antioxidants Article Impact of Seminal Plasma Antioxidants on Donkey Sperm Cryotolerance Jaime Catalán 1,2,3,4,† , Iván Yánez-Ortiz 1,2,3,† , Asta Tvarijonaviciute 5,6 , Luis Guillermo González-Arostegui 5,6 , Camila P. Rubio 6,7 , Marc Yeste 2,3 , Jordi Miró 1, *,‡ and Isabel Barranco 8, *,‡ 1 Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, ES-08193 Cerdanyola del Vallès, Spain; dr.jcatalan@gmail.com (J.C.); ivan.yanez22@gmail.com (I.Y.-O.) 2 Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain; marc.yeste@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 5 Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, University of Murcia, ES-30100 Murcia, Spain; asta@um.es (A.T.); luisgarostegui@gmail.com (L.G.G.-A.) 6 Interdisciplinary Laboratory of Clinical Analysis Interlab-UMU, Faculty of Veterinary Medicine, Regional Campus of International Excellence ‘Campus Mare Nostrum’, University of Murcia, ES-30100 Murcia, Spain; camila.peres@uab.cat 7 Department of Animal and Food Science, Faculty of Veterinary Sciences, Autonomous University of Barcelona, ES-08193 Cerdanyola del Vallès, Spain 8 Department of Veterinary Medical Sciences, University of Bologna, IT-40064 Ozzano dell’Emilia, Italy  * Correspondence: jordi.miro@uab.cat (J.M.); isabel.barranco@unibo.it (I.B.);  Tel.: +34-93-5814293 (J.M.); +34-639923954 (I.B.) Citation: Catalán, J.; Yánez-Ortiz, I.; † These authors contributed equally to this work. Tvarijonaviciute, A.; González- ‡ These authors share senior authorship. Arostegui, L.G.; Rubio, C.P.; Yeste, M.; Miró, J.; Barranco, I. Impact of Abstract: This study investigated whether the activities of the antioxidant components of donkey Seminal Plasma Antioxidants on seminal plasma (SP)—both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione Donkey Sperm Cryotolerance. peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of Antioxidants 2022, 11, 417. total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), https://doi.org/10.3390/ and Trolox equivalent antioxidant capacity (TEAC))—and oxidative stress index (OSI) are related to antiox11020417 sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected Academic Editors: Gwonhwa Song, and split into two aliquots. The first one was used for measuring the activities levels of enzymatic Giulia Collodel and Elena Moretti and non-enzymatic antioxidants and OSI in SP, whereas the other aliquot was cryopreserved. Before cryopreservation, sperm quality parameters (concentration, motility, and viability) were evaluated. Received: 29 December 2021 After thawing, sperm motility, plasma membrane integrity, lipid disorder, mitochondrial membrane Accepted: 16 February 2022 Published: 18 February 2022 potential, reactive oxygen species (ROS), and calcium intracellular levels were also determined. Based on the percentages of total motility (TM) and of sperm with an intact plasma membrane Publisher’s Note: MDPI stays neutral (SYBR14+ /PI− ) after thawing, samples were classified as good-freezability (GFE) or poor-freezability with regard to jurisdictional claims in (PFE) ejaculates through cluster analyses. The SP activity levels of enzymatic (SOD and PON1) and published maps and institutional affil- non-enzymatic antioxidants (CUPRAC, FRAP, and TEAC) were higher (p < 0.05) in GFE than in iations. PFE, whereas SP-OSI was higher (p < 0.05) in PFE than in GFE. In addition, the activity levels of SOD, PON1, GPX, CUPRAC, FRAP, and TEAC were positively (p < 0.05) related to post-thaw sperm motility and plasma membrane integrity and negatively to intracellular ROS levels. The SP-OSI was Copyright: © 2022 by the authors. negatively correlated (p < 0.05) to post-thaw sperm quality parameters and positively to intracellular Licensee MDPI, Basel, Switzerland. ROS levels. It can thus be concluded that donkey SP antioxidants are related to sperm cryotolerance This article is an open access article and that measurements of antioxidants PON1, SOD, CUPRAC, FRAP, and TEAC, as well as SP-OSI, distributed under the terms and could be used as markers of sperm cryotolerance. Further research addressing the relationship of conditions of the Creative Commons these antioxidants and SP-OSI with sperm cryotolerance and their potential use as freezing markers Attribution (CC BY) license (https:// is warranted. creativecommons.org/licenses/by/ 4.0/). Antioxidants 2022, 11, 417. https://doi.org/10.3390/antiox11020417 https://www.mdpi.com/journal/antioxidants Antioxidants 2022, 11, 417 2 of 22 Keywords: seminal plasma (SP); sperm; donkey; cryopreservation; antioxidants; reactive oxygen species (ROS) 1. Introduction In the last few decades, the donkey (Equus asinus) has been rediscovered in an attempt to protect biodiversity, endangered breeds, and develop marginal agricultural areas [1–3]. This fact, together with a growing demand for new uses (milk production, cosmetics, forestry, rural tourism, leisure...) has promoted a growing research interest in studying the reproductive characteristics of this species [3–5]. In this regard, the improvement of breeding management in donkeys with the use of assisted reproductive technologies, such as artificial insemination (AI) and semen cryopreservation, is crucial for their genetic selection and conservation [6]. Despite advances in cryopreservation, the use of frozen-thawed semen has been limited in donkeys due to the low conception rates obtained when jennies (0 to 36%) are inseminated compared to mares (33% to 56%) [7–10], which is attributed to the intense endometrial inflammatory response that occurs when the jenny is inseminated with frozen- thawed sperm [11–13]. In vivo and in vitro experiences have shown that SP, which is removed during the semen-freezing process because of its harmful effects on equids’ sperm [14], may play a key role in modulating this response [12]. Cryopreservation reduces the survival and fertilizing capacity of mammalian sperm [14]. This can be explained by the impact of freeze-thawing on plasma membrane integrity, impairing sperm motility, viability, and morphology [15,16]. Exposure of sperm to a stressful environment during cryopreservation is associated with an overproduction of reactive oxygen species (ROS) [14]. This situation could cause an imbalance in relation to the antioxidant defense, leading to oxidative stress (OS) and subsequent lipid peroxidation (LPO) of the plasma membrane [17–20]. In the case of donkeys, although it also occurs in other species, their sperm are especially sensitive to these events due to the large amount of polyunsaturated fatty acids in their plasma membrane [6,17,19–21]. In addition, the low amount of antioxidant enzymes within the sperm cell, together with the large number of mitochondria, makes the antioxidants (enzymatic and non-enzymatic) present in SP the main defense against the OS-induced damage [6,20,22,23]. The removal of SP before freezing, therefore, may increase sperm susceptibility to OS [6]. In recent years, many efforts have been focused on improving cryopreservation procedures in different species, analyzing the supplementation of freezing/thawing media with different antioxidants and obtaining inconsistent results [24–28]. In donkeys, as in other mammalian species, a high inter- and intra-male variability in the ability of sperm to withstand cryopreservation has been reported [29–34], leading to the classification of males/ejaculates as of “good” (GFE) or “poor” (PFE) freezabil- ity [17,31,35,36]. Differences in the composition of SP surrounding sperm among individu- als and ejaculates could explain such differences [37,38]. In this regard, a recent study from our research group investigated whether the activities of four enzymes with antioxidant properties present in donkey SP (superoxide dismutase (SOD), catalase-like (CAT), glu- tathione peroxidase-like (GPX), and glutathione reductase (GSR)) were related to the sperm resilience to freezing and thawing. The results of this study show that the activity of CAT and GSR and the total and specific activity of SOD in SP were associated with donkey sperm cryotolerance [6]. As far as we are aware, however, it has not yet been reported whether other components of SP with antioxidant properties, beyond these enzymes, are related to sperm freezability in this species, nor has the activity of the previously mentioned enzymes been linked to sperm quality parameters measured by flow cytometry. Related to this, a recent investigation performed in pig SP revealed that the activity of some enzymes, such as paraoxonase (PON1), and the total non-enzymatic antioxidant capacity were positively involved in sperm cryotolerance, minimizing the OS as result of freezing and thawing [39]. Antioxidants 2022, 11, 417 3 of 22 Against this background, this study aimed to determine whether antioxidant com- ponents of donkey SP, including enzymes (SOD, CAT, GPX, and PON1), non-enzymatic antioxidant capacity (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant ca- pacity (TEAC), which are assays that evaluate the total non-enzymatic antioxidant capacity of SP), and the oxidative stress index (OSI), are related to donkey sperm cryotolerance. 2. Materials and Methods 2.1. Animals and Ejaculates This study included 15 ejaculates from Catalonian jackasses (aged 4–11 years old) of proven fertility. All animals used were in good health condition and were housed at the Equine Reproduction Service, Autonomous University of Barcelona (Bellaterra, Cerdanyola del Vallès, Spain), a European Union-approved equine semen collection center (authorization number: ES09RS01E). This center works under strict protocols of health control and animal welfare. Because this Service already works under the approval of the Generalitat de Catalunya (Spain) and given that no manipulation on the animals beyond the collection of semen was conducted, the Ethics Committee of our institution indicated that no further ethical approval was required to carry out this study. All animals were kept in individual paddocks and were fed a standard diet composed of mixed hay and basic concentrate. No antioxidant supplementation was used, and water was available ad libitum. A Hannover artificial vagina was used to collect the ejaculates (Minitüb GmbH, Tie-fenbach, Germany), with a nylon mesh filter connected in- line to remove the gel fraction. After removing the gel fraction, the total semen volume was evaluated, and an aliquot was used for sperm concentration assessment (Neubauer chamber, Paul Marienfeld GmbH and Co. KG; Lau-da-Königshofen, Germany). Then, the total volume of each ejaculate was divided into two equal aliquots. One was used to obtain the SP (see Section 2.2), and the other one was diluted in previously warmed (38 ◦ C) Kenney extender (1:5v:v) [40]. This latter aliquot was utilized to evaluate sperm motility (by computer-assisted semen analysis, CASA; see Section 2.5), morphology, and viability (assessed by eosin-nigrosine staining [41]) immediately after ejaculate collection. All ejaculates fulfilled standard semen quality thresholds (>60% live sperm and >70% morphologically normal sperm). 2.2. Isolation of Seminal Plasma (SP) After collection, semen was immediately centrifuged five times at 1500× g for 10 min (JP Selecta S.A., Barcelona, Spain) at 4 ◦ C. Once each centrifugation cycle finished, the super- natant was examined to ensure the absence of spermatozoa under a phase-contrast micro- scope (Olympus Europa, Hamburg, Germany). Five mL-aliquots of SP were subsequently stored at −80 ◦ C. Before antioxidants measurements, SP samples were thawed on ice. 2.3. Measurement of Antioxidants Activity Levels and OSI in SP The total non-enzymatic antioxidant capacity of SP was assessed in terms of CUPRAC, FRAP, and TEAC, following the protocols described by Li et al. [39], and total thiol following the procedure described by Jocelyn [42] and da Costa et al. [43]. All these procedures were previously adapted to donkey SP. The TEAC method is based on a color change by 2,20 -azinobis-3-ethylbenzothiazoline-6-sulfonate [44], the FRAP method is based on the reduction of Fe3+ to Fe2+ [45], and the CUPRAC method is based on the reduction of Cu2+ to Cu1+ [46]. The analysis of total thiol is based on the fact that thiols interact with 5, 50 -dithiobis- (2-nitrobenzoic acid) (DTNB), forming a highly colored anion with a maximum peak at 412 nm (e412 = 13,600 M−1 cm−1 ) [43]. All these assays were performed using an Olympus AU400 automatic chemistry analyzer (Olympus Europe GmbH, Hamburg, Germany). The results of TEAC, FRAP, and CUPRAC are expressed in mmol/L, and total thiol results are expressed in µmol/L. Antioxidants 2022, 11, 417 4 of 22 Enzymatic SP antioxidants were measured as the activity of CAT, GPX, SOD, and PON1. Activities of CAT, GPX, and SOD were measured using commercially available assays following the manufacturer’s instructions (CAT: Sigma-Aldrich, St. Louis, MO, USA; GPX and SOD: Randox, Crumlin, UK). Activity of PON1 was assessed by measur- ing the hydrolysis of p-nitrophenyl acetate into p-nitrophenol, based on the protocol of Barranco et al. [47] adapted to donkey SP. All measurements, except for the CAT activity, were performed using an Olympus AU400 automatic chemistry analyzer. Activity of CAT was measured using a micro-plate reader (PowerWave XS; Bio-Tek Instruments). Activi- ties of SOD and CAT were expressed as IU/mL, whereas those of PON1 and GPX were expressed as IU/L. To calculate SP-OSI, the total oxidative status (TOS) of SP was previously measured using the method described by Erel [48] and adapted to donkey SP. This assay is based on the oxidation of ferrous ion to ferric ion in the presence of various oxidant species in acidic medium and the measurement of the ferric ion by xylenol orange. This test was also performed using an Olympus AU400 automatic chemistry analyzer. The results of TOS are expressed in µmol H2 O2 equiv./L. Thereafter, SP-OSI value was calculated utilizing the following formula, as described by Wu et al. [49]: OSI (arbitrary unit) = TOS (µmol H2 O2 equiv./L)/TEAC (mmol Trolox equiv./L). Measurements of all analytes were performed in duplicate for each SP sample. In all the analytes, each test showed intra- and inter-assay coefficient variations below 10%. 2.4. Sperm Cryopreservation Sperm cryopreservation was performed as previously described by Flores et al. [50]. For this purpose, before sperm cryopreservation, diluted semen was centrifuged at 600× g for 15 min at 20 ◦ C (Medifriger BL-S, JP Selecta S.A., Barcelona, Spain). After the super- natant removal, the pellets were resuspended in a commercial freezing extender (Botucrio® , Botupharma Animal Biotechnology; Botucatu, Brazil), which contains permeable cryopro- tectants (4% methylformamide and 1% glycerol). Subsequently, sperm concentration, motil- ity, and plasma membrane integrity were reevaluated, and freezing medium (Botucrio® ) was added until reaching a final concentration of 200 × 106 viable sperm per mL (standard- ized in all cases). Prior to their cryopreservation using a controlled-rate freezer (Ice-Cube 14S; Minitüb), samples were packaged into 0.5-mL straws. Three stages of cooling/freezing were performed: (i) from 20 ◦ C to 5 ◦ C for 60 min, at a rate of −0.25 ◦ C/min; (ii) from 5 ◦ C to −90 ◦ C for 20 min, at a rate of −4.75 ◦ C/min; and (iii) from −90 ◦ C to −120 ◦ C for 2.7 min, at a rate of −11.11 ◦ C/min. Once the last freezing step finished, straws were immediately submerged into liquid nitrogen and kept in tanks until their thawing and analysis. The thawing protocol consisted of incubating the straws for 30 s in a water bath at 38 ◦ C followed by dilution with three volumes of Kenney extender, also prewarmed at 38 ◦ C (final concentration: 50 × 106 sperm/mL) 2.5. Assessment of Sperm Motility Before and after cryopreservation, sperm motility was evaluated using a CASA system (Proiser S.L.; Valencia, Spain). For that, 5 µL of each semen sample (diluted in Kenney extender at a concentration of 50 × 106 spermatozoa/mL) was placed onto a previously heated (38 ◦ C) Makler chamber (Sefi Medical Instruments; Haifa, Israel). Then, samples were assessed under a 10× negative phase-contrast objective using an Olympus B × 41 microscope (Olympus, Tokyo, Japan) that also had a plate heated to 38 ◦ C. A minimum of 1000 sperm cells per analysis were counted. Percentages of total (TM, %) and progressive motility (PM, %) were recorded in each evaluation together with kinetic parameters as follows: straight-line velocity (VSL, µm/s), which is the mean path velocity of the sperm head along a straight line from its first to its last position; curvilinear velocity (VCL, µm/s), which is the mean path velocity of the sperm head along its actual trajectory; average path velocity (VAP, µm/s), which is the mean velocity of the sperm head along its average trajectory; percentage of straightness (STR, %), which is the quotient between VSL and VAP Antioxidants 2022, 11, 417 5 of 22 multiplied by 100; percentage of linearity (LIN, %), which is the quotient between VSL and VCL multiplied by 100; percentage of oscillation (WOB, %), which is the quotient between VAP and VCL multiplied by 100; frequency of head displacement (BCF, Hz), which is the frequency at which the head crosses the actual sperm track; and mean amplitude of lateral head displacement (ALH, µm), which is the mean value of the extreme side-to-side movement of the sperm head in each beat cycle. The settings of CASA used were those recommended by the provider, i.e., frames/s: 25 images captured per second; connectivity: 6; particle area >4 and <75 µm2 ; minimum number of images to calculate the ALH: 10. Cut-off value for motile spermatozoa was VAP ≥ 10 µm/s and for progressively motile spermatozoa was STR ≥ 75%. 2.6. Flow Cytometry Analyses Flow cytometry was used to determine the integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential (MMP), and intracellular levels of overall ROS, superoxides, and calcium in post-thawed semen samples, following the recommendations set by the International Society for Advancement of Cytometry [51]. In this study, the Cell Lab Quanta SC™ flow cytometer was used (Beckman Coulter, Fullerton, CA, USA), and particles were excited with an argon laser (488 nm) at a power of 22 mW. Firstly, sperm concentration was adjusted to 1 × 106 sperm/mL. Before starting to use the cytometer, the electronic volume (EV) channel was calibrated with 10-µm-diameter fluorescent beads (Beckman Coulter) following the provider’s instructions. The analyzer threshold was established to exclude cell aggregates (particles with a diameter >12 µm) and cell debris (particles with a diameter <7 µm), and the flow rate was set at 4.17 µL/min. Based on EV and side scatter (SS) distributions, sperm cells were gated. Three different optical filters were used (FL1 for the analysis of H2 DCFDA, SYBR14, YO-PRO-1, JC1 monomers, and Fluo3, detection width: 505–545 nm; FL2 for the analysis of JC1 aggregates, detection width: 560–590 nm; FL3 for the analysis of PI, HE, and M540, detection width: 655–685 nm). Compensation was used when required to minimize fluorescence spill-over into a different channel. All events information was collected in List-mode Data files (EV, SS, FL1, FL2, and FL3) and processed using the Cell Lab Quanta SC MPL Analysis Software (version 1.0; Beckman Coulter). Data were corrected in all assessments based on the percentage of debris particles (SYBR14– /propidium iodide (PI)– ) determined through SYBR14/PI staining, as previously described by Petrunkina et al. [52],. All fluorochromes used were purchased from Molecular Probes® (Invitrogen® , Thermo Fisher Scientific, Waltham, MA, USA) and diluted in dimethyl sulfoxide (DMSO). 2.6.1. Evaluation of Sperm Plasma Membrane Integrity Sperm plasma membrane integrity was assessed using the LIVE/DEAD Sperm Viabil- ity Kit (SYBR14, viable spermatozoa, and PI, non-viable spermatozoa), following a protocol modified for donkey sperm, adapted from Garner and Johnson [53]. Briefly, sperm samples were incubated first with SYBR14 (final concentration: 100 nM) for 10 min at 38 ◦ C and later with PI (final concentration: 12 mM) for 5 min at 38 ◦ C. All incubations were performed protecting samples from the light. To measure the green fluorescence from SYBR14, FL1 was used, and to detect the red fluorescence from PI, FL3 was used. Three sperm popula- tions were identified: (1) sperm with an intact plasma membrane (SYBR14+ /PI− ; viable sperm); (2) sperm with a damaged plasma membrane (SYBR14− /PI+ ); and (3) sperm with a damaged plasma membrane (SYBR14+ /PI+ ). Non-sperm debris particles were identified as those that were not stained with either SYBR14 or PI (SYBR14− /PI− ). SYBR14 spill-over into the PI channel was compensated (2.45%). 2.6.2. Evaluation of Membrane Lipid Disorder Membrane lipid disorder was determined through staining with merocyanine 540 (M540) and YO-PRO-1 fluorochromes. Sperm were incubated with M540 (final concentration: 2.6 mM) and YO-PRO-1 (used as a vital stain; final concentration: 25 nM) at 38 ◦ C for Antioxidants 2022, 11, 417 6 of 22 10 min in the dark [54]. Four sperm populations were detected: (1) viable sperm with low membrane lipid disorder (M540− /YO-PRO-1− ); (2) viable sperm with high mem- brane lipid disorder (M540+ /YO-PRO-1− ); (3) non-viable sperm with low membrane lipid disorder (M540− /YO-PRO-1+ ); and (4) non-viable sperm with high membrane lipid dis- order (M540+ /YO-PRO-1+ ). Percentages of debris particles found in SYBR14/PI staining (SYBR14− /PI− ) were subtracted from those of viable sperm with low membrane lipid disorder (M540− /YO-PRO-1− ); thereafter, the percentages of all sperm populations were recalculated. 2.6.3. Evaluation of Mitochondrial Membrane Potential The MMP was determined using JC-1 (5,50,6,60-tetrachloro-1,10,3,30 tetraethyl-benz- imidazolylcarbocyanine iodide). Under dark conditions, sperm samples were stained with JC-1 (final concentration: 0.5 mM) at 38 ◦ C for 30 min. When mitochondrial membrane potential is low, JC-1 monomers emit green fluorescence (JC-1mon ), which is collected through FL1. If mitochondrial membrane potential is high, JC-1 forms aggregates emitting orange fluorescence (JC-1agg ), which is detected through FL2. Three sperm populations were distinguished: (1) green-stained sperm with low mitochondrial membrane potential (low MMP); (2) orange-stained sperm (high MMP); and (3) sperm with heterogeneous mitochondria stained both green and orange in the same cell (intermediate MMP). Spill- over of FL1 into FL2 channel was compensated (68.50%). Percentages of debris particles found in SYBR14/PI staining (SYBR14− /PI− ) were subtracted from those of sperm with low MMP, and percentages of all sperm populations were recalculated. 2.6.4. Evaluation of Intracellular ROS Levels Intracellular ROS levels were determined through two oxidation-sensitive fluorescent probes: hydroethidine (HE) and 2,7-dichlorodihydrofluorescein and diacetate (H2 DCFDA), which detect superoxide anion (·O2 − ) and overall ROS, respectively [55]. Following a modified procedure from Guthrie and Welch [56], a simultaneous differentiation of viable and non-viable sperm was performed using YO-PRO-1 (HE) or PI (H2 DCFDA). For ·O2 − , sperm samples were mixed with Hydroethidine (HE, final concentration: 4 mM) and YO-PRO-1 (final concentration: 25 nM) and then incubated at 25 ◦ C for 30 min in the dark. Oxidation of HE to ethidium (E+ ) was detected through FL3 (red fluorescence), and green fluorescence from YO-PRO-1 was collected through FL1. Four sperm populations were identified: (1) non-viable sperm with low ·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) viable sperm with high ·O2 − levels (E+ /YO-PRO-1− ). YO-PRO-1 spill-over into the FL3 channel was compensated (5.06%). For overall ROS, sperm samples were stained with H2 DCFDA (final concentration: 140 mM) and PI (final concentration: 12 mM) and incubated at 25 ◦ C for 30 min in the dark. Oxidation of H2 DCFDA to dichlorofluorescein (DCF+ ) was detected through FL1 (green fluorescence), and red fluorescence from PI was collected through FL3. 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+ ). DCF+ spill-over into the FL3 channel was compensated (2.45%). 2.6.5. Evaluation of Intracellular Calcium Levels Previous studies in pig sperm found that Fluo3-acetomethoxyester fluorochrome (Fluo3) mainly stains mitochondrial calcium [57]. For this reason, we combined this fluorochrome with PI (Fluo3/PI), following the method described by Kadirvel et al. [58]. Four sperm populations were identified: (1) viable sperm with low levels of intracellular calcium (Fluo3− /PI− ); (2) viable sperm with high levels of intracellular calcium (Fluo3+ /PI− ); (3) non-viable sperm with low levels of intracellular calcium (Fluo3− /PI+ ); and (4) non-viable sperm with high levels of intracellular calcium (Fluo3+ /PI+ ). The FL3 overflow in the FL1 channel (28.72%) and the FL1 overflow in the FL3 channel (2.45%) were compensated. Antioxidants 2022, 11, 417 7 of 22 2.7. Experimental Design A total of 15 ejaculates from 15 different Catalan donkeys were used. After col- lection, each ejaculate was divided into two aliquots. The first aliquot was centrifuged (see Section 2.2) to obtain SP samples for the measurement of the activity levels of enzy- matic and non-enzymatic antioxidants and OSI. The second aliquot was cryopreserved following the previously described procedure (see Section 2.4). Before cryopreservation, sperm concentration, motility, morphology, and viability were evaluated. Ten minutes after thawing, sperm motility, plasma membrane integrity and lipid disorder, MMP, and intracellular levels of overall ROS, superoxides and calcium were evaluated in each semen sample. Based on the percentage of total motile sperm (TM) and the percentage of viable sperm (SYBR14+ /PI− ) after thawing, donkey ejaculates were classified as GFE and PFE by hierarchical clustering. 2.8. Statistical Analysis Data analyses were performed using the R statistical package (V 4.0.3, R Core Team; Vienna, Austria), and graphs were prepared with the GraphPad Prism software (V 8.4.0, GraphPad Software LLC; San Diego, CA, USA). First, Shapiro–Wilk and Levene tests were run to verify normal distribution and homogeneity of variances, respectively. If necessary, √ data were transformed using the square root of arcsin (arcsin x) to keep parametric assumptions. The minimum level of statistical significance for all analyzes was set at p ≤ 0.05. Results are shown as mean ± standard error of the mean (SEM). 2.8.1. Hierarchical Clusters and Comparison between GFE and PFE Classification of the 15 ejaculates into groups of GFE and PFE was carried out by means of a hierarchical cluster analysis of complete linkage using the Euclidean distances from the percentages of TM and viable sperm (SYBR14+ /PI− ) obtained in each sample after thawing, as described in Morató et al. [59]. The SP activity levels of enzymatic (PON1, CAT, GPX, and SOD) and non-enzymatic antioxidants (total thiol, CUPRAC, FRAP, and TEAC), as well as SP-OSI in donkey ejaculates were compared between GFE and PFE by means of a t-test for independent samples. When, even after being linearly transformed, these data did not match with parametric assumptions, the Mann–Whitney test was used as an alternative. 2.8.2. Correlation Matrix Pearson’s coefficients were calculated to develop the correlation matrix between the SP activity levels of enzymatic (PON1, CAT, GPX, and SOD) and non-enzymatic antioxidants (total thiol, CUPRAC, FRAP, and TEAC), as well as SP-OSI values in donkey ejaculates, and TM, PM, kinematic parameters (VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF), plasma membrane integrity (SYBR14+ /PI− ), MMP (JC-1agg ), intracellular ROS levels (DCF+ /PI− ), intracellular superoxides levels (E+ /YO-PRO-1− ), intracellular calcium levels (Fluo3+ /PI− ), and plasma membrane lipid disorder (M540+ /YO-PRO-1− ), obtained in each sample after thawing. 3. Results 3.1. Classification of Donkey Ejaculates into GFE and PFE Groups According to Their Post-Thaw Sperm Quality and Functionality Parameters According to TM and sperm viability (SYBR14+ /PI− ) obtained after thawing, the 15 ejaculates were classified (by hierarchical clustering, p < 0.01) into GFE (n = 8) and PFE (n = 7). The percentages of TM and viable (SYBR14+ /PI− ) sperm after thawing were significantly higher (p < 0.01 and p < 0.001, respectively; Figure 1) in GFE (48.76 ± 1.58 and 48.85 ± 1.23%, respectively) than in PFE (32.29 ± 3.17 and 30.43 ± 2.64%, respectively). ± 1.23%, respectively) than in PFE (32.29 ± 3.17 and 30.43 ± 2.64%, res The ejaculates classified as GFE presented a range of TM betwe and a range of SYBR14+/PI− between 43.16 and 55.48%, whereas the P Antioxidants 2022, 11, 417 TM and SYBR14+/PI− between 16.09 and 40.14% and between 8 of 22 20.89 tively (Tables S1 and S2). Figure Figure 1. Mean 1. Mean ± SEM of± the SEM of the of percentages percentages of total total sperm motility sperm (TM) and motility of sperm with an (TM) intact and o plasma membrane + (SYBR14 /PI − , viable +sperm) after thawing in donkey ejaculates (n = 15) plasma membrane (SYBR14 /PI−, viable sperm) after thawing inclassified donkey ejacu as good-freezability (GFE) or poor-freezability ejaculates (PFE). The dots represent the TM and as good-freezability (GFE) or poor-freezability ejaculates (PFE). The dots SYBR14+ /PI− individual values in GFE and PFE. ** p ≤ 0.01; *** p ≤ 0.001. SYBR14+/PI− individual values in GFE and PFE. ** p ≤ 0.01; *** p ≤ 0.001. The ejaculates classified as GFE presented a range of TM between 42.35 and 53.27% and a range of SYBR14+ /PI− between 43.16 and 55.48%, whereas the PFE were in ranges of TM 3.2. and Activity SYBR14 + /PI− Levels betweenof Enzymatic 16.09 and 40.14%Antioxidants and between 20.89inand Donkey 37.40%,SP respectively (Tables S1 and S2). The activity levels of PON1 and SOD in SP were significantly hi and 3.2. p < 0.05, Activity Levels ofrespectively; 0.35 in Enzymatic Antioxidants ± Donkey 0.06 andSP 2527 ± 216 IU/mL, respective in The PFE activity (0.14levels of PON1 ± 0.02 andand1760 SOD in± SP 347 were significantly IU/mL, higher in GFE (pOn respectively). < 0.01 the con and p < 0.05, respectively; 0.35 ± 0.06 and 2527 ± 216 IU/mL, respectively; Figure 2a,b) than inshowed PFE (0.14 ±higher 0.02 andactivity 1760 ± 347levels IU/mL, of CAT than respectively). that On the of GFE contrary, (0.35 the SP ± 0.04 v of PFE respectively; showed p