Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils

Abstract

This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 °C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding.

Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm

“…In addition, in horse sperm, LIN, STR, and BCF are decreased in the presence of a GSH concentration ≥2.5 mM; furthermore, VSL and ALH are decreased at ≥5 mM GSH, and VCL and VAP are decreased at ≥7.5 mM GSH [43]. Moreover, four separate subpopulations were herein identified in frozen-thawed donkey sperm, which is in agreement with previous research in this species [27,50,67]. We found that the treatment containing 10 mM GSH significantly increased the proportion of sperm belonging to SP2 (the slowest and least progressive subpopulation), reduced those of sperm belonging to SP3 and SP4 (the subpopulations with intermediate kinematic parameters), and did not affect that of SP1 (the fastest and most progressive subpopulation) with respect to the control.…”

“…In this context, it is worth mentioning that Papas et al [26] showed that the antioxidant capacity of the enzymes present in the SP is significantly higher in donkeys than in horses. In spite of this, the sperm cryopreservation protocols currently applied in donkeys include the elimination of SP by centrifugation [27]. This makes sperm more susceptible to functional damage with lethal consequences [28] and lead them to undergo structural injuries, mainly at the chromatin level, which has a significant impact on male fertility [29].…”

A Novel Approach to Minimising Acute Equine Endometritis That May Help to Prevent the Development of the Chronic State

“…In a recent study with donkey spermatozoa and PMN, enhanced binding to PMN was observed for the SLC-selected sperm samples compared to controls ( 47 ). These results are in agreement with previous studies, which showed that CRISP3 protein in seminal plasma inhibited the binding mechanism between viable spermatozoa and PMNs involved in phagocytosis ( 79 ).…”

“…Therefore, it is problematic to interpret the results of insemination studies in mares with “washed” spermatozoa, unless complete removal of seminal plasma is shown e.g., by seminal plasma markers. In a study in which colloid centrifugation was used to prepare donkey spermatozoa free of seminal plasma, there was increased binding of spermatozoa to PMNs ( 47 ). Two specific fractions of seminal plasma were responsible for these effects: proteins in the ranges of 30–50 kDa and 50–100 kDa had a beneficial effect on sperm motility, membrane integrity and binding to PMNs whereas smaller proteins had either no effect or a detrimental effect on these parameters ( 48 ).…”