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Animal Reproduction Science 138 (2013) 168– 174

Contents lists available at SciVerse ScienceDirect

Animal Reproduction Science

journa l h om epa ge: www.elsev ier .com/ locate /an i reprosc i

he effect of exogenous melatonin during theon-reproductive season on the seminal plasma hormonalrofile and the antioxidant defence system of Rasa Aragonesaams

driana Casaoa,∗, Rosaura Pérez-Péa, José Alfonso Abeciab, Fernando Forcadab,eresa Muino-Blancoa, José Álvaro Cebrián-Péreza

Departamento de Bioquímica y Biología Molecular y Celular, Grupo Biología y Fisiología de la Reproducción (BIOFREZ), Institutoniversitario de Investigación en Ciencias Ambientales de Aragón (IUCA), Facultad de Veterinaria, Universidad de Zaragoza, SpainDepartamento de Producción Animal y Ciencia de los Alimentos, Grupo Biología y Fisiología de la Reproducción (BIOFREZ), Institutoniversitario de Investigación en Ciencias Ambientales de Aragón (IUCA), Facultad de Veterinaria, Universidad de Zaragoza, Spain

a r t i c l e i n f o

rticle history:eceived 2 May 2012eceived in revised form9 December 2012ccepted 9 February 2013vailable online 22 February 2013

eywords:ameminal plasmaelatonin

a b s t r a c t

The aim of this study was to analyze the effect of melatonin implants, during the non-reproductive season, on the content of melatonin, testosterone and 17-� estradiol levels,and the antioxidant enzymes glutathione peroxidase, glutathione reductase, superox-ide dismutase and catalase of seminal plasma in Rasa Aragonesa rams. Five rams wereimplanted with melatonin, and four others were used as a control group. Seminal plasmawas separated from ejaculates collected one week before melatonin treatment until 21weeks after melatonin placement, and the hormonal levels and the antioxidant enzymeactivity were determined. Exogenous melatonin treatment significantly (P < 0.05) increasedthe levels of endogenous melatonin in seminal plasma immediately, and this effect lastedfor 14 weeks. Testosterone and 17-� estradiol levels significantly (P < 0.05) increased

estosteronestradiolntioxidant enzyme

four and eight weeks after melatonin treatment, respectively. As regards the antioxidantenzymes, melatonin treatment significantly increased (P < 0.05) glutathione peroxidase andglutathione reductase activity only, and had no effect on superoxide dismutase and cata-lase. Therefore, melatonin treatment during the non-breeding season modifies the seminalplasma hormonal profile and some antioxidant enzyme activity in Rasa Aragonesa rams.

. Introduction

Sheep are short day breeders and their seasonality is

egulated by melatonin secretion (Malpaux et al., 1996).elatonin treatment during the non-reproductive season,ainly by means of subcutaneous melatonin implants,

∗ Corresponding author at: Departamento de Bioquímica y Biologíaolecular y Celular, Facultad de Veterinaria, Miguel Servet 177, 50013,

aragoza, Spain. Tel.: +34 976761643; fax: +34 976761612.E-mail address: adriana@unizar.es (A. Casao).

378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.anireprosci.2013.02.002

© 2013 Elsevier B.V. All rights reserved.

seems to reverse the reproductive effect of seasonality inboth rams and ewes (Haresign et al., 1990). Out-of-seasonmelatonin treatment in rams has been associated with anincrease in scrotal diameter and an improvement of spermquality and reproductive performances (Casao et al., 2010a;Palacín et al., 2008), although the way this hormone exertsits effect is not widely known. While melatonin seems toexert its function mainly on the hypothalamus-pituitary

axis (Misztal et al., 2002), it can also have an effect on thetesticular and accessory glands, as the presence of mela-tonin receptors in the male reproductive system in otherspecies seems to suggest (Frungieri et al., 2005; Gilad et al.,

duction

A. Casao et al. / Animal Repro

1998). It increases testosterone secretion (Kokolis et al.,2000) and probably regulates antioxidant enzyme activity.A recent study conducted by our group (Casao et al., 2010b)has demonstrated the presence of melatonin in ram semi-nal plasma with seasonal variations, and its correlationwith testosterone levels and antioxidant enzyme activityin the same fluid. Thus, seasonal differences in ram spermquality and fertility may be due not only to the presenceof melatonin in seminal plasma, which can exert a directaction over the spermatozoa (Casao et al., 2010c) by bind-ing to its receptors (Casao et al., 2012), but also to variationsin the composition of the seminal plasma.

Seminal plasma can affect sperm quality and fertilitythrough its composition in organic and inorganic com-pounds, including hormonal levels and also antioxidantenzyme activity (Maxwell et al., 2007). High levels ofreactive oxygen species (ROS) in seminal plasma couldinduce premature sperm capacitation (de Lamirande andGagnon, 1993). along with an increase in apoptosis andDNA damage (Agarwal et al., 2008; Martinez-Pastor et al.,2009), which could lead to infertility. In the male, semi-nal plasma antioxidant enzymes, generated mainly in theepididymis and accessory glands, preserve sperm fromDNA oxidative damage (Wai-sum et al., 2006) and lipidperoxidation of the plasma membrane (Tavilani et al.,2008).

In a recent study (Casao et al., 2010b) we described aseasonal pattern of melatonin and testosterone in seminalplasma and their interplay with the antioxidant system.This suggested that the out-of-season melatonin treatmentof rams could affect the hormonal levels and activity inseminal plasma, and, at least partially explain the effect ofmelatonin on ram fertility and reproductive performance(Casao et al., 2010a; Palacín et al., 2008). Thus, the aim ofthis study was to determine the variation of both the hor-monal levels (melatonin, testosterone and 17-� estradiol)and the antioxidant defence enzyme activities (compris-ing superoxide dismutase (SOD), glutathione reductase(GRD), glutathione peroxidase (GPX) and catalase) in semi-nal plasma after melatonin treatment of rams during thenon-reproductive season.

2. Material and Methods

2.1. Animals and seminal plasma extraction

Seminal plasma was extracted from first ejaculatesof nine Rasa Aragonesa rams maintained under uniformnutritional conditions and natural photoperiod at theExperimental Farm of the University of Zaragoza, Spain(latitude 41◦ 41’ N), in compliance with the requirementsof the European Union for Scientific Procedure Establish-ments. All experimental procedures were performed underthe supervision of the Ethics Committee of the Universityof Zaragoza.

The rams (2–4 years old) belonged to the National Asso-ciation of Rasa Aragonesa Breeding (ANGRA).

The rams were divided into two groups. On February1st,the first group comprising five rams received three 18-mgmelatonin subcutaneous implants (Melovine®, Ceva SantéAnimale, Libourne, France) in the left ear (n = 5; group M);

Science 138 (2013) 168– 174 169

the other four rams constituted the control group (n = 4;group C). The sires were housed individually, and semenwas collected for 21 weeks after melatonin treatment. Firstejaculates of M and C animals were collected between 8:30and 9:00 a.m. every working day using an artificial vagina.In order to eliminate individual differences, ejaculates fromrams of each group were pooled and processed together.Pooled ejaculates provide a uniform, good-quality spermsample suitable for representative studies of ram semen(Ollero et al., 1996). Consequently, all our studies are per-formed with pooled semen and the results obtained can beextrapolated to the ovine species. The ejaculates were keptat 37 ◦C until laboratory analysis.

Seminal plasma was extracted by centrifugation at2400 × g for 10 min in a microfuge at 4 ◦C. The supernatantwas centrifuged again, the seminal plasma was recoveredand, after filtering through a 0.22 �m Millipore membrane(Merk Millipore, Billerica, MA, USA), was aliquoted and keptat −20 ◦C until analysis.

2.2. Melatonin evaluation

Melatonin concentrations in the ram seminal plasmawere measured by means of a commercial competitiveimmunoassay (Direct saliva melatonin ELISA kit, BühlmannLaboratories AG, Schönenbuch, Switzerland), following themanufacturer’s instructions (Casao et al., 2010b). The sen-sitivity of the assay was 0.5 pg/ml and the intra- andinter-assay coefficients of variation were 5.2 and 10.2%,respectively.

2.3. 2.3.Testosterone assays

Testosterone evaluation in the ram seminal plasma wasperformed with a total testosterone commercial ELISA kitassay (Testo-Easia, DiaSource ImmunoAssays S.A., Nivelles,Belgium), following the manufacturer’s instructions (Casaoet al., 2010b). The sensitivity of the assay was 0.05 ng/mland the intra- and inter-assay coefficients of variation were4.8 and 7.2%, respectively.

2.4. Estradiol assays

Estradiol evaluation in the ram seminal plasma wasperformed using a total estradiol commercial ELISA kitassay (E2-EASIA kit, DIAsource Immunoassays, S.A., Niv-elles, Belgium), following the manufacturer’s instructions.Briefly, 50 �l of each sample, control and calibrator, alongwith 50 �l of estradiol labeled with horseradish peroxidase(HRP) and 50 �l of anti-estradiol specific antibody, wereloaded in duplicate onto a microtiter plate coated with anexcess of anti-rabbit-gammaglobulins, and incubated for2 h at room temperature. After incubation, the wells werewashed three times, and 200 �l of chromogenic substrate(TMB-H2O2) were added to each well and incubated for30 min at room temperature, protected from direct lightin a horizontal shaker set at 700 rpm. After incubation,

50 �l of 1.8 N H2SO4 solution were added and absorbancewas measured on a microtiter plate reader (TECAN Spec-trafluor plus, Tecan Group Ltd., Männedorf, Switzerland)at 450 nm. The sensitivity of the assay was 5 pg/ml and the

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ntra- and inter-assay coefficients of variation were 4.3 and.1%, respectively.

.5. Antioxidant enzyme assays

The antioxidant enzymatic activity of four enzymes wasetermined: superoxide dismutase (SOD), gluthatione per-xidase (GPX), gluthatione reductase (GRD) and catalase.easurements were performed by a method used previ-

usly (Marti et al., 2007), adapted for the microtiter plate,s described below:

.5.1. Superoxide dismutase (SOD)All samples were loaded in duplicate and analysed

he same day. Enzymatic activity was measured as aecrease in the XTT (3′-(1-[(Phenylamino)-carbonyl]-3,4-etrazolium)-bis(4-methoxy-6-nitro) benzenesulphoniccid hydrate) reduction by the superoxide anion generatedy xanthine oxidase. The reaction mixture contained0.5 mM sodium phosphate buffer at pH 7.8; 0.15 mManthine; 0.15 mUI xanthine oxidase, 30 mM XTT and0 �l of sample to complete a final volume of 200 �l. Theeaction was initiated by the addition of xantine oxidase,nd the absorbance change at 550 nm was monitored for

min with a microtiter plate reader (TECAN Spectrafluorlus, Tecan Group Ltd., Männedorf, Switzerland).

.5.2. Gluthatione peroxidase (GPX)All samples were loaded in duplicate and analysed the

ame day. Enzymatic activity was measured following thexidation of gluthatione (GSH) to oxidized glutathioneGSSG) catalysed by GPX and using ter-Butylhydroperoxidet-BuO2H) as an electron acceptor, coupled to the recyclingf GSSG back to GSH utilizing GRD and NADPH. The reac-ion mixture contained 300 mM sodium phosphate buffert pH 7.2; EDTA 0.5 mM, 54 mUI of GRD; 85 �M NADPH;

mM GSH; 1.2 mM t-BuO2H and 6 �l seminal plasma toomplete a final volume of 200 �l. The absorbance changet 340 nm was monitored for 3 min with the microtiterlate reader (TECAN Spectrafluor plus, Tecan Group Ltd.,ännedorf, Switzerland).

.5.3. Gluthatione reductase (GRD)All samples were loaded in duplicate and analysed the

ame day. Enzymatic activity was measured following theecrease in absorbance due to NADPH oxidation as a con-equence of the GSSG reduction. The reaction mixtureontained 300 mM sodium phosphate buffer at pH 7.2;.5 mM EDTA; 85 �M NADPH; 0.8 mM oxidized glutathioneGSSG) and 5 �l seminal plasma to complete a final volumef 200 �l. The absorbance change at 340 nm was monitoredor 3 min with the microtiter plate reader (TECAN Spec-rafluor plus, Tecan Group Ltd., Männedorf, Switzerland).

.5.4. CatalaseAll samples were loaded in duplicate and analysed

he same day. The enzymatic activity was determined by

he decrease in absorbance due to t-BuO2H reduction inhe presence of catalase. The reaction mixture contained0 mM sodium phosphate buffer at pH 7; 11 mM t-BuO2Hnd 4 �l seminal plasma to complete a final volume of

Science 138 (2013) 168– 174

200 �l. The absorbance change at 240 nm was monitoredfor 120 s with the microtiter plate reader (TECAN Spec-trafluor plus, Tecan Group Ltd., Männedorf, Switzerland).

2.6. Statistical analysis

The date obtained data from the hormonal and antioxi-dant enzyme activity assays were grouped by week, week0 being the week prior to the melatonin treatment. Afterthe melatonin treatment, seminal plasma samples wereobtained for 21 weeks (week 1–21). The distribution ofthe data was evaluated by the Kolmogorov–Smirnov test.When data showed a normal distribution (i.e. antioxidantenzymes), differences between experimental groups werecompared by means of an unpaired t test. When the studieddata failed the normality test (i.e. hormonal data), dif-ferences between groups were analysed by means of theMann–Whitney test. All statistical analysis was performedwith SPSS (v.15.0, IBM Software, Armonk, New York, U.S.A.).

3. Results

3.1. Hormonal results

There were no statistical differences in hormonal val-ues and antioxidant enzyme activity in seminal plasmabetween the experimental groups before melatonin treat-ment (week 0). After melatonin treatment, the rams ofgroup M experienced a sharp increase in melatonin lev-els. The rise in melatonin levels in treated rams couldbe detected the day after the insertion of the melatoninimplants (60.94 pg/mL vs. 229.45 pg/mL the day before andthe day after melatonin implantation in melatonin-treatedrams, respectively). These differences were statistically sig-nificant (P < 0.05) when compared with the control groupfrom the first week after melatonin treatment to week 17,reaching maximum levels in weeks 8 and 9 after melatonintreatment (Fig. 1a). The melatonin levels in treated malesstarted to decrease from week 17 onwards, and there wereno statistical differences between the groups from week 18to the end of the study.

Testosterone levels in both experimental groups fol-lowed a similar pattern, although the differences betweenthe groups were in this case statistically significant(P < 0.05) from week 4 to week 12, and again in week18, reaching a maximum in week 12 and showing sometendency to significance (P < 0.1) in weeks 13, 17 and 21(Fig. 1b).

Differences in the estradiol levels between the exper-imental groups were not as marked as the melatonin andtestosterone concentrations. The differences showed a ten-dency to significance (P < 0.1) in weeks 8 and 12, and werestatistically significant (P < 0.05) from week 10 to 13. Theeffect lasted for less time than the other studied hormones,and both experimental groups showed similar estradiollevels from week 14 to the end of the study (Fig. 1c).

3.2. Antioxidant enzymes activity

The effect of melatonin treatment on antioxidantenzyme activity was less marked than on the hormonal

A. Casao et al. / Animal Reproduction

Fig. 1. Levels of melatonin (a), testosterone (b) and 17-� estradiol (c)in seminal plasma of melatonin treated (�) and control (�) rams fromweek 0 (before melatonin implant) to week 21 (after melatonin implant)

by binding to its receptors (Roy et al., 2001). This would

during the non-reproductive season. Weekly values are presented asmeans ± S.E.M. *P < 0.1; **P < 0.05; ***P < 0.01.

levels. Initially, there were no significant differencesbetween the experimental groups in the antioxidant enzy-matic activity for all the four enzymes studied. At a laterstage, statistical differences between the groups appearedfor GPX and GRD only. Some differences between thegroups in GPX activity did occur very early after melatonintreatment. These differences, which were statistically sig-nificant (P < 0.05) only in weeks 3 and 5 and which showed atendency to significance (P < 0.1) in weeks 2 and 10 (Fig. 2a),were due to both an increase in GPX enzyme activity ingroup M (P < 0.1 between weeks 0 and 11) and a decreasein group C (P < 0.05 between weeks 0 and 6).

The GRD enzymatic activity showed statistical differ-ences (P < 0.05) between the groups from week 9 to week14, mainly due to an increase in the GRD activity in the

melatonin treated group (P < 0.1 between weeks 1 and 12).From week 15 onwards there were no significant differ-ences between the groups (Fig. 2b).

Science 138 (2013) 168– 174 171

There were no statistical differences for either SOD orcatalase enzymatic activity throughout the duration of thestudy (Fig. 2c and d, respectively).

4. Discussion

Previous studies by our group have demonstratedthe presence of both melatonin and testosterone in ramseminal plasma, their seasonal variation, and the correla-tion between them and with antioxidant enzyme activity(Casao et al., 2010b). Likewise, we have also demonstratedthe presence of melatonin receptors (MT1 and MT2) in ramspermatozoa (Casao et al., 2012). Given these results, thenext step was to assess the effect of melatonin treatment onthe hormonal levels and the antioxidant enzyme activity inram seminal plasma during the non-reproductive season.

The increase in melatonin in the seminal plasma of thetreated rams was almost immediate, and increased steadilyuntil reaching a maximum. It then started to decrease inan irregular fashion until there was no difference betweenthe melatonin-treated and the control animals. Due to thelipophilic nature of melatonin, which allows its rapid dis-tribution through all tissues and body fluids (Reiter et al.,2009), the levels in the ram seminal plasma recorded inour study seem to be a clear reflection of the pharmacoki-netics of the commercial melatonin implants used. Theseimplants were designed to maintain high levels of thispineal hormone for 40–70 days (Haresign et al., 1990),although we have previously reported that they can releasemelatonin for more than 100 days (Zúniga et al., 2002). Inthe treated rams, melatonin levels in seminal plasma werehigher than that of the control rams for nearly 120 days,longer than previously recorded. However, the manufac-turer’s protocol recommends using three melatonin 18 mgimplants per ram, as opposed to only one recommended forewes. This could explain the longer period of action and theirregular pattern observed in the final weeks of this study,probably due to the exhaustion of the melatonin content insome of the injected implants.

The increase in the testosterone levels in the semi-nal plasma of the treated animals appeared later thanthe increase in melatonin levels, and differences betweenthe groups could be seen during a shorter period. Thedelay in the rise in the testosterone levels could be areflection of the increase in the blood testosterone lev-els induced by the melatonin implants (Kaya et al., 2000;Kokolis et al., 2000). The melatonin action over the testos-terone levels, both in blood and seminal plasma, could bedue to the stimulating effect of the pineal hormone onthe hypothalamus-pituitary axis, causing the testosteronelevels to rise through an increase in the GnRH and theLH secretions (Misztal et al., 2002). Although this mech-anism of action is still under study, some recent workshave suggested that melatonin could possibly act in thereproductive axis through direct action on the GnRH geneexpression and regulation of the G-protein coupled mela-tonin receptors on the GnRH neurons of the hypothalamus

cause an increase in the GnRH pulse secretion and animmediate increase in the LH pulsatile secretion (Viguieet al., 1995), which would also lead to an increase in the

172 A. Casao et al. / Animal Reproduction Science 138 (2013) 168– 174

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ig. 2. Levels of GPX (a), GRD (b) SOD (c) and catalase (d) in seminal plasmmplant) to week 21 (after melatonin implant) during the non-reproduct

estosterone release two to four weeks after the changef the photoperiodic conditions (Rosa and Bryant, 2003).owever, recent studies in the rat and the Syrian hamster,hich showed the presence of melatonin MT1 receptor

n the Leydig cells (Frungieri et al., 2005) and its directnvolvement in androgen secretion (Valenti and Giusti,002), suggest a more direct action of the melatonin on theestosterone secretion by the testes. Testosterone found ineminal plasma could come from the fluids of seminifer-us tubules, the intersticium and the rete testis where itan also be measured (Comhaire and Vermeulen, 1976).estosterone in the testis is essential for spermatogene-is and thus male fertility (Walker, 2009) as it seems toegulate the latter phase of spermatogenesis, maintaininghe blood-testis barrier and releasing the mature spermWalker, 2009). Furthermore, testosterone could inhibitpermatocyte and spermatide apoptosis (Ruwanpura et al.,010) and thus increase sperm quality.

Estradiol in ram seminal plasma is originated by theestosterone metabolism through aromatase P450 in theertoli, Leydig and germ cells (Hess et al., 2001; Simpsont al., 1994). Although seasonal estradiol levels in ram semi-al plasma have not previously been evaluated, they haveeen measured in stallions (Braun et al., 1996), bulls andoars (Kozumplik and Vinkler, 1982). In our study, the rise

n estradiol levels in seminal plasma is lower and shortern time than expected due to the increase in testosteroneevels. This could be due to the decrease in aromatasenzymatic activity and gene expression induced by theelatonin treatment (Cos et al., 2005), which could lead to

minor increase in seminal plasma estradiol levels despite

he high increase in the testosterone produced by the pinealormone. Again, the MT1 receptor presence in Leyding cellsFrungieri et al., 2005) suggests a direct action of the exoge-ous melatonin on the estrogen secretion by modulating

elatonin treated (�) and control (�) rams from week 0 (before melatoninn. Weekly values are presented as means ± S.E.M. *P < 0.1; **P < 0.05.

the aromatase activity (Gonzalez et al., 2007). Both estro-gens and aromatase have been linked to sperm motilityin various species, including mouse (Carreau et al., 2007),buffalo (Tiwari et al., 2008), bull (Devkota et al., 2008) andhumans (Lambard et al., 2003). The increase in estradiol inram seminal plasma could partially explain the increasein progressive motility of recently ejaculated spermato-zoa after melatonin treatment observed by our group ina previous work (Casao et al., 2010a).

Regarding antioxidant enzyme activity in seminalplasma, our study showed differences in two enzymesonly, namely GRD and GPX. However, the differencesobserved in the GPX activity at the beginning of the exper-iment were caused by both a slight increase in the GPXactivity in treated males and a decrease in enzymaticactivity in the control group. It cannot, therefore, be saidthat the slight differences observed were caused by themelatonin treatment alone. Moreover, a previous workby our group dealing with seasonal melatonin levels andantioxidant enzyme activity in seminal plasma revealed nocorrelation between melatonin levels and GPX enzymaticactivity (Casao et al., 2010b). Neither were seasonal differ-ences found for this enzyme activity, although the activityduring the breeding season was higher than in the non-breeding season. However, other works (Limon-Pachecoand Gonsebatt, 2010; Rao and Gangadharan, 2008) haveshown a regulation of the glutathione system by melatonin,so the action of the melatonin treatment on the GPX activityin ram seminal plasma cannot be ruled out.

Our previous work (Casao et al., 2010b) also showeda highly significant correlation between seminal plasma

melatonin levels and GRD activity, with significant dif-ferences between the reproductive and non-reproductiveseasons. This result has been corroborated by the presentstudy, in which the commercial melatonin treatment

duction

A. Casao et al. / Animal Repro

during the non-reproductive season increased the GRDenzymatic activity in ram seminal plasma. Although theuse of pooled semen might impair statistical analysisby overestimating differences between treatments andunderestimating the variation between individual rams,the results obtained can be extrapolated to ram semen, arestatistically significant and support the obtained conclu-sions.

We have not found any effect of the melatonin treat-ment on the other two antioxidant enzymes studied,despite our previous findings in which we observed differ-ences in the catalase and the SOD activity levels betweenreproductive and non-reproductive seasons (Marti et al.,2007), and a correlation between the melatonin levels inram seminal plasma and the catalase and SOD enzymaticactivity (Casao et al., 2010b). Thus, the previously observeddifferences could be explained by a long term effect ofmelatonin on the SOD and catalase activities and would nottherefore be induced by the short term rise in melatoninlevels resulting from melatonin treatment.

The antioxidant enzymes found in ram seminal plasmaare mainly secreted by the prostate, although catalaseseems to have a multiglandular origin (Yeung et al.,1998). Melatonin receptors have been identified in benignprostate epithelial cells (Gilad et al., 1996), and seemto be related to cell growth (Tam et al., 2008) but notwith antioxidant enzyme regulation. However, the effectof melatonin on antioxidant enzyme activity may involveother pathways not related with the activation of mela-tonin receptors. Due to its lipophilic nature, which allowsthis pineal hormone to cross the plasma membrane of thecells, melatonin might modulate the calcium-calmodulinesignaling pathway by direct binding to calmoduline(Benitez-King et al., 1993) and regulate antioxidant enzymegene expression through nuclear receptors (Pablos et al.,1997).

5. Conclusions

In conclusion, this study shows the action of commercialmelatonin treatment of rams during the non-reproductiveseason on the hormonal levels of ram seminal plasma,and on some of the enzymes involved in the antioxidantdefence system. These results could partially explain thedifferences observed in seminal quality and reproductiveperformance after out-of-season melatonin treatment.

Competing interests

The authors declare that they have no competinginterests.

Author contributions

Drs. Cebrian-Perez and Casao designed the research, Dr.Abecia implanted the rams, Dr. Casao performed hormonal

assays and drafted the manuscript, Dr. Perez-Pe performedantioxidant enzyme assays, Dr. Muino-Blanco carried outdata interpretation, and Drs. Muino-Blanco, Forcada andAbecia revised and approved the paper.

Science 138 (2013) 168– 174 173

Acknowledgements

Supported by grants CICYT-FEDER AGL 2010-18975 andDGA A-26. The authors thank ANGRA for supplying the siresand S. Morales for the collection of semen samples.

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