Exogenous melatonin after a treatment of artificial long days is able to increase LH secretion during seasonal anoestrus in Payoya goats

Abstract

One experiment was conducted to determine whether the treatment with artificial long days and exogenous melatonin can increase, by itself, luteinizing hormone (LH) secretion during spring (seasonal anoestrus) in Payoya goats and whether this treatment causes a variation in the reactivation of the LH secretion in the normal breeding season. The experiment started on 4 November and finished on 27 October. A total of 22 ovariectomised-oestradiol treated goats were used. Ten goats were exposed to long days (16 h of light/day) from 14 November to 20 February. On 20 February, they received one s.c. melatonin implant and were exposed to natural photoperiodic changes in an open shed. The control group was located in another open shed with any treatment during the whole experiment. For both groups, plasma samples were obtained twice a week for LH assay. The photoperiodic treatment induced an earlier fall of the LH secretion. However, after the melatonin treatment, a clear increase of LH secretion during the natural seasonal anoestrus in the treated group was observed, and no difference in the onset of the LH secretion at the normal breeding season was observed. We can conclude that, in Payoya goats, the photoperiodic–melatonin treatment is by itself able to induce a clear LH increase during the seasonal anoestrus and does not modify the reactivation of the LH secretion at the normal breeding season.

Keywords:

• goat
• LH
• photoperiod
• melatonin
• seasonality

1. Introduction

Seasonal reproduction, in goats as well as in sheep, seems to be the result of a change in the responsiveness of the neuroendocrine system to the inhibitory action of oestradiol. This steroid causes a marked reduction in gonadotropin secretion during the anoestrus season (Zarazaga et al. 2011). Ovariectomised animals bearing oestradiol implants provide an experimental model, commonly used in seasonality research, that allows the changes in the sensitivity of the hypothalamus–hypophyseal axis to oestradiol experienced by normal, non-ovariectomised females to occur, but with the constant release of this hormone (Worthy and Haresign 1983). Thus, this animal model has been widely used to demonstrate the seasonality on goats (Henniawati et al. 1995; Duarte et al. 2008; Zarazaga et al. 2011).

Most breeds of sheep and goats from temperate latitudes and some either from or adapted to, subtropical latitudes exhibit seasonal variations of sexual activity (Chemineau, Daveau, et al. 1992; Delgadillo et al. 1999). The timing of reproductive seasonality in these small ruminants, as well as in most mammals, is controlled by photoperiods (Chemineau, Malpaux, et al. 1992). Melatonin, the main secretory product from the pineal gland, is the neuroendocrine signal that transduces information about the environmental light received by the retina.

Some of the proposed treatments to manipulate the reproductive seasonality are the male effect, the exogenous melatonin treatment, the photoperiodic treatments or a combination. The seasonal variation in reproductive activity observed under natural conditions is profoundly altered when animals are subjected to alternations of 3 months of long and 3 months of short days. Under these conditions, sexual activity of males and females was shown to peak during short days (Chemineau et al. 1988). Under field conditions, the long-day part of the treatment is easy to apply, as extra illumination can be provided indoors or outdoors.

Exogenously administered melatonin from continuous slow-release implants has been shown to advance the onset of the breeding season in goat males and females by mimicking the stimulatory effect of short days (Chemineau, Malpaux, et al. 1992; Zarazaga et al. 2009, 2010). Traditionally in Mediterranean latitudes, exogenous melatonin treatment is implanted around the spring equinox and accompanied by a prior separation of males and females for 45 days, inducing a male effect to optimise the response during the anoestrous period, thus obtaining a higher synchronisation at mount. Due to this isolation between males and females, it has been deemed unnecessary for the male effect to successfully induce ovulation depending on the sexual activity of the familiar and novelty males and sexual activity of the stimulatory males (for details, see Delgadillo et al. 2009). Moreover, this isolation implies more difficult management of the animals and a need for more equipment at the farms.

Therefore, this study had a double aim. The first part was to determine whether, following a treatment with long days from the second fortnight of November to late February, followed by a melatonin implant, it is effective to increase LH secretion in ovariectomised-oestradiol treated female goats during the seasonal anoestrus (short-term effect). The second part was to determine whether this treatment causes a variation in the reactivation of LH secretion in the subsequent natural breeding season (medium-term effect).

2. Materials and methods

The present study was conducted at the University of Huelva experimental farm (latitude 37°15′). All handling and experimental procedures were carried out in strict accordance with Spanish guidelines for the protection of experimental animals (RD 1201/2005), and by trained personnel, conforming to European Union Directive 86/609 regarding the protection of animals used in scientific experiments.

2.1. Animals and management

The animals in the study were 22 adult Payoya female goats (3–4 years old; 47.7±3.5 kg; 2.60±0.09 body condition score). All were ovariectomised 1 month before beginning the experiment and simultaneously implanted subcutaneously with a 3.0-cm Silastic implant (internal diameter 3.3 mm and external diameter 4.6 mm; Karsch et al. 1973) containing crystalline oestradiol (Sigma-Aldrich Chemical Co.). These implants were soaked in physiological serum before insertion to prevent an initial peak of steroid release.

All animals were fed daily with lucerne hay, barley straw (ad libitum) and commercial concentrate to maintain their weight in agreement with Institut National de la Recherche Agronomique (INRA) maintenance requirements (Morand-Fehr and Sauvant 1988). The concentrate was a commercial mixture of maize (26.3%), beans (20%), oats (14.1%), cottonseed (13.7%), peas (13.4%), lupin (7.3%), barley (0.2%), wheat (0.2%), sunflower seeds (0.2%) and a commercial mineral–vitamin complement (4.6%). All animals had free access to water and mineral blocks containing trace elements and vitamins.

2.2. Experimental design

The onset of experiment was 4 November when they were distributed into two groups. On 14 November, 10 females (PHOTO-MEL group, n=10) were exposed to long days (16 h light: 8 h night; lights on 06:00: lights off 22:00) for 98 days, until 20 February. An electric timer that controlled white fluorescent strip lights providing approximately 200 lux at goat eye level during the light phase regulated the photoperiod. A ventilator system was used for air renewal. On 24 February, females were re-exposed to the natural photoperiod, and received a single subcutaneous implant containing 18 mg of melatonin (Melovine®, CEVA Salud Animal, Barcelona, Spain), which was inserted at the base of the left ear. These implants released melatonin for about 10 weeks, raising daytime concentrations to about 100 pg/mL (Delgadillo et al. 2001). The remaining animals (C group, n=12) were kept in a communal yard with an uncovered area and without any supplementary light throughout the experiment.

2.3. Sampling and measurements

The effect of photoperiod treatments on neuroendocrine LH activity was assessed by monitoring long-term plasma LH variations, and by collecting blood samples twice per week by jugular venipuncture at 09:30 h. The samples were immediately centrifuged for 30 min (3000 g) and the plasma stored at −20°C until use.

Plasma LH concentrations were determined using double-antibody ELISA, as previously described in Faure et al. (2005). The sensitivity of the assay was 0.1 ng/mL. The intra- and interassay coefficients of variation of the control were 9.8 and 5.8%, respectively.

2.4. Definitions of reproductive activity

The non-breeding season and the natural breeding season were defined according to the results obtained at the same latitude and with a similar method by Zarazaga et al. (2005, 2009). The activation and cessation of the LH secretion were measured during the non-breeding season induced by the treatment. The onset of reactivation of the LH secretion (breeding season) was measured at the reactivation of reproductive activity in the natural breeding season.

The reproductive state was assessed using characteristics of the LH profile. The onset of the breeding season was defined as the date of the first plasma LH sample above 0.5 ng/mL, in a sequence of three or more samples above 0.5 ng/mL. The end of the breeding season (onset of the seasonal anoestrus) was defined as the date of the last plasma LH sample above 0.5 ng/mL, in a sequence of two or more points below 0.5 ng/mL (Zarazaga et al. 2005, 2009).

2.5. Statistical analysis

The effect of the treatment and time on LH concentrations was analysed using an analysis of variance for repeated measures. The one-way ANOVA was used to evaluate the effect of treatment on the date of the onset and the end of the breeding season. Differences were considered significant at P<0.05. Analysis of data was computed using the Statistical Package for the Social Sciences (SPSS) package (SPSS 2008).

3. Results

Figure 1 shows the changes in LH concentrations during the experiment. The experimental time (P<0.05) and treatment–time interaction (P<0.01) modified the LH evolution. The fall of LH concentrations induced by the photoperiodic treatment was earlier than in the control group (3 February±10.7 days vs. 2 March±4.1 days for PHOTO-MEL and C group, respectively, P<0.05). Thereafter the LH concentrations of the PHOTO-MEL group remained at basal levels and started to rise on March 21st±10.2 days, with values remaining high until 13 June±6.2 days. The mean duration of high LH concentrations induced by the treatment was 84.0±14.6 days. The LH secretion increased rapidly after implant insertion, with a delay of only 29.7±10.2 days. No differences between groups were observed on the reactivation of the LH concentrations at the normal breeding season (23 August±16.8 days vs. 26 August±4.9 days, for PHOTO-MEL and C groups, respectively).

Figure 1.  Weekly means (±SEM) of LH concentrations (ng/mL) of Mediterranean goat females subjected to artificial long days from November to February, followed by treatment with one s.c. melatonin implant (PHOTO-MEL group, ▪) or to natural changes in day length (C group, □). The shaded area indicates the duration of the long-days treatment.

4. Discussion

This study demonstrated that, in Payoya goats, the treatment associating 3 months of long days and exogenous melatonin is able, by itself, to increase the LH concentrations during the natural seasonal anoestrus (short-term effect) and did not induce a retardation on the reactivation of the LH secretion in the natural breeding season (medium-term effect).

The treatment with a photoperiod establishes that this treatment induced an earlier fall of the LH secretion. These results are similar to those described recently by our group with entire goats with the same treatment and in contact with males (Zarazaga et al. 2011a) and suggest that photoperiod controls reproductive activity in goats as it has been recently demonstrated (Gómez-Brunet et al. 2010; Zarazaga et al. 2011). The end of the LH secretion of the control group was very similar to that described by our group using a similar methodology and the same breed (Zarazaga et al. 2005).

The time between the melatonin treatment and the start of the treatment-induced LH secretion was very short and similar to that described in entire goats submitted to the same treatment in contact with males (46.9±2.4 days) (Zarazaga et al. 2011a) and in ovariectomised goats implanted with melatonin around the spring equinox (30.33±10.17 days) (Zarazaga et al. 2009). These results contrast with those described by Chemineau et al. (1986) who observed no ovulation after a period of 70 short days. This discrepancy is probably due to breed differences and/or lactation influences. This increase in the mean values of LH has also been attributed to the effect of melatonin on the hypothalamus–hypophysis (Arendt 1995), increasing the secretion of LH. Afterwards, LH concentrations in the treated group fell to the basal level, probably because of refractoriness to the stimulatory short-days signal provided by melatonin (Almeida and Lincoln 1984; Lincoln and Ebling 1985; Gómez-Brunet et al. 2010).

The LH stimulation during the seasonal anoestrous after melatonin treatment contrasts with those reported in sheep. In this way, Forcada et al. (1995) working with intact females in contact with males, and inserting melatonin implants in April, observed an early onset of the breeding activity but the treatment did not induce reproductive activity during the seasonal anoestrous. Similarly, Forcada et al. (2002) observed in ewes that the melatonin implant inserted around the spring equinox was unable to induce a resumption of reproductive activity before the onset of the natural breeding season. This suggests that goats and sheep may differ in their interpretation of the photoperiodic signal, or that the mechanism of seasonal reproductive control differs between the two species as has been suggested recently in goats (Zarazaga et al. 2011).

The reactivation of the LH secretion in the natural breeding season was no different between groups, which contrast with our previous results using entire females in contact with males (Zarazaga et al. 2011a). However, in that experiment the treated females started their ovarian activity slightly later than control females, but no differences were observed on the onset of the oestrous activity, which could indicate that the negative effect of this treatment on reproductive activity could be limited. In this way, when a similar photoperiodic treatment was performed without melatonin implant, no effect on reactivation of the reproductive at the normal breeding season was observed (Zarazaga et al. 2011b).

Concerning the control group, our results demonstrate unambiguously that Payoya goats express seasonal variations in their reproductive activity. Indeed, they show a clear rest season from February to August characterised by high LH concentrations. This is consistent with results described in the same breed (Zarazaga et al. 2005). In the present experiment, reactivation of the LH secretion was earlier than the first year and later than the second year of the results obtained in the former. The reason for these discrepancies might be down to the year of the breed as it has been demonstrated that it is an important source of variation in reproductive activity (Sierra 1969). Moreover, these results are consistent with other goat breeds at different latitudes. Indeed, local goats in Argentina display seasonal variations in oestrus and ovulation at latitude of 30°S, with a breeding season lasting from February to September (southern hemisphere) (Rivera et al. 2003). Likewise, the breeding season of cashmere goats in Australia at latitudes of 29°S is from March to August (Restall 1992). Similarly, Criolla goats in Northern Mexico at 26°N show a clear contrast between a breeding season lasting from September to February (Duarte et al. 2008; Urrutia-Morales et al. 2009; Rincón et al. 2011).

Conclusions

In conclusion, the results of the present experiment demonstrates that photoperiodic–melatonin treatment, in Payoya goats, is by itself able to induce LH secretion during the seasonal anoestrus and that it did not induce a modification on the LH increase during the normal breeding season.

Acknowledgements

The authors wish to thank the Assay Laboratory of the Station de Physiologie de la Reproduction et des Comportements (INRA, Nouzilly, France) for carrying out the radioimmunoassays. This work was supported by Grant PETRI 95-0964.OP from C.I.C.Y.T. (Spain). The authors are grateful to CEVA Salud Animal, Barcelona, Spain, for providing Melovine® implants.