Effective Cryopreservation of Post Mortem-Collected Roe Deer Gametes by Evaluation of Post-Thaw Oocyte and Sperm Characteristics and In Vitro Fertilization

The aim was to evaluate the effectiveness of semen cryopreservation and oocyte vitrification in roe deer as a potential method of gamete preservation for endangered deer species. Sperm were isolated from the cauda epididymis of fourteen bucks (<i>n</i> = 14). The motility measure (CASA) and morphology of fresh semen (FS) and frozen-thawed semen (TS) were compared. A hyaluronic binding assay was used to distinguish between mature FS spermatozoa expressing hyaluronan receptors and immature FS lacking these receptors, and the mitochondrial membrane potential (MMP) in TS was determined (flow cytometry). A Sperm-Hyaluronan Binding Assay (HBA) showed a viability rate of 61.9% in FS and 78.2% in TS. Oocytes received from eight does (<i>n</i> = 8) underwent a viability test and vitrification, and fresh oocytes from the other eight does (<i>n</i> = 8) were fertilized with TS and embryos were cultured until the blastocyst stage. The number of isolated oocytes, cumulus-oocyte complexes (COCs), cleaved embryos, and expanded blastocysts was evaluated. Higher percentages of morphological factors (acrosome, head, midpiece, and tail shape) were observed in FS compared to TS, whereas the motility and progressive movement were greater in TS (<i>p</i> ≤ 0.001). The viability was 50.5% and MMP was 40.6% in TS. A total of 311 oocytes were collected and from 150 COCs and 125 blastocysts developed. The viability of thawed oocytes after vitrification was 81%. The viability of vitrified oocytes and cryopreserved sperm confirmed the effectiveness of freezing protocols and highlights the potential for their implementation in other deer species.