A simplified fixed‐time insemination protocol using frozen–thawed stallion spermatozoa stored at 17°C for up to 24 h before insemination

Abstract Background Insemination of mares with frozen–thawed spermatozoa requires intensive management and results in 40%–60% per cycle pregnancy rates. Objectives To determine if satisfactory fertility is possible for frozen–thawed semen after processing it through a microfluidic device, followed by storage at 17°C for up to 24 h before fixed‐time insemination. Study design Uncontrolled field trials. Methods A pilot study evaluated the motility of frozen–thawed spermatozoa after centrifugation and storage (17°C) in two different media for up to 48 h. Subsequently, the motility of frozen–thawed semen processed through a microfluidic device, resuspended in two different media during storage (17°C) for up to 24 h was evaluated. The fertility of frozen–thawed spermatozoa, after microfluidic sorting and storage at 17°C for up to 24 h, was evaluated after fixed‐time insemination in a commercial embryo programme. Experiment 1: Frozen–thawed spermatozoa ( N = 5 stallions) were centrifuged and resuspended in Botusemen Gold™ or SpermSafe™ and stored (17°C) for up to 48 h. Sperm motility was evaluated by CASA at 0, 6, 24 and 48 h. Experiment 2: Frozen–thawed spermatozoa ( N = 4 stallions) underwent microfluidic sorting and storage (17°C) for up to 24 h in both media. Sperm concentration and motility were evaluated at 0, 16 and 24 h. Experiment 3: Fertility of frozen–thawed spermatozoa ( N = 3 stallions) was evaluated after insemination of 42 mare cycles at 6, 16 and 24 h after thawing, microfluidic sorting and storage before fixed‐time insemination. Results The stallion significantly influenced sperm motility, but there was no effect of media on motility parameters. Storage time significantly affected sperm motility after centrifugation but not after microfluidic sorting. Storage time had no effect on the overall embryo recovery rate (52%, n = 42). Main limitations Field trial with small mare numbers and no control at time = 0 h. Conclusions Fixed‐time insemination of frozen–thawed spermatozoa after microfluidic sorting and storage at 17°C for up to 24 h produced satisfactory embryo recovery rates.