Effect of melatonin treatment on semen parameters and endocrine function in Black Racka rams out of the breeding season

Small Ruminant Research 116 (2014) 192–198

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Effect of melatonin treatment on semen parameters and endocrine function in Black Racka rams out of the breeding season I. Egerszegi a,∗ , P. Sarlós a , J. Rátky a , L. Solti b , V. Faigl b , M. Kulcsár b , S. Cseh b a Research Group of Reproductive Biology, Research Institute for Animal Breeding and Nutrition, Gesztenyés út 1, 2053 Herceghalom, Hungary b Department and Clinic of Obstetrics and Reproduction, Faculty of Veterinary Science, Szent István University, István u. 2, 1078 Budapest, Hungary

a r t i c l e

i n f o

Article history: Received 18 September 2012 Received in revised form 27 October 2013 Accepted 4 November 2013 Available online 15 November 2013 Keywords: Racka rams Melatonin Semen Testosterone

a b s t r a c t The influence of melatonin implantation on scrotal circumference, semen quantitative and qualitative parameters, plasma melatonin as well as basal and provoked testosterone concentration was evaluated in Black Racka rams in the non-breeding season. Twelve rams were used in the 60-day-long trial; six of them were implanted (M) with Melovine® (Ceva, Libourne, France) subcutaneously on day 0 (10th May) and 30 days later again, while the other six rams remained untreated (C). Scrotal circumference (SC) was measured and semen was collected weekly and assorted to two 30-day-long periods. Blood samples were collected on days 0, 30 and 60 and basal testosterone level and GnRH-induced testosterone response were evaluated. Ejaculate volume (VOL) increased in both groups from the minimum value at the beginning of the trial (0.52 ± 0.08 ml vs. 0.44 ± 0.06 ml) to the maximum at 60 days after the first implantation (0.82 ± 0.07 ml vs. 0.73 ± 0.05 ml, P < 0.05). Significant differences were found in SC, VOL and total spermatozoa number from day 30 to day 60 after the first melatonin implantation between the treatment groups (P < 0.05). The statistical analyses revealed no difference between treated and control groups regarding total motility % (TM%) and progressive motility % (PM%) at the first implantation and the last sampling. However, at the second implantation melatonin treatment had a positive effect on TM% (P < 0.05) and PM% (P < 0.001) compared to group C. Basal testosterone (Tb ) concentrations were not different between groups at the beginning of the treatment; nevertheless, 60 days later a significantly elevated Tb level was measured in group M compared to C (P < 0.05). Testosterone response after the GnRH test was not different between groups at the first and second implantation, while at the third sampling elevated Tb was detected and the provoked testosterone concentrations (Tincr ) were also higher in group M at 30 and 60 min after GnRH treatment (P < 0.05). Plasma melatonin level was increased by implantation to 445.3 ± 91.55 pmol/L measured at day 30 compared to 92.4 ± 9.15 pmol/L on day 0 (P < 0.05), and a further increase of melatonin concentration was recorded after the insertion of the second implants at day 60 (699.45 ± 163.91 pmol/L; P < 0.05). In conclusion, melatonin implantation in the non-breeding season (at the beginning of May) significantly improved the endocrine and exocrine function of testicles and some quantitative as well as quality parameters of the ejaculate in rams of the Hungarian native breed Black Racka. © 2013 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +3623319133/137; fax: +3623319133/120. E-mail address: [email protected] (I. Egerszegi). 0921-4488/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.smallrumres.2013.11.001

I. Egerszegi et al. / Small Ruminant Research 116 (2014) 192–198

1. Introduction Among farm animals living in the temperate climate zone, sheep are regarded as a strongly seasonal species. This phenomenon is controlled primarily by cyclic changes of daylight as well as several other factors, i.e. temperature, feeding, contact with males, lambing time, and lactation length (Rosa and Bryant, 2003). These seasonal changes of reproductive performance can be observed also in rams, in a degree decreasing according to the level of domestication and intensive breeding (Lincoln et al., 1990). However, these changes are less marked in rams than in ewes because spermatogenesis and sexual activity never stop, while females have a definite anoestrous period (Pelletier and Almeida, 1987; Casao et al., 2010a). In seasonal animals, melatonin is the chemical messenger which allows the perception of daylight length changes (Chemineau et al., 1993). Melatonin synthesized by the pineal gland plays important roles in several fields of physiology, e.g. in the nervous system, antioxidant defence mechanism, immune system and gastrointestinal tract, as has been reviewed extensively in recent years (Reiter et al., 2009; Hardeland et al., 2011; Carpentieri et al., 2012). The pattern of melatonin secretion conveys information about light/dark cycles to the physiological centres of the body to enable the organization of seasonal and circadian rhythms. Commercial products have been developed for the manipulation of seasonal breeding in sheep (Arendt, 1998). Melatonin can be used alone as an implant or intravaginal device in ewes, and it is recommended to combine it with other hormonal treatments eventually after an artificial light (photoperiodic) treatment to attain better results (Chemineau et al., 1996; Rathbone et al., 1997). In several studies concerning ewes of different breeds melatonin administration has been found to advance the onset of reproductive activity or prolong sexual activity (English et al., 1986; Kusakari and Ohara, 1997; Abecia et al., 2006; Papachristoforou et al., 2007); furthermore, melatonin has been reported to exert beneficial effects on oocyte quality, ovulation and conception rates (Stellflug et al., 1988; Chemineau et al., 1996; Forcada et al., 2006; deNicolo et al., 2008; Tsiligianni et al., 2009). In young rams under a long daylight period, melatonin administration increased gonadotropin and testosterone concentrations and reduced prolactin level in the blood plasma but did not affect puberty, in contrast to ewes (Kennaway and Gilmore, 1985). In mature rams, the insertion of melatonin implants facilitated testicular growth with an elevated testosterone concentration and improved semen characteristics in different breeds (Chemineau et al., 1996; Garde López-Brea et al., 1996; Kaya et al., 2000; Casao et al., 2010c). Furthermore, melatonin treatment strengthens ram effect and net lamb production of ewes under field conditions (Fitzgerald and Stellflug, 1991; Rosa et al., 2000; Palacín et al., 2008). A direct beneficial action of melatonin on sperm motility (Casao et al., 2010c) and on other sperm characteristics during the non-breeding season has been demonstrated recently with decreased apoptotic-like changes and modulated capacitation and fertilization rates (Casao et al., 2010b). Data of other authors indicate that if no pre-treatment with long days was applied, melatonin

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treatment did not have any effect on testis size and semen parameters (Rosa et al., 2012). The Hungarian Black Racka is a native sheep breed which is mainly kept under extensive conditions and bred strictly seasonally (from August to December; Dunka, 2002). Ram lambs reach puberty at the age of 7 months, whilst ram and virgin ewes are first bred at the age of 1.5 years, and the lambing percentage is 110%. It was observed by progesterone profile investigations that the first ovulation occurred in ewes at the end of August, and open ewes had cyclic ovarian features in late January (Becskei, 2002). The reproductive activity of Black Racka rams runs parallel with the seasonal oestrous patterns of ewes (Sarlós et al., 2013). The freezability of ram semen depends on the season (D’Alessandro and Martemucci, 2003); furthermore, inter-breed differences were observed (El-Alamy and Foote, 2001; Joshi et al., 2005). Despite several weak points of the method, semen freezing has been used in conservation programmes of numerous native sheep breeds (Marco-Jimenez et al., 2005; Nel-Themaat et al., 2006; Sabev et al., 2006). Nowadays there is an increasing demand for ex situ in vitro gene preservation by semen cryopreservation from ejaculates obtained outside the breeding season. The aim of this study was to determine how melatonin treatment affects the endocrine and exocrine functions of the testicles in Hungarian native Black Racka rams in the non-breeding season, with regard to its possible use in conservation programmes. 2. Materials and methods The studies were conducted at the Experimental Farm of the Research Institute for Animal Breeding and Nutrition in Herceghalom (northern latitude: 47◦ 29 , eastern longitude: 18◦ 44 ) during the non-breeding season (from 10 May to 10 July). 2.1. Animals Twelve Black Racka rams (age: 38–48 months, body weight: 55–70 kg) were included in the trial. The animals were housed in groups and fed alfalfa hay and concentrate. They were turned out to pasture daily. The animals had free access to fresh water and mineralized salt blocks. 2.2. Melatonin treatment and GnRH test The rams were randomly assigned to two equal groups. The melatonin group (M) was implanted subcutaneously twice with a single melatonin capsule (18 mg melatonin implant; Melovine® , Ceva, Libourne, France) first at the beginning of the trial (d0) and then 30 days (d30) later. The control group (C) received no implant. The GnRH test was performed on the days of implantation (d0, d30) and 30 days after the last melatonin treatment (d60) with an intravenous injection of 0.008 mg buserelin (Receptal inj.® , Intervet, Angers, France). Blood samples were collected from the jugular vein for the determination of basal hormone levels after sperm collection during the daytime and 30, 60, 90, 120 min after GnRH application. Samplings were repeated according to the timing of the GnRH tests. 2.3. Plasma metabolites To prove the similar energy status of rams, basal samples of GnRH challenges collected on d0, d30 and d60 were also assayed for ␤-OHbutyrate (BHB) and non-esterified fatty acid (NEFA) concentrations. 2.4. Data collected The following parameters were measured in all experimental animals during the 60-day trial.

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2.4.1. Scrotal circumference Scrotal circumference (SC) of the animals was measured at one-week intervals (Coulter Scrotal Tape, Trueman Mfg.) in sitting position at the widest point of the testicles. 2.4.2. Semen evaluation The rams had previously been trained for semen collection using an artificial vagina. Samples were collected weekly into plastic tubes connected to the pre-warmed (41 ◦ C) artificial vagina. Semen volume (VOL) was immediately measured using pipettes and prepared for further investigations. A drop of raw, undiluted semen was put on a glass slide heated to 37 ◦ C and mass motility (MM) was classified as described by Evans and Maxwell (1987) between grades 1 and 5 under the 40× magnification phase contrast objective of an Olympus BX51 microscope. The concentration (CC) was measured by spectrophotometer (IMV, Accucell) after dilution of the samples 1:400 with isotonic saline solution. The total number of sperm cells (TNSC) per ejaculate was calculated. The staining described by Cerovsky (1976) was used after dilution of the samples 1:200 with isotonic saline solution to identify morphological abnormalities of spermatozoa (TMD). Slides were examined under 1000× magnification. The kinematics of semen samples was checked parallel to the GnRH test (d0, d30 and d60) by a computer-assisted semen analyzer (CASA; SpermVision 3.0 software, Minitüb, Tiefenbach, Germany) system. Semen samples were diluted with Andromed (Minitüb, Tiefenbach, Germany) extender to 50–100 × 106 cells/ml concentration and incubated at 37 ◦ C for 10 min before evaluation. A pre-warmed Makler Chamber® (Sefi Medical Instrument, Haifa, Israel) was loaded with 5 ␮l of diluted semen samples and at least 4 non-consecutive, randomly selected microscopic fields per sample were scanned in which at least 500 sperm cells were recorded. The following sperm characteristics were analyzed: total motility (TM %); progressive motility (PM %); curvilinear velocity (VCL, ␮m/s); progressive velocity (VSL, ␮m/s); path velocity (VAP, ␮m/s); amplitude of lateral head displacement (ALH, ␮m) and beat frequency of the tail (BCF, Hz). 2.4.3. Hormonal assays Coat-activated tubes were used for serum and heparinized tubes for plasma collection. All samples were centrifuged within 60 min. Separated plasma was divided into two equal parts for different hormone assays, and the serum was used for BHB and NEFA measurement. Samples were stored at −20 ◦ C until processed further. Plasma testosterone was assayed with a 3 H-radioimmune method (Csernus, 1981), adapted to and validated for small ruminant (sheep, goat, roebuck) plasma and serum (Kulcsár and Huszenicza, unpublished data) (minimal detectable concentration: 1.84–1.98 nmol/L, interassay CV 3.22–15.5%, intra-assay CV: <10%). Melatonin concentration was determined by 125 I-RIA developed for human samples, which was adapted to and validated for small ruminants (Melatonin direct RIA RE29301, IBL-International, Hamburg, Germany). Minimal detectable concentration for melatonin: 19.03 pmol/L, intraassay CV: <10%. BHB and NEFA levels were analyzed with commercially available enzymatic kits (NEFA and BHB: Randox Laboratories Ltd., Ardmore, UK). 2.5. Statistical analysis All data obtained were analyzed using SPSS for Windows 12.0 software. VOL, CC, TNSC, MM and SC data were assorted in three time periods 30 days apart from the first implantation (M0-30, C0-30, M30-60 and C3060, respectively). Means, SEM and ANOVA (LSD-test; P < 0.05) procedures were conducted to compare treatment effects on quantitative and qualitative semen parameters as well as testicular circumferences. Two means of metabolic values and hormone concentrations were compared with Student’s t-test. GnRH-induced testosterone response was also evaluated by comparing the area under the curve (AUC) and the modified area under the curve (modified AUC), meaning total AUC – (t0 value × 120 min).

3. Results Average scrotal circumference increased continuously between the value measured at the first melatonin implantation (31.08 ± 1.12 cm – M vs. 30.5 ± 0.84 cm – C) to the

maximum recorded 30–60 days later (35.00 ± 0.21 cm – M vs. 32.85 ± 0.22 cm – C). Significant differences (P < 0.05) were seen in values between M0-30–M30-60 and C030–C30-60; nevertheless, in SC a significant difference between the treatment groups was found only at the sampling time 30–60 days after the first implantation (P < 0.001). Ejaculate volume increased in both groups from the minimum value at the beginning of the trial (0.52 ± 0.08 ml vs. 0.44 ± 0.06 ml) to the maximum at 60 days after the first implantation (0.82 ± 0.07 ml vs. 0.73 ± 0.05 ml, P < 0.05), in Groups M and C, respectively. Ejaculate volume was significantly higher in M compared to C rams at 30–60 days after the first melatonin implantation. The same trend was demonstrated for sperm concentration; however, in Group M only a slight elevation of CC was noticed in contrast to the significantly increasing CC in Group C animals. As regards TNSC, a growing number of sperm cells per ejaculate was recorded in both groups up to the end of the trial (P < 0.001) and, just like in VOL, a significant difference between groups was observed in TNSC at 30–60 days after the first implantation to the benefit of M – treated rams (P < 0.05). In contrast, mass motility was significantly higher in Group C during the same period (30–60 days). The proportion of abnormal sperm cells was very low during the whole investigation period (less than 10%, in which most defects were acrosome problems – data not shown) and no difference was found between the groups (Table 1). The statistical analyses revealed no difference between treatments at the first implantation and at the last sampling with regard to TM% and PM%; however, at the second implantation melatonin treatment had a positive effect on TM% (P = 0.017) and PM% (P < 0.001) compared to Group C. The mean values of curvilinear velocity (VCL), progressive velocity (VSL) and path velocity (VAP) increased in both groups at the second implantation and decreased by the last analysis (d60); nevertheless, no significant difference was found between groups at the same sampling. Mean values of amplitude of lateral head displacement (ALH) were significantly different between groups at the last investigation (P = 0.026), and a significant variation was observed in beat frequency of the tail (BCF) in control samples during the trial (Table 2). At the beginning of the treatment, basal testosterone (Tb ) concentrations were not different between the groups; nevertheless 60 days later a significantly elevated Tb level was measured in Group M compared to Group C (P < 0.05). The testosterone response after the GnRH test was not different between groups at the first and second implantation days, whereas at the third sampling elevated Tb and provoked testosterone concentrations (Tincr ) were found in Group M at 30 and 60 min after GnRH treatment (P < 0.05; Fig. 1). Assessment of AUC and modified AUC of GnRHinduced testosterone response did not show significant differences between the groups. Metabolic parameters (BHB and NEFA) remained within the physiological limits in both groups throughout the experiment, and there was no energy imbalance or elevation of BHB level (e.g. elevated BHB level was defined as BHB ≥1.60 mmol/L or ≥1.20 mmol/L in two consecutive samples; Henze et al., 1998).

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Table 1 Semen parameters and testicular circumference in melatonin-treated and control rams (mean ± SEM). Parameters

M0-30

Ejaculate volume (ml) Concentration (×109 ml−1 ) Total number of sperm cells (×109 ml−1 ) Mass motility (score 1–5) Testicular circumference (cm) Total number of morphological defects (%)

0.55 4.91 2.68 4.58 32.19 7.5

± ± ± ± ± ±

C0-30 0.04a 0.21A 0.22a 0.16 0.53a 1.19

0.49 4.23 2.20 4.67 31.23 9.19

M30-60 ± ± ± ± ± ±

0.05a 0.23a,B 0.31a 0.14 0.48a 1.28

0.67 4.89 3.40 4.17 35.00 8.83

± ± ± ± ± ±

C30-60 0.06b,A 0.16 0.33a,A 0.23A 0.21b,A 1.09

0.46 4.69 2.13 4.78 32.31 6.5

± ± ± ± ± ±

0.03b,B 0.24a 0.37a,B 0.13B 0.57B 0.50

Different superscripts a, b in sampling period and A, B between treatment groups in the same sampling period mean significant differences (P < 0.05).

Table 2 Mean (±SEM) sperm motility parameters in melatonin-treated and non-treated rams at d0, d30 and d60 after first implantation time. Parameters

M (d0)

Total motility (%) Progressive motility (%) VCL (␮m/s) VSL (␮m/s) VAP (␮m/s) ALH (␮m) BCF (Hz)

96.21 91.97 179.77 64.26 90.03 6.36 27.44

C (d0) ± ± ± ± ± ± ±

0.70 1.03 8.06 4.11 4.33 0.25 0.90

97.05 91.15 186.42 62.69 91.37 6.97 24.88

M (d30) ± ± ± ± ± ± ±

0.25 1.02 10.32 4.68 4.59 0.23 0.65

96.12 92.36 195.00 75.12 98.10 6.20 28.90

± ± ± ± ± ± ±

C (d30) 1.24A 0.79A 15.69 7.61 7.58 0.27 1.00

93.3 86.98 197.17 75.45 98.53 6.13 30.73

M (d60) ± ± ± ± ± ± ±

0.87B 1.18B 8.31 4.66 4.60 0.28 1.59

92.38 85.91 173.20 62.68 85.92 6.53 26.63

± ± ± ± ± ± ±

C (d60) 0.84 0.78 9.42 4.33 4.62 0.33A 1.39

91.93 87.19 186.12 62.15 90.16 7.37 25.22

± ± ± ± ± ± ±

0.25 1.04 8.36 3.87 3.82 0.11B 0.57

Different superscripts A, B between treatment groups in the same sampling period mean significant differences (P < 0.05).

Plasma melatonin level increased after implantation: it was 445.3 ± 91.55 pmol/L at day 30 compared to 92.4 ± 9.15 pmol/L on day 0 (P = 0.03), and a further increase of melatonin concentration was recorded after the insertion of the second implants at day 60 (699.45 ± 163.91 pmol/L; P = 0.02) (Fig. 2). 4. Discussion Slow-release melatonin implants have been widely used to control the reproduction of small ruminants for more than thirty years. However, several issues about their mode of action are still unclear (Abecia et al., 2012). The time of implantation is quite different depending on sheep breeds and regions (from the temperate regions of northern Europe at >45◦ compared to the Mediterranean sheep breeds). The first groups are usually treated around the time of the summer solstice (middle of June) while the others at about the time of spring equinox (middle of March) in order to advance the breeding season (GómezBrunet et al., 2012). Results about the effects of melatonin implants used in rams and bucks are contradictory; nevertheless, it seems that melatonin alters the hormonal secretion pattern of the animals (Fitzgerald and Stellflug, 1991; Chemineau et al., 1992, 1996; Rosa et al., 2012). In our experiment, testicular circumference was significantly increased in melatonin-implanted rams compared to nonimplanted animals during the treatment approx. 30–60 days after the first melatonin implantation (P < 0.05). Animals of Group M had significantly larger SC compared to animals of Group C (P < 0.001). Only a tendency for higher SC in implanted rams was recorded at the beginning of the treatment, but later, at days 30–60 after the first implantation of melatonin, the implanted rams had significantly larger SC compared to animals of Group C (P < 0.001). Rosa et al. (2012) reported the same trend for SC in Charollais and Texel rams without any significant difference. In agreement with our findings, others found that the testicular weight of

the implanted animals increased 2–3 weeks after the onset of melatonin treatment and reached its maximum 5 weeks later. However, the testicular size of the controls remained small (Chemineau et al., 1992). Melatonin-treated Assaf and Manchega rams showed a significant increase in scrotal circumference; however, in Rasa Aragonesa rams no difference was found between the treated and the control groups (Palacín et al., 2008). In Booroola rams, average daytime melatonin concentration was approximately 60% higher in the melatonin-implanted rams than in the non-implanted animals. The melatonin concentration of implanted rams exceeded that of the non-implanted control rams at week 2 and reached a significantly higher level at week 4 (Fitzgerald and Stellflug, 1991). In another trial, melatonin levels of implanted rams were 30-fold and 4-fold higher than those of the untreated rams in the light and dark phase, respectively (Rosa et al., 2012). In Racka rams, hormonal secretion pattern and testosterone response to the GnRH test varied between the groups. In the present study, only 2.5- to 4-fold higher levels of melatonin were detected in the treated rams in the daytime, although it should be mentioned that the control rams also had elevated plasma melatonin concentration (>100 pg/ml). At the beginning of the treatment, basal testosterone (Tb ) concentrations were not different between the groups; nevertheless, 60 days later a significantly elevated Tb level was measured in Group M of Racka rams compared to Group C (P < 0.05). These findings are in accordance with the results obtained in Konya Merino where plasma testosterone levels were significantly higher in melatonin-treated rams on day 70 following implantation (Kaya et al., 2000). However, our data only partially agree with the experimental results obtained in Coloured Mohair bucks where testosterone concentration was higher in the melatonin group compared to the control at 30 and 70 days after implantation, but the difference was non-significant (Dönmez et al., 2004). In our previous study carried out at 47◦ 00 N latitude and 20◦ 00 E longitude, testosterone level at day 0 was

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I. Egerszegi et al. / Small Ruminant Research 116 (2014) 192–198 GnRH-induced testosterone release on day 0

testosterone (nmol/L±SEM)

20

15

10

5

C M

0

0

30

60

90

120

minutes

GnRH induced testosterone release on day 30

testosterone (nmol/L±SEM)

20

15

10

5 C M

0

0

30

60

90

120

minutes

GnRH-induced testosterone release on day 60

*

testosterone (nmol/L±SEM)

20

15

* *

10 C M

5

0

0

30

60

90

120

minutes

Fig. 1. Testosterone concentration after GnRH treatment at d0, d30 and d60 in melatonin-implanted and non-implanted Black Racka rams. * means significant difference between treatment groups at the same sampling time (P < 0.05)

similar in implanted and non-implanted groups of Awassi rams, but basal testosterone level became significantly higher in melatonin-treated animals 49 and 71 days later (Faigl et al., 2009). The intensity of individual testosterone production basically determines the reproductive performance of male animals. The testosterone response elicited by GnRH treatment is a reliable indicator of the animal’s testosterone-producing ability (Schanbacher and Lunstra, 1977). Testosterone response after GnRH test was not different between groups on the first and second implantation days, whereas Tb was elevated at the 3rd sampling and the provoked testosterone concentrations (Tincr ) at 30 and 60 min after GnRH were also higher in Group M (P < 0.05) than in the control group. At the same time, AUC and modified AUC values were statistically not different between groups, which is in accordance with the results published by Faigl et al. (2009) who recorded no variation in GnRH-induced testosterone response throughout the experimental period (February–May) in M-treated versus control Awassi rams. Our results also support the conclusion that GnRH-induced testosterone response was maintained in melatonin-treated animals. As regards semen characteristics after melatonin implantation, a very diverse scale of data was obtained. Melatonin application did not improve the semen parameters in Shall rams out of season (Sookhtehzari et al., 2008), and this was the final conclusion in implanted Awassi, Charollais and Texel rams as well (Faigl et al., 2009; Rosa et al., 2012). In contrast, melatonin implants had positive effects on seminal parameters in Manchego ram lambs when the implant was administered on May 17 (Garde López-Brea et al., 1996). Melatonin-treated Ile-de-France and Préalpes-du-sud rams used for semen production in an AI programme had higher fertility than the control rams (67.6% versus 56.0%). Such treatments have been applied in artificial insemination centres where animals could not be kept in light-proof buildings for financial reasons. Ile-de-France rams treated with melatonin produced 40% more spermatozoa and 100% more AI doses than controls (Chemineau et al., 1992). In our experiment most of the quantitative semen parameters were significantly different between melatonin-treated and control rams from day 30 to day 60 after the first implantation, and an increase was observed in both groups during the study. In Konya Merino rams melatonin treatment enhanced individual sperm motility and the rate of morphologically

Melatonin Melatonin (pmol/L±SEM)

1000 C

800

M

600 400 200 0

aa Day 0

bb

b

1 month

2 months

Fig. 2. Melatonin concentration at d0, d30 and d60 in melatonin-implanted and non-implanted Black Racka rams. a and b mean significant difference between sampling times in the same group (P < 0.05).

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normal spermatozoa (Kaya et al., 2000). Furthermore, in this breed melatonin treatment improved the freezability of sperm cells, resulting in elevated viability and intact acrosome rates in post-thaw samples (Kaya et al., 2001). In another Mediterranean breed, the Rasa Aragonesa, melatonin treatment gradually increased the percentage of progressively motile spermatozoa, starting 30 days after melatonin implantation, reaching its peak between days 46 and 75 and decreasing afterwards. Melatonin treatment induced a significant increase of VCL and a decrease of VSL and VAP of rapid spermatozoa, causing a decrease of linearity, straightness and wobble coefficient starting 76 days after implantation (Casao et al., 2010c). In contrast, we did not observe any difference between the groups in sperm kinematic parameters. However, at the time of the second implantation (day 30) the treatment had a positive effect on TM % (P = 0.017) and PM % (P < 0.001) compared to Group C, which is in partial agreement with results of the last referred study. The background of the differences mentioned is still unclear; however, the pattern of melatonin level appears to be similar in different breeds and probably tissue sensitivity to melatonin may differ between animals (D’Occhio and Suttie, 1992; Chemineau et al., 1992, 1996; Rosa et al., 2012). In conclusion, melatonin implantation in the nonbreeding season (beginning in May) significantly improved scrotal circumference and semen production of rams of the Hungarian native breed Black Racka, with the highest values measured during the period between 30 and 60 days after the first melatonin treatment. Our results indicate that melatonin treatment could be beneficial in in vitro conservation programmes. However, further experiments are needed to prove the supportive action of melatonin in such programmes. Acknowledgements The study was supported by the Hungarian Scientific Research Fund OTKA, project K 76371. The experiments were supported by the TÁMOP-4.2.2.B-10/1 and TÁMOP-4.2.1.B-11/2/KMR-2011-0003 projects. István Egerszegi received the ‘János Bolyai’ research fellowship BO/00635/09 from the Hungarian Academy of Sciences. The authors thank András Molnár, Géza Gyimóthy and Jánosné Szabó for their technical help. References Abecia, J.A., Forcada, F., González-Bulnes, A., 2012. Hormonal control of reproduction in small ruminants. Anim. Reprod. Sci. 130, 173–179. Abecia, J.A., Palacín, I., Forcada, F., Valares, J.A., 2006. The effect of melatonin treatment on the ovarian response of ewes to the ram effect. Domest. Anim. Endocrinol. 31, 52–62. Arendt, J., 1998. Melatonin and the pineal gland: influence on mammalian seasonal and circadian physiology. J. Reprod. Fertil. 3, 13–22. Becskei, Cs., 2002. Investigation of ovarian function and thyreostatic effect of Zineb® in Racka sheep. In: Szent István University, Faculty of Veterinary Science, MSc Thesis (in Hungarian). Carpentieri, A., Diazde Barboza, G., Areco, V., Peralta López, M., Tolosa de Talamoni, N., 2012. New perspectives in melatonin uses. Pharm. Res. 65, 437–444. Casao, A., Cebrián, I., Asumpc¸ão, M.E., Pérez-Pé, R., Abecia, J.A., Forcada, ˜ T., 2010a. Seasonal variations of F., Cebrián-Pérez, J.A., Muino-Blanco,

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