Seminal plasma addition attenuates the dilution effect in bovine sperm

Dilution of semen to low cell numbers/dose can result in a bull-dependent reduction in the post-thaw viability of cryopreserved bovine spermatozoa. It is possible that essential seminal plasma components are lacking at the greater dilution rates, thereby contributing to the deleterious effects of semen dilution. Ejaculates of 6 Holstein bulls were diluted to 120 × 10 6 sperm/mL in an egg yolk citrate extender (EYC). Split samples were further diluted to 80, 40, 20 and 4 × 10 6 sperm/mL in EYC extender with (+SP) and without (−SP) the addition of frozen/thawed seminal plasma previously obtained from a vasectomized bull. Serial dilutions for the +SP treatments were calculated and performed such that each dilution contained a volume of seminal plasma equal to the original 120 × 10 6 sperm/mL dilution. Samples were then loaded into 0.5-mL French straws yielding final sperm concentrations of 30, 20, 10, 5 and 1 × 10 6/dose. Straws from each dilution were analyzed using 2 stain combinations: the sperm viability stain, SYBR-14 and propidium iodide (PI); or the mitochondrial-specific, membrane potential-dependent stain JC-1 along with PI. Split-plot analysis of variance indicated that within bulls, there were greater proportions of viable spermatozoa in aliquots containing added seminal plasma than in aliquots without added seminal plasma (P < 0.05). Contrast analyses showed that sperm viability significantly decreased as sperm concentration decreased in the −SP samples. Although the dilution effect was also observed in the +SP samples, the magnitude of the effect was less than in the −SP samples. At most sperm concentrations, the proportions of spermatozoa that stained with JC-1 were correlated (r > 0.84; P < 0.05) with the percentages of SYBR-14 stained spermatozoa. Furthermore, the proportions of JC-1-stained spermatozoa were greater in the +SP aliquots than in the −SP samples at a concentration of 10 × 10 6 sperm/0.5 mL. These results suggest that the addition of seminal plasma can be beneficial to sperm viability when semen is diluted to low cell numbers/dose.