Dihydromyricetin supplementation during in vitro culture improves porcine oocyte developmental competence by regulating oxidative stress

Dihydromyricetin (DHM), a dihydroflavonoid compound, exhibits a variety of biological activities, including antitumor activity. However, the effects of DHM on mammalian reproductive processes, especially during early embryonic development, remain unclear. In this study, we added DHM to porcine zygotic medium to explore the influence and underlying mechanisms of DHM on the developmental competence of parthenogenetically activated porcine embryos. Supplementation with 5 μM DHM during in vitro culture (IVC) significantly improved blastocyst formation rate and increased the total number of cells in porcine embryos. Further, DHM supplementation also improved glutathione levels and mitochondrial membrane potential; reduced natural reactive oxygen species levels in blastomeres and apoptosis rate; upregulated Nanog, Oct4, SOD1, SOD2, Sirt1, and Bcl2 expression; and downregulated Beclin1, ATG12, and Bax expression. Collectively, DHM supplementation regulated oxidative stress during IVC and could act as a potential antioxidant during in vitro porcine oocytes maturation.     

Impact of heat stress on germinal vesicle breakdown and lipolytic changes during in vitro maturation of bovine oocytes

Two studies were conducted with the overarching goal of determining the extent to which lipolytic changes relate to germinal vesicle breakdown (GVBD) in bovine oocytes matured under thermoneutral or hyperthermic conditions. To this end, cumulus-oocyte complexes underwent in vitro maturation for 0, 2, 4, 6 or 24 h at 38.5 (first study) or 38.5 and 41.0 C (second study; heat stress applied up through first 12 h only, then shifted to 38.5 C). Independent of maturation temperature, triglyceride and phospholipid content decreased markedly by 2 h of in vitro maturation (hIVM; P < 0.0005). Content was lowest at 24 hIVM with no detectable impact of heat stress when exposure occurred during first 12 hIVM. Germinal vesicle breakdown occurred earlier in oocytes experiencing heat stress with effects observed as soon as 4 hIVM (P < 0.0001). Germinal vesicle breakdown was associated with lipolytic changes (R² = 0.2123 and P = 0.0030 for triglyceride content; R² = 0.2243 and P = 0.0026 for phospholipid content). ATP content at 24 hIVM was higher in oocytes experiencing heat stress (P = 0.0082). In summary, GVBD occurs sooner in heat-stressed oocytes. Although marked decreases in triglyceride and phospholipid content were noted as early as 2 hIVM and preceded GVBD, lipolytic changes such as these are not likely serving as an initial driver of GVBD in heat-stressed oocytes because changes occurred similarly in oocytes matured at thermoneutral conditions.     

体外成熟对牛卵母细胞孤雌激活后发育潜力的影响

本试验在卵母细胞体外成熟的研究基础上,研究了培养用水、卵泡液和成熟时间对卵母细胞体外成熟及孤雌激活后发育潜力的影响.结果表明(1)将卵母细胞分别于蒸馏水和Milli-Q超纯水配制的成熟培养液中培养22～24 h, 孤雌激活后, 卵裂率无显著差异(P>0.05); 但孤雌卵在超纯水配制的胚胎培养液中培养,囊胚发育率为22.3%, 明显高于在蒸馏水配制的培养液中的囊胚发育率14.1%(P<0.05).(2)在成熟培养液中添加10%、20%卵泡液,成熟卵母细胞孤雌激活后的囊胚发育率(32.3%, 30.9%)明显高于添加5%和0%卵泡液组的囊胚发育率(21.8%, 22.7%,P<0.05).卵母细胞在添加20%卵泡液的培养液中成熟培养,孤雌激活后囊胚孵化率明显低于添加0、5%、10%卵泡液组的囊胚孵化率(P<0.05).(3)成熟28 h或30 h的卵母细胞孤雌激活后,其囊胚发育率(30.5%或29.4%)明显高于成熟24 h或26 h组的囊胚发育率(20.5%或22.1%,P<0.05).     

Effects of the preservation medium and storage duration of domestic cat ovaries on the maturational and developmental competence of oocytes in vitro

We examined the effectiveness of saline, Euro-Collins solution (EC), and ET-Kyoto solution (ET-K) as preservation media for the cold storage of feline ovaries. Ovaries were maintained in these media at 4°C for 24, 48, or 72 h until oocyte retrieval. The ET-K group exhibited a higher oocyte maturation rate than the saline group after 72 h of storage. Moreover, ET-K could sustain the competence of the feline oocytes to cleave after 48 h, and the morula formation rate of the ET-K group was higher than that of the other groups after 24 and 48 h. Furthermore, the ET-K group exhibited a higher blastocyst formation rate than the other groups after storage for 24 h, and only ET-K retained the developmental competence in blastocysts after 48 h of storage. In addition, regarding the cell numbers of the blastocysts, there was no significant difference among the tested groups. In conclusion, our results indicate that ET-K is a suitable preservation medium for feline ovaries.     

白牦牛卵母细胞的采集方法及卵巢贮存条件对其体外成熟的影响

探讨了白牦牛卵母细胞的采集方法以及卵巢保存温度和时间对其体外成熟的影响。结果表明:先抽吸卵泡,每卵巢平均可获可用卵母细胞4.7枚,再切割卵巢,可显著提高可用卵母细胞数9.8枚/卵巢。将屠宰白牦牛卵巢分别置于20~29℃和30~38℃的生理盐水中,6 h内收集卵母细胞,其成熟率、卵裂率分别为65.7%、41.3%和73.3%、45.7%,两者之间的差异均不显著,但均与<20℃组(16.0%、0)差异显著。在30~38℃下,白牦牛卵巢保存时间超过6 h,卵母细胞的成熟率和卵裂率显著下降,卵巢保存时间在2 h以内最好,最晚不超过6 h。     

Recovery from adverse effects of heat stress on slow-growing chicks in the tropics 1: Effect of ascorbic acid and different levels of betaine

Three hundreds, 21 d-old slow-growing chicks were randomly divided among 5 treatments, of 5 replicates each. Each replicate contained 12 unsexed chicks housed in (1 × 1) a floor pen. A group was kept under thermoneutral condition at 28 ± 4°C and RH was 55 ± 3% during 21–84 d of age (positive control) and fed corn-soybean meal diet. The other four groups were kept for three successive days per week under heat stress (HS) at 38 ± 1.4°C and 49 ± 2% RH from 12.00 to 16.00 pm. Chicks in HS treatments were fed corn-soybean meal diet without (negative control) or with 250 mg AA/kg diet and Bet at 0.5 and 1 g/kg diet. HS decreased productive performance, increased (P < 0.05) meat dry matter, plasma triglyceride and serum calcium whereas decreased (P > 0.05) plasma glucose, serum total protein and water holding capacity (WHC) of meat. AA and 1 g of Bet/kg diet was equally potent for partial relief (P < 0.05) of the negative effect of HS on growth, increased (P < 0.05) feed intake, protein digestibility (P < 0.05), dressing out percentage, liver and giblets, whilst improved (P < 0.05) feed conversion ratio (FCR). Also, a complete recovery from the negative effect (P < 0.05) of HS shown on plasma glucose and partial recovery (P < 0.05) observed in total protein, triglyceride, blood pH, packed cell volume (PCV), hemoglobin (Hgb), rectal temperature (RT) and respiration rate (RR) and improved humoral immune competence to sheep red blood cell (SBRCs) test.     

Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress

Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival.     

茉莉酸浸种对高温胁迫下多年生黑麦草种子发芽的影响

以多年生黑麦草品种(Lolium perenne)‘麦迪’(Mathilde)和‘绅士’(Esquire)为供试材料,采用20、25、30、33℃共4个温度处理,研究不同高温对多年生黑麦草种子发芽的影响及不同浓度(5、10、20、40、60、80、100μmol·L^–1)茉莉酸浸种提高多年生黑麦草在高温胁迫下种子发芽能力的生理效应。结果表明,高温胁迫在一定程度上影响了多年生黑麦草种子的发芽过程,随处理温度的升高,多年生黑麦草种子的发芽率、发芽势和发芽指数均显著下降(P <0.05),特别是对根长和幼苗的影响显著,且‘麦迪’显著优于‘绅士’。在高温胁迫条件下,浓度为40～60μmol·L^–1的茉莉酸浸种显著提高了多年生黑麦草种子的发芽能力,同时种子幼苗的超氧化物歧化酶活性与对照相比显著增高,丙二醛含量显著降低。适宜浓度茉莉酸浸种可以在一定程度上减缓高温胁迫对多年生黑麦草种子发芽的影响,有效提高多年生黑麦草种子在高温胁迫下的发芽能力和种子幼苗活性。     

Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress

Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.     

日粮中添加硫辛酸对热应激条件下母兔的影响

选择体况良好的新西兰母兔20只,随机分为4组,日粮中分别添加硫辛酸200mg/kg、400mg/kg、600mg/kg,对照组不添加。每组5个重复(每个单笼饲养的兔子为一个重复)。探索日粮中添加硫辛酸对热应激条件下母兔影响。试验结果表明,在高温应激情况下,在日粮中添加硫辛酸:①可使母兔血清中丙二醛(MDA)的含量显著降低(P0.05);②可提高母兔的血清中超氧化物歧化酶(SOD)和GSH-Px的活性(P0.05);③可增加母兔血清总抗氧化能力(P0.05)。从相关理化参数可见,以日粮中添加600mg/kg的硫辛酸抗热应激效果最好。     

Effect of chlorogenic acid on intestinal inflammation,antioxidant status,and microbial community of young hens challenged with acute heat stress

Heat stress in poultry is deleterious to productive performance. Chlorogenic acid (CGA) exerts antibacterial, anti-inflammatory, and antioxidant properties. This study was conducted to evaluate the effects of dietary supplemental CGA on the intestinal health and cecal microbiota composition of young hens challenged with acute heat stress. 100-day-old Hy-line brown pullets were randomly divided into four groups. The control group (C) and heat stress group (HS) received a basal diet. HS + CGA₃₀₀ group and HS + CGA₆₀₀ group received a basal diet supplemented with 300- and 600-mg/kg CGA, respectively, for 2 weeks before heat stress exposure. Pullets of HS, HS + CGA₃₀₀, and HS + CGA₆₀₀ group were exposed to 38°C for 4 h while the control group was maintained at 25°C. In this study, dietary CGA supplementation had effect on mitigate the decreased T-AOC and T-SOD activities and the increasing of IL-1β and TNFα induced by acute heat stress. Dietary supplementation with 600 mg/kg CGA had better effect on increasing the relative abundance of beneficial bacterial genera, such as Rikenellaceae RC9_gut_group, Ruminococcaceae UCG-005, and Christensenellaceae R-7_group, and deceasing bacteria genera involved in inflammation, such as Sutterella species. Therefore, CGA can ameliorate acute heat stress damage through suppressing inflammation and improved antioxidant capacity and cecal microbiota composition.     

甜菜碱日粮对热应激条件下肉鸭生长性能、血液指标及盲肠脂肪酸含量的影响

文章旨在评估甜菜碱对热应激条件下肉鸭血液指标、电解质、气体分压和盲肠短链脂肪酸含量的影响。试验选择平均体重为48.6g当天孵化的肉鸭400只,随机分为4组,每组5个重复,每个重复20只。试验日粮处理分为对照组和3个处理组,对照组饲喂基础日粮(自由采食),处理1组为热应激条件下添加1500mg/kg甜菜碱,处理2组为热应激条件下5点~10点以及17点~20点饲喂含1500mg/kg甜菜碱日粮,处理3组为热应激条件下17点~10点饲喂含1500mg/kg甜菜碱日粮,试验共进行42d。结果显示:饲喂甜菜碱日粮的肉鸭较对照组体重显著提高(P<0.05)。对照组肉鸭血液红细胞总数、红细胞压积、血红蛋白、红细胞平均体积、红细胞分布宽度、血小板计数、血小板压积、血小板平均体积均低于甜菜碱组(P<0.05)。不同饲喂时间的甜菜碱组肉鸭电解质浓度均高于对照组(P<0.05)。对照组肉鸭血液二氧化碳分压、氧气分压、细胞外液碱浓度、碳酸氢盐和总二氧化碳浓度均低于甜菜碱组(P<0.05)。与对照组相比,甜菜碱组肉鸭盲肠中总短链脂肪酸、乙酸、丙酸的浓度较高(P<0.05),而丁酸、异丁酸、戊酸和异戊酸含量较低(P<0.05)。因此,与自由采食相比,在上午和下午限制饲喂甜菜碱日粮或17~10点饲喂甜菜碱日粮显著提高了热应激条件下肉鸭的生长性能和生物学参数。     

Effects of cooled floor pads combined with chilled drinking water on behavior and performance of lactating sows under heat stress

This study was conducted to evaluate whether cooled floor pads combined with chilled drinking water could alleviate negative impacts of heat stress on lactating sows. Thirty sows (Landrace × Yorkshire, Parity = 1 to 6) were housed in individual farrowing stalls in two rooms with temperatures being controlled at 29.4°C (0700–1900 hours) and 23.9°C (1900–0700 hours). Sows in one room (Cool), but not in the other room (Control) were provided cooled floor pads (21–22°C) and chilled drinking water (13–15°C). Behavior of sows (15 sows/treatment) was video recorded during farrowing, and days 1, 3, 7, 14, and 21 after farrowing. Videos were viewed continuously to register the birth time of each piglet, from which total farrowing duration and birth intervals were calculated. The number of drinking bouts and the duration of each drinking bout were registered for each sow through viewing videos continuously for 2 h (1530–1730 hours) each video-recording day. Postures (lying laterally, lying ventrally, sitting, and standing) were recorded by scanning video recordings at 5-min intervals for 24 h each video-recording day, and time budget for each posture was calculated. Rectal temperature and respiration rate were measured for all sows the day before and after farrowing, and then once weekly. Sow and litter performance was recorded. Data were analyzed using the Glimmix procedure of SAS. The cooling treatment did not affect sow behavior or litter performance. Sows in the Cool room had lower rectal temperature (P = 0.03) and lower respiration rate (P < 0.001), consumed more feed (P = 0.03), tended to have reduced weight loss (P = 0.07), and backfat loss (P = 0.07) during lactation than sows in the Control room. As lactation progressed, sows increased drinking frequency (P < 0.001) and time spent lying ventrally (P < 0.0001), standing (P < 0.001), and sitting (P < 0.0001), and decreased time spent lying laterally (P < 0.0001) in both Cool and Control rooms. While cooled floor pads combined with chilled drinking water did not affect sow behavior, they did alleviate heat stress partially, as indicated by decreased rectal temperature, respiration rate, weight, and backfat loss, and increased feed intake in lactating sows.     

水杨酸浸种对高羊茅在干旱胁迫下萌发的影响

用不同浓度水杨酸溶液浸泡处理冷季型草坪草高羊茅（Festuca arundinacea）的种子,在PEG 2000模拟干旱胁迫条件下,采用纸上发芽法（TP）研究了水杨酸浸种对盆景2000、猎狗5号、红宝石3个高羊茅品种萌发的影响。结果表明,在干旱条件下,适宜低浓度水杨酸溶液0.12~1.00 mmol/L浸种可以提高高羊茅种子的发芽势、发芽率,促进种子胚芽和胚根的生长,提高高羊茅种子萌发期的抗旱性;而高浓度水杨酸浸种抑制了种子的萌发和生长,3.00 mmol/L达到显著抑制水平（P＜0.05）。其中,能显著增强高羊茅种子萌发阶段抗旱性的水杨酸最适浓度为0.50 mmol/L。     

Effect of ascorbic acid or increasing metabolizable energy level with or without supplementation of some essential amino acids on productive and physiological traits of slow-growing chicks exposed to chronic heat stress

Four hundred and twenty, 21-day-old slow-growing chicks were divided randomly into seven treatments, each containing five replicates. Each replicate was kept in a 1 × 1-m floor pen. One treatment was kept under thermo-neutral conditions in a semi-open house and fed a corn-soybean meal diet (positive control). The other six groups were kept under chronic heat stress (CHS) at 38 °C and 60% RH for 4 h from 12:00 to 16:00 pm for three successive days per week. Chicks in CHS treatments were fed a corn-soybean meal diet without (negative control) or with increasing metabolizable energy (ME) level by oil supplementation alone, or also with increasing some essential amino acids (EAA) such as methionine (Met), methionine and lysine (Met+Lys) or methionine, lysine and arginine (Met+Lys+Arg) or supplemented with 250 mg of ascorbic acid (AA)/kg. CHS impaired (p < 0.05) growth performance, increased plasma triglycerides and total serum Ca while decreasing (p < 0.05) plasma glucose and total serum protein. Meanwhile 250 mg AA/kg diet or an increasing ME without or with some EAA partially alleviated (p < 0.0001) the negative effect of CHS on growth while increasing (p < 0.05) feed intake and improving (p < 0.05) feed:gain ratio (F:G) and crude protein (CP) digestibility (p < 0.05). AA or increasing ME with or without EAA increased (p < 0.05) percentage dressing, liver and giblets to those of the positive control. AA or increasing ME with or without EAA partially alleviated the negative effect of CHS on blood pH, packed cell volume (PCV), haemoglobin (Hgb), total serum protein and total Ca, plasma glucose and triglyceride, rectal temperature and respiration rate. Increasing ME level improved chickens' tolerance to CHS without a significant difference from those supplemented with AA. However, increasing Met, Lys and Arg concentration did not improve performance over that recorded with increasing ME level alone. Under CHS, 250 mg AA/kg diet or increasing ME level by addition of 3% vegetable oil could be an useful approach to improve productive and physiological traits of slow-growing chicks, which may be applicable also to fast-growing one.     

The protective effect of γ‐aminobutyric acid on the development of immune function in chickens under heat stress

This study aimed to investigate the protective effect of γ‐aminobutyric acid (GABA) on the development of immune function in chicks under heat stress (HS). One‐day‐old male Wenchang chicks were randomly divided into control (CK), HS and GABA+HS groups. The GABA+HS group was fed with 0.2 ml GABA solution (50 mg/kg) daily by oral gavage. The HS and GABA+HS groups were placed in 40 ± 0.5 °C environment for 2 h heat treatment from 13:00 each day. Blood samples were routinely taken at 14, 21, 28, 35 and 42 days respectively, and the contents of T and B lymphocyte subsets in the blood and tissue were analysed by flow cytometry after FITC/PE double staining; the plasma levels of interleukin (IL)‐2, immunoglobulin (Ig)A, IgG and IgM were determined using ELISA. The thymus and the bursa of fabricius were also collected to analyse for organ index and observe for the changes in tissue microstructure. In addition, the chicks received primary and secondary immunizations with attenuated Newcastle disease (ND) vaccine (LaSota strain) at 7 and 28 days respectively; conventional hemagglutination inhibition (HI) assay was performed to monitor the titre changes in plasma antibody against ND virus in the birds. Our results indicated that the indices of both thymus and bursa of fabricius, the intactness of tissue structure and development, the plasma levels of IL‐2, IgA, IgG and IgM, the titres of ND antibody, and the levels of B and T lymphocyte subsets in HS group were all significantly lower than those in CK group (p < 0.05). However, all above indices were significantly improved in GABA+HS group compared with those in HS group (p < 0.05). These results demonstrated that while HS seriously affected the development of immune function in Wenchang chicks, GABA effectively alleviated the damages of HS to the development of immune function in chicks.