Donkey Epididymal Transport for Semen Cooling and Freezing

Abstract

The objectives of this study were to assess the cooling and freezing of donkey epididymal semen harvested immediately after castration (Experiment 1, n = 4) or after the shipment (24 or 48 h) of epididymides attached to testicles (Experiment 2, n = 14) or dissected apart (Experiment 3, n = 36). In each experiment, semen was frozen immediately (Non-Centrif) in an egg yolk-based semen extender (EY) or after processing through cushion-centrifugation (Centrif) while extended in a skim milk-based extender (SC). In all three experiments, cooled, pre-freeze, and post-thaw epididymal semen was assessed for total motility (TM), progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP). Data were analyzed with R using mixed models and Tukey’s test as posthoc. Results showed that the cooling of epididymal semen up to 24 h after harvesting did not affect motility parameters or plasma membrane integrity; furthermore, in Experiment 3, the post-thaw evaluation of both Centrif and Non-Centrif achieved similar TM and PM. Collectively, the post-thaw results revealed low motility parameters across groups; while, the PMI and HMMP did not reflect this trend, and the values remained high, suggesting that there was a lack of epididymal sperm activation with either centrifugation or extenders. In summary, freshly harvested and cooled-shipped and cooled semen had satisfactory semen parameters. Future studies need to address donkey epididymal semen fertility in mares and jennies.

Retrograde Flushing Followed by Slicing Float-Up as an Approach to Optimize Epididymal Sperm Recovery for the Purpose of Cryopreservation in Equids

“…The donkey sperm had lower post-thaw motility parameters than horses, the results are consistent with a recent study published by our laboratory [ 8 ]. The high plasma integrity and mitochondrial membrane potential in the presence of low motility parameters suggest that epididymal sperm motility is not activated by standard means such as using an egg yolk commercial extender such as Botucrio used herein or sperm centrifugation [ 7 , 8 ]. It remains to be determined how donkey epididymal sperm harvesting could be activated in vitro.…”

“…In the early 2000s, a wider acceptance of artificial insemination by many horse breeds and advances in semen processing led investigators to turn their focus on epididymal sperm cryobiology. In recent years, epididymal sperm harvesting and cryopreservation evolved remarkably and are widely used in all domesticated mammals [ 3 , 4 , 5 , 6 , 7 , 8 ] and have also been recently applied to captive and non-domesticated species such as giraffes [ 9 ], rhinoceros [ 10 ], and giant pandas [ 11 ].…”

“…Epididymal sperm harvesting is performed after castration, death, or euthanasia. The appropriate storage time and temperature vary with species; for instance, horses seem to tolerate better cold storage of the epididymides before harvesting than donkeys [ 8 , 12 , 13 , 14 ]. Anecdotally, retrograde flushing of the epididymal tail appears to be the technique most used in clinical practice across various domesticated species, and this certainly seems to be the case in equids [ 12 , 15 , 16 ].…”

Seminal Plasma Protein Profile in Indigenous Jacks

“…The seminal plasma was evaluated for protein profile as well as for total protein contents in each sample as shown in Fig. 1 C. Semen Evaluation All semen samples were evaluated immediately after collection (Fordyce et al, 2006;Lago-Alvarez et al, 2020). The volume was recorded and a drop of semen was analyzed by phase-contrast microscopy using a 37 •C stage set.…”

“…An aliquot of 20 μL was diluted into a 0.2% gluteraldehyde, phosphate-buffered saline solution for analysis of sperm morphology. Approximately 1 mL of semen was centrifuged at 700 × g for 15 min and supernatant was snapfrozen in liquid nitrogen for later analysis of seminal plasma proteins (Bucci et al, 2016;Lago-Alvarez et al, 2020;Rego et al, 2014).…”