Oral Administration of a Probiotic Blend Modulates Immuno-Antioxidant Status, Semen Quality, Cytokines, and Endocrine...

Probiotics exert numerous beneficial effects on immunity, metabolic homeostasis, and intestinal health across species; however, their specific role in modulating semen quality and reproductive-related physiological parameters remains poorly understood. This study evaluates the effects of an orally administered multi-strain probiotic blend comprising Lacticaseibacillus casei, Bacillus subtilis, Lactiplantibacillus plantarum, and Bifidobacterium bifidum (1:1:1:1 ratio) on semen quality, antioxidant, immune, and inflammatory responses in rabbit bucks. A total of 33 male New Zealand white rabbits were randomly assigned to three equal experimental groups (n = 11 per group); a control group receiving a basal diet (LB0) or two groups supplemented orally with the probiotic mixture three times weekly at doses of 0.25 × 10⁹ (LB0.25) or 0.5 × 10⁹ (LB0.5) CFU/ animal/dose. Both probiotic doses significantly enhanced semen quality including ejaculate volume, sperm viability, total progressive motility, and individual motility, while markedly reducing sperm abnormalities compared with the control group (P < 0.05). In seminal plasma, total antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were significantly elevated, whereas oxidative stress markers malondialdehyde (MDA) and protein carbonyl (PC) levels were markedly suppressed (P < 0.05) in probiotic-treated groups. Probiotic treatment also upregulated sperm mitochondrial enzyme activities, specifically malate dehydrogenase and succinate dehydrogenase (P < 0.05), without adversely affecting hepatic or renal function markers (P > 0.05). Immunostimulatory benefits of probiotics administration were evidenced by significant increases in serum IgG, IgM, lysozyme activity, and complement C4 levels (P < 0.01). Concurrently, inflammatory cytokines (IL-6, and TNF-α), amyloid A, Toll-like receptor 4 (TLR4), nitric oxide (NO), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were significantly reduced in all probiotic-treated groups (P < 0.05), confirming a robust anti-inflammatory effect. Blood antioxidant profiles (CAT, GPx, and TAC) were elevated alongside significant reductions in MDA (P < 0.05) by probiotics addition. Furthermore, reproductive endocrine profiles (FSH, LH, testosterone) and metabolic adipokines (leptin, adiponectin) were significantly enhanced (P < 0.05). Oral probiotic administration favorably preserved and enhanced the histological architecture of intestinal and testicular tissues (P < 0.05). Intestinal morphometry revealed significantly increased villus height, villus width, and goblet cell density, while testicular morphometry showed significant increases in seminiferous tubule density, diameter, and germinal epithelial height (P < 0.05). Principal Component Analysis (PCA) demonstrated that probiotic treatment positively correlated with immune response, endocrine profiles, and antioxidant capacity, while inversely correlating with oxidative stress and inflammatory markers across both supplemented doses. Overall, oral supplementation with this multi-strain probiotic mixture effectively enhances semen quality and reproductive-related physiological parameters by increasing endocrine profiles, boosting antioxidant defenses and immunity, suppressing inflammation/oxidative signaling, and maintaining intestinal and testicular tissue integrity in rabbit.