Kinematic, bioenergetic and oxidative evaluations of donkey sperm preserved at +4°C

Abstract

Summary Information on donkey sperm bioenergetics, kinetics and oxidative status is scarce even though crucial for development of reproductive technologies and germplasm conservation. For these reasons, it is interesting to monitor sperm kinetics, bioenergetics, and oxidative status during sperm storage at +4°C and with several sperm extenders and concentrations. Donkey semen was collected from three jackasses, three times each. It was diluted with four extenders (Kenney, Equiplus, INRA96 or Hippex), set at three sperm concentrations (30, 50 or 70 × 106 spermatozoa/ml) and evaluated for its functionality after 0, 3, 24, 48 and 72 h storage at +4°C. Sperm kinetics was analyzed by Sperm Computer Analysis; sperm bioenergetics was assessed by mitochondrial membrane potential (MMP); sperm oxidative status was evaluated by lipid peroxidation (LPO), anti-LPO potential and nitroblue tetrazolium (NBT) assays. Incubation produced a progressive (P < 0.01) decline in sperm kinetics and MMP, whereas parameters related to oxidative status either increased (LPO, NBT) or decreased (anti-LPO). The anti-LPO potential was the index better related to sperm motility and kinetics. Extenders proved to be differently (P < 0.01) effective in preserving sperm kinetics, MMP, and oxidative status. The concentration of 30 × 106 spermatozoa/ml provided an optimum preservation of sperm functions. Significant correlations emerged between most parameters examined. This study identified reference criteria for storing donkey spermatozoa at +4°C. A low sperm concentration together with a proper extender are crucial requirements for optimum sperm cryopreservation efficiency. Field trials are, however, required to validate these findings, making them operational in practice.

Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen

“…Despite these results, there is no consistent protocol determining egg yolk concentration or extender formulation to be used in cooled donkey semen. The use of proteindefined extenders without egg yolk improves donkey sperm characteristics compared with milk-based extenders (Di Palma et al, 2020). Similarly, in a preliminary study, an improvement in sperm progressive motility, membrane integrity, and function were observed in cooled semen diluted in a commercial caseinate-base extender supplemented with 2% of egg yolk (Alonso et al, 2017).…”

“…This was most evident after storage over 24-48 h (Pagl et al, 2006). Di Palma et al (2020) report that the use of protein-defined extenders improves donkey sperm characteristics compared with milk-based extenders.…”

Relationships between Seminal Plasma Metabolites, Semen Characteristics and Sperm Kinetics in Donkey (Equus asinus)

“…JC-1 is a dye exhibiting potential-dependent accumulation in mitochondria [19]. MMP was evaluated as previously described [21]. In brief, spermatozoa (1 × 10 6 ) were incubated with 1.5 µM JC-1 in PBS supplemented with 0.5% PVA (PBS-PVA) at 37 • C for 30 min, washed by centrifugation and again incubated for 30 min at 37 • C. Samples were read with a spectrofluorometer (Cary Eclipse, Agilent Technologies, Rome, Italy).…”

“…LPO was determined by measuring C11-BODIPY 581/591 fluorescence, as previously described [21]. Sperm aliquots (1 × 10 6 spermatozoa/mL) were incubated with 2 µM C11-BODIPY 581/591 for 30 min at 37 • C, centrifuged at 230× g for 10 min, incubated in PBS-PVA at 37 • C for 30 min, and read with a spectrofluorometer.…”

“…Anti-LPO potential evaluated the ability of sperm defenses to counteract oxidative stress [21]. In brief, spermatozoa (1 × 10 6 ) were incubated in the dark for 30 min at 37 • C with C11-BODIPY 581/591 and 0.5% OxMix, an oxidant solution containing 2 mM menadione, 1.8 mM CuSO 4 , and 0.25 mM H 2 O 2 [22].…”