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Testis size, gonadotrophin secretion and pituitary responsiveness to GnRH in Romney rams during the transition from the non-breeding to the breeding season
Animal Reproduction Science, 31 ( 1993 ) 99-111
99
Elsevier Science Publishers B.V., Amsterdam
Testis size, gonadotrophin secretion and pituitary responsiveness to GnRH in Romney rams during the transition from the non-breeding to the breeding season Z.Z. Xu', M.F. McDonald, S.N. McCutcheon and H.T. Blair Department of Animal Science, Massey University, Private Bag, Palmerston North, New Zealand (Accepted 13 March 1992 )
ABSTRACT Xu, Z.Z., McDonald, M.F., McCutcheon, S.N. and Blair, H.T., 1993. Testis size, gonadotrophin secretion and pituitary responsiveness to GnRH in Romney rams during the transition from the nonbreeding to the breeding season. Anim. Reprod. Sci., 31: 99-111. Romney rams, which had either an early ( n = 7 ) or a late ( n = 5 ) increase in testis size prior to the onset of the breeding season, were selected from a larger population (n = 60 ). In the following year. the two groups were compared for the pattern of seasonal variation in testis size, circulating gonadotrophin concentrations and pituitary responsiveness to a gonadotrophin releasing hormone (GnRH) challenge during the transitional period from the non-breeding to the breeding season. Rams in the 'early' group had an earlier and greater increase in testis size than rams in the 'late' group, a pattern which was largely repeatable between years. Differences between the groups in the pattern of seasonal variation in testis size were associated with group differences in endocrine function. Thus, rams in the early group had a significantly (P< 0.01 ) higher luteinising hormone (LH) pulse frequency in March (4.4 _+0.4 pulses per 8 h period) than those in the late group ( 1.7 _+0.3 pulses per 8 h period ) and the seasonal increase in plasma follicle stimulating hormone (FSH) concentrations occurred eailier (P< 0.10) in the early group than in the late group. There were also group differences in the pattern of seasonal variation in total LH response to the GnRH challenge. These results show that there are differences among rams of the same breed, in the pattern of seasonal variation in testis size and gonadotrophin secretion, which could potentially be used as criteria to select rams for date of onset of the breeding season in their daughters.
INTRODUCTION
In a previous study (Xu et al., 1991 ), we showed that the Romney and Poll Dorset breeds, which exhibit marked differences in seasonality of female Correspondence to: M.F. McDonald, Department of Animal Science, Massey University, Private Bag, Palmerston North, New Zealand. ~Present address: Department of Animal Science, 163 Animal Science Centre, University of Missouri-Columbia, MO 65211, USA.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0378-4320/93/$06.00
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breeding, also exhibited differences in male reproductive parameters, particularly the pattern of seasonal variation in testis size and gonadotrophin secretion during the transitional period from the non-breeding to the breeding season. These results suggest that seasonal variation in testis size and gonadotrophin secretion prior to onset of the breeding season might be useful predictors of a ram's genetic merit for early onset of the breeding season. Thus, in selection programmes for early onset of the breeding season, it might be possible to select rams based on these indicator traits instead of, or in combination with, direct selection for date of onset of the breeding season in the ewe (Walkley and Smith, 1980; Haresign and McLeod, 1985 ). Because of the greater selection pressure that can be applied on the ram compared with the ewe, the rate of genetic progress could be greatly improved by indirect selection in the ram (Xu, 1991 ). However, while between-breed comparisons are useful in identifying parameters which might be examined as potential indicator traits in a within-breed situation, differences found between breeds do not necessarily guarantee a cause and effect relationship within a breed (Blair et al., 1990) and relationships therefore have to be verified within breeds. Accordingly, the present study was conducted to investigate the extent to which within-breed variation exists, among rams of the highly seasonal Romney breed, in the pattern of seasonal changes in testis size and associated changes in plasma gonadotrophin concentrations and the pituitary responsiveness to a gonadotrophin releasing hormone ( G n R H ) challenge during the transitional period from the non-breeding to the breeding season. The study was based on two groups of Romney rams selected at the extremes of the population to represent animals with early or late seasonal increases in testis diameters, i.e. to parallel differences observed previously in the Poll Dorset vs. Romney comparison (Xu et al., 1991 ). MATERIALS AND METHODS
Animals and selection procedures Twelve Romney rams aged 3 years were used in this study. They were selected, from a group of 60 animals, on the basis of testis diameter measurements made at 2-3 week intervals during the transitional period from the non-breeding to the breeding season ( 1 October 1988-20 February 1989). Only the 7 measurements that were made prior to the summer solstice (22 December) were used for selection. First, values for testis diameters of each ram were expressed as deviations from the mean for all 60 rams, at each measurement time. Next, a linear regression model of deviations on time was fitted to each ram. The regression coefficients from this analysis were then ranked. The six rams with the greatest positive coefficients and the six with
SEASONAL VARIATION IN TESTIS SIZE, LH AND FSH IN RAMS
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the greatest negative coefficients were selected to represent rams with an early and a late increase in testis size, respectively. The normal date of onset of the breeding season for R o m n e y ewes under the local conditions (40. l ° S ) in which this study was conducted is the beginning of March (Knight et al., 1989; Xu, 1991 ).
Experimental procedures Commencing in early October 1989 (the non-breeding season following that in which the initial selection was m a d e ) , testis diameters of the selected rams were measured at intervals of 2-3 weeks (until March 1990) using the technique described by Xu et al. ( 1991 ). On four occasions during this period ( 1 November 1989, 13 December 1989, 31 January 1990 and 20 March 1990), the rams were brought indoors for intensive blood sampling and G n R H challenge studies. Ten days before each planned sampling date, the animals were transferred indoors from pasture and housed in metabolism crates in order to allow them to adjust to the experimental conditions and handling. The room in which the animals were housed had large windows and rams were exposed only to natural light. While indoors, the rams received a maintenance diet of chaffed lucerne hay, supplemented with minerals to counteract possible copper toxicity (Rattray, 1986). Fresh water was available ad libitum. The rams were returned to pasture 3 days after completion of each blood sampling period. While outdoors (i.e. between blood sampling periods), they were run as a single group separate from the ewe flock and grazed on mixed ryegrass and white clover pastures.
Blood sampling and GnRH challenges Jugular vein cannulae were inserted as described previously (Carter et al., 1989 ) 1 day before the date of blood sampling. At each sampling period, 6 ml of blood were collected at 15-min intervals for 8 hours commencing at 09:00 h. After the last sample was taken ( 17:00 h), each animal received an intravenous injection of G n R H at a dose of 50 ng kg- l liveweight. The G n R H was dissolved at a concentration of 1/~g m l - l in saline 1 h prior to injection and administered via the cannulae, followed by 4 ml of saline to flush the cannulae. Further blood samples were collected 10, 20, 30, 45, 60, 80, 100, 120, 150 and 180 min after G n R H injection. All blood samples were collected into heparinised tubes and immediately placed on ice. They were centrifuged within 25 min at 3000 g and 4°C for 20 min. The plasma was harvested and stored frozen at - 2 0 ° C in duplicate vials until hormone analyses were conducted.
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Hormone assays Plasma luteinising hormone (LH) and follicle stimulating hormone (FSH) concentrations were measured by homologous double-antibody radioimmunoassays using kits provided by the National Hormone and Pituitary Program and the National Institute of Diabetic and Digestive and Kidney Diseases ( N I D D K ) , Bethesda, MD. Assay protocols were as described previously (Xu et al., 1991 ). The LH reference preparation was NIADDK-oLH-25 and the assay sensitivity was 0.09 + 0.01 ng m1-1. The intra-assay coefficients of variation (CV) were 4.8%, 3.6% and 5.2%, and the inter-assay CV were 13.9%, 10.4% and 7.0%, at mean LH concentrations of 0.5 ng m1-1, 1.3 ng m l - 1 and 3.0 ng m1-1, respectively. The FSH reference standards was NIAMDD-oFSHRP-I and the assay sensitivity was 0.13 _+0.03 ng ml-1. The intra-assay CV were 10.5%, 11.0% and 5.1%, and the inter-assay CV were 13.4%, 12.5% and 9.8%, at mean FSH concentrations of 0.8 ng ml-1, 2.2 ng ml-~ and 4.4 ng m l - 1, respectively.
Data and statistical analyses Data relating to plasma LH concentrations in serial samples from individual rams at each sampling period were processed by the computer programme DETECT, to identify the presence of secretion pulses (Guardabasso et al., 1988 ). LH pulse frequency was expressed as the number of pulses occurring during an 8 h sampling period. Basal LH concentrations were obtained by averaging values for samples not associated with any LH pulse and mean LH concentrations were calculated by averaging values for all samples taken from each ram during the 8 h sampling period. LH pulse amplitude was determined as the difference between the highest LH concentration associated with an individual pulse and the basal concentration. LH responses to the GnRH challenges were characterised by the peak response (the highest LH concentration post-GnRH injection less the basal concentration) and the total response (measured by the area under the response curve above basal concentration, calculated by triangulation). Analysis of variance for repeated measurements was used to test the overall effects of group ('early' vs. 'late'), sampling time and group X time interaction on testis diameter and the endocrine parameters. In order to apply the repeated measures analysis to the testis diameter data, measurements taken on individual animals during each month were averaged to obtain a monthly mean for testis diameter. This parameter was then used in the analysis. To eliminate the heterogeneity of variance, data for LH pulse amplitude and the peak and total LH responses to G n R H were subjected to logarithm transformation before analysis. Comparison between means at different sampling times was carried out using orthogonal contrasts. Analyses within sampling
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periods were conducted using univariate analysis of variance. All analyses were performed using the SAS statistical package (Statistical Analysis System Institute, 1988 ). RESULTS
Testis diameter Testis diameter and liveweight of the selected rams during the 1988-1989 season are shown in Fig. 1. The mean testis diameter over the entire experim e n t period of rams in the early group (59.5_+0.9 m m ) was significantly (P < 0.01 ) greater than that of rams in the late group ( 53.7 _+0.7 m m ). There were also significant group differences in the pattern of seasonal variation in testis size ( g r o u p × t i m e interaction, P < 0 . 0 1 ). For rams in the early group, testis size increased 25% from a m i n i m u m of 53.4 m m in late October to a m a x i m u m of 66.6 m m in February. For rams in the late group, the corresponding increase was 16% from 50.1 m m in late December to 58.5 m m in February. The liveweight of rams in the early group ( 7 1 . 0 _ 0.7 kg) was sig70
65
90. 80-
70~60-
9--~" 50OCT
DATE OF MEASUREMENT
Fig. 1. Testis d i a m e t e r s ( m e a n + SEM, top ) a n d liveweight ( m e a n + SEM, b o t t o m ) o f rams in the early ( - - O - - , n = 6) a n d late ( - - (3 - - n = 6 ) groups during the period from October 1988 to February 1989.
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nificantly ( P < 0.01 ) greater than that of rams in the late group (59.5 _+0.5 kg). The classification of rams into two groups, based on the method described previously, was repeatable from year to year. Thus the correlation between ranks of the regression coefficients during the 1988-1989 and 1989-1990 seasons was 0.80 (Spearman rank correlation, P < 0.01 ). In the 1988-1989 season, the regression coefficients for rams in the early group were all positive, whereas those of rams in the late group were all negative (as this was the basis on which assignment to groups was m a d e ) . During the 1989-1990 season, the regression coefficients of three rams (one from the early group and two from the late group) changed sign. Because the endocrine study was carried out during the 1989-1990 season, it was considered appropriate to reclassify these animals based on the regression coefficients during the 1989-1990 season. Rams with positive regression coefficients (n = 7) were classified into the early group and those with negative regression coefficients (n = 5 ) were classified into the late group. Consequently, all analyses were based on the newly classified groups. Testis diameter and liveweights during the 1989-1990 season are shown in Fig. 2. The overall mean testis diameter (from October 1989 to March 1990) 706560-
5085 80 75
~ 7o
~
65. 60. 55. OCT
NOV
DEC
JAN
FEB
MAR
APR
DATE OF MEASUREMENT
Fig. 2. Testis d i a m e t e r s ( m e a n + SEM, t o p ) a n d liveweight ( m e a n + SEM, b o t t o m ) of rams in the (reclassified) early ( - - O - - , n = 7 ) a n d late ( - - O - - , n = 5 ) groups during the period from O c t o b e r 1989 to M a r c h 1990.
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of rams in the (reclassified) early group ( 59.2 _+0.8 m m ) was significantly ( P < 0.01 ) greater than that of rams in the late group (54.8 _ 0.5 m m ) . There were significant ( P < 0.01 ) differences between groups in the patterns of seasonal variation in testis diameter. Testis diameter increased 14% for rams in the early group from a minimum of 55.1 mm in late October to a maximum of 62.8 m m in February. The corresponding increase for rams in the late group was 10% from 52.5 m m in December to 57.7 m m in February. The liveweight of rams in the early group (75.6 _+ 1.1 kg) was significantly ( P < 0.01 ) greater than that of rams in the late group (66.9 _+ 1.1 kg). The liveweight varied significantly ( P < 0.01 ) with season, but seasonal changes in testis size did not appear to parallel seasonal changes in liveweight (correlations between testis diameters and liveweights across time were - 0 . 2 1 ( P > 0 . 1 0 ) and - 0 . 7 8 ( P < 0.01 ) for rams in the early and late groups, respectively).
Luteinising hormone The effects of group and sampling time on circulating LH concentrations are presented in Table 1. There were no significant differences, between the TABLE 1 Effect o f sampling date on basal and mean luteinising h o r m o n e ( L H ) concentrations, LH pulse frequency and LH pulse amplitude for rams in the early and late groups I Sampling date
1 November 13 D e c e m b e r 31 January 20 March
Group
Basal concentration (ngml -t )
Mean concentration (ngml -t )
Pulse frequency (per 8 h period)
Pulse amplitude 2 (ngml -I )
Early Late Early Late Early Late Early Late
0.29+0.02 0.27_+0.02 0.32_+0.02 0.29+0.01 0.34-+0.02 0.29_+0.01 0.36_+0.02 0.30-+0.01
0.30_+0.02 0.29_+0.01 0.43_+0.02 0.37_+0.03 0.40-+0.03 0.38_+0.02 0.43+0.03 a 0.33_+0.02 b
0.3_+0.2 0.5_+0.2 1.1 _+0.1 0.9_+0.2 2.8-+0.4 2.3_+0.2 4.4_+0.4 a 1.7_+0.3 c
0.94 0.44_+0.05 1.37_+0.21 1.19+0.08 0.63_+0.03 0.66_+0.06 0.56+_0.04 0.53_+0.04
NS ** NS
NS ** NS
* *** **
NS * NS
Slgnifi¢~ce Group Time G r o u p × time
i Early group n = 7; late group n = 5. Values presented are means + SEM. 2Only one complete LH pulse was present and used for calculating pulse amplitude for rams in the early group on l November. This m e a n therefore has no standard error. a,b'CMeans within each column and sampling date carrying different superscripts differed significantly (a vs. b, P < 0.05; a vs. c, P < 0.01 ). *P < 0.05; * * P < 0.01; ***P < 0.001; NS, not significant.
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early and late groups, in the overall basal LH concentrations (early group, 0.33 + 0.02 ng m l - ~; late group, 0.29 ___0.01 ng m l - ~) or the overall mean LH concentrations (early group, 0.39 + 0.03 ng m l - ~; late group, 0.34 + 0.02 ng m l - l ) . Both the basal and mean LH concentrations varied significantly ( P < 0 . 0 1 ) with sampling time, but there was no significant difference between groups in the pattern of this variation (group×time interaction, P > 0.10). However, the mean LH concentration of rams in the early group was significantly ( P < 0.05 ) higher than that of rams in the late group in March (Table 1 ). LH pulse frequency varied significantly ( P < 0 . 0 0 1 ) with sampling time, increasing as the breeding season approached. There was also a significant group difference in the pattern of seasonal variation in LH pulse frequency (group×time, P<0.01 ). Changes in LH pulse frequency were similar between November and January for both groups. However, LH pulse frequency of rams in the early group increased significantly ( P < 0.01 ) between January and March, whereas that of rams in the late group decreased slightly (Table 1 ). This resulted in rams in the early group having a significantly ( P < 0.01 ) higher LH pulse frequency than those in the late group. LH pulse amplitude varied significantly ( P < 0.05 ) with sampling time, but no significant effects of group or group × time interaction were found.
Follicle-stimulating hormone FSH concentrations varied significantly ( P < 0.01 ) with sampling time and the group difference in the pattern of variation was marginally significant (group× time interaction, P < 0.10, Fig. 3 ). The increase in FSH concentrations appeared to occur earlier in the early group than in the late group. Although there was a general trend towards rams in the early group having higher 8.02.5
~ t~
2 . 0
1.51.00.5OCT D A T E OF S A M P L I N G
Fig. 3. P l a s m a F S H c o n c e n t r a t i o n s ( m e a n + S E M ) o f r a m s in t h e e a r l y ( I O - - , n = 7 ) a n d late ( - - O - - , n = 5 ) g r o u p s d u r i n g t h e p e r i o d f r o m O c t o b e r 1989 to M a r c h 1990.
SEASONAL VARIATION IN TESTIS SIZE, LH AND FSH IN RAMS
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FSH concentrations than those in the late group, the group difference in mean FSH concentrations over all sampling periods was not significant (early group: 2.08+_0.32 ng m l - l ; late group: 1.43+_0.10 ng m l - l ; P > 0 . 1 0 ) . Rams in the early group had marginally higher FSH concentrations in December than rams in the late group (early group, 2.48 _+0.49 ng m l - 1; late group, 1.24 +_0.16 ng ml-I; P < 0 . 1 0 ) . L H responses to G n R H
challenge
Both the peak and total LH responses to GnRH were significantly ( P < 0.01 ) affected by sampling time (Table 2). Generally, both the peak and total LH responses decreased as the breeding season approached in March. There was a difference between groups in the pattern of seasonal variation in the total LH response to GnRH (group×time, P < 0 . 0 5 ) . Among rams in the early group, the total LH response increased slightly between November and December, and then decreased in January. For rams in the late group, there was :~ continu(~us decrease over the whole sampling period. No significant group differences in either the peak or the total LH response to GnRH were apparent. TABLE 2
Effect of sampling date on peak and total luteinising hormone ( L H ) responses to an i n t r a v e n o u s injection of exogenous gonadotrophin-releasing hormone ( G n R H ) ( 50 ng k g - 1 liveweight) for r a m s in the early and late groups 1'2 Sampling date
Group
I November
Early Late
13 December
Early
31 January
Early
20 March
Early
Late Late Late
Peak concentration
Total
( n g m l -I )
( m i n - n g ml -I )
8.36 _+0,99 7.67_+ 1,69 6.71 _+0.82 5.53 _+ 1,11 3.40_+ 0.61 4.25 _+0,83 4.03 _+ 1.35 3.15 "+ 0.62
503.32 _+68.47 465.55"+ 116.87 550.12 "+ 72.18 400.03 "+ 95.63 339.44_+ 51.43 377.00 -+ 96.51 290.63 _+42.67 263.67 "+ 65.8 l
NS ** NS
NS ** *
Significance Group Time
Group × t i m e
Early group n = 7; late group n = 5. Values presented are means _+SEM. 2There were no significant group differences in either the peak or the total LH response to G n R H at any sampling date. * P < 0 . 0 5 ; * * P < 0 . 0 1 ; NS, not significant.
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DISCUSSION
The present study investigated the pattern of seasonal changes in endocrine function of R o m n e y rams which were at the extremes of the breed with respect to patterns of seasonal variation in testis size. To achieve this, two groups of rams differing in timing of the seasonal increase in testis size were selected. The purpose of the selection procedure was to objectively select two extreme groups of rams that had either an early or a late increase in testis size during the transitional period from the non-breeding season to the breeding season and thus to simulate, as far as possible, the situation observed previously in the comparison of Poll Dorset (early increase) vs. Romney (late increase) rams (Xu et al., 1991 ). As the increase in testis size prior to the onset of the breeding season occurs before the s u m m e r solstice in R o m n e y rams (Pelletier and Almeida, 1987; Xu et al., 1991 ), the use of measurements made prior to the s u m m e r solstice should allow separation of rams which had an early or a late increase in testis size. The rationale behind these selection procedures was that if a ram had an earlier increase in testis size than the others in the group, the deviations of testis diameters for the ram would increase with time during the early measurement period. This would result in a positive regression coefficient, the magnitude of which reflected the rate of increase in testis size relative to the group as a whole. The converse was true if a ram had a late increase in testis size. In the present trial, the regression coefficients of three rams changed sign during the 1989-1990 season. However, taking into account the annual variation in the environmental conditions under which the rams were reared and the error associated with testis diameter measurements, the selection procedure appeared to be reasonably repeatable, objective and easy to implement. In both seasons, the increase in testis size occurred earlier for rams in the early group than those in the late group, but reached m a x i m u m values at about similar times of the year (see Figs. 1 and 2 ). The magnitude of seasonal variation in testis size was greater for rams in the early group than for rams in the late group during both seasons, although this effect was less pronounced during the 1989-1990 season than during the 1988-1989 season. Within-breed differences in the magnitude of seasonal variation in testis size have been reported previously (Ringwall et al., 1989 ). Together, these results show that there are differences among rams of the same breed, in the time and magnitude of seasonal variation in testis size, and that these differences appear to be repeatable from year to year. The liveweight of rams in the early group was significantly greater than that of rams in the late group. This was unexpected and it is not clear why the timing of the seasonal increase in testis size is related to liveweight. Such a relationship does not appear to have been reported previously, but could be important in the context of the present study. Thus, further studies are needed
SEASONAL VARIATION IN TESTIS SIZE, LH AND FSH IN RAMS
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to investigate the within-breed relationship between liveweight and the pattern of seasonal variation in testis size. If a consistent relationship exists, then selection for liveweight itself might be more efficient than selection on testis size. Haley et al. (1990) reported that selection for testis size corrected for liveweight in young ram lambs resulted in ewes in the high line being smaller and starting the breeding season earlier than ewes in the low line, However, without knowing the genetic correlations between liveweight and the patten1 of seasonal variation in testis size in the ram on one hand, and date of onset of the breeding season in the female progeny on the other, it is not possible to determine whether adjustment for liveweight is appropriate. In fact, if variation in both liveweight and the pattern of seasonal variation in testis size reflects the same genetic component for date of onset of the breeding season, the adjustment of liveweight may reduce the selection efficiency. Furthermore, a decline in ewe liveweight, such as that observed in the study of Haley et al. (1990), would probably be associated with a decline in carcass weight and wool production. The classification of rams into groups, based on timing of the seasonal increase in testis size prior to the onset of the breeding season, reflected differences in the endocrine function between rams in these groups. Rams in the early group had a significantly higher LH pulse frequency in March than those in the late group. As this group difference in LH pulse frequency occurred after testis size had reached its annual maximum, the relationship between the pattern of seasonal variation in LH pulse frequency and testis size is unclear. The time of the seasonal increase in plasma FSH concentrations was earlier for the early group than for the late group and this appeared to be closely related to the earlier increase in testis size of rams in the early group. The close association between the timing of seasonal variation in testis size and plasma FSH concentrations agrees with our previous study comparing Poll Dorset and Romney rams (Xu et al., 1991 ). This may indicate that, in terms of predicting genetic merit for aseasonality, the measurement of both testis diameters and FSH concentrations would improve accuracy of prediction only marginally compared with measurement of either parameter alone. FSH, acting via its receptors on the Sertoli cells, is an important hormone involved in the control of Sertoli cell hypertrophy and spermatogenesis (Amann and Schanbacher, 1983; Bardin et al., 1988). Although the relative importance of FSH and LH in the control of seasonal increase in testis size in the ram has not been determined, it is likely that both hormones are involved (Lincoln and Short, 1980). FSH secretion is non-pulsatile and its profile in blood can be characterised from a small number of blood samples. Therefore, it should be relatively easy to incorporate plasma FSH concentrations into selection programmes for early onset of the breeding season. However, there were some differences between the results obtained from the present study and those from our previous study comparing Poll Dorset and Romney rams
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(Xu et al., 1991 ). In the present study, an early increase in testis size of rams in the early group was associated with a greater magnitude of seasonal changes in testis size and LH pulse frequency. Conversely, in the between-breed comparison study, the early increase in testis size of Poll Dorset rams was associated with a smaller magnitude of seasonal variation in testis size and LH pulse frequency. As the relationships between the two components (timing and magnitude) of the pattern of seasonal variation in testis size and date of onset of the breeding season are not known, the significance of these differences between the two studies cannot be determined. It is possible that the relationship obtained from between-breed studies may differ from that within a breed (Blair et al., 1990). Nevertheless, the important finding from the present study was that there were large differences in the pattern of seasonal variation in testis size and gonadotrophin secretion among rams of the same breed. If a significant genetic correlation could be found between these parameters in the ram and date of onset of the breeding season in the ewe, it should be possible to incorporate these parameters in breeding programmes for early onset of the breeding season in the ewe. ACKNOWLEDGEMENTS
The authors are indebted to D.L. Burnham, M.F. Scott and Y.H. Cottam for technical assistance, L. Watts and W.B. Parlane for care of the animals, and all those who helped with blood sampling. The materials used in the FSH and LH assays and the G n R H study were kindly donated by the National Hormone and Pituitary Programme, and the National Institute of Diabetic and Digestive and Kidney Diseases, Bethesda, MD, USA. The financial support of the C. Alma Baker Trust is gratefully acknowledged.
REFERENCES Amann, R.P. and Schanbacher, B.D., 1983. Physiology of male reproduction. J. Anim. Sci., 57 (Suppl. 2): 380-403. Bardin, C.W., Cheng C.Y., Musto, N.A. and Gunsalus, G.L., 1988. The Senoli cell. In: E. Knobil and J.D. Neill (Editors), The Physiology of Reproduction. Raven Press, New York, pp. 933-974. Blair, H.T., McCutcheon, S.N. and Mackenzie, D.D.S., 1990. Physiological predictors of genetic merit. Proc. 8th Conf. Australian Association of Animal Breeding and Genetics, 5-9 February 1990, Hamilton and Palmerston North, New Zealand, pp. 133-142. Carter, M.L., McCutcheon, S.N. and Purchas, R.W., 1989. Plasma metabolite and hormone concentrations as predictors of genetic merit for lean meat production in sheep: effects of metabolic challenges and fasting. N.Z.J. Agric. Res., 32: 343-353. Guardabasso, V., Oerter, K.E., Iademarcl, M.F., Veldhuis, J.D. and Rodbard, D., 1988. DETECT. A Program for Detection and Characterization of Peaks and Estimation of Instantaneous Secretory Rate, ISR, in Studies of Pulsatile Hormone Secretion, Version 5.0. Labora-
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tory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, NIH, Bethesda, MD. Haley, C.S., Lee, G.J., Ritchie, M. and Land, R.B., 1990. Direct responses in males and correlated responses for reproduction in females to selection for testicular size adjusted for body weight in young male lambs. J. Reprod. Fertil., 89: 383-396. Haresign, W. and McLeod, B.J., 1985. Physiological criteria in genetic selection for aseasonality. In: R.B. Land and D.W. Robinson (Editors), Genetics of Reproduction in Sheep. Butterworths, London, pp. 291-300. Knight, T.W., Kannegieter, S.G. and Hamilton, G.J., 1989. Breeding season of Romney and Poll Dorset ewes at different locations and the reproductive penalties of a June joining. Proc. N.Z. Soc. Anim. Prod., 49: 265-270. Lincoln, G.A. and Short, R.V., 1980. Seasonal breeding: nature's contraceptive. Recent Prog. Horm. Res., 36: 1-52. Pelletier, J. and Almeida, G., 1987. Short light cycles induce persistent reproductive activity in lie-de-France rams. J. Reprod. Fertil. Suppl., 34:215-226. Rattray, P.V., 1986. Feed requirements for maintenance, gain and production. In: S.N. McCutcheon, M.F. McDonald and G.A. Wickham (Editors), Sheep Production, Vol. 2. Feeding, Growth and Health. New Zealand Institute of Agricultural Science, pp. 75-109. Ringwall, K.A., Wettemann, R.P. and Whiteman, J.V., 1989. Seasonal reproduction in rams: Influence of natural photoperiod on scrotal circumference, sexual flush, body weight and body condition score in mature F2 Finnish Landrace × Dorset rams. Sheep Res. J., 5 ( 3 ): 14-17. Statistical Analysis System Institute, 1988. SAS/STAT User's Guide, Release 6.03 edn. SAS Institute, Cary, NC, 1028 pp. Walkley, J.R.W. and Smith, C., 1980. The use of physiological traits in genetic selection for litter size in sheep. J. Reprod. Fertil., 59: 83-88. Xu, Z.Z., 1991. Endocrine and genetic control of seasonal breeding in sheep. PhD Thesis, Massey University, Paimerston North, New Zealand. Xu, Z.Z., McDonald, M.F., McCutcheon, S.N. and Blair, H.T., 1991. Seasonal variation in testis size, gonadotrophin secretion and pituitary responsiveness to GnRH in rams of two breeds differing in time of onset of the breeding season. Anim. Reprod. Sci., 26:281-292.
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Testis size, gonadotrophin secretion and pituitary responsiveness to GnRH in Romney rams during the transition from the non-breeding to the breeding season Z.Z. Xu', M.F. McDonald, S.N. McCutcheon and H.T. Blair Department of Animal Science, Massey University, Private Bag, Palmerston North, New Zealand (Accepted 13 March 1992 )
ABSTRACT Xu, Z.Z., McDonald, M.F., McCutcheon, S.N. and Blair, H.T., 1993. Testis size, gonadotrophin secretion and pituitary responsiveness to GnRH in Romney rams during the transition from the nonbreeding to the breeding season. Anim. Reprod. Sci., 31: 99-111. Romney rams, which had either an early ( n = 7 ) or a late ( n = 5 ) increase in testis size prior to the onset of the breeding season, were selected from a larger population (n = 60 ). In the following year. the two groups were compared for the pattern of seasonal variation in testis size, circulating gonadotrophin concentrations and pituitary responsiveness to a gonadotrophin releasing hormone (GnRH) challenge during the transitional period from the non-breeding to the breeding season. Rams in the 'early' group had an earlier and greater increase in testis size than rams in the 'late' group, a pattern which was largely repeatable between years. Differences between the groups in the pattern of seasonal variation in testis size were associated with group differences in endocrine function. Thus, rams in the early group had a significantly (P< 0.01 ) higher luteinising hormone (LH) pulse frequency in March (4.4 _+0.4 pulses per 8 h period) than those in the late group ( 1.7 _+0.3 pulses per 8 h period ) and the seasonal increase in plasma follicle stimulating hormone (FSH) concentrations occurred eailier (P< 0.10) in the early group than in the late group. There were also group differences in the pattern of seasonal variation in total LH response to the GnRH challenge. These results show that there are differences among rams of the same breed, in the pattern of seasonal variation in testis size and gonadotrophin secretion, which could potentially be used as criteria to select rams for date of onset of the breeding season in their daughters.
INTRODUCTION
In a previous study (Xu et al., 1991 ), we showed that the Romney and Poll Dorset breeds, which exhibit marked differences in seasonality of female Correspondence to: M.F. McDonald, Department of Animal Science, Massey University, Private Bag, Palmerston North, New Zealand. ~Present address: Department of Animal Science, 163 Animal Science Centre, University of Missouri-Columbia, MO 65211, USA.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0378-4320/93/$06.00
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breeding, also exhibited differences in male reproductive parameters, particularly the pattern of seasonal variation in testis size and gonadotrophin secretion during the transitional period from the non-breeding to the breeding season. These results suggest that seasonal variation in testis size and gonadotrophin secretion prior to onset of the breeding season might be useful predictors of a ram's genetic merit for early onset of the breeding season. Thus, in selection programmes for early onset of the breeding season, it might be possible to select rams based on these indicator traits instead of, or in combination with, direct selection for date of onset of the breeding season in the ewe (Walkley and Smith, 1980; Haresign and McLeod, 1985 ). Because of the greater selection pressure that can be applied on the ram compared with the ewe, the rate of genetic progress could be greatly improved by indirect selection in the ram (Xu, 1991 ). However, while between-breed comparisons are useful in identifying parameters which might be examined as potential indicator traits in a within-breed situation, differences found between breeds do not necessarily guarantee a cause and effect relationship within a breed (Blair et al., 1990) and relationships therefore have to be verified within breeds. Accordingly, the present study was conducted to investigate the extent to which within-breed variation exists, among rams of the highly seasonal Romney breed, in the pattern of seasonal changes in testis size and associated changes in plasma gonadotrophin concentrations and the pituitary responsiveness to a gonadotrophin releasing hormone ( G n R H ) challenge during the transitional period from the non-breeding to the breeding season. The study was based on two groups of Romney rams selected at the extremes of the population to represent animals with early or late seasonal increases in testis diameters, i.e. to parallel differences observed previously in the Poll Dorset vs. Romney comparison (Xu et al., 1991 ). MATERIALS AND METHODS
Animals and selection procedures Twelve Romney rams aged 3 years were used in this study. They were selected, from a group of 60 animals, on the basis of testis diameter measurements made at 2-3 week intervals during the transitional period from the non-breeding to the breeding season ( 1 October 1988-20 February 1989). Only the 7 measurements that were made prior to the summer solstice (22 December) were used for selection. First, values for testis diameters of each ram were expressed as deviations from the mean for all 60 rams, at each measurement time. Next, a linear regression model of deviations on time was fitted to each ram. The regression coefficients from this analysis were then ranked. The six rams with the greatest positive coefficients and the six with
SEASONAL VARIATION IN TESTIS SIZE, LH AND FSH IN RAMS
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the greatest negative coefficients were selected to represent rams with an early and a late increase in testis size, respectively. The normal date of onset of the breeding season for R o m n e y ewes under the local conditions (40. l ° S ) in which this study was conducted is the beginning of March (Knight et al., 1989; Xu, 1991 ).
Experimental procedures Commencing in early October 1989 (the non-breeding season following that in which the initial selection was m a d e ) , testis diameters of the selected rams were measured at intervals of 2-3 weeks (until March 1990) using the technique described by Xu et al. ( 1991 ). On four occasions during this period ( 1 November 1989, 13 December 1989, 31 January 1990 and 20 March 1990), the rams were brought indoors for intensive blood sampling and G n R H challenge studies. Ten days before each planned sampling date, the animals were transferred indoors from pasture and housed in metabolism crates in order to allow them to adjust to the experimental conditions and handling. The room in which the animals were housed had large windows and rams were exposed only to natural light. While indoors, the rams received a maintenance diet of chaffed lucerne hay, supplemented with minerals to counteract possible copper toxicity (Rattray, 1986). Fresh water was available ad libitum. The rams were returned to pasture 3 days after completion of each blood sampling period. While outdoors (i.e. between blood sampling periods), they were run as a single group separate from the ewe flock and grazed on mixed ryegrass and white clover pastures.
Blood sampling and GnRH challenges Jugular vein cannulae were inserted as described previously (Carter et al., 1989 ) 1 day before the date of blood sampling. At each sampling period, 6 ml of blood were collected at 15-min intervals for 8 hours commencing at 09:00 h. After the last sample was taken ( 17:00 h), each animal received an intravenous injection of G n R H at a dose of 50 ng kg- l liveweight. The G n R H was dissolved at a concentration of 1/~g m l - l in saline 1 h prior to injection and administered via the cannulae, followed by 4 ml of saline to flush the cannulae. Further blood samples were collected 10, 20, 30, 45, 60, 80, 100, 120, 150 and 180 min after G n R H injection. All blood samples were collected into heparinised tubes and immediately placed on ice. They were centrifuged within 25 min at 3000 g and 4°C for 20 min. The plasma was harvested and stored frozen at - 2 0 ° C in duplicate vials until hormone analyses were conducted.
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Hormone assays Plasma luteinising hormone (LH) and follicle stimulating hormone (FSH) concentrations were measured by homologous double-antibody radioimmunoassays using kits provided by the National Hormone and Pituitary Program and the National Institute of Diabetic and Digestive and Kidney Diseases ( N I D D K ) , Bethesda, MD. Assay protocols were as described previously (Xu et al., 1991 ). The LH reference preparation was NIADDK-oLH-25 and the assay sensitivity was 0.09 + 0.01 ng m1-1. The intra-assay coefficients of variation (CV) were 4.8%, 3.6% and 5.2%, and the inter-assay CV were 13.9%, 10.4% and 7.0%, at mean LH concentrations of 0.5 ng m1-1, 1.3 ng m l - 1 and 3.0 ng m1-1, respectively. The FSH reference standards was NIAMDD-oFSHRP-I and the assay sensitivity was 0.13 _+0.03 ng ml-1. The intra-assay CV were 10.5%, 11.0% and 5.1%, and the inter-assay CV were 13.4%, 12.5% and 9.8%, at mean FSH concentrations of 0.8 ng ml-1, 2.2 ng ml-~ and 4.4 ng m l - 1, respectively.
Data and statistical analyses Data relating to plasma LH concentrations in serial samples from individual rams at each sampling period were processed by the computer programme DETECT, to identify the presence of secretion pulses (Guardabasso et al., 1988 ). LH pulse frequency was expressed as the number of pulses occurring during an 8 h sampling period. Basal LH concentrations were obtained by averaging values for samples not associated with any LH pulse and mean LH concentrations were calculated by averaging values for all samples taken from each ram during the 8 h sampling period. LH pulse amplitude was determined as the difference between the highest LH concentration associated with an individual pulse and the basal concentration. LH responses to the GnRH challenges were characterised by the peak response (the highest LH concentration post-GnRH injection less the basal concentration) and the total response (measured by the area under the response curve above basal concentration, calculated by triangulation). Analysis of variance for repeated measurements was used to test the overall effects of group ('early' vs. 'late'), sampling time and group X time interaction on testis diameter and the endocrine parameters. In order to apply the repeated measures analysis to the testis diameter data, measurements taken on individual animals during each month were averaged to obtain a monthly mean for testis diameter. This parameter was then used in the analysis. To eliminate the heterogeneity of variance, data for LH pulse amplitude and the peak and total LH responses to G n R H were subjected to logarithm transformation before analysis. Comparison between means at different sampling times was carried out using orthogonal contrasts. Analyses within sampling
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periods were conducted using univariate analysis of variance. All analyses were performed using the SAS statistical package (Statistical Analysis System Institute, 1988 ). RESULTS
Testis diameter Testis diameter and liveweight of the selected rams during the 1988-1989 season are shown in Fig. 1. The mean testis diameter over the entire experim e n t period of rams in the early group (59.5_+0.9 m m ) was significantly (P < 0.01 ) greater than that of rams in the late group ( 53.7 _+0.7 m m ). There were also significant group differences in the pattern of seasonal variation in testis size ( g r o u p × t i m e interaction, P < 0 . 0 1 ). For rams in the early group, testis size increased 25% from a m i n i m u m of 53.4 m m in late October to a m a x i m u m of 66.6 m m in February. For rams in the late group, the corresponding increase was 16% from 50.1 m m in late December to 58.5 m m in February. The liveweight of rams in the early group ( 7 1 . 0 _ 0.7 kg) was sig70
65
90. 80-
70~60-
9--~" 50OCT
DATE OF MEASUREMENT
Fig. 1. Testis d i a m e t e r s ( m e a n + SEM, top ) a n d liveweight ( m e a n + SEM, b o t t o m ) o f rams in the early ( - - O - - , n = 6) a n d late ( - - (3 - - n = 6 ) groups during the period from October 1988 to February 1989.
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nificantly ( P < 0.01 ) greater than that of rams in the late group (59.5 _+0.5 kg). The classification of rams into two groups, based on the method described previously, was repeatable from year to year. Thus the correlation between ranks of the regression coefficients during the 1988-1989 and 1989-1990 seasons was 0.80 (Spearman rank correlation, P < 0.01 ). In the 1988-1989 season, the regression coefficients for rams in the early group were all positive, whereas those of rams in the late group were all negative (as this was the basis on which assignment to groups was m a d e ) . During the 1989-1990 season, the regression coefficients of three rams (one from the early group and two from the late group) changed sign. Because the endocrine study was carried out during the 1989-1990 season, it was considered appropriate to reclassify these animals based on the regression coefficients during the 1989-1990 season. Rams with positive regression coefficients (n = 7) were classified into the early group and those with negative regression coefficients (n = 5 ) were classified into the late group. Consequently, all analyses were based on the newly classified groups. Testis diameter and liveweights during the 1989-1990 season are shown in Fig. 2. The overall mean testis diameter (from October 1989 to March 1990) 706560-
5085 80 75
~ 7o
~
65. 60. 55. OCT
NOV
DEC
JAN
FEB
MAR
APR
DATE OF MEASUREMENT
Fig. 2. Testis d i a m e t e r s ( m e a n + SEM, t o p ) a n d liveweight ( m e a n + SEM, b o t t o m ) of rams in the (reclassified) early ( - - O - - , n = 7 ) a n d late ( - - O - - , n = 5 ) groups during the period from O c t o b e r 1989 to M a r c h 1990.
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of rams in the (reclassified) early group ( 59.2 _+0.8 m m ) was significantly ( P < 0.01 ) greater than that of rams in the late group (54.8 _ 0.5 m m ) . There were significant ( P < 0.01 ) differences between groups in the patterns of seasonal variation in testis diameter. Testis diameter increased 14% for rams in the early group from a minimum of 55.1 mm in late October to a maximum of 62.8 m m in February. The corresponding increase for rams in the late group was 10% from 52.5 m m in December to 57.7 m m in February. The liveweight of rams in the early group (75.6 _+ 1.1 kg) was significantly ( P < 0.01 ) greater than that of rams in the late group (66.9 _+ 1.1 kg). The liveweight varied significantly ( P < 0.01 ) with season, but seasonal changes in testis size did not appear to parallel seasonal changes in liveweight (correlations between testis diameters and liveweights across time were - 0 . 2 1 ( P > 0 . 1 0 ) and - 0 . 7 8 ( P < 0.01 ) for rams in the early and late groups, respectively).
Luteinising hormone The effects of group and sampling time on circulating LH concentrations are presented in Table 1. There were no significant differences, between the TABLE 1 Effect o f sampling date on basal and mean luteinising h o r m o n e ( L H ) concentrations, LH pulse frequency and LH pulse amplitude for rams in the early and late groups I Sampling date
1 November 13 D e c e m b e r 31 January 20 March
Group
Basal concentration (ngml -t )
Mean concentration (ngml -t )
Pulse frequency (per 8 h period)
Pulse amplitude 2 (ngml -I )
Early Late Early Late Early Late Early Late
0.29+0.02 0.27_+0.02 0.32_+0.02 0.29+0.01 0.34-+0.02 0.29_+0.01 0.36_+0.02 0.30-+0.01
0.30_+0.02 0.29_+0.01 0.43_+0.02 0.37_+0.03 0.40-+0.03 0.38_+0.02 0.43+0.03 a 0.33_+0.02 b
0.3_+0.2 0.5_+0.2 1.1 _+0.1 0.9_+0.2 2.8-+0.4 2.3_+0.2 4.4_+0.4 a 1.7_+0.3 c
0.94 0.44_+0.05 1.37_+0.21 1.19+0.08 0.63_+0.03 0.66_+0.06 0.56+_0.04 0.53_+0.04
NS ** NS
NS ** NS
* *** **
NS * NS
Slgnifi¢~ce Group Time G r o u p × time
i Early group n = 7; late group n = 5. Values presented are means + SEM. 2Only one complete LH pulse was present and used for calculating pulse amplitude for rams in the early group on l November. This m e a n therefore has no standard error. a,b'CMeans within each column and sampling date carrying different superscripts differed significantly (a vs. b, P < 0.05; a vs. c, P < 0.01 ). *P < 0.05; * * P < 0.01; ***P < 0.001; NS, not significant.
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early and late groups, in the overall basal LH concentrations (early group, 0.33 + 0.02 ng m l - ~; late group, 0.29 ___0.01 ng m l - ~) or the overall mean LH concentrations (early group, 0.39 + 0.03 ng m l - ~; late group, 0.34 + 0.02 ng m l - l ) . Both the basal and mean LH concentrations varied significantly ( P < 0 . 0 1 ) with sampling time, but there was no significant difference between groups in the pattern of this variation (group×time interaction, P > 0.10). However, the mean LH concentration of rams in the early group was significantly ( P < 0.05 ) higher than that of rams in the late group in March (Table 1 ). LH pulse frequency varied significantly ( P < 0 . 0 0 1 ) with sampling time, increasing as the breeding season approached. There was also a significant group difference in the pattern of seasonal variation in LH pulse frequency (group×time, P<0.01 ). Changes in LH pulse frequency were similar between November and January for both groups. However, LH pulse frequency of rams in the early group increased significantly ( P < 0.01 ) between January and March, whereas that of rams in the late group decreased slightly (Table 1 ). This resulted in rams in the early group having a significantly ( P < 0.01 ) higher LH pulse frequency than those in the late group. LH pulse amplitude varied significantly ( P < 0.05 ) with sampling time, but no significant effects of group or group × time interaction were found.
Follicle-stimulating hormone FSH concentrations varied significantly ( P < 0.01 ) with sampling time and the group difference in the pattern of variation was marginally significant (group× time interaction, P < 0.10, Fig. 3 ). The increase in FSH concentrations appeared to occur earlier in the early group than in the late group. Although there was a general trend towards rams in the early group having higher 8.02.5
~ t~
2 . 0
1.51.00.5OCT D A T E OF S A M P L I N G
Fig. 3. P l a s m a F S H c o n c e n t r a t i o n s ( m e a n + S E M ) o f r a m s in t h e e a r l y ( I O - - , n = 7 ) a n d late ( - - O - - , n = 5 ) g r o u p s d u r i n g t h e p e r i o d f r o m O c t o b e r 1989 to M a r c h 1990.
SEASONAL VARIATION IN TESTIS SIZE, LH AND FSH IN RAMS
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FSH concentrations than those in the late group, the group difference in mean FSH concentrations over all sampling periods was not significant (early group: 2.08+_0.32 ng m l - l ; late group: 1.43+_0.10 ng m l - l ; P > 0 . 1 0 ) . Rams in the early group had marginally higher FSH concentrations in December than rams in the late group (early group, 2.48 _+0.49 ng m l - 1; late group, 1.24 +_0.16 ng ml-I; P < 0 . 1 0 ) . L H responses to G n R H
challenge
Both the peak and total LH responses to GnRH were significantly ( P < 0.01 ) affected by sampling time (Table 2). Generally, both the peak and total LH responses decreased as the breeding season approached in March. There was a difference between groups in the pattern of seasonal variation in the total LH response to GnRH (group×time, P < 0 . 0 5 ) . Among rams in the early group, the total LH response increased slightly between November and December, and then decreased in January. For rams in the late group, there was :~ continu(~us decrease over the whole sampling period. No significant group differences in either the peak or the total LH response to GnRH were apparent. TABLE 2
Effect of sampling date on peak and total luteinising hormone ( L H ) responses to an i n t r a v e n o u s injection of exogenous gonadotrophin-releasing hormone ( G n R H ) ( 50 ng k g - 1 liveweight) for r a m s in the early and late groups 1'2 Sampling date
Group
I November
Early Late
13 December
Early
31 January
Early
20 March
Early
Late Late Late
Peak concentration
Total
( n g m l -I )
( m i n - n g ml -I )
8.36 _+0,99 7.67_+ 1,69 6.71 _+0.82 5.53 _+ 1,11 3.40_+ 0.61 4.25 _+0,83 4.03 _+ 1.35 3.15 "+ 0.62
503.32 _+68.47 465.55"+ 116.87 550.12 "+ 72.18 400.03 "+ 95.63 339.44_+ 51.43 377.00 -+ 96.51 290.63 _+42.67 263.67 "+ 65.8 l
NS ** NS
NS ** *
Significance Group Time
Group × t i m e
Early group n = 7; late group n = 5. Values presented are means _+SEM. 2There were no significant group differences in either the peak or the total LH response to G n R H at any sampling date. * P < 0 . 0 5 ; * * P < 0 . 0 1 ; NS, not significant.
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DISCUSSION
The present study investigated the pattern of seasonal changes in endocrine function of R o m n e y rams which were at the extremes of the breed with respect to patterns of seasonal variation in testis size. To achieve this, two groups of rams differing in timing of the seasonal increase in testis size were selected. The purpose of the selection procedure was to objectively select two extreme groups of rams that had either an early or a late increase in testis size during the transitional period from the non-breeding season to the breeding season and thus to simulate, as far as possible, the situation observed previously in the comparison of Poll Dorset (early increase) vs. Romney (late increase) rams (Xu et al., 1991 ). As the increase in testis size prior to the onset of the breeding season occurs before the s u m m e r solstice in R o m n e y rams (Pelletier and Almeida, 1987; Xu et al., 1991 ), the use of measurements made prior to the s u m m e r solstice should allow separation of rams which had an early or a late increase in testis size. The rationale behind these selection procedures was that if a ram had an earlier increase in testis size than the others in the group, the deviations of testis diameters for the ram would increase with time during the early measurement period. This would result in a positive regression coefficient, the magnitude of which reflected the rate of increase in testis size relative to the group as a whole. The converse was true if a ram had a late increase in testis size. In the present trial, the regression coefficients of three rams changed sign during the 1989-1990 season. However, taking into account the annual variation in the environmental conditions under which the rams were reared and the error associated with testis diameter measurements, the selection procedure appeared to be reasonably repeatable, objective and easy to implement. In both seasons, the increase in testis size occurred earlier for rams in the early group than those in the late group, but reached m a x i m u m values at about similar times of the year (see Figs. 1 and 2 ). The magnitude of seasonal variation in testis size was greater for rams in the early group than for rams in the late group during both seasons, although this effect was less pronounced during the 1989-1990 season than during the 1988-1989 season. Within-breed differences in the magnitude of seasonal variation in testis size have been reported previously (Ringwall et al., 1989 ). Together, these results show that there are differences among rams of the same breed, in the time and magnitude of seasonal variation in testis size, and that these differences appear to be repeatable from year to year. The liveweight of rams in the early group was significantly greater than that of rams in the late group. This was unexpected and it is not clear why the timing of the seasonal increase in testis size is related to liveweight. Such a relationship does not appear to have been reported previously, but could be important in the context of the present study. Thus, further studies are needed
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to investigate the within-breed relationship between liveweight and the pattern of seasonal variation in testis size. If a consistent relationship exists, then selection for liveweight itself might be more efficient than selection on testis size. Haley et al. (1990) reported that selection for testis size corrected for liveweight in young ram lambs resulted in ewes in the high line being smaller and starting the breeding season earlier than ewes in the low line, However, without knowing the genetic correlations between liveweight and the patten1 of seasonal variation in testis size in the ram on one hand, and date of onset of the breeding season in the female progeny on the other, it is not possible to determine whether adjustment for liveweight is appropriate. In fact, if variation in both liveweight and the pattern of seasonal variation in testis size reflects the same genetic component for date of onset of the breeding season, the adjustment of liveweight may reduce the selection efficiency. Furthermore, a decline in ewe liveweight, such as that observed in the study of Haley et al. (1990), would probably be associated with a decline in carcass weight and wool production. The classification of rams into groups, based on timing of the seasonal increase in testis size prior to the onset of the breeding season, reflected differences in the endocrine function between rams in these groups. Rams in the early group had a significantly higher LH pulse frequency in March than those in the late group. As this group difference in LH pulse frequency occurred after testis size had reached its annual maximum, the relationship between the pattern of seasonal variation in LH pulse frequency and testis size is unclear. The time of the seasonal increase in plasma FSH concentrations was earlier for the early group than for the late group and this appeared to be closely related to the earlier increase in testis size of rams in the early group. The close association between the timing of seasonal variation in testis size and plasma FSH concentrations agrees with our previous study comparing Poll Dorset and Romney rams (Xu et al., 1991 ). This may indicate that, in terms of predicting genetic merit for aseasonality, the measurement of both testis diameters and FSH concentrations would improve accuracy of prediction only marginally compared with measurement of either parameter alone. FSH, acting via its receptors on the Sertoli cells, is an important hormone involved in the control of Sertoli cell hypertrophy and spermatogenesis (Amann and Schanbacher, 1983; Bardin et al., 1988). Although the relative importance of FSH and LH in the control of seasonal increase in testis size in the ram has not been determined, it is likely that both hormones are involved (Lincoln and Short, 1980). FSH secretion is non-pulsatile and its profile in blood can be characterised from a small number of blood samples. Therefore, it should be relatively easy to incorporate plasma FSH concentrations into selection programmes for early onset of the breeding season. However, there were some differences between the results obtained from the present study and those from our previous study comparing Poll Dorset and Romney rams
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(Xu et al., 1991 ). In the present study, an early increase in testis size of rams in the early group was associated with a greater magnitude of seasonal changes in testis size and LH pulse frequency. Conversely, in the between-breed comparison study, the early increase in testis size of Poll Dorset rams was associated with a smaller magnitude of seasonal variation in testis size and LH pulse frequency. As the relationships between the two components (timing and magnitude) of the pattern of seasonal variation in testis size and date of onset of the breeding season are not known, the significance of these differences between the two studies cannot be determined. It is possible that the relationship obtained from between-breed studies may differ from that within a breed (Blair et al., 1990). Nevertheless, the important finding from the present study was that there were large differences in the pattern of seasonal variation in testis size and gonadotrophin secretion among rams of the same breed. If a significant genetic correlation could be found between these parameters in the ram and date of onset of the breeding season in the ewe, it should be possible to incorporate these parameters in breeding programmes for early onset of the breeding season in the ewe. ACKNOWLEDGEMENTS
The authors are indebted to D.L. Burnham, M.F. Scott and Y.H. Cottam for technical assistance, L. Watts and W.B. Parlane for care of the animals, and all those who helped with blood sampling. The materials used in the FSH and LH assays and the G n R H study were kindly donated by the National Hormone and Pituitary Programme, and the National Institute of Diabetic and Digestive and Kidney Diseases, Bethesda, MD, USA. The financial support of the C. Alma Baker Trust is gratefully acknowledged.
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