Effects of Supplementing Semen Extenders With Spirulina platensis Extract on Post-Thaw Ram Sperm Quality

Supplementation of semen extender with natural antioxidants represents a promising strategy to mitigate cryopreservation-induced damage in ram spermatozoa.

This study evaluated the dose-dependent effects of Spirulina platensis extract (0-70 µg/mL) supplemented to Tris-based extenders on post-thaw sperm quality in Lori rams.

Semen was collected twice weekly over a four-week period (eight replicates) from four mature rams using an artificial vagina. On each collection day, ejaculates meeting the acceptable criteria were pooled and extended in a Tris-based diluent containing 0, 10, 20, 30, 40, 50, 60, or 70 µg/mL of Spirulina extract, designated as Spir-0 through Spir-70, respectively. All samples were equilibrated at 5°C for 3 h, then cryopreserved in liquid nitrogen and subsequently thawed in a 37°C water bath for 30 s. Post-thaw sperm quality was comprehensively assessed for motility (using CASA), viability, plasma membrane integrity, acrosome morphology, mitochondrial membrane potential (ΔΨm), DNA denaturation (via AO), and oxidative biomarkers, including malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and antioxidant enzyme activities (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GPx]). Data were analysed using a completely randomised design with one-way ANOVA, followed by Tukey's HSD post-hoc test, with statistical significance set at P < 0.05.

Supplementation at the concentration of 50 µg/mL Spirulina extract (Spir-50) yielded the highest post-thaw total (TM; 50.23%) and progressive motility (PM; 33.0%), as compared to the control (TM; 32.30%; PM; 18.8%). Moreover, this concentration significantly enhanced viability (59.16%), plasma membrane functionality (56.95%), and the proportion of spermatozoa exhibiting high mitochondrial membrane potential (hMMP; 54.5%). In addition, Spir-50 markedly increased glutathione peroxidase (GPx) activity (35.49 vs. 23.73 mU/mL in control) and superoxide dismutase (SOD) activity (213.07 vs. 190.74 U/mL in control), along with total antioxidant capacity (TAC; 4.53 µmol/L vs. 3.34 µmol/L in control). Conversely, this treatment substantially reduced malondialdehyde (MDA; 2.60 nmol/mL vs. 7.69 nmol/mL in control), DNA denaturation (2.87% vs. 6.67% in control), and hydrogen peroxide levels (H₂O₂; 16.4% vs. 27.7% in control).

Supplementation with the concentration of 50 µg/mL Spirulina extract exhibited the most effective cryoprotective effects among the tested concentrations by improving sperm motility, viability, membrane and DNA integrity, mitochondrial function, and endogenous antioxidant defences, while attenuating lipid peroxidation and oxidative stress. These findings support the incorporation of Spirulina extract in Tris-based extenders to improve post-thaw ram sperm quality in ram semen cryopreservation protocols.