animals Article Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils Marion Papas 1 , Jaime Catalán 1 , Sandra Recuero 2,3 , Jane M. Morrell 4 , Marc Yeste 2,3, *,† and Jordi Miró 1, *,† 1 Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, E-08193 Bellaterra (Cerdanyola del Vallès), Spain; papas.marion@gmail.com (M.P.); dr.jcatalan@gmail.com (J.C.) 2 Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona, Spain; sandra.recuero@udg.edu 3 Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain 4 Clinical Sciences, Swedish University of Agricultural Sciences, Box 7054, SE-75007 Uppsala, Sweden; jane.morrell@slu.se * Correspondence: marc.yeste@udg.edu (M.Y.); jordi.miro@uab.cat (J.M.); Tel.: +34-972-419514 (M.Y.); +34-93-5814293 (J.M.) † These authors contribute equally.  Received: 26 September 2020; Accepted: 13 November 2020; Published: 16 November 2020  Simple Summary: Donkey Artificial Insemination (AI) with frozen/thawed semen results in poor fertility outcomes. Jennies show a significant post-AI endometrial reaction, with a large amount of defense cells—polymorphonuclear neutrophils (PMN)—migrating to the uterine lumen. Seminal plasma (SP) has a detrimental effect on sperm conservation and its removal is a necessary step in the semen freezing protocol. However, several SP proteins seem to control sperm-PMN binding. Single layer centrifugation (SLC) with colloids, which has been used to select spermatozoa and improve reproductive performance in different species, is known to remove SP proteins attached to the sperm membrane. In this study, two experiments were performed. The first one compared the quality of SLC-selected and non-selected fresh donkey spermatozoa. In the second experiment, PMN obtained from the peripheral blood were co-incubated with selected and unselected spermatozoa, and the interaction between PMN and spermatozoa was analyzed. In conclusion, SLC of fresh donkey semen increases the proportion of functionally intact spermatozoa and appears to remove the SP proteins that inhibit sperm-PMN binding, thus increasing sperm phagocytosis by PMN. 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 Animals 2020, 10, 2128; doi:10.3390/ani10112128 www.mdpi.com/journal/animals Animals 2020, 10, 2128 2 of 17 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: sperm; fresh semen; donkey; single layer centrifugation; sperm–PMN binding 1. Introduction Proper management of breeding and genetic diversity in equine species requires the use of assisted reproduction technologies (ART), which includes artificial insemination (AI), embryo transfer (ET), intracytoplasmic sperm injection (ICSI), and cryopreservation [1,2]. It has been reported that when sperm are selected, fertility outcomes following ART increase [3,4]. This selection is usually based on sperm motility, morphology, and the integrities of plasma membrane, acrosome, and DNA. In addition, sperm selection allows for the removal of seminal plasma, non-viable sperm, pathogens, and debris particles [5]. While seminal plasma has been described to have a detrimental effect on sperm motility and viability during storage, the presence of non-viable, morphologically abnormal spermatozoa may be a source of reactive oxygen species (ROS), which can also be detrimental to sperm survival during storage [6]. Several sperm preparation techniques have been designed to separate male gametes from seminal plasma and to select spermatozoa with better quality. Because simple sperm washing does not remove all seminal plasma components [7], the other two alternatives, i.e., sperm migration and colloid centrifugation, are the most used in equine practice [8]. Sperm migration through swim-up is based on sperm motility and yields low recovery rates [7]. In contrast, centrifugation with colloids uses silane-coated silica particles in a species-specific formulation that allows the separation of heterogeneous sperm into sub-populations according to their density. Not only does this technique select sperm on the basis of their motility, but also on their normal morphology, and plasma membrane and chromatin integrities [8]. Single layer centrifugation (SLC) is a simplification of the density gradient centrifugation as only one layer of colloid is employed [9]. Previous studies have demonstrated that, following SLC, spermatozoa show higher motility, less DNA fragmentation, and higher pregnancy rates in subfertile stallions [10,11]. With respect to jackasses, SLC using Equicoll improves sperm quality parameters, such as motility, viability, and morphology, after 24 h of cooled storage [12], and after thawing of cryopreserved doses [13]. To the best of our knowledge, however, no study with fresh donkey semen has been performed nor has any evaluated the effects of SLC with Equicoll on mitochondrial activity, and intracellular levels of ROS and calcium. Other studies in rams [14], pigs [15], bulls [16], and brown bears [17,18] have demonstrated that centrifugation with SLC increases the proportions of spermatozoa with an intact plasma membrane and acrosome, high mitochondrial activity, an intact DNA, and progressive motility, and has also been found to increase sperm cryotolerance when performed before cryopreservation [19,20]. While elimination of seminal plasma is beneficial for sperm cryopreservation [21] and improves the stability of plasma membrane in fresh and cooled-stored stallion spermatozoa [22], one should bear in mind that several seminal plasma factors are involved in the regulation of the inflammatory response within the female reproductive tract. In addition, studies conducted in other species, such as the pig, showed that SLC removes seminal plasma components, including porcine spermadhesins PSP-I and PSP-II, and some cholesterol molecules from sperm plasma membrane [23]. Remarkably, the presence of seminal plasma has been shown to reduce chemotaxis, sperm-neutrophil binding, phagocytosis, and the formation of DNA-based neutrophil extracellular traps (NETs) [24]. In the donkey, in vitro models suggest that seminal plasma can suppress sperm-PMN attachment [25,26] and Animals 2020, 10, 2128 3 of 17 that, when added to frozen/thawed sperm, downregulates the expression of COX2 in endometrial cells, which leads PMN chemotaxis to reduce [27]. Against this background, we hypothesized that SLC of jackass semen with Equicoll could increase the proportions of donkey spermatozoa exhibiting high motility, an intact and functional plasma membrane and high mitochondrial activity, and modify sperm-PMN binding. The effects of SLC on the proportions of spermatozoa with high levels of intracellular calcium and reactive oxygen species were also investigated. In addition, we tested the effects of SLC with Equicoll on donkey sperm not only after centrifugation, but also after 60, 120, and 180 min of incubation at 38 ◦ C. The rationale of testing SLC-effects over this incubation period is related to the time that sperm are deemed to reside within the uterus (3 h). 2. Materials and Methods 2.1. Animals Semen samples (N = 17) were collected from five Catalonian jackasses, whose age ranged from 3 to 11 years, that were in good health and shown to be fertile. Jackasses underwent a regular semen collection, every other day during the week, and were housed in individual paddocks at the Equine Reproduction Service, Autonomous University of Barcelona (Bellaterra, Cerdanyola del Vallès, Spain), which is an EU-approved equine semen collection center (ES09RS01E). Polymorphonuclear neutrophils were isolated from the peripheral blood of three Catalonian jennies as described previously [26]. These animals, which were also in good health and of proven fertility, were housed in a big paddock at the Equine Reproduction Service, Autonomous University of Barcelona. Animals were handled following the European Union Directive 2010/63/EU for animal experiments and the Animal Welfare Law from the Regional Government of Catalonia (Spain). In addition, this study was approved by the Ethics Committee, Autonomous University of Barcelona (Code: CEEAH 1424). 2.2. Experimental Design This study consisted of two separate experiments. The goal of the first experiment was to determine the effects of SLC with Equicoll on sperm function parameters, such as motility, plasma membrane integrity, membrane lipid disorder, intracellular calcium levels, intracellular ROS levels, and mitochondrial activity. The effects of SLC-selection on donkey sperm subpopulations were also considered. Although previous works evaluated the effects of SLC with Equicoll on donkey semen, they used cooled-stored and cryopreserved sperm [12,13]; in contrast, the current study used fresh semen. Ejaculates (N = 10) were collected through a Hannover artificial vagina (Minitüb GmbH, Tiefenbach, Germany) with an in-line nylon mesh filter to remove gel and debris. Immediately after collection, gel-free semen was extended at a ratio of five volumes of skim milk extender (4.9% glucose, 2.4% skim milk, 100 mL double distilled water), previously warmed at 38 ◦ C, and one volume of semen. After assessing sperm concentration, viability, morphology, and motility, each ejaculate was divided into two fractions. The first one was utilized as a control and was directly adjusted to a final concentration of 25 × 106 sperm/mL. The second fraction was centrifuged through a single layer of a silane-coated silica-based colloid formulation (Equicoll, SLU, Sweden; formerly known as Androcoll-E) according to the protocol for small centrifuge tubes described by Morrell et al. [28]. Briefly, 1.5 mL of extended semen containing up to 100 × 106 sperm/mL was pipetted on top of 4 mL Equicoll in a 15-mL tube. After centrifugation at 300× g and room temperature for 20 min, the supernatant and most of the colloid was discarded and the sperm pellet was transferred to a clean centrifuge tube containing 0.5 mL of the same skim milk extender. Sperm concentration was evaluated and subsequently adjusted to a final concentration of 25 × 106 sperm/mL. Aliquots were incubated at 38 ◦ C under aerobic conditions for 3 h. Sperm motility and other functional parameters evaluated with flow cytometry were determined immediately after SLC-selection (0 min), and after 60 min, 120 min, and 180 min of incubation at 38 ◦ C. Animals 2020, 10, 2128 4 of 17 The second experiment aimed to determine the effects of SLC with Equicoll on the interaction of donkey sperm with polymorphonuclear neutrophils (PMN). With this purpose, each ejaculate (N = 7) was split into two fractions. One fraction was used as a control and its sperm concentration was adjusted to 500 × 106 sperm/mL. The other fraction was centrifuged through a single layer of a silane-coated silica-based colloid formulation (Equicoll) as described for Experiment 1. After centrifugation, sperm concentration was evaluated and subsequently adjusted to a final concentration of 500 × 106 sperm/mL. Treatments consisted of in vitro co-incubation of sperm cells with PMN-rich samples (1:1; v:v) in a water bath at 38 ◦ C for 3 h, as previously described by Miró et al. [26]. Briefly, peripheral blood neutrophils were isolated from healthy jennies. Following incubation in a water bath at 38 ◦ C for 30 min, heparinized blood was subjected to centrifugation at 400× g and 4 ◦ C for 5 min prior to removing plasma. The buffy coat was mixed with an isotonic saline solution (PBS) and again centrifuged at 400× g and 4 ◦ C for 5 min. The PMN-rich buffy coat was collected and re-suspended in PBS to a final concentration of 100 × 106 PMN/mL. All samples (selected and non-selected) were examined for sperm phagocytosis immediately after SLC-selection (0 min), and after 60 min, 120 min, and 180 min of incubation at 38 ◦ C. 2.3. Flow Cytometry Sperm quality parameters were determined using a Cell Laboratory Quanta SCTM flow cytometer (Beckman Coulter; Fullerton, CA, USA). The following sperm parameters were evaluated: plasma membrane integrity (viability), membrane lipid disorder, acrosome integrity, mitochondrial activity (mitochondrial membrane potential), and intracellular levels of calcium (Ca2+ ), superoxides (O2 −• ) and peroxides (H2 O2 ). Prior to any staining, each sample was diluted with HEPES buffered saline solution (10 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 150 mmol/L NaCl, 10% BSA; pH = 7.4) to a final concentration of 1 × 106 spermatozoa/mL. Samples were subsequently incubated with the corresponding fluorochromes under dark conditions. After fluorochrome staining, an argon ion laser was used to excite samples at 488 nm (22 mW). Spermatozoa were selected on the basis of electronic volume (EV) and side scatter (SS) and the sheath flow rate was set at 4.17 µL/min. Based on EV/SS dot-plots, sperm cells were selected and the other events, including subcellular debris (particle diameter <7 µm) and cell aggregates (particle diameter >12 µm), were gated out. The flow cytometer was calibrated every day using 10-µm flow-check fluorospheres (Beckman Coulter), positioning the bead size (10 µm) on the EV channel (EV = 200). Up to 10,000 events were analyzed per replicate and a total of three independent replicates per sample were evaluated. FL1 detector (BP, band pass: 525 nm; DRLP, dichroic long pass: 550 nm) was used for monitoring the green fluorescence emitted by SYBR14, YO-PRO-1 (1-(4-[3-methyl-2,3- dihydro-(benzo-1,3-oxazole)-2-methylidene]-quinolinium)-3trimethylammonium propane diodide), JC1mon (5,50 ,6,60 -tetrachloro-1,10 3,30 tetraethyl-benzimidazolylcarbocyanine iodide monomers), H2 DFCDA (20 ,70 -dichlorodihydrofluorescein diacetate), and Fluo3-AM (acetoxymethyl, ester form) fluorochromes. FL2 detector (BP: 575 nm; DRLP: 600 nm) was used for monitoring the orange fluorescence emitted by JC1agg . FL3 detector (LP, long pass: 670 nm) was used for monitoring the red fluorescence emitted by HE (hydroethidine), PI (propidium iodide), M540 (merocyanine 540), and Rhod5. Post-acquisition analyses were conducted using the Cell Lab Quanta® SC MPL Analysis Software (version 1.0; Beckman Coulter; Fullerton, CA, USA). Data were corrected by subtracting the percentage of debris particles found in SYBR14/PI staining from the percentages of particles appearing at the lower left quadrant in the other tests [29]. The proportions of the other sperm populations were normalized. Animals 2020, 10, 2128 5 of 17 2.3.1. Evaluation of Sperm Membrane Integrity (SYBR14/PI) Sperm viability was assessed using a combination of two fluorochromes (SYBR14 and propidium iodide, PI) available from a commercial kit (Live/Dead Sperm Viability kit, Molecular Probes, ThermoFisher Scientific; Waltham, MA, USA). After adjusting cell concentration to 1 × 106 sperm/mL, spermatozoa were stained with SYBR14 (final concentration: 100 nM) at 38 ◦ C for 10 min. Following this, PI (final concentration: 12 µM) was added and samples were incubated at 38 ◦ C for 5 min. The following three categories of spermatozoa were identified: (i) spermatozoa with an intact plasma membrane, stained in green (viable spermatozoa; SYBR14+ /PI− ); (ii) spermatozoa with an altered plasma membrane, stained in red (non-viable spermatozoa; SYBR14− /PI+ ); and (iii) spermatozoa with an altered plasma membrane, stained in both red and green (non-viable spermatozoa; SYBR14+ /PI+ ). Non-DNA containing particles (debris particles; SYBR14− /PI− ), which appeared at the lower left quadrant, were subtracted from the total number of events and, as aforementioned, were used to correct the other tests. The protocol included compensation of FL1-spill over into the FL3-channel (2.45%). 2.3.2. Evaluation of Sperm Membrane Lipid Disorder (M540/YO-PRO-1) Lipid disorder of plasma membrane was evaluated following the protocol set by Rathi et al. [30] with minor modifications [31], which is based on M540 and YO-PRO-1 fluorochromes (Molecular Probes, ThermoFisher Scientific). Briefly, samples were incubated with M540 (final concentration: 2.6 µM) and YO-PRO-1 (final concentration: 25 nM) at 38 ◦ C for 10 min. A total of four sperm populations was identified in flow cytometry dot-plots: (1) viable spermatozoa with low membrane lipid disorder (M540− /YO-PRO-1− ); (2) viable spermatozoa with high membrane lipid disorder (M540+ /YO-PRO-1− ); (3) non-viable spermatozoa with low membrane lipid disorder (M540− /YO-PRO-1+ ); and (4) non-viable spermatozoa with high membrane lipid disorder (M540+ /YO-PRO-1+ ). Correction was performed following the procedure described previously and data were not compensated. 2.3.3. Evaluation of Mitochondrial Membrane Potential (JC1) Assessment of mitochondrial membrane potential (MMP) was conducted using JC1-staining (Molecular Probes, ThermoFisher Scientific), following the protocol set by Ortega-Ferrusola et al. [32]. With this purpose, samples were incubated with JC1 (final concentration: 0.3 µM) at 38 ◦ C for 30 min. When MMP is low, JC1 remains as monomers (JC1mon ); when MMP is high, JC1 forms aggregates (JC1agg ). Three different sperm populations were identified: (1) spermatozoa with green-stained mitochondria, which had low MMP (JC1mon ); (2) spermatozoa with orange-stained mitochondria; and (3) spermatozoa with green- and orange-stained mitochondria. Populations 2 and 3 were considered as having high MMP (JC1agg ). The protocol included compensation of FL1-spill over into the FL3-channel (68.50%). 2.3.4. Evaluation of Intracellular Calcium Levels (Fluo3/PI and Rhod5/YO-PRO-1) Two different co-staining protocols were used to determine intracellular calcium levels. Fluo3-AM is able to penetrate cell membranes and displays better affinity for the calcium present in the sperm mid-piece [33]. This test, which is combined with PI, was performed as described by Harrison et al. [34] and Kadirvel et al. [35]. Briefly, spermatozoa were incubated with Fluo3-AM (final concentration: 1 µM) and PI (final concentration: 12 µM) at 38 ◦ C for 10 min. Four different sperm populations were identified: (1) viable spermatozoa with low levels of intracellular calcium (Fluo3− /PI− ); (2) viable spermatozoa with high levels of intracellular calcium (Fluo3+ /PI− ); (3) non-viable spermatozoa with low levels of intracellular calcium (Fluo3− /PI+ ); and (4) non-viable spermatozoa with high levels of intracellular calcium (Fluo3+ /PI+ ). The protocol included compensation of FL1-spill over into the FL3-channel (28.72%), and of FL3-spill over into the FL1-channel (2.45%). Animals 2020, 10, 2128 6 of 17 Rhod5 has more affinity for the calcium residing in the sperm head than for that found in the mid-piece. This test was performed following the protocol set by Yeste et al. [33]. In brief, spermatozoa were incubated with Rhod-5N (final concentration: 5 µM) and YO-PRO-1 (final concentration: 25 nM) at 38 ◦ C for 10 min. Four sperm populations were distinguished in flow cytometry dot-plots: (1) viable spermatozoa with low levels of intracellular calcium (Rhod5− /YO-PRO-1− ); (2) viable spermatozoa with high levels of intracellular calcium (Rhod5+ /YO-PRO-1− ); (3) non-viable spermatozoa with low levels of intracellular calcium (Rhod5− /YO-PRO-1+ ); and (4) non-viable spermatozoa with high levels of intracellular calcium (Rhod5+ /YO-PRO-1+ ). The protocol included compensation of FL3-spill over into the FL1-channel (3.16%). 2.3.5. Evaluation of Intracellular Reactive Oxygen Species (ROS) Levels: H2 O2 and O2 −• (H2 DCFDA/PI and HE/YO-PRO-1) The evaluation of intracellular levels of hydrogen peroxides (H2 O2 ) was performed following the protocol set by Morrell et al. [36]. In brief, sperm samples were stained with H2 DCFDA (final concentration: 140 µM) and PI (final concentration: 12 µM) and incubated at 25 ◦ C for 30 min. H2 DCFDA consists of a non-fluorescent probe that is able to penetrate plasma membranes. Upon intracellular oxidation, H2 DCFDA is de-esterified and converted into highly green fluorescent 20 ,70 -dichlorofluorescein (DCF+ ), which is detected through FL1. Flow cytometry dot-plots depicted four sperm populations: (1) viable spermatozoa with high levels of intracellular peroxides (DCF+ /PI− ); (2) viable spermatozoa with low levels of intracellular peroxides (DCF− /PI− ); (3) non-viable spermatozoa with high levels of intracellular peroxides (DCF+ /PI+ ); and (4) non-viable spermatozoa with low levels of intracellular peroxides (DCF− /PI+ ). The protocol included compensation of FL1-spill over into the FL3-channel (2.45%). The determination of intracellular levels of superoxide (O2 −• ) radicals was performed through co-staining with hydroethidine (HE) and YO-PRO-1, following the protocol set by Guthrie and Welch [37]. Briefly, sperm samples were mixed with HE (final concentration: 4 µM) and YO-PRO-1 (final concentration: 25 nM) and incubated at 25 ◦ C for 30 min. In the presence of O2 −• , HE, which is known to pass across plasma membranes, is oxidized to ethidium (E+ ; detected by FL3). A total of four sperm populations were identified: (1) viable spermatozoa with high levels of intracellular superoxides (E+ /YO-PRO-1− ); (2) viable spermatozoa with low levels of intracellular superoxides (E− /YO-PRO-1− ); (3) non-viable spermatozoa with high levels of intracellular superoxides (E+ /YO-PRO-1+ ); and (4) non-viable spermatozoa with low levels of intracellular superoxides (E− /YO-PRO-1+ ). The protocol included compensation of FL1-spill over into the FL3-channel (5.06%). 2.4. Evaluation of Sperm Motility Sperm motility and kinetic parameters were assessed using a computer-assisted sperm motility analysis (CASA) system (ISAS v. 1.0; Proiser S.L., Valencia, Spain) and an Olympus BX41 microscope (Olympus 20x 0.30 PLAN objective; Olympus Europe, Hamburg, Germany). With this purpose, 5 µL of each sample was placed into a Neubauer Chamber and then observed using a phase contrast microscope with a pre-warmed stage (38 ◦ C). For each sample, three fields per drop and at least 1000 spermatozoa were assessed. The following variables were analyzed: percentages of total motile spermatozoa (TMOT), percentages of progressively motile spermatozoa (PMOT), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), linearity index (LIN), straightness index (STR), oscillation index (WOB), amplitude of lateral head displacement (ALH), and beat cross frequency (BCF) [38]. Cut-off values for a given sperm cell to be considered as motile or progressively motile were VAP ≥ 10 µm/s and STR ≥ 75%, respectively. Animals 2020, 10, 2128 7 of 17 2.5. Evaluation of Sperm–PMN Binding Smears were prepared on microscope slides for each treatment and stained with a modified Wright’s dye (Diff Quick® , Quimica Clinica Aplicada, Amposta, Spain). Phagocytosis was determined at 1000× magnification (Olympus Europe, Hamburg, Germany) under immersion oil, and was expressed as the percentage of PMN that ingested at least one spermatozoon [26]. The percentages of spermatozoa in contact with PMN were also counted. Two hundred PMN per slide were counted, and three technical replicates were made. 2.6. Statistical Analysis Data obtained from the analysis of all sperm parameters were first tested for normality (Shapiro–Wilk test) and homogeneity of variances (Levene test). Following this, all variables except those related to the effects of SLC-selection upon sperm motile populations (i.e., % SYBR14+ /PI− spermatozoa, % M540− /YO-PRO-1− spermatozoa, % spermatozoa with high MMP, JC1agg /JC1mon ratios, % Fluo3+ /viable spermatozoa, % Rhod5+ /viable spermatozoa, % DCF+ /viable spermatozoa, % E+ /viable spermatozoa, % TMOT spermatozoa, % PMOT spermatozoa, VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF) were evaluated through a linear mixed model (intra-subjects factor: time of incubation; inter-subjects factor: control vs. SLC-Equicoll), followed by Sidak post-hoc test. In order to classify spermatozoa into separate motile subpopulations, the procedure described by Luna et al. [39] was conducted with minor modifications; this procedure combines Principal Component (PCA) and cluster analyses. First, a PCA with the individual kinematic parameters (VSL, VCL, VAP, LIN, STR, WOB, ALH, and BCF) obtained for each spermatozoon (CASA assessment) was run; data were sorted into PCA components and the matrix was rotated using the Varimax approach and Kaiser standardization. Thereafter, cluster analyses were conducted through a two-way procedure based on Schwarz’s Bayesian Criterion and log-likelihood distance using the individual regression scores of each PCA component. Four separate motile sperm subpopulations were identified, and proportions of spermatozoa belonging to each subpopulation (SP1, SP2, SP3, or SP4) were subsequently calculated. The effects of SLC-selection upon sperm motile populations were evaluated through a Scheirer–Ray–Hare ranked ANOVA followed by Mann–Whitney test for pair-wise comparisons. Proportions of SP1, SP2, SP3, and SP4 spermatozoa were considered as dependent variables. Results are shown as mean ± standard error of the mean (SEM); the minimal level of significance was set at p ≤ 0.05 in all cases. 3. Results 3.1. Integrity and Lipid Disorder of Sperm Plasma Membrane Figure 1a shows (mean ± SEM) the proportions of viable spermatozoa (i.e., spermatozoa with an intact plasma membrane; SYBR14+ /PI− ) in the control and samples selected through SLC with Equicoll. Incubation led to a significant (p < 0.05) decrease in the proportions of viable spermatozoa in both the control and SLC-selected samples. However, proportions of viable spermatozoa were significantly (p < 0.05) higher in samples selected through SLC than in their control counterparts, immediately after selection (0 min) and after 60 min and 120 min of incubation at 38 ◦ C. At 180 min of incubation, no significant differences between treatments were observed. Proportions of viable spermatozoa with low membrane lipid disorder (M540− /YO-PRO-1− ; mean ± SEM) also decreased (p < 0.05) over incubation time (Figure 1b). Again, these percentages were significantly (p < 0.05) higher in SLC-selected samples than in the control immediately after centrifugation (0 min) and after 60 min of incubation at 38 ◦ C. No significant differences between the control and SLC-selected samples were observed after 120 min and 180 min of incubation. immediately after selection (0 min) and after 60 min and 120 min of incubation at 38 °C. At 180 min of incubation, no significant differences between treatments were observed. Proportions of viable spermatozoa with low membrane lipid disorder (M540−/YO-PRO-1−; mean ± SEM) also decreased (p < 0.05) over incubation time (Figure 1b). Again, these percentages were significantly (p < 0.05) higher in SLC-selected samples than in the control immediately after Animals 2020, 10, 2128 8 of 17 centrifugation (0 min) and after 60 min of incubation at 38 °C. No significant differences between the control and SLC-selected samples were observed after 120 min and 180 min of incubation. . Figure 1. Percentages of (a) membrane-intact spermatozoa (SYBR14+ /PI− ) and (b) viable spermatozoa with low2020, Animals membrane 10, x lipid disorder (M540− /YO-PRO-1− ) in control (non-selected samples) and 8 of 17 SLC-selected samples (Equicoll). Different superscript letters (a, b) indicate significant differences Figure (p ≤ 0.05) 1. Percentages between of (a) membrane-intact treatments within a givenspermatozoa time point,(SYBR14 +/PI−) and (b) viable spermatozoa and different superscript numbers (1–3) with low membrane lipid disorder (M540−/YO-PRO-1−) in control (non-selected samples) and SLC- mean significant differences between time points within the control or samples selected with Equicoll. selected samples (Equicoll). Different superscript letters (a, b) indicate significant differences (p ≤ 0.05) Data are shown as mean ± SEM for 10 separate experiments. SLC: single layer centrifugation. between treatments within a given time point, and different superscript numbers (1–3) mean 3.2. significant Mitochondrial differencesPotential Membrane 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. Incubation at 38 ◦ C led to a significant (p < 0.05) decrease in the proportions of spermatozoa with 3.2. Mitochondrial membrane potential high MMP (Figure 2a; mean ± SEM). Upon selection (0 min) and after 60 min of incubation at 38 ◦ C, proportionsIncubation at 38 °C led of spermatozoa to ahigh with significant MMP(pwere < 0.05)significantly < 0.05) higher decrease in the(pproportions of spermatozoa in samples withselected high MMP (Figure 2a; mean ± SEM). Upon selection (0 min) and after 60 min through SLC than in their control counterparts. No significant differences between the control of incubation at 38 °C, and proportions of spermatozoa with high MMP were significantly (p < 0.05) higher in samples◦ selected SLC-selected samples were observed after 120 min and 180 min of incubation at 38 C. In addition, through SLC than in their control counterparts. No significant differences between the control and JC1agg /JC1 mon ratios SLC-selected in thewere samples sperm population observed with after 120 min high MMP and 180 min were at 38 °C.(pIn