Semen samples were collected from crossbred (Holstein Friesian X Sahiwal) breeding bulls (47) maintained at bull rearing unit of ICAR-Central Institute for Research on Cattle, Meerut. The semen samples from each bull were collected twice in a week using an artificial vagina. The bulls were maintained on standard feeding schedule and management conditions under a loose housing system in individual pens. 2 ml of neat semen was collected for trace mineral analysis. Seminal plasma was collected for measurement of oxidative stress parameters. The semen samples were centrifuged (10 min, 8000 × g, 4^°C) and seminal plasma was separated and transferred into 1.5mL tubes and kept frozen (−20^°C) until analysis.

The fresh ejaculates were evaluated for volume, sperm concentration and initial motility as per standard procedures. The concentration of spermatozoa was measured with Accucell bovine photometer (IMV, France). The sperm motility was assessed using an Olympus BX40 phase contrast microscope (Olympus, Tokyo, Japan).

Blood and semen samples were digested with nitric and perchloric acid mixture. The trace elements zinc, copper, cobalt and iron concentrations were estimated using atomic absorption spectrophotometer (GBC Scientific) at the wave length of 213.9, 324.7, 240.7, 248.3 with 7, 6, 7, 5 mA current, respectively. The standards procured (Sisco Research Laboratory, Mumbai, India) for each element were used to calibrate the equipment.

Oxidative stress parameters were measured colorimetrically based on reaction of the target substance and a subsequent UV/VIS spectrophotometric detection at certain wavelength. The lipid peroxidation (LPO) was determined by measuring malondialdehyde (MDA) concentration which is an end product of lipid peroxidation. Superoxide dismutase (SOD) and Catalase activities were estimated as per the method of Marklund (1976) and Bergmeyer and Grabl method (1983) respectively.

The data were analyzed using analysis of variance (ANOVA) to find out the statistical difference between the mean values of various parameters of different group of animals using SPSS 16 (SPSS Incorporated Chicago, IL, USA). The bulls were categorized into three groups as oligozoospermic (sperm concentration < 500 million/ ml, sperm motility- > 70%), ashthenozoospermic (sperm motility- < 40%, sperm concentration > 500 million/ ml) and Normozoospermic (sperm concentration > 500 million/ml, sperm motility- > 70%) A parametric (Pearson) correlation between oxidative stress markers and semen quality parameters was analyzed using standard statistical methods (Snedecor and Cochran, 1994).

The mean Zn and Cu concentrations were significantly lower (p<0.05) in oligozoospermic and ashthenozoospermic bulls as compared to normozoospermic bulls. As regards Co and Fe concentrations, no significant difference was observed among different category of bulls (Table 1). Zn and Cu concentrations were found significantly(p<0.05) lower in seminal plasma of oligozoospermic bulls as compared to normozoospermic bulls however no significant difference was observed in seminal Zn and Cu concentrations of ashtheno and oligozoospermic bulls (Fig. 1). Seminal plasma Co and Fe concentrations did not vary significantly in different group of breeding bulls.

Zn and Cu are important trace elements and act as cofactors in proteins, hormones and numerous enzymatic reactions. Activity of some antioxidant enzymes is enhanced by these minerals to counteract the oxidative stress. The catalytic reaction of Cu/Zn SOD is performed by the cyclic reduction and oxidation of the copper ion (Abolbashari et al., 2019). They are structural ions of SOD and reduce oxidative stress by induction of metallothionein synthesis (Li et al., 2006). Because of their pivotal role in the redox mechanisms, their imbalanced status may lead to an increased susceptibility to oxidative damage (Abolbashari et al., 2019). Our study showed significantly low concentrations of seminal zinc and copper concentrations in oligo and ashthenozoospermic bulls as compared to the normozoospermic bulls. Increased MDA and low SOD acitvity in the seminal plasma of oligo and ashthenozoospermic bulls of present study may explain the decrease of the effective concentration of Zn and Cu, increasing the harmful effects of ROS to sperm cells consequently leading to abnormal semen quality parameters. Studies carried out in human beings mentioned that decrease in Zn and Cu concentration leads to an increase in oxidation of DNA, proteins, and lipids causing reduced semen quality (Hashemi et al., 2018). High concentration of seminal Zn was found to be associated with enhanced sperm count and sperm motility (Marzony and Chaichi, 2009). Zhao and Xiong et al. (2005) also observed a positive relationship between poor production of sperm and poor sperm motility with a lower seminal content of Zn. These findings supported the contribution of seminal Zn and Cu to maintain the physiologic functions of sperm.

The MDA had significant negative correlation with motility (p<0.05, r =−0.303) and sperm concentration (p<0.001, r =−0.473) while SOD and Catalase had significant positive correlation with sperm motility (p<0.05, r = 0.273; p<0.001, r = 0.435) and sperm concentration (p<0.001, r = 0.575; p<0.001, r = 0.631).

Significantly higher MDA and low SOD and catalase activities were present in seminal plasma of oligo and ashthenozoospermic bulls as compared to normozoospermic bulls. It is well-known that spermatozoa themselves contain negligible levels of antioxidants, and thus protected against oxidative stress by antioxidants present in seminal plasma (Gil-Guzman et al., 2001). Seminal plasma contains several antioxidants comprising enzymatic and non-enzymatic systems that play an important role in the normal function of sperm. Studies carried out in past suggested that decreased levels of antioxidants in seminal plasma might be a potential cause of infertility (Mehrotra et al., 2013). Impaired antioxidant status was observed in seminal plasma of oligo and ashtenozoospermic bulls in present study which may be an important risk factor of oxidative damage making the sperm susceptible to lipid per-oxidation.

Increased MDA levels in seminal plasma of oligo and ashthenozoospermic bulls of present study explained the pathologic lipid per-oxidation effects on spermatozoa membrane and consequently on sperm motility and concentration. MDA production reflects the peroxidation of membrane polyunsaturated phospholipids (Colagar et al., 2013). Lipid peroxidative degradation of sperm membrane may be responsible for abnormal sperm motility and concentration. Another study showed that decreasing seminal plasma antioxidants status especially total antioxidant capacity might have significant effect on impaired sperm function. Higher level of ROS was correlated with a decreased number of motile sperm while greater sperm motility was observed in samples with lesser amount of ROS (Colagar et al., 2013).

The findings of the present study are in accordance with previously published observations of the other authors in studies conducted on human beings (Ben Abdallah et al., 2009; Hashemi et al., 2018). Ben Abdallah et al. (2009) reported that MDA content was elevated in oligozoospermic and asthenozoospermic groups. Murawski et al. (2007) also showed significant and positive correlations between seminal SOD activity and sperm concentration and motility, which are regarded as the important criteria for normal fertilizing ability of the spermatozoa. The increased activities of seminal SOD and catalase in normozoospermic bulls of present study and the noted associations between these enzymes and sperm quality proved their ability to remove free radicals and important biological role in controlling the fertilizing potential of spermatozoa.