International Journal of Molecular Sciences Article Seminal Plasma, Sperm Concentration, and Sperm-PMN Interaction in the Donkey: An In Vitro Model to Study Endometrial Inflammation at Post-Insemination Jordi Miró 1, * , Henar Marín 1 , Jaime Catalán 1 , Marion Papas 1 , Sabrina Gacem 1 and Marc Yeste 2,3 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; henarmarin@hotmail.com (H.M.); dr.jcatalan@gmail.com (J.C.); papas.marion@gmail.com (M.P.); swp.sabrina.gacem@gmail.com (S.G.) 2 Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona, Spain; marc.yeste@udg.edu 3 Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain * Correspondence: jordi.miro@uab.cat; Tel.: +34-93-5814273  Received: 1 April 2020; Accepted: 12 May 2020; Published: 13 May 2020  Abstract: In the donkey, artificial insemination (AI) with frozen-thawed semen is associated with low fertility rates, which could be partially augmented through adding seminal plasma (SP) and increasing sperm concentration. On the other hand, post-AI endometrial inflammation in the jenny is significantly higher than in the mare. While previous studies analyzed this response through recovering Polymorphonuclear Neutrophils (PMN) from uterine washings, successive lavages can detrimentally impact the endometrium, leading to fertility issues. For this reason, the first set of experiments in this work intended to set an in vitro model through harvesting PMN from the peripheral blood of jennies. Thereafter, how PMN, which require a triggering agent like formyl-methionyl-leucyl-phenylalanine (FMLP) to be activated, are affected by donkey semen was interrogated. Finally, we tested how four concentrations of spermatozoa (100 × 106 , 200 × 106 , 500 × 106 and 1000 × 106 spermatozoa/mL) affected their interaction with PMN. We observed that semen, which consists of sperm and SP, is able to activate PMN. Whereas there was a reduced percentage of spermatozoa phagocytosed by PMN, most remained attached on the PMN surface or into a surrounding halo. Spermatozoa not attached to PMN were viable, and most of those bound to PMN were also viable and showed high tail beating. Finally, only sperm concentrations higher than 500 × 106 spermatozoa/mL showed free sperm cells after 3 h of incubation, and percentages of spermatozoa not attached to PMN were higher at 3 h than at 1 h, exhibiting high motility. We can thus conclude that semen activates PMN in the donkey, and that the percentage of spermatozoa phagocytosed by PMN is low. Furthermore, because percentages of spermatozoa not attached to PMN were higher after 3 h than after 1 h of incubation, we suggest that PMN-sperm interaction plays an instrumental role in the reproductive strategy of the donkey. Keywords: seminal plasma; sperm; polymorphonuclear neutrophils; donkey 1. Introduction Most European donkey breeds are currently in danger of extinction. However, there has been, in the recent years, an increased global demand for donkey products. As such, donkey skin, which is Int. J. Mol. Sci. 2020, 21, 3478; doi:10.3390/ijms21103478 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 3478 2 of 16 used in China to make a traditional donkey-hide gelatin (Ejiao); meat; and milk, which represents an alternative to its bovine counterpart for infants with some allergic and inflammatory conditions, are also attracting the interest of the cosmetic industry. Despite this, our current knowledge of the donkey reproductive physiology is still scarce and, despite horse reproduction technologies being used, many differences between these two species exist. Jackasses show high spermatogenic efficiency [1], and their sperm concentration, total cell number, sperm kinematic parameters and percentages of progressively motile and morphologically normal spermatozoa are higher than in stallions [2,3]. In addition, while frozen-thawed donkey spermatozoa have good post-thaw viability and motility [4–6], are able to penetrate zona pellucida free bovine oocytes in vitro [7] and yield good conception rates (50%) following artificial insemination (AI) of mares [1], their pregnancy rates are poor (0–28%) when used to inseminate jennies [8]. Oliveira et al. investigated the most suitable jackass sperm concentration for inseminating jennies and mares and concluded that increasing insemination doses up to 109 spermatozoa/mL augmented fertility rates in jennies (four inseminations per cycle), achieving figures similar to those obtained in mares [9]. In addition, these authors demonstrated that in order for optimal fertility of frozen-thawed donkey sperm to be reached, deep-horn intrauterine insemination should be used together with four inseminations per cycle (concentration: 109 spermatozoa/mL). Natural breeding and AI induce a physiological endometrial inflammatory response, entailing neutrophils influx into the uterus. Kotilainen et al. observed that infusing spermatozoa rather than bacteria into the mare uterus provoked the influx of Polymorphonuclear neutrophils (PMN) [10]. They also reported that the highest PMN infiltration occurred when mares were inseminated with frozen-thawed or concentrated fresh semen, and that the intensity of the PMN reaction relied on sperm concentration and/or insemination volume. Therefore, the deposited semen is responsible for the uterine inflammatory response, triggering strong chemotaxis of PMN along with cytokine expression soon after insemination [11–15]. Related with this, it is worth mentioning that when inseminated with frozen-thawed semen, jennies show an exacerbated inflammatory response, higher than in mares [16], which was suggested to lead to reduced pregnancy rates [17]. On the other hand, not only does removing seminal plasma (SP) result in an improvement of quality and survival of cooled-stored donkey semen [18], but is also a required step prior to sperm cryopreservation [4]. However, SP has positive effects on sperm motility and survival into the uterus, since it helps avoid oxidative damage [19] and decreases the binding of sperm to PMN and other phagocytic cells [5,20–22]. Despite this, the mechanism through which SP modulates endometrial inflammation remains to be elucidated. Moreover, and as explained before, sperm concentration and velocity are higher in the donkey than in the horse, and their relationship with the interaction between PMN and spermatozoa is also unknown. In fact, our current knowledge comes from in vitro studies focused on sperm:PMN binding in the donkey, the rich PMN samples being obtained through uterine washings at 6 h post-AI [22,23]. However, performing too many uterine washings may produce significant injuries on the endometrium and reduce the reproductive performance, which is especially critical in the case of endangered breeds. Against this background, the objectives of the present study were: (1) to set a method aimed at obtaining a PMN- rich fraction from jenny peripheral blood; and (2) to determine whether the whole semen, with the presence of SP, and different sperm concentrations affect the percentages of spermatozoa attached to PMN. 2. Results 2.1. Experiment 1: Recovering Donkey PMN from Peripheral Blood Samples We tested seven protocols, as described in Materials and Methods. Protocols 1 to 4 yielded concentrations lower than 100 × 103 PMN/µL. Similarly, protocols 5 and 6 also harvested concentrations lower than 100 × 103 PMN/µL, although they were slightly higher in some biological replicates. Int. J. Mol. Sci. 2020, 21, 3478 3 of 16 In contrast, Protocol 7 showed significantly (p < 0.05) higher PMN concentrations than the others, with values greater than 100 × 103 cells/µL in all cases (Table 1). Table 1. PMN concentration (×103 cells/µL) obtained after conducting different PMN isolation protocols (Experiment 1). Protocol [PMN] (×103 cells/µL) 1 8.23 ± 0.87 a 2 6.20 ± 1.23 a,b 3 3.37 ± 0.95 b 4 56.16 ± 6.31 c 5 93.70 ± 7.07 d 6 126.07 ± 10.01 d 7 307.07 ± 19.77 e Different superscript letters (a, b, c, d, e) mean significant (p < 0.05) differences between protocols. Data are shown as mean ± SEM for seven separate experiments. 2.2. Experiment 2: Evaluation of PMN-sperm Binding (FMLP and DMSO) 2.2.1. Sperm Motility Sperm motility was evaluated through Computer-Assisted Sperm Analysis (CASA). However, we found difficulties while using this device, since CASA systems track the head to determine sperm movement, and a significant number of sperm cells were attached to PMN and showed tail beating. Therefore, it was required for us to distinguish between attached and non-attached (free) sperm populations. Evaluation of sperm motility in the free population, which was evaluated by CASA, evidenced that after 3 h and 4 h of incubation, most spermatozoa of this population were immotile. However, a high percentage of spermatozoa attached to PMN showed high tail beating and, occasionally, some were released and exhibited high velocity and progressiveness (average path velocity, VAP ≥ 95 µm/s; percentage of straightness, STR ≥ 73%; percentage of linearity, LIN ≥ 65%; amplitude of lateral head displacement, ALH ≤ 2.7 µm; and frequency of head displacement, BCF ≥ 13.5 Hz). Interestingly, only treatments with sperm concentrations higher than 500 × 106 sperm/mL showed free motile spermatozoa after 3 h of incubation (Supplementary File 1). For this reason, the relationship between PMN and spermatozoa was investigated in treatments containing 500 × 106 sperm/mL (Table 2; Figures 1–5). Table 2. Sperm viability in different treatments and after incubation at 37 ◦ C for 4 h (Experiment 2). Sperm concentration was set at 500 × 106 sperm/mL in all cases. Treatment 0h 1h 2h 3h 4h PMN + SP 77.9 ± 4.7 1, a 65.2 ± 7.0 1, 2, a 55.8 ± 6.0 2, 3, a 43.2 ± 4.3 3, a 30.5 ± 3.3 4, a PMN + SP + FMLP 73.9 ± 6.8 1, a 62.5 ± 7.1 1, 2, a 53.6 ± 7.1 2, 3, a 38.7 ± 4.5 3, 4, a 28.9 ± 3.4 4, a PMN + SP + DMSO 74.9 ± 7.4 1, a 62.3 ± 8.1 1, 2, a 54.7 ± 6.8 2, 3, a 43.0 ± 8.3 3, 4, a 30.7 ± 7.3 4, a SP + DMSO 74.5 ± 6.1 1, a 64.2 ± 7.0 1, 2, a 55.0 ± 6.8 2, 3, a 43.3 ± 4.8 3, a 31.2 ± 4.9 4, a SP 77.7 ± 6.3 1, a 68.1 ± 5.3 1, 2, a 55.5 ± 5.8 2, 3, a 45.1 ± 6.5 3, a 28.3 ± 4.8 4, a Different superscripts (a, b) mean significant (p < 0.05) differences between treatments within a given time point, and different numbers (1, 2) mean significant (p < 0.05) differences between incubation times within a given treatment. Data are shown as mean ± SEM for seven experiments. PMN: polymorphonuclear neutrophils; SP: seminal plasma; DMSO: dimethyl sulfoxide; FMLP: formyl-methionyl-leucyl-phenylalanine. 2.2.2. Sperm Viability Sperm viability decreased significantly (p < 0.05) after 3 h of incubation, irrespective of the treatment (Table 2). In addition, no significant differences (p > 0.05) in the viability of free spermatozoa, were observed between treatments. On the other hand, dimethyl sulfoxide (DMSO) alone or in the presence of PMN showed no detrimental effects on sperm survival. Int. J. Mol. Sci. 2020, 21, 3478 4 of 16 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 16 At At the the beginning beginning ofof the the experiment experiment (0 (0 h), h), percentages percentages of of viable viable spermatozoa spermatozoa attached to PMN PMN inin the presence of FMLP were significantly (p << 0.05) the presence 0.05) higher higher than than in in the other treatments. treatments. In In addition, addition, although percentages of viable spermatozoa bound to PMN PMN in in the the treatment treatment containing containing DMSO DMSO werewere significantly (p <<0.05) 0.05)lower loweratat11hhand andhigher higherat at33hhthan thanin inthe theother other ones, ones, no no significant significant differences differences between treatments treatments were were observed observedatat22hhand and4 4h h(Figure (Figure1).1).InInthe the case case of of thethe treatment treatment containing containing SP, SP, neither variations along the incubation time, nor significant differences with other neither variations along the incubation time, nor significant differences with other treatments after 2 h treatments after and 42hhofand 4 h of incubation incubation (p > 0.05;(pFigure were observed were observed > 0.05;1).Figure 1). Sperm-PMN Binding 2.2.3. Sperm-PMN Binding Percentages of Percentages of spermatozoa spermatozoa attached attached to to PMN PMN increased increased along along the incubation time, reaching significantly (p significantly (p << 0.05) 0.05) higher higher values values with with regard regard toto the the beginning beginning ofof the the experiment experiment after after 44 hh of incubation. However, incubation. However, the the presence presence ofof FMLP FMLP or or DMSO DMSO did did not not differ differ from from the treatment that did not contain these contain thesetwotwoagents agents(i.e., (i.e.,PMN+SP; PMN+SP;Figure Figure2).2).Moreover, Moreover, the the number number of of spermatozoa spermatozoa attached attached to to PMN (sperm:PMN ratio) showed a progressive decrease along the PMN (sperm:PMN ratio) showed a progressive decrease along the incubation period, and incubation period, and was significantly (p << 0.05) 0.05) higher higher inin the PMN ++SP the PMN SPtreatment treatmentthanthanin inthe theones ones containing containing FMLP FMLP oror DMSO DMSO after 0 h, 2 h and 3 h of incubation (Figure 3). Figure 1. Figure Percentages of 1. Percentages of viable viable spermatozoa spermatozoa bound bound to to PMN PMN in in the the three treatments (PMN ++SP, three treatments SP, PMN PMN +SP + FMLP and PMN + SP + DMSO) throughout the incubation time (4 h; Experiment +SP + FMLP and PMN + SP + DMSO) throughout the incubation time (4 h; Experiment 2). Different 2). Different superscripts (a, superscripts (a, b) b)mean meansignificant (p<<0.05) significant (p 0.05)differences differencesbetween betweentreatments treatmentswithin withinaagiven giventime timepoint, point, and different and different numbers numbers (1, (1, 2) 2) mean mean significant (p < significant (p 0.05) differences < 0.05) differences between between incubation incubation times times within within aa given giventreatment. treatment.Data Dataareare shown shown as as mean mean ± SEM ± SEM for seven for seven experiments. experiments. SpermSperm concentration: concentration: 500 × 500 × 10 6 sperm/mL. PMN concentration: 1006 × 106 PMN/mL. PMN: polymorphonuclear neutrophils; 10 sperm/mL. PMN concentration: 100 × 10 PMN/mL. PMN: polymorphonuclear neutrophils; SP: 6 SP: seminal seminal plasma; plasma; DMSO: DMSO: dimethyl dimethyl sulfoxide; sulfoxide; FMLP:FMLP: formyl-methionyl-leucyl-phenylalanine. formyl-methionyl-leucyl-phenylalanine. 2.2.4. Sperm Attachment to PMN via Their Head or Tail 2.2.4. Sperm Attachment to PMN via Their Head or Tail Percentages of spermatozoa attached to PMN via sperm head varied between 37% and 52% Percentages of spermatozoa attached to PMN via sperm head varied between 37% and 52% on on average (Figure 4). At 0 h, percentages of spermatozoa attached to PMN via sperm head were average (Figure 4). At 0 h, percentages of spermatozoa attached to PMN via sperm head were significantly (p < 0.05) higher in the treatment containing SP than in that containing SP and DMSO. significantly (p < 0.05) higher in the treatment containing SP than in that containing SP and DMSO. In contrast, at 1 h, no significant differences (p > 0.05) in the percentages of spermatozoa attached to In contrast, at 1 h, no significant differences (p > 0.05) in the percentages of spermatozoa attached to PMN via sperm head were observed between treatments. Whereas those percentages were significantly PMN via sperm head were observed between treatments. Whereas those percentages were (p < 0.05) higher in the treatment with SP than in that containing SP and FMLP after 2 h of incubation, significantly (p < 0.05) higher in the treatment with SP than in that containing SP and FMLP after 2 h these differences were not observed at 3 h, when percentages of spermatozoa attached to PMN via of incubation, these differences were not observed at 3 h, when percentages of spermatozoa attached sperm head were significantly (p < 0.05) higher in the treatment containing SP and DMSO than in those to PMN via sperm head were significantly (p < 0.05) higher in the treatment containing SP and DMSO containing either SP, or SP, and FMLP. Finally, at the end of the experiment, data resembled to those of than in those containing either SP, or SP, and FMLP. Finally, at the end of the experiment, data 0 h, since percentages of spermatozoa attached to PMN via sperm head were significantly (p < 0.05) resembled to those of 0 h, since percentages of spermatozoa attached to PMN via sperm head were higher in the treatment containing SP than in that containing SP and DMSO (Figure 4). significantly (p < 0.05) higher in the treatment containing SP than in that containing SP and DMSO (Figure 4). Int. J. Mol. Int. J. Mol. Sci. Sci. 2020, 21, x3478 2020, 21, FOR PEER REVIEW 55 of of 16 16 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 16 Figure 2. Percentages of spermatozoa bound to PMN in the three treatments (PMN + SP, PMN + SP + Figure Figure FMLP 2. and Percentagesofofspermatozoa 2. Percentages spermatozoabound PMN + SP + DMSO) throughout bound toto PMNPMN in the in the the incubation three three treatments treatments time (PMN(PMN (4 h; Experiment + SP,+2). PMN + SP + SP, PMN + Different SP FMLP + FMLP and and PMN PMN + SP+ +SPDMSO) + DMSO) throughout throughout thethe incubation incubation time(4(4h;h; Experiment time Experiment 2). 2). Different Different superscripts (a, b) mean significant (p < 0.05) differences between treatments within a given time point, superscripts superscripts (a, b) significant (p <<0.05) mean significant 0.05)differences differences between between treatments treatments within within aa given given time time point, point, and different(a, b) mean numbers (1, 2) mean (psignificant (p < 0.05) differences between incubation times within and and different different numbers numbers (1, 2) mean significant (p < 0.05) differences between incubation times within a given treatment. Data(1, are2)shown mean as significant mean ± SEM (p < for 0.05) differences seven between experiments. incubation Sperm times within concentration: 500 × aa given given treatment. treatment.Data Dataare areshown shownas as mean mean ± ± SEM SEM for for seven seven experiments. experiments. Sperm Sperm concentration: concentration: 500 × 10 sperm/mL. 6 PMN concentration: 100 × 10 6 PMN/mL. PMN: polymorphonuclear neutrophils; SP: 500 10 × 106 sperm/mL. 6 sperm/mL. PMN PMN concentration: concentration: 100 × 100 10 6× 106 PMN/mL. PMN/mL. PMN: PMN: polymorphonuclear polymorphonuclear neutrophils; neutrophils; SP: seminal plasma; DMSO: dimethyl sulfoxide; FMLP: formyl-methionyl-leucyl-phenylalanine. SP: seminal seminal plasma; plasma; DMSO: DMSO: dimethyl dimethyl sulfoxide; sulfoxide; FMLP:FMLP: formyl-methionyl-leucyl-phenylalanine. formyl-methionyl-leucyl-phenylalanine. Figure Figure 3. Number of 3. Number of spermatozoa spermatozoa bound bound to to PMN PMN (sperm:PMN (sperm:PMN ratio) ratio) inin the the three three treatments treatments Figure (PMN+SP, 3. Number PMN+SP+FMLPof spermatozoa and bound PMN+SP+DMSO) to PMN (sperm:PMN throughout (PMN+SP, PMN+SP+FMLP and PMN+SP+DMSO) throughout the incubation time (4 the ratio) incubation in the time (4three h; h; treatments Experiment Experiment2). (PMN+SP, Different PMN+SP+FMLP superscripts (a, b) and mean PMN+SP+DMSO) significant (p < throughout 0.05) differences the incubation between time treatments 2). Different superscripts (a, b) mean significant (p < 0.05) differences between treatments within a (4 h; Experiment within a given 2). timeDifferent givenpoint, timeand superscripts different point, (a, b) mean and numbers different (1, 2) significant mean (1, numbers 2)(pmean significant (p < < 0.05) differences 0.05) (p between differences significant between < 0.05) treatments within incubation differences timesa between given within time a given incubation point, times and different treatment. within aData numbers aretreatment. given shown (1, as mean Data2) ±mean areSEM shown significant for seven as mean (p± 0.05) (PMN +between SP + 0 DMSO). h, 2 h In and 4 h addition, spermatozoa bound to PMN via the tail were significantly (p < 0.05) higher after 1 h and 3 h than at 0 were observed. whereas After percentages 4 h of of h, incubation, spermatozoa no significant thedifferent bound treatment to PMN containing via the tail differences (pDMSO were showed betweensignificantly significantly > 0.05) (p 2< h 0 h, 0.05) 4 h< were (p andhigher 0.05) higher after percentages 1 h and observed. 3After h than4 hatof of0 spermatozoa h, no significantbound to differentPMN via the differences tail (p than > the 0.05) other between two0 treatments h, 2 h and incubation, the treatment containing DMSO showed significantly (p < 0.05) higher percentages of 4 (Figure h were 5). observed. After 4 h of incubation, spermatozoathe treatment bound to PMN containing via the tail DMSO showed than the other significantly two treatments (p <(Figure 0.05) higher 5). percentages of spermatozoa bound to PMN via the tail than the other two treatments (Figure 5). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 16 Int. J. Mol. Sci. 2020, 21, 3478 6 of 16 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 16 Figure 4. Percentages of viable spermatozoa bound to PMN via sperm head in the three treatments (PMN + SP, PMN + SP + FMLP and PMN + SP + DMSO) throughout the incubation time (4 h; Figure 4.4. Percentages of viable spermatozoa bound to PMN via sperm head in the three three treatments treatments Experiment 2). Different superscripts (a, b) mean significant (p < 0.05) differences between treatments (PMN++SP, (PMN PMN SP, PMN + SP ++ SPFMLP and PMN + FMLP + SP ++DMSO) and PMN throughout SP + DMSO) the incubation throughout time (4 h; Experiment the incubation time (4 h; within a given time point, and different numbers (1, 2) mean significant (p < 0.05) differences between 2). Different superscripts Experiment 2). Different (a, b) mean significant superscripts (a, b) mean(p < 0.05) differences significant between (p < 0.05) treatments differences within between a given treatments incubation times within a given treatment. Data are shown as mean ± SEM for seven experiments. time point, and different numbers (1, 2) mean significant (p < 0.05) differences between incubation within a given time point, and different numbers (1, 2) mean significant (p < 0.05) differences between times Sperm concentration: 500 × 106 sperm/mL. PMN concentration: 100 × 106 PMN/mL. PMN: within a given treatment. Data are shown as mean ± SEM for seven experiments. incubation times within a given treatment. Data are shown as mean ± SEM for seven experiments. Sperm concentration: polymorphonuclear 6 sperm/mL. neutrophils; SP: seminal plasma; DMSO: dimethyl sulfoxide; FMLP: formyl- 6 PMN/mL. 500 × 10concentration: Sperm PMN500concentration: × 106 sperm/mL. 100 × 10 PMN PMN: polymorphonuclear concentration: 100 × 106 PMN/mL. neutrophils; PMN: methionyl-leucyl-phenylalanine. SP: seminal plasma; DMSO: polymorphonuclear dimethyl neutrophils; SP: sulfoxide; FMLP: formyl-methionyl-leucyl-phenylalanine. seminal plasma; DMSO: dimethyl sulfoxide; FMLP: formyl- methionyl-leucyl-phenylalanine. Figure 5. Percentages of viable spermatozoa bound to PMN via sperm tail in the three treatments (PMN Figure 5. Percentages of viable spermatozoa bound to PMN via sperm tail in the three treatments + SP, PMN + SP + FMLP and PMN + SP + DMSO) throughout the incubation time (4 h; Experiment 2). (PMN + SP, PMN + SP + FMLP and PMN + SP + DMSO) throughout the incubation time (4 h; Different Figure 5. superscripts Percentages (a, of b) meanspermatozoa viable < 0.05)todifferences significant (pbound PMN via between sperm tailtreatments within in the three a given treatments Experiment 2). Different superscripts (a, b) mean significant (p < 0.05) differences between treatments time and different numbers (1, 2) mean significant (p < (PMN + SP, PMN + SP + FMLP and PMN + SP + DMSO) throughout the incubation time times point, 0.05) differences between incubation (4 h; within a given time point, and different numbers (1, 2) mean significant (p < 0.05) differences between within a given Experiment 2).treatment. Data are shown Different superscripts asmean (a, b) ± SEM for (p meansignificant seven experiments. < 0.05) differencesSperm betweenconcentration: treatments incubation times within a given treatment. Data are shown as mean ± SEM for seven experiments. 500 × 10a6given within sperm/mL. PMNand time point, concentration: different numbers 6 PMN/mL. 100 × 10(1, PMN: polymorphonuclear 2) mean significant neutrophils; (p < 0.05) differences between Sperm concentration: 500 × 10 sperm/mL. PMN concentration: 100 × 106 PMN/mL. PMN: 6 SP: seminal times incubation plasma; DMSO: within dimethyl a given sulfoxide; treatment. DataFMLP: formyl-methionyl-leucyl-phenylalanine. are shown as mean ± SEM for seven experiments. polymorphonuclear neutrophils; SP: seminal plasma; DMSO: dimethyl sulfoxide; FMLP: formyl- Sperm concentration: 500 × 106 sperm/mL. PMN concentration: 100 × 106 PMN/mL. PMN: 2.3. Experiment 3: Evaluation of PMN-sperm Binding (Sperm Concentration) methionyl-leucyl-phenylalanine. polymorphonuclear neutrophils; SP: seminal plasma; DMSO: dimethyl sulfoxide; FMLP: formyl- methionyl-leucyl-phenylalanine. Sperm-PMN Binding Since results from Experiment 2 showed no significant effects from the presence of FMLP or DMSO, the evaluation of how different sperm concentrations (100 × 106 , 250 × 106 , 500 × 106 , and 1000 × 106 spermatozoa/mL) affected sperm-PMN binding only took into consideration the treatments that did not contain either FMLP or DMSO. Percentages of spermatozoa attached to PMN were significantly (p < 0.05) higher in the treatment containing 1000 × 106 spermatozoa/mL than in those containing lower sperm concentrations, at 0 h Since results from Experiment 2 showed no significant effects from the presence of FMLP or DMSO, the evaluation of how different sperm concentrations (100 × 106, 250 × 106, 500 × 106, and 1,000 × 106 spermatozoa/mL) affected sperm-PMN binding only took into consideration the treatments that did not contain either FMLP or DMSO. Int. J. Mol. Sci. 2020, 21, 3478 7 of 16 Percentages of spermatozoa attached to PMN were significantly (p < 0.05) higher in the treatment containing 1,000 × 106 spermatozoa/mL than in those containing lower sperm concentrations, at 0 h and and 11 h.h. However, these these differences differences between between sperm sperm concentrations concentrations were not not observed observed after after 22 hh of of incubation. incubation. Regardless of sperm concentration, sperm:PMN concentration, sperm:PMN ratios decreased along the incubation decreased along the incubation time, time, reaching reachingthe thelowest lowestlevels levelsafter after33hhof ofincubation incubation(Figure (Figure6). 6). With With regard regard to to sperm sperm viability, viability,percentages percentagesof ofviable viablespermatozoa spermatozoa ranged ranged from from 30% 30% to to 50% 50% on on average, without much variation throughout the incubation time (Figure 7). average, without much variation throughout the incubation time (Figure 7). While samples with the While samples with the highest highest sperm sperm concentration concentration (1000(1,000××10 1066 spermatozoa/mL) spermatozoa/mL) showed showed the the highest highest percentage percentage ofof viable viable spermatozoa spermatozoa attached attached to to PMN PMN at at 00 hh and and 11 h, no significant significant differences differences with with the the other other concentrations concentrations were wereobserved observedafter after22hhof ofincubation. incubation. Figure 6.6. Number of spermatozoa bound to PMN Figure PMN (sperm:PMN (sperm:PMN ratio) ratio) after after incubation incubation with with four four different 6 ,6250 × 106 ,6500 × 106 ,6 and 1000 × 106 spermatozoa/mL) × ×1010 differentsperm spermconcentrations concentrations(100 (100 , 250 × 10 , 500 × 10 , and 1,000 × 106 spermatozoa/mL) forfor 4 h4 (Experiment 3). Different superscripts (a, b) mean significant (p < 0.05) differences h (Experiment 3). Different superscripts (a, b) mean significant (p < 0.05) differences between between treatments within a given treatments time point, within a givenandtime different point,numbers (1, 2) mean and different significant numbers < 0.05)significant (1, 2)(pmean differences(pbetween < 0.05) incubation differences times within between a given times incubation treatment. withinData are shown a given treatment. Data ±are as mean SEM for seven shown experiments. as mean ± SEM for PMN sevenconcentration: experiments. PMN 106 PMN/mL. 100 × 106 PMN/mL. 100 ×concentration: 3. Discussion 3. Discussion Endometrial inflammation in the jenny after insemination was previously investigated in vitro Endometrial inflammation in the jenny after insemination was previously investigated in vitro using uterine flushing collected at 6 h post-AI [22]. Although an enriched PMN fraction from that fluid using uterine flushing3 collected at 6 h post-AI [22]. Although an enriched PMN fraction from that was obtained (100 × 10 cells/mL on average) in the aforementioned study, continued uterine washings fluid was obtained (100 × 103 cells/mL on average) in the aforementioned study, continued uterine can detrimentally impact the female reproductive tract and, in turn, cause infertility. Since the Catalan washings can detrimentally impact the female reproductive tract and, in turn, cause infertility. Since donkey is an endangered breed and in order to avoid the aforementioned undesirable side effect, the Catalan donkey is an endangered breed and in order to avoid the aforementioned undesirable the first aim of the current work was to set an in vitro model by isolating PMN from the peripheral side effect, the first aim of the current work was to set an in vitro model by isolating PMN from the blood. From the seven protocols tested, one (which was designated as Protocol 7) clearly showed the peripheral blood. From the seven protocols tested, one (which was designated as Protocol 7) clearly highest showedrecovery PMN the highest recovery PMN ≥ rate, yielding rate, × 103 cells/mL. 100yielding ≥ 100 ×As10distinct from 3 cells/mL. Asour results, distinct other from protocols our results, tested in other species [24,25], including the equine [26,27], were proven to be unable to isolate other protocols tested in other species [24,25], including the equine [26,27], were proven to be unable enough PMN fromenough to isolate the peripheral PMN from blood. the peripheral blood. In vitro experiments evaluating the activity of PMN in the horse previously used different activating agents, such as formyl-methionyl-leucyl-phenylalanine (FMLP) or recombinant equine interleukin 8 (reqIL-8) [26]. In the current study, while the whole semen (including spermatozoa and SP) was found to be able to stimulate PMN, the presence of FMLP did not alter the percentages of spermatozoa bound to PMN, nor was the viability of free and bound-to-PMN sperm populations over the incubation time affected. These data are in line with previous reports conducted in donkeys, in which SP was found to modulate sperm-PMN binding and sperm motility [22]. On the other hand, the decrease in sperm survival throughout the incubation time was independent from the presence of PMN. While the viability of the sperm population attached to PMN at 0 h was higher using FMLP, perhaps as a result of a fast PMN stimulation, no differences between treatments and over incubation Int. J. Mol. Sci. 2020, 21, 3478 8 of 16 were observed. This suggests that the viability of the sperm population attached to PMN is higher than thatSci. Int. J. Mol. of 2020, the unattached (free) 21, x FOR PEER sperm population. REVIEW 8 of 16 Figure 7. Figure 7. Percentages of viable spermatozoa spermatozoa boundbound to PMN after incubation incubation with four different different sperm sperm concentrations (100 × 10 66, 250 × 1066 , 500 × 1066 , and 1000 × 1066 spermatozoa/mL) for 4 h (Experiment 3). concentrations (100 × 10 250 × , 500 × 10 , and 1,000 × 10 spermatozoa/mL) for 4 h (Experiment Different Differentsuperscripts superscripts(a, (a,b) b) mean mean significant significant (p (p << 0.05) differences differences between between treatments treatments within within aa given given time timepoint, point,and anddifferent differentnumbers numbers(1, (1, 2) mean significant 2) mean (p < (p significant 0.05) differences < 0.05) between differences incubation between times incubation within a given atreatment. times within Data are shown given treatment. Data areas mean shown ± SEM for seven as mean ± SEMexperiments. for seven PMN concentration: experiments. PMN 100 × 106 PMN/mL. concentration: 100 × 10 PMN/mL. 6 Remarkably, In vitro experimentswe observed that few evaluating spermatozoa the activity of PMN appeared to be previously in the horse phagocytosed usedby PMN, different since while many attached the PMN surface, only some sperm activating agents, such as formyl-methionyl-leucyl-phenylalanine (FMLP) or recombinant equine cells were found to be within ainterleukin stained halo surrounding 8 (reqIL-8) [26]. Inless thecolored currentPMN.study,While while one could semen the whole not completely (includingexclude spermatozoathat there and was non-observed SP) was found to be phagocytosis, able to stimulate which PMN,wouldtheneed further presence specific of FMLP did studies not alterincluding time-lapse the percentages of microscopy, spermatozoait boundwas very to apparent PMN, nor that wasan the important viability proportion of free and of spermatozoa bound-to-PMN remained sperm surrounding populations PMN. over thePolymorphonuclear incubation time affected. neutrophils These aredatathearefirst barrier in line within front ofreports previous pathogens, via phagocytosis conducted in donkeys, or in which SP was found to modulate sperm-PMN binding and sperm motility [22]. On(CAT), through releasing their own DNA, histones, and enzymes, including cathepsin the other elastase hand, (ELA) and myeloperoxidase (MPO). In the latter case, this creates the decrease in sperm survival throughout the incubation time was independent from the presencecomplexes known as neutrophil extracellular of PMN. While traps the(NETs; viabilityNETosis), whichpopulation of the sperm prevent theattached dissemination to PMN of at pathogens [28–32]. 0 h was higher According using FMLP, to Branzk et al. [33], whether PMN trigger phagocytosis or NETosis perhaps as a result of a fast PMN stimulation, no differences between treatments and over incubation depends on the size of the pathogen; in effect, while the contact of bacteria with PMN induces were observed. This suggests that the viability of the sperm population attached to PMN is higher phagocytosis, the presence of larger pathogenic than that cells (such(free) of the unattached as thesperm yeast population. ones) activates NETosis. The sperm:PMN binding observed herein Remarkably, we observed that few different warrants further research on two spermatozoa realms. On theto appeared one behand, studies on phagocytosed bythe potential PMN, since ligand/receptor mechanism through which PMN triggers phagocytosis while many attached the PMN surface, only some sperm cells were found to be within a stained halo upon sperm:PMN binding are required to surrounding confirm less colored our reduced PMN. Whilepercentages one could not of sperm phagocytosis completely exclude by that PMN. thereOn wasthe other hand, non-observed and phagocytosis, which would need further specific studies including time-lapse microscopy,further because the formation of NETs was reported on in humans and other species [20,34–37], it was research on whether and how NETs are formed when PMN very apparent that an important proportion of spermatozoa remained surrounding PMN. and sperm are incubated in the donkey is much warranted. NETs Polymorphonuclear are known neutrophils are tothetrap mammalian first barrier in spermatozoa front of pathogens,in the vicinity of PMNs (i.e., via phagocytosis or without direct contact), which results in a significant loss of sperm motility through releasing their own DNA, histones, and enzymes, including cathepsin (CAT), elastase (ELA) [34]. For this reason, we suggest that in a similar(MPO). and myeloperoxidase fashionInto the that latter investigated case, thisin other createsspecies [31,32,38], complexes knownadditional studies as neutrophil focused upon staining of DNA and PMN enzymes should be extracellular traps (NETs; NETosis), which prevent the dissemination of pathogens [28–32].conducted. Remarkably, addressing this issue istoofBranzk According high relevance et al. [33],inwhether this context, PMNsincetriggerthephagocytosis dynamics of or sperm:PMN NETosis depends bindingon observed the size in our study suggests that sperm phagocytosis could be inhibited, which, of the pathogen; in effect, while the contact of bacteria with PMN induces phagocytosis, the presence as indicated by Branzk et al. [33], would lead to the activation of NETosis. Indeed, deficiencies of larger pathogenic cells (such as the yeast ones) activates NETosis. The sperm:PMN binding in the activity of phagocytic receptors observed werehereinsuggested warrants to disrupt further the size-dependent research on two different selectivity realms.of OnNET-release the one hand, duestudies to inefficient on the phagocytosis. Moreover, blocking of Dectin-1, a phagocytic receptor potential ligand/receptor mechanism through which PMN triggers phagocytosis upon sperm:PMN present in human PMN, with abinding specific are antibody required is known to leadour to confirm to lower reduced rates of phagocytosis percentages of spermand higher NETosisby phagocytosis response. PMN. On Based the on the low percentages of phagocytosis, we suggest that seminal plasma other hand, and because the formation of NETs was reported on in humans and other species [20,34– factors could inhibit PMN 37], further research on whether and how NETs are formed when PMN and sperm are incubated in the donkey is much warranted. NETs are known to trap mammalian spermatozoa in the vicinity of PMNs (i.e., without direct contact), which results in a significant loss of sperm motility [34]. For this reason, we suggest that in a similar fashion to that investigated in other species [31,32,38], additional Int. J. Mol. Sci. 2020, 21, 3478 9 of 16 receptors involved in the induction of phagocytosis, which would lead to the activation of NETosis in a similar fashion to that reported by Branzk et al. [33] when Dectin-1 is inhibited. In the present study, we observed that a significant number of spermatozoa attached to PMN were viable and showed tail beating. In addition, not only were the percentages of viable spermatozoa higher in the bound-to-PMN than in the free sperm population, but sperm cells that were apparently released from PMN after 3 h of incubation also showed good motility. Moreover, we also noticed that during the first hours of incubation, spermatozoa tended to attach PMN through the tail rather than through the head. Since we do not know whether this observation has any relationship with the sperm ability to release from PMN, future studies should also contemplate, through time-lapse microscopy, the binding dynamics between spermatozoa and PMN and the relevance of each sperm compartment. All these data suggest that the attachment of spermatozoa to PMN does not always result in the clearance of the formers, but this apparent transient binding could play an important role in the donkey reproduction strategy, since it is quite likely that sperm cells released from PMN are able to fertilize. At the beginning of the experiment, percentages of spermatozoa bound to PMN were significantly higher in the treatment containing the highest sperm concentration (1000 × 106 spermatozoa/mL). This could be explained by the higher likelihood of a given PMN to interact with a given spermatozoon, as sperm concentrations increase. However, it is worth noting that these differences in sperm:PMN binding observed between sperm concentrations were not found after 3 h of incubation. In addition, whereas both sperm:PMN ratios and percentages of viable spermatozoa decreased throughout incubation, percentages of spermatozoa attached to PMN increased over successive incubation times. These data indicate that PMN could be progressively activated throughout incubation, thereby attaching a higher number of spermatozoa. Nevertheless, percentages of viable spermatozoa attached to PMN (approximately 50%) showed no variations between sperm concentrations and incubation times. This suggests that the higher the sperm concentration, the higher the possibility of observing free spermatozoa after 3 h and 4 h of incubation. This hypothesis would be in agreement with our CASA observations, as only concentrations higher than 500 × 106 sperm/mL showed free motile spermatozoa at the end of the experiment. Oliveira et al. observed an increase in fertility rates after inseminating jennies with high concentrated sperm doses [9]. Since jackasses usually show good semen quality with higher sperm concentration and motility than in stallions [1,3], one could suggest that this better sperm quality could play an important physiologic role for the post-breeding endometrial reaction in the jenny. Indeed, as explained before, high sperm concentrations with good motility appear to be required in order for spermatozoa to be released from PMN after binding. However, an important question herein is whether spermatozoa that ultimately fertilize the oocyte belong to the sperm population that are released from PMN. Therefore, future studies should also focus on the relationship between PMN and spermatozoa at post-breeding in the donkey. 4. Materials and Methods 4.1. Animals Three Catalonian jackasses, aged 5–11 years, and three Catalonian jennies, aged 5–8 years, were used in this study. All animals were in good health conditions and of proven fertility. While conducting experiments, they were housed at the experimental farm, Faculty of Veterinary Science, Autonomous University of Barcelona (Bellaterra, Cerdanyola del Vallès; Spain). Jackasses were maintained in individual paddocks, whereas jennies were grouped in a big paddock. Animals were fed grain forage, straw and hay, and water was provided ad libitum. The study was approved by the Ethics Committee, Autonomous University of Barcelona (Bellaterra, Cerdanyola del Vallès, Spain; Code: CEEAH 1424, 23.04.2018). Int. J. Mol. Sci. 2020, 21, 3478 10 of 16 4.2. Experiment 1. Isolation of Donkey PMN from Peripheral Blood Peripheral blood was collected from three different jennies via jugular venipuncture with 10 mL BD Vacutainer® tubes containing 18.0 mg of Ethylenediaminetetraaceticacid (EDTA; BD-Plymouth, UK). Eight tubes were collected per jenny and protocol, and seven protocols were tested. Samples were protected from the light, and immediately transported to the laboratory at room temperature within the following 10 min. To isolate PMN and adjust the concentration to 100 × 103 PMN/mL, different protocols used in other species were evaluated. Each protocol was replicated seven times. 4.2.1. Protocol 1 This protocol was based on the method set by Baumber et al. [26] to isolate equine neutrophils. Briefly, whole blood samples were centrifuged at 200× g and room temperature for 15 min. Supernatants (blood plasma) were discarded and pellets containing leukocytes and erythrocytes (2.5 mL) were layered onto four tubes containing 2.5 mL of 59% and 2.5 mL of 75% Percoll solutions (Sigma-Aldrich® , Merck KGaA, Darmstadt, Germany). Samples were centrifuged at 400× g and room temperature for 20 min. Neutrophils were collected from the interface of the 59/75% Percoll gradient, resuspended in 5 mL PBS and centrifuged at 200× g and room temperature for 10 min. Pellets were resuspended in 1 mL PBS and samples were analyzed through a hematological cytometer (ADVIA 120 Siemens Medical Solutions; Fernwald, Germany) to determine the concentration of PMN per mL. 4.2.2. Protocol 2 This protocol was based on the method set by Siemsen et al. [25] designed to isolate non-human neutrophils. Blood samples were collected in tubes containing EDTA and then incubated at 37 ◦ C in a water bath for 30 min, which was the time required for red blood cells to stack. The leukocyte-rich plasma fraction was aspirated and layered onto four tubes containing 2.5 mL of 70% and 2.5 mL of 85% Percoll solutions. Samples were centrifuged at 400× g and room temperature for 20 min. Neutrophils were collected from the interface of the 70/85% Percoll gradient, resuspended in 2 mL PBS and washed twice at 200× g for 10 min with PBS. The resulting pellets were resuspended in 1 mL PBS and then analyzed through a hematological cytometer (ADVIA 120 Haemathology System, Siemens Healthcare GmbH, Erlangen, Germany) to determine the concentration of PMN per mL. 4.2.3. Protocol 3 The third protocol was based on that of Roth and Kaeberle, which was set to isolate bovine PMN [24]. In brief, blood samples collected with vacutainer tubes were gathered in 15-mL conical tubes and centrifuged at 400× g for 15 min. The leukocyte layer was aspirated with some plasma and placed onto 4 mL of Ficoll (Hystopaque 1077® , Sigma-Aldrich® , Merck KGaA, Darmstadt, Germany); samples were centrifuged at 300× g and room temperature for 30 min. Supernatants were discarded and the white layer just below the Ficoll one was aspirated and transferred into a clean tube. Samples were added with two volumes of distilled water, mixed and incubated at room temperature for 5–7 s to lyse the residual erythrocytes. Following this, samples were immediately mixed in an equal volume of 1.7% NaCl solution to restore isotonicity, and subsequently centrifuged at 250× g for 10 min. Supernatants were discarded and pellets were resuspended in 1 mL of Lactate Ringer (B Braun, Rubí, Barcelona, Spain). Resuspended pellets were analyzed using a hematological cytometer (ADVIA 120 Siemens Medical Solutions) to determine the concentration of PMN per mL. 4.2.4. Protocol 4 This protocol was based on that from Loftus et al. [27], which aims at isolating equine PMN. Blood samples were cooled down and maintained at 4 ◦ C until visibly sedimented, which lasted for approximately 20 min. The leukocyte rich plasma layer was transferred into a new tube and centrifuged at 250× g and 4 ºC for 10 min. Thereafter, the pellet was resuspended in 40 mL of ice-cold 0.9% NaCl, Int. J. Mol. Sci. 2020, 21, 3478 11 of 16 and the resulting cell suspension was layered onto 10 mL Hystopaque 1077® (Sigma- Aldrich® ) and centrifuged at 300× g and room temperature for 40 min. The liquid layer was discarded and the pellet was resuspended in 4 mL ice-cold PBS. Following this, 1 mL of the resulting cell suspension was layered onto 9 mL of 90% Percoll and centrifuged at 650× g and room temperature for 40 min. The neutrophil fraction was collected, mixed with 4 mL PBS, and centrifuged at 300× g for 8 min. Pellets were subsequently resuspended in 1 mL PBS, and then analyzed with a hematological cytometer (ADVIA 120 Siemens Medical Solutions) to determine the concentration of PMN per mL. 4.2.5. Protocol 5 The fifth protocol was a modification from Protocol 1 which as previously mentioned (see Section 4.2.1), was based on Loftus et al. [27]. In brief, blood samples were collected in tubes containing EDTA. Samples were incubated in a water bath at 37 ◦ C for 30 min to allow red blood cells to stack, forming rouleaux. The leukocyte-rich plasma layer was subsequently aspirated, transferred into 15-mL conical tubes, and centrifuged at 402× g and 4 ◦ C for 5 min. Cells were resuspended with 4 mL ice-cold PBS and then centrifuged at 250× g and 4 ◦ C for 5 min. Resulting pellets were resuspended in 500 µL ice-cold PBS containing 2% paraformaldehyde, and maintained on ice for 1 h. Thereafter, cells were centrifuged and resuspended in 500 µL PBS, and the resulting sample was analyzed with a hematological cytometer (ADVIA 120 Siemens Medical Solutions) to determine the concentration of PMN per mL, prior to adjusting the concentration to 100 × 106 PMN/mL in ice-cold PBS. 4.2.6. Protocol 6 This protocol consisted of a modification of Protocol 2, which was based on that of Loftus et al. [27] for isolating equine PMN (see Section 4.2.2). With this purpose, blood samples were collected in tubes containing EDTA and then incubated in water bath at 37 ◦ C for 30 min to allow red blood cells to stack. The leukocyte-rich plasma layer was aspirated, transferred into 15-mL conical tubes and centrifuged at 402× g and 4 ◦ C for 5 min. Samples were resuspended in 4 mL ice-cold PBS and then centrifuged at 250× g and 4 ◦ C for 5 min. Thereafter, cells were resuspended in 500 µL ice-cold PBS, and then analyzed with a hematological cytometer (ADVIA 120 Siemens Medical Solutions) to determine the concentration of PMN per mL. Cell concentration was adjusted to 100 × 106 PMN/mL in ice-cold PBS. 4.2.7. Protocol 7 Blood samples were collected in tubes containing EDTA and incubated in a water bath at 37 ◦C for 30 min, to stack red blood cells. The leukocyte-rich plasma layer was aspirated, transferred into 15-mL conical tubes, and subsequently centrifuged at 402× g and 4 ◦ C for 5 min (modified from Loftus et al. [27]). The supernatant was aspirated and discarded; the pellet was resuspended in 4 mL ice-cold PBS, and centrifuged at 402× g and 4 ◦ C for 5 min. Next, the supernatant was aspirated and discarded, and the upper white layer of the pellet, which contained PMN, was collected and transferred into a 1.5-mL tube. Samples were analyzed with a hematological cytometer (ADVIA 120 Siemens Medical Solutions) to determine the concentration of PMN per mL. Cell concentration was adjusted to 100 × 106 PMN/mL in ice-cold PBS. 4.3. Experiments 2 and 3: Effects of SP and Sperm Concentration on PMN-sperm Binding 4.3.1. Semen Collection and Process Semen was collected with an artificial vagina (Hannover model) equipped with an in-line filter (Minitüb Ibérica SL; Reus, Spain) to get a gel-free semen sample. Semen volume was recorded and diluted 1:1 (v:v) with a skim-milk-based semen extender [39]. Sperm concentration was evaluated using a Neubauer chamber (Paul Marienfeld GmbH & Co. KG; Lauda-Königshofen, Germany), and four treatments containing different sperm concentrations (100 × 106, 200 × 106, 500 × 106, and 1000 × 106 sperm/mL) were prepared. Int. J. Mol. Sci. 2020, 21, 3478 12 of 16 4.3.2. Treatments Eleven treatments containing different sperm concentrations, PMN, FMLP and DMSO were prepared, as follows. In the case of Experiment 2, which tested the role of FMLP activation on PMN [26], these treatments were: (a) PMN + 100 × 106 sperm/mL + SP + FMLP 0.1 mM; (b) PMN+ 200 × 106 sperm/mL + SP + FMLP 0.1 mM; (c) PMN + 500 × 106 sperm/mL + SP+ FMLP 0.1 mM; (d) PMN + 1000 × 106 sperm/mL + SP + FMLP 0.1 mM; (e) PMN + 500 × 106 sperm/mL + SP + DMSO; (f) 500 × 106 sperm/mL + SP + DMSO; and (g) control (500 × 106 sperm/mL + SP sample). Treatments g and f were the controls for DMSO, which was the vehicle for FMLP dilution. Treatment g was the control for sperm viability and motility throughout incubation. In the case of Experiment 3, which aimed at assessing the effects of different sperm concentration on sperm:PMN binding, we tested the following treatments: (a) PMN + 100 × 106 sperm/mL + SP; (b) PMN + 200 × 106 sperm/mL + SP; (c) PMN + 500 × 106 sperm/mL + SP; and (d) PMN + 1000 × 106 sperm/mL + SP. All semen samples were incubated in a water bath at 37 ◦ C, and their viability, sperm-PMN binding and motility evaluated after 0 h, 1 h, 2 h, 3 h and 4 h of incubation. 4.3.3. Evaluation of Sperm Viability Sperm viability was determined through eosin-nigrosin staining [40]. A minimum of 200 spermatozoa/ sample were examined under a bright-field, optical microscope (Olympus Europe, Hamburg, Germany) at 1000 × magnification. We recorded percentages of viable spermatozoa (eosin-negative), and those of viable spermatozoa attached to PMN. 4.3.4. Sperm–PMN Binding Sperm–PMN attachment was determined as previously described [20,41]. In brief, 10 µL of each sample was placed onto a slide prior to mixing with Diff-Quick stain (QCA, Amposta, Spain) and smearing [22]. A minimum of 200 sperm cells were counted under a bright-field, optical microscope at 1000 × magnification. The following three parameters were recorded: (a) percentages of spermatozoa attached to PMN; (b) sperm:PMN ratio (number of spermatozoa attached per PMN); and (c) percentages of spermatozoa attached to PMN either via their head or their tail. 4.3.5. Sperm Motility Sperm motion characteristics in the free (unattached) sperm population were evaluated by means of a computer assisted sperm analysis (CASA) system (Integrated Semen Analysis System, ISAS® Ver.1.0.15; Projects and Services R+D SL, Proiser; Valencia, Spain). This system consisted of a negative phase-contrast microscope (Olympus BH-2) with a yellow light filter and a warm-up plate, and a digital video camera (Basler, Ahrensburg Germany) connected to a computer containing the ISAS software. Samples were placed into a pre-warmed Neubauer chamber (37 ◦ C) and examined at 1000 × magnification, with at least 200 spermatozoa being counted per analysis. 4.4. Statistical Analyses A statistical package (IBM SPSS Statistics 25.0; Armonk, New York, NY, USA) was used to analyze the results obtained in this work. Shapiro-Wilk and Levene tests were conducted to assess data distribution and homogeneity of variances, respectively. When required, data were transformed through √ arcsine x. Following this, one-way ANOVA (experiment 1) or a linear mixed model (experiments 2 and 3) followed by post-hoc Sidak test for pair-wise comparisons were run. The treatment was considered to be the fixed-effects factor, the donkey was the random-effects factor and the incubation time was the intra-subjects factor. When, even after linear transformation, data did not show normal distribution and/or variances were not homogenous, non-parametric alternatives (Kruskal-Wallis and Mann-Whitney tests for Experiment 1; and Friedman and Wilcoxon tests for Experiments 2 and 3) were Int. J. Mol. Sci. 2020, 21, 3478 13 of 16 used. The level of significance was set at p ≤ 0.05, and data are shown as mean ± standard error of the mean (SEM). 5. Conclusions In conclusion, the whole semen, including seminal plasma, activates PMN and induces sperm-PMN binding in jennies. However, we observed low percentages of phagocytosis upon sperm:PMN binding, and not only were the percentages of viable spermatozoa higher in the bound-to-PMN than in the free sperm population, but sperm cells that were apparently released from PMN after 3 h of incubation also showed good motility. Therefore, the fact that sperm previously attached to PMN could be later released, especially when concentration was at least 500 × 106 spermatozoa/mL, suggests that this binding does not necessarily lead to the induction of phagocytosis. For this reason, and together with the fact that NETosis has been extensively reported on in other mammalian species, further research should be aimed at elucidating: (a) through which ligand/receptor spermatozoa and PMN bind; (b) whether the contact between spermatozoa and PMN in the presence of seminal plasma always triggers phagocytosis or rather some sperm cells remain fertile upon releasing from PMN, and (c) if, in the donkey, clearance of spermatozoa through PMN also occurs via NETosis rather than phagocytosis. In the latter case, while one would expect that this could also be the case, the previously reported variations between other species along with the differences related to the semen deposition site support the need for specific experiments in the donkey. Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/10/3478/s1, Supplementary File S1. Movie showing sperm: PMN interaction (sperm concentration: 500 × 106 spermatozoa/mL; PMN concentration: 100 × 106 PMN/mL). It is noticeable that most sperm bound to the PMN surface exhibited high tail activity. Author Contributions: Conceptualization, J.M.; methodology, J.M., H.M., J.C., M.P., S.G. and M.Y.; validation, J.M.; formal analysis, J.M., J.C.; M.P. and M.Y.; investigation, J.M.; H.M., J.C., M.P., S.G. and M.Y.; resources, J.M. and M.Y.; data curation, J.M., H.M. and J.C.; writing—original draft preparation, J.M.; writing—review and editing, M.Y.; supervision, J.M.; project administration, J.M.; funding acquisition, J.M. and M.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Ministry of Science and Innovation, Spain (Grants: RYC-2014-15581 and AGL2017- 88329-R), and the Regional Government of Catalonia (2017-SGR-1229). J.C. was funded by the National Commission of Scientific Research and Technology (CONICYT), Ministry of Education, Chile (Scheme: Becas para Estudios de Postgrado en Chile y en el Extranjero, PFCHA; Grant: 2017/72180128). Acknowledgments: The authors would like to thank the support from Sebastián Bonilla-Correal from Autonomous University of Barcelona, Spain for his technical support. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations AI Artificial insemination ALH Amplitude of lateral head displacement BCF Frequency of head displacement CASA Computer-Assisted Sperm Analysis CAT Cathepsin DMSO Dimethyl sulfoxide EDTA Ethylenediaminetetraacetic acid ELA Elastase FMLP Formyl-methionyl-leucyl-phenylalanine LIN Linearity Int. J. Mol. Sci. 2020, 21, 3478 14 of 16 MPO Myeloperoxidase NETs Neutrophil Extracellular Traps PBS Phosphate buffered saline PFA Paraformaldehyde PMN Polymorphonuclear Neutrophils SEM Standard error of the mean SP Seminal plasma STR Straightness VAP Average path velocity References 1. Canisso, I.F.; Panzani, D.; Miró, J.; Ellerbrock, R.E. Key Aspects of Donkey and Mule Reproduction. Vet. Clin. N. Am. Equine Pract. 2019, 35, 607–642. [CrossRef] 2. 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