veterinary sciences Article Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm Iván Yánez-Ortiz 1,2,3,† , Jaime Catalán 1,2,3,4,† , Ariadna Delgado-Bermúdez 2,3 , Augusto Carluccio 4 , Jordi Miró 1, *,‡ and Marc Yeste 2,3, *,‡ 1 Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, ES-08193 Barcelona, Cerdanyola del Vallès, Spain; ivan.yanez22@gmail.com (I.Y.-O.); dr.jcatalan@gmail.com (J.C.) 2 Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain; ariadna.delgado@udg.edu 3 Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, ES-17003 Girona, Spain 4 Faculty of Veterinary Medicine, University of Teramo, Loc. Piano d’Accio, IT-64100 Teramo, Italy; acarluccio@unite.it * Correspondence: jordi.miro@uab.cat (J.M.); marc.yeste@udg.edu (M.Y.); Tel.: +34-93-5814293 (J.M.); +34-972-419514 (M.Y.) † These authors contributed equally to this work. ‡ These authors share senior authorship. Abstract: In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which   further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm Citation: Yánez-Ortiz, I.; Catalán, J.; plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly Delgado-Bermúdez, A.; Carluccio, A.; found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this Miró, J.; Yeste, M. Addition of fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. Reduced Glutathione (GSH) to The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects Freezing Medium Reduces caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of Intracellular ROS Levels in Donkey different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) Sperm. Vet. Sci. 2021, 8, 302. https:// to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and doi.org/10.3390/vetsci8120302 motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane Academic Editor: Bartosz Kempisty integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2 − ) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM Received: 5 November 2021 or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels Accepted: 30 November 2021 Published: 2 December 2021 compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity Publisher’s Note: MDPI stays neutral suggests that ROS could be important during post-AI and that the impact of using exogenous with regard to jurisdictional claims in antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species. published maps and institutional affil- iations. Keywords: sperm cryopreservation; reactive oxygen species (ROS); superoxide anion (O2 − ); reduced glutathione (GSH); antioxidants; donkey Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article The cryopreservation of genetic material is a milestone for reproductive biotechnology, distributed under the terms and not only in humans but also in other vertebrates [1], since it may be applied together with conditions of the Creative Commons other assisted reproductive technologies (ART) for the dissemination of genes of genetically Attribution (CC BY) license (https:// superior animals [2], as well as for the conservation of biodiversity and the protection of creativecommons.org/licenses/by/ endangered species [3]. This last aspect is very relevant in the donkey because the size 4.0/). Vet. Sci. 2021, 8, 302. https://doi.org/10.3390/vetsci8120302 https://www.mdpi.com/journal/vetsci Vet. Sci. 2021, 8, 302 2 of 17 of the population has suffered a significant decrease in recent years [4], which directly affects the genetic variability of the existing breeds [5]. Research in this species has focused its efforts on improving sperm cryopreservation protocols, precisely to try to safeguard endangered donkey breeds [6–18]. However, the use of cryopreserved donkey sperm has been limited by the low conception rates obtained when jennies are inseminated [7,8,19]. This, which contrasts with the reproductive performance of mares, is largely due to the physiological endometritis that occurs in the jenny after artificial insemination (AI) [20,21]. In addition to the above, cryopreservation is known to induce freezing injuries in sperm due to their exposure to thermal and osmotic stresses and their high sensitivity to an oxidative environment, which leads to subsequent lipid peroxidation in the plasma mem- brane [22]. In the particular case of donkey sperm, and in a similar fashion to other species, this occurs because the plasma membrane contains a large amount of polyunsaturated fatty acids esterified into phospholipids [23]. Oxidative stress can be defined as a consequence of the imbalance between the cellular antioxidant defense system and the production of reactive oxygen species (ROS). Physiologically, sperm release ROS as a by-product of their metabolism; these ROS are scavenged by enzymatic and non-enzymatic antioxidants which play an instrumental role in maintaining the redox balance [24]. Both types of antioxidants, whose function is to prevent oxidative damage, are present in sperm or in seminal plasma (SP) [25]. In this context, it is worth mentioning that Papas et al. [26] showed that the antioxidant capacity of the enzymes present in the SP is significantly higher in donkeys than in horses. In spite of this, the sperm cryopreservation protocols currently applied in donkeys include the elimination of SP by centrifugation [27]. This makes sperm more susceptible to functional damage with lethal consequences [28] and lead them to undergo structural injuries, mainly at the chromatin level, which has a significant impact on male fertility [29]. In addition, it must be considered that, in equids, the presence of sperm during AI causes a powerful endometrial reaction with a large influx of polymorphonu- clear neutrophils (PMN) into the uterine lumen [20,30–32]. These polymorphonuclear neutrophils act mainly via degranulating/extruding their DNA and bactericidal molecules (histones and enzymes), thereby forming neutrophil extracellular traps (NETs) that capture sperm without killing them [33]. Therefore, sperm:PMN binding in donkey activates one of the defense mechanisms of polymorphonuclear neutrophils, NETosis [34], which releases a large amount of ROS. These ROS levels are much greater than those generated by sperm [35] and even by the polymorphonuclear neutrophils themselves [36]. An alternative to counteract the negative effect that ROS generates on sperm from different mammalian species, including the human [37], is the addition of reduced glu- tathione (GSH) to the preservation medium in both cooling [38–41] and freezing [42–46]. Reduced glutathione is one of the most relevant non-enzymatic antioxidants in mammalian cells, and most of its scavenging effect results from the interaction with glutathione re- ductase (GSR) and glutathione peroxidase (GPX) [47]. Reduced glutathione contains a sulfhydryl group (-SH) that protects the disulfide bonds between chromatin fibers from the oxidative damage caused by ROS, thus maintaining the sperm nuclear structure during cryopreservation [47–49]. Despite the antioxidant properties of GSH to maintain the redox balance during cryopreservation, GSH concentrations greater than 2.5 mM have been found to be toxic to horse sperm when added to the freezing extender, as there is a significant reduction in the motility, viability and plasma membrane integrity after thawing [43]. The same effect has been observed in pigs, as the addition of 1 mM GSH to the freezing medium results in greater protection of sperm function than higher concentrations [41]. In donkeys, there is only one study that investigated the effects of supplementing the freezing medium with GSH; however, only one concentration was tested [16]. Testing the resilience of donkey sperm to oxidative stress using different GSH concentrations could shed some light into the reproductive strategy of this species [50–52]. In addition, supplementing freezing media with GSH could improve donkey sperm function and survival after thawing, which would help sperm to fight against the oxidative environment they encounter when reaching the Vet. Sci. 2021, 8, 302 3 of 17 jenny uterus after AI [20]. Therefore, the objective of this study was to evaluate how different GSH concentrations added to the freezing medium affect the cryotolerance of donkey sperm. 2. Materials and Methods 2.1. Donkeys and Sperm Samples A total of 12 ejaculates from 4 adult Catalonian jackasses (age: between 3 and 6 years old) were used. Animals were clinically healthy and of proven fertility (good fertility rates), and were maintained in individual paddocks at the Equine Reproduction Service, Autonomous University of Barcelona (Bellaterra, Cerdanyola del Vallès, Spain). This is a center for the collection of semen from equids that holds an EU approval (authorization number: ES09RS01E) and operates under rigorous protocols which include the control of the health and welfare of animals. Thus, it was not necessary to receive a specific approval from the Ethics Committee of the Autonomous University of Barcelona (Bellaterra, Cerdanyola del Vallès, Spain) to handle animals, since the four jackasses were semen donors. Complying with the sanitary guidelines established by the Council of the European Communities in Directive 82/894/EEC of 21 December 1982, animals were free of equine viral arteritis, equine infectious anemia and equine contagious metritis. Sperm collection was performed on a regular schedule in the morning through an artificial vagina (Hannover model; Minitüb GmbH, Tiefenbach, Germany) with the use of an in-line nylon filter to remove the gel fraction. Once the ejaculate was obtained, it was immediately diluted 1:5 (v:v) in a skim milk-based extender [53], prewarmed to 37 ◦ C. The quality of fresh sperm was checked through the analysis of concentration (Neubauer chamber; Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany), motility (CASA system; see Section 2.5 for details), and viability and morphology (eosin-nigrosin staining; see Section 2.6 for details). All samples were confirmed to be above the standard thresholds before freezing: viable sperm ≥85%, morphologically normal sperm ≥70%, and total motile sperm ≥80%. 2.2. Experimental Design Before cryopreservation, each diluted sperm sample was centrifuged at 600× g and 20 ◦C for 15 min (Medifriger BL-S; JP Selecta SA, Barcelona, Spain); the supernatant was discarded and the pellet was resuspended in a commercial freezing medium containing glycerol and methylformamide as permeable cryoprotectants (BotuCRIO; Botupharma Animal Biotechnology, Botucatu, Brazil). Subsequently, sperm concentration and viability were again analyzed, and the same freezing medium (BotuCRIO) was added to obtain a final concentration of 200 × 106 viable sperm/mL (normalized in all cases). The final volume was divided into 6 aliquots of 3 mL each, and a different concentration of reduced glutathione (GSH; G4251; Saint Louis, MO, USA; final concentrations: 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) was added; the control was not supplemented with GSH. A total of 6 separate 0.5 mL plastic straws were filled with the content of each aliquot (sperm sample with its respective treatment). Each straw was manually labeled with the freezing date, jackass name and treatment as follows: T1 = sperm (control); T2 = sperm + 2 mM GSH; T3 = sperm + 4 mM GSH; T4 = sperm + 6 mM GSH; T5 = sperm + 8 mM GSH; and T6 = sperm + 10 mM GSH. 2.3. Sperm Cryopreservation All straws were cryopreserved in parallel using an automatic controlled-rate freezer (Ice-Cube 14S; Minitüb GmbH, Tiefenbach, Germany). Cooling/freezing was carried out in 3 stages: (1) cooling from 20 ◦ C to 5 ◦ C for 60 min at a rate of −0.25 ◦ C/min, (2) freezing from 5 ◦ C to −90 ◦ C for 20 min at a rate of −4.75 ◦ C/min, and (3) freezing from −90 ◦ C to −120 ◦ C for 2.7 min at a rate of −11 ◦ C/min. Finally, straws were plunged into liquid nitrogen at −196 ◦ C and stored in appropriate tanks for their conservation until analysis. Vet. Sci. 2021, 8, 302 4 of 17 2.4. Sperm Thawing Frozen sperm straws were thawed by incubation at 37 ◦ C for 30 s in a hot water bath. The content of each straw was poured into a 10-mL conical tube for post-thaw sperm analysis. For each treatment and sperm sample, one straw was used for the assessment of motility (CASA system; see Section 2.5 for more details) and viability (eosin-nigrosin staining; see Section 2.6 for more details), and the other two were utilized for the evaluation of sperm parameters through flow cytometry (see Section 2.7 for details). 2.5. Sperm Motility Analysis Sperm motility was evaluated by means of a CASA-Mot module of the ISAS v1.2 sys- tem (Proiser R + D, Valencia, Spain) equipped with a high-resolution digital camera model MQ003MG-CM (Proiser R + D, Valencia, Spain) capable of capturing up to 100 frames per second (fps). Before doing so, the content of each straw was diluted 1:2 (v:v) in a cooling extender based on skim milk prewarmed to 38 ◦ C [53,54]. Briefly, 2 µL of each sperm sample was placed into a reusable Spermtrack10 chamber (Spk 10; Proiser R + D, Valencia, Spain), prewarmed to 38 ◦ C. A minimum of 500 spermatozoa were counted per analysis under a 10× negative phase contrast microscope model UOP200i (Proiser R + D, Valencia, Spain). Total (TM, %) and progressive sperm motility (PM, %) were evaluated together regarding the following kinematic parameters: curvilinear velocity (VCL, µm/s), straight line velocity (VSL, µm/s), average path velocity (VAP, µm/s), linearity coefficient (LIN = [VSL/VCL] × 100, %), straightness coefficient (STR = [VSL/VAP] × 100, %), wob- ble coefficient (WOB = [VAP/VCL] × 100, %), amplitude of lateral head displacement (ALH, µm), and beat-cross frequency (BCF, Hz). The CASA-Mot settings were those rec- ommended by the manufacturer: particle area > 4 and <75 µm2 , connectivity: 6, minimum number of images to calculate ALH: 10. VAP ≥ 10 µm/s and STR ≥ 75% were used as cut-off values for motile sperm and progressively motile sperm, respectively. Three technical replicates were examined. 2.6. Sperm Viability Analysis Sperm viability was analyzed by eosin-nigrosin staining [55]. Briefly, 10 µL of the content of each sperm sample was placed on a slide which was previously preheated to 37 ◦ C. Immediately afterwards, 10 µL of the eosin-nigrosin stain was placed onto the sperm sample and mixed; the mixture was smeared. A bright field light microscope (Carl Zeiss, Göttingen, Germany) at 1000× magnification using immersion oil objective was used to evaluate a minimum of 200 sperm/sample. The percentage of viable sperm (eosin negative) was recorded and three technical replicates were evaluated. 2.7. Evaluation of Sperm through Flow Cytometry The sperm functional parameters evaluated through flow cytometry were plasma membrane integrity (SYBR-14/PI), acrosome membrane integrity (PNA-FITC/PI), mito- chondrial membrane potential (MMP, JC-1), intracellular ROS levels (H2 DCFDA/PI), and intracellular superoxides levels (HE/YO-PRO-1). Fluorochromes were purchased from Molecular Probes (Thermo Fisher Scientific, Waltham, MA, USA). Sperm concentration was adjusted to 1 × 106 sperm/mL before staining. For each sample, a total of 10,000 events were analyzed, and 3 technical replicates were evaluated. A CytoFLEX flow cytometer (Beckman Coulter Fullerton, CA, USA) was used with a sheath flow rate set at 10 µL/min. Samples were excited with an argon ion laser (488 nm) at a power of 50 mW. Cell diameter/volume was assessed through the Coulter principle. To exclude cell aggregates and debris, distributions of two different dot plots were used. Cell aggregates were excluded on the basis of forward scatter height (FSC-H) and altitude (FSC-A) dot plot distribution, whereas subcellular debris were excluded based on the distribution of FSC-A and side scatter altitude (SSC-A) dot plots. A total of 3 different optical filters were used: FITC was used for the analysis of SYBR-14, PNA-FITC, JC-1 monomers (JC-1mon ), DCF+ and YO-PRO-1 (band pass: 525–540 nm); PE for Vet. Sci. 2021, 8, 302 5 of 17 the analysis of JC-1 aggregates (JC-1agg ) and E+ (band pass: 585–542 nm), and PC5.5 for PI analysis (band pass: 690–650 nm). Information about each event (FSC-A, FSC-H, SSC-A, FITC, PE, and PC5.5) was collected in xit files and quantification from each sperm population was obtained using the CytExpert analysis software (Beckman Coulter Fullerton, CA, USA). The analyses were performed following the recommendations of the International Society for the Advancement of Cytometry (ISAC) [56]. For each parameter, the corresponding mean and standard error of the mean (SEM) were calculated. 2.7.1. Plasma Membrane Integrity Analysis (SYBR-14/PI) The analysis of plasma membrane integrity was carried out following the protocol described by Garner and Johnson [57], adapted to donkey sperm, using the LIVE/DEAD sperm viability kit (SYBR-14/PI). Briefly, sperm samples were incubated in the dark at 38 ◦ C with SYBR-14 (final concentration: 31.8 nM) for 10 min and PI (final concentration: 7.6 µM) for a further 5 min. Three sperm populations were identified: (1) sperm with an intact plasma membrane, green-stained (SYBR-14+ /PI− ), (2) sperm with a damaged plasma membrane, red-stained (SYBR-14− /PI+ ), and (3) sperm with a damaged plasma membrane, both green- and red-stained (SYBR-14+ /PI+ ). The unstained sperm (SYBR-14− /PI− ) were considered as non-sperm particles (debris) and were used to correct the data in the other evaluations. The spill over of SYBR-14 in the PC5.5 channel (8.34%) was compensated. 2.7.2. Acrosome Membrane Integrity Analysis (PNA-FITC/PI) The integrity of the acrosome membrane was evaluated following the protocol de- scribed by Rathi et al. [58] adapted to donkey sperm using PNA-FITC and PI. Briefly, sperm samples were incubated in the dark at 38 ◦ C with FITC-conjugated PNA (final concentra- tion: 1.17 µg/mL) and PI (final concentration: 5.6 µm) for 10 min. Four sperm populations were identified: (1) sperm with an intact plasma membrane (PNA-FITC− /PI− ), (2) sperm with a damaged plasma membrane that presented an acrosomal membrane that could not be completely intact (PNA-FITC+ /PI+ ), (3) sperm with a damaged plasma membrane and missing outer acrosomal membrane (PNA-FITC− /PI+ ), and (4) sperm with a damaged plasma membrane (PNA-FITC+ /PI− ). Data were not compensated. 2.7.3. Mitochondrial Membrane Potential (MMP) Analysis (JC-1) The MMP analysis was performed following the protocol described by Ortega-Ferrusola et al. [59] adapted to donkey sperm using JC-1 iodide (5.50 , 6,60 -tetrachloro- 1,10 , 3,30 tetraethylbenzimidazolylcarbocyanine). In brief, sperm samples were incubated in the dark at 38 ◦ C for 30 min with JC-1 (final concentration: 750 nM). In the presence of low MMP, JC-1 molecules remain as green-fluorescent monomers (JC-1mon ), whereas in the presence of high MMP, they form orange-fluorescent aggregates (JC-1agg ). Two sperm pop- ulations were distinguished: (1) low MMP sperm (cells presenting a JC-1mon fluorescence intensity higher than a JC-1agg fluorescence intensity) and (2) high MMP sperm (cells pre- senting a JC-1agg fluorescence intensity higher than a JC-1mon fluorescence intensity). Data were not compensated. The fluorescence intensity of JC-1mon and JC-1agg was recorded in all sperm populations, and the ratio between JC-1agg and JC-1mon was calculated. 2.7.4. Intracellular Reactive Oxygen Species (ROS) Levels Analysis: H2 O2 (H2 DCFDA/PI) and O2 − (HE/YO-PRO-1) The analysis of the intracellular levels of ROS was carried out following the protocol described by Morrell et al. [60] and Guthrie and Welch [61], adapted to donkey sperm. The combination of 20 , 70 -dichlorodihydrofluorescein diacetate (H2 DCFDA) and PI was used to measure overall intracellular ROS levels, and the combination of hydroetidine (HE) and YO-PRO-1 was used to measure intracellular levels of superoxide anion (O2 − ). For ROS evaluation, sperm samples were incubated in the dark at 38 ◦ C with H2 DCFDA (final concentration: 50 µM) for 20 min; thereafter, PI (final concentration: 6 µM) was added and samples were incubated for 5 min. In the presence of ROS, H2 DCFDA is de-esterified Vet. Sci. 2021, 8, 302 6 of 17 and oxidized into the highly fluorescent molecule DCF+ . Therefore, four sperm populations were identified: (1) viable sperm with high ROS levels (DCF+ /PI− ), (2) non-viable sperm with high ROS levels (DCF+ /PI+ ), (3) viable sperm with low ROS levels (DCF− /PI− ), and (4) non-viable sperm with low ROS levels (DCF− /PI+ ). Data were not compensated. The fluorescence intensity of DCF+ was recorded in all sperm populations. For the measurement of the intracellular levels of O2 − , sperm samples were incubated in the dark at 38 ◦ C with HE (final concentration: 5 µM) and with YO-PRO-1 (final concentration: 31.25 nM) for 30 min. In the presence of O2 − , HE is oxidized into fluorescent ethidium (E+ ). This allowed the observation of four sperm populations: (1) viable sperm with high O2 − levels (E+ /YO-PRO-1− ), (2) non-viable sperm with high O2 − levels (E+ /YO- PRO-1+ ), (3) viable sperm with low·O2 − levels (E− /YO-PRO-1− ), and (4) non-viable sperm with low O2 − levels (E− /YO-PRO-1+ ). The spill over of E+ into the FITC channel (3.62%) was compensated. The fluorescence intensity of E+ was recorded in all sperm populations. 2.8. Statistical Analyses Statistical analyses were performed with the R statistical package (V 4.0.3, R Core Team; Vienna, Austria) and graphs were plotted with GraphPad Prism (V 8.4.0, GraphPad Software LLC; San Diego, CA, USA). The Shapiro–Wilk test was run to verify the normal distribution of data and the Levene test checked the homoscedasticity. When necessary, √ data were transformed with arcsin x to match with parametric assumptions. When these assumptions were not met, a non-parametric analysis was performed using the Kruskal–Wallis test followed by the Wilcoxon test (p ≤ 0.05) for pairwise comparisons. The effects of the different GSH concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) on motility, viability (eosin-nigrosin), plasma membrane integrity (SYBR14+ /PI− ), acrosome membrane integrity (PNA-FITC− /PI− ), MPP (JC-1agg ), intracellular H2 O2 lev- els (DCF+ /PI− ), and intracellular O2 − levels (E+ /YO-PRO-1− ), as well as the geometric mean of fluorescence intensity (GMFI) of JC-1agg , DCF+ and E+ after thawing, were ana- lyzed using a one-way ANOVA, followed by the Bonferroni post-hoc test (p ≤ 0.05) for pairwise comparisons. Motile sperm subpopulations were obtained by applying the procedure described by Martí et al. [62]. Briefly, a principal component analysis (PCA) was performed from the kinematic parameters (VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF) of each sperm cell after thawing. The matrix obtained was rotated using the Varimax method with Kaiser nor- malization, where each spermatozoon was assigned a regression score. These values were used to perform a non-hierarchical, multivariate cluster analysis using the k-means model based on Euclidean distances. Finally, the proportion of spermatozoa in each subpopulation was calculated to analyze the effects of the different GSH concentrations through a one-way ANOVA, followed by the Bonferroni post-hoc test (p ≤ 0.05) for pairwise comparisons. In all analyses, the results are expressed as means ± SEM. 3. Results 3.1. Sperm Motility The percentage of total motile sperm at post-thaw (Figure 1A) was significantly lower in the treatments containing 8 mM GSH (29.36% ± 6.88%) and 10 mM GSH (7.85% ± 3.10%) than in the control (62.56% ± 4.81%) and those containing 2 mM GSH (59.18% ± 5.42%) and 4 mM GSH (57.20% ± 5.50%). The concentration of 6 mM GSH (47.81% ± 6.36%) only differed from that of 10 mM (p < 0.05). In addition, the percentage of progres- sively motile sperm (Figure 1B) decreased significantly when GSH concentrations of 8 mM (9.76% ± 3.95%) and 10 mM (1.65% ± 0.96%) were added, compared to the control (34.05% ± 4.61%), 2 mM GSH (37.20% ± 5.04%) and 4 mM GSH (31.41% ± 5.30%). The concentration of 6 mM (27.14% ± 5.70%) was only different from that of 10 mM (p < 0.05). than in the control (62.56% ± 4.81%) and those containing 2 mM GSH (59.18% ± 5.42%) and 4 mM GSH (57.20% ± 5.50%). The concentration of 6 mM GSH (47.81% ± 6.36%) only dif- fered from that of 10 mM (p < 0.05). In addition, the percentage of progressively motile sperm (Figure 1B) decreased significantly when GSH concentrations of 8 mM (9.76% ± Vet. Sci. 2021, 8, 302 3.95%) and 10 mM (1.65% ± 0.96%) were added, compared to the control (34.05% ± 4.61%), 7 of 17 2 mM GSH (37.20% ± 5.04%) and 4 mM GSH (31.41% ± 5.30%). The concentration of 6 mM (27.14% ± 5.70%) was only different from that of 10 mM (p < 0.05). (A) (B) Figure 1. Mean Mean±±SEM SEM of of thethe total (A)(A) total andand progressive (B) (B) progressive motility of frozen-thawed motility of frozen-thaweddonkey sperm donkey following sperm cryopreser- following cryop- vation with with reservation different reduced different glutathione reduced (GSH) glutathione concentrations. (GSH) (a–c)(a–c) concentrations. Different letters Different indicate letters significant indicate differences significant (p ≤ differences 0.05) between the control and GSH treatments. (p ≤ 0.05) between the control and GSH treatments. On On the the other other hand, hand, the the kinematic kinematic parameters parameters ofof frozen-thawed frozen-thawed donkey donkey sperm sperm re-re- mained mained without withoutsignificant significantchanges changesatatconcentrations concentrations ≤ 6≤mM 6 mMGSHGSHcompared comparedto thetocon- the trol (Table control 1). Indeed, (Table VCL,VCL, 1). Indeed, VAP,VAP, WOB and and WOB ALHALH werewere significantly lower significantly in theintreatment lower the treat- containing 10 mM10than ment containing mMin the in than control (p < 0.05), the control whereas (p < 0.05), VSL, LIN, whereas VSL, STR, LIN, and STR,BCFandwere BCF lower than the were lower than control in those the control having in those 8 mM8and having mM10 mM and 10GSH (p < 0.05). mM GSH (p < 0.05). Table 1. Table 1. Mean Mean ±±SEM SEMofofthe kinematic the parameters kinematic of of parameters frozen-thawed donkey frozen-thawed sperm donkey following sperm cryopreservation following withwith cryopreservation dif- ferent reduced glutathione (GSH) concentrations. different reduced glutathione (GSH) concentrations. GSH GSH Concentration Concentration Parameter Parameter Control Control 2 mM 2 mM 4 mM 4 mM 66 mM mM 88 mM mM 10 10 mM mM VCL (µm/s) 134.66 ± 8.53 aa 152.04 ± 6.74 aa 139.16 ± 7.43 aa 138.69 ± 9.47 aa 114.97 ± 13.76 aa 57.03 ± 18.80 bb VCL (µm/s) 134.66 ± 8.53a 152.04 ± 6.74a 139.16 ± 7.43a 138.69 ± 9.47a 114.97 ± 13.76b 57.03 ± 18.80b VSL (µm/s) 53.16 ± 4.55 60.51 ± 4.09 52.71 ± 4.11 50.47 ± 5.20 30.14 ± 4.76 13.53 ± 5.08 VSL (µm/s) 53.16 ± 4.55 aa 60.51 ± 4.09 aa 52.71 ± 4.11 aa 50.47 ± 5.20 aa 30.14 ± 4.76 ab 13.53 ± 5.08 bb VAP (µm/s) 69.42 ± 4.75 a 76.22 ± 3.75 a 70.92 ± 2.98 a 68.26 ± 4.51 a 53.05 ± 5.95 a 27.23 ± 8.54 b VAP (µm/s) 69.42 ± 4.75 a 76.22 ± 3.75 a 70.92 ± 2.98 a 68.26 ± 4.51 a 53.05 ± 5.95 b 27.23 ± 8.54 c LIN (%) 37.02 ± 1.20 37.96 ± 1.26 35.59 ± 1.29 33.96 ± 1.67 23.06 ± 2.61 10.98 ± 3.75 LIN (%) 37.02 ± 1.20 a 37.96 ± 1.26 a 35.59 ± 1.29 a 33.96 ± 1.67 a 23.06 ± 2.61 b 10.98 ± 3.75 c STR (%) STR (%) 70.07 ± 1.91 70.07 ± 1.91 a a 74.07 ± 2.09 74.07 ± 2.09 a a 68.06 ± 3.21 68.06 ± 3.21 a a 67.37 ± 3.21 67.37 ± 3.21 a a 47.72 ± 5.40 47.72 ± 5.40 b b 23.19 ± 23.19 ± 7.92 c 7.92 c WOB WOB (%) (%) 51.61 ± 0.39 51.61 ± 0.39 a a 50.65 ± 0.65 50.65 ± 0.65 a a 51.96 ± 1.50 51.96 ± 1.50 a a 49.95 ± 0.88 49.95 ± 0.88 a a 44.14 ± 4.51 44.14 ± 4.51 a a 24.70 ± 7.55 24.70 ± 7.55 b b ALH (µm) ALH (µm) 1.81 ± 0.08 1.81 ± 0.08 aa 2.06 ± 0.08 2.06 ± 0.08 aa 1.92 ± 0.10 1.92 ± 0.10 aa 1.93 ± 0.11 1.93 ± 0.11 aa 1.62 ± 0.18 1.62 ± 0.18 aa 0.83± 0.83 ± 0.27 b 0.27 b BCF (Hz) BCF (Hz) 28.23 ± 2.28 28.23 ± 2.28 aa 29.27 ± 2.19 29.27 ± 2.19 aa 25.84 ± 1.46 25.84 ± 1.46 aa 23.57 ± 1.91 23.57 ± 1.91 ab ab 15.19 ± 1.90 15.19 ± 1.90 bc bc 8.05 ± 2.52 8.05 ± 2.52 c c VCL (µm/s): curvilinear velocity; VSL (µm/s): straight line velocity; VAP (µm/s): average path velocity; LIN (%): linearity VCL (µm/s): curvilinear velocity; VSL (µm/s): straight line velocity; VAP (µm/s): average path velocity; LIN (%): linearity coefficient; coefficient; STR (%):coefficient; STR (%): straightness straightness coefficient; WOB (%): wobble WOB (%): wobble coefficient; coefficient; ALH (µm): ALH amplitude (µm):head of lateral amplitude of lateral displacement; BCFhead (Hz): displace- beat-cross ment; BCF(a–c frequency. (Hz): beat-cross ) Different lettersfrequency. (a–c) Different indicate significant letters differences (p ≤ indicate significant 0.05) between differences the control and GSH (p ≤ 0.05) between the control treatments. and GSH treatments. 3.2. Motile Sperm Subpopulations Four motile sperm subpopulations were identified in frozen-thawed donkey sperm (Table 2). Subpopulation 1 (SP1) was characterized by being the fastest (higher values of VCL, VSL, and VAP) and most progressive (high values of LIN, STR, ALH and BCF, although WOB was intermediate). Subpopulation 2 (SP2) was the slowest and least progressive, with the lowest ALH and BCF values. Subpopulation 3 (SP3) presented intermediate velocity and progressivity values similar to those of SP1, with an intermediate ALH and a BCF similar to SP1. Finally, subpopulation 4 (SP4) was also characterized by presenting intermediate speed, progressivity values, ALH and BCF. Although these parameters were lower than in SP1 and SP3, SP4 exhibited the highest WOB. The proportions of motile spermatozoa of SP2 identified in frozen-thawed donkey sperm (Figure 2) were significantly higher in the presence of 8 mM (53.65% ± 7.10%) and 10 mM GSH (79.04% ± 9.01%) than in that of 2 mM GSH (24.56% ± 3.37%). On the contrary, SP3 reduced its proportion in the presence of 8 mM (12.88% ± 3.34%) and 10 mM VCL (µm/s) 265.51 ± 0.81 197.90–429.30 69.32 ± 0.35 0.00–147.30 198.68 ± 0.35 127.50–276.00 134.67 ± 0.32 VSL (µm/s) 115.39 ± 0.54 10.80–191.30 15.79 ± 0.13 0.00–54.70 83.56 ± 0.30 3.70–152.10 49.42 ± 0.22 VAP (µm/s) 131.57 ± 0.40 78.60–216.50 33.31 ± 0.19 0.00–91.90 99.91 ± 0.22 44.30–165.90 69.05 ± 0.17 LIN (%) 44.13 ± 0.23 3.90–75.30 24.06 ± 0.19 0.00–92.30 42.75 ± 0.17 1.50–95.60 38.03 ± 0.19 Vet. Sci. 2021, 8, 302 8 of 17 STR (%) 87.42 ± 0.27 7.70–100.00 48.01 ± 0.29 0.00–99.20 83.59 ± 0.23 3.70–100.00 71.71 ± 0.25 WOB (%) 50.09 ± 0.18 27.00–85.30 48.88 ± 0.17 0.00–100.00 50.80 ± 0.13 19.20–100.00 52.32 ± 0.14 ALH (µm) 3.36 ± 0.02 1.50–6.40 1.10 ±compared GSH (7.24% ± 4.66%) 0.00 0.00–2.80 to 2 mM GSH (29.87% 2.77%; p <0.80–4.00 2.61 ±±0.01 0.05). Similarly,1.83 the ± 0.01 BCF (Hz) 36.67 ± 0.31 0.00–73.50 14.39belonging proportion of sperm ± 0.10 to 0.00–52.40 35.29lower SP1 was significantly ± 0.19in the 0.00–72.10 28.39 ± 0.14 treatment containing n (%) 10 1776 (9.97%) mM GSH than in that with 2 mM 5609 (31.50%) GSH (0.72% ± 0.58% vs. 11.52% 4339 (24.36%) ± 2.28%), and the 6085 proportion of SP4-sperm was lower in the presence of 10 mM GSH (13.01% ± 4.92%) than in the control (34.05% ± 1.83%) and treatments with 2 mM (34.11% ± 1.42%), 4 mM (33.23%The proportions ± 1.94%), of motile and 6 mM GSH (29.67%spermatozoa ± 3.83%). of SP2 identified in frozen-th sperm (Figure 2) were significantly higher in the presence of 8 mM (53.65% Table 2. Structure of the four motile sperm subpopulations identified in frozen-thawed donkey sperm following cryopreser- vation with different reduced10 mM GSH glutathione (79.04% (GSH) ± 9.01%) than in that of 2 mM GSH (24.56% ± 3.37%). O concentrations. SP3 reduced its proportion in the presence of 8 mM (12.88% ± 3.34%) an SP1 SP2 SP3 SP4 Parameter Mean ± SEM (7.24% Range ± Mean 4.66%)± SEMcompared Range to Mean 2 mM ± SEM GSH Range(29.87% Mean ± 2.77%; ± SEM p < Range 0.05). Similar VCL (µm/s) 265.51 ± 0.81 197.90–429.30 69.32 ± 0.35 0.00–147.30 198.68 ± 0.35 127.50–276.00 134.67 ± 0.32 66.50–218.70 VSL (µm/s) 115.39 ± 0.54 tion of sperm 10.80–191.30 belonging 15.79 ± 0.13 to SP183.56 0.00–54.70 was ± 0.30significantly 3.70–152.10 lower in the1.80–105.00 49.42 ± 0.22 treatment con VAP (µm/s) 131.57 ± 0.40 78.60–216.50 33.31 ± 0.19 0.00–91.90 99.91 ± 0.22 44.30–165.90 69.05 ± 0.17 25.10–123.90 LIN (%) 44.13 ± 0.23 GSH than24.06 3.90–75.30 in±that 0.19 with 2 mM GSH 0.00–92.30 (0.72%1.50–95.60 42.75 ± 0.17 ± 0.58% vs.38.03 ±11.52% 0.19 ± 2.28%), and 1.20–100.00 STR (%) 87.42 ± 0.27 7.70–100.00 48.01 ± 0.29 0.00–99.20 83.59 ± 0.23 3.70–100.00 71.71 ± 0.25 2.30–100.00 WOB (%) 50.09 ± 0.18 of SP4-sperm 27.00–85.30 was lower 48.88 ± 0.17 0.00–100.00in the50.80presence ± 0.13 of 10 mM52.32 19.20–100.00 GSH ± 0.14(13.01% ± 4.92%) th 17.40–100.00 ALH (µm) 3.36 ± 0.02 1.50–6.40 1.10 ± 0.00 0.00–2.80 2.61 ± 0.01 0.80–4.00 1.83 ± 0.01 0.40–3.50 BCF (Hz) 36.67 ± 0.31 trol (34.05% 0.00–73.50 ± 0.10 14.39 ± 1.83%)0.00–52.40 and treatments 35.29 ± 0.19 with 2 mM (34.11% 0.00–72.10 28.39 ± 0.14 ± 1.42%), 0.00–70.804 mM (33 n (%) 1776 (9.97%) and 6 mM GSH (29.67% ± 3.83%). 4339 (24.36%) 5609 (31.50%) 6085 (34.17) Figure Figure 2. Distribution 2. Distribution of the of the four four motile spermmotile sperm subpopulations subpopulations (SP) identified in (SP) identified in frozen-thawed donkey sperm following cryopreservation with different concentrations donkey sperm following cryopreservation with different concentrations of reduced glutathioneof redu (GSH). (a–c) Different letters indicate significant differences (p ≤ 0.05) between the control and (GSH). (a–c) Different letters indicate significant differences (p ≤ 0.05) between the c GSH treatments. treatments. 3.3. Sperm Viability No significant differences in the viability of frozen-thawed donkey sperm (evaluated with eosin-nigrosin staining) between the different GSH concentrations and the control were found (Figure 3). Vet. Sci. 2021, 8, x FOR PEER REVIEW 3.3. Sperm Viability 9 of 17 No significant differences in the viability of frozen-thawed donkey sperm Vet. Sci. 2021, 8, 302 9 of 17 with eosin-nigrosin staining) between the different GSH concentrations and 3.3. Sperm Viability were found (Figure 3). No significant differences in the viability of frozen-thawed donkey sperm (evaluated with eosin-nigrosin staining) between the different GSH concentrations and the control were found (Figure 3). Figure 3. Mean ± SEM of the viability of frozen-thawed donkey sperm following cryopreservation with different Figure Figure 3. Mean reduced 3. Mean ± SEM glutathione ± SEM (GSH) of the of the viability concentrations. viability No significant of frozen-thawed of frozen-thawed donkey differences donkey sperm followingcry (p ≤ 0.05) sperm be- following cryopreservation tween the control and GSH treatments were found. with different reduced glutathione (GSH) concentrations. No significant differences (p ≤ 0.05) with different reduced glutathione (GSH) concentrations. No significant differences between tween the thecontrol controlandand GSHGSH treatments were found. treatments were found. 3.4. Sperm Quality 3.4. Sperm 3.4.1. Sperm Quality Membrane Integrity 3.4.No 3.4.1. Sperm Sperm Quality Membrane significant Integrity differences in the percentage of frozen-thawed donkey sperm with intactNo significant plasma differences membrane (SYBR14in+/PI the−)percentage were foundofbetween frozen-thawed donkey the control sperm and the with different 3.4.1. Sperm Membrane Integrity + /PI− ) were found between the control and the different intact plasma membrane (SYBR14 GSH concentrations (Figure 4A). No significant GSH concentrations (Figuredifferences 4A). in the percentage of frozen-thawed donkey intact plasma membrane (SYBR14+/PI−) were found between the control and GSH concentrations (Figure 4A). (A) (B) Figure +/PI ; A)−and Figure 4. 4. Mean Mean ±±SEM SEMofofthe thepercentages percentagesofofsperm spermwith anan with intact plasma intact membrane plasma membrane (SYBR14 (SYBR14 + −/PI ; (A))with andan intact with an acrosome membrane (PNA-FITC −/PI−; B) − observed − in frozen-thawed donkey sperm after cryopreservation intact acrosome membrane (PNA-FITC /PI ; (B)) observed in frozen-thawed donkey sperm after cryopreservation with different with GSH concentrations. No significant differences (p ≤ 0.05) between the control and GSH treatments were found. different GSH concentrations. No significant differences (p ≤ 0.05) between the control and GSH treatments were found. 3.4.2. 3.4.2. Acrosome Acrosome Membrane Membrane Integrity Integrity Similarly, no significant differences Similarly, no significant differences in the in the percentage percentage of frozen-thawed of frozen-thawed donkeydonkey sperm (A) sperm with anwith intactanacrosome intact acrosome membrane membrane (PNA-FITC − − (PNA-FITC − /PI ) were/PI observed − ) were observed(B) betweenbetween the the different different GSH concentrations GSH concentrations and the(Figure and the control control4B). (Figure 4B). Figure 4. Mean ± SEM of the percentages of sperm with an intact plasma membrane (SYBR14 /PI ; A) and with + − acrosome membrane (PNA-FITC −/PI−; B) observed in frozen-thawed donkey sperm after cryopreservation with 3.4.3. Mitochondrial Membrane Potential (MPP) GSH concentrations. No significant differences No significant (p ≤ 0.05) differences in between the control the percentage and GSH treatments of frozen-thawed were donkey sperm found. with high-MMP (JC-1agg ) were seen between the different GSH concentrations and the control (Figure 3.4.2. 5A). Similarly,Membrane Acrosome the GMFI of Integrity JC-1agg in the high-MMP sperm population did not differ from the control when GSH was added to the freezing medium (Figure 5B). Similarly, no significant differences in the percentage of frozen-thaw sperm with an intact acrosome membrane (PNA-FITC−/PI−) were observed different GSH concentrations and the control (Figure 4B). 3.4.3. Mitochondrial Membrane Potential (MPP) 3.4.3. Mitochondrial Membrane Potential (MPP) No significant differences in the percentage of frozen-thawed donkey sperm with No significant high-MMP (JC-1agg) differences in the percentage were seen between the differentof GSH frozen-thawed donkey concentrations andsperm with the control Vet. Sci. 2021, 8, 302 high-MMP (JC-1 agg) were seen between the different GSH concentrations and the control (Figure 5A). Similarly, the GMFI of JC-1agg in the high-MMP sperm population did10not of 17 (Figure 5A). Similarly, the GMFI of JC-1 agg in the high-MMP sperm population did not differ from the control when GSH was added to the freezing medium (Figure 5B). differ from the control when GSH was added to the freezing medium (Figure 5B). (A) (B) (A) (B) Figure 5. Mean ± SEM of the percentage of spermatozoa with high mitochondrial membrane potential (high-MMP; higher Figure Figure5.5.Mean proportion Mean ± SEM of JC-1±aggSEMof of thantheJC-1 percentage the percentage of of mon) (A) and spermatozoa spermatozoa geometric with high with mean mitochondrial high mitochondrial fluorescence membrane intensity of JC-1aggpotential membrane (high-MMP; potential (GMFI, channel) higher (high-MMP; PE inhigher the proportion sperm proportion ofofJC-1 population agg than with JC-1 agg than JC-1 high-MMP mon) (B) JC-1 mon (A) ) and (A) andgeometric observed in geometric mean mean fluorescence frozen-thawed donkey intensity sperm fluorescence of JC-1 following intensity of agg JC-1 (GMFI, PE channel) cryopreservation agg (GMFI, PE with in the differ- channel) in the sperm ent sperm population GSHpopulation with concentrations. high-MMP with No (B) significant high-MMP observed in (B) differences frozen-thawed observed in(pfrozen-thawed ≤ 0.05) betweendonkey sperm the control donkey spermandfollowing cryopreservation GSH treatments following with were found. cryopreservation differ- with different ent GSH concentrations. No significant differences (p ≤ 0.05) between the control and GSH treatments were found. GSH concentrations. No significant differences (p ≤ 0.05) between the control and GSH treatments were found. 3.4.4. Intracellular Reactive Oxygen Species (ROS) Levels: H2O2 and O2− 3.4.4. 3.4.4.Intracellular Intracellular Reactive ReactiveOxygen Oxygen Species Species (ROS) Levels: HH 2O2 and O2− − The GSH concentrations of 8 mM and 10(ROS)mM Levels: significantly 2 O2(p and O2 decreased the < 0.05) The GSH The GSH percentage concentrations concentrations of frozen-thawed of 8 mM of 8 mM donkey and sperm 10 andwith mM 10 mM significantly highsignificantly ROS levels (DCF (p < 0.05) /PI−)decreased (p < +0.05) decreasedthe compared the to percentage percentage the control (9.33%ofoffrozen-thawed ± 1.91% anddonkey frozen-thawed 9.36% ±sperm donkey 1.81%with sperm with high high ROS vs. 19.72% ROS levels (DCF /PI+ + levelsrespectively; ± 3.13%, (DCF /PI) compared − − ) Figure compared toto 6A). the thecontrol control Exposure to(9.33% (9.33% ±± any GSH 1.91% 1.91% and and9.36% 9.36%±also concentration 1.81% ± 1.81% vs.vs. reduced19.72%(p <±0.05) 19.72% 3.13%, ± 3.13%,therespectively; respectively; GMFI of DCF Figure + in6A). Figure 6A). the Exposure + − to to any any GSH GSH concentration concentration also alsoreduced reduced (p(p< < 0.05) DCF /PI sperm population (147,217.30 ± 12,735.15 in 2 mM GSH, 132,642.2 ± 5966.27 inthe Exposure 0.05) the the GMFI GMFI ofof DCF DCF + +in the in 4 DCF +/PI − sperm − sperm population (147,217.30 ± 12,735.15 in 2inmM ±GSH, 132,642.2 ± 5966.27 in 4in DCF + /PI population (147,217.30 ± 12,735.15 2 mM GSH, 132,642.2 ±GSH, 5966.27 mM GSH, 145,301.20 ± 9617.71 in 6 mM GSH, 130,855.70 8405.67 in 8 mM and mM4 mM GSH, 133,857.40 GSH, ±145,301.20 in± 10 145,301.20 9184.45 9617.71 ±mM 9617.71in 6inmM GSH) 6with mM GSH, GSH, respect130,855.70 130,855.70 ± 8405.67 to the control ± 8405.67 in in 8 mM (259,537.708 mM ± GSH, GSH,and 49,296.13;and 133,857.40 133,857.40 Figure 6B). ± ±9184.45 9184.45in 10 in mM 10 mM GSH) GSH) with with respect respect totothe the control control (259,537.70 (259,537.70 ± ±49,296.13; 49,296.13; FigureOn6B). Figure 6B). the other hand, no significant differences in either the percentage of frozen- On On thawed donkey the theother other hand, hand, sperm withnohigh·O no significant significant differences differences 2− levels in either in either (E+/YO-PRO-1 thethe −) or theGMFIpercentage percentage of of of frozen- E+ frozen-thawed in the E+/YO- thawed donkey donkey sperm sperm with with high · O − levels high·O 2 − levels (E + /YO-PRO-1 (E +/YO-PRO-1 − ) −or ) or the the GMFI GMFI of of EE++ in the the EE++/YO- /YO- PRO-1− sperm population were 2 observed between the different GSH concentrations and PRO-1 PRO-1 − sperm − sperm population population were were observed observed between between thethedifferent different GSHGSH concentrations concentrations and and the control (Figure 6C,D). the thecontrol control(Figure (Figure6C,D). 6C,D). (A) (B) (A) (B) Figure 6. Cont. Vet.Sci. Vet. Sci.2021, 2021,8,8,x 302 FOR PEER REVIEW 11 11 of of 1717 (C) (D) Figure Figure6.6.Mean Mean± SEM ± SEM of the percentage of the of viable percentage sperm of viable with sperm highhigh with overall ROSROS overall levels (DCF levels +/PI−+ (DCF A),−geometric , /PI mean , (A)), geometric fluorescence intensity of DCF + (GMFI,+ FITC channel) in the DCF +/PI− sperm + population − (B), percentage mean fluorescence intensity of DCF (GMFI, FITC channel) in the DCF /PI sperm population (B), percentage of viable sperma- of viable tozoa with high·O spermatozoa − levels (E+/YO-PRO-1−, C), and geometric mean fluorescence intensity of E+ (GMFI, channel PE) in the with2 high ·O2 − levels (E+ /YO-PRO-1− , (C)), and geometric mean fluorescence intensity of E+ (GMFI, channel E+/YO-PRO-1+− sperm population (D) observed in frozen-thawed donkey sperm following cryopreservation with different PE) in the E /YO-PRO-1− sperm population (D) observed in frozen-thawed donkey sperm following cryopreservation reduced glutathione (GSH) concentrations. (a,b) Different letters indicate significant differences (p ≤ 0.05) between the with different reduced glutathione (GSH) concentrations. (a,b) Different letters indicate significant differences (p ≤ 0.05) control and GSH concentrations in (A,B). No significant differences (p ≤ 0.05) between the control and GSH treatments in between (C,D) werethe control and GSH concentrations in (A,B). No significant differences (p ≤ 0.05) between the control and GSH found. treatments in (C,D) were found. 4. Discussion 4. Discussion The combination of the cellular component (sperm) produced by the germinal epi- The combination of the cellular component (sperm) produced by the germinal ep- thelium of the testes and the liquid component (SP) generated by the epididymis and the ithelium of the testes and the liquid component (SP) generated by the epididymis and the accessory sex glands [63,64] causes semen to become a complex redox system due to the accessory sex glands [63,64] causes semen to become a complex redox system due to the antioxidant action exerted mostly by SP and, to a lesser extent, by sperm, as well as by the antioxidant action exerted mostly by SP and, to a lesser extent, by sperm, as well as by the pro-oxidant action of sperm through ROS production [65]. This last action is more im- pro-oxidant action of sperm through ROS production [65]. This last action is more impor- portant during sperm cryopreservation in equids, because sperm cells lose the antioxidant tant during sperm cryopreservation in equids, because sperm cells lose the antioxidant support of SP before freezing. While intracellular GSH levels in horse sperm have been support of SP before freezing. While intracellular GSH levels in horse sperm have been quantified [66], it is not clear whether this produced amount is high enough for sperm to quantified [66], it is not clear whether this produced amount is high enough for sperm have the ability to have to scavenge the ability to scavenge the oxidative stress the oxidative generated stress generatedby ROS and thus by ROS maintain and thus re- maintain dox balance without the antioxidant support of SP. In the case of donkeys, redox balance without the antioxidant support of SP. In the case of donkeys, it has recently it has recently been beenshown shownthat thatsperm spermare arecapable capableofoftolerating toleratinghigh highROS ROSconcentrations concentrations(H (H2O 2) and that 2 O2 ) and that ininthe the presence of PMN, SP plays a decisive role in the regulation of the ROSproduced presence of PMN, SP plays a decisive role in the regulation of the ROS produced during duringNETosis NETosis[36]. [36].InInthis thisscenario, scenario, the the addition addition of of exogenous exogenous GSH GSHcouldcouldcompensate compensatein inaacertain certainwaywayfor forthe theantioxidant antioxidantcomponent componentthat thatsperm spermloselosewhen whencryopreserved. cryopreserved. The The first hypothesis of this study envisaged that adding GSH to thefreezing first hypothesis of this study envisaged that adding GSH to the freezingmediummedium would would increase the ability of donkey sperm to withstand cryopreservation.However, increase the ability of donkey sperm to withstand cryopreservation. However,our our results resultsindicate indicatethat thatsupplementation supplementationofofthe thefreezing freezingmedium mediumwith withGSHGSHatata aconcentration concentration asashigh highasas1010mM mM does does not notaffect affectthe viability, the viability,plasma plasma and and acrosome acrosome membrane membrane integrity, integrity, and andMMP MMPofofpost-thaw post-thawsperm. sperm.However, However,spermspermmotility motilitywaswasreduced reducedalmost almosttotozero.zero.This This surprising surprisingfinding findingininthe thedonkey donkeyprovides providesususwithwithvaluable valuableinformation informationtotounderstand understandthe the reproductive reproductivestrategy strategy in species,since in this species, sincethethe tolerance tolerance to GSH to GSH observed observed herein herein was was much much higher higher than than that reported that reported in horses in horses [43],[41], [43], pigs pigssheep [41], sheep [42], [45], [42], cattle cattleand[45], and[44]. dogs dogsIn [44]. In effect, in those species, adding high concentrations of GSH effect, in those species, adding high concentrations of GSH to the freezing medium results to the freezing medium results in a significant in a significant decrease decrease in all in all the the aforementioned aforementioned parameters parameters of post-thaw of post-thaw sperm; sperm; in fact, ininfact, some in cases, some cases, these hightheseconcentrations high concentrations have evenhavebeenevenreported been reported to be cytotoxic to be cytotoxic [43]. [43]. Regarding the motility of frozen-thawed donkey sperm, we observed a significant effect Regarding of high the motility ofon GSH concentrations frozen-thawed TM and PM,donkey sperm, weinobserved which decreased the presence a significant of 8 mM effect (29.36% ± 6.88% of high GSH and 9.76% ± 3.95%, concentrations on TM and PM, which respectively) and 10 decreased mM GSHin(7.85% ± 3.10%ofand the presence 8 mM1.65% ± 0.96%, (29.36% respectively) ± 6.88% and 9.76% with±respect 3.95%,torespectively) and 10±mM the control (62.56% 4.81%GSH 34.05%±±3.10% and(7.85% 4.61%, respectively). and 1.65% ± 0.96%, Interestingly, respectively)frozen-thawed with respect donkey to thesperm control were found (62.56% to be more ± 4.81% and tolerant 34.05% ±to exogenous 4.61%, GSH supplementation respectively). Interestingly, than their horse donkey frozen-thawed counterparts, spermaswereTM and found PMtoinbe themore latter Vet. Sci. 2021, 8, 302 12 of 17 are known to be reduced in the presence of 5 mM and 2.5 mM GSH, respectively. Similarly, VSL, LIN, STR, and BCF values were significantly reduced at 8 mM and 10 mM GSH, whereas the other kinematic parameters (VCL, VAP, WOB, and ALH) were affected in the presence of 10 mM GSH. In addition, in horse sperm, LIN, STR, and BCF are decreased in the presence of a GSH concentration ≥2.5 mM; furthermore, VSL and ALH are decreased at ≥5 mM GSH, and VCL and VAP are decreased at ≥7.5 mM GSH [43]. Moreover, four separate subpopulations were herein identified in frozen-thawed donkey sperm, which is in agreement with previous research in this species [27,50,67]. We found that the treatment containing 10 mM GSH significantly increased the proportion of sperm belonging to SP2 (the slowest and least progressive subpopulation), reduced those of sperm belonging to SP3 and SP4 (the subpopulations with intermediate kinematic parameters), and did not affect that of SP1 (the fastest and most progressive subpopulation) with respect to the control. Remarkably, 2 mM, 4 mM, and 6 mM GSH were found not to modify the structure of the frozen-thawed motile sperm subpopulations compared to the control. The analysis of sperm subpopulations once again highlighted the cryotolerance of frozen-thawed donkey sperm as, while the presence of 2 mM GSH is known to modify the structure of those subpopulations in pigs, increasing the fastest subpopulation and decreasing the slowest one [68], no changes were observed herein for concentrations ≤6 mM GSH. While sperm viability and integrities of plasma and acrosome membranes were not af- fected by the presence of high GSH concentrations, no treatment, even the ones containing low concentrations of this antioxidant, improved these sperm parameters post-thaw. This is contrary to what was found by Kumar et al. [16] in frozen-thawed sperm from exotic donkeys (Poitou breed, Martina Franca), where there was a significant increase in motility, viability and plasma membrane integrity when the freezing medium was supplemented with 2.5 mM. Our results, however, agree with the aforementioned study in the percentage of sperm with an intact acrosome membrane, since no differences in this parameter were observed in our study or in that of Kumar et al. [16], even when using two different tech- niques (flow cytometry vs. Giemsa stain + microscopy). Moreover, comparing our results with those found in other species may provide some clues, despite the fact that inconsistent data are seen in the literature. In cattle, Gangwar et al. [45] observed that adding 0.5 mM GSH to the freezing medium increased the viability, plasma and acrosome membrane integrity, and MMP of frozen-thawed sperm. In horses, Oliveira et al. [39] reported that a higher concentration of GSH (2.5 mM) was required to observe an improvement in the percentage of post-thaw sperm with an intact plasma membrane, despite the fact that acrosome integrity was similar to the control. In pigs, Estrada et al. [69] supplemented the freezing medium with 2 mM GSH and observed an increase in the percentages of viable and acrosome-intact sperm, and in fertilizing ability. On the contrary, Silva et al. [42] did not find any improvement in the quality (plasma and acrosome membrane integrity, and MMP) of frozen-thawed sheep sperm with 2 mM GSH in relation to the control, which was in agreement with our observations in the current study. As previously mentioned, whereas TM and PM were found to decrease when 8 mM and 10 mM GSH were added to the freezing medium, viability, plasma and acrosome membrane integrity, and MMP did not differ from the control. Moreover, we observed that the percentage of viable sperm with high intracellular ROS levels was significantly lower in the treatments containing 8 mM and 10 mM GSH than in the control. These observations are in relative or slight agreement with our second hypothesis, which suggested that motility, viability, plasma and acrosome membrane integrity, MMP, and intracellular ROS levels of post-thaw sperm would be affected by the addition of GSH to the freezing medium. ROS production is known to alter the function and structure of sperm membranes [70], causing a detrimental effect on sperm motility and membrane integrity [22]. Our results indicate that the detrimental effects of ROS could be counteracted by the presence of GSH. However, the other parameters of sperm functionality did not improve with the presence of GSH. In fact, the results showed that GSH is effective in scavenging ROS up to 8 mM, and could have an effect on sperm motility. Further studies are needed to address whether fertility is Vet. Sci. 2021, 8, 302 13 of 17 also compromised or if other antioxidants are able to improve the functionality and fertility of donkey sperm. As the post-translational modification of protein thiols is the most likely mechanism to maintain redox balance [41,71], one could speculate that supplementing the freezing medium with exogenous GSH could reverse the oxidative stress generated during the freezing and thawing of donkey sperm. This could maintain the integrity of plasma and acrosome membranes. On the other hand, it is important to highlight that GSH supplementation did not affect the production of intracellular O2 − levels during cryopreservation. Physiologically, O2 − is scavenged by superoxide dismutase (SOD) [72], another antioxidant enzyme present in donkey SP, rather than by the glutathione complex enzymes (GSR and GPX). Freeze- thawing reduces SOD activity in response to an increase in O2 − production [65]; this enzyme converts O2 − into H2 O2 by dismutation [73]. This leads one to posit that if there was high O2 − production in the long run, there would be a greater amount of H2 O2 . This excess of H2 O2 could be scavenged by exogenous GSH and thus reduce the percentage of sperm with high H2 O2 levels, as reported herein. Another important implication of our results is related to what happens in the jenny’s endometrium when AI is performed with frozen-thawed semen. In this scenario, donkey sperm are exposed to an oxidative environment in the uterus, which results from the production of ROS by PMN during the NETosis triggered by the presence of sperm [21]. Based on our results, GSH concentrations were not able to scavenge intracellular sperm ROS levels. Therefore, it seems reasonable to suggest that exogenous GSH supplementation between 2 mM and 6 mM could be a strategy against oxidative stress in this species after cryopreservation. This could be especially relevant after freeze-thawing if one bears in mind that SP, which is the main antioxidant source in semen, is removed before cryopreservation. However, one aspect that must be taken into account is that the degree of reduction in intracellular ROS levels observed at high GSH concentrations (8 mM and 10 mM) was not sufficient to positively affect sperm motility; in fact, it reduced sperm motility. This would not be in agreement with most observations in other mammalian species that indicate that sperm sensitivity to oxidative stress and cryogenic damage results in a reduction in motility after freeze-thawing [45,74,75]. In effect, we observed that, although high concentrations of GSH regulated the intracellular ROS levels, motility decreased. This opposite relationship advises that the impact of ROS on the other functional parameters in donkey sperm differs from other species. 5. Conclusions According to the results obtained, frozen-thawed donkey sperm were found, on the one hand, to have a surprising capacity to tolerate high GSH concentrations compared to other species, maintaining viability, plasma membrane and acrosome integrity, and MMP. On the other hand, supplementation of the freezing medium with GSH appears to be nec- essary to control intracellular ROS levels, mainly H2 O2 , produced during freeze-thawing as well as post-AI, and may improve reproductive performance in the jenny. However, the highest GSH concentrations (8 mM and 10 mM) affected sperm motility, which suggests that how donkey sperm handle ROS generation differs from other species. Therefore, conducting in vivo studies is much warranted to complement the results obtained in this work. Author Contributions: Conceptualization, M.Y. and J.M.; methodology, I.Y.-O., J.C., A.D.-B. and A.C.; validation, M.Y. and J.M.; formal analysis, I.Y.-O.; investigation, I.Y.-O. and J.C.; resources, M.Y. and J.M.; data curation, I.Y.-O.; writing—original draft preparation, I.Y.-O.; writing—review and editing, A.D.-B., M.Y. and J.M.; supervision M.Y. and J.M.; project administration, M.Y. and J.M.; funding acquisition, M.Y. and J.M. All authors have read and agreed to the published version of the manuscript. Funding: I.Y.-O. was funded by the Secretary of Higher Education, Science, Technology and In- novation (SENESCYT), Ecuador (Scheme: Programa de Becas Internacionales de Posgrado 2019; Grant: CZ02-000507-2019). J.C. was funded by the “Demetra” project of the Department of Excel- Vet. Sci. 2021, 8, 302 14 of 17 lence Postdoctoral fellowship, University of Teramo, Italy (Scheme: Assegno di Recerca 2021). The authors also acknowledge the support from the Ministry of Science and Innovation, Spain (Grant: AGL2017-88329-R) and the Regional Government of Catalonia (2017-SGR-1229). Institutional Review Board Statement: The Ethics Committee of the Autonomous University of Barcelona (CEEAH) considered that the study is exempted of clearance. Informed Consent Statement: The informed consent was obtained from owner of Catalonian jack- asses in this study. Data Availability Statement: All data is contained within the article. Acknowledgments: The authors would like to thank Jordi Ribas-Maynou from the Biotechnology of Animal and Human Reproduction (TechnoSperm), University of Girona, Spain, for his support in handling the flow cytometer. Conflicts of Interest: The authors declare no conflict of interest. References 1. Sherman, J.K. Low temperature research on spermatozoa and eggs. Cryobiology 1964, 1, 103–129. [CrossRef] 2. Loomis, P.R. Advanced methods for handling and preparation of stallion semen. Vet. Clin. N. Am. Equine Pract. 2006, 22, 663–676. [CrossRef] [PubMed] 3. Fickel, J.; Wagener, A.; Ludwig, A. Semen cryopreservation and the conservation of endangered species. Eur. J. Wildl. Res. 2007, 53, 81–89. [CrossRef] 4. Camillo, F.; Rota, A.; Biagini, L.; Tesi, M.; Fanelli, D.; Panzani, D. The current situation and trend of donkey industry in Europe. J. Equine Vet. Sci. 2018, 65, 44–49. [CrossRef] 5. Aranguren-Méndez, J.; Jordana, J.; Gomez, M. Genetic diversity in Spanish donkey breeds using microsatellite DNA markers. Genet. Sel. Evol. 2001, 33, 1–10. [CrossRef] 6. Trimeche, A.; Renard, P.; Tainturier, D. A procedure for Poitou jackass sperm cryopreservation. Theriogenology 1998, 50, 793–806. [CrossRef] 7. Vidament, M.; Vincent, P.; Martin, F.X.; Magistrini, M.; Blesbois, E. Differences in ability of jennies and mares to conceive with cooled and frozen semen containing glycerol or not. Anim. Reprod. Sci. 2009, 112, 22–35. [CrossRef] [PubMed] 8. Oliveira, J.V.; Papa, F.O.; Melo-Oña, C.M.; Monteiro, G.A.; Puoli-Filho, J.N.P.; Alvarenga, M.A. New procedures to freeze donkey semen and its influence on mares and jennies fertility. J. Equine Vet. Sci. 2012, 32, 503–504. [CrossRef] 9. Rota, A.; Panzani, D.; Sabatini, C.; Camillo, F. Donkey jack (Equus asinus) semen cryopreservation: Studies of seminal parameters, post breeding inflammatory response, and fertility in donkey jennies. Theriogenology 2012, 78, 1846–1854. [CrossRef] 10. Pukazhenthi, B.S.; Johnson, A.; Guthrie, H.D.; Songsasen, N.; Padilla, L.R.; Wolfe, B.A.; Coutinho da Silva, M.; Alvarenga, M.A.; Wildt, D.E. Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski’s horse (Equus ferus przewalskii). Cryobiology 2014, 68, 205–214. [CrossRef] 11. Ortiz, I.; Dorado, J.; Morrell, J.M.; Crespo, F.; Gosálvez, J.; Gálvez, M.J.; Acha, D.; Hidalgo, M. Effect of single-layer centrifugation or washing on frozen–thawed donkey semen quality: Do they have the same effect regardless of the quality of the sample? Theriogenology 2015, 84, 294–300. [CrossRef] 12. Acha, D.; Hidalgo, M.; Ortiz, I.; Gálvez, M.J.; Carrasco, J.J.; Gómez-Arrones, V.; Dorado, J. Freezability of Andalusian donkey (Equus asinus) spermatozoa: Effect of extenders and permeating cryoprotectants. Reprod. Fertil. Dev. 2016, 28, 1990–1998. [CrossRef] [PubMed] 13. Oliveira, J.V.; Oliveira, P.V.L.F.; Melo-Oña, C.M.; Guasti, P.N.; Monteiro, G.A.; Sancler da Silva, Y.F.R.; Papa, P.M.; Alvarenga, M.A.; Dell’Aqua, J.A.; Papa, F.O. Strategies to improve the fertility of fresh and frozen donkey semen. Theriogenology 2016, 85, 1267–1273. [CrossRef] [PubMed] 14. Bottrel, M.; Acha, D.; Ortiz, I.; Hidalgo, M.; Gósalvez, J.; Camisão, J.; Dorado, J. Cryoprotective effect of glutamine, taurine, and proline on post-thaw semen quality and DNA integrity of donkey spermatozoa. Anim. Reprod. Sci. 2018, 189, 128–135. [CrossRef] 15. Zhang, H.; Ye, H.; Shao, Y.; Wu, S.; Yu, J.; Ji, C.; Wang, S.; Zeng, S. The effects of egg yolk concentration and particle size on donkey semen preservation. J. Equine Vet. Sci. 2018, 65, 19–24. [CrossRef] 16. Kumar, P.; Kumar, R.; Mehta, J.S.; Chaudhary, A.K.; Ravi, S.K.; Chandra Mehta, S.; Ansari, M.M.; Legha, R.A.; Tripathi, B.N.; Talluri, T.R. Ameliorative effect of ascorbic acid and glutathione in combating the cryoinjuries during cryopreservation of exotic jack semen. J. Equine Vet. Sci. 2019, 81, 102796. [CrossRef] 17. Yu, X.; He, S.; Wang, L.; Kang, M.; Zhu, Y.; Wang, S.; Sun, X. Effects of Vitamin C and Vitamin E on cryopreservation of Guanzhong donkey semen. Pak. J. Zool. 2019, 51, 1777–1781. [CrossRef] 18. Li, N.; Yu, J.; Yang, F.; Shao, Y.; Wu, S.; Liu, B.; Li, M.; Wang, T.; Li, J.; Zeng, S. L-proline: An effective agent for frozen and post-thawed donkey semen storage. J. Equine Vet. Sci. 2021, 101, 103393. [CrossRef] [PubMed] Vet. Sci. 2021, 8, 302 15 of 17 19. Canisso, I.F.; Carvalho, G.R.; Davis Morel, M.; Ker, P.G.; Rodrigues, A.L.; Silva, E.C.; Coutinho Da Silva, M.A. Seminal parameters and field fertility of cryopreserved donkey jack semen after insemination of horse mares. Equine Vet. J. 2011, 43, 179–183. [CrossRef] 20. Vilés, K.; Rabanal, R.; Rodríguez-Prado, M.; Miró, J. Effect of ketoprofen treatment on the uterine inflammatory response after AI of jennies with frozen semen. Theriogenology 2013, 79, 1019–1026. [CrossRef] [PubMed] 21. Miró, J.; Papas, M. Post–Artificial Insemination endometrial inflammation and its control in donkeys. J. Equine Vet. Sci. 2018, 65, 38–43. [CrossRef] 22. Yeste, M. Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs. Theriogenol- ogy 2016, 85, 47–64. [CrossRef] [PubMed] 23. Aitken, R.J.; De Iuliis, G.N.; Drevet, J.R. Role of oxidative stress in the etiology of male infertility and the potential therapeutic value of antioxidants. In Oxidants, Antioxidants, and Impact of the Oxidative Status in Male Reproduction; Elsevier: London, UK, 2018; pp. 91–100. 24. Peña, F.J.; O’Flaherty, C.; Ortiz Rodríguez, J.M.; Martín Cano, F.E.; Gaitskell-Phillips, G.L.; Gil, M.C.; Ortega Ferrusola, C. Redox regulation and oxidative stress: The particular case of the stallion spermatozoa. Antioxidants 2019, 8, 567. [CrossRef] 25. Kim, J.G.; Parthasarathy, S. Oxidation and the spermatozoa. Semin. Reprod. Endocrinol. 1998, 16, 235–239. [CrossRef] 26. Papas, M.; Arroyo, L.; Bassols, A.; Catalán, J.; Bonilla-Correal, S.; Gacem, S.; Yeste, M.; Miró, J. Activities of antioxidant seminal plasma enzymes (SOD, CAT, GPX and GSR) are higher in jackasses than in stallions and are correlated with sperm motility in jackasses. Theriogenology 2019, 140, 180–187. [CrossRef] [PubMed] 27. Papas, M.; Catalán, J.; Recuero, S.; Morrell, J.M.; Yeste, M.; Miró, J. Single layer centrifugation improves the quality of fresh donkey semen and modifies the sperm ability to interact with polymorphonuclear neutrophils. Animals 2020, 10, 2128. [CrossRef] [PubMed] 28. Dutta, S.; Majzoub, A.; Agarwal, A. Oxidative stress and sperm function: A systematic review on evaluation and management. Arab J. Urol. 2019, 17, 87–97. [CrossRef] 29. Yeste, M.; Estrada, E.; Casas, I.; Bonet, S.; Rodríguez-Gil, J.E. Good and bad freezability boar ejaculates differ in the integrity of nucleoprotein structure after freeze-thawing but not in ROS levels. Theriogenology 2013, 79, 929–939. [CrossRef] [PubMed] 30. Kotilainen, T.; Huhtinen, M.; Katila, T. Sperm-induced leukocytosis in the equine uterus. Theriogenology 1994, 41, 629–636. [CrossRef] 31. Troedsson, M.H.T.; Loset, K.; Alghamdi, A.M.; Dahms, B.; Crabo, B.G. Interaction between equine semen and the endometrium: The inflammatory response to semen. Anim. Reprod. Sci. 2001, 68, 273–278. [CrossRef] 32. Rodriguez-Martinez, H.; Saravia, F.; Wallgren, M.; Martinez, E.A.; Sanz, L.; Roca, J.; Vazquez, J.M.; Calvete, J.J. Spermadhesin PSP-I/PSP-II heterodimer induces migration of polymorphonuclear neutrophils into the uterine cavity of the sow. J. Reprod. Immunol. 2010, 84, 57–65. [CrossRef] 33. Zambrano, F.; Carrau, T.; Gärtner, U.; Seipp, A.; Taubert, A.; Felmer, R.; Sanchez, R.; Hermosilla, C. Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility. Fertil. Steril. 2016, 106, 1053–1060. [CrossRef] [PubMed] 34. Miró, J.; Marín, H.; Catalán, J.; Papas, M.; Gacem, S.; Yeste, M. Seminal plasma, sperm concentration, and sperm-PMN interaction in the donkey: An in vitro model to study endometrial inflammation at post-insemination. Int. J. Mol. Sci. 2020, 21, 3478. [CrossRef] 35. Baumber, J.; Vo, A.; Sabeur, K.; Ball, B.A. Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa. Theriogenology 2002, 57, 1025–1033. [CrossRef] 36. Yánez-Ortiz, I.; Catalán, J.; Mateo-Otero, Y.; Dordas-Perpinyà, M.; Gacem, S.; Yeste, N.; Bassols, A.; Yeste, M.; Miró, J. Extracellular reactive oxygen species (ROS) production in fresh donkey sperm exposed to reductive stress, oxidative stress and NETosis. Antioxidants 2021, 10, 1367. [CrossRef] 37. Gadea, J.; Molla, M.; Selles, E.; Marco, M.A.; Garcia-Vazquez, F.A.; Gardon, J.C. Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology 2011, 62, 40–46. [CrossRef] [PubMed] 38. Câmara, D.R.; Mello-Pinto, M.M.C.; Pinto, L.C.; Brasil, O.O.; Nunes, J.F.; Guerra, M.M.P. Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen. Small Rumin. Res. 2011, 100, 44–49. [CrossRef] 39. Ansari, M.S.; Rakha, B.A.; Andrabi, S.M.H.; Ullah, N.; Iqbal, R.; Holt, W.V.; Akhter, S. Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa. Reprod. Biol. 2012, 12, 271–276. [CrossRef] 40. Oliveira, R.A.; Piersanti, R.L.; Wolf, C.A.; Viu, M.A.O.; Gambarini, M.L. Glutathione for the freezing of cooled equine semen, using different protocols. Anim. Reprod. 2018, 11, 104–109. 41. Ahmad, E.; Naseer, Z.; Aksoy, M. Glutathione supplementation in semen extender improves rabbit sperm attributes during refrigeration. World Rabbit Sci. 2021, 29, 81. [CrossRef] 42. Gadea, J.; García-Vazquez, F.; Matás, C.; Gardón, J.C.; Cánovas, S.; Gumbao, D. Cooling and freezing of boar spermatozoa: Supplementation of the freezing media with reduced glutathione preserves sperm function. J. Androl. 2005, 26, 396–404. [CrossRef] [PubMed] Vet. Sci. 2021, 8, 302 16 of 17 43. Silva, S.V.; Soares, A.T.; Batista, A.M.; Almeida, F.C.; Nunes, J.F.; Peixoto, C.A.; Guerra, M.M.P. In vitro and in vivo evaluation of ram sperm frozen in tris egg-yolk and supplemented with superoxide dismutase and reduced glutathione. Reprod. Domest. Anim. 2011, 46, 874–881. [CrossRef] 44. Oliveira, R.A.; Wolf, C.A.; Viu, M.A.O.; Gambarini, M.L. Addition of glutathione to an extender for frozen equine semen. J. Equine Vet. Sci. 2013, 33, 1148–1152. [CrossRef] 45. Ogata, K.; Sasaki, A.; Kato, Y.; Takeda, A.; Wakabayashi, M.; Sarentonglaga, B.; Yamaguchi, M.; Hara, A.; Fukumori, R.; Nagao, Y. Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination. J. Reprod. Dev. 2015, 61, 116–122. [CrossRef] [PubMed] 46. Gangwar, C.; Saxena, A.; Patel, A.; Singh, S.P.; Yadav, S.; Kumar, R.; Singh, V. Effect of reduced glutathione supplementation on cryopreservation induced sperm cryoinjuries in Murrah bull semen. Anim. Reprod. Sci. 2018, 192, 171–178. [CrossRef] 47. Luberda, Z. The role of glutathione in mammalian gametes. Reprod. Biol. 2005, 5, 5–17. 48. Irvine, D.S. Glutathione as a treatment for male infertility. Rev. Reprod. 1996, 1, 6–12. [CrossRef] [PubMed] 49. Yeste, M.; Flores, E.; Estrada, E.; Bonet, S.; Rigau, T.; Rodríguez-Gil, J.E. Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze-thawing by stabilising disulfide bonds. Reprod. Fertil. Dev. 2013, 25, 1036–1050. [CrossRef] 50. Miró, J.; Lobo, V.; Quintero-Moreno, A.; Medrano, A.; Peña, A.; Rigau, T. Sperm motility patterns and metabolism in Catalonian donkey semen. Theriogenology 2005, 63, 1706–1716. [CrossRef] 51. 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] 52. Gacem, S.; Papas, M.; Catalan, J.; Miró, J. Examination of jackass (Equus asinus) accessory sex glands by B-mode ultrasound and of testicular artery blood flow by colour pulsed-wave Doppler ultrasound: Correlations with semen production. Reprod. Domest. Anim. 2020, 55, 181–188. [CrossRef] 53. Kenney, M.R. Minimal contamination techniques for breeding mares: Techniques and priliminary findings. Proc. Am. Assoc. Equine Pract. 1975, 327–336. 54. Gacem, S.; Catalán, J.; Valverde, A.; Soler, C.; Miró, J. Optimization of CASA-mot analysis of donkey sperm: Optimum frame rate and values of kinematic variables for different counting chamber and fields. Animals 2020, 10, 1993. [CrossRef] 55. Bamba, K. Evaluation of acrosomal integrity of boar spermatozoa by bright field microscopy using an eosin-nigrosin stain. Theriogenology 1988, 29, 1245–1251. [CrossRef] 56. Lee, J.A.; Spidlen, J.; Boyce, K.; Cai, J.; Crosbie, N.; Dalphin, M.; Furlong, J.; Gasparetto, M.; Goldberg, M.; Goralczyk, E.M.; et al. MIFlowCyt: The minimum information about a flow cytometry experiment. Cytometry 2008, 73, 926–930. [CrossRef] [PubMed] 57. Garner, D.L.; Johnson, L.A. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol. Reprod. 1995, 53, 276–284. [CrossRef] [PubMed] 58. Rathi, R.; Colenbrander, B.; Bevers, M.M.; Gadella, B.M. Evaluation of in vitro capacitation of stallion spermatozoa. Biol. Reprod. 2001, 65, 462–470. [CrossRef] [PubMed] 59. Ortega-Ferrusola, C.; Sotillo-Galán, Y.; Varela-Fernández, E.; Gallardo-Bolaños, J.M.; Muriel, A.; González-Fernández, L.; Tapia, J.A.; Peña, F.J. Detection of “apoptosis-like” changes during the cryopreservation process in equine sperm. J. Androl. 2008, 29, 213–221. [CrossRef] 60. Morrell, J.M.; Winblad, C.; Georgakas, A.; Stuhtmann, G.; Humblot, P.; Johannisson, A. Reactive oxygen species in stallion semen can be affected by season and colloid centrifugation. Anim. Reprod. Sci. 2013, 140, 62–69. [CrossRef] [PubMed] 61. Guthrie, H.D.; Welch, G.R. Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry. J. Anim. Sci. 2006, 84, 2089–2100. [CrossRef] [PubMed] 62. Martí, J.I.; Aparicio, I.M.; García-Herreros, M. Sperm morphometric subpopulations are differentially distributed in rams with different maturity age in cryopreserved ejaculates. Theriogenology 2011, 76, 97–109. [CrossRef] 63. Meyers, S.A. Sperm physiology. In Equine Breeding Management and Artificial Insemination; Samper, J.C., Ed.; Saunders Elsevier: Philadelphia, PA, USA, 2009; pp. 47–55. 64. Rodríguez-Martínez, H.; Kvist, U.; Ernerudh, J.; Sanz, L.; Calvete, J.J. Seminal plasma proteins: What role do they play? Am. J. Reprod. Immunol. 2011, 66, 11–22. [CrossRef] [PubMed] 65. Gadea, J.; Sellés, E.; Marco, M.A.; Coy, P.; Matás, C.; Romar, R.; Ruiz, S. Decrease in glutathione content in boar sperm after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Theriogenology 2004, 62, 690–701. [CrossRef] [PubMed] 66. Ortega-Ferrusola, C.; Martin Muñoz, P.; Ortiz-Rodriguez, J.M.; Anel-López, L.; Balao da Silva, C.; Álvarez, M.; de Paz, P.; Tapia, J.A.; Anel, L.; Silva- Rodríguez, A.; et al. Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: A possible role for GSH regulation in spermatozoa. Biol. Reprod. 2019, 100, 1090–1107. [CrossRef] 67. Catalán, J.; Papas, M.; Trujillo-Rojas, L.; Blanco-Prieto, O.; Bonilla-Correal, S.; Rodríguez-Gil, J.E.; Miró, J.; Yeste, M. Red LED light acts on the mitochondrial electron chain of donkey sperm and its effects depend on the time of exposure to light. Front. Cell Dev. Biol. 2020, 8, 588621. [CrossRef] [PubMed] 68. Estrada, E.; Rivera del Álamo, M.M.; Rodríguez-Gil, J.E.; Yeste, M. The addition of reduced glutathione to cryopreservation media induces changes in the structure of motile subpopulations of frozen-thawed boar sperm. Cryobiology 2017, 78, 56–64. [CrossRef] Vet. Sci. 2021, 8, 302 17 of 17 69. Estrada, E.; Rodríguez-Gil, J.E.; Rocha, L.G.; Balasch, S.; Bonet, S.; Yeste, M. Supplementing cryopreservation media with reduced glutathione increases fertility and prolificacy of sows inseminated with frozen-thawed boar semen. Andrology 2014, 2, 88–99. [CrossRef] 70. Ball, B.A.; Vo, A.T.; Baumber, J. Generation of reactive oxygen species by equine spermatozoa. Am. J. Vet. Res. 2001, 62, 508–515. [CrossRef] [PubMed] 71. Bilodeau, J.F.; Blanchette, S.; Gagnon, C.; Sirard, M.A. Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen. Theriogenology 2001, 56, 275–286. [CrossRef] 72. Fridovich, I. Superoxide radical and superoxide dismutases. Annu. Rev. Biochem. 1995, 64, 97–112. [CrossRef] [PubMed] 73. Ighodaro, O.M.; Akinloye, O.A. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid. Alex. J. Med. 2018, 54, 287–293. [CrossRef] 74. Hammerstedt, R.H. Maintenance of bioenergetic balance in sperm and prevention of lipid peroxidation: A review of the effect on design of storage preservation systems. Reprod. Fertil. Dev. 1993, 5, 675–690. [CrossRef] [PubMed] 75. Holt, W.V. Mechanisms of sperm storage in the female reproductive tract: An interspecies comparison. Reprod. Domest. Anim. 2011, 46, 68–74. [CrossRef] [PubMed]