Seasonal Variations in Semen Quality, Testosterone Levels, and Scrotal Size following Dietary Flaxseed Oil and Ascorbic Acid in South African Indigenous Rams

**Background:** South African indigenous sheep breeds (Zulu, BaPedi, and Namaqua Afrikaner) face the threat of extinction. Conservation programs using reproductive biotechnology require understanding of reproductive biology and environmental influences. Seasonal variations affect ram reproductive efficiency, but the role of nutritional supplementation with omega-3 fatty acids and antioxidants in mitigating seasonal effects has not been studied. Flaxseed oil contains 58% linolenic acid and vitamin E, while ascorbic acid is a natural antioxidant; both may improve semen quality.

**Methods:** Twenty-two South African indigenous rams were randomly distributed into five treatment diets from June 2021 to May 2022 (12 months): negative control (NC, standard feed), positive control (PC, formulated diet), FLAX (5% flaxseed oil), ASCA (4% ascorbic acid), and FLAX + ASCA (5% flaxseed oil + 4% ascorbic acid). Diets were formulated according to National Research Council (1987) standards. Semen was collected after 60 days of dietary supplementation using an artificial vagina. Blood was collected twice weekly using 18-gauge needles and vacutainer tubes for testosterone analysis via ELISA kits (Demeditec, Diagnostics GmbH). Semen and blood collection were repeated eight times each season. Scrotal dimensions (circumference, length, width) were measured using flexible measuring tape and Vernier caliper. Semen evaluation included volume, pH (Oakton pH meter), sperm concentration (spectrophotometer at 534 nm), motility parameters via Computer-Aided Sperm Analyzer (CASA, SCA 5.0), morphology and viability via eosin-nigrosin staining, plasma membrane integrity via Hyper-Osmotic Swelling Test (HOST), and malondialdehyde levels (spectrophotometer at 534 nm). Data were analyzed using General Linear Model (GLM) in Minitab 17® with Fisher's t-test for mean separation (p < 0.05).

**Key Results:** Season significantly affected semen parameters. Sperm concentration was higher in spring (0.93 ± 0.06 ×10⁹) and summer (0.84 ± 0.06 ×10⁹) compared to autumn (0.71 ± 0.05 ×10⁹) and winter (0.70 ± 0.05 ×10⁹) (p < 0.05). Semen volume did not differ significantly across seasons (range 0.88–0.92 mL, p > 0.05). Semen pH was higher in spring (6.44 ± 0.11) and summer (6.32 ± 0.10) than autumn (6.07 ± 0.08) and winter (6.10 ± 0.09) (p < 0.05). Total motility was significantly higher in summer (94.25 ± 1.12%) than spring (91.01 ± 1.29%) (p < 0.05). Progressive motility did not differ significantly across seasons (p > 0.05). The FLAX + ASCA combination produced the highest semen volume during spring (1.20 ± 0.11 mL) and summer (1.20 ± 0.11 mL). Total motility peaked in autumn with FLAX + ASCA (97.54 ± 1.77%) and in winter with FLAX + ASCA (97.15 ± 2.08%). Testosterone levels were significantly higher in autumn (22.04 ± 1.94 ng/mL) compared to spring (16.41 ± 2.00 ng/mL) and summer (14.38 ± 2.04 ng/mL) (p < 0.05). FLAX + ASCA supplementation resulted in non-significant differences in testosterone across seasons (spring 27.52 ± 4.42, summer 20.23 ± 5.11, autumn 25.24 ± 3.96, winter 25.92 ± 4.42 ng/mL). Malondialdehyde levels were highest in NC groups (summer: 0.64 ± 0.10 nmol/mL; winter: 0.68 ± 0.10 nmol/mL) and lowest in FLAX (winter: 0.11 ± 0.10 nmol/mL). Sperm abnormalities (primary, secondary, tertiary) were consistently higher in NC groups and lowest in FLAX + ASCA groups across all seasons. Scrotal circumference did not differ significantly across seasons (p > 0.05), but FLAX + ASCA produced the largest scrotal circumference in winter (33.63 ± 0.62 cm).

**Clinical Implications:** This study demonstrates that seasonal variations significantly affect semen quality and testosterone levels in South African indigenous rams, with autumn and winter being superior reproductive seasons. Dietary supplementation with flaxseed oil (rich in omega-3 fatty acids) and ascorbic acid can effectively reverse seasonal declines in semen quality and maintain reproductive performance year-round. The FLAX + ASCA combination was particularly effective, improving semen volume, total motility, sperm viability, and reducing sperm abnormalities and oxidative stress (malondialdehyde) across all seasons. These findings have direct applications for conservation breeding programs of endangered indigenous sheep breeds, allowing for semen collection and artificial insemination outside the natural breeding season. The results also support the use of nutritional interventions to mitigate climate change impacts on livestock reproduction in developing countries like South Africa.