High-Fat Diet-Induced Obesity Increases Brain Mitochondrial Complex I and Lipoxidation-Derived Protein Damage.

Obesity is a risk factor for highly prevalent age-related neurodegenerative diseases, the pathogenesis of whichinvolves mitochondrial dysfunction and protein oxidative damage. Lipoxidation, driven by high levels of peroxidizable unsaturated fatty acids and low antioxidant protection of the brain, stands out as a significant risk factor. To gain information on the relationship between obesity and brain molecular damage, in a porcine model of obesity we evaluated (1) the level of mitochondrial respiratory chain complexes, as the main source of free radical generation, by Western blot; (2) the fatty acid profile by gas chromatography; and (3) the oxidative modification of proteins by mass spectrometry. The results demonstrate a selectively higher amount of the lipoxidation-derived biomarker malondialdehyde-lysine (MDAL) (34% increase) in the frontal cortex, and positive correlations between MDAL and LDL levels and body weight. No changes were observed in brain fatty acid profile by the high-fat diet, and the increased lipid peroxidative modification was associated with increased levels of mitochondrial complex I (NDUFS3 and NDUFA9 subunits) and complex II (flavoprotein). Interestingly, introducing n3 fatty acids and a probiotic in the high-fat diet prevented the observed changes, suggesting that dietary components can modulate protein oxidative modification at the cerebral level and opening new possibilities in neurodegenerative diseases' prevention.

Validation of the fCAL turbo immunoturbidimetric assay for measurement of calprotectin in porcine and bovine fecal samples.

The concentration of calprotectin in feces is a well-studied marker of gastrointestinal inflammation in humans. However, little is known about fecal calprotectin in farm animals. In this work, we have validated an immunoturbidimetric method for fecal calprotectin (Bühlmann fCAL® turbo assay, Schönenbuch, Switzerland) in porcine and bovine fecal samples. Linearity was evaluated by serial dilution (R2 > 0.97 was obtained for both species). Accuracy was assessed by a recovery study, with results between 80 and 120% for low, medium, and high samples in both species. Intra- and inter-assay variability was <20%. Limit of detection was 6.4 µg/g in pig and 5.3 µg/g in cow. Limit of quantification was 13.4 µg/g (pig) and 11.1 µg/g (cow). Additionally, clinical validation has been included to evaluate the ability of the assay to detect inflammatory status in the intestine under different management conditions. In experiments with porcine, it was found that piglets treated with ZnO had lower concentrations of fecal calprotectin. In a second experiment in bovine, calves with diarrhea had higher concentration of fecal calprotectin. The Bühlmann fCAL® turbo assay is suitable for measurement of calprotectin in porcine and bovine fecal samples. Moreover, fecal calprotectin could be a good biomarker of intestinal inflammation in both species.

Sample stability and heparin interference in ionized calcium and ionized magnesium measurements in horses using the Stat Profile Prime Plus co-oximetry electrolyte analyzer.

BACKGROUND: The determination of iCa and iMg is important in veterinary medicine, but their immediate determination in whole blood is not always possible. Their stability in other sample types and the existence of interferences must be evaluated before its use. OBJECTIVES: We aimed to analyze the effects of storage time on the stability of iCa, iMg, and other analytes in whole blood, plasma, and serum samples in horses and assess the interference of heparin in these measurements. METHODS: Whole blood, heparin-plasma, and serum samples from 10 horses were stored at 4°C and analyzed 1, 2, 3, 4, 5, 6, 7, 8, 24, 48, and 168 hours after sample collection using the Stat Profile Prime Plus Vet equipment (Nova Biomedical, Waltham, MA, USA). Results were analyzed by ANOVA or mixed-effect models. RESULTS: The concentration of iCa, iMg, total calcium (tCa), total magnesium (tMg), and the ratios iCa/tCa and iMg/tMg did not differ up to 168 hours when compared to the initial time. Total Ca, iMg, and tMg were not significantly different among sample types, but iCa concentrations were slightly but significantly lower in plasma. Freezing at -20°C did not affect iCa, iMg, tCa, and tMg. The pH increased in serum and plasma after 8 hours, and a mild negative correlation existed between plasma iCa concentration and pH. A negative correlation was observed also between the ratios iCa/tCa or iMg/tMg and pH in plasma and serum. A significant decrease in iCa and iMg was detected when comparing homemade syringes at high heparin concentration (~200-300 U heparin/mL) and commercial lithium-heparin tubes (20-30 U/mL). CONCLUSIONS: Samples stored at 4°C can be used to determine iCa and iMg concentrations up to 7 days after collection. Other metabolites are stable for up to 8 hours; heparin interference should be taken into account if using homemade heparin syringes.

Acute phase proteins in cattle and swine: A review.

The major acute phase proteins (APPs) in cattle are haptoglobin (Hp) and serum amyloid A (SAA), and in swine, are Hp, SAA, C-reactive protein (CRP), and Pig major acute phase protein (Pig-MAP). Many methodologic assays are presently available to measure these parameters, which are still being improved to increase their specificity, sensitivity, user-friendliness, and economic availability. In cattle, the main applications are the diagnosis and monitoring of frequent diseases such as mastitis and metritis in dairy cows and respiratory problems in young calves. In pigs, APPs are useful in the control of bacterial and viral infections, and they may be used at the slaughterhouse to monitor subclinical pathologies and improve food safety. The utility of APP in animal production must not be forgotten; optimization of protocols to improve performance, welfare, and nutrition may benefit from the use of APPs. Other sample types besides serum or plasma have potential uses; APP determination in milk is a powerful tool in the control of mastitis, saliva is a non-invasive sample type, and meat juice is easily obtained at the slaughterhouse. Increasing our knowledge of reference intervals and the influence of variables such as age, breed, sex, and the season is important. Finally, worldwide harmonization and standardization of analytical procedures will help to expand the use of APPs.

Validation of new automated turbidimetric immunoassays for the measurement of haptoglobin and inter-α-trypsin inhibitor heavy chain H4 specific for the bovine species.

BACKGROUND: Good strategical programs are required for the early detection of disease even in the absence of evident clinical signs, which is crucial in satisfying animal welfare. Haptoglobin (Hp) and inter-α-trypsin inhibitor heavy chain H4 (ITIH4) are acute phase proteins and good biomarkers of early inflammation in cattle, with plasma levels that significantly increase after injury or infection. OBJECTIVES: We aimed to develop and validate two new immunoturbidimetric methods for Hp and ITIH4. METHODS: Species-specific antibodies were obtained and used to develop the immunoassays. For the Hp assay, antibodies were fixed to latex microparticles to enhance detection. The immunoassays were set up in an automated analyzer to carry out validation studies. Reference intervals were calculated using Reference Value Advisor. RESULTS: The Hp immunoturbidimetric method had a linear analytical range up to 0.40 mg/mL. The limit of detection (LoD) was 0.005 mg/mL, and the limit of quantification (LoQ) was 0.007 mg/mL. Total imprecision was less than 7%. Comparison with ELISA and single radial immunodiffusion (SRID) showed good correlation, whereas the comparison with the colorimetric method showed constant and proportional differences. The ITIH4 immunoassay showed linearity up to 5 mg/mL, and the LoD was 0.002 mg/mL. Total imprecision was less than 6%. Method comparison showed a good correlation with single radial immunodiffusion, both methods being equivalent. Bilirubin, triglycerides, and hemoglobin presented no interference in any of the assays. Reference intervals were 0.007-0.017 mg/mL for Hp and 0.2-0.7 mg/mL for ITIH4 in dairy cows 10 days before parturition. CONCLUSIONS: Immunoturbidimetric methods developed for Hp and ITIH4 can measure basal and increased levels of these proteins, showing adequate precision, accuracy, and robustness.

Obesity and Metabolic Traits after High-Fat Diet in Iberian Pigs with Low Birth Weight of Placental Origin.

Intrauterine growth restriction (IUGR) and later obesity and metabolic disorders have classically been associated with maternal malnutrition, but most cases of IUGR are related to placental insufficiency. The current study, using a swine model for IUGR and obesity, aimed to determine the interaction of birth weight (categorized as low birth weight [LBW] or normal birth-weight [NBW]) and postnatal diet (categorized as maintenance diet [MD] or fattening diet [FD]) on body weight, adiposity and metabolic traits. FD induced higher body weight and adiposity (both p < 0.0001), with higher fructosamine levels (p < 0.005) and a trend toward higher HOMA-ß index (p = 0.05). NBW pigs remained heavier than LBW pigs during the early juvenile period (p < 0.005), but there were no differences at later stages. There were no differences in metabolic traits during juvenile development, but there were differences in adulthood, when LBW pigs showed higher glucose and lower insulin levels than NBW pigs (both p < 0.05). These results suggest that (a) FD allows LBW offspring to achieve similar obesity in adulthood as NBW offspring, and (b) glucose metabolism is more compromised in obese LBW than obese NBW pigs. The comparison of our data with previous studies highlights significant differences between offspring with LBW induced by maternal malnutrition or placental insufficiency, which should be considered when studying the condition.

A High-Fat Diet Modifies Brain Neurotransmitter Profile and Hippocampal Proteome and Morphology in an IUGR Pig Model.

Intrauterine Growth Restriction (IUGR) hinders the correct growth of the fetus during pregnancy due to the lack of oxygen or nutrients. The developing fetus gives priority to brain development ("brain sparing"), but the risk exists of neurological and cognitive deficits at short or long term. On the other hand, diets rich in fat exert pernicious effects on brain function. Using a pig model of spontaneous IUGR, we have studied the effect on the adult of a long-term high-fat diet (HFD) on the neurotransmitter profile in several brain areas, and the morphology and the proteome of the hippocampus. Our hypothesis was that animals affected by IUGR (born with low birth weight) would present a different susceptibility to an HFD when they become adults, compared with normal birth-weight animals. Our results indicate that HFD affected the serotoninergic pathway, but it did not provoke relevant changes in the morphology of the hippocampus. Finally, the proteomic analysis revealed that, in some instances, NBW and LBW individuals respond to HFD in different ways. In particular, NBW animals presented changes in oxidative phosphorylation and the extracellular matrix, whereas LBW animals presented differences in RNA splicing, anterograde and retrograde transport and the mTOR pathway.

Plasma-Ionized Magnesium in Hospitalized Horses with Gastrointestinal Disorders and Systemic Inflammatory Response Syndrome.

Magnesium disorders in horses with gastrointestinal disorders or systemic inflammatory response syndrome (SIRS) are scarcely characterized. The purpose of the study was to explore the association of magnesium disorders with diagnosis, SIRS and mortality in horses admitted to a referral equine hospital. In total, 75 sick horses were included in an observational prospective study and classified as: obstructive (n = 17), inflammatory (n = 10) and ischemic gastrointestinal disorders (n = 12), and other non-gastrointestinal systemic disorders (n = 36). All sick horses were also divided according to the presence (n = 26) or absence of SIRS, and survival to discharge from hospital (survivors (n = 61) and non-survivors (n = 14). In addition, 26 horses were included as controls. On admission, mean (iMg) (95% confidence interval (CI)) in the SIRS group (0.47 (0.43-0.50 mmol/L)) was within the normal range (0.4-0.6 mmol/L). The obstructive group had lower (iMg) compared to the control group (0.44 (0.38-0.51 mmol/L) vs. 0.56 (0.50-0.61 mmol/L); p = 0.001). In total, 8 out of 17 (47%) horses with obstructive lesions presented with hypomagnesemia compared to controls (4% (1/26)) (p = 0.001). In conclusion, hypomagnesemia was more prevalent on admission in horses in the obstructive group, and to a lesser extent, in the inflammatory and ischemic groups. In contrast to human ICU patients, the proportion of hospitalized horses with hypomagnesemia was not associated with mortality.

Polyphenols and IUGR Pregnancies: Effects of the Antioxidant Hydroxytyrosol on the Hippocampus Proteome in a Porcine Model.

Supplementation of a mother's diet with antioxidants such as hydroxytyrosol (HTX) has been proposed to ameliorate the adverse phenotypes of foetuses affected by intrauterine growth restriction (IUGR). Our previous studies showed, in a porcine model of IUGR, an effect of maternal HTX supplementation on the neurotransmitter profile of several brain areas and the morphology of the hippocampus in 100 days old foetuses. The present study analyzed the impact of maternal HTX supplementation on the hippocampus proteome at this foetal age by TMT10plex labelling. Eleven differentially abundant proteins were identified by comparing both conditions, and eight of them downregulated and three upregulated in the HTX-treated group. The downregulated proteins were mainly involved in protein synthesis and RNA metabolism and may explain the differences in neuron differentiation in the HTX-treated group. The upregulated proteins were related to cell detoxification and could represent a potential mechanism to explain the neuroprotective effect of HTX.

Polyphenols and IUGR Pregnancies: Intrauterine Growth Restriction and Hydroxytyrosol Affect the Development and Neurotransmitter Profile of the Hippocampus in a Pig Model.

Intrauterine growth restriction (IUGR) refers to poor growth of a fetus during pregnancy due to deficient maternal nutrition or oxygen supply. Supplementation of a mother's diet with antioxidants, such as hydroxytyrosol (HTX), has been proposed to ameliorate the adverse phenotypes of IUGR. In the present study, sows were treated daily with or without 1.5 mg of HTX per kilogram of feed from day 35 of pregnancy (at 30% of the total gestational period), and fetuses were sampled at day 100 of gestation. Fetuses were classified as normal body weight (NBW) or low body weight (LBW) as a consequence of IUGR, constituting four groups: NBW-Control, NBW-HTX, LBW-Control, and LBW-HTX. The brain was removed, and the hippocampus, amygdala, and prefrontal cortex were rapidly dissected. Neuronal markers were studied by immunohistochemistry, and a decrease in the number of mature neurons in the hippocampal Cornu Ammonis subfield 1 (CA1) and the Dentate Gyrus (DG) regions was observed in LBW fetuses together with a higher number of immature neurons and other alterations in neuronal morphology. Furthermore, IUGR conditions altered the neurotransmitter (NT) profile, since an increase in the serotonin (5-HT) pathway was observed in LBW fetuses. Supplementation with HTX was able to reverse the morphological and neurochemical changes, leading both characteristics to values similar to those of NBW fetuses.

A Methodology to Quantify Resilience in Growing Pigs.

There is a growing concern about the genetic determinism of resilience and its possible implementation in breeding programs. The objective of our study was to elaborate novel resilience indicators in growing pigs based on the deviation from the expected growth curve and the increment of the acute-phase protein haptoglobin (HP) after applying a common vaccine. A total of 445 pigs were vaccinated with an attenuated Aujeszky vaccine at 12 weeks of age. Deviation from the expected body weight (ΔBW) given the growth curve of unvaccinated pigs at 28 days post-vaccination (DPV) and the increment of HP at 4 DPV (ΔHP) were suggested as resilience indicators. Challenged pigs that maintained their productivity and had a minor activation of HP were deemed resilient, whereas pigs that had low ∆BW values and a high activation of HP were deemed susceptible. Pigs were also classified based on ∆BW and ∆HP relative to the expected BW at 28 DPV and to the basal level of HP, respectively. The concordance was high between both methods, indicating that ΔBW and ΔHP are not sensitive to the animal's expected BW nor the basal level of HP. The heritability estimates were moderate for ∆BW (0.33) and low-to-moderate for ∆HP (0.16). Our study suggests ΔBW and ΔHP as novel resilience indicators in pigs. The suggested indicators capture different aspects of resilience, are easy to measure, and are genetically controlled. Thus, they may be improved through selective breeding. Further analyses are needed to validate our findings.

Extracellular Reactive Oxygen Species (ROS) Production in Fresh Donkey Sperm Exposed to Reductive Stress, Oxidative Stress and NETosis.

Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The present study aimed to determine the extracellular ROS production in both sperm and PMN. With this purpose, (1) donkey sperm were exposed to reductive and oxidative stresses, through adding different concentrations of reduced glutathione (GSH) and hydrogen peroxide (H2O2), respectively; and (2) PMN were subjected to NETosis in the presence of the whole semen, sperm, seminal plasma (SP) or other activators such as formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular ROS production (measured as H2O2 levels) was determined with the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. Donkey sperm showed more resilience to oxidative stress than to the reductive one, and GSH treatments led to greater H2O2 extracellular production. Moreover, not only did SP appear to be the main inducer of NETosis in PMN, but it was also able to maintain the extracellular H2O2 levels produced by sperm and NETosis.

Polyphenols and IUGR Pregnancies: Effects of the Antioxidant Hydroxytyrosol on Brain Neurochemistry and Development in a Porcine Model.

Supplementation of a mother's diet with antioxidants, such as hydroxytyrosol (HTX), has been proposed to ameliorate the adverse phenotypes of fetuses at risk of intrauterine growth restriction. In the present study, sows were treated daily with or without 1.5 mg of HTX per kilogram of feed from day 35 of pregnancy (at 30% of total gestational period), and individuals were sampled at three different ages: 100-day-old fetuses and 1-month- and 6-month-old piglets. After euthanasia, the brain was removed and the hippocampus, amygdala, and prefrontal cortex were dissected. The profile of the catecholaminergic and serotoninergic neurotransmitters (NTs) was characterized and an immunohistochemical study of the hippocampus was performed. The results indicated that maternal supplementation with HTX during pregnancy affected the NT profile in a brain-area-dependant mode and it modified the process of neuron differentiation in the hippocampal CA1 and GD areas, indicating that cell differentiation occurred more rapidly in the HTX group. These effects were specific to the fetal period, concomitantly with HTX maternal supplementation, since no major differences remained between the control and treated groups in 1-month- and 6-month-old pigs.

Neurobiology of environmental enrichment in pigs: hanges in monoaminergic neurotransmitters in several brain areas and in the hippocampal proteome.

Environmental enrichment in porcine farms improves animal welfare and leads to better public acceptance. To better understand the neurological mechanisms of the response to environmental enrichment, monoaminergic neurotransmitters were quantified in several brain areas from pigs after eight weeks of housing in barren or enriched conditions. Furthermore, iTRAQ labelling combined with LC-MS/MS was used to identify differentially abundant proteins in the hippocampus. Blood biochemical parameters related with stress and welfare were measured. Pigs under enriched conditions showed a decrease in plasma cortisol and lactate. The decrease in noradrenaline in the prefrontal cortex and amygdala, a general decrease in the dopaminergic system and an increase of serotonin in the striatum indicate a lower response to stress in enriched conditions. In the proteomic analysis, 2304 proteins were identified, of which 56 were differential between housing groups (46 upregulated and 10 downregulated). Bioinformatics analysis revealed that they were mainly related to ribosome, translation, microtubules and metabolic mitochondrial processes, indicating that pigs under enriched environments have higher abundance of proteins related to protein synthesis and neuronal activity. Together with previous behavioural studies, our results suggest that environmental enrichment provides a less stressful environment and that pigs cope better with stress conditions like the slaughterhouse. SIGNIFICANCE: Animal welfare has become an important aspect for the sustainability of animal production. The modification of the environment by enriching it with rooting materials and wider space allowance is known to have a positive effect on pigs' welfare. Searching for the underlying neurobiological mechanisms, we found that housing in an enriched environment increased the abundance of proteins related to protein synthesis, microtubule assembly, vesicle-mediated transport and energy metabolism in the hippocampus of pigs. Likewise, changes in the neurotransmitter profile in several brain areas were compatible with a better response to stress. This study expands the knowledge about the biological basis of animal welfare-promoting actions.

Metabolome and proteome changes in skeletal muscle and blood of pre-weaning calves fed leucine and threonine supplemented diets.

In pre-weaning calves, both leucine and threonine play important roles in growth and muscle metabolism. In this study, metabolomics, proteomics and clinical chemistry were used to assess the effects of leucine and threonine supplementation added to milk replacer on 14 newborn Holstein male calves: 7 were fed a control diet (Ctrl) and 7 were fed the Ctrl diet supplemented with 0.3% leucine and 0.3% threonine (LT) from 5.6 days of age to 53.6 days. At this time, blood and semitendinosus muscle biopsies were collected for analysis. Integrated metabolomics and proteomics showed that branched-chain amino acids (BCAA) degradation and mitochondrial oxidative metabolism (citrate cycle and respiratory chain) were the main activated pathways in muscle because of the supplementation. BCAA derivatives and metabolites related to lipid mobilization showed the major changes. The deleterious effects of activated oxidative phosphorylation were balanced by the upregulation of antioxidant proteins. An increase in protein synthesis was indicated by elevated aminoacyl-tRNA biosynthesis and increased S6 ribosomal protein phosphorylation in skeletal muscle. In conclusion, LT group showed greater BCAA availability and mitochondrial oxidative activity; as the muscle cells undergo greater aerobic metabolism, antioxidant defenses were activated to compensate for possible cell damage. Data are available via ProteomeXchange (PXD016098). SIGNIFICANCE: Leucine and threonine are essential amino acids for the pre-weaning calf, being of high importance for growth. In this study, we found that leucine and threonine supplementation of milk replacer to feed pre-weaning calves led to differences in the proteome, metabolome and clinical chemistry analytes in skeletal muscle and plasma, albeit no differences in productive performance were recorded. This study extends our understanding on the metabolism in dairy calves and helps optimizing their nutritional status.

Total and specific activities of superoxide dismutase (SOD) in seminal plasma are related with the cryotolerance of jackass spermatozoa.

This study investigated whether the activities of four antioxidant enzymes present in jackass seminal plasma (SP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) and glutathione reductase (GSR), are related to the sperm ability to withstand cryopreservation. Eighteen ejaculates from 16 healthy jackasses were collected and split into two aliquots. The first one was centrifuged (3,000×g, 4 °C for 10 min) and used to determine the activities of these four enzymes in SP, whereas the other was diluted in a skim-milk extender and then cryopreserved. Assessment of sperm motility and membrane integrity was performed before and after cryopreservation. Based on the percentages of total motile and viable spermatozoa at post-thaw, samples were classified as good (GFE) or poor (PFE) freezability ejaculates through cluster analyses. Total and specific activities of SOD in seminal plasma were higher (P < 0.05) in GFE than in PFE, whereas no significant differences between GFE and PFE were observed regarding total and specific activities of CAT, GPX and GSR. However, post-thaw sperm parameters were positively correlated with total and specific activities of CAT and negatively correlated with those of GSR. In conclusion, determination of total and specific activities of SOD in the seminal plasma of a given jackass ejaculate may predict the sperm ability to withstand cryopreservation. In addition, our results warrant further research on addressing whether SOD activity in seminal plasma does not only allow predicting the sperm cryotolerance of a given ejaculate but also that of all ejaculates from a given jackass.

Specific Activity of Superoxide Dismutase in Stallion Seminal Plasma Is Related to Sperm Cryotolerance.

While the removal of seminal plasma is a routine practice prior to equine sperm cryopreservation, this fluid contains the main source of antioxidant enzymes able to scavenge these reactive oxygen species. Therefore, stallion seminal plasma components may have an impact on ejaculate freezability. Against this background, this study was designed to investigate whether the activities of the main stallion seminal plasma antioxidant enzymes are related to sperm cryotolerance. With this purpose, 16 ejaculates were collected from 14 healthy stallions, and each ejaculate was split into two aliquots. The first one was used to evaluate the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GSR) in seminal plasma. The second aliquot was extended and then processed for cryopreservation. Sperm motility and viability were evaluated before and after cryopreservation, and ejaculates were classified as of good (GFE) or poor freezability (PFE) based on total motile and viable spermatozoa at post-thaw. We observed that, while the specific activities of CAT, GPX, and GSR were similar between GFE and PFE, that of SOD was significantly (p < 0.05) higher in GFE than in PFE. We can thus conclude that, in stallions, the specific activity of SOD in the seminal plasma of a given ejaculate might be related to its freezability.

Activities of antioxidant seminal plasma enzymes (SOD, CAT, GPX and GSR) are higher in jackasses than in stallions and are correlated with sperm motility in jackasses.

This study compared the activities of four antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX; and glutathione reductase, GSR) in the seminal plasma of stallions and jackasses. Eighteen stallion ejaculates and 24 jack ejaculates were collected through an artificial vagina. Seminal plasma was obtained by several centrifugations at 3000×g and 4 °C for 10 min, and activities of SOD, CAT, GPX and GSR were subsequently determined. We also evaluated whether the collecting season had any influence on the activities of these four enzymes in both stallions and jackasses. Antioxidant capacity of seminal plasma was significantly higher in jackasses than in stallions (mean ±â€¯SEM, SOD: 1707.7 ±â€¯195.9 U/mL vs. 231.9 ±â€¯29.6 U/mL; CAT: 9094.7 ±â€¯1292.9 U/L vs.1682.7 ±â€¯525.9 U/L; GPX 845.4 ±â€¯106.0 U/L vs. 469.7 ±â€¯60.3 U/L; GSR: 50.3 ±â€¯5.1 U/L vs. 20.7 ±â€¯4.6 U/L). Furthermore, whereas season had no effect on the activity of these four enzymes in stallions, the activities of CAT and GPX in jack seminal plasma were significantly higher in the summer than in the other seasons. In addition, the activities of SOD and CAT were found to be significantly correlated with the percentages of progressively motile spermatozoa, and with the percentages of linearity and straightness, respectively, in jackasses. In contrast, the activities of these four enzymes were not correlated with sperm quality parameters in stallions. Finally, while SOD, CAT, and GPX activities but not those of GSR were correlated in jackasses, the activities of all four enzymes were correlated each other in stallions. We can thus conclude that the activities of SOD, CAT, GPX and GSR differ between the seminal plasma of stallions and donkeys, and vary between seasons in jackasses.