Introduction

Anestrous is one of the most important functional ovarian disorders in dairy buffaloes affecting adversely economics of milk production. Clinical surveys in India have revealed higher incidences of anestrous due to inactives ovaries in buffaloes in comparison to cattle, and this affects production potential in the form of huge economic losses (Tanwar et al., 2003). India and Pakistan indicate that only 34–49% of buffaloes showed estrus during the first 90 days after calving and 31–42% remained anestrus for more than 150 days (El Wishy, 2006). The poor reproductive performance of dairy herds has become a major concern, especially in buffalo’s herds. Decline in conception rate and increase in calving interval over the last decades have been confirmed by several studies (Royal et al. 2000; Lucy, 2001; Hare et al. 2006). Economic consequences due to a non optimal reproductive performance of a herd consist of extra expenditures and losses in incomes (lower output/input coefficients). Mechanisms involved in the economic consequences of lengthening of calving interval and of extra-culling are quite numerous and complex (Seegers, 2006). In search of fruitful remedy for anoestrus in livestock, many hormonal preparations like estrogen, progesterone, gonadotropins, gonadotropin releasing hormone (GnRH), prostaglandin-F_2α(PGF_2α) and their analogues alone or in combination (Pattabiraman et al., 1986) as well as some non hormonal preparations such as prajna, janova, aloes compounds and clomiphene citrate (Reddy et al., 1994), have been tried with varying degree of success. But, most of these compounds have their own merits and demerits. Application of insulin to modulate reproduction in livestock is fairly a recent development.Several hormonal and non hormonal therapies have been devised with their own merits and demerits. The growth factors and metabolic hormones i.e. somatotropins insulin and insulin like growth factor-1 (IGF-1) have received considerable attention in the regulation of ovarian functions. Thus, the present experiment was designed to study the effect of exogenous insulin alone and in combination with GnRH on estrous induction and restoration of fertility in anestrous buffalo.

Materials and Methods

The work was carried out at the composite livestock farm Adhartal and department of Animal Reproduction, Gynaecology and Obstetrics, college of veterinary science and A.H., Jabalpur. For the experiment 40 healthy, non suckling true, anestrous buffaloes of weighing approx 400-550 kg (measured by electronic digital weighing machine) with the history of normal calving but not resumed estrus to 60 or more days after calving .The confirmation of anestrous was made on the basis of history, gynaecological examination of genitalia twice at weekly interval and serum progesterone estimation before treatment (i.e.<1ng/ml). Animals having smooth ovaries (without palpable corpus luteum and follicle) and normal developed genital tract were selected for the experiment. Animals were divided randomly into 4-groups, each comprising ten (n=10) animals. Animal of  Group-I (G1, n=10)   treated with long acting bovine insulin (Insulin zinc suspension, highly purified long acting Bovine insulin Vinsulin^TM ) at the dose rate of 0.25 IU/ kg bodyweight (dose was calculated individually) subcutaneous once daily for 5 consecutive days (Shukla et. al. 2005), animals of Group-II (G2, n=10) treated with GnRH analogue (Buserline – Acetate Inj. (Receptal^{TM )} where each buffalo received an intramuscular injection of 20  μg of  Receptal^TM  on Day 0; (Nasar et al. 1983), animals of Group-III treated  with insulin plus GnRH (Receptal^TM ) (G3, n=10) in which each received a subcutaneous injection of long  acting bovine insulin at the dose rate of 0.25 IU/ kg bodyweight once daily for 3 consecutive days, followed by an intramuscular injection of 20 μg of GnRH (Receptal^TM )on Day 4 ; and the animals of  control  group (G IV, n=10) in which each animal  receive  an intramuscular injection of 2.5 ml of sterile saline  on Day 0.

Managemental Practices

The buffaloes were stall-fed and housed in pucca sheds with standard management norms of L.S.F. The animals were let loose daily at least for 30 minutes during morning and evening hours in the paddock with good water splashing following milking. Clean drinking water was made available to the animals ad lib. During these hours estrus behaviour of all the buffaloes was observed in the presence of accompanying breeding bull.  Further confirmation of estrus was done by rectal palpation of genitalia. The animals in estrus (heat) were bred by healthy fertile bull.The herd was routinely vaccinated for the diseases, viz. Rinderpest, Foot and Mouth, Anthrax, Haemorrhagic Septicaemia and Black Quarter. The general health check-up and deworming of the animals were done routinely.

Results and Discussion

Results pertaining to the effect of exogenous insulin alone and in combination with GnRH on fertility response in true anestrous buffalos have been presented in Table.

In response to the treatment induction of estrus in higher number of animals using insulin in combination of GnRH (G-3, 60%) as compared to insulin alone (G1, 50.00%) and GnRH alone (G2, 40%), however no animal induced in estrus in control group (G4, 0.0%). The onset of estrus interval (days) following start of treatment was shorter in G-2 (9.5±3.18), as compared to G-3 (15.5 ± 4.61) and  G-1 (25±0.94) ,respectively. The ovulation rate at induced estrus was 100% in all the treatment groups. All the animals bred at induced estrus with the 100% conception i.e. in G-1, G-2 and G-3, respectively.

The beneficial effect of insulin on induction of estrus and fertility in anestrous buffaloes which are comparable to the findings of Ramoun et. al. (2007) and Shukla et. al. (2005) may be due to its effects on follicogenesis and steroidogenesis. In support of this in vitro studies have demonstrated insulin and IGF-1 as important regulators of follicogenesis and steroidogenesis (Gong et al., 1994, Stewart et al., 1995).  Insulin enhances growth and proliferation of granulose, theca and luteal cell present in the ovary (Spicer et al., 1993, Stewart et al., 1995), thus, enhances follicogenesis (Khan, 2000) either acting through specific insulin and IGF-1 or both types of receptors.

Table : Estrous induction and fertility response using insulin, GnRH alone and their combination in anestrous buffaloes

Gonadotopin releasing hormone (GnRH) and its analogue has been used for induction of estrus and fertility in anestrous bovine by the various workers and they have reported onset of estrus 22 to 87% with an average interval of 4 to 29 days, ovulation rate 75 to 100% and conception rate 9 to 66.7% (Dhoble and Gupta 1986; Thompson et al., 1999, Markanadya and Patil, 2003 Sirmour et al. (2006).

The results of induction rate of estrus (60%) and ovulation rate (100%) in G-III (Insulin+GnRH) in the present study are comparable to the findings of Ramoun et. al. (2007) and Shukla et al. (2005) reported nearly similar Induction of estrus (42.85%) and ovulation rate 100% in anestrous cattle. Present study also again supported by findings of   Ramoun et al. (2007) reported that pretreatment with insulin 3 day before GnRH injection increase the diameter of the dominant follicle and estrus induction rate (100%) in acyclic buffaloes.

References

Dhoble, R.L. and S.K. Gupta (1986) Response to synthetic gonadoliberin (GnRH)      in      anestrous cows.Theriogenology 26 (6): 759-765.

El-Wishy, A.B.  (2007).Acyclicity and anestrus postpartum buffaloes ,Animal Reproduction Science;97(3):p216-236

Gong, J.B., D. McBride, T.A. Bramley and R. Webb (1994). Effects of recombinant bovine somatotropin insulin – like growths factors-I and insulin on bovine granulosa cell steroidogenesis in vitro. J. Reprod. Fertil., 110 : 611-622.

Hare E, Norman HD, Wright JR. Trends in calving ages and calving intervals for dairy cattle breeds in the United States. J Dairy Sci, 2006; 89:365

Khan,M.H. (2000). Pretreatment effect of insulin on follicular dynamics and embryo             Production in goats.M.V.Sc. Thesis, submitted to deemed university ,IVRI,Izatnagar,Bareilly,UP (India).

Lucy MC. Reproductive loss in high-producing dairy cattle:where will it end? J Dairy Sci, 2001; 84:1277-1293-370.

Markandeya, N.M. and A.D. Patil (2003). Studies on hormonal therapies for induction of post-partum estrus in buffaloes. Intas Polivet, 4 (11): 167 – 169

Ramoun, A.A, A.D, Khalid, T. Osman, B. Samy, A. Darwish, B. Aly, M. Karen,A. Magdy, H. Gamal (2007). Effect of pretreatment with insulin on the response of buffaloes with inactive ovaries to gonadotrophin-releasing hormone agonist treatment in summer, Reproduction, Fertility and Development, 2007, 19: 351–355

Seegers, Henri (2006).  Economics of the reproductive performance of dairy herds, World Buiatrics Congress – NICE, FRANCE

Shukla, S. N., S. K. Agarwal, U. Shanker, V.P. Varshney and A.C.  Majumdar (2005b). Modulation of ovarian response in anoestrus cattle treated with Insulin alone and in combination with GnRH. Indian Journal of Animal Reproduction., 26 (2):159-164.

Spicer, L.J.,   E.  Alpizar and   S.E.  Echternkamp (1993). Effects of insulin, insulin-like growth factor-I and gonadotropins on bovine granulosa cell proliferation, progesteron production, estradiol production and (or) insulin-like growth factor-I production in vitro. J. Anim. sci., 71 : 1232-1241.

Stewart, R.E., L.J. Spicer, T.D.  Hamilton and B.E. Keefer (1995). Effects of insulin like growth factor-I and insulin on proliferation and on basal luteinizing hormone induced steroidogenosis of bovine thecal cells: involvement of glucose and receptors for insulin-like growth factor-I and luteinizing hormone. J. Aim. Sci., 73: 3719-3731.

Tanwar, P.S., N.K. Rakha and J.B. Phogat (2003). Challenges in buffalo infertility. Intas Polivet, 4 (1): 121 – 127.

Thompson, T. (1999). Follicular, hormonal and pregnancy response of early postpartum Suckled beef cows to GnRH, norgestomet and PGF2 alpha .j. Anim. Sci., 77: 1823-1832.