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Seasonality of semen production and plasma luteinizing hormone, testosterone and prolactin levels in Romney, Merino and polled dorset rams
Animal Reproduction Science, 1 (1978/1979) 213--228
213
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
SEASONALITY OF SEMEN PRODUCTION AND PLASMA LUTEINIZING HORMONE, TESTOSTERONE AND PROLACTIN LEVELS IN ROMNEY, MERINO AND POLLED DORSET RAMS
G.K. BARRELL* and K.R. LAPWOOD
Department of Physiology and Anatomy, Massey University, Palmerston North (New Zealand) *Present address: Department of Veterinary Science, Lincoln College, Canterbury (New Zealand) (Accepted 20 July 1978)
ABSTRACT Barrell, G.K. and Lapwood, K.R., 1978/1979. Seasonality of semen production and plasma luteinizing hormone, testosterone and prolactin levels in Romney, Merino and Polled Dorset rams. Anim. Reprod. Sci., 1 : 213--228. An attempt was made to define the seasonality of reproduction in rams in the southern hemisphere by repeated measurement of semen characteristics and of plasma luteinizing hormone (LH), testosterone and prolactin concentrations. These parameters were studied for 16 months in Romney rams on pasture, with Merino and Polled Dorset rams included for comparison. Semen from all three breeds showed regular seasonal changes in ejaculate volumes, with peak values being recorded during March. A similar autumnal peak of seminal fructose levels was noted for ejaculates from Romney and Merino rams, but not for those from Polled Dorsets. Most of the other semen parameters measured showed little tendency for seasonal variations. However, a change in semen collection technique, from predominantly artificial vagina to entirely electroejaculation, may have masked some seasonal changes. Plasma hormone levels also varied in a regular manner, with peak levels occurring in summer and a u t u m n : highest levels for prolactin were recorded in November--March, for LH in December--February and for testosterone in January--March. An exception to this pattern was recorded from the Merino rams, for which there was no definite peak of LH secretion. It is suggested that these seasonal changes resulted primarily from changes in daily photoperiod.
INTRODUCTION
Observations on the reproductive activity of sheep have indicated that breeds of British origin have a distinct breeding season during the autumn months. Exceptions are Dorset sheep which have an extended season rather like that of Merinos (Hafez, 1952; Belschner, 1971). Recent New Zealand
214 work on the duration of the breeding season of ewes supported the view that Dorsets have a longer breeding season than R o m n e y s (Kelly et al., 1976). Seasonal breeding patterns of rams have received little attention since these animals apparently retain their fertility throughout the year. Despite this fact variations in semen production in rams do occur and can be assessed by repeated observations of semen quality (Ortavant et al., 1964; Smyth and Gordon, 1967). Measurements of plasma concentrations of luteinizing hormone (LH) and testosterone provide some insight into hormonal changes which may be associated with gonadal function in rams (Katongole et al., 1974). Also, the observations that high rates of prolactin release occur just before ovulation in ewes (Davis et al., 1971; Cumming et al., 1972) tend to suggest that this hormone might be involved in reproductive cyclicity in sheep. The work described in this paper was undertaken to define the breeding season of R o m n e y , Merino and Polled Dorset rams. Reproductive activity was investigated in studies of semen production and plasma hormone levels which extended over 16 months and included two breeding seasons. MATERIALS AND METHODS
Experimental animals and procedures Six R o m n e y , five Merino and four Polled Dorset rams were maintained under local farming conditions from February 1972 until June 1973. Initially semen was collected at two-weekly intervals by artificial vagina b u t rams unwilling to m o u n t were ejaculated b y electrical stimulation using a bipolar rectal probe (De la Nichols and Edgar, 1964). After September 1972 all semen was obtained by electroejaculation. Jugular blood samples were collected into heparinized vacutainers between 9.00 h and 10.00 h each Monday. Plasma was separated by centrifugation then frozen to - 1 8 ° C until required for hormone assay procedures. Assays were carried out on samples from every fourth week using the minimum number of assays practicable for each hormone.
Semen appraisal Ejaculate volumes were measured to the nearest 0.1 ml in graduated glass centrifuge tubes. Motility (scale 0--4; Emmens, 1947) and percentage of motile spermatozoa were estimated under a low-power microscope fitted with a 37°C warm stage. Percentages of unstained and morphologically normal spermatozoa were determined from Congo red--nigrosin smears (Blackshaw, 1955) examined under an oil-immersion microscope, while spermatozoal concentrations were obtained by counting a 1 in 200 dilution of semen on the red cell grid of a haemocytometer. Multiplication of the spermatozoal concentration by the ejaculate volume gave the total number of spermatozoa per ejaculate. Immediately after collection a portion of each ejaculate was frozen
215 on dry ice then stored at -18°C for later determination of seminal fructose concentration by AutoAnalyzer (Technicon Corporation, U.S.A.) using a modification of the method of Mann (1964). Total fructose content per ejaculate was calculated by multiplying the seminal fructose concentration by ejaculate volume. Seminal plasma fructose concentration was obtained by dividing the total fructose content by the volume of seminal plasma in each ejaculate. Seminal plasma volume was determined by subtracting from the ejaculate volume the volume occupied by spermatozoa, assuming that each spermatozoan occupied a volume of 6.5 X 10 -11 ml (cited by Glover, 1956). H o r mo n e assays
Plasma LH levels were measured by a double-antibody radioimmunoassay based on that described by Niswender et al. (1969). This assay utilized the following materials: rabbit anti-ovine LH serum (pool No. 15, courtesy Dr. G.D. Niswender), NIH-LH-S18 as assay standards, and highly purified ovine LH (LER-1374A) for radioiodination. Assay standards and samples were assayed in triplicate, while hypophysectomized sheep plasma was added to standard curve tubes. Assay sensitivity was 0.04--0.11 ng/ml, and the intraand inter-assay coefficients of variation (CV) were 8.6 and 18.2%, respectively, for a pool of sheep plasma with a mean LH concentration of 4.17 ng/ml. Plasma testosterone content was determined by a modification of the protein-binding procedure of Anderson (1970). Duplicate plasma samples, or testosterone standards dissolved in wether plasma, were extracted with toluene--hexane (1:4) and the aqueous layer removed by freezing. This assay utilized 1% (w/v) human late pregnancy plasma as source of binding globulin. Testosterone concentrations of samples were read directly from the standard curve obtained by plotting cpm protein-bound steroid versus concentration. Distilled water blanks were indistinguishable from the zero standard and assay sensitivity was 0.5--0.8 ng/ml. Intra-assay CV were 8.6 and 21.6% for plasma with testosterone concentrations in the ranges 4--7 and 1--2 ng/ml, respectively, whilst inter-assay CV were 12.7 and 22.5% for plasma samples containing 10.9 and 1.0 ng/ml testosterone, respectively. Plasma prolactin concentrations were estimated by a double-antibody radioimmunoassay with essentially the same protocol as the LH assay. In this assay the following materials were used: rabbit antiserum to bovine prolactin (NIH-P-B1), NIH-P-Sll as assay standards, and highly purified ovine prolactin (LER-860-2) for radioiodination. Iodination was performed by a modification of the procedure of Koprowski and Tucker (1971), while antiserum specificity was indicated by the lack of cross-reactivity with other ovine anterior pituitary hormones (Fig. 1). Samples and standards were assayed in triplicate and hypophysectomized sheep plasma was added to assay standard tubes. Assay sensitivity was 1.0--2.0 ng/ml. Intra-assay CV were 6.2 and 9.9% and inter-assay CV were 6.4 and 10.2% for plasma samples containing 105.4 and 42.5 ng/ml, respectively.
216 1 TSH.FSH 2 LH. ACTH 3 GH 4 PROLACTIN
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Fig. 1. Cross-reaction of anterior pituitary hormones in the ovine prolactin radioimmunoassay.
Computation of LH and prolactin assay results were performed by an IBM 1620 computer using the method of Burger et al. (1972}, in which hormone concentrations were computed from a best-fit expression of the standard curve.
Statistical analyses All data were subjected to analyses of variance in which orthogonal coefficients were utilized to examine the effects of seasons and breeds, as well as their interaction. The coefficients used in analysis of seasonal effects were obtained from Fisher and Yater {1963}, whilst breed comparisons were made using the coefficients shown in Table I. All coefficients were weighted for disproportionate group size and values for plasma hormone concentrations were transformed to logarithms prior to analysis. TABLE I Orthogonal coefficients used in breed comparisons Contrast
N.Z. R o m n e y
Merino
N.Z. R o m n e y vs Merino and Polled Dorset
+2
--1
--1
0
÷1
--1
Merino vs Polled Dorset
Polled Dorset
217 RESULTS
Meteorological data Meteorological data recorded during the course of the study (Fig. 2) showed that both daily photoperiod and temperature displayed similar sinusoidal patterns throughout the year. Total rainfall also showed a seasonal pattern, but with minimum values during the summer months. Changes in mean relative humidity (not shown) were less marked and corresponded with the changes in total rainfall.
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Fig. 2. Seasonal variations in daily photoperiod (calculated from N.Z. Nautical Almanac, 1972), temperature and total rainfall (recorded at Massey University), between March 1972 and June 1973.
0.90 3.5 68.7 3.76 3.38 5.13 4.65 6.68 81.2 88.4
Volume (ml) Motility (scale 0--4) % Motile Sperm/ml (×109 ) Sperm/ejac (×109) Fr Conc (mg/ml) Fr Cont (rag) S.P. Fr Conc (mg/ml) % Unstained % Normal
0.71 2.9 58.0 3.85 2.80 3.93 2.85 5.14 72.4 79.6
24/5 0.64 2.7 49.3 2.84 1.91 3.41 2.24 4.01 77.4 75.5
5/7 0.55 2.4 45.2 3.70 2.12 2.93 1.53 3.76 74.3 78.8
23/8 1.10 2.4 48.1 1.64 2.17 2.42 2.94 2.77 79.8 76.9
4/10 1.19 2.3 44.2 2.11 3.34 3.21 4.26 3.81 74.8 63.8
22/11 1.08 2.3 48.8 1.40 2.27 2.72 2.59 3.54 81.3 67.3
10/1
1.69 2.4 46.6 1.96 3.77 6.19 11.83 7.15 75.3 58.9
28/2
1.44 2.4 49.2 2.09 3.62 5.37 8.31 6.41 71.9 64.7
11 /4
1.15 2.3 49.4 2.12 2.90 3.74 4.96 4.59 67.9 60.0
30/5
Note: Values are the m e a n calculated from ejaculates collected at three two-weekly samplings. The date given is the midpoint of each collection period. Abbreviations: V o l u m e = ejaculate volume; Motility = motility index; % Motile = percentage of motile spermatozoa; Sperm/ml = concentration of spermatozoa/ml; Sperm/ejac = n u m b e r of spermatozoa/ejaculate; Fr C o n c = fructose concentration of semen; Fr Cont = total ejaculate fructose content; S.P. Fr Conc = fructose concentration of seminal plasma; % Unstained = percentage of unstained spermatozoa; % N o r m a l = percentage of morphologically normal spermatozoa.
5/4
Day/month
Pooled mean values of semen parameters for all three breeds of rams
TABLE II
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219
Semen data
Semen characteristics are recorded in Table II. Those for which regular seasonal patterns of change were recorded were mean ejaculate volume and the three parameters related to seminal fructose levels, all of which reached peak values during the autumn months and one of which is illustrated in Fig. 3. Motility indices, percentages of motile spermatozoa and percentages of morphologically normal spermatozoa declined during March and April, 1972, and showed little further change. On the other hand, numbers of spermatozoa per ejaculate declined during the first two months of the study but increased slightly at the beginning of the following summer (November 1972) (Fig. 4). In the late autumn months of 1973 (April, May and June) percentages of unstained spermatozoa were lower than in the summer of 1972--1973. Abrupt changes in mean values for ejaculate volume, concentration of ROMNEY IO-
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Fig. 3. Seasonal variations in seminal plasma f r u c t o s e c o n c e n t r a t i o n s ( m e a n -+ S.E.) in s e m e n c o l l e c t e d from R o m n e y , Merino and Polled D o r s e t rams b e t w e e n March 1 9 7 2 and June 1 9 7 3 .
220 ROMNEY
MERINO
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MONTHS
Fig. 4. Seasonal variations in numbers of spermatozoa per ejaculate (mean -+ S.E.) in semen collected from Romney, Merino and Polled Dorset rams between March 1972 and June 1973. spermatozoa per ml and total ejaculate fructose content occurred during September 1972 and corresponded with the change in semen collection method, from predominantly artificial vagina to entirely electrical stimulation. Differences between breeds were recorded for some semen parameters. For instance, semen from Romneys had lower overall mean values than that from Merinos and Polled Dorsets for: motility index (2.4 vs 2.7 and 2.6; P < 0.05), percentage of motile spermatozoa (46.2 vs 53.4 and 52.7; P < 0.01) and concentration of spermatozoa per ml (2.13 vs 2.75 and 2.76 X 109; P < 0.01). Also semen from Merinos had higher overall mean values for total ejaculate fructose content (5.79 vs 2.95 mg; P < 0.001), seminal fructose concentration (4.45 vs 3.00 mg/ml; P < 0.001) and seminal plasma fructose concentration (5.52 vs 3.70 mg/ml; P < 0.001) than semen from Polled Dorset rams. The latter differences were due to the absence of an autumnal peak in these parameters for the semen from Polled Dorsets (Fig. 3). Plasma hormone data Plasma LH levels followed a seasonal pattern with maximal levels being recorded during the summer months (November to March), and lower levels
221
during winter (April to August) (Fig. 5). Peak mean levels were 0.88 + 0.31 ng L H per ml plasma while midwinter levels often were below the limit of assay sensitivity.With the orthogonal coefficients used for making contrasts in the Seasons × Breeds interaction, no significant differences were detected, despite the apparently lower summer plasma L H levels in the Merinos than in the R o m n e y or Polled Dorset rams. All three breeds exhibited a marked elevation of plasma testosterone concentrations during January, February and March, 1973, while m i n i m u m levels occurred between May and November in both years (Fig. 6). Declining plasma testosterone levels in March--April 1972 indicated the probable presence of a peak in the previous January--February period, similar to that recorded in 1973. Plasma prolactin levels showed a well
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Fig. 5. Seasonal variations in plasma LH concentrations (mean +- S.E.) recorded from Romney, Merino and Polled Dorset rams between March 1972 and June 1973.
RoMNE
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Fig. 6. Seasonal variations in plasma testosterone concentrations (mean + S.E.) recorded from Romney, Merino and Polled Dorset rams between March 1972 and June 1973. DISCUSSION
Seasonal changes in ram semen production have been reported by a number of workers (see reviews by Emmens and Robinson, 1962; Lodge and Salisbury, 1970). Although the results have tended to be equivocal, generally higher quality semen has been recorded during autumn and early winter. Variations between previous reports mostly can be attributed to factors such as the particular character under study, the breed of sheep, and the location of the experiment. In the present experiment a well-defined seasonal pattern was recorded for ejaculate volume and seminal fructose data, these being the semen parameters which are influenced most by accessory sex gland function (Mann, 1964). This seasonal pattern consisted of a peak in values during autumn and low levels during spring, which supports earlier reports from California (Cupps et al., 1960), Israel (Amir and Volcani, 1965) and Canada (Sanford et al., 1977). All other semen characteristics studied related to the concentration, activity, or morphology of spermatozoa, and did not show any evidence of seasonal changes, except for numbers of spermatozoa per ejaculate
223
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Fig. 7. Seasonal v a m t i o n s in plasma prolaetin concentrations (mean ± 8.E.) recorded from R o m n e y , Merino and Polled Dorset rams between March 1972 and June 1973.
which tended to be elevated during the autumn months. However, it is possible that other seasonal changes may have been masked by the alteration in semen collection method, or alternatively the frequency of collection may have been too low to allow detection of changes in semen production. Polled Dorset rams did not display any seasonality in seminal fructose levels. Also, they showed less distinct seasonal fluctuations in ejaculate volume and spermatozoal numbers which suggested that they had a reduced tendency for seasonal changes in semen production compared to Romney and Merino rams. Although considerable variations were recorded for mean plasma LH levels, the summer peak exhibited by Romney and Polled Dorset rams in particular, coincided with the pattern reported by Hochereau~le Reviers et al. (1976) in France. Most other workers in the northern hemisphere have either failed to
224 demonstrate significant seasonal changes in plasma LH levels (Katongole et al., 1974; Sanford et al., 1974), or have shown that highest concentrations were confined to the autumn months (Pelletier, 1971; Johnson et al., 1973; Schanbacher and Lunstra, 1976). Measurements of episodic secretion of LH in rams have tended to indicate that an increase in frequency of LH discharge is associated with the autumn season (Katongole et al., 1974; Schanbacher and Ford, 1976), or with decreasing daily photoperiods {Lincoln, 1976 a, b). However, episodic secretion patterns were not investigated in this study. Autumnal peaks in plasma testosterone concentrations in rams have been reported by various workers (Attal, 1970; Johnson et al., 1973; Katongole et al., 1974; Sanford et al., 1974, 1977; Schanbacher and Lunstra, 1976). In the present study, however, peak levels were obtained during the months of January, February and March. This finding was more in accord with the results of Gomes and Joyce (1975) who investigated rams of mixed breeds in Ohio. The present and previously described studies have clearly indicated the generally seasonal nature of LH and testosterone output in rams, and small differences in the timing of seasonal events probably were attributable to breed or locality, particularly latitude. The fact that decreasing daily photoperiod is the major stimulus for seasonal changes in reproductive parameters, such as plasma testosterone and seminal fructose levels in rams, has been shown by controlled-lighting studies (Pelletier and Ortavant, 1975 a, b; Barrell, 1976; Lincoln, 1976 a, b); this stimulus probably acts via the pineal gland at the level of the hypothalamus. Plasma prolactin levels followed a seasonal pattern similar to those described for rams (Ravault, 1976), goats (Buttle, 1974) and bulls {Schams and Reinhardt, 1974), with elevated levels during the summer months. In each study the animals were subjected to seasonal changes in both daily photoperiod and temperature, so the relative importance of these environmental factors could not be determined. However, Pelletier (1973) showed that there was a direct relationship between length of the daily photoperiod and plasma prolactin levels in rams and wethers subjected to artificial lighting regimes. An interesting feature of the results of the present experiment was the successive timing of seasonal peaks of plasma prolactin, LH, testosterone and seminal fructose concentrations (Fig. 8). This experiment was not designed to investigate the reproductive role of prolactin in rams, but it is possible to conjecture that prolactin may have acted as a 'conditioning hormone' (Nicoll, 1973) which prepared the testes for the subsequent rise in LH. Evidence from rodent studies has shown that prolactin increases Leydig cell sensitivity to LH (Hafiez et al., 1972; Bartke et al., 1975) and increases testicular binding of LH (Bex and Bartke, 1977). Recent work with rams indicated that prolactin also increases seminal vesicle sensitivity to testosterone (Ravault et al., 1977). In turn LH undoubtedly stimulated androgen production which led to increased accessory gland activity. It can be presumed that this sequence of events was controlled by seasonal changes in pituitary or hypothalamic activity, and/or by seasonal changes in testicular responsiveness to
225
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Fig. 8. Interrelationships between seasonal variations in plasma prolactin, LH and testosterone levels, and in seminal plasma fructose concentrations, for Romney ( Merino ( - - - - - ) and Polled Dorset ( . . . . . ) rams between March 1972 and June 1973.
LH, similar to those described for the male goat (Racey et al., 1975). The probable role of the pineal gland in mediating these responses to altered daily photoperiod may provide a worthwhile avenue for further research (Reiter, 1974; Barrell, 1976).
226
The data indicated that rams of all three breeds probably were capable of reproduction throughout the year. However, plasma hormone levels and the parameters measuring accessory sex gland function did exhibit marked seasonal patterns which might have been reflected by subtle variations in the relative levels of fertility which were not detectable by the semen parameters measured. It might therefore be worthwhile to investigate ram fertility either in vivo by inseminating ewes, or by use of more stringent in vitro semen viability tests. It is worth noting that the change in semen collection technique in the winter of 1972 was necessitated by a decline in ram libido; an effect of this type could severely limit the success of out-of-season breeding programmes. In contrast to Romney rams, Merinos and Polled Dorsets did not exhibit variations in plasma LH and in seminal fructose levels, respectively, which indicated that breed differences in seasonality may exist which are similar to those described in ewes of like breeds in New Zealand (Kelly et al., 1976). ACKNOWLEDGEMENTS
We gratefully acknowledge the following for providing radioimmunoassay reagents: N.I.A.M.D.D., N.I.H., U.S.A., for ovine-LH, highly purified ovine-LH and ovine prolactin; Dr L.E. Reichert, Jr., Emory University, U.S.A., for highly purified ovine prolactin; Dr G.D. Niswender, Colorado State University, U.S.A., for antiserum to ovine-LH; and Prof. D.S. Flux, Massey University for antiserum to bovine prolactin. Prof. R.E. Munford provided advice and assisted in data analysis, while Mrs H. Carter, Mr R.H. Telfer and Mr R.N. Ward were competent technical assistants.
REFERENCES
Amir, D. and Volcani, R., 1965. Seasonal fluctuations in the sexual activity of Awassi, German Mutton Merino, Border Leicester and Dorset Horn rams. J. Agric. Sci.,64: 115--134. Anderson, D.C., 1970. A simple and clinicallyuseful method for plasma testosterone-like substances by competitive protein binding. Clin. Chim. Acta, 29: 513--522. Attal, J., 1970. Mesure des oestrog~nes et des androg~nes testiculaireset plasmatiques dans l'esp~ce ovine par des microm~thodes de chromatographie en phase gazeuse: influence de l'age,de la saison et du cycle diurne. Th~se de Doctorate d'Etat es'Sciences Naturelles, Paris. C.N.R.S.: A.O. 4145. Barrell, G.K., 1976. Studies of neuroendocrine mechanisms influencing seasonal variations in semen production and plasma hormone levels in rams. Ph.D. Thesis, Massey University. Bartke, A., Croft, B.T. and Dalterio, S., 1975. Prolactin restores plasma testosterone levels and stimulates testiculargrowth in hamsters exposed to short day-length. Endocrinology, 97: 1601--1604. Belschner, H.G., 1971. Sheep Management and Diseases. 9th edit. Angus and Robertson, Sydney, pp. 166--167. Bex, F.J. and Bartke, A., 1977. Testicular L H binding in the hamster: modification by photoperiod and prolactin. Endocrinology, 100: 1223--1226. Blackshaw, A.W., 1955. Factors affecting the revivalof bull and ram spermatozoa after freezing to --79°C. Aust. Vet. J., 31: 238--241.
227 Burger, H.G., Lee, V.W.K. and Rennie, G.C., 1972. A generalized computer program for the treatment of data from competitive protein-binding assays including radioimmunoassays. J. Lab. Clin. Med., 80: 302--312. Buttle, H.L., 1974. Seasonal variation of prolactin in plasma of male goats. J. Reprod. Fert., 37: 95--99. Cumming, I.A., Brown, J.M., Goding, J.R., Bryant, G.D. and Greenwood, F.C., 1972. Secretion of prolactin and luteinizing hormone at oestrus in the ewe. J. Endocrinol., 54: 207--213. Cupps, P.T., McGowan, B., Rahlmann, D.F., Reddon, A.R. and Weir, W.C., 1960. Seasonal changes in the semen of rams. J. Anita. Sci., 19: 208--213. Davis, S.L., Reichert, L.E., Jr. and Niswender, G.D., 1971. Serum levels of prolactin in sheep as measured b y radioimmunoassay. Biol. Reprod., 4: 145--153. De la Nichols, G. and Edgar, D.G., 1964. A transistorized rectal probe for ejaculating rams. N.Z. Vet. J., 12: 145--146. Emmens, C.W., 1947. The motility and viability of rabbit spermatozoa at different hydrogen ion concentrations. J. Physiol. (London), 106: 471--481. Emmens, C.W. and Robinson, T.J., 1962. Artificial insemination in the sheep. In: J.P. Maule (Editor), The Semen of Animals and Artificial Insemination, Comm. Agric. Bur., Farnham Royal, pp. 205--251. Fisher, R.A. and Yates, F., 1963. Statistical Tables for Biological, Agricultural and Medical Research. 6th edit. Oliver and Boyd, London, p. 98. Glover, T.D., 1956. The effect of scrotal insulation and the influence of breeding season upon fructose concentration in the semen of the ram. J. Endocrinol., 13: 235--242. Gomes, W.R. and Joyce, M.C., 1975. Seasonal changes in serum testosterone in adult rams. J. Anita. Sci., 41: 1373--1375. Hafez, E.S.E., 1952. Studies on the breeding season and reproduction of the ewe. J. Agric. Sci., 42: 189--265. Hafiez, A.A., Lloyd, C.W. and Bartke, A., 1972. The role of prolactin in regulation of testis function: the effects of prolactin and luteinizing hormone on the plasma levels of testosterone and androstenedione in hypophysectomized rats, J. Endocrinol., 52: 327--332. Hochereau:deReviers, M.T., Loir, M. and Pelletier, J., 1976. Seasonal variations in the responses of the testis and LH levels to hemicastration of adult rams. J. Reprod. Fert., 46: 203--209. Johnson, B.H., Desjardins, C. and Ewing, L.L., 1973. Seasonal effects on testis function in rams. J. Anita. Sci., 37 : 247, Abstr. Katongole, C.B., Naftolin, F. and Short, R.V., 1974. Seasonal variations in blood luteinizing hormone and testosterone levels in rams. J. Endocrinol., 60: 101--106. Kelly, R.W., Allison, A.J. and Shackell, G.H., 1976. Seasonal variation in oestrous and ovarian activity of five breeds of ewes in Otago. N.Z.J. Exp. Agric., 4: 209--214. Koprowski, J.A. and Tucker, H.A., 1971. Failure of oxytocin to initiate prolactin or luteinizing hormone release in lactating dairy cows. J. Dairy Sci., 54: 1675--1680. Lincoln, G.A., 1976a. Seasonal variation in the episodic secretion of luteinizing hormone and testosterone in the ram. J. Endocrinol., 69: 213--226. Lincoln, G.A., 1976b. Secretion of LH in rams exposed to two different photoperiods. J. Reprod. Fert., 47: 351--353. Lodge, J.R. and Salisbury, G.W., 1970. Seasonal variation and male reproductive efficiency. In: A.D. Johnson, W.R. Gomes and N.L. VanDemark (Editors), The Testis, Vol. III. Academic Press, New York and London, pp. 139--167. Mann, T., 1964. The Biochemistry of Semen and of the Male Reproductive Tract. Methuen, London, p. 239 and pp. 37--78. Nicoll, C.S., 1973. 'Spinoff' from comparative studies on prolactin physiology of significance to the clinical endocrinologist. In: J.L. Pasteels and C. Robyn (Editors), Human Prolactin. Excerpta Medica Foundation, Amsterdam, pp. 119--136. Niswender, G.D., Reichert, L.E., Jr. and Midgely, A.R., Jr., 1969. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology, 84: 1166--1173.
228 Ortavant, R., Mauleon, P. and Thibault, C., 1964. Photoperiodic control of gonadal and hypophyseal activity in domestic animals. Ann. N.Y. Acad. Sci., 117: 157--193. Pelletier, J., 1971. Influence du photop~riodisme et des androg~nes sur la synth~se et la liberation de LH chez le b~lier. Th~se de Doctorat d']~tat es'Sciences Naturelles, Paris. C.N.R.S.: A.O. 5441. Pelletier, J., 1973. Evidence for photoperiodic control of prolactin release in rams. J. Reprod. Fert., 35: 143--147. Pelletier, J. and Ortavant, R., 1975a. Photoperiodic control of LH release in the ram. I. Influence of increasing and decreasing light photoperiods. Acta Endocrinol. (Copenhagen), 78: 435--441. PeUetier, J. and Ortavant, R., 1975b. Photoperiodic control of LH release in the ram. II. Light--androgens interaction. Acta endocrinol. (Copenhagen), 78: 442--450. Racey, P.A., Rowe, P.H. and Chesworth, J.M., 1975. Changes in the luteinizing hormone-testosterone system of the male goat during the breeding season. J. Endocrinol., 65: 8P--9P, Abstr. Ravault, J.P., 1976. Prolactin in the ram: seasonal variations in the concentration of blood plasma from birth until three years old. Acta Endocrinol. (Copenhagen), 83: 720--725. Ravault, J.P., Courot, M., Gamier, D., PeUetier, J. and Terqui, M., 1977. Effect of 2-bromo~-ergocryptine (CB 154) on plasma prolactin, LH and testosterone levels, accessory reproductive glands and spermatogenesis in lambs during puberty. Biol. Reprod., 17: 192--197. Reiter, R.J., 1974. Circannual reproductive rhythms in mammals related to photoperio~ and pineal function: a review. Chronobiologia, 1: 365--395. Sanford, L.M., Palmer, W.M. and Howland, B.E., 1974. Seasonal variation of serum levels of LH and testosterone in the ram. Can. J. Anita. Sci., 54: 247--249. Sanford, L.M., Palmer, W.M. and Howland, B.E., 1977. Changes in the profiles of serum LH, FSH and testosterone, and in mating performance and ejaculate volume in the ram during the ovine breeding season. Can. J. Anim. Sci., 45: 1382--1391. Schams, D. and Reinhardt, V., 1974. Influence of season on plasma prolactin level in cattle from birth to maturity. Horm. Res., 5: 217--226. Schanbacher, B.D. and Ford, J.J., 1976. Seasonal profiles of plasma luteinizing hormone, testosterone and estradiol in the ram. Endocrinology, 99: 752--757. Schanbacher, B.D. and Lunstra, D.D., 1976. Seasonal changes in sexual activity and serum levels of LH and testosterone in Finnish Landrace and Suffolk rams. J. Anita. Sci., 43 : 644--650. Smyth, P. and Gordon, I., 1967. Seasonal and breed variations in the semen characteristics of rams in Ireland. Ir. V e t . J., 21 : 222--233.
213
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
SEASONALITY OF SEMEN PRODUCTION AND PLASMA LUTEINIZING HORMONE, TESTOSTERONE AND PROLACTIN LEVELS IN ROMNEY, MERINO AND POLLED DORSET RAMS
G.K. BARRELL* and K.R. LAPWOOD
Department of Physiology and Anatomy, Massey University, Palmerston North (New Zealand) *Present address: Department of Veterinary Science, Lincoln College, Canterbury (New Zealand) (Accepted 20 July 1978)
ABSTRACT Barrell, G.K. and Lapwood, K.R., 1978/1979. Seasonality of semen production and plasma luteinizing hormone, testosterone and prolactin levels in Romney, Merino and Polled Dorset rams. Anim. Reprod. Sci., 1 : 213--228. An attempt was made to define the seasonality of reproduction in rams in the southern hemisphere by repeated measurement of semen characteristics and of plasma luteinizing hormone (LH), testosterone and prolactin concentrations. These parameters were studied for 16 months in Romney rams on pasture, with Merino and Polled Dorset rams included for comparison. Semen from all three breeds showed regular seasonal changes in ejaculate volumes, with peak values being recorded during March. A similar autumnal peak of seminal fructose levels was noted for ejaculates from Romney and Merino rams, but not for those from Polled Dorsets. Most of the other semen parameters measured showed little tendency for seasonal variations. However, a change in semen collection technique, from predominantly artificial vagina to entirely electroejaculation, may have masked some seasonal changes. Plasma hormone levels also varied in a regular manner, with peak levels occurring in summer and a u t u m n : highest levels for prolactin were recorded in November--March, for LH in December--February and for testosterone in January--March. An exception to this pattern was recorded from the Merino rams, for which there was no definite peak of LH secretion. It is suggested that these seasonal changes resulted primarily from changes in daily photoperiod.
INTRODUCTION
Observations on the reproductive activity of sheep have indicated that breeds of British origin have a distinct breeding season during the autumn months. Exceptions are Dorset sheep which have an extended season rather like that of Merinos (Hafez, 1952; Belschner, 1971). Recent New Zealand
214 work on the duration of the breeding season of ewes supported the view that Dorsets have a longer breeding season than R o m n e y s (Kelly et al., 1976). Seasonal breeding patterns of rams have received little attention since these animals apparently retain their fertility throughout the year. Despite this fact variations in semen production in rams do occur and can be assessed by repeated observations of semen quality (Ortavant et al., 1964; Smyth and Gordon, 1967). Measurements of plasma concentrations of luteinizing hormone (LH) and testosterone provide some insight into hormonal changes which may be associated with gonadal function in rams (Katongole et al., 1974). Also, the observations that high rates of prolactin release occur just before ovulation in ewes (Davis et al., 1971; Cumming et al., 1972) tend to suggest that this hormone might be involved in reproductive cyclicity in sheep. The work described in this paper was undertaken to define the breeding season of R o m n e y , Merino and Polled Dorset rams. Reproductive activity was investigated in studies of semen production and plasma hormone levels which extended over 16 months and included two breeding seasons. MATERIALS AND METHODS
Experimental animals and procedures Six R o m n e y , five Merino and four Polled Dorset rams were maintained under local farming conditions from February 1972 until June 1973. Initially semen was collected at two-weekly intervals by artificial vagina b u t rams unwilling to m o u n t were ejaculated b y electrical stimulation using a bipolar rectal probe (De la Nichols and Edgar, 1964). After September 1972 all semen was obtained by electroejaculation. Jugular blood samples were collected into heparinized vacutainers between 9.00 h and 10.00 h each Monday. Plasma was separated by centrifugation then frozen to - 1 8 ° C until required for hormone assay procedures. Assays were carried out on samples from every fourth week using the minimum number of assays practicable for each hormone.
Semen appraisal Ejaculate volumes were measured to the nearest 0.1 ml in graduated glass centrifuge tubes. Motility (scale 0--4; Emmens, 1947) and percentage of motile spermatozoa were estimated under a low-power microscope fitted with a 37°C warm stage. Percentages of unstained and morphologically normal spermatozoa were determined from Congo red--nigrosin smears (Blackshaw, 1955) examined under an oil-immersion microscope, while spermatozoal concentrations were obtained by counting a 1 in 200 dilution of semen on the red cell grid of a haemocytometer. Multiplication of the spermatozoal concentration by the ejaculate volume gave the total number of spermatozoa per ejaculate. Immediately after collection a portion of each ejaculate was frozen
215 on dry ice then stored at -18°C for later determination of seminal fructose concentration by AutoAnalyzer (Technicon Corporation, U.S.A.) using a modification of the method of Mann (1964). Total fructose content per ejaculate was calculated by multiplying the seminal fructose concentration by ejaculate volume. Seminal plasma fructose concentration was obtained by dividing the total fructose content by the volume of seminal plasma in each ejaculate. Seminal plasma volume was determined by subtracting from the ejaculate volume the volume occupied by spermatozoa, assuming that each spermatozoan occupied a volume of 6.5 X 10 -11 ml (cited by Glover, 1956). H o r mo n e assays
Plasma LH levels were measured by a double-antibody radioimmunoassay based on that described by Niswender et al. (1969). This assay utilized the following materials: rabbit anti-ovine LH serum (pool No. 15, courtesy Dr. G.D. Niswender), NIH-LH-S18 as assay standards, and highly purified ovine LH (LER-1374A) for radioiodination. Assay standards and samples were assayed in triplicate, while hypophysectomized sheep plasma was added to standard curve tubes. Assay sensitivity was 0.04--0.11 ng/ml, and the intraand inter-assay coefficients of variation (CV) were 8.6 and 18.2%, respectively, for a pool of sheep plasma with a mean LH concentration of 4.17 ng/ml. Plasma testosterone content was determined by a modification of the protein-binding procedure of Anderson (1970). Duplicate plasma samples, or testosterone standards dissolved in wether plasma, were extracted with toluene--hexane (1:4) and the aqueous layer removed by freezing. This assay utilized 1% (w/v) human late pregnancy plasma as source of binding globulin. Testosterone concentrations of samples were read directly from the standard curve obtained by plotting cpm protein-bound steroid versus concentration. Distilled water blanks were indistinguishable from the zero standard and assay sensitivity was 0.5--0.8 ng/ml. Intra-assay CV were 8.6 and 21.6% for plasma with testosterone concentrations in the ranges 4--7 and 1--2 ng/ml, respectively, whilst inter-assay CV were 12.7 and 22.5% for plasma samples containing 10.9 and 1.0 ng/ml testosterone, respectively. Plasma prolactin concentrations were estimated by a double-antibody radioimmunoassay with essentially the same protocol as the LH assay. In this assay the following materials were used: rabbit antiserum to bovine prolactin (NIH-P-B1), NIH-P-Sll as assay standards, and highly purified ovine prolactin (LER-860-2) for radioiodination. Iodination was performed by a modification of the procedure of Koprowski and Tucker (1971), while antiserum specificity was indicated by the lack of cross-reactivity with other ovine anterior pituitary hormones (Fig. 1). Samples and standards were assayed in triplicate and hypophysectomized sheep plasma was added to assay standard tubes. Assay sensitivity was 1.0--2.0 ng/ml. Intra-assay CV were 6.2 and 9.9% and inter-assay CV were 6.4 and 10.2% for plasma samples containing 105.4 and 42.5 ng/ml, respectively.
216 1 TSH.FSH 2 LH. ACTH 3 GH 4 PROLACTIN
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Computation of LH and prolactin assay results were performed by an IBM 1620 computer using the method of Burger et al. (1972}, in which hormone concentrations were computed from a best-fit expression of the standard curve.
Statistical analyses All data were subjected to analyses of variance in which orthogonal coefficients were utilized to examine the effects of seasons and breeds, as well as their interaction. The coefficients used in analysis of seasonal effects were obtained from Fisher and Yater {1963}, whilst breed comparisons were made using the coefficients shown in Table I. All coefficients were weighted for disproportionate group size and values for plasma hormone concentrations were transformed to logarithms prior to analysis. TABLE I Orthogonal coefficients used in breed comparisons Contrast
N.Z. R o m n e y
Merino
N.Z. R o m n e y vs Merino and Polled Dorset
+2
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--1
0
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Polled Dorset
217 RESULTS
Meteorological data Meteorological data recorded during the course of the study (Fig. 2) showed that both daily photoperiod and temperature displayed similar sinusoidal patterns throughout the year. Total rainfall also showed a seasonal pattern, but with minimum values during the summer months. Changes in mean relative humidity (not shown) were less marked and corresponded with the changes in total rainfall.
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0.90 3.5 68.7 3.76 3.38 5.13 4.65 6.68 81.2 88.4
Volume (ml) Motility (scale 0--4) % Motile Sperm/ml (×109 ) Sperm/ejac (×109) Fr Conc (mg/ml) Fr Cont (rag) S.P. Fr Conc (mg/ml) % Unstained % Normal
0.71 2.9 58.0 3.85 2.80 3.93 2.85 5.14 72.4 79.6
24/5 0.64 2.7 49.3 2.84 1.91 3.41 2.24 4.01 77.4 75.5
5/7 0.55 2.4 45.2 3.70 2.12 2.93 1.53 3.76 74.3 78.8
23/8 1.10 2.4 48.1 1.64 2.17 2.42 2.94 2.77 79.8 76.9
4/10 1.19 2.3 44.2 2.11 3.34 3.21 4.26 3.81 74.8 63.8
22/11 1.08 2.3 48.8 1.40 2.27 2.72 2.59 3.54 81.3 67.3
10/1
1.69 2.4 46.6 1.96 3.77 6.19 11.83 7.15 75.3 58.9
28/2
1.44 2.4 49.2 2.09 3.62 5.37 8.31 6.41 71.9 64.7
11 /4
1.15 2.3 49.4 2.12 2.90 3.74 4.96 4.59 67.9 60.0
30/5
Note: Values are the m e a n calculated from ejaculates collected at three two-weekly samplings. The date given is the midpoint of each collection period. Abbreviations: V o l u m e = ejaculate volume; Motility = motility index; % Motile = percentage of motile spermatozoa; Sperm/ml = concentration of spermatozoa/ml; Sperm/ejac = n u m b e r of spermatozoa/ejaculate; Fr C o n c = fructose concentration of semen; Fr Cont = total ejaculate fructose content; S.P. Fr Conc = fructose concentration of seminal plasma; % Unstained = percentage of unstained spermatozoa; % N o r m a l = percentage of morphologically normal spermatozoa.
5/4
Day/month
Pooled mean values of semen parameters for all three breeds of rams
TABLE II
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219
Semen data
Semen characteristics are recorded in Table II. Those for which regular seasonal patterns of change were recorded were mean ejaculate volume and the three parameters related to seminal fructose levels, all of which reached peak values during the autumn months and one of which is illustrated in Fig. 3. Motility indices, percentages of motile spermatozoa and percentages of morphologically normal spermatozoa declined during March and April, 1972, and showed little further change. On the other hand, numbers of spermatozoa per ejaculate declined during the first two months of the study but increased slightly at the beginning of the following summer (November 1972) (Fig. 4). In the late autumn months of 1973 (April, May and June) percentages of unstained spermatozoa were lower than in the summer of 1972--1973. Abrupt changes in mean values for ejaculate volume, concentration of ROMNEY IO-
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220 ROMNEY
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Fig. 4. Seasonal variations in numbers of spermatozoa per ejaculate (mean -+ S.E.) in semen collected from Romney, Merino and Polled Dorset rams between March 1972 and June 1973. spermatozoa per ml and total ejaculate fructose content occurred during September 1972 and corresponded with the change in semen collection method, from predominantly artificial vagina to entirely electrical stimulation. Differences between breeds were recorded for some semen parameters. For instance, semen from Romneys had lower overall mean values than that from Merinos and Polled Dorsets for: motility index (2.4 vs 2.7 and 2.6; P < 0.05), percentage of motile spermatozoa (46.2 vs 53.4 and 52.7; P < 0.01) and concentration of spermatozoa per ml (2.13 vs 2.75 and 2.76 X 109; P < 0.01). Also semen from Merinos had higher overall mean values for total ejaculate fructose content (5.79 vs 2.95 mg; P < 0.001), seminal fructose concentration (4.45 vs 3.00 mg/ml; P < 0.001) and seminal plasma fructose concentration (5.52 vs 3.70 mg/ml; P < 0.001) than semen from Polled Dorset rams. The latter differences were due to the absence of an autumnal peak in these parameters for the semen from Polled Dorsets (Fig. 3). Plasma hormone data Plasma LH levels followed a seasonal pattern with maximal levels being recorded during the summer months (November to March), and lower levels
221
during winter (April to August) (Fig. 5). Peak mean levels were 0.88 + 0.31 ng L H per ml plasma while midwinter levels often were below the limit of assay sensitivity.With the orthogonal coefficients used for making contrasts in the Seasons × Breeds interaction, no significant differences were detected, despite the apparently lower summer plasma L H levels in the Merinos than in the R o m n e y or Polled Dorset rams. All three breeds exhibited a marked elevation of plasma testosterone concentrations during January, February and March, 1973, while m i n i m u m levels occurred between May and November in both years (Fig. 6). Declining plasma testosterone levels in March--April 1972 indicated the probable presence of a peak in the previous January--February period, similar to that recorded in 1973. Plasma prolactin levels showed a well
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Fig. 6. Seasonal variations in plasma testosterone concentrations (mean + S.E.) recorded from Romney, Merino and Polled Dorset rams between March 1972 and June 1973. DISCUSSION
Seasonal changes in ram semen production have been reported by a number of workers (see reviews by Emmens and Robinson, 1962; Lodge and Salisbury, 1970). Although the results have tended to be equivocal, generally higher quality semen has been recorded during autumn and early winter. Variations between previous reports mostly can be attributed to factors such as the particular character under study, the breed of sheep, and the location of the experiment. In the present experiment a well-defined seasonal pattern was recorded for ejaculate volume and seminal fructose data, these being the semen parameters which are influenced most by accessory sex gland function (Mann, 1964). This seasonal pattern consisted of a peak in values during autumn and low levels during spring, which supports earlier reports from California (Cupps et al., 1960), Israel (Amir and Volcani, 1965) and Canada (Sanford et al., 1977). All other semen characteristics studied related to the concentration, activity, or morphology of spermatozoa, and did not show any evidence of seasonal changes, except for numbers of spermatozoa per ejaculate
223
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which tended to be elevated during the autumn months. However, it is possible that other seasonal changes may have been masked by the alteration in semen collection method, or alternatively the frequency of collection may have been too low to allow detection of changes in semen production. Polled Dorset rams did not display any seasonality in seminal fructose levels. Also, they showed less distinct seasonal fluctuations in ejaculate volume and spermatozoal numbers which suggested that they had a reduced tendency for seasonal changes in semen production compared to Romney and Merino rams. Although considerable variations were recorded for mean plasma LH levels, the summer peak exhibited by Romney and Polled Dorset rams in particular, coincided with the pattern reported by Hochereau~le Reviers et al. (1976) in France. Most other workers in the northern hemisphere have either failed to
224 demonstrate significant seasonal changes in plasma LH levels (Katongole et al., 1974; Sanford et al., 1974), or have shown that highest concentrations were confined to the autumn months (Pelletier, 1971; Johnson et al., 1973; Schanbacher and Lunstra, 1976). Measurements of episodic secretion of LH in rams have tended to indicate that an increase in frequency of LH discharge is associated with the autumn season (Katongole et al., 1974; Schanbacher and Ford, 1976), or with decreasing daily photoperiods {Lincoln, 1976 a, b). However, episodic secretion patterns were not investigated in this study. Autumnal peaks in plasma testosterone concentrations in rams have been reported by various workers (Attal, 1970; Johnson et al., 1973; Katongole et al., 1974; Sanford et al., 1974, 1977; Schanbacher and Lunstra, 1976). In the present study, however, peak levels were obtained during the months of January, February and March. This finding was more in accord with the results of Gomes and Joyce (1975) who investigated rams of mixed breeds in Ohio. The present and previously described studies have clearly indicated the generally seasonal nature of LH and testosterone output in rams, and small differences in the timing of seasonal events probably were attributable to breed or locality, particularly latitude. The fact that decreasing daily photoperiod is the major stimulus for seasonal changes in reproductive parameters, such as plasma testosterone and seminal fructose levels in rams, has been shown by controlled-lighting studies (Pelletier and Ortavant, 1975 a, b; Barrell, 1976; Lincoln, 1976 a, b); this stimulus probably acts via the pineal gland at the level of the hypothalamus. Plasma prolactin levels followed a seasonal pattern similar to those described for rams (Ravault, 1976), goats (Buttle, 1974) and bulls {Schams and Reinhardt, 1974), with elevated levels during the summer months. In each study the animals were subjected to seasonal changes in both daily photoperiod and temperature, so the relative importance of these environmental factors could not be determined. However, Pelletier (1973) showed that there was a direct relationship between length of the daily photoperiod and plasma prolactin levels in rams and wethers subjected to artificial lighting regimes. An interesting feature of the results of the present experiment was the successive timing of seasonal peaks of plasma prolactin, LH, testosterone and seminal fructose concentrations (Fig. 8). This experiment was not designed to investigate the reproductive role of prolactin in rams, but it is possible to conjecture that prolactin may have acted as a 'conditioning hormone' (Nicoll, 1973) which prepared the testes for the subsequent rise in LH. Evidence from rodent studies has shown that prolactin increases Leydig cell sensitivity to LH (Hafiez et al., 1972; Bartke et al., 1975) and increases testicular binding of LH (Bex and Bartke, 1977). Recent work with rams indicated that prolactin also increases seminal vesicle sensitivity to testosterone (Ravault et al., 1977). In turn LH undoubtedly stimulated androgen production which led to increased accessory gland activity. It can be presumed that this sequence of events was controlled by seasonal changes in pituitary or hypothalamic activity, and/or by seasonal changes in testicular responsiveness to
225
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Fig. 8. Interrelationships between seasonal variations in plasma prolactin, LH and testosterone levels, and in seminal plasma fructose concentrations, for Romney ( Merino ( - - - - - ) and Polled Dorset ( . . . . . ) rams between March 1972 and June 1973.
LH, similar to those described for the male goat (Racey et al., 1975). The probable role of the pineal gland in mediating these responses to altered daily photoperiod may provide a worthwhile avenue for further research (Reiter, 1974; Barrell, 1976).
226
The data indicated that rams of all three breeds probably were capable of reproduction throughout the year. However, plasma hormone levels and the parameters measuring accessory sex gland function did exhibit marked seasonal patterns which might have been reflected by subtle variations in the relative levels of fertility which were not detectable by the semen parameters measured. It might therefore be worthwhile to investigate ram fertility either in vivo by inseminating ewes, or by use of more stringent in vitro semen viability tests. It is worth noting that the change in semen collection technique in the winter of 1972 was necessitated by a decline in ram libido; an effect of this type could severely limit the success of out-of-season breeding programmes. In contrast to Romney rams, Merinos and Polled Dorsets did not exhibit variations in plasma LH and in seminal fructose levels, respectively, which indicated that breed differences in seasonality may exist which are similar to those described in ewes of like breeds in New Zealand (Kelly et al., 1976). ACKNOWLEDGEMENTS
We gratefully acknowledge the following for providing radioimmunoassay reagents: N.I.A.M.D.D., N.I.H., U.S.A., for ovine-LH, highly purified ovine-LH and ovine prolactin; Dr L.E. Reichert, Jr., Emory University, U.S.A., for highly purified ovine prolactin; Dr G.D. Niswender, Colorado State University, U.S.A., for antiserum to ovine-LH; and Prof. D.S. Flux, Massey University for antiserum to bovine prolactin. Prof. R.E. Munford provided advice and assisted in data analysis, while Mrs H. Carter, Mr R.H. Telfer and Mr R.N. Ward were competent technical assistants.
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