antioxidants Article Seminal Plasma Antioxidants Are Related to Sperm Cryotolerance in the Horse Jaime Catalán 1,2,3,4,† , Iván Yánez-Ortiz 1,2,3,† , Asta Tvarijonaviciute 5,6 , Luis Guillermo González-Aróstegui 5,6 , Camila P. Rubio 6,7 , Isabel Barranco 8 , Marc Yeste 2,3,9, *,‡ and Jordi Miró 1, *,‡ 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 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 Animal Medicine and 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; isabel.barranco@unibo.it 9 Catalan Institution for Research and Advanced Studies (ICREA), ES-08010 Barcelona, Spain * Correspondence: marc.yeste@udg.edu (M.Y.); jordi.miro@uab.cat (J.M.); Tel.: +34-972-419514 (M.Y.); +34-93-5814293 (J.M.) † These authors contributed equally to this work. Citation: Catalán, J.; Yánez-Ortiz, I.; ‡ These authors share senior authorship. Tvarijonaviciute, A.; González- Aróstegui, L.G.; Rubio, C.P.; Barranco, I.; Yeste, M.; Miró, J. Seminal Plasma Abstract: The objective of this study was to determine the relationship of enzymatic (superoxide Antioxidants Are Related to Sperm dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and Cryotolerance in the Horse. non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; Antioxidants 2022, 11, 1279. https:// cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as doi.org/10.3390/antiox11071279 well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm Academic Editor: Giulia Collodel to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to harvest the SP and assess the activity levels of antioxidants and the Received: 1 June 2022 OSI, and the second one was cryopreserved. The following post-thaw sperm quality parameters Accepted: 25 June 2022 were evaluated: sperm motility, plasma membrane and acrosome integrity, mitochondrial membrane Published: 28 June 2022 potential, intracellular levels of reactive oxygen species (ROS), and plasma membrane lipid disorder. Publisher’s Note: MDPI stays neutral Based on post-thaw total motility (TM) and plasma membrane integrity (SYBR14+ /PI− ), ejaculates with regard to jurisdictional claims in were hierarchically (p < 0.001) clustered into two groups of good (GFE) and poor (PFE) freezability. published maps and institutional affil- The SP activity levels of PON1, SOD, and TEAC were higher (p < 0.05) in GFE than in PFE, showing a iations. positive relationship (p < 0.05) with some sperm motility parameters and with plasma membrane (PON1 and TEAC) and acrosome (SOD and TEAC) integrity. In contrast, OSI was higher (p < 0.05) in the SP of PFE than in that of GFE, and was negatively correlated (p < 0.05) to some sperm motility parameters and to plasma membrane and acrosome integrity, and positively (p < 0.05) to the percent- Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. age of viable sperm with high plasma membrane lipid disorder. In conclusion, enzymatic (PON1 This article is an open access article and SOD) and non-enzymatic (TEAC) antioxidants of SP are related to horse sperm cryotolerance. distributed under the terms and In addition, our results suggest that PON1 could be one of the main antioxidant enzymes involved conditions of the Creative Commons in the control of ROS in this species. Further investigation is needed to confirm the potential use of Attribution (CC BY) license (https:// these SP-antioxidants and OSI to predict sperm cryotolerance in horses. creativecommons.org/licenses/by/ 4.0/). Antioxidants 2022, 11, 1279. https://doi.org/10.3390/antiox11071279 https://www.mdpi.com/journal/antioxidants Antioxidants 2022, 11, 1279 2 of 20 Keywords: antioxidants; cryopreservation; horse; oxidative stress; reactive oxygen species; seminal plasma; sperm 1. Introduction Sperm cryopreservation has acquired great importance in recent decades, due to the benefits offered by its use [1–3]. Freeze-thawing procedures, however, impact negatively on sperm motility, morphology, functionality, and survival [4,5], as well as on their fertilizing capacity [6]; this leads to lower pregnancy rates after artificial insemination compared to fresh or cooled semen [7,8]. During cryopreservation, a stressful environment is gener- ated for sperm [9], mainly associated with an excess of reactive oxygen species (ROS) [6] that overwhelms the sperm antioxidant defense capacity. This situation drives the oxida- tive/antioxidant balance to shift towards the oxidative state [10,11]. In this circumstance, oxidative stress underlies several sperm injuries at the level of plasma membrane (lipoper- oxidation) [12–14] and DNA (fragmentation) [11,15,16], and affects functional parameters (such as motility, viability, acrosome membrane integrity and mitochondrial membrane potential) [9,17,18]. Horse sperm are particularly susceptible to oxidative stress due to the large amount of polyunsaturated fatty acids (PUFAs) in plasma membrane phospholipids [13,19,20], and their low intracellular antioxidant activity [21–24]. Under this scenario, seminal plasma (SP) represents the main antioxidant defense source, as it is endowed with enzymatic and non-enzymatic antioxidants that scavenge excessive ROS [25–28]. Yet, current equine semen cryopreservation protocols remove SP by centrifugation [14,29]. While this step is carried out to improve post-thaw sperm quality [1,30], it entails the removal of antioxidants, thus increasing further the susceptibility of sperm to oxidative damage [24,31]. In the horse, as in other mammalian species, differences in sperm cryotolerance between breeds [32], individuals [33] and even between ejaculates from the same animal [6] have been reported. Because these differences, which allow ejaculates to be classified as with “good” (GFE) or “poor” freezability (PFE) [14,28,34], are related to the composition of SP [35,36], elucidating the function of antioxidants is very relevant. In horses, some SP antioxidant enzymes (such as superoxide dismutase (SOD), catalase (CAT) and the glutathione peroxidase/glutathione reductase system (GPX/GSR)) are responsible for reducing oxidative stress [14,26,37]. Of these enzymes, total and specific activities of SOD in horse SP are related to sperm cryotolerance [14]. Moreover, paraoxonase type 1 (PON1) is another antioxidant enzyme that has been found in the SP of humans [38,39], pigs [40] and donkeys [28]. This enzyme could also play a function, as its activity levels are known to be positively associated to several sperm quality and functionality parameters in liquid- stored pig semen [41], and frozen-thawed pig [42] and donkey [28] sperm. Regarding the non-enzymatic antioxidant capacity of SP, it has been analyzed in other mammalian species using different methodological approaches, such as cupric-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP) and Trolox equivalent antioxidant capacity (TEAC). Recently, these non-enzymatic antioxidant capacities were found to be directly involved in the resilience of pig [42] and donkey [28] sperm to freeze-thawing. A balance between ROS production and antioxidants is crucial for sperm function and survival [9,43]. In this sense, it has been established that the measurement of the oxidative stress index (OSI) accurately shows the oxidant/antioxidant ratio in a biological sample; hence, an increase in this ratio would indicate the risk of oxidative stress due to an increase in ROS production or a decrease in antioxidants [44]. This new concept has been applied to SP in other species, such as pigs [45] and donkeys [28]. In both cases, the levels of SP-OSI appear to be related to the sperms’ ability to withstand liquid storage in pigs [45] and cryopreservation in donkeys [28]. Against this background, the present study aimed to evaluate whether OSI, activities of enzymatic antioxidants (including PON1, SOD, CAT, and GPX) and capacity of non- Antioxidants 2022, 11, 1279 3 of 20 enzymatic antioxidants (measured in terms of: CUPRAC, FRAP, and TEAC) are related to horse sperm cryotolerance. 2. Materials and Methods 2.1. Stallions and Samples Ejaculates were collected from 21 stallions (one ejaculate per individual) of different breeds, mature (between 5 and 15 years old) and of proven fertility. All stallions were clinically healthy and their standard diet included mixed hay and basic concentrate, without antioxidant supplementation and with ad libitum water availability. They were housed in individual paddocks at the facilities of the Equine Reproduction Service, Autonomous University of Barcelona (Bellaterra, Cerdanyola del Vallès, Spain). This is a center, approved (authorization number: ES09RS01E) by the European Union for the collection of equine semen, that operates under rigorous health and animal welfare protocols. This Service already works under the approval of the Regional Government of Catalonia, Spain; 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 to carry out this study was necessary. Likewise, the health guidelines established by the Council of the European Communities in Directive 82/894/CEE of 21 December 1982 were complied with, as stallions were free from equine viral arteritis, equine infectious anemia and equine contagious metritis. The collection was performed using a Hannover model artificial vagina (Minitüb GmbH, Tiefenbach, Germany), previously heated to a temperature between 48 ◦ C and 50 ◦ C and coupled with an in-line nylon filter to remove the gel fraction. Once the ejaculate was obtained, the gel fraction was removed and 10 µL were taken to evaluate the sperm concentration using a Neubauer chamber (Paul Marienfeld GmbH & Co. KG, Lauda- Königshofen, Germany). After that, each ejaculate was divided into two aliquots. The first was used to harvest the SP (see Section 2.2 for more details), and the second was diluted 1:5 (v:v) in a commercial extender based on skimmed milk [46] and subsequently cryopreserved (see Section 2.5 for more details). Before cryopreservation, this second aliquot was used for sperm quality analysis (which included sperm motility, morphology and viability). Sperm motility was analyzed using a Computer-Aided Sperm Analysis (CASA) system (see Section 2.6 for more details), and sperm viability and morphology through an eosin-nigrosine staining [47]. All semen samples used in this study fulfilled the standard thresholds for sperm quality before freezing (>80% total motile sperm, >60% viable sperm, and >70% morphologically normal sperm). 2.2. Seminal Plasma (SP) Collection Right after collection, ejaculates were centrifuged five times at 1500× g and 4 ◦ C for 10 min (Medifriger BL-S; JP Selecta S.A., Barcelona, Spain). The supernatant was examined under a phase contrast microscope (Olympus Europe; Hamburg, Germany) at 200× to verify the absence of sperm. Finally, 5 mL of each SP sample was stored at −80 ◦ C until analysis. Once all SP samples were procured and stored, they were thawed on ice to measure the activity levels of enzymatic and non-enzymatic antioxidants at the same time. 2.3. Measurement of Enzymatic and Non-Enzymatic Antioxidant Levels in SP Enzymatic antioxidants assessed in SP were PON1, SOD, CAT, and GPX. Activity levels of PON1 were measured following the protocol described by Barranco et al. [40] adapted to horse SP, by measuring the hydrolysis of p-nitrophenyl acetate into p-nitrophenol. For the measurement of SOD, CAT, and GPX activity, commercially available assays were used following the manufacturer’s instructions (CAT: Merck, Darmstadt, Germany; GPX and SOD: Randox, Crumlin, UK). Determination of PON1, SOD, and GPX activity was conducted using an Olympus AU400 automated chemistry analyzer (Olympus Europe GmbH, Hamburg, Germany), whereas that of CAT was performed using a microplate reader Antioxidants 2022, 11, 1279 4 of 20 (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA). Activity levels of PON1 and GPX were expressed as IU/L, whereas those of SOD and CAT were expressed as IU/mL. Non-enzymatic antioxidant capacity of SP was measured in terms of CUPRAC, FRAP, and TEAC. All of them were assessed following the protocol described by Li et al. [42], adapted to horse SP. These assays are based on the reduction of Cu2+ to Cu+ (CUPRAC) [48] and of Fe3+ to Fe2+ (FRAP) [49], as well as a color change by 2,20 - azinobis-3-ethylbenzothiazoline-6-sulfonate (TEAC) [10]. All these determinations were performed using an Olympus AU400 automated chemistry analyzer. Activity levels of CUPRAC, FRAP, and TEAC were expressed as mmol Trolox equivalent/L. The analytes from each SP sample were measured in duplicate. In each test, the intra and inter-assay coefficient showed a variation below 10%. 2.4. Measurement of Oxidative Stress Index (OSI) in SP The levels of SP-OSI were calculated as follows: OSI (arbitrary unit) = Total oxidative status (TOS, µmol H2 O2 equivalent/L)/TEAC (mmol Trolox equivalent/L) [28,50]. TOS was measured following the protocol described by Erel [51], adapted to horse SP. This assay is based on the oxidation of the ferrous ion to the ferric ion in the presence of oxidants in an acid medium, and the measurement of the ferric ion by xylenol orange. An Olympus AU400 automated chemistry analyzer was used for this evaluation. TOS results were expressed as µmol H2 O2 equivalent/L. 2.5. Sperm Cryopreservation Prior to cryopreservation, each extended semen sample was centrifuged at 660× g and 20 ◦ C for 15 min. Thereafter, the supernatant was discarded and the pellet was resuspended in a commercial freezing extender (BotuCRIO® ; Botupharma Animal Biotech- nology, Botucatu, Brazil) containing 1% glycerol and 4% methylformamide as permeable cryoprotectants. After analyzing the sperm concentration and viability in each sample, the same freezing medium was added to obtain a final concentration of 200 × 106 viable spermatozoa/mL. Finally, samples loaded into 0.5-mL straws were cooled/frozen using an automatic controlled-rate freezer (Ice-Cube 14S; Minitüb GmbH, Tiefenbach, Germany); the freezing curve included the following three stages: (1) cooling of 20 ◦ C to 5 ◦ C for 60 min at a rate of −0.25 ◦ C/min, (2) freezing of 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 −11.11 ◦ C/min. Once this process was completed, straws were plunged into liquid nitrogen at −196 ◦ C and stored in appropriate tanks for conservation. Thawing was carried out in a circulating water bath at 37 ◦ C for 30 s; the content of each straw was poured into a 10-mL conical tube and further diluted (1:2, v/v) with Kenney extender [46], pre-warmed at 37 ◦ C. Sperm quality and functionality parameters assessed in each frozen-thawed sample were: (1) motility, (2) plasma membrane and (3) acrosome integrity, (4) membrane lipid disorder, (5) mi- tochondrial membrane potential (MMP), and intracellular levels of (6) overall ROS and (7) superoxides (see Sections 2.6 and 2.7 for more details). 2.6. Sperm Motility Analysis Sperm motility analysis was performed using the CASA-Mot module of the ISAS® v1.2 system (Proiser R + D, Valencia, Spain). This system consists of a high-resolution digital camera (model MQ003MG-CM; Proiser R + D) that captures up to 100 frames per second (fps). A reusable Spermtrack® 10 chamber (Spk 10; Proiser R + D) pre-warmed to 37 ◦ C was used, in which 2 µL of each sample was placed. Using a 10× negative phase contrast microscope (model UOP200i; Proiser R + D), a minimum of 500 spermatozoa were counted per analysis. The analysis included total (TM, %) and progressive (PM, %) motility, together with the kinematic parameters that define sperm movement: straight line velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), average path velocity (VAP, µm/s), straightness coefficient (STR = [VSL/VAP] × 100, %), linearity coefficient (LIN = [VSL/VCL] × 100, %), wobble coefficient (WOB = [VAP/VCL] × 100, %), beat-cross frequency (BCF, Hz), and Antioxidants 2022, 11, 1279 5 of 20 amplitude of lateral head displacement (ALH, µm). In all the analyses, the CASA-Mot settings recommended by the manufacturer were used (particle area > 4 and < 75 µm2 , connectivity = 6, minimum number of images to calculate ALH = 10) and cut-off values were taken for total (VAP ≥ 10 µm/s) and progressive (STR ≥ 75%) motility. Three replicates per sample were examined. 2.7. Sperm Functionality Analysis Sperm functionality parameters were analyzed by flow cytometry and included: plasma membrane integrity (SYBR14/PI), acrosome integrity (Arachis hypogaea (peanut) agglutinin-fluorescein isothiocyanate (PNA-FITC)/Propidium iodide (PI)), mitochondrial membrane potential (MMP; 5,50 ,6,60 -tetrachloro-1,10 3,30 tetraethyl-benzimidazolylcarbo- cyanine iodide (JC-1)), intracellular levels of ROS (2,7-dichlorodihydrofluorescein and diac- etate (H2 DCFDA)/PI) and superoxides (hydroethidine (HE)/1-(4-[3-methyl-2,3-dihydro- (benzo-1,3-oxazole)-2-methylidene]-quinolinium)-3-trimethylammonium propane diodide (YO-PRO-1)), and plasma membrane lipid disorder (Merocyanine 540 (M540)/YO-PRO-1). The flow cytometer used was a CytoFLEX (Beckman Coulter Fullerton, CA, USA) with a sheath flow rate set at 10 µL/min. Fluorochromes were purchased from Molecular Probes® (Thermo Fisher Scientific, Waltham, MA, USA) and were resuspended in dimethyl sulfoxide (DMSO; Merck). Analyses were performed following the recommendations of the International Society for Advance Cytometry (ISAC) [52]. Prior to staining, sperm concentration was adjusted to 1 × 106 sperm/mL. In each sample, a total of 10,000 events were analyzed and three technical replicates were evaluated. Samples were excited with an argon ion laser (488 nm) at a power of 50 mW. Dis- tributions of two different dot plots were used to exclude (1) cellular aggregates, based on the dot plot distribution of forward scatter height (FSC-H) and altitude (FSC-A), and (2) cellular debris, as a function of FSC-A distribution and side scatter altitude (SSC-A) dot plots. Four different optical filters were used: (1) FITC with a band-pass of 525-540 nm for analysis of SYBR14, PNA-FITC, JC-1 monomers (JC-1mon ), dichlorofluorescein (DCF+ ) and (YO-PRO-1), (2) PE with a band-pass of 585-542 nm for analysis of JC-1 aggregates (JC-1agg ) and fluorescent ethidium (E+ ), (3) ECD with a band-pass of 610–620 nm for analysis of M540, and (4) PC5.5 with a band-pass of 690–650 nm for analysis of PI. The information obtained regarding each event (FSC-A, FSC-H, SSC-A, FITC, PE and PC5.5) was collected in xit files and analyzed using the CytExpert analysis software (Beckman Coulter Fullerton) to quantify sperm populations. For each parameter, the corresponding mean and standard error of the mean (SEM) were calculated. 2.7.1. Plasma Membrane Integrity (SYBR14/PI) Plasma membrane integrity of sperm was analyzed using the LIVE/DEAD sperm viability kit (SYBR14/PI), according to the protocol described by Garner and Johnson [53], adapted to horse sperm. Briefly, semen samples were incubated with SYBR14 (final concen- tration: 31.8 nM) for 10 min and then with PI (final concentration: 7.6 µM) for 5 min in the dark at 37 ◦ C. Three sperm populations were identified: (1) sperm with a damaged plasma membrane (SYBR14+ /PI+ ), (2) sperm with a damaged plasma membrane (SYBR14− /PI+ ), and (3) sperm with an intact plasma membrane (SYBR14+ /PI− ; viable sperm). Particles without staining (SYBR14− /PI− ) were considered as non-sperm debris and were used to correct the data in the other assessments. SYBR14 overflow in channel PC5.5 (8.34%) was compensated. 2.7.2. Acrosome Integrity (PNA-FITC/PI) Acrosome integrity of sperm was analyzed using the combination of PNA-FITC and PI, according to the protocol described by Rathi et al. [54]. Briefly, semen samples were incubated in the dark at 37 ◦ C for 10 min with PNA conjugated with FITC (final concen- tration: 1.17 µg/mL) and with PI (final concentration: 5.6 µM). Four sperm populations were identified: (1) sperm with damaged plasma membrane (PNA-FITC+ /PI− ), (2) sperm Antioxidants 2022, 11, 1279 6 of 20 with damaged plasma membrane together with fully-lost outer acrosome membrane (PNA- FITC− /PI+ ), (3) sperm with a damaged plasma membrane that exhibited an outer acrosome membrane that could not be completely intact (PNA-FITC+ /PI+ ), and (4) viable sperm with an intact acrosome membrane (PNA-FITC− /PI− ). No compensation was needed. 2.7.3. Mitochondrial Membrane Potential (JC-1) Mitochondrial membrane potential (MMP) of sperm was analyzed using JC-1, accord- ing to the protocol described by Ortega-Ferrusola et al. [55]. In brief, semen samples were incubated with JC-1 (final concentration: 750 nM) at 37 ◦ C for 30 min in the dark. JC-1 molecules form orange fluorescent aggregates (JC-1agg ) in the presence of high MMP, while remaining as green fluorescent monomers (JC-1mon ) in the presence of low MMP. Two sperm populations were identified: (1) sperm exhibiting low MMP (JC-1mon fluorescence intensity higher than JC-1agg ), and (2) sperm exhibiting high MMP (JC-1agg fluorescence intensity higher than JC-1mon ). In each population, the fluorescence intensity of JC-1mon and JC-1agg was recorded and the ratio between them was calculated. Data were not compensated. 2.7.4. Intracellular Reactive Oxygen Species: Total ROS (H2 DCFDA/PI) and O2 − (HE/YO-PRO-1) Intracellular ROS levels of sperm were analyzed using oxidation-sensitive fluores- cent probes: H2 DCFDA to assess overall ROS and HE to measure superoxide anion (O2 − ) [56]. The differentiation of viable and non-viable spermatozoa was performed using PI (for H2 DCFDA) or YO-PRO-1 (for HE), following the modified protocol of Guthrie and Welch [57]. For overall ROS measurement, semen samples were incubated with H2 DCFDA (final concentration: 50 µM) at 37 ◦ C in the dark for 20 min, and then with PI (final concentration: 6 µM) for 5 min. In presence of ROS, there is a de-esterification and oxidation of H2 DCFDA to DCF+ , which is a highly fluorescent molecule. Four sperm populations were identified: (1) non-viable sperm with low ROS levels (DCF− /PI+ ), (2) viable sperm with low ROS levels (DCF− /PI− ), (3) non-viable sperm with high ROS levels (DCF+ /PI+ ), and (4) viable sperm with high ROS levels (DCF+ /PI− ). DCF+ fluorescence intensity was recorded in all sperm populations. Data were not compensated. For O2 − analysis, semen samples were incubated with HE (final concentration: 5 µM) and YO-PRO-1 (final concentration: 31.25 nM) at 37 ◦ C in the dark for 30 min. In the pres- ence of O2 - , HE is oxidized into E+ . Four sperm populations were identified: (1) non-viable sperm with low O2 − levels (E− /YO-PRO-1+ ), (2) viable sperm with low O2 − levels (E− /YO- PRO-1− ), (3) non-viable sperm with high O2 − levels (E+ /YO-PRO-1+ ), and (4) viable sperm with high O2 − levels (E+ /YO-PRO-1− ). Fluorescence intensity of E+ was recorded in all sperm populations. The overflow of E+ to the FITC channel (3.62%) was compensated. 2.7.5. Plasma Membrane Lipid Disorder (M540/YO-PRO-1) Plasma membrane lipid disorder of sperm was analyzed using the combination of M540/YO-PRO-1, according to the protocol described by Rathi et al. [54] with minor modifications [58], adapted to horse sperm. Briefly, semen samples were incubated in the dark at 37 ◦ C for 10 min with M540 (final concentration: 2.5 µM) and YO-PRO-1 (final concentration: 25 nM). Four sperm populations were identified: (1) viable sperm with high plasma membrane lipid disorder (M540+ /YO-PRO-1− ), (2) non-viable sperm with high plasma membrane lipid disorder (M540+ /YO-PRO-1+ ), (3) viable sperm with low plasma membrane lipid disorder (M540− /YO-PRO-1− ), and (4) non-viable sperm with low plasma membrane lipid disorder (M540− /YO-PRO-1+ ). Data were not compensated. 2.8. Statistical Analysis The analysis of the data and the elaboration of the figures was carried out using the statistical package R (V 4.0.3, R Core Team; Vienna, Austria) and the GraphPad Prism software (V 8.4.0, GraphPad Software LLC; San Diego, CA, USA), respectively. The first step Antioxidants 2022, 11, 1279 7 of 20 was to check the normality of data using the Shapiro–Wilk test, as well as the homogeneity √ of variances using the Levene test. When necessary, arcsine x was applied to transform data and thus obtain a normal distribution. In all cases, the minimum level of statistical significance was set at p ≤ 0.05. 2.8.1. Classification of Ejaculates Based on Their Cryotolerance Classification of the 21 horse ejaculates based on their cryotolerance (GFE and PFE) was performed following the procedure described by Morató et al. [34]. In brief, post- thaw percentages of TM and of sperm with an intact plasma membrane (sperm viability, SYBR14+ /PI− ) recorded in each sample were taken to perform a complete linkage hierar- chical cluster analysis using Euclidean distances from the mentioned parameters. Results in the text are expressed as means ± SEM. 2.8.2. Comparison of Antioxidant Variables Measured in SP between Good (GFE) and Poor Freezability Ejaculates (PFE) Comparison between GFE and PFE regarding activity levels of enzymatic (PON1, SOD, CAT, and GPX) and non-enzymatic (CUPRAC, FRAP, and TEAC) antioxidants in SP, and SP-OSI was performed using a t-test for independent samples. When, even after transforming the data, a normal distribution was not obtained, the Mann–Whitney test was used as a non-parametric alternative. Results in the text were expressed as means ± SEM. 2.8.3. Correlations of Antioxidant Variables with Post-Thaw Semen Parameters The relationship of activity levels of enzymatic antioxidants (PON1, SOD, CAT, and GPX) and non-enzymatic antioxidants (CUPRAC, FRAP, and TEAC) in SP, and SP-OSI with motility parameters (TM, PM, VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF) and with sperm functionality parameters (SYBR14+ /PI− , PNA-FITC− /PI− , JC-1agg , DCF+ /PI− , E+ /YO-PRO-1− , and M540+ /YO-PRO-1− ) was evaluated by calculating Pearson’s correla- tion coefficients. 3. Results 3.1. Classification of Horse Ejaculates Based on Their Cryotolerance Classifying the 21 horse ejaculates through hierarchical clusters (p < 0.001) based on post-thaw TM and sperm viability (SYBR14+ /PI− ) separated 13 GFE from 8 PFE. Figure 1 shows that the ejaculates classified as GFE exhibited significantly higher val- ues (p < 0.001) of both TM and sperm viability (SYBR14+ /PI− ) compared to those classified as PFE (66.44 ± 2.61% vs. 35.54 ± 4.36%, and 67.90 ± 1.54% vs. 42.03 ± 2.49%, respec- tively). The ranges of TM and sperm viability (SYBR14+ /PI− ) in both groups of ejaculates are shown in Tables S1 and S2, respectively. 3.2. Activity Levels of Enzymatic Antioxidants in the SP of Horse Ejaculates Classified as GFE and PFE The ejaculates classified as GFE showed significantly higher activity levels (p < 0.05) of PON1 and SOD in their SP than those classified as PFE (0.80 ± 0.08 IU/L vs. 0.58 ± 0.06 IU/L, and 245.23 ± 35.52 IU/mL vs. 171.38 ± 24.19 IU/mL, respectively, Figure 2a,b). No differences regarding the activity levels of CAT and GPX in SP (Figure 2c,d) were found between GFE and PFE. The mean ± SEM and ranges of the activity levels of enzymatic antioxidants measured in the SP of all horse ejaculates are shown in Table S3. Antioxidants 2022, 11, x FOR PEER REVIEW 8 of 20 Antioxidants 2022, 11, 1279 8 of 20 Figure 1. Box-whisker plot showing the percentages of total motile (TM) and viable (SYBR14+/PI−) sperm after thawing in horse ejaculates classified as with good (GFE, n = 13; blue) or poor freezabil- ity (PFE, n = 8; orange). The boxes enclose the 25th and 75th percentiles, the whiskers extend to the 5th and 95th percentiles, and the line indicates the median. (***) p ≤ 0.001. 3.2. Activity Levels of Enzymatic Antioxidants in the SP of Horse Ejaculates Classified as GFE and PFE The ejaculates classified as GFE showed significantly higher activity levels (p < 0.05) of PON1 Figure and SOD inplot 1. Box-whisker their SP thanthe showing those classifiedofastotal percentages PFEmotile (0.80 ±(TM) 0.08 IU/L vs. 0.58 and viable ± 0.06 + /PI− ) (SYBR14 IU/L, and 245.23 ± 35.52 IU/mL vs. 171.38 ± 24.19 IU/mL, respectively, Figure 2a,b). No sperm after thawing in horse ejaculates classified as with good (GFE, n = 13; blue) or poor freezability differences regarding the activity levels of CAT and GPX in SP (Figure 2c,d) were found (PFE, n = 8; orange). The boxes enclose the 25th and 75th percentiles, the whiskers extend to the 5th between GFE and PFE. The mean ± SEM and ranges of the activity levels of enzymatic and 95th percentiles, and the line indicates the median. (***) p ≤ 0.001. antioxidants measured in the SP of all horse ejaculates are shown in Table S3. Antioxidants 2022, 11, x FOR PEER REVIEW 9 of 20 (a) (b) (c) (d) Figure Figure 2. 2. Box-whisker Box-whisker plot plot showing showing the the activity levels (IU/mL) activity levels (IU/mL) ofofenzymatic enzymatic antioxidants antioxidants measured measured 1in the seminal plasma of horse ejaculates classified as with good (GFE, n = 13; blue) or poor freeza- in the seminal plasma of horse ejaculates classified as with good (GFE, n = 13; blue) or poor freezability bility (PFE, n = 8; orange): paraoxonase type 1 (PON1, a), superoxide dismutase (SOD, b), catalase (PFE, n = 8; orange): paraoxonase type 1 (PON1, a), superoxide dismutase (SOD, b), catalase (CAT, c), (CAT, c), and glutathione peroxidase (GPX, d). The boxes enclose the 25th and 75th percentiles, the and glutathione whiskers extend peroxidase (GPX, to the 5th and The boxes and d).percentiles, 95th enclose the 25th the line and 75th indicates percentiles, the median. (*) pthe whiskers ≤ 0.05. extend to the 5th and 95th percentiles, and the line indicates the median. (*) p ≤ 0.05. 3.3. Activity Levels of Non-Enzymatic Antioxidants in the SP of Horse Ejaculates Classified as GFE and PFE The SP-levels of non-enzymatic antioxidants measured in terms of CUPRAC and FRAP did not differ between GFE and PFE (Figure 3a,b). The levels of TEAC in SP, how- (c) (d) Figure 2. Box-whisker plot showing the activity levels (IU/mL) of enzymatic antioxidants measured in the seminal plasma of horse ejaculates classified as with good (GFE, n = 13; blue) or poor freeza- bility (PFE, n = 8; orange): paraoxonase type 1 (PON1, a), superoxide dismutase (SOD, b), catalase Antioxidants 2022, 11, 1279 (CAT, c), and glutathione peroxidase (GPX, d). The boxes enclose the 25th and 75th percentiles, 9 ofthe 20 whiskers extend to the 5th and 95th percentiles, and the line indicates the median. (*) p ≤ 0.05. 3.3. Activity 3.3. Activity Levels of Non-Enzymatic Levels of Non-Enzymatic Antioxidants Antioxidants in in the the SP SP of of Horse Horse Ejaculates Ejaculates Classified Classified asas GFE and PFE The SP-levels of non-enzymatic antioxidants measured in terms of CUPRAC and FRAP diddid not notdiffer differbetween betweenGFE GFEand PFE and (Figure PFE 3a,b). (Figure The levels 3a,b). of TEAC The levels of TEACin SP, inhow- SP, ever, werewere however, significantly higher significantly (p < 0.001) higher in ejaculates (p < 0.001) classified in ejaculates as GFE classified than than as GFE in those clas- in those sified as PFE classified (0.83 as PFE (0.83 ± 0.06 ± 0.06 mmol/L vs. 0.47 mmol/L 0.05±mmol/L, vs. ±0.47 respectively; 0.05 mmol/L, FigureFigure respectively; 3c). The 3c).mean The ± SEM±and mean SEM ranges of non-enzymatic and ranges antioxidants of non-enzymatic measured antioxidants in the measured inSP theofSPallofejaculates are all ejaculates are shownshown in Table in Table S3. S3. Antioxidants 2022, 11, x FOR PEER REVIEW 10 of 20 (a) (b) (c) Figure 3.3. Box-whisker Figure Box-whisker plot plot showing showing non-enzymatic non-enzymatic antioxidants antioxidants in in the the seminal seminal plasma plasma ofof horse horse ejaculates classified as with good (GFE, n = 13; blue) or poor freezability (PFE, n = 8; orange), meas- ejaculates classified as with good (GFE, n = 13; blue) or poor freezability (PFE, n = 8; orange), ured as cupric-reducing antioxidant capacity (CUPRAC, a), ferric-reducing ability of plasma (FRAP, measured as cupric-reducing antioxidant capacity (CUPRAC, a), ferric-reducing ability of plasma b), and Trolox equivalent antioxidant capacity (TEAC, c). The boxes enclose the 25th and 75th per- (FRAP, and b),the centiles, Trolox equivalent whiskers antioxidant extend to the capacity 5th and 95th (TEAC, c). percentiles, andThe theboxes enclose the line indicates the25th and 75th median. (***) percentiles, p ≤ 0.001. the whiskers extend to the 5th and 95th percentiles, and the line indicates the median. (***) p ≤ 0.001. 3.4. Oxidative Stress Index in the SP of Horse Ejaculates Classified as GFE and PFE 3.4. Oxidative Stress Index in the SP of Horse Ejaculates Classified as GFE and PFE Ejaculates classified as GFE showed significantly lower SP-OSI levels (p < 0.001) com- Ejaculates classified as GFE showed significantly lower SP-OSI levels (p < 0.001) pared to those classified as PFE (6.35 ± 0.48 vs. 13.03 ± 1.80; Figure 4). The SP levels of TOS compared to those classified as PFE (6.35 ± 0.48 vs. 13.03 ± 1.80; Figure 4). The SP levels of in ejaculates classified as GFE were 4.99 ± 0.18 µ mol/L, and in those classified as PFE were TOS in ejaculates classified as GFE were 4.99 ± 0.18 µmol/L, and in those classified as PFE 5.53 ±5.53 were 0.31±µ mol/L (p > 0.05). 0.31 µmol/L (p >The mean 0.05). ± SEM The meanand ranges ± SEM andofranges levels OSI and TOS of levels OSImeasured and TOS in the SP of all horse ejaculates are shown in Table S3. measured in the SP of all horse ejaculates are shown in Table S3. 3.4. Oxidative Stress Index in the SP of Horse Ejaculates Classified as GFE and PFE Ejaculates classified as GFE showed significantly lower SP-OSI levels (p < 0.001) com- pared to those classified as PFE (6.35 ± 0.48 vs. 13.03 ± 1.80; Figure 4). The SP levels of TOS in ejaculates classified as GFE were 4.99 ± 0.18 µ mol/L, and in those classified as PFE were Antioxidants 2022, 11, 1279 10 of 20 5.53 ± 0.31 µ mol/L (p > 0.05). The mean ± SEM and ranges of levels OSI and TOS measured in the SP of all horse ejaculates are shown in Table S3. Figure4.4.Box-whisker Figure Box-whiskerplot plotshowing showingoxidative oxidativestress stressindexes indexes(OSI) (OSI)(arbitrary (arbitraryunits) units)measured measuredininthe the seminal plasma of horse ejaculates classified as with good (GFE, n = 13; blue) or poor freezability seminal plasma of horse ejaculates classified as with good (GFE, n = 13; blue) or poor freezability (PFE, n = 8; orange). The boxes enclose the 25th and 75th percentiles, the whiskers extend to the 5th (PFE, n = 8; orange). The boxes enclose the 25th and 75th percentiles, the whiskers extend to the 5th and 95th percentiles, and the line indicates the median. (***) p ≤ 0.001. and 95th percentiles, and the line indicates the median. (***) p ≤ 0.001. 3.5.Correlations 3.5. Correlationsbetween betweenSP SPAntioxidants Antioxidants(Enzymatic, (Enzymatic,Non-Enzymatic Non-Enzymaticand andOSI) OSI)and andPost-Thaw Post- Thaw Sperm Motility Sperm Motility Parameters Parameters Figure5 5shows Figure showsthethe correlations correlations of enzymatic of enzymatic andand non-enzymatic non-enzymatic antioxidants, antioxidants, and and OSI OSI levels measured in the SP of horse ejaculates with post-thaw sperm levels measured in the SP of horse ejaculates with post-thaw sperm motility parameters. motility parame- ters. Activity Activity levels levels of four of four enzymatic enzymatic antioxidants antioxidants (PON1, SOD,(PON1, SOD, CAT, and CAT, GPX) and wereGPX) were positively positively(pcorrelated correlated (p