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.

Keywords: donkey; fresh semen; single layer centrifugation; sperm; sperm–PMN binding.

Figure 1

Percentages of (a) membrane-intact spermatozoa (SYBR14⁺/PI⁻) and (b) viable spermatozoa with low membrane lipid disorder (M540⁻/YO-PRO-1⁻) in control (non-selected samples) and SLC-selected samples (Equicoll). Different superscript letters (a, b) indicate significant differences (p ≤ 0.05) between treatments within a given time point, and different superscript numbers (1–3) mean significant differences between time points within the control or samples selected with Equicoll. Data are shown as mean ± SEM for 10 separate experiments. SLC: single layer centrifugation.

Figure 2

(a) Percentages of spermatozoa with high mitochondrial membrane potential (MMP) and (b) ratios between JC1_agg and JC1_mon of the sperm population with high MMP in control (non-selected samples) and SLC-selected samples (Equicoll). Different superscript letters (a, b) indicate significant differences (p ≤ 0.05) between treatments within a given time point, and different superscript numbers (1–3) mean significant differences between time points within the control or samples selected with Equicoll. Data are shown as mean ± SEM for 10 separate experiments. SLC: single layer centrifugation.

Figure 3

Proportions of viable spermatozoa with high intracellular calcium levels evaluated with two separate fluorochromes: Fluo3 (a) and Rhod5 (b) in control (non-selected samples) and SLC-selected samples (Equicoll). Data are given considering the viable sperm population. Different superscript letters (a, b) indicate significant differences (p ≤ 0.05) between treatments within a given time point, and different superscript numbers (1–3) indicate significant differences between time points within the control or samples selected with Equicoll. Results are shown as mean ± SEM for 10 separate experiments. SLC: single layer centrifugation.

Figure 4

Proportions of viable spermatozoa with high intracellular levels of peroxides (DCF⁺; a) and superoxides (E⁺; b) in control (non-selected samples) and SLC-selected samples (Equicoll). Data are given considering the viable sperm population. Different superscript letters (a, b) indicate significant differences (p ≤ 0.05) between treatments within a given time point, and different superscript numbers (1–3) mean significant differences between time points within the control or samples selected with Equicoll. Results are shown as mean ± SEM for 10 separate experiments. SLC: single layer centrifugation.

Figure 5

Proportions of (a) total and (b) progressively motile spermatozoa in control (non-selected samples) and SLC-selected samples (Equicoll). Different superscript letters (a, b) indicate significant differences (p ≤ 0.05) between the control and SLC-selected samples, and different superscript numbers (1–3) mean significant (p ≤ 0.05) differences between time points within the control or samples selected with Equicoll. Data are shown as mean ± SEM for 10 separate experiments. SLC: single layer centrifugation.

Figure 6

Proportions of spermatozoa belonging to one of each sperm subpopulations ((a), SP1; (b), SP2; (c), SP3; (d), SP4) in control (non-selected samples) and SLC-selected samples (Equicoll). Different superscript letters (a, b) indicate significant differences (p ≤ 0.05) between treatments within a given time point, and different superscript numbers (1–3) mean significant differences between time points within the control or samples selected with Equicoll. Data are shown as box-whisker plots for 10 separate experiments. SLC: single layer centrifugation. SP1: subpopulation 1; SP2: subpopulation 2; SP3: subpopulation 3; SP4: subpopulation 4.

Figure 7

Percentages of (a) spermatozoa bound to PMN and (b) sperm phagocytosed by PMN in control (non-selected samples) and SLC-selected samples (Equicoll). Different superscript letters (a, b) indicate significant differences (p ≤ 0.05) between treatments within a given time point, and different superscript numbers (1, 2) mean significant differences between time points within the control or samples selected with Equicoll. Data are shown as mean ± SEM for 7 separate experiments. SLC: single layer centrifugation. PMN: Polymorphonuclear neutrophils.