Pre-incubation prior to semen processing and the subsequent effect on the quality of fresh-cooled and cryopreserved ram semen

For artificial insemination (AI) in the pig, semen is routinely maintained at room temperature for 2–4 h prior to extending—to reduce the cooling damage to sperm during cryopreservation. In the sheep industry, however, semen is diluted and cooled immediately after collection. This trial evaluated the effect of a 4 h pre-incubation period for semen at room temperature on the subsequent quality parameters of ram sperm prepared for AI. Immediately following collection, ram semen was divided in 2 aliquots—one was left undiluted for 4 h at room temperature (20 °C; pre-incubation) and the other (control) was diluted with an egg-yolk-based extender and either cooled to 5 °C (n = 8 different ejaculates) for short-term fresh conservation or cryopreserved (n = 6 different ejaculates). After 4 h at room temperature, the pre-incubated semen was then diluted and either cooled to 5 °C or cryopreserved, as was the control. Sperm motility, viability and chlortetracycline (CTC) pattern distribution of the pre-incubated semen were compared to the control. For fresh semen conserved at 5 °C, total sperm motility and the proportion of CTC pattern F sperm (referring to non-capacitated, non-acrosome reacted cells) were reduced by the 4 h incubation at room temperature, compared to the control. The effect of pre-incubation at room temperature was more evident in the cryopreserved semen in terms of total and progressive sperm motility, with the viability being reduced following pre-incubation. For the cryopreserved semen, the percentage of CTC pattern F sperm declined, while the pattern of AR sperm (referring to acrosome-reacted cells) increased, compared to the controls. In conclusion, pre-incubation of ram semen for 4 h at room temperature prior to preparation for AI is not beneficial to the subsequent functionality of the sperm. Furthermore, this pre-incubation period is more harmful to frozen-thawed than to fresh-cooled sperm.