Effects of α-tocopherol and Ascorbic Acid on Equine Semen Quality after Cryopreservation

This study investigated the effects of ascorbic acid and α-tocopherol supplementation on semen quality parameters of equine thawed-frozen semen. Semen was divided in seven different treatments in a final concentration of 100 × 10⁶ sperm/mL by using Gent extender containing no supplements (control) and the following supplements with three different concentrations: α-tocopherol (0.5, 1, and 2 mM) and ascorbic acid (0.45, 0.9, and 1.8 g/L). After thawing, all samples were maintained at 37°C, while analyses were performed at 0, 60, and 120 minutes. Evaluation of viability and acrosome status (using Pisum sativum agglutinin conjugated to fluorescein isothiocyanate and propidium iodide), mitochondrial membrane potential (5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolyl carbocyanine iodine [JC-1]), membrane lipid peroxidation (LPO; C₁₁-BODIPY^581/591), and stability of the plasmatic membrane (merocyanine 540 and Yo-Pro-1) of each sample was determined by flow cytometry. Relative to the control group, supplementation with α-tocopherol improved (P ≤ .05) postthaw membrane LPO, yet the higher concentrations of ascorbic acid (0.9 and 1.8 g/L, respectively) showed a negative effect on membrane LPO. Neither antioxidant significantly increased (P > .05) the acrosome integrity and mitochondrial membrane potential of frozen-thawed spermatozoa, although supplementation with α-tocopherol and ascorbic acid (0.9 and 1.8 g/L, respectively) had a positive effect on membrane integrity and stability (P ≤ .05). For all semen parameters, the lower concentration of ascorbic acid (0.45 g/L) did not show significant differences (P > .05) compared with the control. In conclusion, α-tocopherol seems to be an efficient antioxidant for reducing the oxidative stress provoked by cryopreservation, decreasing lipid peroxidation on equine spermatozoa.     

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.     

Antioxidative Effects of Astaxanthin against Nitric Oxide‐Induced Oxidative Stress on Cell Viability and Gene Expression in Bovine Oviduct Epithelial Cell and the Developmental Competence of Bovine IVM/IVF Embryos

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co‐culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide‐induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl‐2, Caspase‐3 and Bax) by RT‐PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co‐cultured with BOEC pre‐treated with astaxanthin (500 μm ) in the presence or absence of sodium nitroprusside (SNP, 1000 μm ) for 24 h. Cell viability in BOEC treated with SNP (50–2000 μm ) lowered, while astaxanthin addition (50–500 μm ) increased it in a dose‐dependent manner. Cell viability in astaxanthin plus SNP (1000 μm ) gradually recovered according to the increase in astaxanthin additions (100–500 mm ). The LPO in astaxanthin group (50–500 μM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 μM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT‐PCR. Bcl‐2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase‐3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6–7 days under co‐culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 μm astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.     

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.     

Effect of Alpha-Tocopherol and Ascorbic Acid on Bovine Oocyte in Vitro Maturation

In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus-oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence.     

The Effect of Added Serum and Glucose, and Some Inherent Factors, on Phagocytosis in vitro by Milk Leukocytes from Several Cows

On four occasions leukocytes were obtained from mammary secretions of four cows following irritation by sterile distilled water.

The effects of three levels of bovine immune serum, three levels of glucose, and combinations of these on the ability of the leukocytes to ingest staphylococci in vitro were studied. Phagocytosis was estimated by examination of stained slides prepared after four hours contact at 37°C.

While 1% immune serum increased the percentage of milk leukocytes ingesting staphylococci, no improvement resulted from the addition of 3% or 5%. Addition of 20mg% glucose produced a larger increase than did 1% serum, and 40 and 60mg% gave further increases. Little advantage was obtained by combining serum and glucose at any of the levels studied.

Overall significant differences were found in the ability to ingest staphylococci by milk leukocytes from the four cows, and the previous observation that cells from some cows in vitro do not respond fully to added serum and glucose was confirmed. Neither of these phenomena was related to glycogen levels in blood or milk leukocytes as determined by the anthrone method, blood glucose levels, leukocyte viability, relative proportions of PMN's in circulating blood white cells, nor the amount of homologous blood serum in the mammary secretions. In two cows whose milk cells did not respond fully, very few blood PMN's were active.

     

Electrostatic spraying of antioxidants on the oxidative quality of ground beef

To prevent oxidative quality changes, a few selected antioxidants, ascorbic acid, or an ascorbic acid plus α-tocopherol combination were electrostatically sprayed on the surface of ground beef patties. Color, metmyoglobin, oxidation-reduction potential, lipid oxidation, and volatiles of the samples were determined during the 8 d of aerobic storage. Spraying of ascorbic acid at 500 mg/kg was the most effective in controlling discoloration of ground beef. Spraying of ascorbic acid at 500 mg/kg was also effective in reducing 2-thiobarbituric acid-reactive substances and volatile aldehydes such as hexanal and heptanal related to lipid oxidation. Spraying of phenolic antioxidants such as tocopherol, sesamol, or rosemary oleoresin showed significant (P < 0.05) antioxidant effects, but had no effects (P < 0.05) in stabilizing the color of ground beef. Sesamol at 100 mg/kg showed the most potent antioxidant activities among the antioxidants, and its antioxidant effect was as strong as that of 500 mg/kg of ascorbic acid. It was concluded that electrostatic spray of ascorbic acid on the surface of ground beef at 500 mg/kg was an efficient and economical way to prevent both lipid oxidation and color changes in ground beef.     

Effect of α-Tocopherol on Bovine In Vitro Fertilization

The objectives of this work are to determine if exogenous supplementation with α-tocopherol increases the in vitro fertilization (IVF) rate of bovine oocytes and improves viability of selected spermatozoa after 'swim-up'. The percentage of fertilized oocytes was significantly but negatively correlated ( r  = −0.941, p < 0.01) with the concentration of α-tocopherol. The control resulted in 95% of fertilized oocytes, which decreased as follows: 25 μM α-tocopherol (α25) 86%, 50 μM α-tocopherol (α50) 74%, 100 μM α-tocopherol (α100) 66% and 200 μM α-tocopherol (α200) 56%. Relatively to sperm viability after 'swim-up' with α-tocopherol supplementation, this antioxidant proved to have a beneficial effect as its concentration increased up to α50, decreasing for the concentrations of α100 and α200. Control resulted in 83% of live cells and 16% of dead cells; α25 resulted in 88% of live cells and 12% of dead cells; α50 resulted in 91% of live cells and 9% of dead cells; α100 resulted in 67% of live cells and 33% of dead cells; and finally α200 resulted in 57% of live cells and 42% of dead cells. In summary, the present study allows to conclude that, in our conditions, supplementation with the antioxidant α-tocopherol in IVF of bovine oocytes has a detrimental effect on fertilization rates. Nevertheless, exogenous supplementation with α-tocopherol at a concentration of 50 mM in the sperm-TALP media during the 'swim-up' technique has a significant beneficial effect on the selected spermatozoa viability.     

Status of Lipid Peroxidation,Some Antioxidant Enzymes and Erythrocytic Fragility of Crossbred Cattle Naturally Infected with <Emphasis Type="Italic">Theileria annulata</Emphasis>

Erythrocytic lipid peroxidation, activities of some antioxidant enzymes and osmotic fragility of red blood cells was studied in adult (>1 year) crossbred cattle naturally infected with Theileria annulata. Twenty clinically healthy animals (group I) and 15 clinical cases (group II) of tropical theileriosis were selected. Cattle suffering from theileriosis had significantly higher (p<0.01) erythrocytic lipid peroxidation and osmotic fragility. Activities of antioxidant enzymes, viz. glucose-6-phosphate dehydrogenase (G6PD) and glutamate peroxidase (GPx) were also significantly increased (p<0.01) in group II. However, superoxide dismutase and catalase did not show significant changes. The results indicated that infection with theileria led to increased oxidative stress to the animals, and even a significant rise in the activities of antioxidant enzymes. G6PD and GPx could not lower this oxidative stress. However, the increase in the activities of antioxidant enzymes pointed towards the body’s defence mechanism against lipid peroxidation during oxidative stress in theileriosis.     

Transporting bovine oocytes in a medium supplemented with different macromolecules and antioxidants: Effects on nuclear and cytoplasmic maturation and embryonic development in vitro

We investigated whether supplementing the medium used to transport bovine oocytes with different macromolecules [foetal calf serum (FCS) or bovine serum albumin (BSA)] or a mixture of antioxidants (cysteine, cysteamine and catalase) affects their nuclear and cytoplasmic maturation and thereby affects their subsequent embryonic development and cryotolerance. Oocytes were transported for 6 hr in a portable incubator and then subjected to standard in vitro maturation (IVM) for 18 hr. The oocytes in the control groups were cultured (standard IVM) for 24 hr in medium containing 10% FCS (Control FCS) or 10% FCS and the antioxidant mixture (Control FCS+Antiox). The intracellular concentrations of reactive oxygen species (ROS) at the end of IVM period were lower in the oocytes subjected to simulated transport in the presence of a macromolecular supplement or the antioxidant mixture than that of the control group (FCS: 0.62 and BSA: 0.66 vs. Control FCS: 1.00, p < .05; and Transp: 0.58 and Transp Antiox: 0.70 vs. Control FCS: 1.00, p < .05). After IVM, the mitochondrial membrane potentials of the transported oocytes were lower than those of the non‐transported oocytes (FCS: 0.41 and BSA: 0.57 vs. Control FCS: 1.00, p < .05; and Transp: 0.48 and Transp Antiox: 0.51 vs. Control FCS: 1.00 and Control Antiox: 0.84, p < .05). The blastocyst formation rates (36.9% average) and the re‐expansion rates of vitrified‐warmed blastocysts (53%, average) were unaffected (p > .05) by the treatments. In conclusion, supplementing the medium in which bovine oocytes are transported with antioxidants or different macromolecules did not affect their in vitro production of embryos or their cryotolerance.     

Dietary Ascorbyl Monophosphate Depresses Lipid Peroxidation in Rainbow Trout Spermatozoa

Abstract

This study was designed to analyze lipid peroxidation in spermatozoa of rainbow trout Oncorhynchus mykiss by an optimized thiobarbituric acid (TBA) method, and to evaluate the effect of graded levels of dietary antioxidant (ascorbic acid in the form of ascorbyl monophosphate, AP) on TBA values of spermatozoa. Sperm from rainbow trout fed diets supplemented with AP at 0, 110, or 870 mg/kg were sampled during the reproductive season. The group given the unsupplemented diet had the lowest ascorbic acid concentration in seminal plasma during most of the spermiation season compared with groups fed diets with AP While the ascorbic acid concentration in seminal plasma decreased toward the end of the reproductive season, spermatozoa malondialdehyde production, indicative of increasing peroxidation, tended to increase. At the end of the season, significant differences (P < 0.05) were found in peroxidation levels in spermatozoa from fish fed different levels of ascorbic acid. The most abundant polyunsaturated fatty acid in sperm lipids, docosahexaenoic acid (C22:6), was significantly lower in lipids from spermatozoa of the AP-devoid (control) group than in the 870-mg/kg supplement group. By the end of the reproductive season, spermatozoa from the control group had significantly higher peroxidation levels than did spermatozoa from fish given AP (110 and 870 mg/kg). Thus, it is the first evidence in fish that dietary AP supplements can increase seminal plasma ascorbic acid concentrations, and it suggests that peroxidative damage to spermatozoa can be reduced during the reproductive season, which in turn may affect sperm fertilizing ability.     

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.     

Antioxidant Status and Biomarkers of Oxidative Stress in Dogs with Lymphoma

Background: Oxidative stress might play a role in carcinogenesis, as well as impacting morbidity and mortality of veterinary cancer patients. The purpose of this study was to evaluate antioxidant concentrations and biomarkers of oxidative stress in dogs with newly diagnosed lymphoma before treatment and once in remission, with comparison with healthy controls.
Hypothesis: Dogs with lymphoma have increased oxidant and reduced antioxidant concentrations compared with healthy controls, and that these abnormalities normalize once remission is achieved.
Animals: Seventeen dogs with lymphoma and 10 healthy controls.
Methods: Prospective, observational study. Measures of oxidative stress [malondialdehyde and total isoprostanes (isoP)] and antioxidants [α-tocopherol, γ-tocopherol, oxygen radical absorbance capacity (ORAC), and glutathione peroxidase (GSHPx)] were assessed in dogs with newly diagnosed lymphoma before treatment compared with healthy control dogs. The same parameters were measured in the dogs with lymphoma on week 7 of the chemotherapy protocol when all dogs were in remission.
Results: At baseline, dogs with lymphoma had significantly lower α-tocopherol ( P <.001) and γ-tocopherol ( P = .003) but higher GSHPx ( P = .05), ORAC ( P = .001), and isoP ( P < .001) compared with healthy controls. In the dogs with lymphoma, α-tocopherol concentrations were higher ( P = .005) and ascorbic acid were lower ( P = .04) after treatment.
Conclusions and Clinical Importance: Results suggest that dogs with lymphoma have alterations in oxidant and antioxidant concentrations and that the status of some of these biomarkers normalize after remission. Further studies are warranted to determine whether antioxidant interventions to correct these are beneficial in the treatment of canine lymphoma.     

Status of Lipid Peroxidation, Antioxidants, and Oxidation Products of Nitric Oxide in Equine Babesiosis: Status of Antioxidant and Oxidant in Equine Babesiosis

Equine babesiosis is a tick-borne protozoal disease of horses caused by Theileria equi and Babesia caballi. The disease is endemic in most tropical and subtropical areas. The aim of this paper is to assess the antioxidant status, lipid peroxidation, and oxidation products of nitric oxide (NO) in horses and mules naturally infected with T. equi and B. caballi. East and Southeast Anatolian horses and mules living in rural region of the Eastern border of Turkey were used as the material for this study. These animals are used as pack animal (3–7 years of age). Infected animals were in acute or subacute infection period. In the current study, malondialdehyde (MDA), oxidation products of NO (nitrate and nitrite), serum glutathione (GSH), vitamin E, and retinol levels were analyzed in 58 equids (horse and mule) infected with T. equi and B. caballi as well as in 44 healthy equids. Compared with controls, the level of MDA and nitrate increased significantly (P < .01, P < .05, respectively), whereas GSH concentration and levels of vitamin E decreased significantly (P < .01). There was no significant change in the level of nitrite and retinol between two groups. The results of the current study suggest that in equids infected with T. equi and B. caballi, this alteration in the lipid peroxidation, oxidants, and antioxidants may be related to the host's defenses against parasitic infection and may play a central role in the pathologic conditions associated with babesiosis.     

The Effect of Coenzyme Q10 and α-Tocopherol in Skim Milk–Based Extender for Preservation of Caspian Stallion Semen in Cool Condition

The purpose of this study was to determine the effects of different concentrations of coenzyme Q₁₀ (CoQ₁₀) and α-tocopherol (T) along with their interaction effects on the quality of preserved stallion semen at 5°C for a period of 48 hours. Semen was collected and diluted with skim milk–based extender that was supplemented with different antioxidants: no antioxidant (negative control [NC]), 0.9% (vol/vol) dimethyl sulfoxide (positive control [PC]), α-tocopherol (5 [T5] or 10 [T10] mM), CoQ₁₀ (1 [C1] or 2 [C2] μM), 1 μM CoQ₁₀ + 5 mM α-tocopherol (C1T5), 1 μM CoQ₁₀ + 10 mM α-tocopherol (C1T10), 2 μM CoQ₁₀ + 5 mM α-tocopherol (C2T5), and 2 μM CoQ₁₀ + 10 mM α-tocopherol (C2T10), then kept at 5°C. The results showed that C1 extender resulted in higher total motility (62.44 ± 3.82) and plasma membrane integrity (65.16 ± 3.63%) compared with NC after 48 hours of storage (P < .05). Different concentrations of α-tocopherol had no significant effects on sperm quality, with the exception of plasma membrane integrity, compared with NC and PC extenders (P > .05). Also, C1T5 extender improved total and progressive motility, plasma membrane integrity and functionality, and decreased lipid peroxidation compared with NC and C2T10 extenders over 48 hours of storage at 5°C (P < .05). The C1T5 extender was similar to C1 and T5 extenders in all semen parameters evaluated during storage time. In conclusion, between previously mentioned extenders, C1T5 could improve stallion sperm quality during 48 hours of storage. In the present study, none of extenders had effect on sperm quality until 24-hour storage.     

Polyphenol compounds in the chicken/animal diet: from the past to the future

Animal feed provides a range of antioxidants that help the body building an integrated antioxidant system responsible for a prevention of damaging effects of free radicals and products of their metabolism. Vitamin E is considered to be the main chain‐breaking antioxidant located in the membranes and effectively protecting them against lipid peroxidation. Recently, various polyphenol compounds, especially flavonoids, have received substantial attention because of their antioxidant activities in various in vitro systems. However, it was shown that flavonoid compounds are poorly absorbed in the gut and their concentrations in target tissues are too low to perform an effective antioxidant defences. The aim of the present paper is to review existing evidence about possible roles of various plant extracts provided with the diet in animal/poultry nutrition with a specific emphasis to their antioxidant activities.     

Effect of Freezing Rates and Supplementation of α-Tocopherol in the Freezing Extender in Equine Sperm Cryosurvival

This study was conducted to determine the impact of antioxidant (α-tocopherol) supplementation in the cryopreservation extender and three different freezing rates (FRs) on quality of post-thaw semen to elaborate a new protocol for stallion semen cryopreservation. Six ejaculates from each of four stallions were subjected to cryopreservation with a commercial extender (Gent, Minitub Iberia, Spain), without any supplementation (control) or supplemented with 2 mM α-tocopherol. The semen was exposed to three different freezing rates between 5°C and −15°C: slow (5°C/min), moderate (10°C/min), and fast (20°C/min). After thawing, the sperm viability (Sybr-14 and propidium iodide [PI]), mitochondrial membrane potential (MMP) (5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolyl carbocyanine iodine), lipid membrane peroxidation (LPO; C₁₁-BODIPY^581/591), and apoptosis status (fluorescein isothiocyanate–conjugated annexin V and PI) of the plasmatic membrane of each sample were determined by flow cytometry. For both extenders, the percentage of viable cells was higher for spermatozoa cooled at 5°C/min than at 10°C/min and 20°C/min (P ≤ .05). The FR of 20°C/min demonstrated a lower value of MMP than the FR of 5°C/min and 10°C/min (P ≤ .05). The α-tocopherol extender improved (P ≤ .05) post-thaw membrane LPO; however, it did not improved viability and the apoptosis status of the sperm plasmatic membrane after thawing. In conclusion, results clearly indicate that the cryosurvival of stallion spermatozoa is improved when FR of 5°C/min is used from 5°C to −15°C.     

Methyl‐β‐Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida‐Binding Ability of Frozen/Thawed Bovine Spermatozoa

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein‐free media. However, the additive capacitating effect of methyl‐β‐cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen–thawed bovine spermatozoa in a BSA‐containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose‐dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre‐incubation of spermatozoa in MβCD‐supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)‐binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen–thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.     

唇形科植物提取物对单胃动物的抗氧化作用及其机制

动物在患病、应激或特殊生理条件下,机体内会大量产生自由基,自由基与机体内的脂质、ＤＮＡ等产生反应,可对动物造成氧化损伤。唇形科植物提取物中的抗氧化物主要活性成分是酚类化合物,它和抗坏血酸及类胡萝卜素能够阻止自由基引发的氧化伤害。本文阐述了唇形科植物提取物对猪、鸡体外和体内抗氧化作用的实例与抗氧化功能,分析了其抗氧化作用机制。     

Effects of Prolonged in vitro Culture and Cryopreservation on Viability,DNA Fragmentation,Chromosome Stability and Ultrastructure of Bovine Cells from Amniotic Fluid and Umbilical Cord

The objective of this work was to study cellular types that did not participated in the gastrulation process, amniotic fluid cells (AFCs) and umbilical cord cells (UCCs), in conditions of long‐term culture and cryopreserved with different solutions. The AFCs and UCCs were used in a comparative study with ear fibroblast cells (EFCs) that were cultured in vitro until 20 cellular passages and cryopreserved in 10% dimethylsulphoxide (DMSO), 5% dimethyl formamide (DMF) and 7% glycerol (Gly) solutions. The cellular viability, ultrastructure, DNA fragmentation and chromosome stability were evaluated to determine the cellular type most resistant. In all cell types, it was possible to evaluate the AFCs until 15 passages and UCCs until 20 passages with different periods of cellular growth to reach the confluence phase. Solutions containing 10% DMSO ensured viability of 90.33 ± 5.58%, 90.56 ± 4.40% and 81.90 ± 3.31%, respectively for EFCs, AFCs and UCCs, being significantly more efficient and with less variation than other cryoprotectant solutions. The AFCs were more sensitive to cryopreservation and presented low viability rate at the passage 20 (17.2 ± 8.87%). There was no change in karyotype and nuclear fragmentation was low in all cellular passages studied. With the scanning electron analysis was possible the characterization of AFCs and UCCs in suspension. The three cellular types of cells presented different shapes and characteristics on the surface. The results demonstrate that bovine AFCs and UCCs can be isolated, cultured in vitro and cryopreserved in 10% DMSO, not causing damage to DNA and chromosomes. The UCCs were more resistant than AFCs in all aspects.