reprod. 67, 1099–105.
Kezele, P., Nilsson, E.E. & Skinner, M.K. (2005). Keratinocyte growth factor acts as a mesenchymal factor that promotes ovarian primordial to primary follicle transition. Biol. Reprod. 73, 967–73.
Leong, I.P. & Shui, G. (2002). An investigation of antioxidant capacity of fruits in Singapore markets. Food Chem. 76, 6975.
Li, R., Philips, D.M. & Mather, J.P. (1995). Activin promotes ovarian follicle development in vitro. Endocrinology 136, 849–56.
Martins, F.S., Santos, R.R., Celestino, J.J.H., Matos, M.H.T., Silva, G A., Ferreira, F.V.A. & Figueiredo, J.R. (2004). Development of goat preantral follicles in coconut water solution. Acta Scientiae Veterinariae 32 (Supplement), 136.
Nandi, S., Ravindranatha, B.M., Gupta, P.S.P. & Sarma, P.V. (2001). Effect of somatic cells monolayer on maturation of buffalo oocytes in vitro. Indian J. Anim. Sci. 71, 936–7.
Nandi, S., Raghu, H.M., Ravindranatha, B.M., Gupta, P.S.P. & Sarma, P.V. (2002). Effect of somatic cell co-culture and synthetic oviductal fluid on development of buffalo embryos in vitro. Online J. Physiol. 1, 2632.
Nilsson, E.E., Doraiswamy, V. & Skinner, M.K. (2003). Transforming growth factor-beta isoform expression during bovine ovarian antral follicle development. Mol. Reprod. Dev. 66, 237–46.
Parrot, J.A. & Skinner, M.K. (1997). Direct actions of KL on theca cell growth and differentiation during follicle development. Endocrinology 138, 3819–27.
Prochazka, R., Naggova, E., Brem, G., Schellander, K. & Motlik, J. (1998). Secretion of cumulus expansion enabling factor (CEEF) in porcine follicles. Mol. Reprod. Dev. 49, 141–9.
Santos, S.S., Biondi, F.C., Corderio, M.S., Miranda, M.S., Dantas, J.K., Figuerido, J.R. & Ohashi, O.M. (2006). Isolation, follicular density, and culture of preantral follicles of buffalo fetuses of different ages. Anim. Reprod. Sci. 95, 115.
Silva, J.R.V., Lucci, C.M., Carvalho, F.C.A., Bao, S.N., Costa, S.H.F., Santos, R.R & Figueiredo, J.R. (2000). Effect of coconut water and Braun-Collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro. Theriogenology 54, 809–22.
Silva, J.R.V., Van Den Hurk, R., Costa, S.H.F., Andrade, E.R., Nunes, A.P.A., Ferreira, F.V.A., Lobo, R.N.B., & Figueiredo, J.R. (2004). Survival and growth of primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water. Anim. Reprod. Sci. 81, 273–86.
Songsasen, N. & Apimeteetumrong, M. (2002). Effects of beta-mercaptoethanol on formation of pronuclei and developmental competence of swamp buffalo oocytes. Anim. Reprod. Sci. 71, 193202.
Spears, N., Boland, N.I., Murray, A.A. & Gosden, R.G. (1994). Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile. Hum. Reprod. 9, 527–32.
Vanderstichele, H., Delaey, B., De Winter, J., De Jong, F., Rombauts, I., Verhoeven, G., Dello, C., Van de Voorde, A. & Briers, T. (1994). Secretion of steroids, growth factors, and cytokines by immortalized mouse granulosa cell lines Biol. Reprod. 50, 1190–202.
Vigne, J.L., Lisa, I., Halburnt, & Skinner, M.K. (1994). Characterization of bovine ovarian surface epithelium and stromal cells: identification of secreted proteins. Biol. Reprod. 51, 1213–21.
Wu, J., Carrel, D.T. & Wilcox, A.L. (2001a). Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection. Biol. Reprod. 65, 1579–85.
Wu, J., Emery, B.R. & Carrel, D.T. (2001b). In vitro growth, maturation, fertilization and embryonic development of oocytes from porcine preantral follicles. Biol. Reprod. 64, 375–81.
Wu, M.F., Huang, W.T., Tsay, C., Hsu, H.F., Liu, B.T., Chiou, C.M., Yen, S.C., Cheng, S.P. & Ju, J.C. (2002). The stage-dependent inhibitory effect of porcine follicular cells on the development of preantral follicles. Anim. Reprod. Sci. 73, 7388.



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed