Indian Journal of Animal Research

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Effect of Melatonin Administration on Libido, Body Weight and Serum Biochemical Profile in Magra Rams During Non-breeding Season

Pawan Kumar Sharma1, Amit Kumar1, Ashok Kumar2,*, Pramod Kumar1, S.S. Dangi3, R.A. Legha3, Ashish Chopra2, Arun Kumar3
1Department of Veterinary Gynaecology and Obstetrics, Rajasthan University of Veterinary and Animal Sciences, Bikaner-334 001, Rajasthan, India.
2ICAR-Central Sheep and Wool Research Institute, Arid Region Campus, Bikaner-334 006, Rajasthan, India.
3ICAR-Central Sheep and Wool Research Institute, Avikanagar, Tonk-304 501, Rajasthan, India.

ABSTRACT

Background: The present study was designed to evaluate the effects of single subcutaneous melatonin administration on body weight, libido and serum biochemical profile in Magra rams during non-breeding season.

Methods: Magra (n=12) rams, (average age: 26 months, Avg. body weight: 35 kg) were randomly divided in to two groups and rams received single subcutaneous melatonin @18 mg/ram dissolved in 1 ml corn oil were considered as treatment group (n=6) and ram received corn oil (1 ml) sans melatonin were considered as control group (n=6). Post four weeks of experiment, reaction time was observed during the semen collection @ once in a week for next six consecutive weeks for quantification of libido. Initial and final body weight was recorded to evaluate the effect of melatonin on the body weight gain. Blood was collected at fortnightly intervals i.e., 0, 15, 30, 45, 60 and 75th day of experiment to evaluate serum biochemical profiles.

Result: In the present study, overall mean values of reaction time were observed significantly (P<0.05) lower in treatment group as compared to control group, however, mean values during weeks didn’t differ significantly between groups. Analysis of variance didn’t reveal significant interaction between the mean values of reaction time and weeks in both groups. The mean values of initial body weight, final body weight, body weight gain, daily weight gain, serum aspartate aminotransferase (AST), alanine transaminase (ALT), AST/ALT ratio, A/G ratio and cholesterol didn’t differ significantly between groups. The overall mean values of serum total protein, albumin and globulin were significantly (P<0.05) higher in treatment as compared to the control group. Analysis of variance didn’t reveal significant interaction between serum total protein, albumin globulin and weeks in rams of both groups. The results of the study inferred that subcutaneous administration of melatonin (@18 mg/ram) ameliorated the libido, serum total protein, albumin and globulin but has no significant effect on the body weight gain, AST, ALT, AST/ALT ratio, A/G ratio and cholesterol of Magra rams during non-breeding season.

INTRODUCTION

Among livestock, sheep are of vital importance to the Indian agriculture based economy as they support the livelihoods of most small and medium farmers and landless workers.  In India, sheep population is increased by 14.13% from 74.26 million over previous census of 2012 (DADF, BAHS, 2019). Seasonality in breeding behavior of sheep is a marked limitation to sheep production and regulated primarily by annual variations in photoperiod and several other factors (Abecia et al., 2012; Sarlos et al., 2013). Longer photoperiod is characterized by suppression of melatonin secretion resulting in reduction of libido, sperm quality and quantity leading to a period of low productivity (Cevik et al., 2017; Ramírez-Ramírez et al., 2021). Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine derived neurohormone, naturally synthesized and secreted by the pineal gland during the dark hours (Malpaux et al., 1997; Teng et al., 2017).  Shorter photoperiods are associated with a longer pattern of melatonin secretion, which exerts a stimulatory effect on the reproductive axis in short day breeders like sheep (Castle-Miller et al., 2017) which results in increased reproductive hormone promoting libido, semen quality and quantity (Deng et al., 2018).

Exogenous melatonin administration can overturn the effect of seasonality on reproductive performance of sheep during non-breeding season (Rosa et al., 2012; Cevik et al., 2017; Deng et al., 2018). Total number of matings and reaction time are generally used as main criteria for assessing the libido levels of male animals (Estienne and Harper, 2000). In general, the longer reaction time is considered as an indication of poor-quality of semen in cross bred bulls (Pathak et al., 1990). Melatonin is also associated with fat deposition in fat tissues of obese animal and effects body weight (Piccinetti et al., 2010; Cardinali et al., 2011a). According to study of Gundogan et al., (2004) some reproductive parameters of rams showed a correlation with some serum biochemical properties and may be used to assess reproductive performance of a ram. Magra sheep is recognized as important carpet wool breed of western Rajasthan, India. Institute distributes superior quality rams for genetic improvement of field flock under different schemes. It is observed that some of rams show poor libido during non-breeding season at field level. To the best of our knowledge and literature cited, no data are available on effect of subcutaneous melatonin injection on libido parameters, body weight and serum biochemical profile of rams concerning tropical sheep breed. Therefore, the aim of the present work was to evaluate the impact of subcutaneous melatonin injection on body weight, reaction time and serum biochemical profile of Magra ram during non-breeding season.

MATERIALS AND METHODS

The present study was conducted at ICAR -Central Sheep and Wool Research Institute, Arid Region Campus, Bikaner, Rajasthan, India in collaboration with Department of Veterinary Gynaecology and Obstetrics, College of Veterinary and Animal Science, Bikaner, Rajasthan, India during the non-breeding season (June to August, 2022). During the study period, the average temperature and per cent humidity were 33.83°C and 47, respectively. All the experimental procedures involving rams were done following the standard guidelines issued by the Institute Animal Ethics Committee (IAEC).

Experimental animals

Twelve Magra rams (average age: 26 months, Avg. body weight: 35 kg) trained to donate semen in artificial vagina were included in the study. All of the rams were kept in separate groups in open-sided, asbestos-roofed sheds with well ventilation. Before commencement of the study, all animals were clinically inspected and were found free from any kind of disease. The general health management practices for deworming, vaccination etc. were followed as per health calendar of the research institute. Animals were allowed to graze for at least 7 hours per day. The animals were fed of a concentrate mixture in evening to fulfil the protein, energy and other nutrients requirements after returning from the pasture and fresh water was offered twice in a day (ad libitum). All of the rams were kept in identical dietary and management conditions.

Experimental design

Experimental rams were divided equally into treatment (group I, n=6) and control (group II, n=6) group on the basis of body weight, pre semen evaluation, testicular biometry and mounting ability. Each ram of treatment group received single subcutaneous injection of 18 mg melatonin (Sigma Aldrich, St. Louis, MO, USA) which was dissolved in 1 ml corn oil (Sangrur Agro Ltd., Sangrur, Punjab, India), whereas each ram of control received corn oil (1ml) subcutaneously sans melatonin (El-Shalofy et al., 2022). Reaction time was started to record during the semen collection after 4 weeks of melatonin administration for next 6 consecutive weeks (@once in a week) for quantification of libido. Initial and final body weight were recorded to evaluate the effect on the body weight gain by using digital weigh machine. Blood samples were collected aseptically at every fortnightly interval i.e., day 0, 15, 30, 45, 60, 75 of treatment in sterilized 10 ml tubes from all the rams by jugular vein puncture using 20 G needle followed by separation of serum by centrifugation (Eppendorf, 5430R, Hamburg, Germany) @3000 rpm for 10 minutes and was stored at -20°C until biochemical analysis. Serum biochemical analysis of  samples was done to estimate total proteins, albumin, globulin, albumin-globulin ratio, alanine aminotransferase (ALT), aspartate aminotransferase (AST), ALT-AST ratio and cholesterol using Kinetic UV test employing commercially available test kits (Accurex Biomedical Pvt Ltd, India) and the results were read on a colorimeter (Spectrostar Nano, BMG LABTECH, Germany) as per described procedures.

Statistical analysis

The experimental data were analyzed statistically by using one way and three ways analysis of variance having 2×6 factorial design using SPSS computer programme (version 20.0). The mean values were compared using Duncan’s multiple range test (Duncan, 1955).

RESULTS AND DISCUSSION

In the present study, the overall mean value of reaction time (seconds) differed significantly (P<0.05) between both the groups having lower value in the treatment as compared to control group (Table 1).

Table 1. Effect of melatonin administration on reaction time (seconds) of Magra rams during non-breeding season (Mean ± SE).



However, during the weeks, the mean values of reaction time didn’t differ significantly in both the groups. Analysis of variance also didn’t reveal significant interaction between groups and weeks. The results of the study are in accordance with Ramadan et al. (2009) and Sarswat (2021) who reported a significantly (P<0.05) lower reaction time in melatonin treated Damascus and Sirohi bucks. Similarly, Zarazaga et al., (2019) noticed significant effect of melatonin administration on libido score in Mediterranean bucks. On contrary to present findings, Vince et al., (2017) and Pool et  al. (2020) did not observe significant increase in libido intensity by administration of melatonin in French Alpine bucks and Pole Dorset and Merino breeds of ram, respectively. In the present study, the mean values of initial body weight (Kg), final body weight (Kg) and body weight gain (Kg) didn’t differ significantly between treatment and control groups (Table 2), are in accordance with observation of Kleemann et al., (2014) in Border Leicester rams.

Table 2: Effect of melatonin administration on body weight (Kg) of Magra rams during non-breeding season (Mean± SE).



In the current study, melatonin treated group exhibited non-significant effect on serum total protein, albumin and globulin during the days of experiment, however, overall effect was significant (p<0.05) on serum total protein, albumin and globulin between both the groups (Table 3). There was a non-significant (p >0.05) effect of melatonin administration on serum AST, ALT, AST: ALT ratio, Albumin: Globulin ratio and cholesterol levels on the experimental rams as compared to control groups (Table 3, 4).

Table 3: Effect of melatonin administration on serum total protein, albumin and globulin of magra rams during non-breeding season (Mean± SE)


Table 4. Effect of melatonin administration on serum aspartate amino transaminase, serum alanine transaminase, serum AST/ALT ratio and cholesterol of Magra rams during non-breeding season (Mean± SE)



There is a paucity of literature to compare our results with previous finding in sheep and goat. The results of the study are in accordance with Ramadan et al., (2019) who observed significantly higher total protein and albumin in seminal plasma after administration of melatonin implant in buffaloes during non-breeding season. Kassim et al., (2008) observed sharp decline in melatonin, when buffalo heifers exposed to long photoperiod, but total serum protein did not show any significant difference like our finding. Similarly, Oqetark et al. (2004) also reported that melatonin treatment did not cause a significant change in total protein levels in rat. On the basis of the results obtained by Piccione et al., (2011) it is conceivable that albumin production from liver is strictly dependent on this circadian clock and, may be, on melatonin release (Alila-Johansson et al., 2001; Piccioneet_al2008). Melatonin implantation 18 mg, 36 mg/lamb/day significantly (P<0.05) increased total protein and decreased globulin level in serum in Awassi Lambs (Ismail Mahmud and Karadaş, 2023).

Melatonin, as being a potent antioxidant, the present data are in accordance with observations of previous researchers who also noted a significant increase in serum globulin concentration treated by non-enzymatic antioxidant (ascorbic acid) in ewes and lactating dairy cows (Monzaly, 2000; Abu El-Hamad et al., 2007). However, melatonin treatment has non-significant effect on seminal plasma alanine transaminase level but significant effect on seminal plasma cholesterol in buffaloes during non-breeding season (Ramadan et al., 2019). Oleshchuk et al., (2019) found that melatonin @ 10 mg/kg body weight significantly decreased activities of serum AST and ALT in Sprague-Dawley male rats. In contrary to this, Zhang et al., (2017) recorded that oral administration of melatonin @ 5 mg/kg body weight for 30 days in rats alleviated the increases in the plasma of the ALT and AST activity.

Melatonin treatment mimics short photoperiods and also associated with changes in sexual behaviors, particularly libido (Daramola et al., 2006; Abecia et al., 2011). The constant-release of melatonin implants in rams may be a sufficient stimulus to activate responses at the hormonal and sexual behavioral levels (Rosa et al., 2012) which favors that melatonin dissolved in corn oil has similar effect as implant. Melatonin is secreted from the pineal gland into the blood stream which can inhibit fat deposition in fat tissues of obese animal models (Cardinali et al., 2011a) and has shown reduced body weight (Piccinetti et al., 2010). In humans, melatonin effectively regulates body weight and effectively used in treatment of metabolic problems (Cardinali et al., 2011b). Melatonin can physiologically regulate brown adipose tissue and metabolic activity in mammals and humans (Tan et al., 2011). But in conflict, the present experiment didn’t observe the effect of melatonin on body weight and needs further research trials on metabolic disordered animals at large scale. Blood serum biochemical estimates are helpful in semen evaluation as semen attributes estimation alone are not completely satisfactory for semen appraisal in the current practice of commercial artiûcial insemination (Ashworth et al., 1994; Perez et al., 1997; Barrios et al., 2000). A link has been examined by previous researchers among biochemical parameters and specific reproductive parameters of rams (Gundogan et al., 2004).

CONCLUSION

In conclusion, findings of the present study revealed that the administration of single subcutaneous melatonin (@ 18 mg/ram) has positive effect on reaction time and biochemical parameters like serum total protein, albumin and globulin whereas have no significant effect on body weight and serum biochemical parameters like AST, ALT, AST/ALT ratio, A/G ratio and cholesterol on Magra rams.  However, further studies are warranted to prove its efficacy at large scale as well as in clinically poor libido rams as well.

ACKNOWLEDGEMENT

Authors are thankful to Dean and Dean PGS, CVAS, Bikaner; Head, VGO, CVAS, Bikaner for providing financial and technical support. Authors are also thankful to the Director, ICAR-CSWRI, Avikanagar, Project Coordinator, NWPSI and PI on Magra Field Unit; Head, ICAR-CSWRI, ARC, Bikaner for providing animals and laboratory facilities during the research period.

CONFLICT OF INTEREST

All authors declare that  they have no conflict of interest.

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