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Active immunization against inhibin-based peptides to increase ovulation rate in non-prolific Malpura ewes
Small Ruminant Research 81 (2009) 163–166
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Technical note
Active immunization against inhibin-based peptides to increase ovulation rate in non-prolific Malpura ewes S.M.K. Naqvi a,∗ , A. Joshi a , R. Gulyani a , V.P. Maurya a , R.S. Manik b , P. Palta b a b
Central Sheep and Wool Research Institute (CSWRI), Avikangar 304501, India National Dairy Research Institute (NDRI), Karnal 132001, India
a r t i c l e
i n f o
Article history: Received 26 August 2008 Received in revised form 11 December 2008 Accepted 11 December 2008 Available online 29 January 2009 Keywords: Inhibin Immunization Ovulation rate Sheep
a b s t r a c t The aim of this study was to explore the possibility of increasing the ovulation rate of Malpura, a non-prolific tropical breed of sheep by immunization against inhibin-based peptide immunogens. Ewes were divided into three groups (n = 5 each) and actively immunized against the synthetic peptides from the ␣C [bI␣(1–29)-Tyr30 ] or ␣N [bI-43 Tyr152 (153–167)Cys168 ] area of the bovine inhibin ␣-subunit conjugated to ovalbumin or against ovalbumin (control). Each ewe received a primary immunization of 400 g immunogen and 3 boosters, each of 200 g immunogen at 4-week intervals. Estrus was synchronized using a double PGF2 ␣ injection schedule and laparoscopy was performed after each estrus to determine the ovulation response. Immunization against both the peptides did not affect the interval from PGF treatment to the onset of estrus, the duration of estrus and the number of large unovulated follicles. In contrast to the complete absence of multiple ovulations in the controls, all the ewes immunized against ␣C or ␣N peptides showed multiple ovulations (range 2–7) in all the three estrous cycles evaluated, except for one ewe immunized against the ␣N peptide, which exhibited multiple ovulations in only 1 out of the 3 estrous cycles. Compared to that of the controls (1.0 ± 0.9, 1.0 ± 0.0 and 0.6 ± 0.2, respectively), the mean ovulation rate was higher (P < 0.01) in the ewes immunized against the ␣C (4.8 ± 1.02, 5.0 ± 1.05 and 5.0 ± 0.45, respectively) or against ␣N (4.5 ± 1.19, 2.5 ± 0.87 and 2.7 ± 0.75, respectively, P < 0.05) peptide in estrous cycles numbers 1, 2 and 3. These results show that active immunization against inhibin-based peptide immunogens is effective in increasing ovulation rate in Malpura, a non-prolific breed of sheep and that it may be an alternative to conventional superovulation regimes. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The majority of native Indian sheep populations are found in the arid and semi-arid regions and contribute significantly to meat and wool production, despite prevailing harsh conditions like high ambient temperatures, scarcity of feed and water. However, the reproductive efficiency of these sheep breeds is relatively low (Arora and Garg, 1998). The Malpura sheep are native to a semi-arid tropical region
∗ Corresponding author. Tel.: +91 1437 220165; fax: +91 1437 220163. E-mail address: [email protected] (S.M.K. Naqvi). 0921-4488/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2008.12.010
of India and are known for their superior quality mutton and carpet-type wool (Acharya, 1982). It is a monotoccus breed with an ovulation rate of 1 and a very low incidence of twinning (<5%). The average litter size at birth has been reported to be 1.05 for the Malpura sheep (Mishra, 2008). Thus, there is a need to improve the reproductive potential for enhancing the mutton and wool production of the Malpura sheep. Inhibin, a glycoprotein hormone produced by the granulosa cells in the ovarian follicles suppresses the production and secretion of pituitary gonadotropins, preferentially FSH through negative feedback mechanism (Burger, 1992). It is also involved in the regulation of folliculogenesis through
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autocrine and/or paracrine control (Findlay, 1993). Active immunization against inhibin, either in its native form or in the form of inhibin-based synthetic peptides, has been shown to increase the ovulation rate in cattle (Akagi et al., 2002; Medan et al., 2004), goats and sheep (Medan et al., 2003; Padilla et al., 2007). However, in all the studies in sheep, the animals have been of temperate breeds with high fecundity, viz. the Romney (Wrathall et al., 1990; McLeod et al., 1992), Suffolk (Schanbacher et al., 1991; Wheaton et al., 1992), Merino (Anderson et al., 1998) or crossbreds (Wrathall et al., 1992; Tannetta et al., 1997). Lately, there has been an increasing interest in the development of inhibinbased fecundity vaccines (Han et al., 2007; Padilla et al., 2007). There is however, no report available on the effects of immunization against inhibin in any tropical or non-prolific breeds of sheep. The objective of the present study was thus to explore the possibility of increasing the ovulation rate in the Malpura, a tropical breed of sheep with low fecundity, by active immunization against synthetic peptides based on the sequence of the ␣-subunit of bovine inhibin. 2. Materials and methods 2.1. Animals and their management Fifteen non-pregnant adult ewes of Malpura breed, 4–7 years of age and weighing 32.1 ± 0.53 kg were randomly allocated in equal numbers (n = 5) to three treatment groups. All the animals were managed under the standard farm practices at the CSWRI’s sheep farm at Avikanagar, which is located at a longitude of 75◦ 28 E, latitude of 26◦ 26 N and an altitude of 320 m above mean sea level in a semi-arid region of India. The climate in this region is essentially tropical with the highest temperatures occurring from April to June with a mean monthly temperature of 42 ◦ C. The minimum and maximum ambient temperatures range from 7 to 37 ◦ C and from 25 to 46 ◦ C, respectively—while the mean relative humidity varies between 15% and 90%. The rainfall is erratic and mainly concentrated during July to August with a precipitation of 200–500 mm per annum. 2.2. Immunization protocol Synthetic peptides mimicking the amino acid sequences of fragments of the bovine inhibin ␣-subunit were commercially prepared by Sigma–Genosys, England. The peptides were conjugated to ovalbumin, with the peptide-to-ovalbumin molar ratio being approximately 20:1 to increase their antigenicity. The synthetic peptides included a 30-mer peptide based on the ␣C [bI␣(1–29)-Tyr30 ] and a 17-mer peptide based on the ␣N region [bI-43 -Tyr152 (153–167)Cys168 ] of the ␣-subunit of bovine inhibin. The Malpura ewes of Group I and II were immunized against the 30-mer and the 17-mer peptide, respectively, whereas those of the control group were immunized against ovalbumin. Primary immunization involved the injection of 400 g of peptide–ovalbumin conjugate (Groups I and II) or ovalbumin (control) dissolved in 1 ml isotonic saline and emulsified with an equal volume of Freund’s complete adjuvant. Each animal was given 3 boosters, each of 200 g peptide–ovalbumin conjugate or ovalbumin dissolved in 1 ml isotonic saline and emulsified with an equal volume of Freund’s incomplete adjuvant, at 4-week intervals during the natural breeding season (August to October). 2.3. Estrus synchronization Estrus was synchronized using a double injection schedule of PGF2␣ (Lutalyse, Pharmacia N.V./S.A., Puurs-Belgium, 7.5 g per treatment) on Days 35 and 45 of the trial (Day 0 = Day of primary immunization). Subsequently, the animals were allowed to undergo normal cyclicity until the end of trial. Estrus was detected by using an aproned ram per group (2–4 years of age) of high sexual vigour at 6 h intervals. Blood samples were collected at 08:00 twice weekly from the onset to the end of the trial for determination of plasma progesterone concentrations for confirmation of cyclicity (Kamboj and Prakash, 1989). The intra- and inter-assay co-efficients of variation were both less than 13%.
2.4. Ovarian examination for ovulation rate For evaluating the ovarian response to immunization, the ovaries were examined by laparoscopy 3–6 days after each estrus (Naqvi and Gulyani, 1995; Naqvi et al., 2000). In brief, after fasting the animals for at least 24 h prior to laparoscopy, all ewes were sedated with xylazine hydrochloride and locally anaesthetized with lidocaine hydrochloride. After visualization of the reproductive tract through the laparoscope, the ovary was grasped with an atraumatic forceps and the number of corpora lutea (CL) and large follicles (LF) recorded. 2.5. Data analysis The data were expressed as the mean ± S.E.M. Data on estrus response, ovulation rate and the numbers of large follicles were compared between different groups by analyses of variance (ANOVA), using the SYSTAT version 7.0 (SPSS Inc., USA, 1997).
3. Results Immunization against the peptides bI␣(1–29)-Tyr30 and bI-43 -Tyr152 (153–167)Cys168 did not have any adverse effect on the interval from PGF to the onset of estrus and the duration of estrus (Table 1). Also, the number of large unovulated follicles recorded was not significantly different in the ewes immunized against the inhibin-based peptides, compared to those in the control group in the estrous cycle numbers 2 and 3 (Table 2). The incidence of multiple ovulations increased significantly by active immunization against inhibin. In contrast to the complete absence of multiple ovulations in the control group, all the ewes immunized against bI␣(1–29)Tyr30 showed multiple ovulations in all the three estrous cycles examined in the study – with the ovulation rates ranging from 2 to 7. In the ewes immunized against bI152 (153–167)Cys168 , except for ewe No. 4, which 43 -Tyr exhibited multiple ovulations in only one estrous cycle, while all the other ewes had multiple ovulations – ranging between 2 and 6 in all the 3 estrous cycles examined. In the ewes immunized against bI␣(1–29)-Tyr30 , the mean ovulation rate was nearly 5-fold higher (P < 0.01) than that for the controls in all the three estrous cycles examined (Table 2). In the ewes immunized against bI-43 -Tyr152 (153–167)Cys168 also, the ovulation rate was significantly higher (P < 0.05) than that for the controls in estrous cycles 1 and 3. 4. Discussion To current knowledge, this is the first report on the effects of immunization against inhibin in any non-prolific breed of sheep. It has shown that active immunization against peptides (based on the ␣-subunit of inhibin), can be used for inducing superovulation in the Malpura, a tropical breed of sheep, that otherwise has an ovulation rate of approximately 1 and a very low incidence of twinning. Inhibin is a heterodimer composed of an ␣- and a -subunit. During the post-translational processing, the prepro-␣-subunit of inhibin gives rise to three peptides: the carboxy-terminal peptide ␣C , which forms the 32 kDa form of inhibin after combining with the -subunit; the amino-terminal peptide ␣N , which gives rise to the 57 kDa form of inhibin after combining with the ␣C and -subunit; a pro-␣C peptide (Bernanrd et al., 2001). Active immuniza-
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Table 1 Estrus response of Malpura ewes immunized against inhibin-based peptides. Treatment
Control (ovalbumin) Group I [bI␣(1–29)-Tyr30 ] Group II [bI-43 -Tyr152 (153–167)Cys168 ] a
Number of ewes treated (n)
5 5 4a
Mean ± S.E. Interval from PGF treatment to onset of estrus (h)
Duration of estrus (h)
49.2 ± 2.94 48.0 ± 3.46 40.0 ± 5.29
28.8 ± 3.50 38.0 ± 2.00 48.0 ± 3.46
One animal (M-387) in Group II diagnosed as pregnant and hence omitted.
Table 2 Ovarian response in Malpura ewes immunized against inhibin peptides. Treatment
Estrous cycle number 1
Control (ovalbumin) Group I [bI␣(1–29)-Tyr30 ] Group II [bI-43-Tyr152 (153–167)Cys168 ]
2
3
Ovulations
Large follicles
Ovulations
Large follicles
Ovulations
Large follicles
1.00 ± 0.0 4.8 ± 1.02* 4.5 ± 1.19*
0.0 ± 0.0 0.2 ± 0.20 0.7 ± 0.25*
1.0 ± 0.0 5.0 ± 1.05** 2.5 ± 0.87
0.4 ± 0.24 0.8 ± 0.37 0.0 ± 0.00
0.6 ± 0.24 5.0 ± 0.45** 2.7 ± 0.75*
0.6 ± 0.24 0.2 ± 0.2 0.5 ± 0.5
Means ± S.E. within a column are significantly different from controls. * P < 0.05. ** P < 0.01.
tion against the 30-mer peptide bI␣(1–29)-Tyr30 , which is from the ␣C region was found to induce multiple ovulations ranging from 2 to 7 and to increase the ovulation rate almost 5-fold, compared to the controls. These results are in agreement with earlier studies in which immunization against this peptide was found to increase the ovulation rate in many prolific breeds of sheep like e.g. the Romney (Wrathall et al., 1990; McLeod et al., 1992) and Blue-faced Leicester × Swadale crosses (Wrathall et al., 1992; Tannetta et al., 1997). Also, among the many inhibinbased peptides compared, this specific peptide had been reported to be the most effective in increasing ovulation rate in cattle (Bleach et al., 1996). The 17-mer peptide bI152 (153–167)Cys168 , which is from the ␣ region of N 43 -Tyr bovine inhibin, was selected in view of its reported efficacy in increasing the ovulation rate in cattle (Morris et al., 1995). Active immunization against this peptide resulted in over a 2.5-fold increase in ovulation rate in the present study. Interestingly, immunization of sheep against recombinant fusion protein immunogens containing the full length of ␣N sequences has been found to lower fertility by suppressing ovulation (Findlay et al., 1994; Russell et al., 1995). Between the two peptides, the ␣C 30-mer peptide bI␣(1–29)-Tyr30 was found to be more effective in increasing the ovulation rate than the ␣N 17-mer peptide bI-43 -Tyr152 (153–167)Cys168 . The efficacy of different peptide immunogens has been reported to vary depending upon their location within the inhibin molecule. Peptides derived from the amino-terminal end of the ␣-subunit are more immunogenic than those from other regions of the ␣-subunit (Saito et al., 1989), as amino acids 13–26 contain important antigenic sites (Groome et al., 1990; Funaba et al., 1996). Addition of residues 7–12 has been reported to be necessary for further increasing the immunogenicity and ovulatory response to immunization (Anderson et al., 1998). The size of the peptide also plays an important role in determining its immunogenicity. In an exhaustive study on Merino sheep, Anderson et al. (1998) reported
that, whereas immunization against the larger synthetic peptides 1–32, 1–26, 7–26 and 8–30 of porcine inhibin gave a 3–5-fold increase in ovulation rate, the medium sized peptides 10–26 and 13–26 gave a reasonable ovulatory response. It was difficult to obtain a response with the shorter peptides 1–16, 8–22, 13–25, 8–19 and 10–19. The higher efficacy of bI␣(1–29)-Tyr30 than that of peptide bI152 (153–167)Cys168 in increasing the ovulation rate, 43 -Tyr as observed in the present study, may be due to its location on the amino-terminal end of inhibin and its larger size. Despite a dramatic increase in the ovulation rate by immunization against the inhibin-based peptides, reproductive parameters like the number of large unovulated follicles, the interval from PGF treatment to the onset of estrus, duration of estrus, etc. were found to be normal. The peripheral serum progesterone profiles were also found to be normal in the inhibin immunized animals (data not shown). Many reports have provided evidence that ovarian inhibin involves in the negative feedback regulation of follicle-stimulating hormone (FSH) secretion in the ewe (Findlay et al., 1993; Terqui et al., 1995). The increase in the ovulation rate associated with inhibin immunization is accompanied by and is believed to be due to an increase in FSH concentrations in sheep (Wrathall et al., 1990), goat (Sasaki et al., 2006) and cattle (Glencross et al., 1992). The considerable inconsistence found between the antibody titres against different portions of the ␣C-subunit, FSH concentrations and ovulation rate (O’Shea et al., 1991) has led some researchers hypothesizing a local role of ␣C or ␣N inhibin in controlling atresia at the ovarian level, independent of the concentrations of the circulating FSH (Dhar et al., 2001). To conclude, the results of the present study show that active immunization against inhibin-based peptide immunogens is effective in increasing the ovulation rate in Malpura, a non-prolific breed of sheep that otherwise has a very low incidence of twinning and that it may be used as an alternative to conventional superovulation techniques.
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Acknowledgements The authors are grateful to the Director of the institute for providing all the research facilities and to the Dept. of Biotechnology, Govt. of India for providing financial support for this study. The technical support rendered by Mr. S.R Pareek, Mr. Q. Sheikh and Mr. N.L. Gouttam in carrying out this study is also acknowledged. References Arora, C.L., Garg, R.C., 1998. Sheep Production and Breeding. International Book Distribution Company, Lucknow, India. Acharya, R.M., 1982. Sheep and goat breeds of India, Anim. Prod. Health Report (30) 37, FAO, Rome. Akagi, S., Shi, F., Kaneko, H., Watanabe, G., Tsonis, C.G., Taya, K., 2002. Ovarian response and hormonal profiles in heifers after immunization and reimmunization against inhibin ␣-subunit. J. Reprod. Dev. (48), 599–605. Anderson, S.T., Bindon, B.M., Hillard, M.A., O’Shea, T., 1998. Increased ovulation rate in Merino ewes immunized against small synthetic peptide fragments of the inhibin alpha subunit. Reprod. Fertil. Develop. (10), 421–431. Bernanrd, D.J., Chapman, S.C., Woodruff, T.K., 2001. Mechanism of inhibin signal transduction. Recent Prog. Horm. Res. (56), 417–450. Bleach, E.C.L., Muttukrishna, S., Cunnigham, F.J., Knight, P.G., Glencross, R.G., 1996. Effect of inhibin immunization using different synthetic peptide fragments of the bovine ␣c -subunit on plasma anti-inhibin titres, plasma FSh concentrations and the incidence of multiple ovulation in heifers. Anim. Reprod. Sci. (41), 1–12. Burger, H.G., 1992. Inhibin. Reprod. Med. Rev. (1), 1–20. Dhar, A., Doughton, B.W., Pruysers, E., Brown, R.W., Findlay, J.K., 2001. Effect of immunization against the alpha N (␣N) and alpha C (␣C) peptides of the alpha43 subunit of inhibin on antral follicular growth and atresia and the patterns of gonadotropin secretion in ewes. Reproduction (121), 707–718. Findlay, J.K., Russel, D.L., Doughton, B., Tsonis, C.G., Borchers, C., Forage, R.G., 1994. Effect of active immunization against the amino-terminal peptide (␣N ) of the alpha 43 kDa subunit of inhibin (␣43 ) on fertility of ewes. Reprod. Fertil. Develop. (6), 265–267. Findlay, J.K., 1993. An update on the roles of inhibin, activin and follistatin as local regulators of folliculogenesis. Biol. Reprod. (48), 15–23. Findlay, J.K., Doughton, B.W., Tsonis, C.G., Brown, R.W., Hungerford, J.W., Greenwood, P.E., Forage, R.G., 1993. Inhibin as a fecundity vaccine. Anim. Reprod. Sci. (33), 325–343. Funaba, M., Murata, T., Fujimura, H., Murata, E., Abe, M., Takahashi, M., Torii, K., 1996. Unique recognition of activin and inhibin by polyclonal antibodies to inhibin subununits. J. Biochem. (119), 953–960. Glencross, R.G., Bleach, E.C.L., McLeod, B.J., Beard, A.J., Knight, P.G., 1992. Effect of active immunization of heifers against inhibin on plasma FSH concentrations, ovarian follicular development and ovulation rate. J. Endocrinol. (134), 11–18. Groome, N., Hancock, J., Betteridge, A., Lawrence, M., Craven, R., 1990. Monoclonal and polyclonal antibodies reactive with the 1–32 amino terminal sequence of the alpha subunit of human 32 K inhibin. Hybridoma (9), 31–42. Han, L., Mao, D.G., Zhang, D.K., Liang, A.X., Fang, M., Moaeen-Ud-Din, M., Yang, L.G., 2007. Development and evaluation of a novel DNA vaccine expressing inhibin alpha (1–32) fragment for improving the fertility in rats and sheep. Anim. Reprod. Sci. (109), 251–265. Kamboj, M., Prakash, B.S., 1989. Relationship of progesterone in plasma and whole milk in buffaloes during cyclicity and early pregnancy. Trop. Anim. Health Prod. (25), 185–192.
McLeod, B.J., Hunter, M.G., Bleach, E.C.L., Glencross, R.G., Wrathall, J.H.M., 1992. Preovulatory follicle development and luteal function in ewes immunized against a synthetic peptide sequence on the ␣ subunit of bovine inhibin. J. Endocrinol. (133), 413–419. Medan, M.S., Akagi, S., Kaneko, H., Watanabe, G., Tsonis, C.G., Taya, K., 2004. Effects of re-immunization of heifers against inhibin on hormonal profiles and ovulation rate. Reproduction (128), 475–482. Medan, M.S., Watanabe, G., Sasaki, K., Nagura, Y., Sakaime, H., Fujita, M., Sharawy, S., Taya, K., 2003. Ovarian and hormonal response of female goats to active immunization against inhibin. J. Endocrinol. (177), 287–294. Mishra, A.K., 2008. Malpura: a promising breed for enhancing mutton production. In: Karim, S.A., Tripathi, M.K., Chaturvedi, O.H., Shinde, A.K. (Eds.), Small Ruminant Production in India. Satish Serial Publication House, Delhi, India, pp. 295–304. Morris, D.G., McDermott, M.G., Grealy, M., Diskin, M.G., Morrison, C.A., Swift, P.J., Sreenan, J.M., 1995. Effect of immunization against synthetic peptide sequences of the ␣-N -subunit of bovine inhibin on ovulation rate, gonadotropin concentrations and fertility in heifers. J. Reprod. Fertil. (103), 285–291. Naqvi, S.M.K., Gulyani, R., Pareek, S.R., 2000. Effect of superovulatory regimens on ovarian response and embryo production in fine wool sheep in tropics. Asian-Aust. J. Anim. Sci. (13), 595–599. Naqvi, S.M.K., Gulyani, R., 1995. A quick method for embryo transplantation in sheep. Indian J. Small Rumin. (1), 22–24. O’Shea, T., Andrews, C.M., Bindon, B.M., Hllard, M.A., Miyamoto, K., Sinosich, M.J., 1991. Immunisation of Merino ewes with a synthetic peptide or with preparations obtained from bovine and porcine follicular fluids by immunoaffinity chromatography result in different effects on ovulation rate and on plasma gonadotropin concentrations. Reprod. Fertil. Develop. (3), 659–670. Padilla, G., Knight, P.G., Holtz, W., 2007. Superovulation and embryo collection in nulliparous Boer goat does immunized against a recombinant ovine ␣-subunit inhibin. Small Rumin. Res. (74), 159–164. Russell, D.L., Salamonsen, L.A., Findlay, J.K., 1995. Immunization against the N-terminal peptide of the inhibin alpha 43-subunit (alpha N) disrupts tissue remodeling and the increase in matrix metalloproteinase-2 during ovulation. Endocrinology (136), 3657–3664. Saito, S., Roche, P.C., McCormick, D.J., Ryan, R.J., 1989. Synthetic peptide segments of inhibin ␣ and -subunits: preparation and characterization of polyclonal antibodies. Endocrinology (125), 898–905. Sasaki, K., Medan, M.S., Watanabe, G., Sharawy, S., Taya, K., 2006. Immunization of goats against inhibin increased follicular development and ovulation rate. J. Reprod. Dev. (52), 543–550. Schanbacher, B.D., Schemn, S.R., Rhind, S.M., 1991. Gonadotropin concentrations and ovulation rates in Suffolk ewes actively or passively immunized against inhibin alpha. J. Reprod. Fertil. (93), 133–139. Tannetta, D.S., Fray, M.D., Wrathall, J.H., Bleach, E.C., Glencross, R.G., Knight, P.G., 1997. Effects of supplementary treatment with bovine growth hormone on hormonal and ovulatory responses to inhibin immunization in ewes. J. Reprod. Fertil. (110), 255–262. Terqui, M., Wrathall, J.H.M., Driancourt, M.A., Knight, P.G., 1995. Modulation of ovarian function by steroid and inhibin immunization. Livestock Prod. Sci. (42), 181–192. Wrathall, J.H., McLeod, B.J., Glencross, R.G., Knight, P.G., 1992. Effects of active immunization against a synthetic peptide sequence of the inhibin ␣-subunit on plasma gonadotrophin concentration, ovulation rate and lambing rate in ewes. J. Reprod. Fertil. (95), 75–82. Wrathall, J.H., McLeod, B.J., Glencross, R.G., Beard, A.J., Knight, P.G., 1990. Inhibin immunoneutralization by antibodies raised against synthetic peptide sequences of inhibin alpha subunit: effects on gonadotrophin concentrations and ovulation rate in sheep. J. Endocrinol. (124), 167–176. Wheaton, J.E., Karlson, K.M., Kusina, N.T., 1992. Active and passive immunoneutralization of inhibin increases follicle-stimulating hormone levels and ovulation rate in ewes. Biol. Reprod. (47), 361–367.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Technical note
Active immunization against inhibin-based peptides to increase ovulation rate in non-prolific Malpura ewes S.M.K. Naqvi a,∗ , A. Joshi a , R. Gulyani a , V.P. Maurya a , R.S. Manik b , P. Palta b a b
Central Sheep and Wool Research Institute (CSWRI), Avikangar 304501, India National Dairy Research Institute (NDRI), Karnal 132001, India
a r t i c l e
i n f o
Article history: Received 26 August 2008 Received in revised form 11 December 2008 Accepted 11 December 2008 Available online 29 January 2009 Keywords: Inhibin Immunization Ovulation rate Sheep
a b s t r a c t The aim of this study was to explore the possibility of increasing the ovulation rate of Malpura, a non-prolific tropical breed of sheep by immunization against inhibin-based peptide immunogens. Ewes were divided into three groups (n = 5 each) and actively immunized against the synthetic peptides from the ␣C [bI␣(1–29)-Tyr30 ] or ␣N [bI-43 Tyr152 (153–167)Cys168 ] area of the bovine inhibin ␣-subunit conjugated to ovalbumin or against ovalbumin (control). Each ewe received a primary immunization of 400 g immunogen and 3 boosters, each of 200 g immunogen at 4-week intervals. Estrus was synchronized using a double PGF2 ␣ injection schedule and laparoscopy was performed after each estrus to determine the ovulation response. Immunization against both the peptides did not affect the interval from PGF treatment to the onset of estrus, the duration of estrus and the number of large unovulated follicles. In contrast to the complete absence of multiple ovulations in the controls, all the ewes immunized against ␣C or ␣N peptides showed multiple ovulations (range 2–7) in all the three estrous cycles evaluated, except for one ewe immunized against the ␣N peptide, which exhibited multiple ovulations in only 1 out of the 3 estrous cycles. Compared to that of the controls (1.0 ± 0.9, 1.0 ± 0.0 and 0.6 ± 0.2, respectively), the mean ovulation rate was higher (P < 0.01) in the ewes immunized against the ␣C (4.8 ± 1.02, 5.0 ± 1.05 and 5.0 ± 0.45, respectively) or against ␣N (4.5 ± 1.19, 2.5 ± 0.87 and 2.7 ± 0.75, respectively, P < 0.05) peptide in estrous cycles numbers 1, 2 and 3. These results show that active immunization against inhibin-based peptide immunogens is effective in increasing ovulation rate in Malpura, a non-prolific breed of sheep and that it may be an alternative to conventional superovulation regimes. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The majority of native Indian sheep populations are found in the arid and semi-arid regions and contribute significantly to meat and wool production, despite prevailing harsh conditions like high ambient temperatures, scarcity of feed and water. However, the reproductive efficiency of these sheep breeds is relatively low (Arora and Garg, 1998). The Malpura sheep are native to a semi-arid tropical region
∗ Corresponding author. Tel.: +91 1437 220165; fax: +91 1437 220163. E-mail address: [email protected] (S.M.K. Naqvi). 0921-4488/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2008.12.010
of India and are known for their superior quality mutton and carpet-type wool (Acharya, 1982). It is a monotoccus breed with an ovulation rate of 1 and a very low incidence of twinning (<5%). The average litter size at birth has been reported to be 1.05 for the Malpura sheep (Mishra, 2008). Thus, there is a need to improve the reproductive potential for enhancing the mutton and wool production of the Malpura sheep. Inhibin, a glycoprotein hormone produced by the granulosa cells in the ovarian follicles suppresses the production and secretion of pituitary gonadotropins, preferentially FSH through negative feedback mechanism (Burger, 1992). It is also involved in the regulation of folliculogenesis through
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autocrine and/or paracrine control (Findlay, 1993). Active immunization against inhibin, either in its native form or in the form of inhibin-based synthetic peptides, has been shown to increase the ovulation rate in cattle (Akagi et al., 2002; Medan et al., 2004), goats and sheep (Medan et al., 2003; Padilla et al., 2007). However, in all the studies in sheep, the animals have been of temperate breeds with high fecundity, viz. the Romney (Wrathall et al., 1990; McLeod et al., 1992), Suffolk (Schanbacher et al., 1991; Wheaton et al., 1992), Merino (Anderson et al., 1998) or crossbreds (Wrathall et al., 1992; Tannetta et al., 1997). Lately, there has been an increasing interest in the development of inhibinbased fecundity vaccines (Han et al., 2007; Padilla et al., 2007). There is however, no report available on the effects of immunization against inhibin in any tropical or non-prolific breeds of sheep. The objective of the present study was thus to explore the possibility of increasing the ovulation rate in the Malpura, a tropical breed of sheep with low fecundity, by active immunization against synthetic peptides based on the sequence of the ␣-subunit of bovine inhibin. 2. Materials and methods 2.1. Animals and their management Fifteen non-pregnant adult ewes of Malpura breed, 4–7 years of age and weighing 32.1 ± 0.53 kg were randomly allocated in equal numbers (n = 5) to three treatment groups. All the animals were managed under the standard farm practices at the CSWRI’s sheep farm at Avikanagar, which is located at a longitude of 75◦ 28 E, latitude of 26◦ 26 N and an altitude of 320 m above mean sea level in a semi-arid region of India. The climate in this region is essentially tropical with the highest temperatures occurring from April to June with a mean monthly temperature of 42 ◦ C. The minimum and maximum ambient temperatures range from 7 to 37 ◦ C and from 25 to 46 ◦ C, respectively—while the mean relative humidity varies between 15% and 90%. The rainfall is erratic and mainly concentrated during July to August with a precipitation of 200–500 mm per annum. 2.2. Immunization protocol Synthetic peptides mimicking the amino acid sequences of fragments of the bovine inhibin ␣-subunit were commercially prepared by Sigma–Genosys, England. The peptides were conjugated to ovalbumin, with the peptide-to-ovalbumin molar ratio being approximately 20:1 to increase their antigenicity. The synthetic peptides included a 30-mer peptide based on the ␣C [bI␣(1–29)-Tyr30 ] and a 17-mer peptide based on the ␣N region [bI-43 -Tyr152 (153–167)Cys168 ] of the ␣-subunit of bovine inhibin. The Malpura ewes of Group I and II were immunized against the 30-mer and the 17-mer peptide, respectively, whereas those of the control group were immunized against ovalbumin. Primary immunization involved the injection of 400 g of peptide–ovalbumin conjugate (Groups I and II) or ovalbumin (control) dissolved in 1 ml isotonic saline and emulsified with an equal volume of Freund’s complete adjuvant. Each animal was given 3 boosters, each of 200 g peptide–ovalbumin conjugate or ovalbumin dissolved in 1 ml isotonic saline and emulsified with an equal volume of Freund’s incomplete adjuvant, at 4-week intervals during the natural breeding season (August to October). 2.3. Estrus synchronization Estrus was synchronized using a double injection schedule of PGF2␣ (Lutalyse, Pharmacia N.V./S.A., Puurs-Belgium, 7.5 g per treatment) on Days 35 and 45 of the trial (Day 0 = Day of primary immunization). Subsequently, the animals were allowed to undergo normal cyclicity until the end of trial. Estrus was detected by using an aproned ram per group (2–4 years of age) of high sexual vigour at 6 h intervals. Blood samples were collected at 08:00 twice weekly from the onset to the end of the trial for determination of plasma progesterone concentrations for confirmation of cyclicity (Kamboj and Prakash, 1989). The intra- and inter-assay co-efficients of variation were both less than 13%.
2.4. Ovarian examination for ovulation rate For evaluating the ovarian response to immunization, the ovaries were examined by laparoscopy 3–6 days after each estrus (Naqvi and Gulyani, 1995; Naqvi et al., 2000). In brief, after fasting the animals for at least 24 h prior to laparoscopy, all ewes were sedated with xylazine hydrochloride and locally anaesthetized with lidocaine hydrochloride. After visualization of the reproductive tract through the laparoscope, the ovary was grasped with an atraumatic forceps and the number of corpora lutea (CL) and large follicles (LF) recorded. 2.5. Data analysis The data were expressed as the mean ± S.E.M. Data on estrus response, ovulation rate and the numbers of large follicles were compared between different groups by analyses of variance (ANOVA), using the SYSTAT version 7.0 (SPSS Inc., USA, 1997).
3. Results Immunization against the peptides bI␣(1–29)-Tyr30 and bI-43 -Tyr152 (153–167)Cys168 did not have any adverse effect on the interval from PGF to the onset of estrus and the duration of estrus (Table 1). Also, the number of large unovulated follicles recorded was not significantly different in the ewes immunized against the inhibin-based peptides, compared to those in the control group in the estrous cycle numbers 2 and 3 (Table 2). The incidence of multiple ovulations increased significantly by active immunization against inhibin. In contrast to the complete absence of multiple ovulations in the control group, all the ewes immunized against bI␣(1–29)Tyr30 showed multiple ovulations in all the three estrous cycles examined in the study – with the ovulation rates ranging from 2 to 7. In the ewes immunized against bI152 (153–167)Cys168 , except for ewe No. 4, which 43 -Tyr exhibited multiple ovulations in only one estrous cycle, while all the other ewes had multiple ovulations – ranging between 2 and 6 in all the 3 estrous cycles examined. In the ewes immunized against bI␣(1–29)-Tyr30 , the mean ovulation rate was nearly 5-fold higher (P < 0.01) than that for the controls in all the three estrous cycles examined (Table 2). In the ewes immunized against bI-43 -Tyr152 (153–167)Cys168 also, the ovulation rate was significantly higher (P < 0.05) than that for the controls in estrous cycles 1 and 3. 4. Discussion To current knowledge, this is the first report on the effects of immunization against inhibin in any non-prolific breed of sheep. It has shown that active immunization against peptides (based on the ␣-subunit of inhibin), can be used for inducing superovulation in the Malpura, a tropical breed of sheep, that otherwise has an ovulation rate of approximately 1 and a very low incidence of twinning. Inhibin is a heterodimer composed of an ␣- and a -subunit. During the post-translational processing, the prepro-␣-subunit of inhibin gives rise to three peptides: the carboxy-terminal peptide ␣C , which forms the 32 kDa form of inhibin after combining with the -subunit; the amino-terminal peptide ␣N , which gives rise to the 57 kDa form of inhibin after combining with the ␣C and -subunit; a pro-␣C peptide (Bernanrd et al., 2001). Active immuniza-
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Table 1 Estrus response of Malpura ewes immunized against inhibin-based peptides. Treatment
Control (ovalbumin) Group I [bI␣(1–29)-Tyr30 ] Group II [bI-43 -Tyr152 (153–167)Cys168 ] a
Number of ewes treated (n)
5 5 4a
Mean ± S.E. Interval from PGF treatment to onset of estrus (h)
Duration of estrus (h)
49.2 ± 2.94 48.0 ± 3.46 40.0 ± 5.29
28.8 ± 3.50 38.0 ± 2.00 48.0 ± 3.46
One animal (M-387) in Group II diagnosed as pregnant and hence omitted.
Table 2 Ovarian response in Malpura ewes immunized against inhibin peptides. Treatment
Estrous cycle number 1
Control (ovalbumin) Group I [bI␣(1–29)-Tyr30 ] Group II [bI-43-Tyr152 (153–167)Cys168 ]
2
3
Ovulations
Large follicles
Ovulations
Large follicles
Ovulations
Large follicles
1.00 ± 0.0 4.8 ± 1.02* 4.5 ± 1.19*
0.0 ± 0.0 0.2 ± 0.20 0.7 ± 0.25*
1.0 ± 0.0 5.0 ± 1.05** 2.5 ± 0.87
0.4 ± 0.24 0.8 ± 0.37 0.0 ± 0.00
0.6 ± 0.24 5.0 ± 0.45** 2.7 ± 0.75*
0.6 ± 0.24 0.2 ± 0.2 0.5 ± 0.5
Means ± S.E. within a column are significantly different from controls. * P < 0.05. ** P < 0.01.
tion against the 30-mer peptide bI␣(1–29)-Tyr30 , which is from the ␣C region was found to induce multiple ovulations ranging from 2 to 7 and to increase the ovulation rate almost 5-fold, compared to the controls. These results are in agreement with earlier studies in which immunization against this peptide was found to increase the ovulation rate in many prolific breeds of sheep like e.g. the Romney (Wrathall et al., 1990; McLeod et al., 1992) and Blue-faced Leicester × Swadale crosses (Wrathall et al., 1992; Tannetta et al., 1997). Also, among the many inhibinbased peptides compared, this specific peptide had been reported to be the most effective in increasing ovulation rate in cattle (Bleach et al., 1996). The 17-mer peptide bI152 (153–167)Cys168 , which is from the ␣ region of N 43 -Tyr bovine inhibin, was selected in view of its reported efficacy in increasing the ovulation rate in cattle (Morris et al., 1995). Active immunization against this peptide resulted in over a 2.5-fold increase in ovulation rate in the present study. Interestingly, immunization of sheep against recombinant fusion protein immunogens containing the full length of ␣N sequences has been found to lower fertility by suppressing ovulation (Findlay et al., 1994; Russell et al., 1995). Between the two peptides, the ␣C 30-mer peptide bI␣(1–29)-Tyr30 was found to be more effective in increasing the ovulation rate than the ␣N 17-mer peptide bI-43 -Tyr152 (153–167)Cys168 . The efficacy of different peptide immunogens has been reported to vary depending upon their location within the inhibin molecule. Peptides derived from the amino-terminal end of the ␣-subunit are more immunogenic than those from other regions of the ␣-subunit (Saito et al., 1989), as amino acids 13–26 contain important antigenic sites (Groome et al., 1990; Funaba et al., 1996). Addition of residues 7–12 has been reported to be necessary for further increasing the immunogenicity and ovulatory response to immunization (Anderson et al., 1998). The size of the peptide also plays an important role in determining its immunogenicity. In an exhaustive study on Merino sheep, Anderson et al. (1998) reported
that, whereas immunization against the larger synthetic peptides 1–32, 1–26, 7–26 and 8–30 of porcine inhibin gave a 3–5-fold increase in ovulation rate, the medium sized peptides 10–26 and 13–26 gave a reasonable ovulatory response. It was difficult to obtain a response with the shorter peptides 1–16, 8–22, 13–25, 8–19 and 10–19. The higher efficacy of bI␣(1–29)-Tyr30 than that of peptide bI152 (153–167)Cys168 in increasing the ovulation rate, 43 -Tyr as observed in the present study, may be due to its location on the amino-terminal end of inhibin and its larger size. Despite a dramatic increase in the ovulation rate by immunization against the inhibin-based peptides, reproductive parameters like the number of large unovulated follicles, the interval from PGF treatment to the onset of estrus, duration of estrus, etc. were found to be normal. The peripheral serum progesterone profiles were also found to be normal in the inhibin immunized animals (data not shown). Many reports have provided evidence that ovarian inhibin involves in the negative feedback regulation of follicle-stimulating hormone (FSH) secretion in the ewe (Findlay et al., 1993; Terqui et al., 1995). The increase in the ovulation rate associated with inhibin immunization is accompanied by and is believed to be due to an increase in FSH concentrations in sheep (Wrathall et al., 1990), goat (Sasaki et al., 2006) and cattle (Glencross et al., 1992). The considerable inconsistence found between the antibody titres against different portions of the ␣C-subunit, FSH concentrations and ovulation rate (O’Shea et al., 1991) has led some researchers hypothesizing a local role of ␣C or ␣N inhibin in controlling atresia at the ovarian level, independent of the concentrations of the circulating FSH (Dhar et al., 2001). To conclude, the results of the present study show that active immunization against inhibin-based peptide immunogens is effective in increasing the ovulation rate in Malpura, a non-prolific breed of sheep that otherwise has a very low incidence of twinning and that it may be used as an alternative to conventional superovulation techniques.
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