The effect of freezing and thawing techniques on the in vitro quality of pellet-frozen ram semen

The post-thaw quality of pellet-frozen ram semen was evaluated based on (i) sperm concentration, (ii) thawing diluent, and (iii) the number of pellets thawed simultaneously. Ejaculates from three Merino rams were collected, with four replicates per ram (n = 12 ejaculates) for each experiment. In Experiment 1, ejaculates were frozen at 200, 400, 600 or 800 × 10⁶ sperm/ mL. In Experiment 2, ejaculates were frozen at 600 × 10⁶ sperm/ mL and thawed in a dry test tube (control) or with 1 + 3 tris-citrate-fructose, tris-citrate-fructose+egg yolk, PBS+dye, PBS alone or IVF media (Emcare). In Experiment 3, groups of 1, 2 or 3 pellets were thawed in tris-citrate-fructose or a dry test tube (control). Post-thaw samples were tested for motility, acrosome and membrane integrity (FITC-PNA/PI) at 0, 3, and 6 h (all Experiments) and morphology at 0 h (Experiment 1). At 3 and 6 h post-thaw, motility was reduced at 200 × 10⁶ sperm/mL compared to other concentrations (P < 0.05). Across all time points, samples frozen at 800 × 10⁶ sperm/mL (P < 0.05) showed lower viability, while freezing at 200 × 10⁶ sperm/mL increased morphological abnormalities (P < 0.05). Thawing pellets with tris-citrate-fructose media resulted in higher motility, acrosome and membrane integrity 0, 3 and 6 h post-thaw (P > 0.05). Thawing one-pellet in tris-citrate-fructose improved motility and viability post-thaw (P < 0.05) compared to multiple pellets, while the number of pellets thawed had no significant effect when dry-thawed (P > 0.05). The results suggest that the optimal post-thaw quality is achieved when semen is frozen between 400 and 600 × 10⁶ sperm/mL and thawed in tris-citrate-fructose media, one pellet at a time.