The active immunization of sheep against oestrone, andostenedione or testosterone to increase twinning.

Abstract

Animal Production in Australia THE ACTIVE IMMUNIZATION OF SHEEP AGAINST OESTRONE, ANDROSTENEDIONE OR TESTOSTERONE TO INCREASE TWINNING R.I. COX*, PATRICIA A. WILSON*, R.J. SCARAMUZZI*, R.M. HOSKINSON*, J.M. GEORGE** and B.M. BINDON**. SUMMARY Procedures have been developed to produce consistent increases in ovulation rate in Merino ewes by immunization against the steroids, androstenedione, testosterone and oestrone. The procedures were based on experiments in the field over the period 1978-1981 using groups of 50 ewes for each treatment. The method uses selected steroid-human serum albumin conjugates, together with DEAEdextran adjuvant, administered subcutaneously in two treatments, three weeks apart. Joining with rams then commences three to five weeks later. With Merino ewes, increases in ovulation rate of 0.2 to 0.4 can be obtained whilst maintaining full oestrus. This results in increases in lambing rate of about 20%, essentially confined to twins. INTRODUCTION Most sheep in Australia, predominantly Merinos, have low ovulation rates (0-R-s) and, hence, low lambing rates. Often this is considered desirable for animals maintained under extensive grazing conditions. However, for many producers it would be a useful management option to be able to increase fecundity in adequately nourished sheep, and cattle, by a simple treatment. Potentially, such a procedure is immunization against specific steroids, an example of new developments in immunophysiology. Earlier work reported that increases in 0-R. of sheep followed immunization against steroid oestrogens or androgens (Cox et al. 1976; Scaramuzzi et al. 1976). A later study with larger numbers of sheep confirmed increases in O.R. and also in anoestrus, the latter caused by too high levels of circulating antiThus, to take practical body following immunization (Martin et al. 1979). advantage of the enhanced 0-R. the elimination of associated anoestrous behaviour was of critical importance. In order to obtain antibodies to steroids, suitable steroid derivatives need to be covalently linked to an immunogenic protein, and these conjugates injected into sheep with an adjuvant. Although Freunds complete adjuvant is DEAE-dextran suitable for experimental work, it tends to produce local lesions. is a possible alternative adjuvant, producing little local reaction and giving moderate antibody responses to steroid-protein conjugates (Cox and Wilson 1976). From the results of these earlier studies it seemed useful to attempt to develop more practical procedures for the immunization of :sheep against steroids, and to test these procedures with larger groups of animals in the field; careful control of antibody response by selection of immunogens and adjuvants needed to The aims of this study were to develop more effective and simpler be explored. immunization treatments and to carry out observations over several years to assess the reproducibility of the increases in 0-R. MATERIALS AND METHODS Merino ewes (two-three years old at first treatment) were first immunized with 1.1-1.5 mg steroid-protein conjugates, together with either Freunds complete *CSIRO, Division of Animal Production, at P-0. Box 239, Blacktown, N.S.W., 2148, and at **Private Bag, Armidale, N.S.W., 2350. 511 Animal Production in Australia adjuvant in two treatments four weeks apart, or with DEAE-dextran adjuvant in two treatments two-four weeks apart. The 'Standard' immunization treatment was about 0.9 ml injected at two intramuscular sites in the hind legs and 0.2 ml at each of six subcutaneous sites across the back (total dose 3.0 ml). The 'simplified' treatment used in 1981 was 1.5 ml at each of two subcutaneous sites in the neck. Immunization was in February-March with a five-six week joining in April-May. Immunogens used were oestrone-6-carboxymethyloximino:human serum albumin (E.6CMO: HSA); oestrone-3-carboxymethylether:HSA (E.3CM:HSA); androstenedione-7-carboxyethylthioether:HSA (A.7:HSA) and testosterone-3-carboxymethyloximino:HSA (T.3CMO: These were synthesized with steroid hapten contents in the range of 17-24 HSA). moles steroid/mole protein. Immunogens were pre-tested in six to eight sheep to determine the antibody titre response. Selected immunogens were used in the Antibody titre was determined by measuring the dilution of larger trial groups. serum or plasma which bound 50% of the maximal specific binding of 3H-labelled steroid at 4OC under standardized conditions. RESULTS In the first field experiment in 1978, groups of 50 Merino ewes were immunized with the conjugates and adjuvants shown in Table 1. This experiment was designed to define, more precisely, the range of acceptable antibody titres required to produce significant increases in 0-R. and to determine the response level at which anoestrus became evident. TABLE 1 Reproductive performance in immunized Merino ewes in April-May 1978 *P < 0.05; **p < 0.01, for ovulation rates and lambs born. Substantial increases in 0-R. were obtained in the androstenedione and oestrone immunized groups. However, a considerable degree of anoestrus was evident, particularly in the androstenedione group, indicating that antibody responses were too high. To produce lower antibody responses, steroid protein conjugates of lower steroid hapten content and, hence, lower immunogenicity were prepared for the next trials. Also, the use of the milder and preferred adjuvant Optimum conditions for the use of DEAEDEAE-dextran, was clearly possible. dextran with steroid-protein conjugates were determined. Two injection treatments were found to be best spaced three weeks apart. Antibody titres were maximal about one week after the second treatment and thereafter slowly declined until they were very low by lambing. In the next trial in 1979, with new groups of ewes, DEAE-dextran solely was used as an adjuvant. Also, the oestrone derivative was changed to the E.3CM compound since this is more readily synthesised chemically than the E.GCMO compound. The 0-R-s following androstenedione immunizations were satisfactory, but the responses with oestrone immunizations were weaker than desired (Table 2). The incidence of oestrus was maintained. These groups were retreated in 1980 and 1981. The differences between the groups in percent lambs born was maintained over the three years of treatment. 1980 was a drought year, and under the grossly adverse nutritional conditions, no increase in 0-R. was noted that year. The general experience with this technique has been that the response obtained in the initial year sets the response for subsequent years due to 512 Animal Production in Australia TABLE 2 Reproductive performance in immunized Merino ewes, joining April-May, each season, 1979 to 1981 *kp < 0.05; **p < 0.01, assessed for flock ovulation rate and performance. 'immunological memory'. From this and subsequent tests, a satisfactory ovulatory response without anoestrus was obtained when antibody titres for the steroids were between 1 in 500 and 1 in 3,000 at the start of joining. In 1981, fresh ewes were immunized with pre-tested steroid conjugates that were considered optimal. Included was a group of ewes immunized against testosterone, since such immunization had increased 0.R.s in smaller trials. In this 1981 experiment, the simplified injection protocol was used for half of each group. Ovulatory and antibody titre responses were not significantly different between the stardard and simplified immunization protocols. Good increases in 0-R. at the conception cycle occurred in all groups and full oestrus was maintained (Table 3). Testosterone immunization produced good results. There were TABLE 3 Reproductive performance in Merino ewes joined in April-May 1981 *kp < 0.05; **p < 0.01, assessed for flock ovulation rate and performance. 513 Animal Production in Australia gains at lambing, although in the androstenedione group the difference between O.R. and lambs born/ewe lambing suggests a higher reproductive wastage in this group. Again, there were no triplets born. DISCUSSION The tests demonstrate that immunization of sheep against androstenedione, oestrone and testosterone can be effective in producing increases in 0-R. without loss of oestrus, under field conditions. The antibodies produced by the immunization are known to bind the appropriate circulating endogenous steroid and render part or all the steroid biologically unavailable. Such an immunophysiological action is thought to alter the hormonal feed-back systems influencing ovarian function. Hypotheses for the possible mechanisms of action involved have been considered elsewhere (Scaramuzzi, 1979). DEAE-dextran has proved an effective adjuvant. The current simplified injection procedure is straightforward and is likely that it may be simplified further. The work described in this paper was with Merino sheep in New South Wales. Good results have also been obtained using similar immunogens and procedures with Merinos in Western Australia (Croker, Cox, Johnson and Wilson, unpublished observations), and with Dorset Horn ewes in New South Wales (Scaramuzzi et al. 1982), and with Romney, Coopworth and Perendale ewes in New Zealand (Smith et al. 1981; Smith and Cox 1981). From these experiments on some 544 Merino ewes between 1978-1981, there has evolved an immunization procedure that regularly gives increases in ovulation rate and lambing rate. The experimental protocol now being used is close to one which could be acceptable under farming conditions. Although more work remains to be done to establish fully the applicability of the techniques under practical conditions, we believe the immunization procedure is ready for initial on-farm trials. REFERENCES J. Reprod. Fert. 22: 524. COX, R.I., and WILSON, P.A. (1976). Theriogenology, 6: 607. COX, R-I., WILSON, P-A., and MATTNER, P.E. (1976). MARTIN, G.B., SCARAMUZZI, R.J., COX, R.I., and GHERARDI, P.B. (1979).= Aust. J. Expt. Agric. An. Husb. &4: 673. SCARAMUZZI, R.J. (1976). In 'Physiological Effects of Immunity against Reproductive Hormones', p. 67, editors R.G- Edwards and M.H. Johnson. (Cambridge Univ. Press, Cambridge, England). SCARAMUZZI, R.J. (1979). J. Steroid Biochem. A&: 957. SCARAMUZZI, R.J., COX, R-I., and HOSKINSON, R.M. (1982). Proceedings of World Congress on Sheep and Beef Cattle Breeding, 2 (in press). Proceedings, Ruakura Farmers Conference, SMITH, J.F., and COX, R.I. (1981). 22: 19. SMITH, J-F., COX, R-I., MCGOWAN, L.T., WILSON, P., and HOSKINSON, R.M. (1981). Proc. N.Z. Soc. Anim. Prod. 41: 193. == 514