Abstract: From a commercial flock, 15 prepuberal does of the Alpine breed were selected, body weight and age averaged 35.8 kg and 8 months, respectively. The experiment was carried out during the months of May and June when anoestrus is more incipient in this latitude (19°13'N). Feeding and handling was conventional with water ad libitum. In groups of 5 chosen at random, they were treated as follows: Group A was used as control, group B received an implant of 15 mg naloxone. And group C received an i.m. injection of 0.5 mg naloxone i.m. at 12 h intervals for 15 days. To observe the effect of this experimental treatment on the pattern of secretion of LH, on day 0 a blood sample (2 mL) was obtained through a cannulae implanted in the jugular vein every 20 min for 7 h continuous. Blood samples were allowed to coagulate at room temperature and thereafter, centrifuged at 2.500 rpm for 15 min; plasma was decanted into small plastic vials and stored frozen until LH assay was carried out. Blood samples were obtained on day 0, 5, 10, 15 and 20. Plasma concentration of LH was determined by RIA as previously reported. And the statistical analysis of LH concentrations was studied using a descriptive method followed by an inferential screening and a variance analysis. It was observed that the plasmatic concentrations of LH changed through time in all groups, changes were more pronounced in the naloxone treated animals as compared with the control group. It was observed that there was no significance on the interaction between treatment and the sampling day. An influence of time upon the plasmatic levels of LH was observed, showing a high degree of significance (f = 12.58 (4.44) p = 0.0000005). There was tendency of an increase in LH secretion through time observed in all groups. A large difference was detected when comparing plasma LH from the first 2 samples with samples obtained in days 10, 15 and 20 of the experiment. It was observed that the effect of naloxone through time did not show significant differences until after the second week of treatment. This results give further support of the important role that endogenous opioids on the control of reproduction of sheep.

INTRODUCTION

The survival of species is determined through their capacity for reproduction and this event is controlled by the interaction of different biological systems characteristic of each species. Reproductive behaviour is under the control of external and internal factors. All of which permit birth to happen during the appropriate time of the year, in order to insure the survival of the new born (Fabre-Nys et al., 1993; Mori, 1989).

Among the many species that are under the influence of ambient factors, the ewe and goat are good examples. These small ruminants have a period of anestrus followed by a period of sexual activity (oestrus). The expression of sexual behaviour is stimulated during the beginning of the short days, while, the period of anoestrus is onset during the long days (Fuentes, 1988). This behaviour is more determinant in the north hemisphere, where the European breeds originated (Chemineau et al., 1988). In countries near the ecuator where photoperiod changes are not so marked, European goat breeds behave in much the same way (Silva et al., 1994; Mellado, 1997).

The period of anestrus is of interest because productivity in intensive production units is not optimal and many research teams are studying the mechanisms that determine the presence of periods of anoestrus and oestrus, using small ruminants as a model for such observations (Currie et al., 1991a; Chemineau et al., 1988; Malven, 1995). The capacity for connecting and disconnecting sexual behaviour in these species, means that there is an internal mechanism that through external and internal sensors are able to change the pattern of activity of the hypothalamic-hypophisial-gonadal axis. Modulating the secretion of the gonadotrophic hormones such as LH (Edqvist and Stabenfeldt, 1993a b; Fabre-Nys et al., 1993). During anoestrus the pulsatile secretion of LH is dimished (Schall et al., 1991) and during the breeding season pulsatile secretion of LH is increased (Scaramuzzi and Baird, 1977). The secretion of gonadotrophins is under the influence of releasing hormones that are originated in the median eminence of the hypothalamus. Furthermore, releasing hormones are secreted at rates determined by the influence of central nervous transmitters and neurohormones. Among the latter there is evidence that melatonin (Malpaux et al., 1998), dopamine and peptide opioids are important modulators of GnRH secretion (Rossier and Chapouthier, 1981; Currie, 1989; Currie and Rawlings, 1991b; Malven, 1995; Malpaux et al., 1998; Fuentes et al., 1998a).

Trough the manipulation of opioidergic receptors with opioid antagonists it is possible to induce changes in the pattern of secretion of gonadotrophic hormones in several species (Brooks et al., 1986; Cosgrove et al., 1993; Fuentes, 1988; Aurich et al., 1995; Grossman, 1983). Some have considered the pattern and magnitude of naloxone-induced changes in endocrine function to facilitate identification of sexually active and inactive rams during the breeding season. Prediction accuracy of the naloxone-based test was 69-85% (Stellflug et al., 2004). In the latter experiment, Rams were treated with 1.5 mg of naloxone kg^-1 BW. In experiments with different animal species, de dose used was in average 1 mg body^-1 weight. Some report undesirable side effects and death of experimental animals after the acute administration of such high doses (Nanda et al., 1989; Yang et al., 1988). In bucks naloxone was used to study opioid interaction with LH and testosterone secretion, with doses of 1 mg kg^-1 BW (Baljeet et al., 2000). In previous research, we have observed that small doses produce significant changes in LH, Testosterone and prolactin plasma levels in different species (Fuentes et al., 1997, 2007).

It is well known that hypothalamic opioid receptors are sensitive to opioid antagonists, the higher the dose the higher effect on different opioid receptors. Naloxone administered in high doses affect various opioid receptors and it is possible to postulate that small doses show a tendency to affect selectively μ-receptors (Fuentes et al., 2007).

In previous researches, it was observed that small doses of opioid antagonists with selective affinity for μ-endorphinergic receptors, such as naloxone, induced changes in the sexual behaviour and blood hormones levels of sheep and goats (Fuentes, 1988). The administration of naloxone is limited by the pharmacological characteristic of its absorption and distribution through the experimental animal body. It is administered solely by the parenteral route, therefore all previous experiments with this opioid antagonist have being carried out using the intramuscular and intravenous routes. It was considered of interest to study the effect of a subcutaneous implant containing 15 mg naloxone on the plasmatic concentrations of LH of the goat during the anoestrus period.

MATERIALS AND METHODS

From a commercial flock, 15 does of the Alpine breed were selected, body weight and age averaged 35.8 kg and 8 months, respectively. The experiment was carried out during the months of May and June when anoestrus is more incipient in this latitude (19°13'N). Feeding and handling was conventional with water ad libitum. In groups of 5 chosen at random, they were treated as follows: Group A was used as control, group B received an implant of 15 mg naloxone. And group C received an i.m. injection of 0.5 mg naloxone i.m. at 12 h intervals for 15 days.

To observe the effect of treatment on the pattern of LH secretion, on day 0 a blood sample (2 mL) was obtained through a cannulae implanted in the jugular vein every 20 min for 7 continuous h. Blood samples were allowed to coagulate at room temperature and thereafter centrifuged at 2.500 rpm for 15 min, plasma was stored frozen until LH assay was carried out.

Blood samples were obtained on day 0, 5, 10, 15 and 20. Plasma concentration of LH was determined by RIA as previously reported (Fuentes et al., 1998b). And the statistical analysis of LH concentrations were studied using a descriptive method followed by an inferential screening and a variance analysis.

RESULTS

It was observed that the plasmatic concentrations of LH changed through time in all groups, changes were more pronounced in the naloxone treated animals as compared with the control group. This changes can be appreciated observing Fig. 1-5. Where plasma concentrations of all groups can be compared during the different times of the experiment.

After performing the variance analysis, it was observed that there was no significance on the interaction between treatment and the sampling day. But there is a treatment influence on the plasmatic levels of LH. An influence of time upon the plasmatic levels of LH was observed, showing a high degree of significance (f = 12.58 (4.44), p = 0.0000005).

Fig. 1:	The effect of nalexone on plasmatic levels of LH on the 1st day of the experiment

Fig. 2:	Plasma levels of LH in nalexone injected, implanted and control goats on

Fig. 3:	Plasma levels of LH on the 14 day of the treatment in nalexone injected

Fig. 4:	Comparative level of plasma LH on day 21, in between groups

Fig. 5:	Accumulated plasma levels of LH according to the day of sampling and group

This interaction is clearly illustrated in Fig. 5, where a contrast of the obtained averages is observed in the different groups and also is observed a tendency of an increase in LH secretion through time observed in all groups.

In order to study, the influence of treatment upon time on the plasmatic levels of LH a large difference was detected when comparing the first 2 samples with the samples obtained in days 10, 15 and 20 of the experiment. It was observed that the effect of naloxone through time did not show significant differences until after the second week of treatment.

DISCUSSION

The observed results in this study show that the plasmatic concentrations of LH are in agreement with the reports of similar observations in the ewe (Currie et al., 1991a, b; Fuentes et al., 1998a, b; Malven, 1995; Schall et al., 1991); goats (Fuentes et al., 1997; Cheminenau, 1988).

Naloxone is an opioid antagonist that has being used as a tool to study the interaction of endogenous opioids with sexual function, but the dose used is the first reports is extremely high, in the order of 0.5 mg kg^-1 (Currie and Rawlings, 1989; Schall et al., 1991) and in this research, the dose used is 1.0 mg total dosis day^-1. Furthermore, in previous research Fuentes (1988) and Fuentes et al. (1998a, b) using the same dose (1 mg day^-1) has reported changes in sexual behaviour and changes in the pulsatile release of LH. In this research, the use of a subcutaneous implant with 15 mg naloxone produces much the same changes as those observed with injections of 0.5 mg at 12 h intervals and it was observed that the concentration of LH increased with time of treatment. When Tuckey’s test is used to study this changes it was observed a significant difference between the plasmatic levels of control goats with those of the naloxone treated animals. The latter observation shows that the effect of naloxone is not inmediate, it takes time for naloxone to express the antagonist effect on μ receptors. When naloxone was administered to adult bucks during the anoestrus season, testosterone levels were not affected until after 7 days of treatment (Fuentes et al., 1997). Furthermore, in this research, significant changes are not present until after 7 days of treatment. It is interesting that when naloxona is injected epidrurally for the treatment of chronic pain, the effect of one single injection persists for several weeks (Blaise, 2003). The latter suggests that there is a time effect on the endocrine response to naloxona treatment (Fuentes et al., 2003). There are reports, in which the administration of high doses in acute or continuous intravenous infusions of naloxona does not consistently affect blood hormone concentrations (Ebling and Lincoln, 1985; Barb et al., 1991). The latter might be due to effect of high doses of naloxone on different opioid receptors (Barb et al., 1991).

CONCLUSION

This observation further supports that naloxone administered in low doses produces changes of significance both in sexual behaviour and hormone concentrations (LH). The effect of an implant of naloxone is similar to the effect observed when the opioid antagonist is administered in small doses at 12 h intervals for 15 days.

This findings show the need of further research using low doses of opioid antagonists in order to permit continuous interaction with opioid receptors with the aim of inducing physiologic changes as those needed to express sexual behaviour.