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Effect of immunizing prepuberal lambs of low and high ovulation rate genotypes with inhibin partially purified from bovine follicular fluid
THERIOGENOLOGY
EFFECT OF IMMUNIZING PREPUBERAL LAMBS OF LQW AND HIGH OWLATION RATE GENOTYPES WITH INHIBIN PARTIALLY PURIFIED FROM BOVINE FOLLICULAR FLUID D.G. Morris, M.G. McDermott and J.M. Sreenan Animal Reproduction Department, Agriculture and Food Development Authority, Belclare, Tuam, Co. Galway, Ireland Received for publication: July 6, 1990 Accepted: November 14, 1990 ABSTRACT Active immunization of prepuberal lambs with a partially purified inhibin preparation, isolated from bovine follicular fluid, increased the ovulation rate. In ewe lambs of a low fecundity breed (Suffolk x Galway), the ovulation rate rose from 1.15 to 1.95 (PcO.05) compared with that of the controls. An ovulation rate of 3.38 was recorded for immunized ewe lambs of a high fecundity breed (Finn x Dorset Horn), while the rate for mature ewes from the same flock was 2.29. Immunization did not affect the time of onset of puberty or estrous cycle length. Following immunization, antibodies were produced that bound to a pure preparation of 68kDa bovine inhibin. This report demonstrates the production of antibody to a 68kDa preparation of inhibin following active immunization of sheep using a partially purified preparation. It was concluded that the increased ovulation rate was due to the production of antibodies to inhibin, which may have reduced its negative feedback effect on FSH secretion. Key words:
immunization, inhibin, ovulation rate, prepuberal lambs INTRODUCTION
The synthesis and secretion of gonadotrophins are influenced by the negative feedback effect of ovarian steroids and inhibin on the hypothalamic-pituitary axis. Altering this feedback has resulted in an increased ovulation rate in sheep. For example, immunization of sheep Acknowledgments The authors thank Dr. J.P. Hanrahan, Belclare, for assistance with the statistical analysis: Mr. G. Morris for technical support: and Mr. G. Burke for care of the animals. The work was supported in part by Bioresearch Ireland, Farm Business Development, Ltd., Ireland and by the Commission of the European Communities.
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against a range of steroid hormones has increased ovulation rate (1,2) and litter size (3,4). Similarly, the active immunization of sheep against partially purified bovine follicular fluid preparations enriched in inhibin has also increased the ovulation rate (5,6). While an immune response to the partially purified follicular fluid preparations was demonstrated by immunodiffusion in agar in these latter studies the production of antibody specific to inhibin was not demonstrated. The purpose of our study in Experiment 1 was to determine whether active immunization with a partially purified inhibin preparation derived from bovine follicular fluid would elicit an immune response to inhibin in prepubertal lambs. Experiment 2 was carried out to determine the possible effects of such immunization on the onset of puberty and ovulation rate in low and high fecund breeds. MATERIALS AND METHODS Collection
of Follicular
Fluid
Cow ovaries were obtained at a local abattoir and were held in ice-cold 0.9% (w/v) saline until arrival at the laboratory. Bovine follicular fluid (bFF) was aspirated from follicles of 5 to 20 mm in diameter using a syringe and 20-gauge needle and was stored at -2OOC until required. Steroid Removal Frozen aliguots of follicular fluid were thawed, pooled and centrifuged at 1000 g for 30 min at 4OC to remove cell debris. The supernatant was then decanted and treated with dextran-coated charcoal (1:lO) at a concentration of 1 mg/ml, by stirring at 4'C overnight. The charcoal was then removed by centrifugation at 1000 g for 30 min, followed by pressure filtration through Whatman No.1 filter paper. Steroid removal was monitored by measuring the progesterone concentration in the follicular fluid before and after the charcoal treatment (7) using a previously described and validated enzymeimmunoassay (8) and with reagents supplied by Noctech Ltd., Galway, Ireland. The sensitivity of the assay was 0.30 ng/ml with an ED50 of 1.33 ng/ml. The within assay coefficients of variation were l.l%, 2.6% and 6.4% for samples having mean progesterone concentrations of 1.44, 4.76 and 9.88 ng/ml, respectively. Charcoal treatment was effective in reducing the mean progesterone concentration of a pool of follicular fluid by more than 95%, from 240 to 10 ng/ml. Inhibin Purification A partially purified inhibin was isolated from the charcoal treated bFF by sequential chromatography on Red
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Sepharosea followed by Lentil Lectin Sepharosea as described previously (9). The partially purified inhibin was lyophilized and stored at -2OOC until required for immunization. An essentially homogenous inhibin was then obtained by sequential chromatography on affinity and hydrophobic matrices (10) followed by ion exchange chromatography on a Mono Qa column at pH 9.2 (11). The biological activity of all the inhibin preparations in this study was measured using an in vitro bioassay model, based upon the inhibition of FSH secretion by rat pituitary cells, as described previously (12). Potency estimates were calculated using the computer program ALLFIT (13), while assay statistics were calculated for parallel line bioassays (14). The dose response curves (n = 3) had a precision index 0,) of 0.142+0.026 (MkSEM), while the dose of charcoal-treated follicular fluid required to half maximally inhibit FSH secretion was 0.83kO.03 pg/well. Using this bioassay, the partially purified inhibin was purified 31- to 49-fold compared with that in the original follicular fluid. The homogenous inhibin was purified 79fold following chromatography on affinity and hydrophobic matrices. Following chromatography on Mono Q, the homogenous inhibin was assayed in the bioassay at one dose level only, where it reduced (PcO.05) basal FSH secretion with no effect (P>O.O5) on LH secretion (15). Electrophoresis of the homogenous inhibin was carried out on prgcast 4 to 30% (w/v) gradient polyacrylamide gels (PAA under nonreducing conditions. Silver 4/30) using SDS-P%E staining (Gel Code) of the gel revealed the presence of one protein which migrated, with an apparent molecular mass of 68kDa (15). Immunization
of Prepuberal
Lambs
The partially purified inhibin (2.5 mg/lamb) was dissolved in 5 ml of saline, emulsified in an equal quantity of Freund's complete adjuvant (FCA) (16) and then injected subcutaneously into four sites in the axillary region. Booster injections (1.0 mg) similarly prepared but emulsified in Freund's incomplete adjuvant (FIA) were injected at the time intervals outlined below. Antigen
Binding Studies
The homogenous 68kDa inhibin was iodinated using the iodogen procedure (17). Two micrograms of inhibin in 50 ~1 of 0.05 M phosphate buffer (PB; pH 7.4) were added to a 1.5-ml Eppendorf microvial precoated with 20 ps iodogen. b Five microlitres of 125-I (IMS 30, 100 mCi/ml) were then added and the reaction was allowed to proceed with E Pharmacia Biotechnology, Pierce, Cambridge, U.K. c Amersham, Bucks, U.K.
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agitation every minute for 10 min. The reaction was stopped by adding 200 ~1 of PB to the microvial and then removing the contents by pipetting. The free iodine was separated from the bound by gel filtration on a PD-10 column which had been pre-equilibrated with 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). All antigenbinding studies were carried out using one iodinated preparation which had a specific activity of 13.6 pCi/pg (15). The antigen binding capacity of serum from two of the immunized lambs was determined by incubating 10 ~1 of immune serum in 100 ~1 of 10% normal serum with 100 ~1 (30,000 cpm) of radioiodinated inhibin (37500 dpm, approx 18 fmole) for 24 h, at room temperature in a total volume of 300 ~1 in 0.05 M of PBS. The bound activity was separated from the free (18) by precipitating pith two volumes of 20% polyethylene glycol (PEG 4000). This was followed by centrifugation at 4000 g for 30 min, by further washing of the precipitate with 16% PEG in PBS and by centrifugation again. The precipitates were then counted in a Beckman 5500 automatic gamma counter for 1 min. Antigen binding capacity was expressed as the percentage of the total counts added per tube bound by immune serum after correcting for non-specific binding by pre-immune serum. Ovulation Rate and Estrous Cycle Length Measurements Following the first booster injection, vasectomized rams fitted with harnesses and crayons were used to check for standing estrus to establish the time of onset of puberty and estrous cycle length. Ovulation rate was determined at mid-cycle by direct laparoscopic observatign using a Wolf 180° direct vision 495x5.5-mm bronchoFcope and a Downs fiber light source (Mk.ii projector). Experiment
1
Because of the limited amount of material available for immunization at this time, only two lambs, born in August, were immunized with the partially purified inhibin at 22 wk of age, followed by booster injections at 27, 33, 48 and 57 wk of age. Blood samples for antibody measurement were
taken immediately before (pre-immune) and at regular intervals after immunization (immune) by jugular venipuncture into lo-ml tubes without additive (Vacutainer).g The blood samples were allowed to clot for 1 h at 37OC and then centrifuged at 1000 g and 4OC to separate the serum, which was then stored at -2OOC until assay. The ovulation rate was also measured on the two lambs over three cycles following the onset of puberty. u BDH Chemical Co. Poole, Dorset, U.K. F Wolf GmBH, W. Germany Down Bros and Mayer L Phelps Ltd., U.K. g Becton and Dickinson, U.K.)
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THERIOGENOLOGY Experiment
2
Suffolk x Galway ewe lambs born in April were randomly allotted to either an immunization (n = 9) or control (n = 9) group at 19 wk of age. Booster injections were given at 25, 35 and 40 wk of age. Finn x Dorset Horn ewe lambs born in April were similarly immunized (n=4) at 22 wk of age, with booster injections at 28, 38 and 43 wk of age. While controls were not available for the Finn x Dorset Horn ewe lambs, ovulation data recorded over two cycles in mature ewes (n = 12), from the same flock, was used for comparison. Data on the onset of puberty and estrous cycle length, reported by Quirke (19), was used for the comparison of these traits. Statistical
Analysis
The ovulation rate and estrous cycle measurements between groups were compared by analysis of variance. Statistical significance of the ovulation rate was tested following log transformation. In the model used, ewes were treated as a random effect nested within treatments, and time was fitted as a fixed effect. The mean square for ewes was used to test the significance of the treatment difference. Antigen binding was analyzed by Wilcoxon rank sum test (20). RESULTS Experiment
1
The antigen binding capacity of the serum from immunized lambs increased (PCO.002) in comparison with the preimmunization serum level (Figure 1). Radioactive binding by pre-immune sera was less than 0.5%. The number of ovulations measured for each of the first three cycles of the season starting approximately 1 wk after the final boost, was one, seven and four for one of the lambs and one, two and six for the other. Experiment
2
One immunized ewe lamb in the Suffolk x Galway group was injured and was excluded from the experiment. There was no effect of immunization on the age at puberty or on estrous cycle length in either breed (Table 1). While direct controls were not available, published data (19) suggests that immunization did not affect these traits in the Finn x Dorset Horn ewe lambs. Immunization increased the ovulation rate in the Suffolk cross lambs by 70% (PcO.05; Table 2). There was no time x treatment interaction on ovulation rate (PBO.44). The ovulation rate in the immunized Finn x Dorset Horn ewe lambs was 3.38kO.76. This was 47% higher than the ovulation rate (2.2920.16) recorded over two cycles for mature ewes (n = 12) from the same flock measured over the same time period (Table 3).
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I- 35 -
\
m 30 ; 25 z 20 m
20
28
36
44 AGE (weeks)
52
60
Figure l.Percentage of binding of radioiodinated 68kDa bovine inhibin by serum from ewe lambs immunized with a partially purified preparation of bovine follicular fluid. Arrows indicate time of immunization.
Table l.Effect of immunization of prepuberal lambs on the onset of puberty and estrous cycle length (M+SEM) Breed type
Status
n
Onset of puberty Datea
Age(days)
Cycle
No. of estrous (days) cycles
Suffolk x Galway
Immunized Control
a 9
344+10 334+07
224+10 214+07
15.3kO.4 15.5kO.3
Finn x Dorset Born
Immunized 4 Control 89
327206 321+03
235-+05 252+03
15.8kO.5 21 17.820.3 109 b
E Date is referenced to January Data from Quirke (19).
33 44
1 = Day 1.
There was no effect of immunization on the number of Suffolk x Galway lambs showing estrus or on the number ovulating (Table 2). However, one inhibin-immunized lamb failed to ovulate despite showing estrous activity on two occasions. A frequency distribution for ovulation rate is shown in Table 4 for all breeds.
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THERIOGENOLOGY Table 2. The number of corpora lutea (CL) of control and inhibin immunized Suffolk x Galway ewe lambs at different ages and the arithmetic mean+SEM No. of CL at different Group
n
Control
9
Inhibin 8 immunized
ages
(weeks) 38
Ovulation rate
28
30
31
32
33
42
(9,
A
(Z)
(:)
(66) K)
(9,
A
A
A
(:)
1.15+0.09 1.95+0.28* ("1) (:)
t:)
yalues in parentheses are the number of lambs that ovulated, Significantly higher than for control group lambs (PqO.05) from analysis of variance. Table 3. The number of corpora lutea (CL) for inhibinimmunized Finn x Dorset Horn ewe lambs at different ages and the arithmetic meanfSEM No. of CL at different Group
n
Inhibin Immunized
4
Values
32
33
35
(4)
in parentheses
ages (weeks) Ovulation rate
36
41
45
(:)
16 (3)
17 (3)
3.38kO.76
are the number of lambs that ovulated.
Table 4. The frequency distribution of the ovulation rate for inhibin-immunized and control Suffolk x Galway and Finn x Dorset Horn ewe lambs
Group
No. of cycles
Inhibin immunized Suffolk x Galway
20
Control Suffolk x Galway
26
Inhibin immunized Finn x Dorset Horn
13
Control Finn x Dorset Horn
24
Values
1
Ovulation
rate class
2
4
3
5
(4:) (3:) (2:) (Z)
(f,
in parentheses
(::) (2:)
are percentages.
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A
9
-
(41)
(2:) (341) (1:) (A
6
(81) (81) (i)
-
(1:) -
-
THERIOGENOLOGY
DISCUSSION Antibodies to inhibin were detected as early as 30 d following primary immunization with a partially purified preparation of bFF. Subsequent booster injections resulted in a rapid increase in the percentage of radiolabeled 68kDa inhibin bound by serum from the immunized lambs. The six and seven ovulations recorded for each of the immunized lambs fall outside the range recorded for ewe lambs for this breed (21). Direct evidence of an immune response to inhibin following immunization with partially purified inhibin has also been reported recently (22). These authors detected antibodies to a 3lkDa bovine inhibin following immunization of adult Merino ewes with partially purified inhibin preparations from bFF. A greater and more rapid response with respect to inhibin antibody titre was obtained with preparations of increasing purity. Previously, immune responses had only been demonstrated to the partially purified preparations used but not specifically to inhibin (5‘6). Active immunization of the prepuberal lambs increased the ovulation rate (Experiment 2) in the Suffolk x Galway breed (P~0.05). The ovulation rate of immunized Finn x Dorset Horn ewe lambs was increased by 47% over that recorded for mature ewes from the same flock. Increases in the ovulation rate have been reported following active immunization of low fecund adult ewes against partially purified preparations from bFF (5,6). An increase in the ovulation rate has also been reported following frequent (approximately monthly) immunization of ewe lambs of a low fecund breed with a partially purified bovine inhibin preparation, starting from either 3 or 9 wk of age and continuing up to 35 wk of age (23). It is not clear, however, from the data of these reports or from the data presented here whether such increases in the ovulation rate are due to the production of antibodies to inhibin or to other proteins present in the immunogen. A significant correlation between antibody titre to 31 kDa inhibin and the ovulation rate, has been reported (22) after the immunization of adult Merino ewes with bovine follicular fluid inhibin: greater and more rapid responses in the ovulation rate followed immunization with preparations of increasing purity. Other reports have shown that active immunization of sheep against either a recombinant bovine (24) or human (25) inhibin a subunit, or a synthetic peptide (a I-29-Tyr 30) of bovine inhibin (26), raised antibodies which bound to pure 32kDa bovine inhibin, and which resulted in an increased ovulation rate. While FSH concentrations were not measured in this experiment, nevertheless it is likely that the increased ovulation rate recorded is due to the production of antibodies to inhibin, with a consequent increase in FSH secretion. There is evidence that immunization against inhibin increases FSH secretion (22,25-28), although not all studies have found this (5,23,29). It has been suggested that the day and
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frequency of blood sampling, the radioimmunoassays used, group size and records of basal and gonadotrophin-releasing hormone (GnRH)-stimulated secretion in the different studies may explain the reported differences in FSH secretion in inhibin immunized animals (30). The increase in ovulation rate in the immunized Suffolk x Galway breed was due to an increase in the proportion of ovulatory cycles, with more than one ovulation (11/20) compared with that of the control group (3/26). while one immunized lamb had six ovulations the remainder had either two or three. The increases in ovulation rate in the Finn x Dorset Horn ewe lambs was mainly due to the fact that one ewe had nine ovulations at each of two consecutive cycles. In one study (6), a large variation was found in the response of adult Merino ewes to immunization against a partially purified bovine follicular fluid preparation, both between ewes and within individual ewes at different times. A large variation in the ovulation rate following immunization of both prepuberal and adult Merino ewe lambs with a similar preparation has also been reported (23,27). Following immunization, starting at 19 wk of age (Experiment 2), there was no effect of active immunization against partially purified inhibin on the age at puberty or on estrous cycle length in either breed. An advancement of puberty has been reported following frequent immunization of ewe lambs with a partially purified bovine inhibin preparation when the immunization started as early as 3 or 9 wk of age and continued up to 35 wk of age (23). In the same study, when lambs were immunized three times between 3 and 9 wk of age, the advancement in puberty was less pronounced. A recent study (31) reported an advancement in puberty in ewe lambs following immunization with a synthetic inhibin fragment (porcine a l-32), beginning at 3 wk of age. Advancement of puberty may require immunization at an early age. The present study has shown that active immunization of prepuberal ewe lambs against a partially purified preparation of bFF raised antibodies capable of binding to an homogenous preparation of bovine inhibin. Active immunization increased the ovulation rate without affecting the onset of puberty or estrous cycle length. Even though FSH secretion was not measured, these results suggest that immunization against inhibin increases the ovulation rate in sheep, possibly by acting to increase FSH secretion. REFERENCES 1. Scaramuzzi, R.J., Davidson, W.G. and van Look,P.F.A. The effect of active immunisation against androstenedione on oestrus and ovulation in sheep. Nature (London) =:817-818 (1977).
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2. Webb, R., Land, R.B., Pathiraja, N. and Morris, B.A. Passive immunization against steroid hormones in the female. u: Crighton, D.B. (ed), Immunological Aspects of Reproduction in Mammals. Butterworths, London, 1984, PP- 475-499. 3.
Cox, R.I., Wilson, P.A., Scaramuzzi, R.J., Hoskinson, R.M., George, J.M. and Bindon, B.M. The active immunization of sheep against oestrone, androstenedione, or testosterone to increase twinning. Proc. Aust. Sot. Anim. Prod. =:511-514 (1982).
4.
Land, R.B., Morris, B.A., Baxter, G., Fordyce, M. and Forster, J. Improvement of sheep fecundity by treatment with antisera to gonadal steroids. J. Reprod. Fertil. s:625-634 (1982).
5.
Henderson, K.M., Franchimont, P., Lecomte-Yerna, M.J., Hudson, N. and Ball, K. Increase in ovulation rate after active immunisation of sheep with inhibin partially purified from bovine follicular fluid. J. Endocrinol. m:305-309 (1984).
6.
Cummins, L.J., O'Shea, T., Al-Obaidi, S.A.R., Bindon, B.M. and Findlay, J.K. Increase in ovulation rate after immunization of Merino ewes with a fraction of bovine follicular fluid containing inhibin activity. J. Reprod. Fertil. x:365-372 (1986).
7. Johnson, S.K. and Smith , M.F. Effects of charcoalextracted, bovine follicular fluid on gonadotrophin concentrations, the onset of estrus and luteal function in heifers. J. Anim. Sci. a:203-209 (1985). 8.
Cleere, W.F., Gosling, J.P., Morris, M.C., Charleton, M.F., Maloney, B.T., Fottrell, P.F. and Sreenan, J.M. A high performance, high throughput enzymeimmunoassay for analysis of progesterone in plasma or milk. Ir. Vet. J. =:6-14 (1985).
9.
de Jong, F.H, Jansen, E.H.J.M. and van der Molen Purification and characterization of inhibin. In: Franchimont, P. and Channing, C.P. (eds), Intragonadal Regulation of Reproduction. Academic Press, London, 1981, pp. 229-250.
10. Jansen, E.H.J.M., Steenbergen, J., de Jong, F.H. and van der Molen, H.J. The use of affinity matrices in the purification of inhibin from bovine follicular fluid. Mol. Cell. Endocrinol. a:109-117 (1981). 11.
348
van Dijk, S., de Jong, F.H. and van der Molen, H.J. Use of fast protein liquid chromatography in the purification of inhibin from bovine follicular fluid. Biochem. Biophys. Res. Commun. 125:307-314 (1984).
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12. Channing, C.P., Gordon, W.L., LiU, W.K. and Ward, D.N. Physiology and biochemistry of ovarian inhibin. Proc. Sot. exp. Biol. Med. m:339-361 (1985). 13. de Lean, A., Munson, P.J. and Rodbard, D. Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose response curves. Am. J. Physiol. 23512):E97-El02 (1978). 14. Finney, D.J. Parallel line assay. &l: Statistical Method in Biological Assay. Charles Griffin, London, 1964, pp. 101-138. 15. Morris, D.G. Bovine Inhibin Studies. Ph. D. Thesis, University of Dublin, Trinity College, Dublin, Ireland, 1989. 16. Freund, J., Thomson, K.J., Hough, H.B., Sommer, H.E. and Pisani, T.M. Antibody formation and sensitization with the aid of adjuvants. J.Immunol. a:383-398 (1948). 17. Fraker, F.J. and Speck, J.C. Protein and cell membrane iodinations with a sparingly soluble chloramide, 1,3,4,6-tetrachloro-3&,6a-diphenylglycoluril. Biochem. Biophys. Res. Commun. m:849-857 (1978). 18. Desbuguois, B. and Aurbach, G.D. Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoasssays. J. Clin. Endocrinol. =:732-738 (1971). 19. Quirke, J.F. Onset of puberty and oestrous activity in Galway, Finnish Landrace and Finn-cross ewe lambs during their first breeding season. Ir. J. Agric. Res. =:15-23 (1978). 20. SAS, SAS User's Guide: Statistics. SAS Institute Inc., Cary, NC, 1988. 21. Quirke, J.F., Hanrahan, J.P., Loughnane, W. and Triggs, R. Components of the breeding and non-breeding seasons in sheep: breed effects and repeatability. Ir. J. Agric. Res. =:167-172 (1986). 22. O'Shea, T., Bindon, B.M., Hillard, M.A., Piper, L.R., Findlay, J.K. and Miyamoto K. Increase in ovulation rate in Merino ewes after active immunisation with inhibin preparations obtained by immunoaffinity chromatography. Reprod. Fertil. Dev. 1:347-355 (1989).
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THERIOGENOLOGY
23.
Al-Obaidi, S.A.R., Bindon, B.M., Hillard, M.A. and O'Shea, T. Reproductive characteristics of lambs actively immunized early in life with inhibin-enriched preparations from follicular fluid of cows. J. Reprod. Fertil. =:403-414 (1987).
24.
Forage, R.G., Brown, R.W., Oliver, K.J., Atrache, B.T., Devine, P.L., Hudson, G.C., Goss, N.H., Bertram, K.C., Tolstoshev, P., Robertson, D.M., de Kretser, D.M., Doughton, B., Burger, H.G. and Findlay, J.K. Immunization against an inhibin subunit produced by recombinant DNA techniques results in increased ovulation rate in sheep. J. Endocrinol. u:Rl-R4 (1987).
25.
Mizumachi, M., Voglmayr, J.K. Washington, D.W., Chen, C.C.-L. and Bardin, C.W. Immunisation of ewes against the a-subunit of human recombinant inhibin increases FSH levels and ovulation rates. Biol. Reprod. &Q(Suppl.):56 abstr. (1989).
26.
Wrathall, J.H.M., McLeod, B.J., Glencross, R.G., Beard, A.J. and Knight P.G. Immunization of Romney ewes against a synthetic inhibin fragment raises ovulation rate. J. Reprod. Fertil. (abstr. series no. 3):37 abstr. (1989).
27.
Al-Obaidi, S.A.R., Bindon, B-M., Findlay, J-K., Hillard, M.A. and O'Shea, T. Plasma follicle stimulating hormone in Merino ewes immunized with an inhibin-enriched fraction from bovine follicular fluid. Anim. Reprod. Sci. u:39-51 (1987).
28.
Mann, G.E., Campbell, B.K., McNeilly, A.S. and Baird, D.T. Passive immunisation of sheep against inhibin raises plasma FSH concentration. J. Reprod. Fertil. (abstr. series no. 3):37 abstr. (1989).
29.
Schanbacher B.D. Increased ovulatory response of Suffolk ewes vaccinated against a synthetic fragment of porcine inhibin. Biol. Reprod. x(Suppl):62 abstr. (1988).
30.
Findlay, J.K., Doughton, B., Robertson, D.M. and Forage Effects of immunization against recombinant R.G. bovine inhibin a subunit on circulating concentrations of gonadotrophins in ewes. J. Endocrinol. m:59-65 (1989).
31.
O'Shea, T., Anderson, S.T., Bindon, B.M., Hillard M.A. and Munro R.K. Immunisation against a synthetic inhibin fragment advances onset of puberty in female Merino lambs. J. Reprod. Fertil. (abstr. series no. 4.):6 (1989).
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EFFECT OF IMMUNIZING PREPUBERAL LAMBS OF LQW AND HIGH OWLATION RATE GENOTYPES WITH INHIBIN PARTIALLY PURIFIED FROM BOVINE FOLLICULAR FLUID D.G. Morris, M.G. McDermott and J.M. Sreenan Animal Reproduction Department, Agriculture and Food Development Authority, Belclare, Tuam, Co. Galway, Ireland Received for publication: July 6, 1990 Accepted: November 14, 1990 ABSTRACT Active immunization of prepuberal lambs with a partially purified inhibin preparation, isolated from bovine follicular fluid, increased the ovulation rate. In ewe lambs of a low fecundity breed (Suffolk x Galway), the ovulation rate rose from 1.15 to 1.95 (PcO.05) compared with that of the controls. An ovulation rate of 3.38 was recorded for immunized ewe lambs of a high fecundity breed (Finn x Dorset Horn), while the rate for mature ewes from the same flock was 2.29. Immunization did not affect the time of onset of puberty or estrous cycle length. Following immunization, antibodies were produced that bound to a pure preparation of 68kDa bovine inhibin. This report demonstrates the production of antibody to a 68kDa preparation of inhibin following active immunization of sheep using a partially purified preparation. It was concluded that the increased ovulation rate was due to the production of antibodies to inhibin, which may have reduced its negative feedback effect on FSH secretion. Key words:
immunization, inhibin, ovulation rate, prepuberal lambs INTRODUCTION
The synthesis and secretion of gonadotrophins are influenced by the negative feedback effect of ovarian steroids and inhibin on the hypothalamic-pituitary axis. Altering this feedback has resulted in an increased ovulation rate in sheep. For example, immunization of sheep Acknowledgments The authors thank Dr. J.P. Hanrahan, Belclare, for assistance with the statistical analysis: Mr. G. Morris for technical support: and Mr. G. Burke for care of the animals. The work was supported in part by Bioresearch Ireland, Farm Business Development, Ltd., Ireland and by the Commission of the European Communities.
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against a range of steroid hormones has increased ovulation rate (1,2) and litter size (3,4). Similarly, the active immunization of sheep against partially purified bovine follicular fluid preparations enriched in inhibin has also increased the ovulation rate (5,6). While an immune response to the partially purified follicular fluid preparations was demonstrated by immunodiffusion in agar in these latter studies the production of antibody specific to inhibin was not demonstrated. The purpose of our study in Experiment 1 was to determine whether active immunization with a partially purified inhibin preparation derived from bovine follicular fluid would elicit an immune response to inhibin in prepubertal lambs. Experiment 2 was carried out to determine the possible effects of such immunization on the onset of puberty and ovulation rate in low and high fecund breeds. MATERIALS AND METHODS Collection
of Follicular
Fluid
Cow ovaries were obtained at a local abattoir and were held in ice-cold 0.9% (w/v) saline until arrival at the laboratory. Bovine follicular fluid (bFF) was aspirated from follicles of 5 to 20 mm in diameter using a syringe and 20-gauge needle and was stored at -2OOC until required. Steroid Removal Frozen aliguots of follicular fluid were thawed, pooled and centrifuged at 1000 g for 30 min at 4OC to remove cell debris. The supernatant was then decanted and treated with dextran-coated charcoal (1:lO) at a concentration of 1 mg/ml, by stirring at 4'C overnight. The charcoal was then removed by centrifugation at 1000 g for 30 min, followed by pressure filtration through Whatman No.1 filter paper. Steroid removal was monitored by measuring the progesterone concentration in the follicular fluid before and after the charcoal treatment (7) using a previously described and validated enzymeimmunoassay (8) and with reagents supplied by Noctech Ltd., Galway, Ireland. The sensitivity of the assay was 0.30 ng/ml with an ED50 of 1.33 ng/ml. The within assay coefficients of variation were l.l%, 2.6% and 6.4% for samples having mean progesterone concentrations of 1.44, 4.76 and 9.88 ng/ml, respectively. Charcoal treatment was effective in reducing the mean progesterone concentration of a pool of follicular fluid by more than 95%, from 240 to 10 ng/ml. Inhibin Purification A partially purified inhibin was isolated from the charcoal treated bFF by sequential chromatography on Red
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Sepharosea followed by Lentil Lectin Sepharosea as described previously (9). The partially purified inhibin was lyophilized and stored at -2OOC until required for immunization. An essentially homogenous inhibin was then obtained by sequential chromatography on affinity and hydrophobic matrices (10) followed by ion exchange chromatography on a Mono Qa column at pH 9.2 (11). The biological activity of all the inhibin preparations in this study was measured using an in vitro bioassay model, based upon the inhibition of FSH secretion by rat pituitary cells, as described previously (12). Potency estimates were calculated using the computer program ALLFIT (13), while assay statistics were calculated for parallel line bioassays (14). The dose response curves (n = 3) had a precision index 0,) of 0.142+0.026 (MkSEM), while the dose of charcoal-treated follicular fluid required to half maximally inhibit FSH secretion was 0.83kO.03 pg/well. Using this bioassay, the partially purified inhibin was purified 31- to 49-fold compared with that in the original follicular fluid. The homogenous inhibin was purified 79fold following chromatography on affinity and hydrophobic matrices. Following chromatography on Mono Q, the homogenous inhibin was assayed in the bioassay at one dose level only, where it reduced (PcO.05) basal FSH secretion with no effect (P>O.O5) on LH secretion (15). Electrophoresis of the homogenous inhibin was carried out on prgcast 4 to 30% (w/v) gradient polyacrylamide gels (PAA under nonreducing conditions. Silver 4/30) using SDS-P%E staining (Gel Code) of the gel revealed the presence of one protein which migrated, with an apparent molecular mass of 68kDa (15). Immunization
of Prepuberal
Lambs
The partially purified inhibin (2.5 mg/lamb) was dissolved in 5 ml of saline, emulsified in an equal quantity of Freund's complete adjuvant (FCA) (16) and then injected subcutaneously into four sites in the axillary region. Booster injections (1.0 mg) similarly prepared but emulsified in Freund's incomplete adjuvant (FIA) were injected at the time intervals outlined below. Antigen
Binding Studies
The homogenous 68kDa inhibin was iodinated using the iodogen procedure (17). Two micrograms of inhibin in 50 ~1 of 0.05 M phosphate buffer (PB; pH 7.4) were added to a 1.5-ml Eppendorf microvial precoated with 20 ps iodogen. b Five microlitres of 125-I (IMS 30, 100 mCi/ml) were then added and the reaction was allowed to proceed with E Pharmacia Biotechnology, Pierce, Cambridge, U.K. c Amersham, Bucks, U.K.
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Milton Keynes, U.K.
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agitation every minute for 10 min. The reaction was stopped by adding 200 ~1 of PB to the microvial and then removing the contents by pipetting. The free iodine was separated from the bound by gel filtration on a PD-10 column which had been pre-equilibrated with 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). All antigenbinding studies were carried out using one iodinated preparation which had a specific activity of 13.6 pCi/pg (15). The antigen binding capacity of serum from two of the immunized lambs was determined by incubating 10 ~1 of immune serum in 100 ~1 of 10% normal serum with 100 ~1 (30,000 cpm) of radioiodinated inhibin (37500 dpm, approx 18 fmole) for 24 h, at room temperature in a total volume of 300 ~1 in 0.05 M of PBS. The bound activity was separated from the free (18) by precipitating pith two volumes of 20% polyethylene glycol (PEG 4000). This was followed by centrifugation at 4000 g for 30 min, by further washing of the precipitate with 16% PEG in PBS and by centrifugation again. The precipitates were then counted in a Beckman 5500 automatic gamma counter for 1 min. Antigen binding capacity was expressed as the percentage of the total counts added per tube bound by immune serum after correcting for non-specific binding by pre-immune serum. Ovulation Rate and Estrous Cycle Length Measurements Following the first booster injection, vasectomized rams fitted with harnesses and crayons were used to check for standing estrus to establish the time of onset of puberty and estrous cycle length. Ovulation rate was determined at mid-cycle by direct laparoscopic observatign using a Wolf 180° direct vision 495x5.5-mm bronchoFcope and a Downs fiber light source (Mk.ii projector). Experiment
1
Because of the limited amount of material available for immunization at this time, only two lambs, born in August, were immunized with the partially purified inhibin at 22 wk of age, followed by booster injections at 27, 33, 48 and 57 wk of age. Blood samples for antibody measurement were
taken immediately before (pre-immune) and at regular intervals after immunization (immune) by jugular venipuncture into lo-ml tubes without additive (Vacutainer).g The blood samples were allowed to clot for 1 h at 37OC and then centrifuged at 1000 g and 4OC to separate the serum, which was then stored at -2OOC until assay. The ovulation rate was also measured on the two lambs over three cycles following the onset of puberty. u BDH Chemical Co. Poole, Dorset, U.K. F Wolf GmBH, W. Germany Down Bros and Mayer L Phelps Ltd., U.K. g Becton and Dickinson, U.K.)
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THERIOGENOLOGY Experiment
2
Suffolk x Galway ewe lambs born in April were randomly allotted to either an immunization (n = 9) or control (n = 9) group at 19 wk of age. Booster injections were given at 25, 35 and 40 wk of age. Finn x Dorset Horn ewe lambs born in April were similarly immunized (n=4) at 22 wk of age, with booster injections at 28, 38 and 43 wk of age. While controls were not available for the Finn x Dorset Horn ewe lambs, ovulation data recorded over two cycles in mature ewes (n = 12), from the same flock, was used for comparison. Data on the onset of puberty and estrous cycle length, reported by Quirke (19), was used for the comparison of these traits. Statistical
Analysis
The ovulation rate and estrous cycle measurements between groups were compared by analysis of variance. Statistical significance of the ovulation rate was tested following log transformation. In the model used, ewes were treated as a random effect nested within treatments, and time was fitted as a fixed effect. The mean square for ewes was used to test the significance of the treatment difference. Antigen binding was analyzed by Wilcoxon rank sum test (20). RESULTS Experiment
1
The antigen binding capacity of the serum from immunized lambs increased (PCO.002) in comparison with the preimmunization serum level (Figure 1). Radioactive binding by pre-immune sera was less than 0.5%. The number of ovulations measured for each of the first three cycles of the season starting approximately 1 wk after the final boost, was one, seven and four for one of the lambs and one, two and six for the other. Experiment
2
One immunized ewe lamb in the Suffolk x Galway group was injured and was excluded from the experiment. There was no effect of immunization on the age at puberty or on estrous cycle length in either breed (Table 1). While direct controls were not available, published data (19) suggests that immunization did not affect these traits in the Finn x Dorset Horn ewe lambs. Immunization increased the ovulation rate in the Suffolk cross lambs by 70% (PcO.05; Table 2). There was no time x treatment interaction on ovulation rate (PBO.44). The ovulation rate in the immunized Finn x Dorset Horn ewe lambs was 3.38kO.76. This was 47% higher than the ovulation rate (2.2920.16) recorded over two cycles for mature ewes (n = 12) from the same flock measured over the same time period (Table 3).
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THERIOGENOLOGY
I- 35 -
\
m 30 ; 25 z 20 m
20
28
36
44 AGE (weeks)
52
60
Figure l.Percentage of binding of radioiodinated 68kDa bovine inhibin by serum from ewe lambs immunized with a partially purified preparation of bovine follicular fluid. Arrows indicate time of immunization.
Table l.Effect of immunization of prepuberal lambs on the onset of puberty and estrous cycle length (M+SEM) Breed type
Status
n
Onset of puberty Datea
Age(days)
Cycle
No. of estrous (days) cycles
Suffolk x Galway
Immunized Control
a 9
344+10 334+07
224+10 214+07
15.3kO.4 15.5kO.3
Finn x Dorset Born
Immunized 4 Control 89
327206 321+03
235-+05 252+03
15.8kO.5 21 17.820.3 109 b
E Date is referenced to January Data from Quirke (19).
33 44
1 = Day 1.
There was no effect of immunization on the number of Suffolk x Galway lambs showing estrus or on the number ovulating (Table 2). However, one inhibin-immunized lamb failed to ovulate despite showing estrous activity on two occasions. A frequency distribution for ovulation rate is shown in Table 4 for all breeds.
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THERIOGENOLOGY Table 2. The number of corpora lutea (CL) of control and inhibin immunized Suffolk x Galway ewe lambs at different ages and the arithmetic mean+SEM No. of CL at different Group
n
Control
9
Inhibin 8 immunized
ages
(weeks) 38
Ovulation rate
28
30
31
32
33
42
(9,
A
(Z)
(:)
(66) K)
(9,
A
A
A
(:)
1.15+0.09 1.95+0.28* ("1) (:)
t:)
yalues in parentheses are the number of lambs that ovulated, Significantly higher than for control group lambs (PqO.05) from analysis of variance. Table 3. The number of corpora lutea (CL) for inhibinimmunized Finn x Dorset Horn ewe lambs at different ages and the arithmetic meanfSEM No. of CL at different Group
n
Inhibin Immunized
4
Values
32
33
35
(4)
in parentheses
ages (weeks) Ovulation rate
36
41
45
(:)
16 (3)
17 (3)
3.38kO.76
are the number of lambs that ovulated.
Table 4. The frequency distribution of the ovulation rate for inhibin-immunized and control Suffolk x Galway and Finn x Dorset Horn ewe lambs
Group
No. of cycles
Inhibin immunized Suffolk x Galway
20
Control Suffolk x Galway
26
Inhibin immunized Finn x Dorset Horn
13
Control Finn x Dorset Horn
24
Values
1
Ovulation
rate class
2
4
3
5
(4:) (3:) (2:) (Z)
(f,
in parentheses
(::) (2:)
are percentages.
FEBRUARY 1991 VOL. 35 NO. 2
A
9
-
(41)
(2:) (341) (1:) (A
6
(81) (81) (i)
-
(1:) -
-
THERIOGENOLOGY
DISCUSSION Antibodies to inhibin were detected as early as 30 d following primary immunization with a partially purified preparation of bFF. Subsequent booster injections resulted in a rapid increase in the percentage of radiolabeled 68kDa inhibin bound by serum from the immunized lambs. The six and seven ovulations recorded for each of the immunized lambs fall outside the range recorded for ewe lambs for this breed (21). Direct evidence of an immune response to inhibin following immunization with partially purified inhibin has also been reported recently (22). These authors detected antibodies to a 3lkDa bovine inhibin following immunization of adult Merino ewes with partially purified inhibin preparations from bFF. A greater and more rapid response with respect to inhibin antibody titre was obtained with preparations of increasing purity. Previously, immune responses had only been demonstrated to the partially purified preparations used but not specifically to inhibin (5‘6). Active immunization of the prepuberal lambs increased the ovulation rate (Experiment 2) in the Suffolk x Galway breed (P~0.05). The ovulation rate of immunized Finn x Dorset Horn ewe lambs was increased by 47% over that recorded for mature ewes from the same flock. Increases in the ovulation rate have been reported following active immunization of low fecund adult ewes against partially purified preparations from bFF (5,6). An increase in the ovulation rate has also been reported following frequent (approximately monthly) immunization of ewe lambs of a low fecund breed with a partially purified bovine inhibin preparation, starting from either 3 or 9 wk of age and continuing up to 35 wk of age (23). It is not clear, however, from the data of these reports or from the data presented here whether such increases in the ovulation rate are due to the production of antibodies to inhibin or to other proteins present in the immunogen. A significant correlation between antibody titre to 31 kDa inhibin and the ovulation rate, has been reported (22) after the immunization of adult Merino ewes with bovine follicular fluid inhibin: greater and more rapid responses in the ovulation rate followed immunization with preparations of increasing purity. Other reports have shown that active immunization of sheep against either a recombinant bovine (24) or human (25) inhibin a subunit, or a synthetic peptide (a I-29-Tyr 30) of bovine inhibin (26), raised antibodies which bound to pure 32kDa bovine inhibin, and which resulted in an increased ovulation rate. While FSH concentrations were not measured in this experiment, nevertheless it is likely that the increased ovulation rate recorded is due to the production of antibodies to inhibin, with a consequent increase in FSH secretion. There is evidence that immunization against inhibin increases FSH secretion (22,25-28), although not all studies have found this (5,23,29). It has been suggested that the day and
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THERIOGENOLOGY
frequency of blood sampling, the radioimmunoassays used, group size and records of basal and gonadotrophin-releasing hormone (GnRH)-stimulated secretion in the different studies may explain the reported differences in FSH secretion in inhibin immunized animals (30). The increase in ovulation rate in the immunized Suffolk x Galway breed was due to an increase in the proportion of ovulatory cycles, with more than one ovulation (11/20) compared with that of the control group (3/26). while one immunized lamb had six ovulations the remainder had either two or three. The increases in ovulation rate in the Finn x Dorset Horn ewe lambs was mainly due to the fact that one ewe had nine ovulations at each of two consecutive cycles. In one study (6), a large variation was found in the response of adult Merino ewes to immunization against a partially purified bovine follicular fluid preparation, both between ewes and within individual ewes at different times. A large variation in the ovulation rate following immunization of both prepuberal and adult Merino ewe lambs with a similar preparation has also been reported (23,27). Following immunization, starting at 19 wk of age (Experiment 2), there was no effect of active immunization against partially purified inhibin on the age at puberty or on estrous cycle length in either breed. An advancement of puberty has been reported following frequent immunization of ewe lambs with a partially purified bovine inhibin preparation when the immunization started as early as 3 or 9 wk of age and continued up to 35 wk of age (23). In the same study, when lambs were immunized three times between 3 and 9 wk of age, the advancement in puberty was less pronounced. A recent study (31) reported an advancement in puberty in ewe lambs following immunization with a synthetic inhibin fragment (porcine a l-32), beginning at 3 wk of age. Advancement of puberty may require immunization at an early age. The present study has shown that active immunization of prepuberal ewe lambs against a partially purified preparation of bFF raised antibodies capable of binding to an homogenous preparation of bovine inhibin. Active immunization increased the ovulation rate without affecting the onset of puberty or estrous cycle length. Even though FSH secretion was not measured, these results suggest that immunization against inhibin increases the ovulation rate in sheep, possibly by acting to increase FSH secretion. REFERENCES 1. Scaramuzzi, R.J., Davidson, W.G. and van Look,P.F.A. The effect of active immunisation against androstenedione on oestrus and ovulation in sheep. Nature (London) =:817-818 (1977).
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2. Webb, R., Land, R.B., Pathiraja, N. and Morris, B.A. Passive immunization against steroid hormones in the female. u: Crighton, D.B. (ed), Immunological Aspects of Reproduction in Mammals. Butterworths, London, 1984, PP- 475-499. 3.
Cox, R.I., Wilson, P.A., Scaramuzzi, R.J., Hoskinson, R.M., George, J.M. and Bindon, B.M. The active immunization of sheep against oestrone, androstenedione, or testosterone to increase twinning. Proc. Aust. Sot. Anim. Prod. =:511-514 (1982).
4.
Land, R.B., Morris, B.A., Baxter, G., Fordyce, M. and Forster, J. Improvement of sheep fecundity by treatment with antisera to gonadal steroids. J. Reprod. Fertil. s:625-634 (1982).
5.
Henderson, K.M., Franchimont, P., Lecomte-Yerna, M.J., Hudson, N. and Ball, K. Increase in ovulation rate after active immunisation of sheep with inhibin partially purified from bovine follicular fluid. J. Endocrinol. m:305-309 (1984).
6.
Cummins, L.J., O'Shea, T., Al-Obaidi, S.A.R., Bindon, B.M. and Findlay, J.K. Increase in ovulation rate after immunization of Merino ewes with a fraction of bovine follicular fluid containing inhibin activity. J. Reprod. Fertil. x:365-372 (1986).
7. Johnson, S.K. and Smith , M.F. Effects of charcoalextracted, bovine follicular fluid on gonadotrophin concentrations, the onset of estrus and luteal function in heifers. J. Anim. Sci. a:203-209 (1985). 8.
Cleere, W.F., Gosling, J.P., Morris, M.C., Charleton, M.F., Maloney, B.T., Fottrell, P.F. and Sreenan, J.M. A high performance, high throughput enzymeimmunoassay for analysis of progesterone in plasma or milk. Ir. Vet. J. =:6-14 (1985).
9.
de Jong, F.H, Jansen, E.H.J.M. and van der Molen Purification and characterization of inhibin. In: Franchimont, P. and Channing, C.P. (eds), Intragonadal Regulation of Reproduction. Academic Press, London, 1981, pp. 229-250.
10. Jansen, E.H.J.M., Steenbergen, J., de Jong, F.H. and van der Molen, H.J. The use of affinity matrices in the purification of inhibin from bovine follicular fluid. Mol. Cell. Endocrinol. a:109-117 (1981). 11.
348
van Dijk, S., de Jong, F.H. and van der Molen, H.J. Use of fast protein liquid chromatography in the purification of inhibin from bovine follicular fluid. Biochem. Biophys. Res. Commun. 125:307-314 (1984).
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THERIOGENOLOGY
12. Channing, C.P., Gordon, W.L., LiU, W.K. and Ward, D.N. Physiology and biochemistry of ovarian inhibin. Proc. Sot. exp. Biol. Med. m:339-361 (1985). 13. de Lean, A., Munson, P.J. and Rodbard, D. Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose response curves. Am. J. Physiol. 23512):E97-El02 (1978). 14. Finney, D.J. Parallel line assay. &l: Statistical Method in Biological Assay. Charles Griffin, London, 1964, pp. 101-138. 15. Morris, D.G. Bovine Inhibin Studies. Ph. D. Thesis, University of Dublin, Trinity College, Dublin, Ireland, 1989. 16. Freund, J., Thomson, K.J., Hough, H.B., Sommer, H.E. and Pisani, T.M. Antibody formation and sensitization with the aid of adjuvants. J.Immunol. a:383-398 (1948). 17. Fraker, F.J. and Speck, J.C. Protein and cell membrane iodinations with a sparingly soluble chloramide, 1,3,4,6-tetrachloro-3&,6a-diphenylglycoluril. Biochem. Biophys. Res. Commun. m:849-857 (1978). 18. Desbuguois, B. and Aurbach, G.D. Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoasssays. J. Clin. Endocrinol. =:732-738 (1971). 19. Quirke, J.F. Onset of puberty and oestrous activity in Galway, Finnish Landrace and Finn-cross ewe lambs during their first breeding season. Ir. J. Agric. Res. =:15-23 (1978). 20. SAS, SAS User's Guide: Statistics. SAS Institute Inc., Cary, NC, 1988. 21. Quirke, J.F., Hanrahan, J.P., Loughnane, W. and Triggs, R. Components of the breeding and non-breeding seasons in sheep: breed effects and repeatability. Ir. J. Agric. Res. =:167-172 (1986). 22. O'Shea, T., Bindon, B.M., Hillard, M.A., Piper, L.R., Findlay, J.K. and Miyamoto K. Increase in ovulation rate in Merino ewes after active immunisation with inhibin preparations obtained by immunoaffinity chromatography. Reprod. Fertil. Dev. 1:347-355 (1989).
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THERIOGENOLOGY
23.
Al-Obaidi, S.A.R., Bindon, B.M., Hillard, M.A. and O'Shea, T. Reproductive characteristics of lambs actively immunized early in life with inhibin-enriched preparations from follicular fluid of cows. J. Reprod. Fertil. =:403-414 (1987).
24.
Forage, R.G., Brown, R.W., Oliver, K.J., Atrache, B.T., Devine, P.L., Hudson, G.C., Goss, N.H., Bertram, K.C., Tolstoshev, P., Robertson, D.M., de Kretser, D.M., Doughton, B., Burger, H.G. and Findlay, J.K. Immunization against an inhibin subunit produced by recombinant DNA techniques results in increased ovulation rate in sheep. J. Endocrinol. u:Rl-R4 (1987).
25.
Mizumachi, M., Voglmayr, J.K. Washington, D.W., Chen, C.C.-L. and Bardin, C.W. Immunisation of ewes against the a-subunit of human recombinant inhibin increases FSH levels and ovulation rates. Biol. Reprod. &Q(Suppl.):56 abstr. (1989).
26.
Wrathall, J.H.M., McLeod, B.J., Glencross, R.G., Beard, A.J. and Knight P.G. Immunization of Romney ewes against a synthetic inhibin fragment raises ovulation rate. J. Reprod. Fertil. (abstr. series no. 3):37 abstr. (1989).
27.
Al-Obaidi, S.A.R., Bindon, B-M., Findlay, J-K., Hillard, M.A. and O'Shea, T. Plasma follicle stimulating hormone in Merino ewes immunized with an inhibin-enriched fraction from bovine follicular fluid. Anim. Reprod. Sci. u:39-51 (1987).
28.
Mann, G.E., Campbell, B.K., McNeilly, A.S. and Baird, D.T. Passive immunisation of sheep against inhibin raises plasma FSH concentration. J. Reprod. Fertil. (abstr. series no. 3):37 abstr. (1989).
29.
Schanbacher B.D. Increased ovulatory response of Suffolk ewes vaccinated against a synthetic fragment of porcine inhibin. Biol. Reprod. x(Suppl):62 abstr. (1988).
30.
Findlay, J.K., Doughton, B., Robertson, D.M. and Forage Effects of immunization against recombinant R.G. bovine inhibin a subunit on circulating concentrations of gonadotrophins in ewes. J. Endocrinol. m:59-65 (1989).
31.
O'Shea, T., Anderson, S.T., Bindon, B.M., Hillard M.A. and Munro R.K. Immunisation against a synthetic inhibin fragment advances onset of puberty in female Merino lambs. J. Reprod. Fertil. (abstr. series no. 4.):6 (1989).
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