antioxidants Article Measurement of Oxidative Stress Index in Seminal Plasma Can Predict In Vivo Fertility of Liquid-Stored Porcine Artificial Insemination Semen Doses Isabel Barranco 1 , Camila P. Rubio 2,3 , Asta Tvarijonaviciute 3,4 , Heriberto Rodriguez-Martinez 5 and Jordi Roca 4, * 1 Department of Veterinary Medical Sciences, University of Bologna, 40064 Ozzano dell’Emilia, Bologna, Italy; isabel.barranco@unibo.it 2 Department of Animal and Food Science, School of Veterinary Science, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain; camila.peres@uab.cat 3 Interdisciplinary Laboratory of Clinical Analysis Interlab-UMU, Faculty of Veterinary Medicine, Regional Campus of International Excellence ‘Campus Mare Nostrum’, University of Murcia, 30100 Murcia, Spain; asta@um.es 4 Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, University of Murcia, 30100 Murcia, Spain 5 Department of Biomedical & Clinical Sciences (BKV), BKH/Obstetrics & Gynaecology, Faculty of Medicine and Health Sciences, Linköping University, 58185 Linköping, Sweden; heriberto.rodriguez-martinez@liu.se * Correspondence: roca@um.es   Abstract: The study evaluated the relation between the oxidative stress index (OSI) in porcine Citation: Barranco, I.; Rubio, C.P.; seminal plasma (n = 76) with sperm resilience and in vivo fertility (farrowing rate and litter size Tvarijonaviciute, A.; Rodriguez- of 3137 inseminated sows) of liquid-stored artificial insemination (AI) semen doses. The OSI was Martinez, H.; Roca, J. Measurement of assessed as the ratio of advanced oxidation protein products to Trolox-equivalent antioxidant capacity, Oxidative Stress Index in Seminal both measured using an automated analyzer. Sperm motility (computer-assisted sperm analyzer) Plasma Can Predict In Vivo Fertility and viability (flow cytometry) were evaluated in semen AI-doses at 0 and 72 h of storage at 17 ◦ C. of Liquid-Stored Porcine Artificial Sperm resilience was defined as the difference between storage intervals. Semen AI-doses were Insemination Semen Doses. hierarchically clustered as having high, medium and low seminal OSI (p < 0.001) with those of Antioxidants 2021, 10, 1203. low displaying higher resilience (p < 0.01). Boars were hierarchically clustered into two groups https://doi.org/10.3390/ (p < 0.001) as having either positive or negative farrowing rate and litter size deviation; the negative antiox10081203 one showing higher seminal OSI (p < 0.05). In sum, seminal OSI was negatively related to sperm Academic Editor: Nicole McPherson motility and the in vivo fertility of liquid-stored boar semen AI-doses, with the receiver operating characteristic curve presenting seminal OSI as a good predictive biomarker of in vivo fertility of Received: 2 July 2021 AI-boars (area under the curve: 0.815, p < 0.05). Accepted: 26 July 2021 Published: 27 July 2021 Keywords: in vivo fertility; oxidative stress index; pig; seminal plasma; sperm quality Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. The aerobic metabolism of spermatozoa generates reactive oxygen species (ROS) which, in controlled amounts, are essential for sperm physiological processes, including capacitation, acrosome reaction, sperm–oocyte interaction, and further subsequent implan- tation and embryo development [1,2]. However, when ROS generation is excessive beyond Copyright: © 2021 by the authors. the control of seminal plasma antioxidant mechanisms, spermatozoa experience oxidative Licensee MDPI, Basel, Switzerland. stress (OS), which impairs their functionality and even leads to their death [2]. Specifically, This article is an open access article this imbalance between ROS and antioxidants induces the peroxidation of lipids of the distributed under the terms and sperm plasma membrane (lipid peroxidation, LPO), resulting in a loss of sperm motility conditions of the Creative Commons and membrane fluidity and integrity [3]. Moreover, the highly reactive molecules produced Attribution (CC BY) license (https:// as result of LPO are also capable of causing sperm DNA damage, which have harmful creativecommons.org/licenses/by/ effects on their fertilizing ability and subsequent embryo development [4]. 4.0/). Antioxidants 2021, 10, 1203. https://doi.org/10.3390/antiox10081203 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 1203 2 of 12 Boar spermatozoa are especially vulnerable to OS due to their limited antioxidant defense mechanism in their cytoplasm [5] and the high concentration of polyunsaturated fatty acids in their plasma membrane [6], which, due to their double bonds, makes them particularly sensitive to ROS-induced damage mediated by LPO [2]. In this scenario, seminal plasma antioxidants play an essential role in scavenging excessive ROS and thus avoiding OS in spermatozoa [5,7]. Recent studies in this realm have reported that boar seminal plasma, a complex fluid mainly secreted by accessory sex glands, is endowed with a plethora of antioxidants (enzymatic and non-enzymatic), playing an essential role in sperm functional performance [8–12]. Artificial insemination (AI) is the most applied biotechnology for pig breeding in pork- producing countries, and is a key tool for genetic and production improvement [13,14]. Liquid storage at 17 ◦ C is still the most widely used method for preserving AI-doses of pig semen, as it is easy to apply and preserves sperm functionality for 3–5 days, which is plenty of time for successful use in production farms [14]. Semen extenders play a key role in this success and their enrichment with additives having antioxidant capacity is encouraged with the ultimate purpose of minimizing potential OS and thus extending sperm functional life [13]. However, it should be noted that an excess of antioxidants may also lead to OS, induced by the “antioxidant paradox”, or reductive stress, decreasing the ROS levels required for sperm physiological function [15]. For this reason, the maintenance of an adequate balance between ROS and antioxidants levels is essential for optimal sperm function [2]. Measurement of the OS level in semen AI-doses has not yet been performed. This is even though it should be standard practice in AI-centers as it is in human andrology laboratories. This measurement would improve the current understanding of the causes of the low sperm performance of some semen AI-doses and would also be an objective criterion to determine the value of exogenous antioxidants in semen extenders. Oxidative stress in semen samples is usually assessed by the direct measurement of ROS (e.g., using the d-ROM test or by flow cytometry) or products resulting from ROS attack (e.g., LPO markers) [16]. These tests have some drawbacks, as they do not consider seminal plasma antioxidants that scavenge ROS, minimizing their possible damaging effects. Thus, labora- tory tests able to measure the balance between oxidants and antioxidants in seminal plasma would be more objective to determine OS in semen samples. Among these tests would be the oxidative stress index (OSI); a quick, easy and inexpensive technique to accurately display oxidant/antioxidant ratio in biological samples [17]. In humans, seminal OSI values were related to male infertility issues [18–21]. However, as far as we know, no studies have been conducted in livestock assessing OSI in seminal plasma and evaluating its relationship with sperm performance and male fertility. In this context, this study aimed to test the usefulness of seminal OSI measurements in pig AI-centers. For this purpose, the relationship of seminal OSI with (1) the resistance of spermatozoa from semen AI-doses to withstand liquid storage at 17 ◦ C for 72 h, and (2) the in vivo fertility outcomes of these liquid-stored semen AI-doses were evaluated. 2. Materials and Methods 2.1. Reagents and Media All reagents and media were purchased from Merck (KgaA, Darmstadt, Germany) and fluorochromes from Molecular Probes Europe BV (Leiden, The Netherlands), unless otherwise indicated. 2.2. Boars, Ejaculates and Seminal Plasma Samples Healthy, mature (1.5–3 years of age) and fertile boars of different breeds (Duroc, Landrace, Large White, and Pietrain) underwent regular semen collection to produce commercial semen AI-doses were used in the study. The boars belonged to the company AIM Ibérica (Topigs Norsvin Spain SLU), which fulfilled the Spanish (ES300130640127, August 2006) and European (ES13RS04P, July 2012) directions for animal health and welfare Antioxidants 2021, 10, 1203 3 of 12 and ejaculate collection. All boars were housed in an AI-center (Calasparra, Murcia, Spain) in individual pens with controlled temperature (15–25 ◦ C) and light (16 h/per day, artificial plus natural). Entire ejaculates were collected from these AI-boars using a semi-automatic semen collection method (Collectis® , IMV Technologies, L’Aigle, France) at a frequency of two collections per week. Only ejaculates that satisfied the sperm quality requirements to produce commercial semen AI-doses (namely sperm concentration >200 × 106 sperm/mL, sperm motility >70% and morphologically normal sperm >75%) were included in the study. For seminal plasma harvesting, entire ejaculates were twice centrifugated (1500× g for 10 min at room temperature (Rotofix 32A, Hettich Centrifuge UK, Newport Pagnell, Buckinghamshire, England, UK)), immediately after their collection. The resultant super- natants (seminal plasma samples) were analyzed in an Eclipse E400 microscope (Nikon, Tokyo, Japan) to assess it was sperm-free. Then, seminal plasma samples were sent in insulated containers (15–17 ◦ C) to the Animal Andrology Laboratory of the Veterinary Teaching Hospital of the University of Murcia, where they arrived within three hours of ejaculate collection. Once in the laboratory, seminal plasma samples were immediately stored at −80 ◦ C (Ultra Low Freezer; Haier Inc., Qingdao, China) until OSI measurements. 2.3. Oxidative Stress Index Measurement in SP Advanced oxidation protein products (AOPPs) and the Trolox equivalent antioxidant capacity (TEAC) of four seminal plasma samples were measured to calculate the OSI. Both assessments, AOPP and TEAC, were performed using an automated biochemistry analyzer (Olympus AU600 Automatic Chemistry Analyzer, Olympus Europe GmbH, Germany) and displayed an intra- and inter-assay coefficient variations <10%, showing linearity in serial dilutions. Each measurement was performed per duplicate in each seminal plasma sample. The AOPPs, as markers of oxidant-mediated protein damage, were measured follow- ing the procedure described by Witko-Sarsat et al. [22], adapted to porcine seminal plasma. Briefly, 10 µL of the seminal plasma sample was mixed with 160 µL of 0.074 M potassium iodide and 25 µL of acetic acid (50%) and the change in absorbance was measured at 340 nm after incubation at 37 ◦ C for 40 s. This assay was calibrated using chloramine-T (0–500 µM) and the results were expressed in µmol/L of chloramine-T equivalents. The TEAC, as a marker of total antioxidant capacity, was measured following the previously described method [23], and previously adapted to porcine seminal plasma samples. This assay was based on the ability of the non-enzymatic antioxidants to scavenge radical 2, 20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) enzymatically pre- generated using an acid medium. Briefly, 12 µL of seminal plasma sample were mixed with 40 mmol/L acetate buffer (reagent 1) and with 2 mmol/L H2 O2 and 10 mmol/L ABTS in a 30 mmol/L acetate buffer solution (reagent 2). The change in absorbance was measured at 600 nm. A concentration between 0.0 and 2.0 mmol/L of 6-hydroxy-2,5,7,8- tetramethylchromane-2-carboxylic acid (Trolox) was used to calibrate the assay and the results were expressed as mmol/L Trolox equivalent. The OSI value was calculated using the formula described by Venturini et al. [24]: OSI (arbitrary unit) = AOPP (µmol/L) × 100 /TEAC (µmol/L). 2.4. Assessment of Sperm Quality Sperm quality parameters were assessed in extended semen samples (30 × 106 sperm/mL), in terms of (1) sperm motility (total and progressive) and (2) sperm viability (integrity of plasma and acrosomal sperm membranes). Sperm motility was assessed using a computer-assisted sperm analyzer (ISASV1® , Proiser R+D S.L., Paterna, Spain). For that, semen samples (5 µL) were deposited in a pre-warmed (38 ◦ C) Makler chamber (Sefi Medical Instruments, Haifa, Israel) and ten fields (>600 spermatozoa/sample) were microscopically assessed. Spermatozoa with an average path velocity >20 µm/s were classified as motile and those with a straight-line velocity >40 µm/s as displaying progressive motility, and both were expressed as percentage. Sperm viability was assessed using a flow cytometry (BD FACS Antioxidants 2021, 10, 1203 4 of 12 Canto II flow cytometer, Becton Dickinson & Company, Franklin Lakes, NJ, USA). For that, semen samples (100 µL) were incubated at 37 ◦ C for 10 min (Sanyo MIR-153 incubator, Gemini BV, Apeldoorn, The Netherlands) with 3 µL of Hoechst 33342 (H-42; 0.05 mg/mL in phosphate buffered saline (PBS)), 2 µL of propidium iodide (PI, 0.5 mg/mL in PBS) and 2 µL fluorescein-conjugated peanut agglutinin (PNA-FITC, 100 µg/mL in PBS). Thereafter, samples were diluted in 400 µL of PBS for subsequent flow cytometric analysis. The sperm that exhibited intact plasma and acrosome membranes (H-42-positive/PI-negative/PNA- FITC-negative) were recorded as viable and results were expressed in percentage. A total of three technical replicates (with 10,000 H-42-positive events per each replicate) were performed. 2.5. Experimental Design The experiments were authorized by the Bioethics Committee of the University of Murcia (research code: 639/2012). 2.5.1. Experiment 1: Relationship between Seminal OSI and the Sperm Quality of Semen AI-Doses Stored at 17 ◦ C for 72 h Fifty-eight entire ejaculates (one ejaculate per boar) were collected and split into two aliquots: (1) the first aliquot was used to harvest seminal plasma (as detailed above) and stored at −80 ◦ C until OSI measurement; (2) the second aliquot was extended in Beltsville Thaw Solution (BTS, Kubus, Las Rozas Madrid, Spain) alike an semen AI-dose (30 × 106 sperm/mL), send to the Animal Andrology Laboratory of the Veterinary Teaching Hospital of the University of Murcia, where were stored at 17 ◦ C during 72 h, assessing sperm quality (motility and viability) on arrival (0 h) and at 72 h of storage. Sperm re- silience was defined as the difference between the percentages of sperm quality parameters measured at the 0 and 72 h storage time interval. 2.5.2. Experiment 2: Relationship between Seminal OSI and In Vivo Fertility of Semen AI-Doses Stored at 17 ◦ C Eighteen Landrace and Large White boars included in a commercial AI-program provided ejaculates. For OSI measurements, seminal plasma samples were harvested from four ejaculates per boar collected over one year, at a rate of one ejaculate every four months. Ejaculates collected during the same year were extended in Biosem+ (Magapor, Ejea de los Caballeros, Zaragoza, Spain) at the rate of 30 × 106 sperm/mL to produce AI-doses of 40 mL (1200 × 106 total sperm) that were stored at 17 ◦ C and used for intra-uterine AI (twice per estrus) of 3137 Landrace and Large White multiparous sows (>100 sows inseminated per boar). These sows were housed in different Spanish farms subjected to same standard management, including two inseminations per estrus. The fertility of the inseminated sows was recorded in terms of: (1) farrowing rates (%): number of sows farrowed/number of sows inseminated × 100; (2) litter size: total number (alive plus dead) of piglets born per litter; and (3) a fertility index, defined as the total number of piglets born as a proportion of the number of sows inseminated. 2.6. Statistical Analysis Data were analyzed with the statistical software packages IBM SPSS Statistics 24.0 (IBM, Armonk, NY, USA) and GraphPad Prism 9.1.2 (GraphPad Software, Inc., La Jolla, CA, USA). Shapiro–Wilk test was conducted to assess the assumption of normality of the dataset. In the first experiment, Pearson correlation coefficients between SP-OSI and sperm parameters were calculated. Then, a hierarchical cluster analysis was carried out in order to classify ejaculates into three groups according to the seminal OSI (low, medium or high). A Kruskal–Wallis test, followed by Dunn’s post hoc test for multiple comparisons, was performed to evaluate putative differences on sperm quality parameters (sperm resiliency) among the three seminal OSI groups. In the second experiment, fertility records were subjected to a multivariate statistical model [25], in order to identify the direct boar effect for each fertility parameter (farrowing rate and litter size). Fertility data were expressed order to classify ejaculates into three groups according to the seminal OSI (low, medium or high). A Kruskal–Wallis test, followed by Dunn’s post hoc test for multiple compari- sons, was performed to evaluate putative differences on sperm quality parameters (sperm resiliency) among the three seminal OSI groups. In the second experiment, fertility records Antioxidants 2021, 10, 1203 were subjected to a multivariate statistical model [25], in order to identify the direct boar 5 of 12 effect for each fertility parameter (farrowing rate and litter size). Fertility data were ex- pressed as deviation from the average fertility for the breed. Pearson correlation coeffi- cients betweenfrom as deviation seminal the OSI and fertility average fertility outcomes were calculated. for the breed. Then, a hierarchical Pearson correlation coefficients cluster between seminal-OSI and fertility outcomes were calculated. Then, adisplaying analysis was carried out to classify the boars into two groups hierarchicalnegative cluster oranalysis positivewasdeviations in farrowing rate and litter size. A Mann–Whitney carried out to classify the boars into two groups displaying negative test was con-or ducted to evaluate the putative differences on seminal OSI values positive deviations in farrowing rate and litter size. A Mann–Whitney test was conductedbetween the two groups. A receiver to evaluate operating the putative characteristic differences (ROC)OSI on seminal curve wasbetween values performedtheto evaluate two groups. theA predictive ability of the seminal OSI to discriminate between AI-boars receiver operating characteristic (ROC) curve was performed to evaluate the predictive with higher or lower fertility index (OSI threshold was 3). Areas under the ROC curve (AUC) ability of the seminal OSI to discriminate between AI-boars with higher or lower fertility and cut-off values werethreshold index (OSI calculatedwas by 3). theAreas program. underDiscrimination the ROC curvestrength wascut-off (AUC) and measuredvaluesbywere the AUC value following criteria: 1.00–0.90 (excellent), 0.90–0.80 (good), calculated by the program. Discrimination strength was measured by the AUC value 0.80–0.70 (fair), 0.70– 0.60 (poor),criteria: following 0.60–0.50 (fail) and 1.00–0.90 ˂0.50 (no discriminating). (excellent), 0.90–0.80 (good),Significant statistical 0.80–0.70 (fair), differences 0.70–0.60 (poor), were defined 0.60–0.50 from (fail) andp<0.50 < 0.05. (no discriminating). Significant statistical differences were defined from p < 0.05. 3. Results 3. Results 3.1. Experiment 1: Relationship between Seminal OSI and Sperm Quality of Semen AI-Doses 3.1. Experiment Stored at 17 °C for1:72Relationship h between Seminal OSI and Sperm Quality of Semen AI-Doses Stored at 17 ◦ C for 72 h The OSI in the 58 seminal plasma samples ranged from 0.75 to 5.54. The Pearson cor- The OSI in the 58 seminal plasma samples ranged from 0.75 to 5.54. The Pearson relation coefficients showed that seminal OSI was not related to the sperm quality param- correlation coefficients showed that seminal OSI was not related to the sperm quality eters of semen AI-doses assessed at either 0 or 72 h of storage at 17 °C◦ (Supplementary parameters of semen AI-doses assessed at either 0 or 72 h of storage at 17 C (Supplementary Figure S1). Therefore, the ejaculates were grouped according to their seminal OSI by hier- Figure S1). Therefore, the ejaculates were grouped according to their seminal OSI by archical clustering, and three groups were generated (p < 0.01) as having either low (be- hierarchical clustering, and three groups were generated (p < 0.01) as having either low tween 0.75 and 1.89; n = 16), medium (between 2.01 and 2.91; n = 22) or high (between 3.04 (between 0.75 and 1.89; n = 16), medium (between 2.01 and 2.91; n = 22) or high (between and 5.54; n = 20) seminal OSI (Figure 1). 3.04 and 5.54; n = 20) seminal OSI (Figure 1). Valuesofofoxidative Figure1.1.Values Figure oxidativestress stressindex index(OSI) (OSI)ininseminal seminalplasma plasmasamples samples(n (n==58) 58)from fromporcine porcine entireejaculates entire ejaculates(n(n = 58; = 58; oneone perper boar). boar). TheThe ejaculates ejaculates werewere clustered clustered according according to their to their seminal seminal OSI OSI values values (hierarchical (hierarchical clustering, clustering, p < as p < 0.01) 0.01) as having having low (between low (between 0.751.89; 0.75 and and n1.89; n =medium = 16), 16), medium (be- tween 2.01 2.01 (between and and 2.91;2.91; n = 22) n =or high 22) (between or high 3.04 3.04 (between and and 5.54;5.54; n = 20) n =OSI 20) values. The line OSI values. The indicates the line indicates the median, the whiskers extend to the 5th and 95th percentiles, and dots represent the seminal OSI values. **** p < 0.00001; *** p < 0.0001; ** p < 0.001. Sperm motility resilience differed among seminal OSI groups (p < 0.01). Semen AI-doses from ejaculates with low seminal OSI showed less differences in total motility between 0 and 72 h of storage at 17 ◦ C than those with high seminal OSI (Figure 2A). The differences ranged from 0 to 11% in the semen AI-doses from ejaculates classified with low seminal OSI values (only 2 of the 16 semen AI-doses had a difference ≥10%), and from 3 to 36% in those classified with high seminal OSI values (13 of the 20 semen AI-doses had values. **** p < 0.00001; *** p < 0.0001; ** p < 0.001. Sperm motility resilience differed among seminal OSI groups (p < 0.01). Semen AI- doses from ejaculates with low seminal OSI showed less differences in total motility be- tween 0 and 72 h of storage at 17 °C than those with high seminal OSI (Figure 2A). The Antioxidants 2021, 10, 1203 6 of 12 differences ranged from 0 to 11% in the semen AI-doses from ejaculates classified with low seminal OSI values (only 2 of the 16 semen AI-doses had a difference ≥10%), and from 3 to 36% in those classified with high seminal OSI values (13 of the 20 semen AI-doses had aadifference difference≥10%). ≥10%).TheThedifferences differencesininthe thepercentages percentagesofofprogressive progressivemotile motileand andviable viable spermatozoa spermatozoawith with intact intact acrosome acrosome were similar similar among among semen semenAI-doses AI-dosesofofthe thethree threeseminal sem- inal OSIOSI groups groups (Figure (Figure 2B,C). 2B,C). Figure 2. Box-whisker plot showing the differences (sperm resilience) in the percentages of (A) total Figure 2. Box-whisker plot showing the differences (sperm resilience) in the percentages of (A) total motile sperm; (B) progressive motile sperm; and (C) viable sperm with intact acrosome between motile sperm; (B) progressive motile sperm; and (C) viable sperm with intact acrosome between both evaluation time-points (0 and 72 h of storage at 17 ◦ C) of artificial insemination semen doses both evaluation time-points (0 and 72 h of storage at 17 °C) of artificial insemination semen doses (n==58) (n 58)ofofboar boarejaculates ejaculates clustered clustered intointo three three groups groups (hierarchical (hierarchical clustering, clustering, p < 0.01) p < 0.01) according according to to the the oxidative oxidative stress stress index index (OSI) (OSI) of seminal of seminal plasma. plasma. Low Low seminal seminal OSI ranged OSI ranged from from 0.75 0.75 to to(16 1.89 1.89 (16 semen semen doses), doses), medium medium fromfrom 2.012.01 to 2.91; to 2.91; (22 (22 semen semen doses) doses) andand highhigh fromfrom 3.043.04 to to 5.54 5.54 (20(20semen semen doses). doses).Boxes Boxesenclose enclosethe the25th 25thand and75th 75thpercentiles, percentiles,whiskers whiskersextend extendtotothe the5th 5thand and95th 95thpercentiles percentiles and andthe theline lineindicates indicatesthe themedian. median.****p p< <0.001; 0.001;ns: ns:not significant. not significant. 3.2.Experiment 3.2. Experiment2:2:Relationship Relationshipbetween betweenSeminal SeminalOSIOSIand andInInVivo VivoFertility FertilityofofSemen SemenAI-Doses AI-Doses Stored at 17 ◦C Stored at 17 °C Fertilitydata Fertility datawere werestatistically statisticallycorrected correctedtotodetermine determinethethedirect directeffect effectofofthe theboar boarand and the results for each boar were recorded as the deviation from the mean of boar populationof the results for each boar were recorded as the deviation from the mean of boar population the same genetic line. The deviation ranged between +4.54 and −2.79 for the farrowing rate and between +0.83 and −0.43 for the number of piglets born per farrowing. The OSI values of the 72 seminal plasma samples (four per boar) ranged from 0.81 to 7.42. The correlation coefficient of Pearson evidenced that seminal OSI was weakly related with farrowing rate deviation (p < 0.05; R = −0.54) and was not related with litter size deviation (p > 0.05; R = −0.34). Then, the 18 AI-boars included in this study were hierarchically clustered (p < 0.001) into two groups as having either a negative (ranging from −2.79 to −0.16; n = 8) or positive (ranging from +0.94 to +4.54; n = 10) farrowing rate deviation (Figure 3A). Similarly, the boars were also clustered into two groups as having either negative (ranging Similarly, the boars were also clustered into two groups as having either negative (ranging from −0.43 to −0.05; n = 8) or positive (ranging from +0.01 to +0.83; n = 10) litter size devi- ation (Figure 3B). The seminal OSI values were different (p < 0.05) between the two groups of boars for both the farrowing rate and litter size. Boars with negative deviations showed Antioxidants 2021, 10, 1203 higher (p < 0.05) seminal OSI than those with positive deviations (Figure 4A,B). 7 of 12 Like the first experiment, the boars were grouped according to their seminal OSI (mean of four seminal plasma samples from four different ejaculates collected over a year) by hierarchical clustering, and two groups were generated (p < 0.01) with either a low (n from −0.43 to −0.05; n = 8) or positive (ranging from +0.01 to +0.83; n = 10) litter size = 9) or high (n = 9) seminal OSI (OSI threshold was 3). The ROC-curve showed that the deviation (Figure 3B). The seminal OSI values were different (p < 0.05) between the two seminal OSI had good discriminating ability to predict the in vivo fertility of AI-boars, groups of boars for both the farrowing rate and litter size. Boars with negative deviations measured in terms of fertility index (AUC = 0.815; p < 0.05) (Figure 5). showed higher (p < 0.05) seminal OSI than those with positive deviations (Figure 4A,B). Figure 3. Distribution by hierarchical cluster analysis (p < 0.001) of the 18 boars used in artificial insemination pro- Figureinto grams 3. Distribution two groups by for hierarchical cluster analysis having (A) negative (p <−0.001) (between of the 2.79 and 18 boars −0.16; n = 8)used in artificial or positive insemination (between programs +0.94 and +4.54; into two groups for having (A) negative (between −2.79 and −0.16; n = 8) or positive (between +0.94 and +4.54; n = 10) n = 10) farrowing rate deviations; and (B) negative (between −0.43 and −0.05; n = 8) or positive (between +0.01 and +0.83; farrowing Antioxidants 2021,rate deviations; and (B) negative (between −0.43 and −0.05; n = 8) or positive (between +0.01 and +0.83; n = 10) 8 of 13 10, 1203 n = 10) litter size deviations. The line indicates the median, the whiskers extend to the 5th and 95th percentiles, and the dots litter size deviations. The line indicates the median, the whiskers extend to the 5th and 95th percentiles, and the dots represent represent the boars.**** the boars. ****p p<<0.0001; ***pp<<0.001. 0.0001;*** 0.001. Figure 4. Box-whisker plot showing oxidative stress index (OSI) values assessed in the seminal plasma of ejaculates collected Figure from 4. Box-whisker artificial insemination plot showing boars oxidative(pstress hierarchically index < 0.001) (OSI) as grouped values assessed showing in the seminal (A) negative (rangingplasma from −of2.79 ejaculates col- to −0.16; lected from artificial insemination boars hierarchically (p < 0.001) grouped as showing (A) negative (ranging from −2.79 to n = 8) or positive (ranging from +0.94 to +4.54; n = 10) farrowing rate deviations and (B) negative (ranging from −0.43 −0.16; n = 8) or positive (ranging from +0.94 to +4.54; n = 10) farrowing rate deviations and (B) negative (ranging from −0.43 to −0.05; n = 8) or positive (ranging from +0.01 to +0.83; n = 10) litter size deviations. Boxes enclose the 25th and 75th to −0.05; n = 8) or positive (ranging from +0.01 to +0.83; n = 10) litter size deviations. Boxes enclose the 25th and 75th percentiles, percentiles, thethe line indicates line thethe indicates median median and andthethe whiskers whiskersextend to to extend thethe 5th5th and 95th and percentiles. 95th * Indicates percentiles. significant * Indicates significant differences (p < 0.05) in seminal OSI values between differences (p < 0.05) in seminal OSI values between groups.groups. Like the first experiment, the boars were grouped according to their seminal OSI (mean of four seminal plasma samples from four different ejaculates collected over a year) by hierarchical clustering, and two groups were generated (p < 0.01) with either a low (n = 9) or high (n = 9) seminal OSI (OSI threshold was 3). The ROC-curve showed that the seminal OSI had good discriminating ability to predict the in vivo fertility of AI-boars, measured in terms of fertility index (AUC = 0.815; p < 0.05) (Figure 5). Figure 4. Box-whisker plot showing oxidative stress index (OSI) values assessed in the seminal plasma of ejaculates col- lected from artificial insemination boars hierarchically (p < 0.001) grouped as showing (A) negative (ranging from −2.79 to −0.16; n = 8) or positive (ranging from +0.94 to +4.54; n = 10) farrowing rate deviations and (B) negative (ranging from −0.43 to −0.05; n = 8) or positive (ranging from +0.01 to +0.83; n = 10) litter size deviations. Boxes enclose the 25th and 75th Antioxidants 2021, 10, 1203 8 of 12 percentiles, the line indicates the median and the whiskers extend to the 5th and 95th percentiles. * Indicates significant differences (p < 0.05) in seminal OSI values between groups. Nonparametric Figure5.5.Nonparametric Figure receivers receivers operating operating characteristic characteristic (ROC) (ROC) curvecurve showing showing the ability the ability of of oxi- oxidative stress index (OSI) assessed in seminal plasma to predict in vivo fertility index dative stress index (OSI) assessed in seminal plasma to predict in vivo fertility index of boars whoseof boars whose as semen, semen, as liquid-stored liquid-stored semen(17 semen doses doses was◦ C), °C),(17 usedwas forused for artificial artificial insemination. insemination. AUC: AUC: area area under the ROC under curve. the ROC curve. 4.4.Discussion Discussion Asfar As faras as we we are are aware, aware, this this is is the the first first report reportconducted conductedin inaalivestock livestockspecies speciesmeasur- meas- ing OSI in seminal plasma and assessing its putative influence on sperm uring OSI in seminal plasma and assessing its putative influence on sperm quality and quality and the the fertility of semen AI-doses. The results showed that seminal fertility of semen AI-doses. The results showed that seminal OSI values OSI values were negatively were negatively relatedwith withthe thesperm spermmotility motilityofofsemen semenAI-doses AI-doses storage ◦ C for 72 h, as well as related storage at at 1717°C for 72 h, as well as in in vivo vivo fertility, fertility, in terms in terms of farrowing of farrowing rates rates andand litter litter size. size. OSI is not currently included in the battery of tests used for ejaculate selection in AI OSI is not currently included in the battery of tests used for ejaculate selection in AI centers for livestock species, despite the fact it is well known that fresh semen samples centers for livestock species, despite the fact it is well known that fresh semen samples generate ROS that impair sperm functionality [26]. In pigs, seminal ROS levels increase during semen liquid storage, compromising the functional lifespan of stored spermatozoa, including their fertilizing ability [13,26–28]. In addition, the extension of semen prior to liquid storage lowers the concentration of seminal plasma antioxidants. The increase in ROS together with the low antioxidant concentration in semen AI-doses results in both an unbalanced ROS: antioxidants ratio and negative influence on the sperm performance. Supplementing semen extender with antioxidants is one of the most accepted options to avoid this undesirable scenario [7,13]. However, this supplementation often has a detri- mental effect on sperm functionality, especially when unsuitable high concentrations are used [27,29–32], as they lead to a complete inhibition of ROS, which negatively impairs key sperm functions such as capacitation [33,34]. Taken together, these studies support the argument that measuring seminal OSI could be a good option to improve the selec- tion of ejaculates used for AI. Different assays have been proposed in the literature to evaluate the ROS/antioxidant ratio in human semen (reviewed by Agarwal et al., [35]). The oxidation–reduction potential, based on the measurement of the potential of electrons to change from one chemical species to another, and OSI or ROS-TAC score, a measure of oxidation/antioxidation ratio, will be among the best-known direct assays [35]. To calculate OSI, both the oxidant and antioxidant capacities can be measured using different procedures. For instance, ferric reducing antioxidant potential, total antioxidant activity or TEAC for measuring antioxidant capacity; and total oxidative capacity, total oxidative status or AOPP for measuring oxidative capacity [18–21]. In porcine, Wang et al. [27] mea- sured OSI in the supernatant of extended liquid-stored semen samples using the ratio of the total oxidative status to TEAC. Although these assays would be suitable to calculate OSI, Antioxidants 2021, 10, 1203 9 of 12 Venturini et al. [24] suggested that the AOPP/TEAC ratio could be a better indicator for OS. Accordingly, we opted for this test because it was simple, quick to perform and rather inexpensive, the latter reason being an important requirement for porcine insemination centers due to their narrow profit margins. The seminal OSI values varied widely between ejaculates and between boars. This variation was expected, since both ROS levels and antioxidant concentrations in seminal plasma also vary widely between ejaculates and boars [8–12]. Unfortunately, we cannot discuss whether the seminal OSI values are high or low since there are no previous studies that have evaluated this seminal plasma index in semen from porcine or other livestock species. The seminal OSI values were not related with the loss of sperm viability, measured in terms of plasma and acrosomal membrane integrity during liquid storage. In agreement with this finding, previous studies reported that this sperm parameter is little affected by the 72 h liquid storage of porcine semen [11,28,36,37]. However, seminal OSI values were related to the loss of total sperm motility during semen storage time. It has been extensively reported that an excess of ROS has detrimental effects on sperm motility [38,39]. The ROS:antioxidants unbalance would cause mitochondrial dysfunction, leading to adenosine triphosphate depletion, decreasing energy availability and thereby impairing sperm motility [40]. An excess of ROS also negatively influences the activity of some enzymes essential for sperm motility, such as glucose-6-phosphate dehydrogenase [41]. Studies conducted in pig semen have linked the loss of sperm motility in liquid-stored semen samples to an increase in malondialdehyde, one of the end-products of the ROS-induced peroxidation of sperm membrane lipids [26,42]. This, and other end-products of LPO of sperm membranes such as 4-hydroxynonenol, can also cause a decrease in sperm motility because they modify the permeability of the sperm plasma membrane and thus their ability to regulate intracellular ions [43]. The seminal OSI differed between boars with high and low fertility outcomes, for both farrowing rates and litter sizes. There are no previous studies evaluating the putative association between seminal OSI and in vivo fertility in livestock species. Human studies have reported the usefulness of seminal OSI in predicting male fertility, showing greater predictive ability than the measurements of ROS or total antioxidant capacity alone in discriminating between fertile and infertile men [18–21,44]. The negative relationship between seminal OSI and in vivo fertility would be related to the negative effect of seminal OSI on total sperm motility, since it is well-known that the percentage of total motile spermatozoa is positively related to in vivo fertility of liquid-stored pig semen doses [45]. In addition, the aforementioned peroxidation of sperm membrane lipids, associated with excessive ROS due to an unbalance between ROS and antioxidants, leads to a decrease in membrane fluidity, which is necessary for sperm–oocyte fusion and fertilization [46]. Excessive ROS levels could also adversely affect fertility by inducing sperm nuclear DNA fragmentation [39]. A high DNA fragmentation may reduce the fertilization rates, implan- tation, embryo development, increasing pregnancy loss [35]. In this regard, Boe–Hansen et al. [47] reported an increase in sperm nuclear DNA fragmentation in boar semen samples after 72 h of liquid storage. These authors also observed that the percentage of affected sperm varied significantly between ejaculates, which could be related to the differences in seminal OSI between ejaculates. Pig AI-centers are calling for the development of predictive tests to identify subfertile boars prior to their inclusion in AI-programs, as it is estimated that 5–7% of boars included in insemination programs are subfertile even though their ejaculates show good sperm quality [48]. Under field conditions, the two measures that define in vivo fertility in swine, namely farrowing rate and litter size, cannot be isolated from each other. Therefore, the fertility index, combining both measures, would be a more accurate measurement of boar in vivo fertility. The ROC-curve showed that seminal OSI had a good predicting ability. Accordingly, seminal OSI could be a good candidate as an in vivo fertility biomarker for boars included in artificial insemination programs. Antioxidants 2021, 10, 1203 10 of 12 This study has some limitations that should be considered in future studies. The first would be related to the sperm assessments. In this study, with the purpose of approach- ing the reality of the laboratories of swine AI-centers, we only evaluated those sperm parameters that are usually evaluated in AI-centers to select valid ejaculates for use in AI-programs. Including other sperm functional tests such as LPO and nuclear DNA frag- mentation would help better understand the relationship between seminal OSI and sperm function. The second limitation would be related to the AI trial, which was performed under field conditions, also with the purpose of assessing the usefulness of measuring seminal OSI in the operating work context of AI-centers. Thus, the semen doses used in the AI-trial were those produced and marketed by the AI-center, using a semen extender of unknown public composition (Biosem+). The use of semen extenders of known elemental composition would exclude external factors that may influence fertility associated with semen extender components. 5. Conclusions In conclusion, the seminal OSI was positively related with sperm motility loss in liquid-stored extended boar semen. In addition, seminal OSI was negatively related with the in vivo fertility of liquid-stored semen doses, measured in terms of farrowing rate and the litter size of sows receiving intra-uterine inseminations. The ROC-curve showed seminal OSI, an easy, quick, and inexpensive method, which could be a good predictive biomarker for in vivo fertility. Therefore, it could be economically profitable to include it in the battery of tests routinely used in pig AI-centers. The measurement of seminal OSI would help identify subfertile boars prior to their inclusion in AI-programs. It would also be useful in identifying ejaculates that would require antioxidant supplementation to retain functional sperm life during preservation. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antiox10081203/s1, Figure S1: Heat map showing Pearson’s correlation coefficients between seminal plasma oxidative stress index (OSI) and sperm quality parameters of porcine artificial insemination semen doses (semen AI-doses, n = 58). The semen quality parameters were assessed at 0 and 72 h of storage at 17 ◦ C and the correlation coefficients between seminal OSI and the sperm resilience (difference in each sperm quality parameter between evaluation time-points: 0 and 72 h) was also assessed. Author Contributions: Conceptualization, I.B., H.R.-M. and J.R.; methodology, I.B., C.P.R. and A.T.; software, C.P.R. and A.T.; validation, C.P.R. and A.T.; formal analysis, I.B., C.P.R. and A.T.; resources, A.T. and J.R.; data curation, I.B., J.R.; writing—original draft preparation, I.B.; review and editing, H.R.-M. and J.R.; visualization, H.R.-M. and J.R.; supervision, J.R.; project administration, J.R.; funding acquisition, J.R. All authors have read and agreed to the published version of the manuscript. Funding: This experimental study was supported by ANPSTAND Operating Group (Madrid, Spain) and FEDER (European Union) and the Spanish Ministry of Agriculture, Fisheries and Food (Madrid, Spain). Grant number: 20190020007328. Institutional Review Board Statement: Animal husbandry and handling were conducted in com- pliance with the European directive 2010/63/EU. The experiments were approved by the Bioethics Committee of the University of Murcia (research code: 639/2012). Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article and Supplementary Materials. Acknowledgments: The authors of this manuscript would like to thank the staff of ANSPTAND for their cooperation in handling the semen samples and collecting the fertility data. I.B. was financially supported by European Union’s Horizon 2020 research and innovation program (H2020- MSCA-IF-2019-891382), C.P.-R. was financially supported by the Ministry of Science and Innovation (Spain; FJC2019-042475-I) and A.T. was financially supported by the Ministry of Economy and Competitiveness (RYC-2017-22992), State Research Agency (AEI, Spain), and the European Social Fund (ESF). Antioxidants 2021, 10, 1203 11 of 12 Conflicts of Interest: The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. 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