Effect of L-citrulline supplementation on sperm characteristics and hormonal and antioxidant levels in blood and seminal plasma of rams

Abstract

With the aim of providing a theoretical basis for the application of L-citrulline (L-Cit) in animal husbandry, the effects of L-Cit on reproductive hormone levels, antioxidant capacity and semen quality of rams were studied by feeding them varying doses of L-Cit. A total of 32 rams were randomly divided into four groups with eight rams each. After all rams were trained to donate sperm normally, the control group was fed a basic diet, whereas the experimental groups I, II and III were provided with feed supplemented with 4, 8 and 12 g/d of L-Cit respectively. The experiment was conducted for 70 days, during which blood samples were collected from the jugular vein on days 0, 15, 30, 45 and 60, and semen samples were collected on days 0, 20, 40 and 60. In the same group, 100 µl of semen was used to test for quality, The rest of the semen sample and blood samples were centrifuged at 600 g for 15 min, and the supernatant and serum, respectively, were used to determine the levels reproductive hormones and antioxidant indices. Ram semen samples were also collected on day 70 and used to study sperm plasma membrane, substitution and mitochondrial membrane potential. Compared with the control group, the groups receiving L-Cit showed an increase in sperm concentration and number of linear motile sperm (p < .01); a decrease in the number of dead sperm (p < .01); an increase in sperm viability, particularly in groups II and III (p < .01); and an increase in sperm mitochondrial membrane potential (p < .01). Moreover, groups I, II and III showed significantly higher levels of serum gonadotropin-releasing hormone (GnRH), glutathione peroxidase (GSH-Px) and nitric oxide (NO) (p < .01). Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels increased in groups I (p < .05), II (p < .05) and III (p < .01), whereas testosterone (T), catalase (CAT) and superoxide dismutase (SOD) levels increased in groups I and II (p < .01). Serum total antioxidant capacity (T-A) increased (p < .05), whereas both hydroxyl radical (·OH) and peroxy radical ( $O_2^{·-}$ ) levels decreased (p < .01). Compared with the control, all groups had significantly higher SOD and GSH-Px in their seminal plasma (p < .01), and groups I, II (p < .05 for both) and III (p < .01) had higher levels of GnRH and FSH. LH, CAT and NO levels increased in group I (p < .05), II and III (p < .01 for both); malondialdehyde levels decreased in groups I, II (p < .05 for both) and group III (p < .01); and $O_2^{·-}$ levels decreased in groups I, II and III (p < .01). Under our experimental conditions, GnRH, FSH, LH, T, CAT, SOD, T-A, GSH-P_X and NO levels in the serum and seminal plasma of rams receiving L-Cit increased, whereas Oestradiol (E₂ ), $O_2^{·-}$ and ·OH levels in the seminal plasma decreased; this improved the semen quality of rams supplemented with L-Cit. Moreover, supplementation with 12 g/d gave the best results.

Keywords: L-cit; antioxidant capacity; hormone levels; rams; semen quality.