Effect of astaxanthin on the quality of boar sperm stored at 17°C,incubated at 37°C or under in vitro conditions

The aim of the study was to investigate the effect of the antioxidant astaxanthin on boar semen. Twenty ejaculates from 10 boars (two ejaculates/boar) were extended and split in three groups: semen control (SC), solvent control (C; semen with dimethyl sulfoxide, the diluent of astaxanthin) and semen with astaxanthin (A) in concentration 0.5 μmol/L. Sperm quality parameters (motility and kinetics, morphology, viability, functional integrity of sperm plasma membrane by Hypo‐Osmotic Swelling Test [HOST] and DNA integrity) were assessed at 0, 24 and 48 hr of storage at 17°C (experiment I), before (0 hr) and after (1 hr) of sperm thermal resistance assay at 37°C (experiment II) and finally before (0 hr) and after (1 hr) sperm in vitro incubation (38.5°C, 5% CO₂, maximum humidity [experiment III]). In experiment I, group A performed overall better than group SC and as a tendency better than group C regarding viability. Total motility, rapid spermatozoa and HOST remained constant across time in group A, whereas they decreased in the remaining groups. In experiment II, regarding motility and viability, group A displayed better results across time than the other two groups. In experiment III, viability and total motility decreased in groups SC and C, while in group A, these parameters were not significantly different between the examination time points. In conclusion, astaxanthin has a beneficial and protective effect on boar semen quality under the investigated conditions.     

Epigallocatechin‐3‐gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24–72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin‐3‐gallate (EGCG) at 20, 60 and 120 μm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.     

Use of protocatechuic acid as the sole antioxidant in the base medium for in vitro culture of ovine isolated secondary follicles

This study evaluated the effect of the protocatechuic acid (PCA) as the sole antioxidant in the base medium for in vitro culture of ovine secondary follicles. Secondary follicles (200‐230 μm) were isolated and cultured in α‐minimal essential medium supplemented with BSA, insulin, glutamine and hypoxanthine (α‐MEM: antioxidant‐free medium) or α‐MEM also added by transferrin, selenium and ascorbic acid (α‐MEM+: with antioxidant) or α‐MEM added by PCA (56.25; 112.5; 225; 450; or 900 μg/ml). Moreover, after culture, oocytes were matured and the chromatin configuration and DNA fragmentation were evaluated. After 12 days, the treatment containing 56.25 μg/ml PCA showed higher percentage of normal follicles than control medium or the other treatments (p < .05), except for 900 μg/ml PCA (p > .05). The antrum formation was significantly higher in treatments containing 56.25, 112.5 or 900 μg/ml PCA, compared to the α‐MEM and similar (p > .05) to the other treatments. The rates of fully grown oocytes (≥110 μm) were similar (p > .05) among all treatments containing PCA and α‐MEM+, and those were superior (p < .05) than α‐MEM, except for 450 μg/ml PCA (p > .05). GSH levels and mitochondrial activity were higher (p < .05) in α‐MEM+ than in α‐MEM and similar (p > .05) to all PCA treatments. The rates of meiotic resumption and DNA fragmentation were similar (p > .05) among α‐MEM+ and 56.25 μg/ml PCA. In conclusion, PCA at 56.25 μg/ml as the sole antioxidant added to the medium for ovine isolated secondary follicle culture maintains follicular survival, GSH and active mitochondria levels, meiotic developmental competence and DNA integrity of cultured oocytes.     

Effect of dietary α‐tocopherol on the bioavailability of lutein in laying hen

Lutein and its isomer zeaxanthin have gained considerable interest as possible nutritional ingredient in the prevention of age‐related macular degeneration (AMD) in humans. Egg yolk is a rich source of these carotenoids. As an oxidative sensitive component, antioxidants such as α‐tocopherol (T) might contribute to an improved accumulation in egg yolk. To test this, chickens were fed lutein esters (LE) with and without α‐tocopherol as an antioxidant. After depletion on a wheat–soya bean‐based lutein‐poor diet for 21 days, laying hens (n = 42) were equally divided into three groups and fed the following diets for 21 days: control (basal diet), a LE group (40 mg LE/kg feed) and LE + T group (40 mg LE plus 100 mg T/kg feed). Eggs and blood were collected periodically. Carotenoids and α‐tocopherol in yolk and blood plasma were determined by HPLC. Egg yolk was also analysed for total carotenoids using a one‐step spectrophotometric method (iCheck^(?)). Lutein, zeaxanthin, α‐tocopherol and total carotenoids in egg yolk were highest after 14 days of feeding and decreased slightly afterwards. At the end of the trial, eggs of LE + T group contained higher amount of lutein (13.72), zeaxanthin (0.65), α‐tocopherol (297.40) and total carotenoids (21.6) compared to the LE group (10.96, 0.55, 205.20 and 18.0 mg/kg, respectively, p < 0.05). Blood plasma values of LE + T group contain higher lutein (1.3), zeaxanthin (0.06) and tocopherol (20.1) compared to LE group (1.02, 0.04 and 14.90 mg/l, respectively, p < 0.05). In conclusion, dietary α‐tocopherol enhances bioavailability of lutein reflecting higher content in egg yolk and blood plasma. Improved bioavailability might be due to increased absorption of lutein in the presence of tocopherol and/or a greater stability of lutein/zeaxanthin due to the presence of α‐tocopherol as an antioxidant.     

Influence of idebenone on ram semen quality stored at 4°C

The current study was designed to investigate the effect of idebenone (Id), an antioxidant on ram semen quality. Semen samples were collected, pooled and diluted in a Tris‐based extender supplemented with 0, 1, 2, 4 and 8 µM idebenone. Computer‐assisted sperm analysis was used to evaluate spermatozoa kinematics. Sperm viability and membrane functionality were assessed respectively, by eosin‐nigrosin staining and HOS test. Biochemical assays were carried out to measure different metabolites in spermatozoa and medium at 0, 24, 48 and 72 hr. Total and forward progressive motility were greater in 1, 2 and 4 µM idebenone treated groups compared to control at 24, 48 and 72 hr time points (p < 0.05). Semen supplementation with Id significantly increased viability and functionality of spermatozoa membrane during storage (p < 0.05). Lower amounts of lipid hydroperoxides in medium and spermatozoa were observed in Id‐treated groups compared to control one at 24 and 48 hr of storage (p < 0.05). Medium and spermatozoa amounts of malondialdehyde and nitric oxide were less in Id 4 µM group compared to the control at 72 hr (p < 0.05). Total antioxidant capacity values and superoxide dismutase activity of spermatozoa and medium were greater in 2 and 4 µM idebenone treated groups in comparison with the control at 72 hr (p < 0.05). Results of the current study indicated that ram semen supplementation with Id at 4 µM level improved quality by ameliorating nitrosative and peroxidative stress, hence could be considered as an antioxidant additive during storage at 4°C.     

Deoxynivalenol induces oxidative stress,inflammatory response and apoptosis in bovine mammary epithelial cells

Deoxynivalenol (DON) is a toxic secondary metabolite produced by Fusarium graminearum. It is one of the most common feed contaminants that poses a serious threat to the health and performance of dairy cows. This study investigated the in vitro cytotoxicity of DON on bovine mammary epithelial cells (MAC‐T). DON at different concentrations (0.25, 0.3, 0.5, 0.8, 1 or 2 μg/ml) inhibited the growth of MAC‐T cells after 24 hr of exposure (p < .001). DON at 0.25 μg/ml increased lactate dehydrogenase (LDH) leakage (p < .05); decreased glutathione (GSH) levels (p < .001), total superoxide dismutase (T‐SOD) activity and total antioxidant capacity (T‐AOC; p < .01); and increased malondialdehyde (MDA) concentration (p < .01) in MAC‐T cells after 24 hr of exposure. We also observed that DON increased reactive oxygen species (ROS) levels in cells incubated for 9, 15 and 24 hr (p < .001). DON at 0.25 μg/ml triggered oxidative damage in MAC‐T cells. Furthermore, it induced an inflammatory response in the cells incubated for 9, 15 and 24 hr (p < .05) by increasing the mRNA expression levels of nuclear factor kappa B, myeloid differentiation factor 88 (MyD88), tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, cyclooxygenase‐2 and IL‐8. We further examined the effect of DON on apoptosis. DON prevented normal proliferation of MAC‐T cells by blocked cell cycle progression in 24 hr (p < .001). In addition, the apoptosis rate measured using annexin V‐FITC significantly increased (p < .05) with increase in the mRNA expression level of Bax (p < .01) and increase in the Bax/Bcl‐2 ratio (p < .01) in cells incubated for 24 hr. In summary, DON exerts toxic effects in MAC‐T cells by causing oxidative stress, inducing an inflammatory response, affecting cell cycle and leading to apoptosis.     

Antioxidants protect proteins' anchorage to the bilayer by improving plasma membrane integrity of ram spermatozoa during liquid preservation in a soya lecithin‐based diluent

Antioxidants are known to prevent the reactive oxygen species (ROS)‐mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid–protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll^®. Sperm pellet was resuspended in soya lecithin–Tris–fructose diluent (400 × 10⁶ cells/ml) containing either antioxidants (100 IU/ml catalase + 10 mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3–5°C for 72 hr. Sperm motility, viability, lipid peroxidation (LPO) and hypo‐osmotic swelling test (HOST) were performed at 0, 24, 48 and 72 hr. Sperm proteins extracted with 0.5% Triton X‐100 were resolved by SDS‐PAGE and quantified using Quantity One software (Bio‐Rad, USA). The rapid motility, linearity and straight‐line velocity (VSL) were found significantly (p < .05) higher in the antioxidant‐treated group compared to the control at 48 hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant‐treated sample compared to the control both at 48 and at 72 hr. Overall, the proteins P1 (106.09 kDa), P2 (87.00 kDa) and P4 (51.14 kDa) were lower (p < .05) in the sperm extract of antioxidant‐treated group compared to the control. The content of P4 (51.14 kDa) in sperm extract was found to increase (p < .05) earlier (48 vs. 72 hr) in the control group compared to the antioxidant‐treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72 hr of storage.     

Effects of vitamin E and selenium supplementation on blood lipid peroxidation and cortisol concentration in dairy cows undergoing omentopexy

Twenty dairy cows with left abomasal displacement were used to investigate the effects of vitamin E and selenium treatment on thiobarbituric acid reactive substances (TBARS) and blood cortisol in dairy cows stressed by omentopexy. The cows were randomly divided into two groups. Ten hours before surgery 6 g of DL‐α‐tocopheryl acetate (6 mg/kg) and 67 mg of natrium selenite (0.1 mg/kg) in volume of 40 ml (Vitaselen^®) were administered subcutaneously to 10 cows; the control animals (n = 10) received an equivalent volume of injectable water (40 ml). The injection of vitamin E and selenium produced a rapid rise (p < .05) in blood α‐tocopherol and selenium concentrations. The serum vitamin E increased several times 10 hr after vitamin E and Se injection and raised continuously to the highest average concentration 21.6 mg/L at hr 24 after the surgery. The highest selenium concentration was seen 10 hr after selenium administration with holding the increased concentrations in comparison with initial ones during the whole study. Two‐way ANOVA did not show significant treatment effect on plasma concentrations TBARS in the study. The plasma concentrations of thiobarbituric acid reactive substances reached the maximum value of 0.18 μmol/L in the control group 5 hr after the surgery. Twenty‐four hours after the surgery, the TBARS values returned to the initial ones. Serum cortisol increased in both groups after surgery. The highest cortisol concentrations were reached at 1 hr after surgery in the experimental and control group (56.7 ± 28.8 and 65.3 ± 26.1 μg/L respectively). A return to the levels similar to the initial ones was recognized 24 hr after the surgery. The ANOVA revealed a significant effect of vitamin E and selenium injection on plasma cortisol (p < .05). In conclusion, we have demonstrated that abdominal surgery resulted in typical stress changes with no significant effects of a single vitamin E/Se injection on blood lipid peroxidation. In addition, a weaker cortisol response to the abdominal surgery was recognized in animals treated with vitamin E and selenium.     

Effects of ascorbic acid 2‐glucoside and alpha‐tocopherol on the characteristics of equine spermatozoa stored at 5°C

The aim of this experiment was to evaluate the effects of adding ascorbic acid 2‐glucoside (AA2G), a water‐soluble antioxidant and stable derivative of ascorbate, to the semen extender and compare it to the addition of vitamin C (Vit. C) and the fat‐soluble antioxidant α‐tocopherol (α‐Toh), both individually and in combination, on the seminal variables of equine sperm submitted to cooling for 72 h. We used two ejaculates from 10 stallions and evaluated them for motility, membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation. In the analysis of lipid peroxidation, the control group showed 2506.2 ± 796.4 ng malondialdehyde/10⁸ sperm, which was higher (P < 0.05) than the groups treated with antioxidants. The average value of motility in the AA2G group was 68.4 ± 18.1%, which was higher (P < 0.05) than that observed in the control group (62.1 ± 16.2%). The variables membrane integrity, chromatin fragmentation and mitochondrial activity did not show significant difference (P > 0.05) between treatments. It was concluded that the antioxidants protected sperm cells from lipid peroxidation and that AA2G was effective during the cooling process of equine semen at 5°C for72 h, providing increased levels of total motility.     

Epigallocatechin‐3‐Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm–Zona Pellucida Heterologous Binding

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin‐3‐gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2‐h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm , 500 nm and 5 μm ) and EGCG (10, 20 and 60 μm ), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm ) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm , the presence of EGCG at all the concentrations tested (10, 20 and 60 μm ) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.     

橙皮苷对3种霉菌毒素处理的HepG2细胞增殖和迁移的影响

探讨橙皮苷对3种霉菌毒素处理的HepG2细胞增殖和迁移作用的影响。采用MTT、Griess、细胞划痕愈合试验分别检测细胞活力、细胞分泌NO、细胞迁移率。结果3种毒素对HepG2细胞增殖的影响随毒素浓度的增加呈现先促进后抑制的作用,低浓度DON、OTA(0.04μmol/L和0.08μmol/L),ZEN(<2μmol/L)显著提高HepG2细胞活力及促进增殖,高浓度霉菌毒素明显降低HepG2细胞活力及抑制细胞增殖。与霉菌毒素组比较,橙皮苷组HepG2细胞活力降低及增殖减弱,表示橙皮苷能缓解霉菌毒素对细胞的毒性。与空白对照组比较,霉菌毒素组HepG2分泌NO量显著降低,不同浓度及不同霉菌毒素对NO分泌没有明显影响;与霉菌毒素组比较,橙皮苷组不同浓度DON处理的细胞分泌NO差异不显著。低浓度DON毒素(0.08μmol/L^2μmol/L)处理HepG2细胞24、36、48 h后,DON毒素能极显著促进HepG2细胞迁移,随着霉菌毒素对细胞处理时间延长细胞迁移快,而高浓度DON毒素(10μmol/L、50μmol/L)对促细胞迁移影响显著低于低浓度DON。结果低浓度DON霉菌毒素促进HepG2细胞增殖及迁移,表明DON毒素对HepG2具有潜在的致癌性,橙皮苷能抑制HepG2细胞增殖及缓解霉菌毒素毒性的作用。     

Quercetin attenuates H2O2‐induced toxicity of rooster semen during liquid storage at 4°C

Current study was carried out to examine the protective effects of quercetin against toxicity induced by hydrogen peroxide in rooster semen in vitro. Semen samples were collected from ten roosters (Ross 308 broiler breeder males, 32 weeks old) twice a week by abdominal massage method. Samples with ≥70% progressive motility were selected, pooled, diluted and used for the study. Experimental groups consisted of negative control, control that received solvent of quercetin, H₂O₂ (40 μM) and combination groups which incubated with constant dose of H₂O₂ (40 μM) plus various levels of quercetin (20, 40 and 80 μM). Measurement of total hydroperoxide (HPO), malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (TAC) and superoxide dismutase activity as well as routine sperm tests were done at 0, 24 and 48 hr of storage at 4°C. Results revealed that exposure to hydrogen peroxide significantly increased HPO (138.43 ± 7.32 vs. 66.08 ± 3.97 μmol/g protein), MDA (7.21 ± 0.08 vs. 5.71 ± 2.16 μmol/g protein) and NO (0.367 ± 0.013 vs. 0.215 ± 0.011 μmol/g protein) levels and decreased sperm progressive motility (27.28 ± 1.21 vs. 47.49 ± 1.29%), and amounts of TAC (11.49 ± 0.39 vs. 15.70 ± 0.79 mmol/g protein) compared to control at 24 hr (p < 0.05). Changes at mentioned variables were repeated at 48 hr of storage. Also, co‐administration of quercetin (especially at 40 and 80 μM) with hydrogen peroxide restored the toxic effects of hydrogen peroxide on rooster semen parameters such as primary and secondary lipid peroxidative indicators and other evaluated variables. The study concluded that rooster semen enrichment with quercetin would protect lipid peroxidative and nitrosative hydrogen peroxide‐mediated damage during cold liquid storage of rooster semen.     

N‐Acetyl‐d‐galactosamine prevents soya bean agglutinin‐induced intestinal barrier dysfunction in intestinal porcine epithelial cells

Soya bean agglutinin (SBA) is a glycoprotein and the main anti‐nutritional component in most soya bean feedstuffs. It is mainly a non‐fibre carbohydrate‐based protein and represents about 10% of soya bean‐based anti‐nutritional effects. In this study, we sought to determine the effects of N‐Acetyl‐D‐galactosamine (GalNAc or D‐GalNAc) on the damage induced by SBA on the membrane permeability and tight junction proteins of piglet intestinal epithelium (IPEC‐J2) cells. The IPEC‐J2 cells were pre‐cultured with 0, 0.125 × 10^?4, 0.25 × 10^?4, 0.5 × 10^?4, 1.0 × 10^?4 and 2.0 × 10^?4 mmol/L GalNAc at different time period (1, 2, 4 and 8 hr) before being exposed to 0.5 mg/ml SBA for 24 hr. The results indicate that pre‐incubation with GalNAc mitigates the mechanical barrier injury as reflected by a significant increase in trans‐epithelial electric resistance (TEER) value and a decrease in alkaline phosphatase (ALP) activity in cell culture medium pre‐treated with GalNAc before incubation with SBA as both indicate a reduction in cellular membrane permeability. In addition, mRNA levels of the tight junction proteins occludin and claudin‐3 were lower in the SBA‐treated groups without pre‐treatment with GalNAc. The mRNA expression of occludin was reduced by 17.3% and claudin‐3 by 42% (p < 0.01). Moreover, the corresponding protein expression levels were lowered by 17.8% and 43.5% (p < 0.05) respectively. However, in the GalNAc pre‐treated groups, occludin and claudin‐3 mRNAs were reduced by 1.6% (p > 0.05) and 2.7% (p < 0.01), respectively, while the corresponding proteins were reduced by 4.3% and 7.2% (p < 0.05). In conclusion, GalNAc may prevent the effect of SBA on membrane permeability and tight junction proteins on IPEC‐J2s.     

Biological and biochemical characteristics of male reproductive system,serum metabolites and carcass quality of Japanese quails by the supplementation of Pinus ponderosa leaves and α‐tocopherol acetate

The aim of the current study was to evaluate the effect of dietary supplementation of Pinus ponderosa leaves ( pine leaves) and α‐tocopherol acetate (vitamin E) powder on male reproductive system, serum metabolites and carcass characteristics of Japanese quails. A total of 360‐day‐old male quails were purchased from the open market and kept at poultry shed for ninety‐four days. After ten days of adaptation, all quails were randomly assigned into 4 groups, control (IC); supplemented with α‐tocopherol acetate (IE) at the rate of 150 mg/L; Pinus ponderosa leaves (IP) at the rate of 150 mg/L; and 70 mg α‐tocopherol acetate and 70 mg Pinus ponderosa leaves (IEP). Pinus ponderosa leaves and α‐tocopherol acetate supplementation had not significantly (p > .05) effected on final body weight gain, feed intake and feed conversion ratio of quails. The high‐density lipoprotein cholesterol (HDLC) and total cholesterol (TC) were significantly (p > .05) affected by IE and IP groups as compared to IC and IEP groups. Triglyceride (TG), glutathione peroxidase (GPx) and superoxide dismutase (SOD) significantly (p < .05) increased in all treatment groups except for the IC group. Aspartate transaminase (AST) significantly (p > .05) decreased in treatment groups as compared to control group. Overall, the mineral levels significantly (p < .05) increased in treatment groups as compared to control. Cloacal gland index values, the quantity of foam production and testis weight were significantly (p < .05) increased in treatment groups. It was concluded that the supplementation of Pinus ponderosa leaves and α‐tocopherol acetate improved the testis weight, foam production, serum antioxidant enzymes and mineral level especially zinc in Japanese quail considered an indicative characteristic of higher sperm production rate and improved sexual activity. Further, higher gametogenesis rate, sperm production or reproductive behaviour including different hormonal level will be analysed in future study.     

Administration of RRR‐α‐tocopherol to pregnant mares stimulates maternal IgG and IgM production in colostrum and enhances vitamin E and IgM status in foals

This study assessed the effect of a vitamin E supplement given to pregnant mares on immunoglobulins (Ig) levels in foals. In addition, the fatty acid (FA) content and composition of the mares’ milk was assessed. Milk α‐tocopherol concentrations were compared between pregnant Danish Warmblood mares (n = 17) given a daily oral supplement of 2500 international units (IU) RRR‐α‐tocopherol in the last 4 weeks of pregnancy and a group of unsupplemented mares (n = 17) receiving 170–320 IU vitamin E daily originating from the feed. Milk α‐tocopherol was higher in supplemented mares (36.7, 12.4 and 9.8 μmol/l respectively) in relation to control mares (13.1, 6.4 and 5.8 μmol/l on days 1, 2 and 3 respectively; p < 0.001). Milk IgG was higher on days 2 and 3 post‐partum (PP) in supplemented mares (1.03 and 0.73 mg/ml respectively) in relation to control mares (0.79 and 0.56 mg/ml respectively; p < 0.05). Milk IgM was higher on days 2 and 3 post‐partum (PP) in supplemented mares (0.19 and 0.17 mg/ml) in relation to control mares (0.13 and 0.11 mg/ml respectively; p < 0.05). Plasma α‐tocopherol in foals was higher from supplemented mares on days 1, 2 and 3 (5.7, 14.8 and 19.2 μmol/l respectively) in relation to foals from control mares (3.6, 6.1 and 7.6 respectively; p < 0.001). Foal plasma IgM was higher from supplemented mares on day 3 (0.50 mg/ml) in relation to foals from control mares (0.32 mg/ml; p < 0.001). The total FA content in milk was highest on day 1 (21.6 g FA/kg milk) in relation to days 2 and 3 (13.6 and 13.5 g FA/kg milk respectively; p < 0.001). In conclusion, a daily oral supplement of 2500 IU RRR‐α‐tocopherol increased α‐tocopherol content in mare milk and foal plasma, IgG and IgM in mare milk and IgM in foal plasma.     

Pharmacokinetics of cefquinome in healthy and Pasteurella multocida‐infected rabbits

The pharmacokinetics of cefquinome were studied in healthy and Pasteurella multocida‐infected rabbits after a single intramuscular (IM) injection at 2 mg/kg of its sulfate salt. Twelve female New Zealand white rabbits (2.0–2.5 kg) were used; six of them served as controls, and the other six had been infected with P. multocida; the experiments were conducted 1–2 days after nasal inoculation of P. multocida when rabbits showed the signs of respiratory infection. Plasma concentrations of cefquinome were determined using high‐performance liquid chromatography. The values of elimination half‐life, area under the curve, area under the first moment curve, and mean residence time were significantly lower in infected rabbits (0.48 hr, 4.54 hr*μg/ml, 3.63 hr* hr*μg/ml and 0.8 hr, respectively) than healthy rabbits (0.72 hr, 9.11 hr*μg/ml, 9.85 hr* hr*μg/ml and 1.1 hr, respectively), whereas total body clearance was significantly higher in infected than healthy rabbits. Therefore, P. multocida infection caused significant changes in some of the pharmacokinetic parameters of cefquinome in rabbits. These pharmacokinetic changes may affect dose regimen when used in P. multocida‐infected rabbits.     

Effect of bovine seminal plasma on bovine endometrial epithelial cells in culture

The purpose of this study was to investigate the effect of seminal plasma (SP) from bulls of known fertility on bovine endometrial epithelial cells (bEEC) in culture. The bEEC from passage 5, approximately 5.0–13 × 10⁵ cells per flask, were challenged with SP from bulls of high or low fertility (n = 3 and 2, respectively) or PBS (control), at 1% (75 μl) or 4% (300 μl) and were incubated for 72 hr (n = 13 per challenge). Total cell number and viability of bEEC after challenge with 1% SP from either high‐ or low‐fertility bulls (75H or 75L, respectively) did not differ from controls. In contrast, challenge with 4% of SP from high‐ or low‐fertility bulls (300H or 300L) negatively affected bEEC cell number and viability. Challenge with 300 L had a greater adverse effect than 300H. These results suggest that the negative effect of bovine SP on bEEC is both dose‐dependent and fertility‐dependent.     

Effect of olive-derived antioxidants (3,4-dihydroxyphenylethanol and 3,4 dihydroxyphenylglycol) on sperm motility and fertility in liquid ram sperm stored at 15°C or 5°C

The aim of the present study was to evaluate the effect of the addition of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during liquid storage at 5°C and 15°C. Semen was collected, pooled, diluted and then divided into aliquots supplemented with different concentrations (5 μg/ml, 10 μg/ml, 50 μg/ml and 100 μg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. Sperm motility characteristics were assessed in the different samples at 0, 6, 24, 48, 72 and 96 hr after cooling, and a fertility trial was also conducted. The results showed that the antioxidant addition did not significantly improve total and progressive motility in ovine cooled sperm maintained at 15° or 5°C. However, in samples stored at 5°C, LIN (48, 72, 96 hr), STR (0 hr) and WOB (0, 48, 72, 96 hr) values significantly decreased in comparison with control treatment when high antioxidant concentrations were added (MIX100 or HT100). When samples were maintained at 15°C, MIX50 showed significantly higher VCL values than the control treatment after 6 hr cooling, and MIX100 showed significantly lower VCL values at 96 hr after cooling. According to the artificial insemination trial, no significant differences were observed when antioxidants were added. In conclusion, the use of HT and DHPG showed small impact in sperm motility and fertility was not affected (nor detrimentally nor positively) when insemination was carried out using antioxidant-supplemented liquid sperm.     

Protective Effects of Quercetin on Selected Oxidative Biomarkers in Bovine Spermatozoa Subjected to Ferrous Ascorbate

Quercetin (QUE) is a natural flavonol‐type flavonoid with antibacterial, anti‐inflammatory and anti‐aggregatory properties. It is also a powerful reactive oxygen species (ROS) scavenger and chelating agent. The aim of this study was to assess the effectiveness of QUE to reverse ROS‐mediated alterations to the motility, viability and intracellular antioxidant profile of bovine spermatozoa. Spermatozoa were washed out of fresh bovine semen, suspended in 2.9% sodium citrate and subjected to QUE treatment (7.5, 25, 50 and 100 μmol/l) in the presence or absence of a pro‐oxidant, that is ferrous ascorbate (FeAA; 150 μmol/l FeSO4 and 750 μmol/l ascorbic acid) during a 6‐h in vitro culture. Spermatozoa motion characteristics were assessed using the SpermVision computer‐aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified via luminometry, and the nitroblue tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the in vitro culture to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). FeAA treatment led to a reduced sperm motility (p < 0.001), viability (p < 0.001) and decreased the antioxidant parameters of the sperm samples (p < 0.001) but increased the ROS generation (p < 0.001), superoxide production (p < 0.001) and lipid peroxidation (p < 0.001). QUE administration resulted in a preservation of the spermatozoa vitality and antioxidant characteristics (p < 0.01 with respect to the enzymatic antioxidants, p < 0.001 in relation to GSH) with a concentration range of 50–100 μmol/l QUE revealing to be the most effective. Our results suggest that QUE exhibits significant ROS‐scavenging and metal‐chelating properties which may prevent spermatozoa alterations caused by ROS, and preserve the functionality of male reproductive cells.     

Effect of vitamin E supplementation in milk replacer and Shiga toxoid vaccination on serum α‐tocopherol,performance, haematology and blood chemistry in male Holstein calves

Vitamin E (vit E), an essential antioxidant for maintaining the stability of biological membranes and the function of the immune system, is considered to support adaptive immune responses and performance in cattle. The principal virulence factor of Shiga toxin (Stx)‐producing Escherichia coli (STEC), the eponymous Stx, modulates cellular immune responses in cattle, the primary STEC reservoir. Active and passive immunization of calves with Shiga toxoids (rStx_MUT) was recently shown to reduce the STEC shedding. Here, we examined the influence of vit E on calves' serum α‐tocopherol, performance, haematology, blood chemistry and its interaction with rStx_MUT immunization. Data from calves having received passive (colostrum from immunized cows) and active (intramuscularly at 5th and 8th weeks of life) vaccination with rStx_MUT (n = 24) were compared to unvaccinated controls (n = 24; fed with low anti‐Stx colostrum, placebo injected). For each vaccination group, data were analysed according to the level of vit E supplementation offered by milk replacer (188 IU all‐rac‐α‐tocopheryl acetate daily [VitE_M] vs. 354 IU [VitE_H]). An increase by 79% in daily vit E supplementation led to slightly higher serum α‐tocopherol level and earlier concentrate intake at the beginning of the experiment without significant differences in live weight gain, haematology, blood chemistry parameters and peripheral CD4⁺ and CD8⁺ T‐cell subpopulations. rStx_MUT vaccination modulated the CD4⁺/CD8⁺ ratio irrespective of vit E supplementation but decreased concentrate intake in VitE_H in a time‐dependent manner. Results of our study indicate that an increase in daily vit E supplementation vastly fails to exert effects on laboratory parameters and growth performance. However, observed interactive effects of vit E supply and vaccination on the regulation of feed intake deserves further attention.