Hanging Drop Monoculture for Selection of Optimal Antioxidants During In Vitro Maturation of Porcine Oocytes

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre‐culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 μg/ml of L‐carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non‐presence of oxidant stress induced by H₂O₂ supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 μg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H₂O₂. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture.     

Effect of L‐carnitine on maturation,cryo‐tolerance and embryo developmental competence of bovine oocytes

In this study, the effects of the addition of L‐carnitine in in vitro maturation (IVM) medium for bovine oocytes on their nuclear maturation and cryopreservation were investigated; they were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of L‐carnitine (control, 0.3, 0.6 and 1.2 groups, respectively) and some of them were vitrified by Cryotop. Moreover, the effects of L‐carnitine during in vitro fertilization (IVF) and in vitro culture (IVC) on the developmental potential and quality of IVF embryos were also examined. A significantly higher maturation rate of oocytes was obtained for 0.3 and 0.6 mg/mL groups compared with the control (P < 0.05). The blastocyst formation rate in the 0.6 group was significantly improved, whereas the rate in the 1.2 group was significantly decreased when compared with the control group (P < 0.05). No significant difference was found in embryo development between the control and the L‐carnitine group after oocyte vitrification. Supplementation of IVF and IVC media with L‐carnitine had no effect on development to the blastocyst stage of IVM oocytes treated with 0.6 mg/mL L‐carnitine. In conclusion, the supplementation of L‐carnitine during IVM of bovine oocytes improved their nuclear maturation and subsequent embryo development after IVF, but when they were vitrified the improving effects were neutralized.     

Influence of co‐culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus‐enclosed porcine oocytes in a defined system

Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system.     

Effect of estradiol‐17β during in vitro growth culture on the growth,maturation, cumulus expansion and development of porcine oocytes from early antral follicles

Growing porcine oocytes from early antral follicles can acquire meiotic and developmental competence under suitable culture conditions, but at lower rates compared to full‐grown oocytes. We postulated that estradiol‐17β (E₂) supported the acquisition of meiotic and developmental competence as well as cumulus‐expansion ability during growth culture. Growing oocytes from early antral follicles (1.2 to 1.5 mm in diameter) were grown in vitro for 5 days in a medium containing 0, 10^?7, 10^?6, 10^?5 or 10^?4 mol/L E₂; after in vitro maturation, 35, 58, 47, 74 and 49% of oocytes matured to metaphase II, 25, 79, 77, 90 and 97% acquired cumulus‐expansion ability, and 23, 54, 63, 89 and 64% were fully surrounded by cumulus cells, respectively. Following maturation, electro‐stimulation was applied to the oocytes grown with 10^?5 mol/L E₂. After 6 days of culture, in vitro‐grown oocytes developed to the blastocyst stage at a rate similar to that for full‐grown oocytes (31% and 40%, respectively). Therefore, we suggest that the use of E₂ during growth culture improves the meiotic and developmental competence of oocytes, cumulus‐expansion ability, and cumulus cell attachment to the oocytes.     

Effects of Co‐culture of Cumulus Oocyte Complexes with Denuded Oocytes During In Vitro Maturation on the Developmental Competence of Cloned Bovine Embryos

This study evaluated the effects of co‐culture of immature cumulus oocyte complexes (COCs) with denuded immature oocytes (DO) during in vitro maturation on the developmental competence and quality of cloned bovine embryos. We demonstrated that developmental competence, judged by the blastocyst formation rate, was significantly higher in the co‐cultured somatic cell nuclear transfer (SCNT+DO, 37.1 ± 1.1%) group than that in the non‐co‐cultured somatic cell nuclear transfer (SCNT‐DO, 25.1 ± 0.9%) group and was very similar to that in the control IVF (IVF, 38.8 ± 2.8%) group. Moreover, the total cell number per blastocyst in the SCNT+DO group (101.7 ± 6.2) was higher than that in the SCNT‐DO group (81.7 ± 4.3), while still less than that in the IVF group (133.3 ± 6.0). Furthermore, our data showed that mRNA levels of the methylation‐related genes DNMT1 and DNMT3a in the SCNT+DO group were similar to that in the IVF group, while they were significantly higher in the SCNT‐DO group. Similarly, while the mRNA levels of the deacetylation‐related genes HDAC2 and HDAC3 were significantly higher in the SCNT‐DO group, they were comparable between the IVF and SCNT+DO groups. However, the mRNA levels of HDAC1 and DNMT3B were significantly higher in the SCNT+DO group than in the other groups. In conclusion, the present study demonstrated that co‐culture of COCs with DO improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell number.     

Maternal Liver Damage Delays Meiotic Resumption in Bovine Oocytes through Impairment of Signalling Cascades Originated from Low p38MAPK Activity in Cumulus Cells

The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p < 0.05). When cumulus cells were removed from cumulus‐oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7 h of in vitro maturation (IVM) (p < 0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0 h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p < 0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.     

Induction of CIRBP expression by cold shock on bovine cumulus–oocyte complexes

The aim of this study was to induce the cold‐inducible RNA‐binding protein (CIRBP) expression on cumulus–oocyte complexes (COCs) through exposure to a sub‐lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold‐inducible RNA‐binding protein (CIRBP) were assessed after 24 hr of in vitro maturation of control (38.5°C) and cold‐stressed oocytes (33.5°C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold‐stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold‐stressed groups, cold‐stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes.     

All‐trans retinoic acid improves goat oocyte nuclear maturation and reduces apoptotic cumulus cells during in vitro maturation

All‐trans retinoic acid (t‐RA) is a natural component and representative physiologically active metabolite of vitamin A, having multiple physiologic functions. The objective of this study was to evaluate the effect of t‐RA on goat oocyte maturation and cumulus cell apoptosis during in vitro maturation (IVM). Immature goat cumulus‐oocyte complexes (COCs) were matured in vitro in the absence or presence of t‐RA at concentrations of 10 nmol/L, 100 nmol/L and 1000 nmol/L. Oocyte maturation and embryo development were assessed by polar body formation and parthenogenetic activation, respectively. Oocyte survival was checked by Trypan blue staining. Apoptosis of cumulus cells was analyzed by terminal deoxynucleotidyl transferase nick end labeling staining and quantitative real‐time PCR. In comparison with the control group, 100 nmol/L and 10 nmol/L t‐RA significantly improved goat nuclear oocyte maturation and survival (P < 0.05). Addition of 1000 nmol/L t‐RA improved nuclear maturation (P < 0.05), but had no effect on survival of goat oocytes. t‐RA had no positive effect on goat parthenogenetic embryonic cleavage, blastocyst formation or total cell numbers. However, t‐RA inhibits the apoptosis of cumulus cells (P < 0.01). t‐RA treatment up‐regulated the expression of B‐cell lymphoma 2 (BCL‐2), catalase (CAT) (P < 0.05) and down‐regulated the expression of Caspase‐8 (P < 0.05). In conclusion, t‐RA has positive effects on goat oocyte nuclear maturation and reduces apoptotic cumulus cells during IVM.     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

Cytoskeletal and mitochondrial properties of bovine oocytes obtained by Ovum Pick‐Up: the effects of follicle stimulation and in vitro maturation

Follicle stimulation by follicular stimulating hormone (FSH) is known to improve developmental competence of bovine oocytes obtained by Ovum Pick‐Up (OPU); however, the exact factors in oocytes affected by this treatment have remained unclear. We compared in vitro matured (IVM) oocytes obtained at the immature stage from cows by OPU either without or with stimulation with FSH (non‐stimulated and stimulated OPU, respectively) to those obtained by superstimulation and in vivo maturation in terms of cytoskeleton morphology, mitochondrial distribution, intracellular adenosine triphosphate (ATP) content and H₂O₂ levels at the metaphase‐II stage and intracellular Ca²⁺ levels after in vitro fertilization (IVF). Confocal microscopy after immunostaining revealed reduced size of the meiotic spindle, associated with increased tendencies of microfilament degradation and insufficient mitochondrial re‐distribution in non‐stimulated OPU‐derived IVM oocytes compared with those collected by stimulated OPU, which in turn resembled in vivo matured oocytes. However, there was no difference in mitochondrial functions between oocytes obtained by stimulated or non‐stimulated OPU in terms of ATP content, cytoplasmic H₂O₂ levels, base Ca²⁺ levels and the frequencies and amplitudes of Ca²⁺ oscillations after IVF. Larger size of metaphase spindles in oocytes obtained by stimulated OPU may reflect and potentially contribute to their high developmental competence.     

Effect of permeable cryoprotectant‐free vitrification on DNA fragmentation of equine oocyte–cumulus cells

DNA fragmentation of cumulus cells could be used as an indicator of oocyte vitrification success as an indirect indicator of the quality of the oocyte. This study was designed to compare the DNA fragmentation of post‐mortem equine cumulus cells before or after vitrification in the absence of permeable cryoprotectant agents. Cumulus–oocyte complexes (COCs; n = 56) were recovered from slaughterhouse ovaries and subjected to in vitro maturation (42 hr/38.2°C/5%CO₂) before (control group) or after a permeable cryoprotectant‐free vitrification method using 1 M sucrose (vitrification group). After in vitro maturation, COCs were denuded, and cumulus cells were washed and stored at ?80°C until thawing. Cumulus cell samples were processed with the chromatin dispersion test (Ovoselect, Halotech DNA, Spain). Low, high and total DNA fragmentation percentages of cumulus cells were recorded and compared between the two groups by Student's t test. Results were expressed as mean ± SEM. The vitrified group resulted in significantly higher (p < 0.05) percentages for low (16.81 ± 1.62 vs. 6.63 ± 0.77) and total (21.14 ± 1.84 vs. 12.76 ± 1.48) DNA fragmentation of cumulus cells. There were no significant differences between groups for high DNA fragmentation of cumulus cells. In conclusion, permeable cryoprotectant‐free vitrification of equine oocytes increased the total DNA fragmentation rate of cumulus cells but protected them against high DNA fragmentation rates. Further studies are needed to examine the relationship between DNA fragmentation of cumulus cells and the developmental competence of equine oocytes.     

Time Course of In Vitro Maturation of Compact Cumulus Horse Oocytes after Roscovitine‐Induced Meiotic Inhibition: Effects on the Coordination Between Nuclear and Cytoplasmic Maturation

This study was carried out to evaluate the usefulness of a pre‐maturation step in improving the coordination between cytoplasmic and nuclear maturation of horse compact cumulus oocytes by the addition of roscovitine (ROSC). Oocytes were collected by scraping and pre‐cultured for 18 h in a maturation medium TCM199 supplemented with pyruvate, LH, FSH, insulin growth factor (IGF), epidermal growth factor (EGF), insulin, transferrin and selenium (IVM‐ROSC) or in a simple medium (M199‐ROSC). After pre‐maturation, oocytes from both the groups were in part denuded and fixed‐stained and in part in vitro matured to assess the kinetic of in vitro maturation (IVM). The nuclear progression and the cytoskeletal organization of microfilaments and cortical granules (CG) of treated and untreated oocytes were assessed by fluorescent probes. Oocytes immediately fixed after recovery and oocytes pre‐cultured in M199‐ROSC for 18 h did not show metaphase II (MII) plates, whereas in IVM‐ROSC group, 6/69 oocytes (8.7%) showed MII plates. After inhibition, during maturation kinetics at 11, 18 and 29 h, maturation rate of M199‐ROSC group progressively increased and at 29 h of IVM, reached the maturation rate of control group (13/66, 19.7% vs 31/125, 24.8%). No statistically significant differences in cytoplasmic maturation were found. The number of MII plates after 29 h of IVM, was significantly higher (p < 0.05) in IVM‐ROSC group (34/90) compared with M199‐ROSC (13/66) and control groups (31/125) as well as the number of oocytes with microfilaments and CG distributed in cortical region (25/34 vs 3/13 and 7/31 respectively). Our results showed that pre‐culturing in the presence of Roscovitine in a fully supplemented maturation medium containing gonadotropins and growth factors partially suppressed the meiotic maturation, but established a more suitable environment for improving cytoplasmic maturation of horse compact cumulus oocytes as defined by microfilaments and CG configuration.     

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.     

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.     

Chlorogenic acid supplementation during in vitro maturation improves maturation,fertilization and developmental competence of porcine oocytes

Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200 μM). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7 day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50 μM CGA were significantly (p < .05) higher than those of the control oocytes. Hydrogen peroxide (H₂O₂) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1 mM H₂O₂ to assess the protective effect of CGA, 50 μM CGA supplementation improved the maturation rate and the proportion of DNA‐fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50 μM CGA (control) or caffeic acid (10, 50 and 100 μM), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50 μM CGA were similar to those of oocytes matured with 10 and 50 μM caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50 μM CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.     

绵羊卵丘细胞对裸卵体外成熟及孤雌发育影响的研究

本研究探讨绵羊卵丘细胞对裸卵体外成熟和后期发育的影响。实验分为4组,对照组为卵丘-卵母细胞复合体(COCs);裸卵(DO)组;COCs与DO共培养组(CODO);卵丘细胞(CC)与DO共培养组(CCDO)。体外成熟培养18 h后,检测各组卵子成熟质量并进行孤雌发育研究。结果表明:CODO和CCDO组中裸卵活率显著高于DO组(P<0.05),但显著低于COCs组(P<0.05),且COCs组中卵子活率极显著高于DO组(P<0.01);在极体排出率方面,CODO、CCDO和DO3组差异不显著(P>0.05),但是它们都显著低于COCs组(P<0.05)。COCs、CODO、CCDO和DO 4组卵裂率差异均不显著(P>0.05)。然而,DO组的囊胚率显著低于CODO和CCDO组(P<0.05),极显著低于COCs组(P<0.01),但CODO和CCDO 2组中裸卵的囊胚率差异不显著(P>0.05)。总之,无论散在的卵丘细胞还是COCs均能提高裸卵体外成熟及后期发育潜力。     

Low-protein diets supplemented with glutamic acid or aspartic acid ameliorate intestinal damage in weaned piglets challenged with hydrogen peroxide

Glutamic acid (Glu) and aspartic acid (Asp) are acidic amino acids with regulatory roles in nutrition, energy metabolism, and oxidative stress. This study aimed to evaluate the effects of low-protein diets supplemented with Glu and Asp on the intestinal barrier function and energy metabolism in weaned piglets challenged with hydrogen peroxide (H₂O₂). Forty piglets were randomly divided into 5 groups: NC, PC, PGA, PG, and PA (n = 8 for each group). Pigs in the NC and PC groups were fed a low-protein diet, while pigs in the PGA, PG, or PA groups were fed the low-protein diet supplemented with 2.0% Glu +1.0% Asp, 2.0% Glu, or 1.0% Asp, respectively. On day 8 and 11, pigs in the NC group were intraperitoneally injected with saline (1 mL/kg BW), while pigs in the other groups were intraperitoneally administered 10% H₂O₂ (1 mL/kg BW). On day 14, all pigs were sacrificed to collect jejunum and ileum following the blood sample collection in the morning. Notably, low-protein diets supplemented with Glu or Asp ameliorated the intestinal oxidative stress response in H₂O₂-challenged piglets by decreasing intestinal expression of genes (P < 0.05) (e.g., manganese superoxide dismutase [MnSOD], glutathione peroxidase [Gpx]-1, and Gpx-4) encoding oxidative stress-associated proteins, reducing the serum concentration of diamine oxidase (P < 0.05), and inhibiting apoptosis of the intestinal epithelium. Glu and Asp supplementation attenuated the upregulated expression of energy metabolism-associated genes (such as hexokinase and carnitine palmitoyltransferase-1) and the H₂O₂-induced activation of acetyl-coenzyme A carboxylase (ACC) in the jejunum and adenosine monophosphate-activated protein kinase–acetyl-ACC signaling in the ileum. Dietary Glu and Asp also ameliorated intestinal barrier damage as indicated by restored intestinal histology and morphology. In conclusion, low-protein diets supplemented with Glu and Asp protected against oxidative stress-induced intestinal dysfunction in piglets, suggesting that this approach could be used as a nutritional regulatory protectant against oxidative stress.     

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.