Trivalent Cr is considered an essential nutrient for insulin, sugar, and lipid metabolism (Cefalu and Hu, 2004). While trivalent Cr is considered non-toxic, hexavalent Cr is toxic and carcinogenic and affects reproductive system negatively (Ernst, 1990). Therefore, what is used as a dietary supplement in human and animal nutrition is trivalent type of Cr with organic or inorganic forms. Organic forms of trace elements have advantages over inorganic forms; some of those benefits are soluble, stable, absorbable and more metabolizable and these characteristics directly impacts the animal’s health and physiology (Spears, 1996). It has been reported that dietary supplementation of Cr alone or in combination with some trace elements such as zinc and Se, can be act as metabolic modifiers and there was a great interest in studding the interaction between these elements in physiological processes in domestic animals (Domínguez-Vara et al., 2009). Despite increasing scientific reports on the effects of Se on semen quality, but to the best of our knowledge no study until today has reported on the effect of dietary supplementation of organic Cr alone or combined with organic Se on semen quality, seminal plasma biochemical composition and antioxidant status in ram. Therefore, when there were no sheep studies, we had to use the results of other animal species or human studies to match and discuss the results of our study.
The animals used in this study were adults and therefore the purpose of measuring changes in BW and DMI was not to evaluate their growth performance because their time of growth has passed. The purpose of measuring BW changes and DMI and ADG was only to evaluate the effect of Se and Cr intake on changes in animal BW during the experiment which was likely to affect other parameters or any effect of Cr and Se intake on weight loss in adult animals. Human studies has reported that Cr intake can result in weight loss by elevating resting energy expenditure, decreasing body fat and increasing lean body mass (Diaz, et al., 2008). Some reports suggest that Cr could suppress appetite and stimulate thermogenesis through sensitization of insulin-sensitive glucoreceptors in the brain (Wang, et al., 2007). Therefore, Cr is a nutritional supplement that has garnered interest for use as a weight loss aid in human (Cefalu, et al., 2004). In addition, the results of various studies on the effects of Se supplementation on metabolic hormones profile and body fat composition have shown that Se could exert beneficial effects not only in reducing peripheral and central leptin resistance (through its antioxidant activity, by increasing selenoproteins activity, and by interacting with inflammatory biomarkers), but also may act via a direct effect on adipose tissue (Cavedon, et al., 2020). It is reported that Se-yeast combined with Cr-yeast had positive effects on performance and carcass composition of finishing lambs and modified blood hormones and metabolites which could improve lamb meat quality (Domínguez-Vara, et al., 2009). However, in this study BW, TG, DMI and ADG were not affected by Se or Cr intake. Therefore, it is possible that metabolic hormones or carcass composition have been affected by Cr and Se intake. Although, these parameters may be affected by breeds, age, animal physiological state, duration of experimental period and Cr and Se content of basal diet.
Semen is developed and produced in the lumen of tubuli seminiferi in the testis; therefore, there is correlation between body weight, age, and the volume of testis as well as the SC with EV. Scrotal circumference is an essential part of the breeding soundness evaluation and sperm parameters (Perumal, 2014). Whereas, in our study SC was not affected by Cr or Se intake (Table 3), therefore, a significant impact of Cr and Se on EV was not expected. In line with the results of this experiment, no significant change in SC of Ossimi rams was observed by dietary supplementation with organic Se at 0.2 and 0.5 ppm (Baiomy et al. 2009). It was reported that different levels of inorganic Se supplementation of Brazil’s ram did not influence EV (Piagentini et al., 2017). In contrast, short-term dietary supplementation of Se-enriched yeast increased significantly goat EV during the breeding season (Shi et al., 2010). No evidence was found on the effect of Cr on EV or SC in ruminants, but it was showed that organic Cr or Se alone or their co-supplementation in cocks from Dokki-4 strain increased EV (Attia et al 2015). According to the results obtained from this study, co-supplementation of Cr and Se increased slightly EV but it was not statistically significant (Table 4). Since there was no change in BW, it was not expected to observe significant increase in EV. However, we believe that EV is more influenced by, animal species, BW and breeds.
In our study sperm concentration decreased by Cr adding to Se contained diet, in other words, sperm concentration decreased by co-supplementation of Cr and Se (2.72 vs. 3.07×109 ml, Table 7) but this reduction did not happen with Cr or Se alone and difference was not significant with control. It may be due to slightly the greater effect of Se on sperm concentration. Therefore, it can be concluded that the combined Cr and Se had no negative effect on sperm concentration; in particular, the total TSE did not differ between groups. Conflicting results on reproductive effects of Cr compounds have been reported. No negative effects of organic Cr (chromium picolinate or chromium nicotinate) have been observed on male reproductive tract in previous reports (World Health Organization, 2020). Chromium picolinate in the diet of male rats for 3 months did not produce adverse effects on reproductive tissues, as assessed by organ weights, gross and histopathological examinations, sperm count and sperm motility (Rhodes, et al., 2005). All the negative effects reported in the previous documents are related to the inorganic forms (Cr-sulfate or Cr-chloride in drinking water). For example, decreased spermatogenesis was observed in Balb/c mice treated with 9.1 mg as Cr-sulfate /kg/day in drinking water for 7 weeks. In addition, significantly lower absolute weight of testes, seminal vesicles, and preputial glands was observed in male Sprague-Dawley rats with intake of 40 mg Cr-sulfate /kg/day. It is unclear if differences in results are related to experimental methods, including exposure media (drinking water versus feed), or to differences in toxicity of the specific trivalent chromium compounds evaluated (World Health Organization, 2020). Considering that in our study we used the organic form of Cr (CrMet) with very lower dose compared to previous studies, it is unlikely can link the reduced sperm count to toxicity effect of CrMet however, it may need more detailed toxicological study in ram.
The positive effects of Cr or Se alone or their combination on semen quality were appeared in higher sperm viability, membrane integrity and lower abnormal spermatozoa (Table 4). Similar to results of Cr treated group, Horký et al. (2011) reported that Cr supplementation of boars diet ( 181.81 µg per kg of diet), after 95 days, sperm concentration and motility were absolutely the same in boars of control group and the beneficial effect of Cr was seen in the reduced count of pathological sperm in boars. In Se treated group, results of sperm survival and abnormality were in agreement and sperm motility and concentration were disagreement with previous human and animal studies (Kendall et al., 2000, Hawkes and Turek, 2001 and Shi et al., 2010). It is reported that hot weather, such as long periods of temperatures over 32 0C, or short spells of very high temperatures (38 0C or higher), affects the production of viable sperm in ram. As the climatologically data of this experiment shows (Table 2), animals of this study were more likely to experience temperatures above 32 0C in the summer. Therefore, the presence of heat stress in animals of this study is likely. It is reported that heat stress alters post-absorptive carbohydrate (basal and stimulated) metabolism, characterized primarily by increased basal insulin concentrations and insulin response to a glucose tolerance test (O’brien et al., 2010). The positive effects observed in this study for sperm cells viability, membrane integrity and reduced abnormality may be linked to antioxidant properties of Cr and Se. It is reported that, dietary antioxidant supplementation of stallions (Deichsel et al., 2008), rats (Wu et al., 1979) and goats (Shi et al., 2010) could lead to a significant reduction of morphologically abnormal spermatozoa. There are growing evidences show that Se or Cr supplementation could improve antioxidant status and can be used as effective treatment strategy for minimize adverse effects of free radicals in rats (Sundaram et al., 2013), diabetic patients (Cheng et al., 2004) and infertile men (Alahmar et al., 2021). Scientific documents emphasize that Se and Cr are a promising agent for combating the adverse effects of stress in animals and is a strong antioxidant that prevents heat stress-induced lipid per-oxidation, improves nutrient metabolism and cortisol hormone activity, promotes insulin action in responsive tissues, thereby increasing farm animal productivity (Bin-Jumah, et al., 2020). Recent findings suggest that dietary high chromium-methionine supplementation in summer-exposed animals improves blood antioxidant status and feed to gain efficiency without adverse effects on lambs’ health and metabolism (Seifalinasab et al., 2021). Accompanying these studies, Biswas et al. (2014) reported that 500 or 750 µg/kg Cr-picolinate in diet of adult male turkeys in summer improve sperm concentration, progressive motility and fertility. In our study the higher FRAP and lower TBARS concentrations with Cr and Se or their co-supplementation (Table 6) indicate the positive effects of Cr and Se on blood antioxidant status in rams, which in turn may be responsible for these positive effects on viability, membrane integrity and reduced abnormally. Oxidative stress is common cause of low sperm quality. In high levels, reactive oxygen species (ROS) can affect sperm motility, increase sperm abnormality as well as impair sperm capacitation, acrosome reaction, and the fertilizing ability (Agarwal et al., 1994). In contrast to our results, it was reported that supplementation of chromium picolinate (3.5 mg of Cr/cow daily) under hot conditions did not have an effect on antioxidant activities such as serum total antioxidant capacity, superoxide dismutase (SOD) activity, or MDA levels of lactating cows (Qi et al., 2018). In calves receiving Cr-methionine at levels of 0.05 mg Cr/kg of body weight at weaning, Cr pplementations do not influence malondialdehyde (MDA) or glutathione peroxidase (GPx) serum levels, but the concentration of superoxide dismutase (SOD) tended to increase (Jin et al., 2017). In line with our study, it was found that Cr supplementation reduced serum concentration of MDA, in stressed hens (Sahin et al,m 2002). In addition, Anderson et al. (2001) reported that 6 months of Cr supplementation (400 µg of chromium pidolate) in people with type 2 Diabetic patient could significantly reduce plasma TBARS and not significantly change antioxidant enzymes such as SOD and GPX. The mechanism by which Cr acted as an antioxidant is still not totally understood. Contradictory results in different studies are significantly related to the amount of Cr used, duration of Cr intake, severity of stress, Cr concentration in basal diet, forms of Cr and physiologic condition could affect results.
In our study daily Cr intake (1 mg/ram/ day for 60 days) increases sperm linearity (VSL/VCL) as a measure of a curvilinear path, in addition VSL and VAP had numerically higher mean value but intake of combined Cr and Se reduced VCL compared to Se or Cr alone. This reduction did not happen with each of Cr or Se alone and difference was not significant with control. It may be due to slightly the greater effect of Cr and Se alone on VCL. Therefore, it can be concluded that the combined Cr and Se had no negative effect on VCL. Total motility and FPM did not changed significantly but FPM tended to increase by Cr intake. In agreement with our results, Shanmugam et al (2020) reported that organic Cr supplementation did not improve sperm total motility, fertility and hatchability in layer breeders. Beyond, 2.5% Cr-yeast supplementation of rabbit diet improved sperm progressive motility (Shabaan et al., 2014). It was reported that during stress conditions the requirement of Cr increases and supplementation of Cr may have some positive effects (spears, 2019). In the present study ram experienced summer heat stress but stress intensity may not to be so severe to affect parameters significantly.
Rams in Cr or combined Cr and Se supplemented groups were expressed more sexual behaviors and readily court and ejaculate when expose to stimulus as showed with lower reaction time (Table 4). It is reported that, most adult rams readily court, mount, and ejaculate when exposed for a brief periods to females in estrus. Minimal threshold concentrations of testosterone are required for reaction of ram to stimulus (D'Occhio and Brooks, 1982). Testosterone and its androgenic components and estrogenic signaling pathways in the nervous system are critical for the expression of sexual behaviors (Sachs and Meisel, 1994). It is reported that testosterone levels were significantly increased with Cr supplementation in streptozotocin treated rats (Abu-zaiton et al 2021). In addition study with goats showed that Se supplementation, after 60 days, increased serum testosterone concentration (Kumar et al. 2013). Testosterone measurement conditions were not met in our experiment but, the lower reaction time with Cr was significant (P ≤ 0.05). The insignificant difference in the Cr group with control may be related to the animal’s health and the adequacy of the basic dietary Cr concentration for maintains normal testosterone biosynthesis and libido. In general, the more positive effects of Cr supplementation on libido are likely to be seen in patient or animals with impaired glucose metabolism (Abu-zaiton et al 2021).
In this study, seminal plasma ALP reduced significantly by Cr or combined Cr and Se intake but no effect on the level of ALT and AST was detected. In addition, activities of these enzymes did not affected by Se alone. In contrast to results of our study, Cr as Cr-picolinate, at 500 and 750 µg/kg levels in diet of male turkeys, did not affect seminal plasma ALP (Biswas et al., 2014). Alkaline phosphatase, ALT and AST are enzymes that are associated with the activities of cell division, metabolism and energy transfer. The measures of the alteration of these enzymes are markers that indicate the degree of damage to the testicular tissue (Long et al., 2017). The decline in ALP activity in Cr or their combination groups may relate to protective action of Cr and Se on parenchyma of testis or sperm cell membrane which leads to a decline in the release of ALP in seminal plasma (Ebrahimzadeh et al., 2013). The rate of generation of free radicals in testis or spermatozoa appears to be temperature-dependent. Spontaneous lipid peroxidation and generation of TBARS, has been reported to increase with temperature elevation (Alvarez and Storey, 1985). Antioxidant treatment, which enhances the endogenous antioxidant defense, can inhibit germ cells apoptosis and semen quality and decrease enzyme release in to seminal plasma (Verhaegen et al., 1995). It was reported that rabbit seminal plasma ALP activity is negatively correlated with sperm concentration but there was no significant correlation with CASA detected parameters (Viudes-De-Castro et al., 2015).
Selenium or Cr supplementation in our study had no significant effect on seminal plasma TG, TC, TP Alb, calcium and phosphorus concentrations but glucose concentration reduced by Cr intake. In line to results of our study, Cr as Cr-picolinate, at 500 and 750 µg/kg levels in diet of male turkeys reduced glucose concentration, in contrast increase TP and TC concentrations (Biswas et al., 2014). Glucose in seminal plasma is important for ATP production, sperm capacitation and acrosome reaction and low content of seminal proteins is associated with poor semen quality (Baas et al., 1983). Chromium is glucose tolerance factor (GTF) and potentiates insulin action and increases the uptake of glucose by cells (Weksler-Zangen et al., 2012). Therefore lower seminal plasma glucose concentration in Cr treated rams in the current study may support this suggestion.