Freezing Protocol Optimization for Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm under Field Conditions

Abstract

Creating germplasm banks of wild species, such as the Iberian red Deer (Cervus elaphus hispanicus) can be challenging. One of the main difficulties is the obtention and cryopreservation of good-quality reproductive cells when the spermatozoa are obtained from epididymides after death. To avoid a loss of seminal quality during transport, developing alternative methods for cooling and freezing sperm samples under field conditions is necessary. The objective of this study was to evaluate the effects of different durations of equilibrium and different techniques of cooling and freezing on Iberian red deer epididymal sperm quality after thawing to optimize the processing conditions in this species. Three experiments were carried out: (I) evaluation of refrigeration in straws or tubes of 15 mL; (II) study of equilibration period (0, 30, 60, or 120 min); and (III) comparison of four freezing techniques (liquid nitrogen vapor in a tank (C), liquid nitrogen vapor in a polystyrene box (B), dry ice (DY), and placing straws on a solid metallic plate floating on the surface of liquid nitrogen (MP)). For all experiments, sperm motility and kinematic parameters, acrosomal integrity, sperm viability, mitochondrial membrane potential, and DNA integrity were evaluated after thawing. All statistical analyses were performed by GLM-ANOVA analysis. Samples refrigerated in straws showed higher values (p ≤ 0.05) for mitochondrial activity and lower values (p ≤ 0.05) for apoptotic cells. Moreover, the acrosome integrity showed significant differences (p ≤ 0.05) between 0 and 120 min, but not between 30 and 60 min, of equilibration. Finally, no significant differences were found between freezing in liquid nitrogen vapors in a tank or in a box, although there was a low quality after thawing when the samples were cryopreserved in dry ice or by placing straws on a solid metallic plate floating on the surface of liquid nitrogen. In conclusion, under field conditions, it would be possible to refrigerate the sperm samples by storing them in straws with a 120 min equilibration period and freezing them in liquid nitrogen vapors in a tank or box.

The Effect of Different Extenders on the Quality Characteristics of European Red Deer Epididymal Sperm Stored at 5 °C

“…Sperm harvested from the tail of epididymis differ from ejaculated spermatozoa in motility, membrane integrity, and DNA integrity [ 6 , 7 ], but they retain their fertilizing capacity [ 1 , 8 ]. Epididymal spermatozoa can be stored in both liquid and frozen state [ 9 , 10 ] before they are used for insemination [ 8 , 11 ]. Cervid spermatozoa are usually frozen in liquid nitrogen with the use of various extenders.…”

DNA integrity and viability of testicular cells from diverse wild species after slow freezing or vitrification

“…Despite slow freezing provided better results for elongated spermatids/spermatozoa than vitrification, the viability of these testicular cells was remarkably lower than fresh ones. Perhaps, the inclusion of an equilibration step in the slow freezing protocol in a future could improve the post-thaw quality of elongated germ cells, since the equilibration period contributes to membrane lipid adaptation to cooler temperatures ( 34 ).…”

Fluorescence Microscopy and Flow-Cytometry Assessment of Substructures in European Red Deer Epididymal Spermatozoa after Cryopreservation

“…Sperm collected from hunter-harvested wild-living animals is a valuable source of genetic material that can be used for the conservation and genetic improvement of animal populations in breeding farms [ 1 , 2 , 3 ]. Sperm can be cryopreserved in liquid nitrogen to preserve its fertilizing potential for a long period of time (many years), which has important implications for species conservation [ 4 , 5 , 6 ].…”

“…In wild-living animals, post-mortem sperm sampling from the epididymides is the easiest sperm-harvesting method, which, unlike electroejaculation, does not require specialist equipment or complex procedures [ 8 , 9 ]. The epididymal sperm for reproductive purposes can be stored in a liquid state or cryopreserved [ 4 , 10 , 11 ]. However, epididymal spermatozoa cannot always be collected directly from hunter-harvested animals because the hunting grounds are usually situated in remote locations.…”