Red wolf (Canis rufus) sperm quality and quantity is affected by semen collection method, extender components, and post-thaw holding temperature

Oviductal Extracellular Vesicles Improve Post-Thaw Sperm Function in Red Wolves and Cheetahs

“…However, freezing and thawing procedures are known to cause damages that impair sperm function and survival in several species [25,27,55,[58][59][60][61][62]. Sperm cryoinjuries can include insults to the plasma membrane (integrity, changes in ion channels and lipid compositions, and alterations of membrane fluidity), nucleus (DNA fragmentation), perinuclear theca (changes to cytoskeletal proteins and nuclear decondensation), mitochondrial function (reduced activity, increased lipid peroxidation), acrosome (premature exocytosis), motility (reduction), mRNAs and microRNAs (degradation), and tyrosine phosphorylation of sperm proteins [27,28,60,[63][64][65][66][67]. Previous studies have demonstrated that cheetah sperm is highly sensitive to the thawing and cryoprotectant removal processes, which cause a more than 35% reduction in sperm with intact acrosomes and more than a 15% decline in gamete motility [25,28].…”

“…Previous studies have demonstrated that cheetah sperm is highly sensitive to the thawing and cryoprotectant removal processes, which cause a more than 35% reduction in sperm with intact acrosomes and more than a 15% decline in gamete motility [25,28]. Similarly, cryopreserved red wolf spermatozoa exhibit 50% and 40% decline in motility and acrosome integrity, respectively, compared to fresh ejaculates [27]. In the present study, we observed a 19 ± 12% and 37 ± 9% decrease in motility immediately post-thaw for red wolf and cheetah sperm, respectively.…”

“…This was likely due to differences in the susceptibility to cryoinjuries between the cheetah and red wolf rather than a deviation in the function of dog and cat oEVs, as thawing of cheetah sperm in the presence of dog oEVs (PostO) displayed no improvements on cheetah sperm motility (data not shown, n = 3 males). Because the species' low heterozygosity [5], male cheetah consistently ejaculate sperm with high proportions of morphologically abnormal gametes (>60%) [68,69], whereas red wolf ejaculates contain 50% to 60% normal cells [27]. Previous studies have shown that sperm from teratospermic donors (>60% of cells in the ejaculates are abnormal) are more susceptible to cryoinjury than normospermic counterparts [70].…”

“…To avoid urine contamination in semen samples, the urinary bladder was emptied by inserting a 3.5 fr × 22 in polypropylene urinary catheter into the urethra and urine aspirated and bladder flushed with saline solution. Semen collection was performed using an electroejaculation method as previously described for the red wolf [27] via insertion of a lubricated rectal probe (1.9-2.1 cm) and application of about 60 to 90 total electrical stimulations (2-6 volts) over a period of 20 min. Intensity of the stimulation was adjusted as appropriate based on individual animal's physical response.…”

“…The motility of cryopreserved red wolf sperm decreases by approximately 50% compared to fresh ejaculates [24,27], and the motility index of cheetah sperm is reduced by approximately 30% after freezing and thawing [25,28]. Moreover, the numbers of red wolf and cheetah sperm with intact acrosomes are reduced by approximately 40 and 45%, respectively, after freezing and thawing [24,25,27,28]. Thus, there is still a need to improve post-thaw survival of wild canid and felid sperm before AI can be reliably incorporated into breeding programs.…”