22-Title: Effect of growth factors on in vitro maturation and in vitro culture of buffalo oocytes

Authors: Vijay Singh, AK Misra, Suresh Kumar and Vijay Kumar

Source: Ruminant Science (2015)-4(2):219-224.

How to cite this manuscript: Singh Vijay, Misra AK, Kumar Suresh and Kumar Vijay (2015). Effect of growth factors on in vitro maturation and in vitro culture of buffalo oocytes. Ruminant Science 4(2):219-224.


The objective of this work was to study the effect of growth factors (EGF and IGF-I) on in vitro maturation, fertilization and development of buffalo (Bubalus sp.) cumulus oocyte complexes (COCs). Buffalo ovaries were collected from abattoir and transported to laboratory within 2 h of slaughter. Oocytes aspirated from 3-10mm follicles through 18 G needle using 10 ml syringe in oocyte collection medium, were classified as A, B, C and D grades (depending upon the cumulus cells attached and even granulation) and only grades A, B and C were used for in vitro maturation. The oocytes were matured in two groups [six treatment groups comprised of 10, 20, 30 ng/ml of EGF or 10, 50 or 100 ng/ml of IGF-I in the IVM media whereas seventh group (without EGF /IGF-I) served as control]. Matured oocytes were co-incubated with ~ 1×106/ml sperms of Murrah bull in mSOF medium with heparin (10µg/ml) and after 22 h of oocyte-sperm incubation, fertilized oocytes were stripped of cumulus cells and cultured in mSOF medium for 8 days to study embryo development. Oocytes maturation rate improved significantly (P<0.05) at all concentrations of EGF or IGF-I compared to control. Following IVF, cleavage was significantly (P<0.05) high at 20 or 30 ng/ml EGF/50 or 100 ng/ml IGF-I, compared to control .Development of embryos to morula stage was significantly (P<0.05) improved with 20 ng/ml EGF or all the three concentrations of IGF-I. Significantly (P<0.05) more blastocyst developed at 100 ng/ml of IGF-I. It was concluded that supplementation of IVM media with either EGF or IGF-1 significantly improves the in vitro maturation and subsequent development of buffalo oocytes.


Agrawal JK, Kumar N, Singh MM, Sharma A and Kumar P (2015). Seasonal variations in ovarian follicular growth in sheep and goats of himachal pradesh. Ruminant Science 4(1):33-35.

Bridges TS, Davidson TR, Chamberlain CS, Geisert RD and Spicer CJ (2002). Changes in follicular fluid steroids, insulin-like growth factors IGF and IGF-binding protein concentration and proteolytic activity during equine follicular development. Journal of Animal Sciences 80:179-190.

Chauhan MS, Singla SK, Palta P, Manik RS and Madan ML (1999). Effect of EGF on cumulus expansion, meiotic maturation and development of buffalo oocytes in vitro. Veterinary Record 144:266-267.

Chauhan MS, Singla SK, Palta P, Manik RS and Tomar OS (1998). IGF-II stimulation of in vitro maturation, in vitro fertilization and subsequent development of buffalo (Bubalus bubalis) oocytes in vitro. Veterinary Record 142:727-728.

Das K, Stout LE, Hensleigh HC, Tagatz GE, Phippa WR and Leungh BS (1991). Direct positive effect of epidermal growth factor on cytoplasmic maturation of mouse and human oocytes. Fertility Sterility 55:1000-1004.

Driancourt MA and Thuel B (1998). Control of oocyte growth and maturation by follicular cells and maturation by follicular cells and molecules present in follicular fluid. A review. Reproduction Nutrition Development 38:345-362.

Guler A, Poulin N, Mermillod P, Terqui M and Cognie Y. (2000). Effect of growth factors, EGF, IGF-I and estradiol on in vitro maturation of sheep oocytes. Theriogenology 54:209-218.

Gupta PSP, Nandi S, Ravindranatha BM and Sarma PV. (2002). In vitro maturation of buffalo oocytes with epidermal growth factor and fibroblast growth factor. Indian Journal of Animal Sciences 72:20-23.

Jaskulski D, Gatti C, Travali S, Calabretta B and Baserga R. (1988). Regulation of the proliferating cell nuclear antigen cyclin and thymidine kinase mRNA levels by growth factors. Journal of Biological Chemistry 263:1075-1079.

Jones JKI and Clemmons DR (1995). Insulin-like growth factors and their binding proteins: Biological action. Endocrinology Reviews 16:3-34.  

Khamsi F and Armstrong DT. (1997). Interactions between follicle stimulating hormone and growth factors in regulation of deoxyribonucleic acid synthesis in bovine granulosa cells. Biology of Reproduction 57:684-688.

Kumar D and Purohit GN (2004). Effect of epidermal and insulin like growth factor-1 on cumulus expansion, nuclear maturation and fertilization of buffalo cumulus oocyte complexes in simple serum free media DMEM and Ham’s F-10. Veterinarski Arhiv 74 (1):13-25.

Lonergan P, Carolan C, Van Langendouck FA, Donnay I, Khalir H and Mermillod P (1996). Role of epidermal growth factor in bovine oocyte maturation and pre-implantation embryo development in vitro. Biology of Reproduction 54:1420-1429.   

Lorenzo PL, Illera MJ, Illera JC and Illera M. (1994). Enhancement of cumulus expansion and nuclear maturation during bovine oocyte maturation in vitro by the addition of epidermal growth factors and insulin-like growth factors. Journal of Reproduuction and Fertility 101:697-701.

Nandi S, Raghu HM, Ravindranatha BM, Gupta PS and Sharma PV. (2002). In vitro development of buffalo oocytes in media containing fluids from different size class follicles. Theriogenology  57(3):1151-1159.

Nandi S, Ravindranatha BM, Gupta PSP, Raghu HM and Sarma PV (2003). Developmental competence and post thaw survivability of buffalo embryo produced in vitro: Effect of growth factors in oocyte maturation medium and embryo culture system. Theriogenology 60:1621-1631.

Palta P and Chauhan MS. (1998). Laboratory production of buffalo (Bubalus bubalis) embryos. Reproduction Fertility and Development 10:379-91.

Park DH, Yang BK, Kim JK, Choung HT, Park CK, Kim JB and Kim CI (1997). Effect of b-Mercaptoethanol and cysteamine with bovine oviduct epithelial cells on development and intracellular glutathione concentrations of bovine IVM/IVF embryos. Korean Journal of Embryo Transfer 12 (3): 269-276.

Park KW, Choi SH, Song XX, Funahashi H and Niwa K (1999). Effect of epidermal growth factor on production of PAS in oocytes and cumulus cells. Biology of Reproduction 61:298-304.

Pawshe CH, Appa Rao KPCA and Totey SM. (1998). Effect of insulin-like growth factor I and its interaction with gonadotrophins on in vitro maturation and embryonic development, cell proliferation and biosynthetic activity of cumulus-oocyte complexes and granulosa cell in buffalo. Molecular Reproduction Development 49:277-289.

Puri Gopal and Bag Sadhan (2012). Development of embryonic stem cell clone from in vitro derived buffalo embryos in feeder and feeder free culture conditions. Ruminant Science 1(2):109-112.

Purohit GN, Brady MS and Sharma SS (2005). Influence of epidermal growth factor and insulin like growth factor-1 on nuclear maturation and fertilization of buffalo cumulus complexes in serum free media and their subsequent development in vitro. Animal Reproduction Science 87:229-239.

Raghu HM, Nandi S and Reddy SM (2002). Effect of insulin transferin and selenium and epidermal growth factor on development of buffalo oocytes to the blastocyst stage in vitro in serum-free, semi-defined media. Veterinary Record 151(9):260-265.

Sadeesh EM, Shah Fozia, Balhara AK, Thirumaran SMK, Balhara Sunesh and Yadav PS (2013). Effect of thiol compound supplemented culture medium on in vitro maturation of buffalo oocytes. Ruminant Science (2013)-2(1):59-62.

Sakaguchi M, Dominko T, Yamauchi N, Leibfried-Rutledge ML, Nagai T and First NL (2002). Possible mechanism for acceleration of meiotic progression of bovine follicular oocytes by growth factors in vitro. Reproduction 123:135-142.

Sharma Manjinder, Kumar Rajesh and Sharma G Taru (2015). Potential of allogeneic feeders to support the development of buffalo embryonic stem cells. Ruminant Science 4(1):21-27.

Snedecor GW and Cochran WG (1989). Statistical Methods. 8th Edn, The Iowa State University Press, Amnes, Iowa, USA.

Wandji SA, Srsen V, Voss AK, Eppig JJ and Fortune JE (1996). Initiation of in vitro  growth of bovine primordial follicles. Biology of Reproduction 55:942-948.