An Investigation of Equine Sperm Quality Following Cryopreservation at Low Sperm Concentration and Repeated Freeze-Thawing

Stallion spermatozoa are typically cryopreserved at 200 to 300 million sperm/ml; however recent ad-vances such as intracytoplasmic sperm injection (ICSI) requires only one spermatozoon, wasting many, after thawing a whole straw. Cryopreserving at concentrations less than the current standard or refreez-ing thawed spermatozoa could maximize the use of genetically valuable animals and reduce waste. This investigation aimed to identify if lowering the sperm concentration for cryopreservation affected post -thaw quality after one and two freeze-thaw cycles. Nine ejaculates were collected from three fertile, "good freezer" stallions (post-thaw motility >35%) for experiment 1. Each ejaculate was split into eight treatments: five, 10, 20, 50, 100, 200, 300, 400 million sperm/ml and cryopreserved. Post-thaw: motility, viability, acrosome integrity and oxidative stress were assessed. Experiment 2, straws from experiment 1 (300 million sperm/ml) were thawed, diluted to 20 million sperm/ml or left undiluted (control) and refrozen. Post-thaw motility and viability were assessed. In experiment 1 sperm concentration did not affect post-thaw total motility (TM), progressive motility (PM) or viability at 50 to 400 million sperm/ml ( P > .05). Whilst sperm concentrations of five to 20 million/ml did differ (post-thaw TM and PM). Both refreezing and reducing spermatozoa concentration, decreased TM, PM and viability ( P < .05) after two freeze-thaw cycles. These results suggest cryopreserving at sperm concentrations as low as 50 million/ml maintains spermatozoa quality in good freezer stallions. Spermatozoa maintained some motility and via-bility when initially cryopreserved at 20 million sperm/ml and after two freeze-thaw cycles but research should investigate more optimal conditions.(c) 2022 Elsevier Inc. All rights reserved.