Shielding effect of melatonin improves seminal quality and oxidative stress indices during chilled storage of ram semen

Supplementation of antioxidant to semen extender maintains seminal quality by reducing oxidative stress during preservation time period. The main aim of this study was to investigate the effect of different concentrations of melatonin supplementation on liquid storage of Magra ram semen. This study was performed on adult Magra ram (n = 8), and seminal ejaculates (48) were collected and evaluated for various macroscopic and microscopic seminal quality parameters for further processing. After preliminary evaluation, ejaculates of each collection session were mixed and divided into four equal aliquots. All the aliquots were diluted (1:10) with Tris-citric fructose egg yolk extender contained sans melatonin served as control, whereas the other three aliquots were supplemented with 0.5, 1 and 2 mM MLT which were grouped as MLT0.5, MLT1 and MLT2, respectively. Thereafter, the samples were stored at 4 ºC for 72 h, and various seminal parameters (individual sperm progressive motility, viability, abnormalities, plasma membrane functionality) along with oxidative stress parameters (total antioxidant capacity (TAC), malondialdehyde (MDA)) were evaluated at 0, 24, 48 and 72 h of preservation. The results indicated that the mean percent values for progressive sperm motility, viability, plasma membrane functionality and TAC were significantly higher (p < 0.05) in treated groups with the highest values in MLT1 group. Significantly lower (p < 0.05) percentage of total sperm abnormalities and MDA level was observed in treatment groups compared to control group. The results report that supplementation of 1 mM melatonin efficiently maintains the seminal quality and ameliorates oxidative stress during preservation at 4 ºC to 72 h.


Introduction
Artificial insemination (AI) is considered as one of the most pragmatic approaches to overcome, hurdles associated with low productive potential and scarcity of elite germplasm of sheep (Redden and Thorne 2020). The major problem is that ovine semen collapse fast at ambient temperature, making transportation almost impossible in the remote areas which limit application of AI in field conditions. Hence, it is a need of the day to extend semen storage life without reducing fertility potential to facilitate the transport and widespread use of AI technique in areas located far from the semen production centres. Long-term storage of ram semen is still a researchable issue as ram semen stored at ultralow temperature presents more damaged sperms and highly reduced ability to fertilize ova compared to liquid storage. Therefore, it is still advised to use liquid chilled semen within 24 h post-collection for the AI to achieve better lambing rates (O'Hara et al. 2010). Storage of the semen at refrigerator temperature for longer times would assist in rectifying in reproductive management of large flocks in field conditions. This scenario imposes researchers to work on storage of semen at 4 ºC as inexpensive and valid alternative of cryopreserved semen. Compared to other mammalian species, ram semen is more sensitive to cooling process and produce more reactive oxygen species (ROS) and lipid peroxidation (LPO) causing compromised sperm fertility (Çoyan et al. 2012;Shafiei et al. 2015). This situation paved the way for inclusion of antioxidant in semen extender to increase the storage life span of sperm by combating the excess ROS and LPO production during liquid semen storage (Santiani et al. 2014).
Melatonin (MLT) (N-acetyl-5-methoxytryptamine) is a potent non-enzymatic antioxidant synthesized and secreted in the pineal gland of all mammalian species (Reiter and Fraschini 1969;Jang et al. 2010). In recent years, the antioxidant ability of MLT and its metabolic derivatives has been uncovered which act synergistically with other classic antioxidants to protect organisms from oxidative stresses (Reiter et al. 2014;Pang et al. 2016). Recent studies have shown that MLT in semen extender could protect seminal quality by preventing oxidative stress damage and augmenting antioxidant activity (Ashrafi et al. 2013). The information on the effect of addition of MLT on seminal quality and oxidative stress indices during chilled liquid storage of the ram semen is little explored. Considering the importance of sheep husbandry in Indian rural economy, hurdle faced by the farmers and urgent need of elite germplasm for sheep improvement breeding programmes, the present study was undertaken to assess the effect of this antioxidant on Magra ram seminal quality and biomarkers of oxidative stress during chilled storage at 4 °C up to 72 h.

Materials and methods
The experimental procedures related to animals were executed following the standard guidelines issued by the Institute Animal Ethics Committee (IAEC). All the chemicals used in this study, were purchased from Sigma Aldrich (St. Louis, MO, USA), unless specified.

Experimental animals
The present study was conducted at the Artificial Insemination Lab., Indian Council of Agricultural Research-Central Sheep & Wool Research Institute, Arid Region Campus, Bikaner, Rajasthan (India), during July to September of the year 2019. During the research period, the average temperature, percent humidity and thermal humidity index (THI) were 32.44 °C, 55.16 and 30.17, respectively. Eight breeding Magra rams, with an average age of 1.5-3.0 years and body weight of 38 ± 5 kg, trained to donate semen by artificial vagina technique, were selected for the study. Throughout the study period, the rams were housed iso-managerial in a shed having fenced open yard and allowed to graze on the improved pasture of Sewan grass (Lasiurus scindicus) for at least 7 h per day. Then the animals were fed of a concentrate mixture in evening to fulfil the protein, energy and other nutrients requirements. Fresh water was offered twice in a day (ad libitum) after returning from the pasture. Before commencement of the study, all animals were clinically inspected and were found free of any kind of disease. The general health management practices for deworming, vaccination etc. were followed as per health calendar of the research institute.

Semen extender preparation
The semen extender was prepared on each day of semen collection as per Evans and Maxwell (1987). Stock solution of melatonin was composed by solvating melatonin powder in dimethyl sulfoxide (DMSO) (Succu et al. 2011) and supplemented in the extender as per the following described protocols.

Semen collection and processing
Each day of semen collection, pre-stimulation in terms of two false mountings, was adopted to get optimum quantity and quality of semen production. A total of six ejaculates from each ram were collected, twice a week. Soon after collection, collection cups were docketed and transferred to water bath at 37 °C and evaluated for acceptability of semen in terms of concentration > 2.5 × 10 9 spermatozoa/ ml; mass motility > 3 + ; individual motility > 70%; and total abnormality < 10%) (Gil et al. 2003). Acceptable ejaculates of each day were mixed to eliminate individual variation and was treated as a single ejaculate. This pooled ejaculate was further divided into four equal parts using split sample technique. These aliquots were step-wise extended with prewarmed (37 ºC) extender to reach a final concentration of approximately 150 × 10 6 sperm/ml. The aliquot extended sans melatonin served as control, and the remaining three aliquots were added stock solution of MLT to make final concentration of 0.5, 1 and 2 mM melatonin and grouped as MLT0.5, MLT1 and MLT2, respectively. Diluted semen aliquots were kept in a glass tube stand and chilled from 37 to 4 ºC at a rate of 0.2-0.3 ºC/min and stored at 4 ºC up to 72 h. The time when the temperature of aliquots reached at 4 ºC was considered as 0 h.

Semen quality evaluation
All the chilled semen aliquots (in duplicate) were thawed at 37 ºC for 5 min in water bath and evaluated at 24-h intervals during the preservation time up to 72 h for following parameters.

Sperm physical parameters
Individual sperm motility In all extended samples, sperm motility was assessed subjectively in five random microscopic fields by placing each sample on a pre-warmed glass slide, covered with pre-warmed cover slip (37 ºC) under microscope at 40 × magnification (Dewinter Binocular Microscope, Italy). The mean of these different microscopic fields was recorded in terms of percentage progressive motile sperms (0-100) (Gil et al. 2003).

Percentage of sperm viability and abnormality
Sperm viability (live sperm percentage) and abnormalities were examined using differential staining techniques. The stain was prepared using eosin (1gm) and nigrosin (5gms) in 100 ml of buffer solution (2.94% sodium citrate dihydrate solution in double glass distilled water). The solution was heated in water bath at 37 ºC for 30 min and filtered after cooling through Whatman's filter paper No. 40 and stored at room temperature for further use. Sperm suspension smear was prepared by gentle mixing of small drop (30 µl) of semen with same amount of eosin-nigrosin stain and spreading the mixture with another pre-warmed slide followed by air drying. The sperm viability and abnormality were determined by counting 400 spermatozoa in different microscopic fields at 40 × and 100 × , respectively. Sperm displaying partial or complete purple staining represented dead sperm, while live had complete exclusion of stain. The morphological deformity observed in structures of sperm like head, body or tail was considered as abnormality (Evans and Maxwell 1987).

Sperm functional parameter
Hypo-osmotic swelling test (HOST) was carried out to assess the sperm functionality by evaluating sperm plasma membrane integrity. The test was assayed by mixing 0.1 ml of semen with 1.0 ml of a 150 mOsm/l hypo-osmotic solution (1.351 g fructose + 0.735 g sodium citrate dehydrate/100 ml of distilled water) followed by incubation at 37 °C for 60 min in water bath. To obtain better visibility, 0.2 ml eosin solution (0.5% w/v in sodium citrate 2.9% + 10% formalin) was added to the test tube after incubation and 0.2 ml mixture placed on slide and mounted with cover slip. A total of 200 spermatozoa were evaluated in five different fields at 40 × magnification, and spermatozoa with swollen and curled tails were counted as HOS responsive (Mosaferi et al. 2005).

Oxidative stress assay
For determination of oxidative stress, 2 ml of each aliquot at times parallel to semen quality evaluation was centrifuged at 150 g for 10 min at 4 ºC (Eppendorf, 5430R, Hamburg, Germany). The supernatant was aspirated and stored at − 20 ºC until used. Malondialdehyde (MDA) (by-product of LPO) and total antioxidant capacity (TAC) were assayed in duplicate to determine oxidative stress in seminal plasma using ELISA Kits (SinoGeneClon Biotech Co., Ltd. China) according to the manufacturer's instructions.

Statistical analysis
Variables were first checked by Shapiro-Wilk test for adherence to the assumptions of normal distribution for metric variables. One-way ANOVA was used to compare the mean differences among the groups for sperm motility, plasma membrane integrity, sperm viability, sperm abnormality, TAC and MDA levels. To compare each treatment mean, Duncan's multiple range test served as post hoc test and was used for analysis of data. The results are expressed as mean ± SE. The data analysis was done with SPSS software (IBM® SPSS® statistics, Version 25.0).

Results
The effect of supplementing MLT in semen extender was found to be significant on seminal parameters and oxidative stress indices up to 72 h of storage of liquid semen at 4 ºC which are tabulated under Table 1 and Figs. 1 and 2. The results of the same were analysed in the following sections:

Effect of supplementing MLT on sperm motility, viability and abnormality
Sperm motility, viability and abnormality are considered as major vital parameters for defining quality semen. Melatonin supplemented groups exhibited significantly (p < 0.05) higher sperm motility as compared to control group at each time point of the study (Table 1). The study revealed that supplementation of MLT in extender could maintain individual sperm motility up to 72 h post-storage period and showed most profound effect at the level of 1 mM MLT concentration (p < 0.05) ( Table 1). The sperm viability and abnormality were comparable (p > 0.05) among all the groups at 0 h, but it exerted significant (p < 0.05) positive effect on both the attributes 24 h onwards. The most significant effect of MLT was observed at 1 mM MLT concentration (significantly higher sperm viability and lower sperm abnormality percentage) (p < 0.05) ( Table 1).

Functional attributes of spermatozoa
The effect of supplementing MLT on functional attributes was assessed by observing plasma membrane integrity of spermatozoa. The plasma membrane integrity was assayed by HOST. The percentage of osmolarity responsive spermatozoa did not differ significantly (p > 0.05) among all the groups at 0 h of incubation period; however, it was significantly improved by inclusion of MLT in extender 24 h onwards compared to control group with the highest values in MLT1 group (p < 0.05) ( Table 1).

Oxidative stress indices
The storage potential of MLT supplemented semen extender was assessed as the level of TAC and MDA in the seminal plasma up to 72 h of storage period. The TAC level was significantly (p < 0.05) higher in MLT treated group compared to control groups during storage period (Fig. 1). MDA values were significantly lower (p < 0.05) in MLT treated group compared to control groups during the incubation period (Fig. 2). Among the treatment groups, significantly (p < 0.05) higher TAC and lower MDA value in 1 mM MLT treated group indicated the potent antioxidant potential of MLT at 1 mM concentration in semen extender. 6.33 ± 0.33 6.67 a ± 0.33 8.33 a ± 0.33 10.00 a ± 0.37

Discussion
Refrigerated stored ram semen is practical alternative to ultralow frozen semen, as AI in field conditions with refrigerated semen is inexpensive, non-invasive and easy to carry out with satisfactory semen quality. Nowadays, antioxidants gained great attention of semen biologists for the improvement of semen quality during preservation. In recent past, the substantial antioxidant capacity of melatonin has been recognized (Hardeland et al. 2009). Therefore, we evaluated the antioxidant effect of different concentrations of MLT on seminal attributes, commonly observed to represent seminal quality and oxidative stress indices during the chilled storage of Magra ram semen. Sperm motility is an important parameter to assess seminal quality during storage period which decreases with the advancement of the incubation period at refrigerated temperature. The highest motility of the sperm was observed in the semen supplemented with 1 mM MLT concentration. Further, the proportion of live sperm with high sperm membrane integrity and least sperm abnormalities was better sustained in 1 mM MLT treated group compared to other MLT treated as well as control group, which may be one of the reasons for optimum motility in this group (du Plessis et al. 2010). These seminal quality findings are consistent with previous findings in rams (Khalifa 2017;Dai et al. 2018) and buck (El-Battawy and El-Nattat 2013; Daramola et al. 2016) where addition of MLT in semen extender improved seminal characteristics compared to control. Gwayi and Bernard (2002) and Ashrafi et al. (2013) reported dose-dependent negative effects of MLT on sperm motility in rat and bull which may be due to different processing procedure and sperm membrane composition in species (Zhang et al. 2015). Shoae and Zamiri (2008) and Ashrafi et al. (2011) showed that the higher level of antioxidants caused high fluidity of plasma membrane above the desired point, making sperm more prone to acrosomal damages. In addition, the excess quantity of antioxidant may be detrimental to spermatozoa due to physiological changes in the semen extender. Destruction of integrity causes a rise in the membrane permeability and a decrease in the ability of sperm to control the intracellular concentrations of ions that in turn are involved in sperm motion (Baumber et al. 2000), which further may negatively affect other seminal parameters. Our study also revealed that when the threshold level of MLT was increased beyond 1 mM, it significantly reduced the spermatozoa viability and functionality. These observations were in agreement with findings in bull where freezing of sperm at a higher concentration of MLT decreased the viability and quality of spermatozoa (Ashrafi et al. 2013;Asma-ul-Husna et al. 2017).
In the present study, antioxidant capacity of MLT in protection of spermatozoa from detrimental effects of ROS was indirectly assessed by measuring MDA and TAC level in seminal plasma. MDA is a by-product of LPO and used as an index of ROS-induced lipid peroxidative damage in spermatozoa (Makker et al. 2009), whereas TAC activity is an important indicator for determining seminal antioxidant capacity. Hence, TAC and MDA were analysed as indicator of oxidative stress level. Compared to control group, TAC activity owned significant improvement, whereas MDA was significantly lower in MLT supplemented group indicating positive effect of MLT on seminal quality during incubation period. These results were in agreement with previous reports in ram (Dai et al. 2018;Mohamadi et al. 2018), buck (Dessouki and Sakr 2019) and bull (Ashrafi et al. 2013;El-Raey et al. 2015). MLT is more potent antioxidant compared to vitamin E, glutathione and mannitol in scavenging ROS (Hardeland et al. 1993;Pieri et al. 1994). Moreover, the increased concentration of TAC along with decreased levels of MDA in MLT supplemented group confirms and explains the antioxidant potential of MLT in protecting seminal quality throughout the chilled storage. The excess generation of ROS utilizes antioxidants, thus leading to reduction in TAC and increase in MDA level with advancement of the incubation period. The higher TAC and lower MDA level in treatment group may also be attributed to stimulatory effects of MLT on the activity of enzymes involved in intrinsic antioxidant defence and reduced production of free radicals, thereby maintaining quality of semen (Tsantarliotou et al. 2008;Izadpanah et al. 2015).
Ram sperm membrane is rich in polyunsaturated fatty acids which render it more prone to lipid peroxidation and generation of ROS leading to reduced sperm motility, viability and plasma membrane integrity during the liquid storage (Falchi et al. 2018). In addition to this, intrinsic scavenger mechanism of ram semen is not enough to neutralize the ROS due to dilution of semen and small size of sperm, and intrinsic scavenger mechanism decreased with advancement of incubation period. This further accelerates impairment in sperm functions (Bilodeau et al. 2000;Alvarez and Storey 2005;Camara et al. 2011). Sperm mitochondria encase the axosomes, connect with dense fibres in the middle piece and produce adenosine triphosphate (ATP) which may be damaged by a high level of ROS (Aitken and Clarkson 1987), resulting in poor sperm motility and other seminal quality parameters. It has been proved that seminal fluid contains melatonin and its receptors are present on the sperm membrane (Van Vuuren et al. 1992;Reppert et al. 1995). Therefore, addition of MLT is beneficial in semen extender and helps in maintaining sperm motility, viability, plasma membrane integrity and reduced sperm abnormality and oxidative stress by virtue of its antioxidant potential (Perumal et al. 2013).
In conclusion, the findings of the present study revealed that supplementation of melatonin in semen extender improves semen quality parameters and counteracts oxidative stress as evident from oxidative stress indices during liquid storage at 4 ºC. In addition, 1 mM melatonin was found to be optimum concentration for preservation of liquid semen. However, further study is warranted to prove its efficacy in vivo by conducting fertility trails and also to determine its role in protecting the seminal quality in cryopreserved semen.