Effect of naloxone on the plasma levels of LH, FSH, prolactin and testosterone in Beetal bucks

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1 Small Ruminant Research 37 (2000) 51±55 Effect of naloxone on the plasma levels of LH, FSH, prolactin and testosterone in Beetal bucks Baljeet Singh, V.D. Dixit, Pardeep Singh *, G.C. Georgie, V.P. Dixit Department of Animal Production Physiology, CCS Haryana Agricultural University, Hisar , India Accepted 21 September 1999 Abstract Ten adult male Beetal goats were used for the study to elucidate the modulation of gonadotrophin, prolactin and testosterone secretion by endogenous opioid peptides. An indwelling catheter was placed in the jugular vein of each buck 20 h before the onset of the experiment. Bucks were divided randomly into two groups: Group I (n ˆ 5) received naloxone at a dose rate of 1 mg/kg body weight (BW) and Group II (n ˆ 5) received naloxone at a dose rate of 2 mg/kg BW intravenous. Blood samplings were done from 2 h before treatment until 2 h after treatment at 15 min intervals. Blood samples were quanti ed for plasma LH, FSH and prolactin concentration using a heterologous double antibody radioimmunoassay (RIA) and testosterone concentration was quanti ed by coat-a-count RIA kit. The mean plasma LH levels during pretreatment phase were ng/ml in Group I and ng/ml in Group II which signi cantly (p < 0.05) increased to ng/ml in Group I and ng/ml in Group II. The mean plasma FSH levels did not show a difference in pre- and posttreatment animals in both groups. A signi cant (p < 0.05) increase in plasma testosterone concentration was observed in both groups after naloxone treatment, whereas, a decrease (p < 0.05) was observed in plasma prolactin levels after naloxone treatment. Thus, it can be concluded that endogenous opioids do play an important role in modulating plasma LH, prolactin and testosterone concentrations in male goats. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Naloxone; Gonadotrophin; Prolactin; Testosterone; Male goat 1. Introduction Endogenous opioid modulation of reproductive activity is well documented for female animals in several domesticated species (Whisnant et al., 1986; Gregg et al., 1986; Currie and Rawling, 1987; Byerley et al., 1992; Xia Orong, 1996). However, in the male * Corresponding author. Tel.: /ext. 4235; fax: address: hau@hau.hry.nic.in (Pardeep Singh) animal of various species the information in this regard is sparse. In adult rats b-endorphins have been shown to inhibit testosterone secretion possibly because of its effect on synthesis of testosterone precursors (Chandershekar and Baskte, 1992). Earlier Cicero et al. (1989) reported an increase in serum LH and a dose independent increase in testosterone concentration in rats after naloxone treatment. Naloxone treatment was found to elevate plasma FSH levels but not plasma LH levels in immature pigs (Trudeau et al., 1989). Naloxone has been found to signi cantly /00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S (99)

2 52 B. Singh et al. / Small Ruminant Research 37 (2000) 51±55 increase the release of LH and prolactin in stallions whereas, it has no effect in either plasma LH or prolactin levels in geldings (Aurich et al., 1996). In growing bull calves it has been indicated that the effect of naloxone on LH was age related, and a decrease of opioidergic inhibition of pulse frequency of LH at 12± 18 weeks of age contributed to the overall increase in the circulating serum concentrations of LH (Evans et al., 1993). A signi cant correlation between basal testosterone levels and increase in LH in stallions has been reported (Pluschke, 1994) indicating that opioid inhibition of LH release is related to the presence of gonadal steroids in this species. In male goats, (Fuentes et al., 1997, 1998) have reported an increase in libido and testosterone levels during non-breeding season. The caprine species has received little attention with respect to opioid modulation of reproductive or endocrine activity. The objective of the present study was to characterise the endocrine response of the male goat to naloxone. 2. Material and methods Ten adult Beetal male goats aged 4±6 year were randomly selected from the University goat ock (45± 60 kg body weight (BW)) and used in this study. The experiment was conducted during the month of May (non-breeding season) The goats were fed a legume hay grain diet and had free access to water. The bucks were divided randomly into two groups. The goats in Group I were given naloxone hydrochloride intravenous (Sigma, USA) at a rate of 1 mg/ kg BW, while the bucks in the other group were given naloxone at a rate of 2 mg/kg BW as a bolus dose intravenously. An indwelling catheter was placed in the jugular vein of each buck 20 h prior to the onset of blood sampling. Blood samples were collected at 15 min intervals from 2 h before to 2 h after the naloxone treatment. The blood samples were stored in an ice bath prior to centrifugation at 3000 rpm in a refrigerated centrifuge (58C). The plasma was separated within 30 min of sample collection and stored at 758C, pending hormonal analysis. The plasma LH, FSH and prolactin levels were quanti ed using the heterologus double antibody radioimmunoassay techniques standardised in the laboratory (Kaker et al., 1980; Kaker et al., 1982; Razdan et al., 1982). The sensitivities, intra-assy and inter-assay coef cients of variation were ng/ tube, 4.5 and 4.9% for plasma LH, 0.25 ng/tube, 9.3 and 10.2% for plasma FSH and 2.5 ng/tube, 7.6 and 9.4% for the prolactin assay systems, respectively. Plasma testosterone concentrations were also determined by the direct solid phase 125 I RIA kit (Coat-A- Count, Diagnostic Products, Los Angeles, CA, USA). The data obtained were analysed using a general linear model two way Manova (multivariate anova), treating dose and phase (pre-treatment and post-treatment) as xed independent variables and hormone concentrations as dependent variables with sample sequences as a covariate using a statistical software package on a PC (SPSS version 7.51, release of 1996; SPSS; Chicago, USA). 3. Results Plasma concentration of hormones (range and mean SEM) at two dose levels of naloxone treatment prior to and after administration of the opioid antagonist are presented in Table 1. The mean and SEM of the hormone levels at each 15 min sampling is set out in Fig. 1. It was evident from the statistical analysis that only in the case of plasma LH and testosterone the difference in the hormone concentrations at the two dose levels of naloxone were signi cant (p < 0.05). The testosterone levels started rising in both groups 15 min after the increased plasma LH concentration resulting from naloxone treatment. Thus, by only these two hormones exhibited naloxone dose related responses in their circulating plasma concentrations. Plasma LH was elevated by the higher (2 mg/kg BW) dose of naloxone as was the case with the gonadal hormone testosterone. The increase in plasma LH was signi cant (p < 0.05) and amounted to a 4-fold increase in level by 30 min after naloxone treatment, compared to the prenaloxone period concentrations. The increase was only 3-fold in the case of 1 mg/kg BW naloxone injection after 30 min. The circulating levels of FSH did not change as a result of naloxone administration (Fig. 1). It can be seen that plasma FSH was not affected by the treatment with naloxone. On the other hand, plasma LH and testosterone concentrations in the circulation was elevated and plasma prolactin concentrations decreased. The

3 B. Singh et al. / Small Ruminant Research 37 (2000) 51±55 53 Table 1 Plasma concentrations of LH, FSH, prolactin and testosterone before and after naloxone treatment in bucks Pre-naloxone a Post-naloxone a Pre-naloxone a Post-naloxone a 1 mg/kg BW (Group I) 2 mg/kg BW (Group II) LH (ng/ml) Range 0.17± ± ± ±2.92 Mean SEM 0.41 A BC A BD 0.07 FSH (ng/ml) Range 14.25± ± ± ±37.26 Mean SEM A A A A 1.69 Prolactin (ng/ml) Range 83.94± ± ± ± Mean SEM B A B A 3.89 Testosterone (ng/dl) Range 17.67± ± ± ± Mean SEM A BC A BD 4.79 a Mean bearing dissimilar letters differ signi cantly from each other (p < 0.05). Fig. 1. Effect of opioid antagonist naloxone on plasma (a) LH, (b) FSH, (c) prolactin and (d) testosterone in male goats. (zero on X-axis is the time of administration of naloxone.

4 54 B. Singh et al. / Small Ruminant Research 37 (2000) 51±55 statistical analysis also indicated that the sampling sequence did not affect the hormone concentrations. 4. Discussion The consistent increase in plasma LH immediately following naloxone injection in these bucks (Fig. 1, Table 1) demonstrated a considerable involvement of endogenous opioids in the modulation of plasma LH levels in the blood circulation. As naloxone is relatively speci c as an endogenous opioid receptor antagonist (Goldstein, 1984), it can be concluded that endogenous opioids are inhibitory to LH release in male goats. The LH release by naloxone treatment due to disinhibition of the opioid effect has also been observed in female goats (Xia Orong, 1996). The plasma LH levels reported in this study are in agreement with other reports in steers (Peck et al., 1990) in rats (Cicero et al., 1989 and El-Sheltawi et al., 1995), in stallions (Aurich et al., 1996), in prepubertal bull calves (Rawlings et al., 1991 and Evans et al., 1993). However, Trudeau et al. (1989) and Prunier et al. (1990) did not nd any increase in plasma LH secretion after naloxone injection in immature male pigs and suggested that endogenous opioids are not involved in regulation of LH in the immature male pig. Plasma FSH concentrations have been reported to be increased by naloxone treatment in intact and castrated rats (El-Sheltawi et al., 1995), in young ram lamb (Rawlings et al., 1991) and in immature male pigs (Trudeau et al., 1989). In contrast to this study, naloxone administration did not increase plasma FSH concentration in Group I, however there was slight increase in FSH concentration in Group II, which was not signi cant. Lincoln (1988) reported no in uence of naloxone on serum FSH secretion and Rawlings et al. (1993) suggested that in the adult ewe, naloxone does not appear to in uence FSH secretion. This leads to the conclusion that the opioid antagonist acts directly on the LH producing cells at the hypophysis level and is not affected by the GnRH releasing mechanism at the hypothalmic level. However, it cannot be assessed from this study as to whether the synthesis of LH or its release from hypophyseal cells or both are in uenced by naloxone. It has been hypothesized from studies in rats (Miller et al., 1986) that the mechanism of action may be through interference with the GnRH pulse generator. However, lack of response in FSH and response only in plasma LH obtained in this study on bucks do not support this hypothesis. Administration of naloxone decreased plasma prolactin concentrations in ewes (Gregg et al., 1986) and intact and castrated male rats (El-Sheltawi et al., 1995). Aurich et al. (1996) found no effect of naloxone on plasma prolactin levels in geldings, but increased prolactin concentrations after naloxone was reported in stallions by the same researchers. In the present study, plasma prolactin concentration decreased signi cantly (p< 0.05) after naloxone injection in both treatment groups. A signi cant (p< 0.05) increase in plasma testosterone levels as reported in this study, is in agreement with results reported earlier (Cicero et al., 1989; Pluschke, 1994; Fuentes et al., 1997; Fuentes et al., 1998). The effect of naloxone on plasma testosterone levels can be attributed to the after effect of elevated LH levels. Similar conclusion was reported by Miller et al. (1986). Naloxone probably acts by blocking the inhibitory effect on testosterone precursor synthesis by the endogenous opioids, as is reported to occur in rats (Chandershekar and Baskte, 1992). It was evident that endogenous opioids do play an important role in modulating LH, prolactin and testosterone concentrations in bucks of the Indian subtropical breed of Beetal goats. There is also an indication that at least in this species, naloxone has acted independently of its antagonistic effect on the action of endogenous opioid peptides on GnRH pulse generator. Acknowledgements The authors acknowledge the donation of naloxone by Prof. Nahid Parvizi, Institute of Animal Breeding and Ethology, Mariensee, Federal Republic of Germany. The RIA reagents for LH, FSH and prolactin were donated by NHPP, MIDDK, NIH, Bethesda, MD, USA. References Aurich, C., Burgmann, F., Hoppe, H., Opioid regulation of luteinizing hormone and prolactin release in the horse-identical or independent endocrine pathways? Anim. Reprod. Sci. 44 (2), 127±134.

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