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Comparison of seasonal changes in reproductive parameters of adult male European fallow deer (Dama dama dama) and hybrid Mesopotamian × European fallow deer (D. d. mesopotamica × D. d. dama)
SCIENCE EISEVIER
Animal Reproduction Science 45 ( 1996) 20 I-2 15
Comparison of seasonal changes in reproductive parameters of adult male European fallow deer ( Dama dama dama) and hybrid Mesopotamian X European fallow deer ( D. d. mesopotamica X D. d. dama) G.W. Asher av*, D.K. Berg b, S. Beaumont b, C.J. Morrow ‘, K.T. O’Neill a, M.W. Fisher a aAgReResearch,Invermay Agricultural Centre. Private Bag 50034, Mosgiel, New Zealand b AgResearch, Rukuru Agricultural Centre. Private Bag 3123. Hamilton, New Zeuiund ’Nationul Zoological Purk, Conservation und Research Center. Smithsonian Institution, Front Royal VA 22630. USA
Accepted 9 April 1996
Abstract In a study, aimed at comparing seasonal reproductive development of European fallow deer dama dama) with Mesopotamian (D.d. mesopotumicu) X European F, hybrids, five adult males of each genotype, which had been raised together since birth, were maintained as a bachelor group. Morphometric (body weight, neck circumference and testis diameter), endocrine (plasma testosterone concentrations) and seminal (ejaculate volume, spermatozoa per ejaculate and spermatozoa motility) parameters were recorded at fortnightly or monthly intervals for a 15-month period, and antler status was noted daily during the general periods of casting and velvet stripping. In addition, two bucks of each genotype were blood sampled via indwelling jugular catheters every 30 min for 24-h periods on five occasions (2-3 months intervals) during the year, and plasma was analysed for concentrations of testosterone and LH. Parameter profiles of the two genotypes were compared by global and time series ante-dependence covariance analysis to investigate overall profile similarity and the seasonal nature of any observed differences. Plasma hormone profiles from high-frequency blood sampling were subjected to PULSAR analysis to determine pulse frequency and amplitude. Throughout the study hybrid males were = 30% (Dama
* Corresponding author. 0378-4320/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)015771
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heavier than European males. However, both genotypes exhibited dramatic but parallel patterns of body weight change (global P = 0.054). Neck circumference was correlated with body weight throughout (P < O.OS),with similar regression slopes between the genotypes at any sampling time (P > 0.10). Covariance adjustment to a common initial body weight was performed to eliminate the effects of large body weight differences on muscle hypertrophy and regression. While profiles of corrected neck circumference were significantly different at the global level (P < O.Ol>, analysis by time revealed differences occurring only during the latter period of muscular regression in spring. However, profiles of other parameters, including testis diameter, plasma testosterone concentrations, spermatozoa per ejaculate and percentage motile spermatozoa, exhibited significant displacement between genotypes (global P < 0.05) evident as 2-4 weeks advancement in the sexual development (late summer/autumn) and quiescence (spring) phases for hybrid males relative to European males. Furthermore, mean dates of antler casting and velvet stripping were significantly earlier by 2-3 weeks for hybrid males than European males (P < 0.05). High frequency blood sampling revealed markedly seasonal patterns of secretion of testosterone and LH, with hybrid males exhibiting an apparent earlier onset of high-amplitude testosterone ‘surges’ in February (late summer) compared to those occurring in April (autumn) for European males. When viewed collectively, the data indicate strongly that the Mesopotamian influence is evident in the earlier attainment of sexual development and fertility in late summer and autumn, and earlier onset of sexual quiescence in spring. This is in accord with anecdotal information on earlier reproductive patterns in purebred Mesopotamian fallow deer. Keywords:
Fallow deer; Dama dama; Genotype; Hybridisation; Breeding season
1. Introduction Amongst the cervidae there are numerous taxa with close genetic affiliations (Whitehead, 1972). Recent trends into domestication and captive propagation have been associated with deliberate or inadvertent hybridisation between taxa (Short, 1985). In some instances, this may confer considerable economic advantage through additive genetic effects and putative heterotic effects of hybridisation on growth performance of farmed deer, as demonstrated with subspecies hybridisation of red deer (Cervus e&&us scoticus) with North American wapiti (C. e. nelsoni, C. e. rooseueltii or C. e. manitobensis) (Moore and Littlejohn, 1989; Fennessy et al., 1991). The farming of fallow deer (Dama dama) has gained acceptance and momentum in Australasia and North America within the last decade. While this is based largely on the common European subspecies (D. d. dumu), a significant proportion of farms have incorporated a component of hybridisation into their operations, using various levels of crossbreeding with the rare Mesopotamian subspecies (D. d. mesopotumicd to improve production (Morris, 1993). The Fl hybrid (Mesopotamian sire x European dam) and subsequent maternal and paternal backcrosses are reputed to exhibit increased growth rates and overall mature body size over the purebred European subspecies (Lenz et al., 1993). It is also mooted that there is a significant shift in reproductive seasonality with hybrids that reflects a generally earlier breeding season of the Mesopotamian subspecies (Otway, 1993). However, there are little objective data on reproductive seasonality of either Mesopotamian fallow deer or their hybrids. In contrast, there is considerable
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recent information on seasonality of European fallow deer (Asher, 1985; Asher et al., 1989; Schnare and Fischer, 1987). The aim of the present study was to compare seasonal reproductive development of adult male fallow deer of the European subspecies with the F, hybrid by monitoring somatic and testicular growth, secondary sexual characteristics, semen production and hormone secretory patterns.
2. Materials and methods 2.1. Animals and management
Ten mature (4 years old) fallow deer bucks, born and raised on the Ruakura Agricultural Centre (37”46’S, 175”20’E), were used in this study. Five bucks were of the European subspecies, progeny from natural mating of does and bucks derived from wild New Zealand stock. The remaining five bucks were F, hybrids between the European and Mesopotamian subspecies, progeny from artificial insemination of European does with semen of Mesopotamian bucks imported into New Zealand from Germany in 1987. The ten bucks were managed on pasture as a single group throughout the study, having been raised together since birth. While maintained as a bachelor group, the bucks were never more than 150 m distant from females of the same species. 2.2. Gross morphometry and semen collection At fortnightly or monthly intervals from 1 September 1992 to 20 November 1993 (5: 15 months) the bucks were mustered into covered handling yards. They were guided individually into a restraining cradle where they received an intramuscular injection of ketamine hydrochloride (5 mg kg-’ liveweight, Ketaset, Aveco Co., Fort Dodge, IA, USA) and xylazine hydrochloride (2.5 mg kg- ‘, Rompun, Bayer Leverkusen, German) to induce disassociative anaesthesia. Upon recumbancy they were weighed, blood sampled by jugular venepuncture into heparinised vacutainers, their neck circumference was measured cranial to the larynx with a plastic tape, and their testis diameter was measured with electronic calipers (Asher et al., 1987; Asher et al., 1989). Semen was collected on each occasion by electroejaculation (Asher et al., 1987; Asher et al., 1992). A sine-wave electroejaculator (AC 20-60 Hz) and rectal probe (Lane Pulsematic III; Lane Manufacturing, Co., USA) were used to administer 15-30 incremental stimuli of between 1.5 and 5.0 volts. Stimuli were administered in pulses of 3-5 s and were continued until either fluids ceased to be presented for 3-4 consecutive pulses or the ejaculate became clear (aspermatic). Semen was collected into pre-warmed glass cups and kept at 31°C in a warmed container until transferred to the laboratory. Following semen collection, anaesthesia was reversed by intravenous injection of yohimbine hydrochloride (0.4 mg kg- ’ liveweight, Recervyl, Aspiring Animal Services. Wanaka, NZ) and the bucks returned to pasture. Antler status of the bucks (i.e. hard, cast or velvet) was recorded daily during the general expected periods of casting and velvet stripping.
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2.3. Semen evaluation The semen was transferred to a water bath at 37°C immediately after collection and assessed for volume in a graduated glass pipette. Concentration of spermatozoa was determined by haemocytometer count following dilution of 20 ~1 raw semen in 200 ml 2% saline solution plus 0.01% formaldehyde. Percentage motility (to nearest 5%) was appraised visually under phase-contrast optics ( X 200) based on a 10 ~1 sample diluted in 100 ml of phosphate buffered saline solution. 2.4. Intensive blood sampling On five occasions during the year (2-3 month intervals) four of the bucks (two European and two hybrid bucks) were separated from the group in the yards and fitted with jugular catheters (Asher and Peterson, 1991) before being returned to pasture. On the following morning they were again mustered into the yards, where they remained for the next 24 h. During this time they were repeatedly restrained and blood sampled (5 ml) at 30 min intervals. Water and meadow hay were provided ad libitum. Blood samples were centrifuged within 30 min of collection and the plasma stored at - 10°C until assayed. 2.5. Hormone radioimmunoassays Plasma LH concentrations were determined in duplicate using a heterologous radioimmunoassay procedure described for ovine plasma (Scaramuzzi et al., 1970) and validated for fallow deer plasma (Asher et al., 1986). The LH antibody, raised in a rabbit against NIH-LH-S , , , was used in the assay at a final dilution of 1:200 000. Cross-reactivity with other proteins has been described previously (Kelly et al., 1982; Asher et al., 1986). The inter-assay coefficients of variation, calculated from determinations of cervine control samples, were 15.8% for low (mean = 1.l 1 ng ml-’ ), 6.7% for medium (5.25 ng ml-‘) and 8.2% for high (10.40 ng ml-‘) samples. The intra-assay coefficient of variation was 12.7%, 6.2% and 7.3% for the three control samples respectively and sensitivity of the standard curve was 0.03 ng NIH-LH-S, , per tube (0.30 ng ml- ’>. Plasma testosterone concentrations were determined in duplicate using an extraction radioimmunoassay similar to that described by Asher et al. (1987). The antiserum, raised in an entire ewe against testosterone-human serum cy globulin conjugate, cross-reacted with androstenedione (1.5%) and dihydrotestosterone (5.5%) but exhibited negligible binding (< 0.6%) with cholesterol, oestradiol, progesterone and a range of other androgens. Efficiency of recovery by extraction of radioactive ligand was 84.1% and parallelism was demonstrated between buffer standards, charcoal-stripped plasma standards and serially diluted plasma samples containing high concentrations of immunoreactive testosterone. Control plasma samples, with mean values of 32.8, 16.2, 8.4 and 3.5 nmol testosterone, were placed at regular intervals in each assay. Inter-assay coefficients of variation were 16.8, 10.0, 13.2 and 9.9%, respectively and the intra-assay coefficients of variation were 10.1, 8.2, 9.4 and 7.9%, respectively. Sensitivity of the standard curve was 1.4 nmol testosterone.
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2.6. Statistical analysis
Data summaries of morphometric, plasma testosterone and semen data in the form of graphs of the means of the two genotypes with the standard errors of differences (SED) for each time point measurements were prepared for the 1%month observation period. The profiles were compared using the ante-dependence covariance structure as proposed by Kenward (1987). The method is ideally suited to this situation in which no specific features of the profiles are, a priori, known to be of interest. After establishing the order of the ante-dependence structure and performing a global test of the difference between the two profiles, the technique was used to investigate the form of the difference amongst the profiles. This provides an improvement over the procedure of calculating a separate r-test for each time of measurement. LH and testosterone profile from high-frequency blood sampling were analysed separately for each buck at each sampling session using the PULSAR pulse identification routine (Merriam and Wachter, 1982). Pulse analyses were carried out for 50 points, with inputs for PULSAR including intra-assay standard deviation and a smoothing time of 180 min. A cut-off value of 2.7 standard deviation units was used for peak splitting.
3. Results At all times during the study period hybrid males were significantly heavier in body weight than European males (P < 0.05 for individual t-tests), with an overall difference between respective means of between 18 and 25 kg (Fig. 1). The proportional change in body weight from the start of the study appeared to follow similar trends for the two genotypes. While a global test of ante-dependence covariance indicated a marginally significant difference between the profiles of the two genotypes (P = 0.0541, further analysis indicated only one time in early January when a significant difference occurred (P = 0.028; Fig. 1). In general, the patterns of body weight change were characterised by rapid gains (130- 170 g day- ’) between October and February (spring and summer), precipitous reductions in weight (350-420 g day-‘) over a 3-4 week period in April/May (autumn rut> and maintenance through the period from June to September (winter). The periods of retention of hard antler stubbs (buttons) were of similar duration (= 7 months) for the two genotypes (Fig. 1). However, the hybrid males initiated velvet stripping 2 weeks earlier than did the European males (8 February vs 21 February; P < 0.05) with this event being highly synchronised (i.e. within 24 h) within each genotype. Similarly, antler casting was on average 2-3 weeks earlier for hybrid males (5 September f 2.2 days vs 24 September + 1.8 days; P < 0.05) but was less synchronous within genotype than was velvet stripping (Fig. 1). Profiles of mean testis diameter (Fig. 2) reveal parallel patterns of seasonal changes having occurred between the two genotypes, although significant differences between means were observed around the times of complete testicular regression (SeptemberNovember) and maximal testicular development (March-April). The global test of ante-dependence covariance highlighted a highly significant difference between geno-
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Fig. 1. Profiles of mean liveweight and liveweight as a proportion of initial liveweight, superimposed on the individual time sequences of hard antler retention, for adult male European fallow deer (0; n = 5) and F, hybrid Mesopotamian XEuropean fallow deer (0; n = 5). Vertical bars and associated asterisks indicate the SED and significant differences (P < 0.09, respectively, based on pooled variance and individual r-tests. Significance and order of the global ante-dependence covariance test are presented for each pair of profiles, and crosses indicate times when the profiles were shown subsequently to be significantly different (P < 0.05).
types in the overall profiles of mean testis diameter (P = 0.003) and subsequent analysis revealed differences occurring around peak testicular development and recrudescent phases (Fig. 2). These differences indicate a temporal displacement of testicular growth profiles of about 2 weeks. Although the maximal mean testis diameter was similar ( = 52 mm) between the hybrid and European males (albeit 2 weeks apart), hybrids exhibited significantly smaller mean testis diameter during the spring nadir (e.g. 27.4 + 0.8 mm vs 31.5 * 0.9 mm; P < 0.05). There were no significant within-genotype correlations between body mass and testis diameter at any time during the study (P > 0.05).
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Fig. 2. Profiles of mean testes diameter, liveweight-corrected neck circumference and plasma testosterone concentrations for adult male European fallow deer (0; n = 5) and F, hybrid Mesopotamian X European fallow deer (0; n = 5). Vertical bars and associated asterisks indicate the SED and significant differences (P < 0.05). respectively, based on pooled variance and individual t-tests. Significance and order of the global ante-dependence covariance test are presented for each pair of profiles, and crosses indicate times when the profiles were shown subsequently to be significantly different (P < 0.05).
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Absolute neck circumference (not shown) differed significantly between the two genotypes at all times during the study. However, there were significant positive within-genotype correlations between body weight and neck circumference throughout the study period (P < 0.05). Furthermore, regression slopes on any sampling date were not significantly different for the two genotypes (P > O.lO), with exceptions to this occurring on only two sampling dates in July. Covariance adjustment to a common initial live weight enabled analysis of corrected neck circumference (CNC) that largely eliminated the effects of the between-genotype difference in absolute body weight (Fig. 2). Both genotypes exhibited a markedly seasonal pattern of neck muscle hypertrophy and regression that closely paralleled testicular changes. Although the global test of ante-dependence covariance of both corrected and non-corrected neck circumference indicated a highly significant difference between the mean profiles (P < O.OOl>,analysis by time revealed differences occurring only during the period of muscular regression towards the end of the study period (September and October). Similarly, the global test of mean plasma testosterone concentration profiles (Fig. 2) indicated a highly significant difference between genotypes (P = 0.003) although time analysis revealed sporadic points of significance. Most notable was the apparent displacement of peak mean concentrations occurring in early March (hybrids males) and early April (European males). Of 277 attempts at electroejaculation, fluids were collected on 275 occasions. Ejaculates varied from opaque spermatozoa-rich collections to clear aspermatic fluid collections, depending on season. On three occasions ejaculates were contaminated with copious quantities of urine and were immediately discarded. Statistically, analysis was based on 272 valid collections fairly evenly distributed across the two genotypes (134: 138). Semen parameters (Fig. 3) showed distinct seasonal patterns reflecting alternating periods of testicular activity and quiescence. For both genotypes, mean ejaculate volume was lower during the testicular regression phase (October-December) than at other times of the year (P < 0.05). However, mean volumes were significantly different between genotypes on only one date in January and the global test of ante-dependence covariance revealed no difference in overall profiles (P = 0.923). Other seminal parameters of spermatozoa population and viability displayed significant differences between genotypes at both a global level and at various times during the study (Fig. 3). In particular, mean total spermatozoa per ejaculate and mean percentage motility differed during the testicular recrudescence phase (November-February) and at the initiation of testicular regression (July-August). In both periods, hybrids appeared to initiate events 2-4 weeks earlier than European males. Interestingly, both genotypes exhibited a temporary decline in both the mean number of spermatozoa per ejaculate and the mean proportion of motile spermatozoa presented in ejaculates during the period of peak rutting activity (i.e. vocalisations), being early April for hybrids and late April-early May for European males (Fig. 3). Seasonal patterns of LH and testosterone secretion were evident from the highfrequency blood sampling programme (Fig. 4). There were low incidences of LH and testosterone pulses (i.e. O-l pulses per 24 h) for both genotypes in September and December profiles. Pulse frequency of both hormones increased dramatically in the February profiles (i.e. during the transition into the breeding season). However, LH
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pulse frequency was greater for the European bucks (S-12 pulses per 24 h) than for the hybrid bucks (4-9 pulses per 24 h), with pulsable secretion of testosterone tending to mirror LH pulse patterns for European bucks. While testosterone secretion was similar in hybrid bucks, the pulses were of higher amplitude (0.5 vs 1.0 nmol) and of longer duration. There was a notable absence of detectable LH pulses in April (early breeding season) for all bucks. However, episodic secretion of testosterone was evident as 3-4 h ‘surges’ for the European bucks, but such events were absent from the profiles of hybrid bucks. Lower amplitude testosterone surges were again observed in June for European bucks but were not evident for hybrid bucks (Fig. 4).
4. Discussion While it is clear that both fallow deer genotypes in the present study exhibited pronounced reproductive seasonality, the data collectively indicate a degree of temporal displacement between them. The magnitude of this displacement, however, was only in the order of 2-3 weeks, representing a single sampling interval for most parameters investigated. Nonetheless, some of the morphometric, endocrine and seminal parameters measured highlighted differences between the two genotypes at key times during the annual reproductive cycle despite possible social interaction effects (e.g. proximity of oestrous does) that may have served to promote within-herd synchrony. The annual cycle of retention, casting and regrowth of antlers provides the most conclusive indicator of seasonality displacement. The highly seasonal nature of these events has been well described for various temperate cervid species, including European fallow deer (Chapman and Chapman, 1975; Schnare and Fischer, 1987). In the present study there was a startling degree of within-genotype synchrony in the velvet stripping process in late summer, with all hybrid bucks cleaning their antlers on 8 February and all European bucks doing likewise on 21 February. A similar disparity in mean antler casting dates (i.e. about 2 weeks) was also evident but this event was not subject to such synchrony. Clearly, the antler cycle is indicative of underlying endocrine changes, principally those related to testicular development and regression. The annual antler cycle has been correlated with annual fluctuations in testicular secretion of testosterone for numerous cervid species (Wislocki, 1949; Lincoln, 1971; Chapman, 1975). In the present study on fallow deer, endocrine parameters did reflect differences between the two genotypes. In particular, there was a 2-week (single sampling interval) displacement in peak mean concentrations of plasma testosterone from single samples collected during the study, despite the markedly episodic pattern of testosterone secretion described previously for fallow deer (Asher et al., 1989; Asher and Peterson, 1991). Furthermore, high-frequency blood sampling at various times during the year indicated differences between genotypes in the patterns of episodic secretion of both LH and testosterone during the testicular recrudescence phase in summer and at the approximate time of the rut in autumn. The characteristic ‘surge’ patterns of testosterone secretion occurring at the peak of sexual development (Asher et al., 1989) appeared to be instigated in summer in hybrids, but were not apparent until autumn in the European bucks. Collectively,
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antler and endocrine data indicate earlier attainment of sexual development in the hybrid than the European animals. With the exception of body weight, other morphometric and seminal parameters indicated, to varying degrees, temporal displacement between the two genotypes. Absolute body weight differences were very obvious, with hybrids being approximately 30% larger than their European herdmates throughout the study. This undoubtedly represents a genetic effect on growth and mature body size (Morris, 1993; Lenz et al., 1993). However, proportional change in body weight was essentially similar for the two genotypes and did not reflect any seasonal disparity despite dramatic seasonal patterns being evident. It is probable that changes in body mass are also determined by factors other than endogenous endocrine regulation (e.g. fluctuations in feed supply, temperature and time of day), factors that may mask the breeding season disparity. Furthermore, the rapid decline in body weight during the rut may have been influenced strongly in both genotypes by the proximity of oestrous European fallow deer does on the same farm, leading to a strong social inductive effect on the precise timing of rutting behaviour (and hence, inappetence) of bucks. For both genotypes, profiles of testes diameter and of neck circumference showed approximately parallel seasonal changes, highlighting the influence of the testes on annual cycles of neck muscle hypertrophy and regression (Field et al., 1985). Testicular development was displaced temporarily by about 2 weeks between the two genotypes, with developmental differences being particularly pronounced during phases of testicular enlargement and regression. However, neck circumference changes measured in this study were not markedly different between the two genotypes. Seasonal changes in body weight clearly had a major influence on neck dimensions over and above any testicular influences, and the obvious difference between genotypes in overall body weight (= 30%) was similarly reflected in absolute neck circumference on any sampling date. However, covariance adjustment largely eliminated the body weight effect but still revealed little seasonal difference in neck muscle hypertrophy and regression between the two genotypes. This is somewhat unexpected given the known association between the testes and specific musculature of the neck. We speculate that, either, the measurement of neck dimensions is subject to imprecision (although the very obvious protruding larynx of male fallow deer does allow repeatable siting of the measuring tape>, there are additional influences of changing body mass on neck muscle developments that could not be eliminated by covariance analysis, or there exists a different interaction between testes development and muscular hypertrophy/regression between the two genotypes. This does not lessen, however, the profound nature of seasonal changes in neck musculature associated with the reproductive cycle in adult male fallow deer (Asher et al., 1987; Schnare and Fischer, 1987). It is interesting to note that testicular size was essentially similar between the two genotypes despite the obvious disparity in body weight. In fact, the larger hybrid males had significantly smaller testes at the nadir of testicular cycle. While this was in no way reflected in fertility in this or other studies (Asher et al., 19921, the phenomenon has also been observed in hybridisation between P&e David’s deer (EZuphurus dauidiunus) and red deer (D. Goosen, personal communication). Unfortunately, we have little or no information on the relationship between testis size and body mass in the paternal taxa,
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the purebred Mesopatamian fallow deer and the P&e David’s deer, and therefore cannot conclude whether or not it is a consequence of hybridisation. The seminal parameters provided interesting insights into testicular function of the two fallow deer genotypes. Distinct temporal displacement was evident for indices of spermatozoa1 output (e.g. spermatozoa per ejaculate) and viability (e.g. % motile spermatozoa). This was perhaps accentuated by the fact that both genotypes exhibited a distinct period of aspermatogenesis in early summer, as indicated by ejaculates completely devoid of spermatozoa. The reinstigation of spermatogenesis occurred about 2 weeks earlier in the hybrids. However, the subsequent cessation of spermatogenic activity in late spring was not as well separated temporally between the two genotypes, with both presenting aspermatic ejaculates for the first time on the same sampling date. Interestingly, both genotypes exhibited a distinct temporary reduction (30-N%) in mean spermatozoa per ejaculate and mean percentage motile spermatozoa early into the breeding season, with an apparent genotype displacement of 2-4 weeks. In the case of the European subspecies, at least, this reduction in spermatozoa output and quality corresponded with the expected timing of peak rutting activity in April, and is a phenomenon that the authors have encountered previously in commercial semen collection operations. We speculate that this event may have been due to either a high secretory output of testosterone during the immediate pre-rut and rut periods (Asher et al., 1989) having an inhibitory influence on spermatogenesis, as seen in rams treated with exogenous testosterone (Courot et al., 1979; Schanbacher, 1980), or was a consequence of repeated natural ejaculation during rutting fervour (despite being in a bachelor group) leading to deminished semen reserves. In summary, collective data on the morphometric, endocrine and seminal traits indicate strongly that the Mesopotamian influence is evident in the earlier attainment of sexual development and fertility in late summer and autumn. However, the data are more equivocal in relation to the onset of sexual quiescence, although the overall impression is that the entire seasonal repertoire of the two genotypes in the study displays a phase-shift of about 2-3 weeks between them. These data support our unpublished studies on females, whereby hybrids (25% Mesopotamian, 75% European; maternal backcross) exhibited earlier initiation and cessation of oestrous cycles relative to European does. Unfortunately, there is little information on the seasonal reproductive patterns of the paternal species. However, in accord with anecdotal information (Otway, 19931, it is likely that purebred Mesopotamian fallow deer exhibit considerable disparity with European fallow deer (i.e. 4-5 weeks earlier) in reproductive seasonality. This is, perhaps only one example of many where genetically related cervid taxa with differing patterns of reproductive seasonality produce hybrids that exhibit intermediate seasonality. For example, Loudon et al. (1989) demonstrated convincingly the widely disparate seasonality of red deer and P&e David’s deer. Recently these two species (assigned to different genera) have been hybridised to produce fertile offspring (Asher et al., 1988; Fennessy et al., 1991). It will be interesting to see if the hybrids exhibit predictable reproductive seasonality. Such studies on these, and other hybrids, may provide interesting insights into the central control mechanisms of seasonality, as well as providing a useful genetic resource that will enable selection of farmed deer better suited to different seasonal environments.
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Acknowledgements
We thank P. Johnstone and D. Duganzich for statistical advice, T. Manley, R. Porteous and B.G. Mockett for assistance with testosterone radioimmunoassays and staff of the Ruakura Deer Unit for animal management. We acknowledge the contribution of the late Dr Whitley Otway in establishing the mesopotamian fallow deer in the southern hemisphere.
References Asher, G.W., 1985. Gestrous cycle and breeding season of farmed fallow deer, Dama dama. J. Reprod. Fertil., 75: 521-529. Asher, G.W. and Peterson, A.J., 1991. Pattern of LH and testosterone secretion of adult male fallow deer (Dama dama) during the transition into the breeding season. J. Reprod. Fertil., 91: 649-654. Asher, G.W., Adam, J.L., Otway. W., Bowmar, P., van Reenan, Cl., Mackintosh, C.G. and Dratch, P., 1988. Hybridization of P&e David’s deer (Elaphurus dauidianus) and red deer (Cerous elaphus) by artificial insemination. J. Zool., Lond., 215: 197-203. Asher, G.W. Barrell, G.K. and Peterson, A.J., 1986. Hormonal changes around oestrus of farmed fallow deer, Dama dama. J. Rep&. Fertil., 78: 487-496. Asher, G.W. Day, A.M. and Barrell, G.K., 1987. Annual cycle of liveweight and reproductive changes of farmed male fallow deer (Dama dama) and the effect of daily oral administration of melatonin in summer on the attainment of seasonal fertility. J. Reprod. Fertil., 79: 353-362. Asher, G.W. Morrow, C.J. Jabbour, N, Mulley, R.C. Veldhiusen, F.A. and Langridge, M., 1992. Laparoscopic intra-uterine insemination of fallow deer with frozen-thawed or fresh semen after synchronisation with CIDR devices. N. 2. Vet. J., 40: 8-14. Asher, G.W. Peterson, A.J. and Bass, J.J., 1989. Seasonal pattern of LH and testosterone secretion in adult male fallow deer, Dama damn. J. Reprod. Fertil., 85: 657-665. Chapman, D.I., 1975. Antlers: bones of contention. Mamm. Rev., 5: 121-172. Chapman, D.I. and Chapman, N., 1975. Fallow deer: their history, distribution and biology. Terence Dalton, Lavenham. Courot, M. Hochemau-de Reviers, M.T. Monet-Kuntz, C. Locatelli, A. Pisselet, C. Blanc, M.R. and Dacheux, J.L., 1979. Endocrinology of spennatogenesis of the hypophysectomised ram. J. Reprod. Fertil. Suppl., 26: 165-173. Fennessy, P.F. Tate, M.L. and Johnstone, P.D., 1991. Hybridisation between ted deer (Ceruus elaphus) and other deer species. Proc. Aust. Assoc. Anim. Breed. Gen., 9: 469-472. Field, R.A., Young, O.A., Asher, G.W. and Foote, M., 1985. Characteristics of male fallow deer muscle at a time of sex-related muscle growth. Growth, 49: 190-201. Kelly, R.W. McNatty, K.P. Moore, G.H. Ross, D. and Gibb, M., 1982. Plasma concentrations of LH, prolactin, oestradiol and progesterone in female red deer (Ceruus elaphus) during pregnancy. J. Reprod. Fertil., 64: 475-483. Kenwood, M.G., 1987. A method for comparing profiles of repeated measurements. Appl. Stat. 36: 296-308. Lenz, M.L. English, A.W. and Dradjat, A., 1993. Initial studies on the growth performance of Mesopotamian fallow deer hybrids. Proceedings of the First World Forum on Fallow Deer Farming, Mudgee, Australia, pp. 157-164. Lincoln, G.A., 1971. The seasonal reproductive changes in the red deer stag (Cervus elaphus). J. Zool., 163: 105-125. Loudon, A.S.I., Mime, J.A., Curlewis, J.D. and McNeilly, A.S., 1989. A comparison of the seasonal hormone changes and patterns of growth, voluntary food intake and reproduction in juvenile and adult red deer (Ceruus elaphus) and P&c David’s deer (Elaphurus dauidianus) hinds. J. Endocr., 122: 733-745.
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Merriam, G.R. and Wachter, K W., 1982. Algorithms for the study of episodic hormone secretion. Am. J. Physiol., 243: E3 lo-E318. Moore, G H. and Littlejohn, R.P., 1989. Hybridisation of farmed wapiti (Ceruus eluphus mcmitobensis) and red deer (Crrous elaphus). N. 2. J. Zool., 16: I9 I - 198. Morris, C.A., 1993. Genetic improvement programmes for farmed fallow deer. Proceedings of the First World Forum on Fallow Deer Farming, Mudgee, Australia, pp. l37- 145. Otway, W., 1993. Mesopotamian fallow deer. Proceedings of the First World Forum on Fallow Deer Farming, Mudgee, Australia, pp. I5 I - 155. Scaramuzzi, R.J., Caldwell B.V. and Moor R.M., 1970. Radioimmunoassay of LH and estrogen during the estrous cycle of ewe. Biol. Reprod., 3: I lo- 119. Schanbacher, B D., 1980. Dose-dependent inhibition of spermatogenesis in mature rams with exogenous testosterone. Int. J. Androl., 3: 563-573. Schnare. H. and Fischer, K., 1987. Secondary sex characteristics and connected physiological values in male fallow deer ( Damu dama) and their relationship to changes of the annual photoperiod: doubling the frequency. J. Exp. Zool., 244: 463-471. Short, R.V., 1985. Deer: yesterday, today and tomorrow. Biology of deer production, The Royal Society of New Zealand, Bulletin, 22: 461-469. Whitehead, G.K., 1972. Deer of the World. Constable, London. Wislocki, G.B., 1949. Seasonal changes in the testes, epididymis and seminal vesicles of deer investigated by histochemical methods. Endocrinology, 44: l67- 189.
Animal Reproduction Science 45 ( 1996) 20 I-2 15
Comparison of seasonal changes in reproductive parameters of adult male European fallow deer ( Dama dama dama) and hybrid Mesopotamian X European fallow deer ( D. d. mesopotamica X D. d. dama) G.W. Asher av*, D.K. Berg b, S. Beaumont b, C.J. Morrow ‘, K.T. O’Neill a, M.W. Fisher a aAgReResearch,Invermay Agricultural Centre. Private Bag 50034, Mosgiel, New Zealand b AgResearch, Rukuru Agricultural Centre. Private Bag 3123. Hamilton, New Zeuiund ’Nationul Zoological Purk, Conservation und Research Center. Smithsonian Institution, Front Royal VA 22630. USA
Accepted 9 April 1996
Abstract In a study, aimed at comparing seasonal reproductive development of European fallow deer dama dama) with Mesopotamian (D.d. mesopotumicu) X European F, hybrids, five adult males of each genotype, which had been raised together since birth, were maintained as a bachelor group. Morphometric (body weight, neck circumference and testis diameter), endocrine (plasma testosterone concentrations) and seminal (ejaculate volume, spermatozoa per ejaculate and spermatozoa motility) parameters were recorded at fortnightly or monthly intervals for a 15-month period, and antler status was noted daily during the general periods of casting and velvet stripping. In addition, two bucks of each genotype were blood sampled via indwelling jugular catheters every 30 min for 24-h periods on five occasions (2-3 months intervals) during the year, and plasma was analysed for concentrations of testosterone and LH. Parameter profiles of the two genotypes were compared by global and time series ante-dependence covariance analysis to investigate overall profile similarity and the seasonal nature of any observed differences. Plasma hormone profiles from high-frequency blood sampling were subjected to PULSAR analysis to determine pulse frequency and amplitude. Throughout the study hybrid males were = 30% (Dama
* Corresponding author. 0378-4320/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)015771
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heavier than European males. However, both genotypes exhibited dramatic but parallel patterns of body weight change (global P = 0.054). Neck circumference was correlated with body weight throughout (P < O.OS),with similar regression slopes between the genotypes at any sampling time (P > 0.10). Covariance adjustment to a common initial body weight was performed to eliminate the effects of large body weight differences on muscle hypertrophy and regression. While profiles of corrected neck circumference were significantly different at the global level (P < O.Ol>, analysis by time revealed differences occurring only during the latter period of muscular regression in spring. However, profiles of other parameters, including testis diameter, plasma testosterone concentrations, spermatozoa per ejaculate and percentage motile spermatozoa, exhibited significant displacement between genotypes (global P < 0.05) evident as 2-4 weeks advancement in the sexual development (late summer/autumn) and quiescence (spring) phases for hybrid males relative to European males. Furthermore, mean dates of antler casting and velvet stripping were significantly earlier by 2-3 weeks for hybrid males than European males (P < 0.05). High frequency blood sampling revealed markedly seasonal patterns of secretion of testosterone and LH, with hybrid males exhibiting an apparent earlier onset of high-amplitude testosterone ‘surges’ in February (late summer) compared to those occurring in April (autumn) for European males. When viewed collectively, the data indicate strongly that the Mesopotamian influence is evident in the earlier attainment of sexual development and fertility in late summer and autumn, and earlier onset of sexual quiescence in spring. This is in accord with anecdotal information on earlier reproductive patterns in purebred Mesopotamian fallow deer. Keywords:
Fallow deer; Dama dama; Genotype; Hybridisation; Breeding season
1. Introduction Amongst the cervidae there are numerous taxa with close genetic affiliations (Whitehead, 1972). Recent trends into domestication and captive propagation have been associated with deliberate or inadvertent hybridisation between taxa (Short, 1985). In some instances, this may confer considerable economic advantage through additive genetic effects and putative heterotic effects of hybridisation on growth performance of farmed deer, as demonstrated with subspecies hybridisation of red deer (Cervus e&&us scoticus) with North American wapiti (C. e. nelsoni, C. e. rooseueltii or C. e. manitobensis) (Moore and Littlejohn, 1989; Fennessy et al., 1991). The farming of fallow deer (Dama dama) has gained acceptance and momentum in Australasia and North America within the last decade. While this is based largely on the common European subspecies (D. d. dumu), a significant proportion of farms have incorporated a component of hybridisation into their operations, using various levels of crossbreeding with the rare Mesopotamian subspecies (D. d. mesopotumicd to improve production (Morris, 1993). The Fl hybrid (Mesopotamian sire x European dam) and subsequent maternal and paternal backcrosses are reputed to exhibit increased growth rates and overall mature body size over the purebred European subspecies (Lenz et al., 1993). It is also mooted that there is a significant shift in reproductive seasonality with hybrids that reflects a generally earlier breeding season of the Mesopotamian subspecies (Otway, 1993). However, there are little objective data on reproductive seasonality of either Mesopotamian fallow deer or their hybrids. In contrast, there is considerable
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recent information on seasonality of European fallow deer (Asher, 1985; Asher et al., 1989; Schnare and Fischer, 1987). The aim of the present study was to compare seasonal reproductive development of adult male fallow deer of the European subspecies with the F, hybrid by monitoring somatic and testicular growth, secondary sexual characteristics, semen production and hormone secretory patterns.
2. Materials and methods 2.1. Animals and management
Ten mature (4 years old) fallow deer bucks, born and raised on the Ruakura Agricultural Centre (37”46’S, 175”20’E), were used in this study. Five bucks were of the European subspecies, progeny from natural mating of does and bucks derived from wild New Zealand stock. The remaining five bucks were F, hybrids between the European and Mesopotamian subspecies, progeny from artificial insemination of European does with semen of Mesopotamian bucks imported into New Zealand from Germany in 1987. The ten bucks were managed on pasture as a single group throughout the study, having been raised together since birth. While maintained as a bachelor group, the bucks were never more than 150 m distant from females of the same species. 2.2. Gross morphometry and semen collection At fortnightly or monthly intervals from 1 September 1992 to 20 November 1993 (5: 15 months) the bucks were mustered into covered handling yards. They were guided individually into a restraining cradle where they received an intramuscular injection of ketamine hydrochloride (5 mg kg-’ liveweight, Ketaset, Aveco Co., Fort Dodge, IA, USA) and xylazine hydrochloride (2.5 mg kg- ‘, Rompun, Bayer Leverkusen, German) to induce disassociative anaesthesia. Upon recumbancy they were weighed, blood sampled by jugular venepuncture into heparinised vacutainers, their neck circumference was measured cranial to the larynx with a plastic tape, and their testis diameter was measured with electronic calipers (Asher et al., 1987; Asher et al., 1989). Semen was collected on each occasion by electroejaculation (Asher et al., 1987; Asher et al., 1992). A sine-wave electroejaculator (AC 20-60 Hz) and rectal probe (Lane Pulsematic III; Lane Manufacturing, Co., USA) were used to administer 15-30 incremental stimuli of between 1.5 and 5.0 volts. Stimuli were administered in pulses of 3-5 s and were continued until either fluids ceased to be presented for 3-4 consecutive pulses or the ejaculate became clear (aspermatic). Semen was collected into pre-warmed glass cups and kept at 31°C in a warmed container until transferred to the laboratory. Following semen collection, anaesthesia was reversed by intravenous injection of yohimbine hydrochloride (0.4 mg kg- ’ liveweight, Recervyl, Aspiring Animal Services. Wanaka, NZ) and the bucks returned to pasture. Antler status of the bucks (i.e. hard, cast or velvet) was recorded daily during the general expected periods of casting and velvet stripping.
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2.3. Semen evaluation The semen was transferred to a water bath at 37°C immediately after collection and assessed for volume in a graduated glass pipette. Concentration of spermatozoa was determined by haemocytometer count following dilution of 20 ~1 raw semen in 200 ml 2% saline solution plus 0.01% formaldehyde. Percentage motility (to nearest 5%) was appraised visually under phase-contrast optics ( X 200) based on a 10 ~1 sample diluted in 100 ml of phosphate buffered saline solution. 2.4. Intensive blood sampling On five occasions during the year (2-3 month intervals) four of the bucks (two European and two hybrid bucks) were separated from the group in the yards and fitted with jugular catheters (Asher and Peterson, 1991) before being returned to pasture. On the following morning they were again mustered into the yards, where they remained for the next 24 h. During this time they were repeatedly restrained and blood sampled (5 ml) at 30 min intervals. Water and meadow hay were provided ad libitum. Blood samples were centrifuged within 30 min of collection and the plasma stored at - 10°C until assayed. 2.5. Hormone radioimmunoassays Plasma LH concentrations were determined in duplicate using a heterologous radioimmunoassay procedure described for ovine plasma (Scaramuzzi et al., 1970) and validated for fallow deer plasma (Asher et al., 1986). The LH antibody, raised in a rabbit against NIH-LH-S , , , was used in the assay at a final dilution of 1:200 000. Cross-reactivity with other proteins has been described previously (Kelly et al., 1982; Asher et al., 1986). The inter-assay coefficients of variation, calculated from determinations of cervine control samples, were 15.8% for low (mean = 1.l 1 ng ml-’ ), 6.7% for medium (5.25 ng ml-‘) and 8.2% for high (10.40 ng ml-‘) samples. The intra-assay coefficient of variation was 12.7%, 6.2% and 7.3% for the three control samples respectively and sensitivity of the standard curve was 0.03 ng NIH-LH-S, , per tube (0.30 ng ml- ’>. Plasma testosterone concentrations were determined in duplicate using an extraction radioimmunoassay similar to that described by Asher et al. (1987). The antiserum, raised in an entire ewe against testosterone-human serum cy globulin conjugate, cross-reacted with androstenedione (1.5%) and dihydrotestosterone (5.5%) but exhibited negligible binding (< 0.6%) with cholesterol, oestradiol, progesterone and a range of other androgens. Efficiency of recovery by extraction of radioactive ligand was 84.1% and parallelism was demonstrated between buffer standards, charcoal-stripped plasma standards and serially diluted plasma samples containing high concentrations of immunoreactive testosterone. Control plasma samples, with mean values of 32.8, 16.2, 8.4 and 3.5 nmol testosterone, were placed at regular intervals in each assay. Inter-assay coefficients of variation were 16.8, 10.0, 13.2 and 9.9%, respectively and the intra-assay coefficients of variation were 10.1, 8.2, 9.4 and 7.9%, respectively. Sensitivity of the standard curve was 1.4 nmol testosterone.
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2.6. Statistical analysis
Data summaries of morphometric, plasma testosterone and semen data in the form of graphs of the means of the two genotypes with the standard errors of differences (SED) for each time point measurements were prepared for the 1%month observation period. The profiles were compared using the ante-dependence covariance structure as proposed by Kenward (1987). The method is ideally suited to this situation in which no specific features of the profiles are, a priori, known to be of interest. After establishing the order of the ante-dependence structure and performing a global test of the difference between the two profiles, the technique was used to investigate the form of the difference amongst the profiles. This provides an improvement over the procedure of calculating a separate r-test for each time of measurement. LH and testosterone profile from high-frequency blood sampling were analysed separately for each buck at each sampling session using the PULSAR pulse identification routine (Merriam and Wachter, 1982). Pulse analyses were carried out for 50 points, with inputs for PULSAR including intra-assay standard deviation and a smoothing time of 180 min. A cut-off value of 2.7 standard deviation units was used for peak splitting.
3. Results At all times during the study period hybrid males were significantly heavier in body weight than European males (P < 0.05 for individual t-tests), with an overall difference between respective means of between 18 and 25 kg (Fig. 1). The proportional change in body weight from the start of the study appeared to follow similar trends for the two genotypes. While a global test of ante-dependence covariance indicated a marginally significant difference between the profiles of the two genotypes (P = 0.0541, further analysis indicated only one time in early January when a significant difference occurred (P = 0.028; Fig. 1). In general, the patterns of body weight change were characterised by rapid gains (130- 170 g day- ’) between October and February (spring and summer), precipitous reductions in weight (350-420 g day-‘) over a 3-4 week period in April/May (autumn rut> and maintenance through the period from June to September (winter). The periods of retention of hard antler stubbs (buttons) were of similar duration (= 7 months) for the two genotypes (Fig. 1). However, the hybrid males initiated velvet stripping 2 weeks earlier than did the European males (8 February vs 21 February; P < 0.05) with this event being highly synchronised (i.e. within 24 h) within each genotype. Similarly, antler casting was on average 2-3 weeks earlier for hybrid males (5 September f 2.2 days vs 24 September + 1.8 days; P < 0.05) but was less synchronous within genotype than was velvet stripping (Fig. 1). Profiles of mean testis diameter (Fig. 2) reveal parallel patterns of seasonal changes having occurred between the two genotypes, although significant differences between means were observed around the times of complete testicular regression (SeptemberNovember) and maximal testicular development (March-April). The global test of ante-dependence covariance highlighted a highly significant difference between geno-
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types in the overall profiles of mean testis diameter (P = 0.003) and subsequent analysis revealed differences occurring around peak testicular development and recrudescent phases (Fig. 2). These differences indicate a temporal displacement of testicular growth profiles of about 2 weeks. Although the maximal mean testis diameter was similar ( = 52 mm) between the hybrid and European males (albeit 2 weeks apart), hybrids exhibited significantly smaller mean testis diameter during the spring nadir (e.g. 27.4 + 0.8 mm vs 31.5 * 0.9 mm; P < 0.05). There were no significant within-genotype correlations between body mass and testis diameter at any time during the study (P > 0.05).
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Fig. 2. Profiles of mean testes diameter, liveweight-corrected neck circumference and plasma testosterone concentrations for adult male European fallow deer (0; n = 5) and F, hybrid Mesopotamian X European fallow deer (0; n = 5). Vertical bars and associated asterisks indicate the SED and significant differences (P < 0.05). respectively, based on pooled variance and individual t-tests. Significance and order of the global ante-dependence covariance test are presented for each pair of profiles, and crosses indicate times when the profiles were shown subsequently to be significantly different (P < 0.05).
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Absolute neck circumference (not shown) differed significantly between the two genotypes at all times during the study. However, there were significant positive within-genotype correlations between body weight and neck circumference throughout the study period (P < 0.05). Furthermore, regression slopes on any sampling date were not significantly different for the two genotypes (P > O.lO), with exceptions to this occurring on only two sampling dates in July. Covariance adjustment to a common initial live weight enabled analysis of corrected neck circumference (CNC) that largely eliminated the effects of the between-genotype difference in absolute body weight (Fig. 2). Both genotypes exhibited a markedly seasonal pattern of neck muscle hypertrophy and regression that closely paralleled testicular changes. Although the global test of ante-dependence covariance of both corrected and non-corrected neck circumference indicated a highly significant difference between the mean profiles (P < O.OOl>,analysis by time revealed differences occurring only during the period of muscular regression towards the end of the study period (September and October). Similarly, the global test of mean plasma testosterone concentration profiles (Fig. 2) indicated a highly significant difference between genotypes (P = 0.003) although time analysis revealed sporadic points of significance. Most notable was the apparent displacement of peak mean concentrations occurring in early March (hybrids males) and early April (European males). Of 277 attempts at electroejaculation, fluids were collected on 275 occasions. Ejaculates varied from opaque spermatozoa-rich collections to clear aspermatic fluid collections, depending on season. On three occasions ejaculates were contaminated with copious quantities of urine and were immediately discarded. Statistically, analysis was based on 272 valid collections fairly evenly distributed across the two genotypes (134: 138). Semen parameters (Fig. 3) showed distinct seasonal patterns reflecting alternating periods of testicular activity and quiescence. For both genotypes, mean ejaculate volume was lower during the testicular regression phase (October-December) than at other times of the year (P < 0.05). However, mean volumes were significantly different between genotypes on only one date in January and the global test of ante-dependence covariance revealed no difference in overall profiles (P = 0.923). Other seminal parameters of spermatozoa population and viability displayed significant differences between genotypes at both a global level and at various times during the study (Fig. 3). In particular, mean total spermatozoa per ejaculate and mean percentage motility differed during the testicular recrudescence phase (November-February) and at the initiation of testicular regression (July-August). In both periods, hybrids appeared to initiate events 2-4 weeks earlier than European males. Interestingly, both genotypes exhibited a temporary decline in both the mean number of spermatozoa per ejaculate and the mean proportion of motile spermatozoa presented in ejaculates during the period of peak rutting activity (i.e. vocalisations), being early April for hybrids and late April-early May for European males (Fig. 3). Seasonal patterns of LH and testosterone secretion were evident from the highfrequency blood sampling programme (Fig. 4). There were low incidences of LH and testosterone pulses (i.e. O-l pulses per 24 h) for both genotypes in September and December profiles. Pulse frequency of both hormones increased dramatically in the February profiles (i.e. during the transition into the breeding season). However, LH
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pulse frequency was greater for the European bucks (S-12 pulses per 24 h) than for the hybrid bucks (4-9 pulses per 24 h), with pulsable secretion of testosterone tending to mirror LH pulse patterns for European bucks. While testosterone secretion was similar in hybrid bucks, the pulses were of higher amplitude (0.5 vs 1.0 nmol) and of longer duration. There was a notable absence of detectable LH pulses in April (early breeding season) for all bucks. However, episodic secretion of testosterone was evident as 3-4 h ‘surges’ for the European bucks, but such events were absent from the profiles of hybrid bucks. Lower amplitude testosterone surges were again observed in June for European bucks but were not evident for hybrid bucks (Fig. 4).
4. Discussion While it is clear that both fallow deer genotypes in the present study exhibited pronounced reproductive seasonality, the data collectively indicate a degree of temporal displacement between them. The magnitude of this displacement, however, was only in the order of 2-3 weeks, representing a single sampling interval for most parameters investigated. Nonetheless, some of the morphometric, endocrine and seminal parameters measured highlighted differences between the two genotypes at key times during the annual reproductive cycle despite possible social interaction effects (e.g. proximity of oestrous does) that may have served to promote within-herd synchrony. The annual cycle of retention, casting and regrowth of antlers provides the most conclusive indicator of seasonality displacement. The highly seasonal nature of these events has been well described for various temperate cervid species, including European fallow deer (Chapman and Chapman, 1975; Schnare and Fischer, 1987). In the present study there was a startling degree of within-genotype synchrony in the velvet stripping process in late summer, with all hybrid bucks cleaning their antlers on 8 February and all European bucks doing likewise on 21 February. A similar disparity in mean antler casting dates (i.e. about 2 weeks) was also evident but this event was not subject to such synchrony. Clearly, the antler cycle is indicative of underlying endocrine changes, principally those related to testicular development and regression. The annual antler cycle has been correlated with annual fluctuations in testicular secretion of testosterone for numerous cervid species (Wislocki, 1949; Lincoln, 1971; Chapman, 1975). In the present study on fallow deer, endocrine parameters did reflect differences between the two genotypes. In particular, there was a 2-week (single sampling interval) displacement in peak mean concentrations of plasma testosterone from single samples collected during the study, despite the markedly episodic pattern of testosterone secretion described previously for fallow deer (Asher et al., 1989; Asher and Peterson, 1991). Furthermore, high-frequency blood sampling at various times during the year indicated differences between genotypes in the patterns of episodic secretion of both LH and testosterone during the testicular recrudescence phase in summer and at the approximate time of the rut in autumn. The characteristic ‘surge’ patterns of testosterone secretion occurring at the peak of sexual development (Asher et al., 1989) appeared to be instigated in summer in hybrids, but were not apparent until autumn in the European bucks. Collectively,
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antler and endocrine data indicate earlier attainment of sexual development in the hybrid than the European animals. With the exception of body weight, other morphometric and seminal parameters indicated, to varying degrees, temporal displacement between the two genotypes. Absolute body weight differences were very obvious, with hybrids being approximately 30% larger than their European herdmates throughout the study. This undoubtedly represents a genetic effect on growth and mature body size (Morris, 1993; Lenz et al., 1993). However, proportional change in body weight was essentially similar for the two genotypes and did not reflect any seasonal disparity despite dramatic seasonal patterns being evident. It is probable that changes in body mass are also determined by factors other than endogenous endocrine regulation (e.g. fluctuations in feed supply, temperature and time of day), factors that may mask the breeding season disparity. Furthermore, the rapid decline in body weight during the rut may have been influenced strongly in both genotypes by the proximity of oestrous European fallow deer does on the same farm, leading to a strong social inductive effect on the precise timing of rutting behaviour (and hence, inappetence) of bucks. For both genotypes, profiles of testes diameter and of neck circumference showed approximately parallel seasonal changes, highlighting the influence of the testes on annual cycles of neck muscle hypertrophy and regression (Field et al., 1985). Testicular development was displaced temporarily by about 2 weeks between the two genotypes, with developmental differences being particularly pronounced during phases of testicular enlargement and regression. However, neck circumference changes measured in this study were not markedly different between the two genotypes. Seasonal changes in body weight clearly had a major influence on neck dimensions over and above any testicular influences, and the obvious difference between genotypes in overall body weight (= 30%) was similarly reflected in absolute neck circumference on any sampling date. However, covariance adjustment largely eliminated the body weight effect but still revealed little seasonal difference in neck muscle hypertrophy and regression between the two genotypes. This is somewhat unexpected given the known association between the testes and specific musculature of the neck. We speculate that, either, the measurement of neck dimensions is subject to imprecision (although the very obvious protruding larynx of male fallow deer does allow repeatable siting of the measuring tape>, there are additional influences of changing body mass on neck muscle developments that could not be eliminated by covariance analysis, or there exists a different interaction between testes development and muscular hypertrophy/regression between the two genotypes. This does not lessen, however, the profound nature of seasonal changes in neck musculature associated with the reproductive cycle in adult male fallow deer (Asher et al., 1987; Schnare and Fischer, 1987). It is interesting to note that testicular size was essentially similar between the two genotypes despite the obvious disparity in body weight. In fact, the larger hybrid males had significantly smaller testes at the nadir of testicular cycle. While this was in no way reflected in fertility in this or other studies (Asher et al., 19921, the phenomenon has also been observed in hybridisation between P&e David’s deer (EZuphurus dauidiunus) and red deer (D. Goosen, personal communication). Unfortunately, we have little or no information on the relationship between testis size and body mass in the paternal taxa,
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the purebred Mesopatamian fallow deer and the P&e David’s deer, and therefore cannot conclude whether or not it is a consequence of hybridisation. The seminal parameters provided interesting insights into testicular function of the two fallow deer genotypes. Distinct temporal displacement was evident for indices of spermatozoa1 output (e.g. spermatozoa per ejaculate) and viability (e.g. % motile spermatozoa). This was perhaps accentuated by the fact that both genotypes exhibited a distinct period of aspermatogenesis in early summer, as indicated by ejaculates completely devoid of spermatozoa. The reinstigation of spermatogenesis occurred about 2 weeks earlier in the hybrids. However, the subsequent cessation of spermatogenic activity in late spring was not as well separated temporally between the two genotypes, with both presenting aspermatic ejaculates for the first time on the same sampling date. Interestingly, both genotypes exhibited a distinct temporary reduction (30-N%) in mean spermatozoa per ejaculate and mean percentage motile spermatozoa early into the breeding season, with an apparent genotype displacement of 2-4 weeks. In the case of the European subspecies, at least, this reduction in spermatozoa output and quality corresponded with the expected timing of peak rutting activity in April, and is a phenomenon that the authors have encountered previously in commercial semen collection operations. We speculate that this event may have been due to either a high secretory output of testosterone during the immediate pre-rut and rut periods (Asher et al., 1989) having an inhibitory influence on spermatogenesis, as seen in rams treated with exogenous testosterone (Courot et al., 1979; Schanbacher, 1980), or was a consequence of repeated natural ejaculation during rutting fervour (despite being in a bachelor group) leading to deminished semen reserves. In summary, collective data on the morphometric, endocrine and seminal traits indicate strongly that the Mesopotamian influence is evident in the earlier attainment of sexual development and fertility in late summer and autumn. However, the data are more equivocal in relation to the onset of sexual quiescence, although the overall impression is that the entire seasonal repertoire of the two genotypes in the study displays a phase-shift of about 2-3 weeks between them. These data support our unpublished studies on females, whereby hybrids (25% Mesopotamian, 75% European; maternal backcross) exhibited earlier initiation and cessation of oestrous cycles relative to European does. Unfortunately, there is little information on the seasonal reproductive patterns of the paternal species. However, in accord with anecdotal information (Otway, 19931, it is likely that purebred Mesopotamian fallow deer exhibit considerable disparity with European fallow deer (i.e. 4-5 weeks earlier) in reproductive seasonality. This is, perhaps only one example of many where genetically related cervid taxa with differing patterns of reproductive seasonality produce hybrids that exhibit intermediate seasonality. For example, Loudon et al. (1989) demonstrated convincingly the widely disparate seasonality of red deer and P&e David’s deer. Recently these two species (assigned to different genera) have been hybridised to produce fertile offspring (Asher et al., 1988; Fennessy et al., 1991). It will be interesting to see if the hybrids exhibit predictable reproductive seasonality. Such studies on these, and other hybrids, may provide interesting insights into the central control mechanisms of seasonality, as well as providing a useful genetic resource that will enable selection of farmed deer better suited to different seasonal environments.
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Acknowledgements
We thank P. Johnstone and D. Duganzich for statistical advice, T. Manley, R. Porteous and B.G. Mockett for assistance with testosterone radioimmunoassays and staff of the Ruakura Deer Unit for animal management. We acknowledge the contribution of the late Dr Whitley Otway in establishing the mesopotamian fallow deer in the southern hemisphere.
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