Recovery of epididymal spermatozoa from bull and red deer, stored at different times and temperatures before freezing - thawing

Abstract

In order to preserve male germoplasm, the recovery and cryopreservation of spermatozoa from the epididymides of hunted animals represents an accessible source of gametes. As a first experimental model, epididymal spermatozoa from slaughtered bulls were recovered at 30, 54, 78 and 102 h after death. The scrotal contents were stored at either 5 or 20°C. The sperm cells of each treatment (time + temperature combinations) were frozen with Triladyl (T) or Triladyl + Trehalose (TT) diluents. In order to assess sperm viability and integrity, post-thawing evaluation included individual motility, supravital stain, hyperosmotic swelling test (E+), acrosome status and sperm chromatin structure assay. Both at raw and post-refrigerated states, the sperm motility rate was higher in sperm obtained from epidydmes stored at 5°C, compared with those stored at 20°C for all collection times. Sperm collected at 102 h after death from epididymides stored at 5°C maintained a motility of 20% (120 h, raw state). When comparisons were carried out after thawing, motility was higher in the 5°C group, achieving the best results with TT diluent (7.5%) at 102 h. However, when supravital stain and E+ tests were observed, viability and membrane integrity were well preserved even at 102 h post mortem (30 and 36%, respectively, with TT diluent at 5°C). These results suggest that frozen-thawed epididymal spermatozoa could have a low motility rate while most of them remain alive. Acrosome status was not greatly affected by storage time. In a second experiment, epididymal spermatozoa from hunted red deer stags (Cervus elaphus) were recovered at 4 and 30 h after death. The scrotal contents were stored at 20°C, because that temperature is closer to field and shipment conditions. The sperm cells were frozen with TT diluent. Post-thawing evaluation included the same parameters indicated for bull spermatozoa. The assessment of spermatozoa collected at 30 hours post mortem and then subsequently frozen and thawed indicated that at this time an acceptable motility rate (35%) and viability (39.7%) were achieved. Frozen and subsequently thawed epididymal spermatozoa showed 47.9% of membrane integrity, 59.3% of acrosome integrity and 26.5% of chromatin damage, using TT diluent. A preliminary in vivo trial demonstrated that the pregnancy rate in artificially inseminated deer decreased when sperm were obtained at 30 h post mortem. According to these results, it may be concluded that storage at 5°C is better than 20°C to obtain well preserved epididymal spermatozoa from bulls, and that TT could be a useful cryoprotectant to preserve viable and fertile sperm cells after the freezing–thawing process. Before these results can be applied to assisted reproduction programs in endangered deer species, some adaptations must be developed.

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.…”

Extended abstracts

“…Time elapsed between the death and the samples recovery was about 7 days. Sperm cells were diluted in a Tris-glycerol-egg yolk extender, loaded in 0.25 mL straws, and frozen and stored in liquid nitrogen (Malcotti et al 2012). At thawing, sperm motility (SM), plasma membrane functionality (HOST), plasma membrane fluidity (M540), cell viability (CFDA), live cells with intact acrosome (LCIC), mitochondrial activity (MA), DNA integrity (DNA), area of sperm head (ASH), length of sperm head (LSH), width of sperm head (WSH) and ellipticity of sperm head (ESH) were assessed.…”

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

“…Previous studies have shown that the sperm of many animal species, including mice [ 12 ], rams [ 13 ], dogs [ 14 ], and bulls [ 15 , 16 , 17 ], is resistant to low storage temperatures in the range of 0–6 °C. Iberian red deer spermatozoa can be stored in the epididymides for up to several days at a temperature 4–5 °C [ 7 , 18 ] before cryopreservation [ 19 , 20 ].…”