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

Abstract

BackgroundInsemination of mares with frozen–thawed spermatozoa requires intensive management and results in 40%–60% per cycle pregnancy rates.ObjectivesTo 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 designUncontrolled field trials.MethodsA 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.ResultsThe 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 limitationsField trial with small mare numbers and no control at time = 0 h.ConclusionsFixed‐time insemination of frozen–thawed spermatozoa after microfluidic sorting and storage at 17°C for up to 24 h produced satisfactory embryo recovery rates.

Advances in equine reproduction

“…In addition, frozen semen needs to be stored and transported in liquid nitrogen and thawed onsite, all of which increases costs to the mare owner. To tackle these logistical challenges, Morris et al 5 examined the feasibility of shortterm (≤24 h) post-thaw storage of stallion semen, which would allow thawing at one location followed by transport to a subsidiary site where a mare could be inseminated in accordance with a fixed-time AI protocol. The authors report that frozen-thawed semen was able to maintain acceptable motility and viability over a 24 h period at 17 C if, immediately after thawing, it was centrifuged or, better still, passed through a microfluidic chip to select motile sperm, before resuspension in an extender designed for storage at either 4 C or 17 C. Even though sperm selection via the microfluidic device markedly reduced the total number of motile sperm, mares inseminated 6-24 h post-thaw, deep in the uterine horn 40 h after induction of ovulation with injectable deslorelin acetate still demonstrated acceptable embryo recovery rates, even with AI doses as low as 9 million motile spermatozoa.…”