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Theriogenology 68 (2007) 663–672 www.theriojournal.com
Twin efficiency for reproductive variables in monozygotic twin sheep P. Celi a,*, S.W. Walkden-Brown a,b, D. Blache a, A.Z. Sze´ll c, H.M. Wilkinson a, G.B. Martin a a
Faculty of Natural & Agricultural Sciences, School of Animal Biology, The University of Western Australia, Crawley, WA 6009, Australia b School of Rural Science and Agriculture, University of New England, Armidale, NSW 2351, Australia c Sheep Industries Branch, Western Australian Department of Agriculture, PO Box 757, Katanning, WA 6317, Australia Received 27 February 2007; received in revised form 14 May 2007; accepted 22 May 2007
Abstract The aim of this study was to determine whether the number of animals used in experiments examining reproductive variables could be reduced without loss of statistical efficiency by using monozygotic twin (MT) sheep. In a series of four experiments, we measured the reproductive responses to changes in nutritional, opioidergic, and calcium status and calculated values for twin efficiency (TE) for each variable. In Experiment 1, we monitored the changes in gonadotrophin and testosterone secretion, scrotal circumference and live weight, of MT rams after an acute change in nutritional regime. In Experiment 2, we examined the changes in ovulation rate and gonadotrophin secretion in MT ewes following treatment with bovine follicular fluid. In Experiment 3, we determined responses to naloxone and exogenous calcium on gonadotrophin secretion in MT rams. In Experiment 4, we investigated the effects of naloxone and exogenous calcium on the hypothalamus–pituitary–ovarian axis of MT ewes. The TE values were high only for live weight and scrotal circumference; the other reproductive traits had less variation between than within MT pairs, suggesting that randomly selected animals were just as efficient as genetically identical twins in experiments examining physiological reproductive traits. # 2007 Elsevier Inc. All rights reserved. Keywords: Nutrition; Naloxone; Calcium; Gonadotrophins; Sheep
1. Introduction Monozygotic twins (MT) have been used in research with the aim of reducing the number of animals needed to provide an adequate statistical base, provided that there is uniformity within twin pairs for the specific trait measured [1]. This strategy is attractive as it
* Corresponding author. Present address: School of Rural Science and Agriculture, University of New England, Armidale, NSW 2351, Australia. Tel.: +61 2 93511782; fax: +61 2 93511693. E-mail address:
[email protected] (P. Celi). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.05.056
could simultaneously minimize animal utilization [2] and maximize cost effectiveness. In addition, the technology to produce monozygotic twins in vitro by microsurgical dissection of embryos is available on a large scale [3]. This experimental approach would be of particular value in sheep reproductive research, which often requires the use of a large number of randomly selected animals (e.g. experiments determining ovulation rate) or utilize expensive measurements techniques (e.g. hormone assays for serial blood samples). The major sources of environmental variation in the reproductive traits of Merino sheep are photoperiod,
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nutrition and social cues [4,5]; the relative importance of the environmental and genetic components of variance differs between traits. Monozygotic twins would thus be expected to have more environmental variations in traits that have a low genetic component of variance than in traits with a high genetic component of variance. In sheep, genetic variance for reproduction is most evident in differences between breeds. For example, there are significant genotypic differences in scrotal circumference in rams, ovarian activity in ewes, and gonadotrophin secretion in both genders [6– 9]. These observations suggest a considerable genetic component in the control of the secretion of reproductive hormones. The relative value of MT sheep for measuring reproductive variables has not yet been reported. We therefore tested whether the number of animals used in experiments examining reproductive variables could be reduced without loss of statistical power by using MT using four experiments examining the reproductive responses of sheep to changes in nutritional, opioidergic, and calcium status. In Experiment 1, we challenged rams with an acute change in nutrition (lupin grain supplementation) to increase the activity of the reproductive axis (review: [10]). In Experiment 2, we administered bovine follicular fluid (bFF) to increase ovulation rate in sheep [11]. In Experiments 3 and 4, we measured reproductive hormones in male and female sheep after the injection of exogenous calcium and/or an opioid antagonist. Opioid peptides in the brain represent one of many systems that inhibit the activity of GnRH neurons in the sheep and it is very likely that they play roles in the responses of gonadal steroids and photoperiod [12]. Some of these roles appear to depend on intracellular calcium status [13,14]. The inhibitory effect exerted by opioid peptides on LH secretion has been well defined by studying the responses to the opioid antagonist, naloxone [15]. When administered in low doses, naloxone has more affinity for the mreceptors which are responsible for gonadotrophin secretion [16]. Low doses can reverse the anoestrous condition in lactating ewes [17], stimulate LH secretion in Soay rams [18], and facilitate estrous behaviour in ewes [19].
2.1. Animals All animals used in this study were obtained by embryo splitting and then transferred individually into single recipients; multiparous Merino ewes were used as embryo donors [3]. Recipient and donor ewes, of similar live weight and body condition score, were drawn from a large flock of Merino ewes aged 2.5 years and older, which had lambed in previous years. After birth, the animals were subjected to similar environments throughout their lifetime. For the studies described here, the animals were housed indoors in individual pens under natural photoperiod in an animal house at The University of Western Australia (328S, 1158E). Between experiments, the animals were kept outdoors in an animal facility at the University of Western Australia. 2.2. Experiment 1: reproductive variables in monozygotic twin rams responding to an acute change in nutrition We determined the twin efficiency (TE) for the increases in LH pulse frequency, FSH and testosterone secretion, testicular size and live weight in seven pairs of mature MT rams (age, 3–4 years; average live weight, 73.5 1.5 kg) following an acute nutritional supplement. During the month of July, the rams were fed for 14 days with a maintenance diet (MD) comprising 1 kg oaten chaff containing 10% lupin grain (Lupinus angustiflolius) and a complete mineral mix (Siromin; Narrogin Mineral Stockmix, Narrogin, WA, Australia). This regime was designed to maintain constant body mass by providing approximately 8.4 MJ/d of metabolizable energy and 50 g protein/d (as described [20]). Water was provided ad libitum. On Day 0, the rams were fed the MD plus 750 g lupin grain (high diet (HD); about twice the maintenance energy requirement) for 10 days, after which they returned to the MD for a further 14 days. Live weight and scrotal circumference were measured on Days 14, 0, 10, and 24. On Days 0, 10, and 24, blood was sampled every 20 min for 24 h via an indwelling jugular venous catheter. Plasma was separated and stored at 20 8C pending hormone assays.
2. Materials and methods All experimental protocols conformed to the Code of Practice formulated by the National Health & Medical Research Council of Australia and implemented by the Animal Ethics Committee of The University of Western Australia (AAA61/96/96).
2.3. Experiment 2: reproductive variables in monozygotic twin ewes following bovine follicular fluid treatment We administered bFF to determine values for TE for ovulation rate and gonadotrophin secretion in nine pairs
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of MT ewes (age, 3–6 years; average live weight, 67 1.5 kg) during the month of May. The ewes were fed an MD and water was provided ad libitum. To synchronize the estrous cycles, the ewes were first given an intravaginal progestagen device (CIDR, InterAg, Hamilton, New Zealand) that was left in place for 13 days and removed on Day 1 (estrous on Day 0). The next estrous cycle was further synchronized by intramuscular treatments of 250 mg cloprostenol (Estrumate, Coopers, NSW, Australia) on Days 13 and 28 to induce luteolysis. Cycle synchrony was verified with males; all ewes came into estrous within 24 h in both cycles. A linear design was used in which all ewes received the same treatment at the same time. Bovine follicular fluid (bFF) was aspirated from the large, non-cystic follicles in ovaries collected from a local abattoir, pooled, and extracted with charcoal to remove steroids [11]. The bFF was stored in 80 mL aliquots at 20 8C until required. All ewes were injected subcutaneously with 4 mL bFF every 8 h for 5 days (Days 8–12) during the first synchronized cycle. The animals were weighed weekly throughout the experiment. On Days 7, 22, and 37, the number of corpora lutea (ovulation rate) was determined at laparoscopy. On Days 8–13, blood was sampled every 8 h and plasma was retained for an FSH assay. Blood was also sampled via indwelling jugular catheter every 20 min for 12 h on Days 12 and 27 (luteal phase), and every 10 min for 6 h on Days 14 and 29 (follicular phase) for LH pulse analysis. 2.4. Experiment 3: endocrine variables following treatments with naloxone and calcium in monozygotic twin rams The aim of this study was to determine the TE for gonadotrophin secretion using monozygotic twin rams treated with exogenous calcium and naloxone. Three pairs of mature MT rams (age, 5–6 years; average live weight, 67.5 0.5 kg) were studied during March and April. They were fed the MD and water was provided ad libitum. The rams were randomly allocated to a 2 2 factorial design with four cross-over cycles (each ram received each treatment). The rams received five daily (injection time, 0900 h) intravenous injections, as follows (n = 6 per treatment): (a) calcium borogluconate 0.02 g/kg + naloxone hydrochloride 0.02 mg/kg (Nal + Ca2+); (b) naloxone hydrochloride 0.02 mg/kg (Nal); (c) calcium borogluconate 0.02 g/kg (Ca2+) and (d) 0.1 mL/kg NaCl 0.9% (saline). Naloxone hydrochloride was purchased from Sigma–Aldrich Pty. Ltd. (NSW, Australia) and calcium borogluconate was
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purchased from WA Drug Company (Perth, WA, Australia). The naloxone was dissolved either in physiological saline or calcium borogluconate and all treatments were administered via indwelling jugular catheter. The doses were chosen on the basis of a previous study with sheep [17]. Blood was sampled every 20 min for 12 h on Days 0, 1, and 5 of the treatment period; plasma was separated and frozen pending assay for LH and FSH. 2.5. Experiment 4: endocrine variables following treatments with naloxone and calcium in monozygotic twin ewes The aim of this study was to determine the TE for gonadotrophin secretion in MT ewes treated with exogenous calcium and naloxone. Eight pairs of adult monozygotic twin ewes (age, 5–8 years; average live weight, 49.5 1.5 kg) were studied during the months of July and August. Before the beginning of the experiment, they were subjected to laparoscopy to confirm the presence of a corpus luteum. Estrous cycles were synchronized by two intramuscular injections of 250 mg cloprostenol (Estrumate Coopers, NSW, Australia) 11 days apart and the following estrous cycle was synchronized with intravaginal devices (CIDR, InterAg, Hamilton, New Zealand). Cycle synchrony was verified with rams; all ewes came into estrous within 24 h in both cycles. The ewes were randomly allocated to 2 2 factorial design, with two cross-over cycles. They received five intravenous injections during the follicular phase, at 0900 h on Days 8–12 of the estrous cycle, under the following protocol (n = 8 for all treatments): (a) Ca2+ 0.02 g/ kg + Nal 0.02 mg/kg; (b) Nal 0.02 mg/kg; (c) Ca2+ 0.02 g/kg; and (d) 0.1 mL/kg NaCl 0.9% (saline). At laparotomy on Day 13, the number of corpora lutea was counted and the follicular fluid was aspirated from the largest visible ovarian follicles with Hamilton syringes. The volume of fluid from each follicle was noted and the follicular fluid was diluted in 1 mL NaCl 0.9% and stored at –20 8C until assayed for 17-b estradiol (FFE2). The sampled follicles were then classified according to their estrogenic activity on the basis of their FFE2 [21]: non-estrogenic (NO; FFE2 < 10 mg/L), estrogenic (O; 10 mg/L < FFE2 < 50 mg/L), or estrogenic potentially ovulatory (OPO; FFE2 > 50 mg/L). Blood was sampled via an indwelling jugular catheter every 20 min for 24 h on Days 0 and 5 of the treatment period (Days 7 and 12 of the estrous cycle). Plasma was separated and stored prior to assays for LH and FSH.
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2.6. Hormone assays All serial plasma samples were assayed for LH; pooled samples were used to measure all other hormones. Duplicate aliquots were assayed for LH with a doubleantibody RIA [22], based on a preparation CNRS-M3 of ovine LH (biopotency 1.8 IU NIH-LH-S1/mg) that was used for iodination and standards and had been kindly supplied by M. Jutisz (College de France, Paris, France). The limit of detection was 0.24 0.05 ng/mL (mean S.E.M.). The intra-assay coefficient of variation was estimated in each assay using six replicates of three control samples containing 0.85 ng/mL (9.6%), 2.15 ng/mL (9.3%) and 4.05 ng/mL (14%). The interassay coefficients of variation were 9.6, 9.3, and 14.3%. Plasma was assayed for FSH in duplicate by a doubleantibody RIA [23] using NIAMDD-oFSH-RP-1 (biopotency 75 NIH-FSH-S1) and NIADDK-anti-oFSH-1 serum. The limit of detection was 0.12 ng/mL. Included in the assay were six replicates of three control samples containing 1.5, 2.6, and 3.6 ng/mL, which were used to estimate the intra-assay coefficients of variation (7, 8, and 9%). Plasma was assayed for testosterone using a nonextraction radioimmunoassay with 1,2,6,7-3H-testosterone (Amersham, Sydney, NSW, Australia) as tracer and an antibody that had been raised in our laboratory against testosterone-3-CMO-HSA [24]. Cross-reactions were 100% with testosterone, 70% with dihydrotestosterone, 3.7% with androstenedione, and less than 0.05% with progesterone, estradiol-17b, estrone, and estriol. The limit of detection of the assay was 0.12 ng/mL and the within-assay coefficients of variation were 14, 14, and 12% for quality controls containing 0.5, 2.1, and 6.5 ng/ mL. Estradiol in follicular fluid was measured without extraction using a double-antibody RIA described previously [25]. Cross-reactions were 16% with estrone, 1.2% with testosterone, 3% with estriol, and 0.1% with 5a-dihydrotestosterone. The limit of detection of the assay was 0.4 pg/mL and the within-assay coefficients of variation were 11, 8, and 6% for quality controls containing 1.7, 5.25, and 10.44 ng/mL. 2.7. LH pulse analysis The LH data were analysed for pulses with a modified version of the ‘‘Pulsar’’ algorithm developed by Merriam and Wachter [26] and modified for the Apple Macintosh computer (‘‘Munro’’, Zaristow Software, West Morham, Haddington, East Lothian, UK). The G parameters (the number of standard deviations by which a peak must exceed the baseline in order to be accepted) were set at 3.98, 2.4, 1.68, 1.24, and 0.93 for
G1 to G5, respectively; those were the requirements for pulses composed of one to five successive samples that exceed the baseline. The Baxter parameters, describing the parabolic relationship between the concentration of a hormone in a sample and the standard deviation (assay variation) about that concentration were 0.30853 (b1, is the y intercept), 0.00213 (b2, the x coefficient) and 0.00268 (b3, is the x2 coefficient). The pulse frequency, the mean pulse amplitude (the difference between pulse peak and preceding nadir) and the mean concentration of LH were calculated for each profile. 2.8. Statistical analysis Repeated measures ANOVA was applied to all variables. When main effects or interactions were significant, one-way ANOVA was applied and Fisher’s protected LSD was used for comparison between treatment groups. Ovulation rate was analysed using the x2 method. For Experiments 3 and 4, the experimental design, the effects of randomization of rams into groups and the order of the cross-over cycles were obtained by using the GenStat 8 for Windows statistical package [27]. The main effects of treatment, time and their interaction were analysed by ANOVA directive within GenStat. The usefulness of monozygotic twins as an experimental tool was evaluated by calculating the following uniformity statistics (derived from ANOVA) using the model previously reported [1]. Briefly, the percentage variation between twin pairs was defined as the proportion of total variation that is attributed to variation between twin pairs as a percentage: s 2b % variation between twin pairs ¼ 100 s 2v þ s 2b where s 2v is within twin pair variation and s 2b is between twin pair variation. The percentage of variation within twin pairs was defined as the proportion of total variation attributed to variation within twin pairs as a percentage: s2 v % variation within twin pairs ¼ 100 s 2b þ s 2v Finally, twin efficiency (TE) was defined as the ratio between the information available per experimental unit recognizing twins and the information available per experimental unit ignoring twins: TE = 1% (1 – variation between twin pairs) In practice, TE is the number of animals chosen randomly that one twin pair can replace without loss of statistical power.
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3. Results 3.1. Experiment 1: reproductive variables in monozygotic twin rams responding to an acute change in nutrition Compared to their initial values, both live weight and scrotal circumference increased (P < 0.05) after 10 days of HD feeding and then declined when the rams were returned to MD (Fig. 1). Similar responses (P < 0.05) were observed for LH pulse frequency and mean plasma concentrations of FSH and testosterone (Fig. 2). In contrast, LH pulse amplitude decreased after 10 days of lupin supplementation (P < 0.05) and then increased when rams returned to the MD (Fig. 2). Mean LH concentration increased (P < 0.05) after 10 days of lupin supplementation, but did not change significantly thereafter (Fig. 2). High TE values, ranging from 9 to 79 and 3 to 12, were observed for live weight and scrotal circumfer-
Fig. 2. Mean (S.E.M.) effect of lupin supplementation on LH pulse frequency, LH pulse amplitude, and plasma concentrations of LH, FSH and testosterone in monozygotic twin rams (n = 14). *P < 0.05.
ence (Table 1). For LH pulse frequency, LH, and testosterone concentrations, the values equal to one. Among the endocrine variables, LH pulse amplitude had a TE value greater one.
FSH were only than
3.2. Experiment 2: reproductive variables in monozygotic twin ewes following bovine follicular fluid treatment
Fig. 1. Mean (S.E.M.) effect of lupin supplementation on changes (%) in live weight and scrotal circumference in monozygotic twin rams (n = 14). *P < 0.05.
The ewes had a slight decrease in live weight at the beginning of the experiment (Fig. 3). Ovulation rate was not increased after bFF treatment (Fig. 3), but it decreased by 20% between the first (Day 22) and second (Day 37) cycles following bFF treatment (P < 0.05). There was no effect of bFF treatment on LH pulse frequency (Fig. 4). During the luteal phase, LH pulse amplitude and mean LH concentration were not affected by bFF treatment (Fig. 4). However, during the follicular phase of the treatment cycle, the ewes had a higher (P < 0.05) LH pulse amplitude and LH mean concentration than during the control cycle. In both the treatment and control cycles, there was a higher (P < 0.05) LH pulse frequency and lower LH pulse amplitude in the follicular phase than in the
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Table 1 Mean (S.E.M.) twin efficiency values in monozygotic twin sheep Variable
Experiment 1
Experiment 2
Live weight Scrotal circumference LH pulse frequency LH pulse amplitude Mean LH Mean FSH Mean testosterone No. of ovulations
37 15 82 10 20 10 10 10
23 2
luteal phase, but no difference in mean LH concentration. Plasma FSH concentration was initially reduced by bFF treatment, but this suppression was not sustained throughout the 5-days treatment (Fig. 3). When the second batch of bFF was begun (the third day of treatment), plasma FSH concentrations began to rise and returned to pre-treatment value at the time of luteolysis. For LW, the TE values were again high, ranging between 21 and 26. For LH pulse amplitude and mean concentrations of LH and FSH, the TE values were approximately two. For ovulation rate and LH pulse frequency, TE values were equal to one (Table 1).
10 20 21 21
Experiment 3
Experiment 4
21 31 31 92
21 42 20 42
10
3.3. Experiment 3: endocrine variables following treatments with naloxone and calcium in monozygotic twin rams Neither Nal nor Ca2+ increased the pulsatile secretion of LH on any day of observation and no synergism between these treatments was detected (Fig. 5). Similarly, the treatments did not have any effect on any of the other LH pulse variables measured (Fig. 5), nor on FSH concentrations (Fig. 5). The TE values were again low for all the variables that described LH secretion (Table 1). In contrast with Experiments 1 and 2, a relatively high TE value was recorded for mean FSH concentration (Table 1).
Fig. 3. Mean (S.E.M.) ovulation rate, change in live weight before (Day 7) and after (Days 22 and 37) administration of exogenous bovine follicular fluid (bFF) and the effect of bFF on plasma FSH concentrations during the luteal phase of the treatment cycle in monozygotic twin ewes (n = 18). *P < 0.05.
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Fig. 5. Mean (S.E.M.) effect of five daily injections of calcium and naloxone on LH pulse frequency, LH pulse amplitude and LH concentration in monozygotic twin rams (n = 6). (&)Ca2+ + Nal; ( ) Nal; ( ) Ca2+; (& )saline.
Fig. 4. Mean (S.E.M.) effect of bovine follicular fluid (bFF) on LH pulse frequency, LH pulse amplitude and mean LH concentration during the luteal and follicular phase of the treatment (&) and control (&) cycle in monozygotic twin ewes (n = 18). *P < 0.05.
3.4. Experiment 4: endocrine variables following treatments with naloxone and calcium in monozygotic twin ewes There was no effect of time or treatment on LH secretion, so LH pulse frequency, LH pulse amplitude and mean LH concentration had similar values in all groups on both days of observation (Fig. 6). Similarly,
there was no effect of treatment or time on plasma FSH concentration (Fig. 6). Overall, there was no effect of treatment on the total number of large visible follicles or on follicular fluid volume (data not shown). The treatments produced a similar proportion of the different classes of follicles (data not shown). The TE values for endocrine variables were again low (Table 1). 4. Discussion The aim of this study was to determine whether the number of animals used in reproductive experiments
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Fig. 6. Mean (S.E.M.) effect of five daily injections of calcium and naloxone on LH pulse frequency, LH pulse amplitude, LH and FSH concentration in monozygotic twin ewes (n = 8). (& )Ca2+ + Nal; ( ) Nal; ( ) Ca2+; (& )saline.
could be reduced without loss of statistical efficiency when genetically identical twins are used. Monozygotic twins had a clear advantage for live weight and testicular size, but very little advantage for the endocrine variables that are typically measured in such studies. In other words, randomly selected sheep were just as efficient as identical twin sheep in experiments designed to test reproductive hormonal pathways. Variation in live weight was much lower within than between twin pairs, suggesting that the expression of mature live weight depended to a considerable extent on genetic factors. The TE values for live weight also varied between studies (37 in Experiment 1 and 23 in Experiment 2) so any change in the environmental variance component will affect the value of using genetically identical twins and TE values should be based on more that a single observation. Nevertheless, theoretically, one pair of monozygotic twin sheep can replace 30 randomly selected sheep in experiments where live weight is a major endpoint. This agreed with the findings of [28] who estimated a similar value of 26 in monozygotic twin cows. An intermediate TE value was observed for testicular circumference, suggesting that one pair of monozygotic twin ram can replace eight randomly selected rams in experiments where testicular size is a major variable. The same conclusion could be extended to daily sperm production, as this measure of gamete output is very strongly related to testicular mass [29]. The small variation within twin pairs suggested that acquired individuality is an unimportant source of variation in animals with a common nutritional background. This outcome agreed with those of [30] who found that testicular size in ram Merino lambs was highly heritable and was strongly correlated with live weight. In contrast, there was little difference in the variation within and between twin pairs of ewes for the female equivalent of gamete production, ovulation rate, for which the genetic component of variance seems to be low. The contrasting outcome between genders was not expected, due to the positive correlation between scrotal circumference and ovulation rate in sheep [31]. Conversely, our results were supported by the finding that heritability for ovulation rate is low in Merino ewes [32]; this contrasts with other, more fecund breeds such as the Romanov, Galway and Finn, in which heritability for ovulation rate is moderately high [33,34]. That the TE for ovulation rate may also be higher in highfecundity breeds should be tested experimentally. Gonadotrophin secretion had very little variation between twin pairs; therefore, TE values were low. This suggested that, in general, gonadotrophin secretion
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depends more on environmental than genetic factors in MT sheep. On occasion, some TE values were greater than unity, but these observations were not consistent across the four experiments. Only LH pulse amplitude consistently had TE values greater than one, consistent with [35] who reported that LH pulse amplitude had a heritability of 0.5, suggesting that this endpoint has a significant component of genetic variance. However, even in this case, a preference for MT animals would not be warranted for three reasons. First, LH pulse amplitude is never measured exclusively as in reproductive experiments, being always accompanied by LH concentration and pulse frequency. Second, the physiological importance of LH pulse amplitude is debatable because it is poorly related to gonadal activity [36]. Third, the secretory patterns for LH failed to exhibit the same TE value across experiments. In Experiment 2, bFF failed to persistently decrease FSH concentrations or increase ovulation rate, perhaps due to a problem in the second batch of bFF. During treatment with the first batch of bFF, the FSH response was similar to that reported in previous studies in our hands [37,38], but the second batch did not maintain low FSH concentrations; this could have accounted for the lack of effect on ovulation rate. With respect to our aim to measure TE, however, this is of little consequence. In Experiments 3 and 4, we tested whether exogenous calcium and naloxone would have synergistic effects on gonadotrophin secretion. In neither experiment did naloxone evoke an increase in LH and FSH secretion, probably because the dose (0.02 mg/ kg) was too low to compete adequately for opioid receptors. Other studies with sheep have shown a stimulation of LH secretion with doses of 0.14– 3.44 mg/kg [18] and the facilitation of sexual behaviour in ewes and bucks with doses of 0.4 and 0.5 mg/kg [19,39]. It is generally accepted that an opioidergic mechanism is involved in the control of gonadotrophin secretion during the breeding season, whereas this pathway seems to be insensitive to naloxone administration during the non-breeding season [40]. For the present study, we chose the much lower dose because we expected a strong synergism with calcium, based in vitro studies showing that Ca2+ can improve the competitiveness of naloxone over b-endorphin for binding to the opioid receptor [16,41]. The complete absence of any trend indicating a response to exogenous calcium lead us to question the relevance of this for gonadotrophin secretion in Merino sheep. Alternatively, the five daily injections of calcium might not have affected calcium homeostasis adequately, because the peripheral concentration of this ion is tightly regulated.
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In Experiment 4, we expected an effect of calcium plus naloxone on ovarian activity because the same treatment has been suggested to reverse anovulation in lactating ewes [17], in which opioidergic tone markedly inhibits the resumption of reproductive activity [42,43]. The lack of effect exogenous calcium plus naloxone on ovulation rate or follicular activity (Experiment 4) was almost certainly due to absence of a stimulation of the hypothalamic–pituitary axis, as was also seen in male sheep (Experiment 3). This is of little consequence with respect to our aim to estimate TE, but further studies are needed to verify calcium–opioid interactions in vivo. In conclusion, monozygotic twin sheep offered considerable advantages for experiments where live weight and testicular mass were major endpoint variables. However, for the internal physiological variables that are involved in reproductive function, randomly selected animals were just as efficient as genetically identical twins. Acknowledgments The animal experiments described here would not have been possible without help willingly provided by everyone in the Animal Science Group. This work was supported by the National Health & Medical Research Council. Pietro Celi was supported by a UWA International Postgraduate Research Scholarship. References [1] Biggers JD. The potential use of artificially produced monozygotic twins for comparative experiments. Theriogenology 1986;26:1–25. [2] Festing MF, Altman DG. Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR J 2002;43:244–58. [3] Sze´ll A, MacLeod IM, Windsor DP, Kelly RW. Production of identical twin lambs by embryo splitting. Theriogenology 1994;41:1643–52. [4] Blache D, Chagas L, Blackberry MA, Vercoe PE, Martin GB. Metabolic factors affecting the reproductive axis in male sheep. J Reprod Fertil 2000;120:1–11. [5] Martin GB, Rodger J, Blache D. Nutritional and environmental effects on reproduction in small ruminants. Reprod Fertil Dev 2004;16:491–501. [6] Poulton AL, Robinson TJ. The response of rams and ewes of three breeds to artificial photoperiod. J Reprod Fertil 1987;79:609–26. [7] Thomas GB, Pearce DT, Oldham CM, Martin GB, Lindsay DR. Effects of breed, ovarian steroids and season on the pulsatile secretion of LH in ovariectomized ewes. J Reprod Fertil 1988;84:313–24. [8] Martin GB, Ho¨tzel MJ, Blache D, Walkden-Brown SW, Blackberry MA, Boukhliq RC, et al. Determinants of the annual pattern of reproduction in mature male Merino and Suffolk
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