Dextran sulfate protects porcine but not bovine cultured endothelial cells from free radical injury

Previous studies demonstrated that the polyanion dextran sulfate (DS) protects rat coronary and porcine aortic endothelium (PAE) from oxygen-derived free radical (OFR) injury due to hydrogen peroxide (H₂O₂) or xanthine/xanthine oxidase (X/XO). To determine if DS has a similar protective effect in bovine aortic endothelium (BAE) and bovine brain microvascular endothelium (BBME), H₂O₂ or X/XO was added to confluent cultures. Cell injury was assessed 1 d later by measuring the percentage of viable cells (by trypan blue exclusion) and the release of lactate dehydrogenase (LDH) into the medium. After H₂O₂ doses of 6.0 mM for BAE and BBME and 0.8 mM for PAE, and after X doses of 10 μM and XO doses of 0.3 U/mL for all cell types, approximately 50% of cells were viable. Cultures were pretreated with DS (0.001 to 500 μg/mL) 24 to 26 h prior to H₂O₂ or X/XO exposure. Pretreatment at concentrations of 0.5, 5, and 50 μg/mL significantly increased the percentage of viable cells and reduced LDH release in cultures of PAE, but not BAE or BBME, treated with H₂O₂. Similarly, pretreatment with DS concentrations of 5 and 50 μg/mL significantly increased the percentage of viable cells and reduced LDH release in cultures of PAE, but not BAE or BBME, treated with X/XO. Thus, DS protected porcine but not bovine endothelium. Catalase (10 U/mL) increased the percentage of viable cells and reduced LDH release in H₂O₂-treated BAE and BBME, suggesting that DS likely acts by a different mechanism and does not neutralize H₂O₂. These results suggest that the protective effect of DS on OFR-injured endothelium is species-dependent.     

Periplaneta americana peptide decreases apoptosis of pig-ovary granulosa cells induced by H2O2 through FoxO1

Oxidative stress can induce apoptosis of granulosa cells and lead to follicular atresia, thereby reducing the number of pigs giving birth. The aim of this study was to investigate the protective effect of Periplaneta americana peptide (PAP) on the apoptosis of the granulosa cells of pig ovaries (PGCs) induced by hydrogen peroxide (H₂O₂) via FoxO1. PGCs were treated with H₂O₂ to establish a cell apoptosis model. Cell viability was measured using the cell counting kit-8 (CCK-8) assay, and cell apoptosis was detected using flow cytometry. The malondialdehyde (MDA) level and nitric oxide (NO) content were detected to reflect the oxidative stress. Western blotting, qRT-PCR and overexpression were undertaken to determine the expression of FoxO1 and caspase-3, and immunofluorescence was used to detect FoxO1 in the nucleus and cytoplasm. PGCs were treated with 100 μM H₂O₂ for 6 hr, which resulted in oxidative damage and apoptosis and an apoptosis rate for PGCs of 32.95%. Next, PGCs were treated with 400 μg/ml PAP for 24 hr to repair the apoptosis induced by H₂O₂. PAP improved cell viability in H₂O₂-stimulated PGCs, the increased MDA level and NO content caused by H₂O₂ stimulation were reversed and the apoptotic rate of PGCs was reduced. The qRT-PCR and Western blotting results indicated that PAP decreased the H₂O₂-induced apoptosis and the expression of FoxO1 and caspase-3 in PGCs. The effect of PAP was the same following FoxO1 overexpression. FoxO1 was expressed in the nucleus when stimulated by H₂O₂ or overexpression; however, it migrated to the cytoplasm following PAP treatment. PAP decreased the apoptosis of PGCs induced by H₂O₂ by regulating FoxO1 expression and nuclear translocation.     

Hypoxia induces FGF2 production by vascular endothelial cells and alters MMP9 and TIMP1 expression in extravillous trophoblasts and their invasiveness in a cocultured model

The role of fibroblast growth factor 2 (FGF2) secretion by vascular endothelial cells during trophoblast invasion was assessed. The human extravillous trophoblast cell line, TEV-1, and umbilical vein endothelial cell line, HUVE-12, were cocultured under normal and hypoxic conditions. FGF2 expression in HUVE-12 cells and matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) expression in TEV-1 cells were analyzed using quantitative RT-PCR and Western blot analyses. TEV-1 cell invasion was also examined. FGF2 expression in the HUVE-12 cells cocultured with TEV-1 cells was significantly increased under hypoxic conditions. In the TEV-1 cells cocultured with HUVE-12, hypoxia reduced MMP9 expression and increased TIMP1 expression; it also reduced cell invasion by 43%. However, the expression of MMP9 and TIMP1 and ratio of MMP9/TIMP1 were increased when the TEV-1 cells were cultured alone under hypoxic conditions. These findings suggest that FGF2 release by stressed endothelial cells of uterine spiral arteries play roles in decreasing MMP9 and increasing TIMP1 production in extravillous trophoblasts (EVT) in response to stress, resulting in reduced EVT invasion and possibly shallow implantation of the placenta.     

Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H₂O₂)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H₂O₂ in a dose-dependent manner. Additionally, H₂O₂ treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H₂O₂ significantly attenuated the H₂O₂-induced decrease of cell viability. H₂O₂ exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H₂O₂-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H₂O₂-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H₂O₂-induced apoptosis by inhibiting ROS generation.     

Effect of tea catechins on regulation of cell proliferation and antioxidant enzyme expression in H2O2‐induced primary hepatocytes of goat in vitro

Tea catechins (TC) are polyphenols that have potent antioxidant activity. The objectives of this study were to determine the effects of TC on antioxidant status of hepatocytes challenged with H₂O₂. Primary hepatocytes of goat were exposed to 1 mm H₂O₂ without or with 5, 50 and 500 μg/ml TC. The cells were harvested at 48 h post‐treatment to determine effects of TC on proliferation, apoptotic features and membrane integrity of cells, and expression of genes and activities of antioxidant enzymes. H₂O₂ exposure caused damage to cells (p < 0.001). A lower concentration of TC (5 μg/ml) displayed a protective effect by inhibiting exorbitant cell proliferation and DNA degradation. Both H₂O₂ exposure and TC pre‐incubation affected expression of antioxidant enzymes at mRNA and protein levels (p < 0.001). The activities of catalase (CAT) (p = 0.027), CuZn‐superoxide dismutase (CuZn‐SOD) (p < 0.001) and glutathione peroxidase (GPx) (p < 0.001) increased with TC pre‐incubation followed by H₂O₂ challenge. Changes of CuZn‐SOD activity induced by H₂O₂ and TC basically paralleled the changes in the corresponding mRNA and protein levels, but the correlation in CAT and GPx expression displayed slightly different patterns at different concentrations of TC. These findings infer that oxidative stress can induce deleterious cellular responses and this unfavourable condition may be alleviated by treatment with TC.     

Effect of dihydrotestosterone on mouse embryonic stem cells exposed to H2O2-induced oxidative stress

Oxidative stresses induced by reactive oxygen species (ROS) have been shown to be involved in several physiological and pathophysiological processes, such as cell proliferation and differentiation. Steroid hormones can protect cells against apoptosis or induce cell proliferation by several mechanisms. Among androgenic hormones, dihydrotestosterone (DHT) is generated by a 5α-reduction of testosterone. Unlike testosterone, DHT cannot be aromatized to estradiol, therefore DHT is considered a pure androgenic steroid. This study was conducted to examine the effect of DHT (10^-7 M) on H₂O₂ (10^-3 M) -induced injuries in mouse embryonic stem (ES) cells. H₂O₂ induced ROS generation and increased lipid peroxide formation and DNA fragmentation. These effects of H₂O₂ were inhibited by pretreatment with DHT. H₂O₂ also increased the phosphorylation of p38 MAPK, SAPK/JNK and nuclear factor kappa B (NF-κB), but DHT blocked these effects. Moreover, H₂O₂ decreased DNA synthesis and the levels of cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4]. These effects of H₂O₂ were inhibited by pretreatment with DHT. In conclusion, DHT may partially prevent H₂O₂-induced cell injury through inhibition of ROS and ROS-induced activation of p38 MAPK, SAPK/JNK and NF-κB in mouse ES cells.     

Dietary taurine attenuates hydrogen peroxide-impaired growth performance and meat quality of broilers via modulating redox status and cell death signaling

Oxidative stress seriously affects poultry production. Nutritional manipulations have been effectively used to alleviate the negative effects caused by oxidative stress. This study investigated the attenuating effects and potential mechanisms of dietary taurine on the growth performance and meat quality of broiler chickens challenged with hydrogen peroxide (H₂O₂). Briefly, a total of 192 male Arbor Acres broilers (28 d old) were randomly categorized into three groups: non-injection of birds on basal diets (control), 10.0% H₂O₂ injection of birds on basal diets (H₂O₂), and 10.0% H₂O₂ injection of birds on basal diets supplemented with 5 g/kg taurine (H₂O₂ + taurine). Each group consisted of eight cages of eight birds per cage. Results indicated that H₂O₂ administration significantly reduced growth performance and impaired breast meat quality by decreasing ultimate pH and increasing shear force value (P < 0.05). Dietary taurine improved the body weight gain and feed intake and decreased feed/gain ratio of H₂O₂-challenged broilers. Meanwhile, oxidative stress induced by intraperitoneal injection of H₂O₂ suppressed the nuclear factor-κB (NF-κB) signaling and initiated autophagy and apoptosis. Compared with the H₂O₂ group, taurine supplementation restored the redox status in the breast muscle by decreasing levels of reactive oxygen species and contents of oxidative products and increasing antioxidant capacity (P < 0.05). Moreover, upregulated mRNA expression of NF-κB signaling-related genes, including NF-κB subunit 1 (p50) and B-cell CLL/lymphoma 2 (Bcl-2), and enhanced protein expression of NF-κB were observed in the H₂O₂ + taurine group (P < 0.05). Additionally, dietary taurine decreased the expression of caspase family, beclin1, and microtubule-associated protein 1light chain 3 beta (LC3-II; P < 0.05), thereby rescuing autophagy and apoptosis in breast muscle induced by H₂O₂. Collectively, dietary supplementation with taurine effectively improves growth performance and breast meat quality of broilers challenged with H₂O₂, possibly by protecting against oxidative injury and modulating cell death signaling.     

L‐carnitine improves hydrogen peroxide‐induced impairment of nuclear maturation in porcine oocytes

We investigated the effect of oxidative stress induced by hydrogen peroxide (H₂O₂) on lipid peroxide (LPO) level and nuclear maturation in porcine oocytes cultured with or without cumulus cells. After 22 h of pre‐culture, oocytes with attached cumulus cells (COC group) or denuded oocytes (DO group) were cultured with H₂O₂, and intra‐oocyte H₂O₂ and LPO levels were quantitatively analyzed using immunofluorescence. This is the first report evaluating LPO levels in porcine oocytes. After H₂O₂ supplementation, the DO group showed severe accumulation of H₂O₂ and LPO in the oocytes. Similarly, while inhibition of progression of nuclear maturation was observed in both groups, the effect was more severe in the DO group. These results demonstrate that cumulus cells reduce the accumulation of H₂O₂ stress in oocytes. Furthermore, we attempted to reduce the oxidative stress by H₂O₂ with L‐carnitine, a H₂O₂ scavenger. L‐carnitine decreased H₂O₂ and LPO levels in the oocytes in both groups, and improvement in the progression of impaired nuclear maturation was observed. These effects were different by the presence of cumulus cells. Our results provide that L‐carnitine is useful for alleviating H₂O₂‐induced oxidative stress by reducing LPO levels and improving the progression of nuclear maturation.     

Effect of hypoxia on generation of neurospheres from adipose tissue-derived canine mesenchymal stromal cells

Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are good candidates for cell therapy due to the accessibility of fat tissue and the abundance of AT-MSCs therein. Neurospheres are free-floating spherical condensations of cells with neural stem/progenitor cell (NSPC) characteristics that can be derived from AT-MSCs. The aims of this study were to examine the influence of oxygen (O₂) tension on generation of neurospheres from canine AT-MSCs (AT-cMSCs) and to develop a hypoxic cell culture system to enhance the survival and therapeutic benefit of generated neurospheres.AT-cMSCs were cultured under varying oxygen tensions (1%, 5% and 21%) in a neurosphere culture system. Neurosphere number and area were evaluated and NSPC markers were quantified using real-time quantitative PCR (qPCR). Effects of oxygen on neurosphere expression of hypoxia inducible factor 1, α subunit (HIF1A) and its target genes, erythropoietin receptor (EPOR), chemokine (C-X-C motif) receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF), were quantified by qPCR. Neural differentiation potential was evaluated in 21% O₂ by cell morphology and qPCR.Neurospheres were successfully generated from AT-cMSCs at all O₂ tensions. Expression of nestin mRNA (NES) was significantly increased after neurosphere culture and was significantly higher in 1% O₂ compared to 5% and 21% O₂. Neurospheres cultured in 1% O₂ had significantly increased levels of VEGF and EPOR. There was a significant increase in CXCR4 expression in neurospheres generated at all O₂ tensions. Neurosphere culture under hypoxia had no negative effect on subsequent neural differentiation. This study suggests that generation of neurospheres under hypoxia could be beneficial when considering these cells for neurological cell therapies.     

Oxygen Concentration and Cysteamine Supplementation During In vitro Production of Buffalo (Bubalus bubalis) Embryos Affect mRNA Expression of BCL‐2, BCL‐XL,MCL‐1, BAX and BID

This study examined the effects of O₂ concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm , respectively; IVM, IVF and IVC carried out in 20% O₂), on blastocyst rate and relative mRNA abundance of some apoptosis‐related genes measured by real‐time qPCR in immature and in vitro‐matured buffalo oocytes and in embryos at 2‐, 4‐, 8‐ to 16‐cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL‐positive cells was significantly lower (p < 0.05) under 5% O₂ than that under 20% O₂. The mRNA expression of anti‐apoptotic genes BCL‐2 and MCL‐1 was significantly higher (p < 0.05) and that of pro‐apoptotic genes BAX and BID was lower (p < 0.05) under 5% O₂ than that under 20% O₂ concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL‐XL and MCL‐1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O₂ groups and in cysteamine supplemented vs controls. At the 8‐ to 16‐cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL‐2 and MCL‐1 was highest under 5% O₂ concentration and that of BAX and BID was highest (p < 0.05) under 20% O₂ concentration. These results suggest that one of the mechanisms through which beneficial effects of low O₂ concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti‐apoptotic and a decrease in the expression of pro‐apoptotic genes.     

外源H2O2对沙打旺抗黄矮根腐病的影响

本研究以沙打旺抗病和感病品种为材料,接种埃里砖格孢孢子悬浮液后分别施加外源H₂O₂及其清除剂AsA,通过统计发病率、病情指数及测定POD、PPO、CAT、SOD、Glu和Cht六种病程相关蛋白酶活性,研究H₂O₂对沙打旺抗黄矮根腐病的影响。结果表明,接种埃里砖格孢后2种沙打旺H₂O₂含量高于未接种植株,且感病品种在38 h H₂O₂含量最高,为929 μmol/g,抗病品种最高值出现在24 h,为986 μmol/g。抗病、感病沙打旺发病率、病情指数在H₂O₂处理后最低,施加AsA最高。H₂O₂处理各种酶活性都有不同程度的增加,且对抗病品种酶活性提高大于感病品种,表明外施H₂O₂可以减缓沙打旺黄矮根腐病的发生和侵染。表明H₂O₂与沙打旺抗病性紧密相关,并通过调节病程相关酶活性来增强沙打旺对黄矮根腐病的抗性,减少侵染率。     

Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide,and Importance of Individual Male Variability

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H₂O₂) levels on red deer spermatozoa after cryopreservation, and the role of male‐to‐male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μm H₂O₂ for 2 h at 37°C. Intracellular reactive oxygen species (H₂DCFDA‐CM) increased with H₂O₂ concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μm . Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H₂O₂ and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm H₂O₂ for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H₂O₂ presence. We found that the kinematic parameters reflected male‐to‐male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H₂O₂ or after incubation with H₂O₂. Red deer spermatozoa are relatively resilient to H₂O₂ after thawing, but it seems to be a great male‐to‐male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.     

Hydrogen peroxide produced by a low‐molecular‐weight compound in milk with high electrical conductivity in dairy cows

In dairy cows, hydrogen peroxide (H₂O₂) produced from a low‐molecular‐weight compound in milk from inflamed quarters was lower than that in milk from un‐inflamed quarters. In milk of delivery grade, characteristics of H₂O₂ production in milk with high electrical conductivity (EC) were examined in this study. Milk samples were collected from a total of 230 cows at 1‐month intervals, and the EC of skimmed milk was determined. Based on the highest and the lowest EC of a cow's quarter milk, the inter‐quarter difference of ≥0.6 mS/cm (mean + t_0.01 SE) was taken as a high EC. Milk with high EC was found in 52 quarters. In cows with milk of high EC, H₂O₂ production in milk with normal EC was higher than that in milk with high EC in the same animal but was lower than that in the control population. In milk with high EC, the decrease of H₂O₂ production correlated with the increase in EC. The production of H₂O₂ decreased in particular when the inter‐quarter difference exceeded 0.8 mS/cm. In milk collected from the same quarter 1 month before, EC changed from normal to high, and H₂O₂ production decreased. In milk from the other three quarters, EC remained normal and H₂O₂ production remained unchanged. We concluded that milk with high EC appeared in low H₂O₂‐producing cows. The results suggest that the degree of decrease in H₂O₂ production reflects the extent of quarter abnormality.     

Cholesterol Protects <Emphasis Type="Italic">Acholeplasma laidlawii</Emphasis> Against Oxidative Damage Caused by Hydrogen Peroxide

The aim of this study was to determine whether cholesterol, added to the cell growth medium or to cell suspension buffer, could protect Acholeplasma laidlawii cells against the toxic effects of hydrogen peroxide (H₂O₂). Variable concentrations of cholesterol (0.05–1.0 mg/ml) were added to the A. laidlawii suspension buffer and to the growth medium. Cells were then washed carefully and incubated with 0.001% (v/v) H₂O₂ at 37°C for 30 min and the viability was determined. The results indicated that cells were more viable in the presence of cholesterol than were cells grown in the absence of cholesterol. In addition, the oxygen uptake rate resulting from the oxidation of 5.5 mmol/L glucose was 2-fold and 4-fold higher for cells grown in medium supplemented with 0.05 and 0.50 mg/ml cholesterol, respectively, compared to cells grown in a medium with no added cholesterol. These findings indicate that cholesterol might play a role in protecting Mollicutes against the oxidative damage caused by H₂O₂. Deceased October 25, 2001     

Exhaled breath condensate hydrogen peroxide and pH for the assessment of lower airway inflammation in the horse

Measurement of hydrogen peroxide (H₂O₂) concentration and pH in exhaled breath condensate (EBC) is useful for detection and monitoring of asthma in humans. In contrast, limited information on the use of these parameters for the investigation of lower airway inflammation (LAI) is available for horses. Aims of the current study were to investigate the intra- and inter-day variations of EBC H₂O₂ concentration and pH in horses and establish any relationship(s) with LAI. Both intra- and inter-day variability of EBC H₂O₂ concentration were large, while those of pH were small. No significant difference in the intra-day or inter-day H₂O₂ concentrations or pH measurements were found in control or LAI horses, except for inter-day H₂O₂ concentration in horses with LAI (p = 0.019). There was no significant difference in EBC pH or H₂O₂ concentration between control and LAI horses, however a trend for a reduced pH in horses with LAI was observed.     

Stanniocalcin-1 protects bovine intestinal epithelial cells from oxidative stress-induced damage

Chronic enteritis can produce an excess of reactive oxygen species resulting in cellular damage. Stanniocalcin-1(STC-1) reportedly possesses anti-oxidative activity, the aim of this study was to define more clearly the direct contribution of STC-1 to anti-oxidative stress in cattle. In this study, primary intestinal epithelial cells (IECs) were exposed to hydrogen peroxide (H₂O₂) for different time intervals to mimic chronic enteritis-induced cellular damage. Prior to treatment with 200 µM H₂O₂, the cells were transfected with a recombinant plasmid for 48 h to over-express STC-1. Acridine orange/ethidium bromide (AO/EB) double staining and trypan blue exclusion assays were then performed to measure cell viability and apoptosis of the cells, respectively. The expression of STC-1 and apoptosis-related proteins in the cells was monitored by real-time PCR and Western blotting. The results indicated that both STC-1 mRNA and protein expression levels positively correlated with the duration of H₂O₂ treatment. H₂O₂ damaged the bovine IECs in a time-dependent manner, and this effect was attenuated by STC-1 over-expression. Furthermore, over-expression of STC-1 up-regulated Bcl-2 protein expression and slightly down-regulated caspase-3 production in the damaged cells. Findings from this study suggested that STC-1 plays a protective role in intestinal cells through an antioxidant mechanism.     

Ameliorative Effects of Melatonin against Hydrogen Peroxide‐Induced Oxidative Stress on Boar Sperm Characteristics and Subsequent In Vitro Embryo Development

Melatonin, the major secretory product of the pineal gland, scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, indicating that melatonin is a potent function as an antioxidant. The objective of this study was to investigate the effect of melatonin in the presence or absence of hydrogen peroxide (H₂O₂) on sperm characteristics (motility, viability, survival rate, membrane integrity, lipid peroxidation (LPO) and mitochondria activity) and also to examine the developmental rates to the blastocysts stage of porcine oocytes fertilized in vitro with semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ). The sperm were treated with melatonin in the presence or absence of H₂O₂ for 3, 6, 9 and 12 h at 37°C and then analysed for the sperm characteristics. The porcine embryos were produced by in vitro maturation and in vitro fertilization (IVM/IVF) using semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ) for 6 h. The semen characteristics, including motility, viability, survival rate, membrane integrity and mitochondria activity, were higher in the groups that were treated with melatonin in comparison to other groups, irrespective of incubation periods. Malondialdehyde levels in control, melatonin and melatonin + H₂O₂ groups were lower than H₂O₂ only group. A positive correlation was shown among motility, viability, survival rate and membrane integrity, but a negative correlation was observed between LPO and the other evaluation methods. The developmental rates to blastocysts of IVM/IVF porcine oocytes fertilized by semen treated with melatonin were significantly increased compared with any other groups, with the cell number of blastocysts shown to have a similar trend to the developmental rates. These results demonstrate that melatonin can improve the semen characteristics during in vitro storage and support the developmental ability of IVM/IVF embryos in pigs.     

Similar developmental fluctuations of hepato-renal xanthine oxidoreductase gene expression and xanthine oxidase activity in layer and broiler chicken embryos

1. Xanthine oxidase (XO) has many physiological functions associated with the synthesis of both antioxidant (uric acid: UA) and numerous oxidants (e.g. H₂O₂), which makes it an important regulator of the cellular redox potential involving organogenesis. The ontogenetic study of hepatic and renal XO makes a better understanding of the putative role of this enzyme in the development of these tissues.

2. Developmental changes of gene expression of xanthine oxidoreductase (XOR), XO activity and UA content of liver and kidney tissues in both broiler and layer chicken embryos were examined during incubation d 14–21.

3. In both strains, hepatic XOR gene expression peaked on d 21 while renal XOR gene expression did not change.

4. The XO activity was higher in kidney than liver in both strains. Hepatic XO activity of both strains peaked on d 18 and thereafter was decreased on d 21. Renal XO activity peaked on d 18 and from then on did not show any significant changes until d 21 in both strains.

5. The UA content was higher in kidney vs. liver in both strains. The hepatic and renal UA values of the both strains increased significantly from d 14 to d 21.

6. The present results showed dissimilar behaviour of XOR gene expression, XO activity and UA content of liver and kidney tissues in both broiler and layer chicken embryos.     

Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°C In Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H₂O₂ was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 10⁸ sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H₂O₂ per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H₂O₂. Among the different levels of H₂O₂, the 50‐μm H₂O₂‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H₂O₂‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 10⁹ spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H₂O₂ and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H₂O₂ was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.     

DNA Fragmentation as an Early Indicator of Extender Efficacy: A Preliminary Study

Evaluation of new potential semen extenders is a field of economic and scientific importance, but assessing motility alone may not be sufficient. The objectives of this study were to examine the effect of oxidative damage by short-term exposure to H₂O₂ on stallion sperm motility and DNA fragmentation and to correlate motility to the percentage of DNA damage as assessed by both terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and single-cell gel electrophoresis assays. Motility and DNA fragmentation were determined immediately before cooling (0 hour) and at 24 hours postcooling. The addition of H₂O₂ (300 μM) to the extender had no effect on either total or progressive motility (P > .05). DNA fragmentation as determined by both Comet and TUNEL assays did not differ between 0 hour and those cells stored for 24 hours in the absence of H₂O₂ (P > .05). However, the addition of H₂O₂ to the extender plus incubation for 24 hours resulted in greater total Comet length, tail length, and tail moment as well as an increase in percentage of sperm cells with DNA damage detected by TUNEL compared to 0 hour (P < .05). Motility was not correlated with DNA damaged cells detected by TUNEL or Comet assays (P > .05). In conclusion, although both the Comet assay and TUNEL detected significant DNA fragmentation in cells exposed to H₂O_2, there was not a significant or appreciable effect of H₂O₂ on motility. Therefore, motility alone is likely not the best laboratory assay with which to assess cooled extender efficacy.