Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse

Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P