Effects of FSH and LH on Steroid Production by Buffalo (Bubalus bubalis) Granulosa Cells Cultured In Vitro Under Serum‐Free Conditions

The objective of this study was to examine the effects of FSH and LH on oestradiol‐17β and progesterone production by buffalo granulosa cells cultured under serum‐free conditions. Granulosa cells (3 × 10⁵) from small (≤5 mm diameter) follicles were cultured for up to 4 days in 48‐well plates coated with 3.3 μg/cm² fibronectin in Dulbecco's modified Eagle's medium (DMEM) : nutrient mixture F‐12 Ham (1 : 1 ratio) supplemented with 10^?7 m androstenedione, 5 μg/ml human apo‐transferrin and 0.1% bovine serum albumin, in the presence or absence of FSH or LH (0, 1, 2, 4, 8, 16, 32 or 64 ng/ml each). Basal oestradiol‐17β production by granulosa cells from small follicles reduced (p < 0.01) from days 1 to 2 of culture and became undetectable by day 3 and basal progesterone production increased (p < 0.05) from day 1 through day 4 of the culture. Although there was no effect of FSH on day 1 of the culture, FSH at 2, 4, 8 and 16 ng/ml increased (p < 0.05) oestradiol‐17β production by granulosa cells from small follicles on day 2. Progesterone secretion was increased (p < 0.05) by all doses of FSH on all days of culture. All doses of LH had no effect on oestradiol‐17β or progesterone production by granulosa cells from small follicles on any day of the culture. The results of this study demonstrate a serum‐free culture system for buffalo granulosa cells and stimulatory effect of FSH but not LH on steroid hormone production by buffalo granulosa cells under these conditions.     

Expression of Heparin‐Binding EGF‐Like Growth Factor (HB‐EGF) in Bovine Endometrium: Effects of HB‐EGF and Interferon‐τ on Prostaglandin Production

Heparin‐binding EGF‐like growth factor (HB‐EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB‐EGF, prostaglandins (PGs) and interferon‐τ (IFN‐τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB‐EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB‐EGF and IFN‐τ on PGE2 and PGF2‐α production by endometrial cells were investigated. RT‐PCR analysis revealed that HB‐EGF mRNA was greater at the mid‐luteal stage than at the early and regressed luteal stages (p < 0.05). ErbB1 mRNA expression was greater at the mid‐ and late luteal stages than at the other luteal stages (p < 0.05). IFN‐τ increased the expression of HB‐EGF, ErbB1 and ErbB4 mRNA in epithelial cells (p < 0.05). HB‐EGF did not affect PGF2‐α or PGE2 production by bovine endometrial epithelial cells, but increased PGF2‐α and PGE2 production by bovine endometrial stromal cells (p < 0.05). IFN‐τ significantly decreased HB‐EGF‐stimulated PGF2‐α (p < 0.05), but not PGE2 (p > 0.05) production by stromal cells. These results indicate that HB‐EGF and its receptors expression changed in bovine endometrium throughout the oestrous cycle. IFN‐τ increased their expression in cultured endometrial cells. HB‐EGF and IFN‐τ have the ability to regulate PGs production by stromal cells and therefore may play a role in the local regulation of uterine function at the time of implantation in cattle.     

N-乙酰半胱氨酸对山羊子宫内膜基质细胞增殖及相关基因表达的影响

旨在探究N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)对山羊子宫内膜基质细胞(goat endometrial stro-mal cells,gESCs)的影响.本研究以山羊子宫内膜基质细胞为对象,体外培养基中添加浓度梯度分别为100、200、400μmol·L-1的NAC,将0μmol·L1 NA...     

脂多糖对体外培养奶牛子宫内膜上皮细胞子宫容受性因子表达的影响

试验旨在通过脂多糖（lipopolysaccharide,LPS）诱导奶牛子宫内膜上皮细胞构建子宫内膜炎体外感染模型,研究子宫内膜炎对奶牛子宫容受性因子大分子转膜黏蛋白-糖蛋白1（MUC-1）、高度保守的分泌型WNT家族亚型糖蛋白（Wnt-7a）、β受体1（IFNAR1）、IFNAR2、Integrin αvβ3 mRNA及蛋白相对表达量的影响,进而阐明子宫内膜炎引起奶牛屡配不孕和繁殖率低下的机制。采用组织块法分离培养奶牛子宫内膜上皮细胞,免疫荧光方法鉴定细胞纯度,不同浓度LPS刺激子宫内膜上皮细胞,在不同时间点用CCK8测定细胞存活率,ELISA检测炎性因子白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、IL-6、IL-8的分泌变化,实时荧光定量PCR和Western blotting分别检测子宫内膜感染模型中子宫容受性因子MUC-1、Wnt-7a、IFNAR1、IFNAR2、Integrin αvβ3 mRNA及蛋白的表达变化。结果显示,通过组织块法纯化获得的奶牛子宫上皮细胞数量较多,经免疫荧光角蛋白染色证实纯度较高。采用CCK8测定细胞存活率发现,与对照组相比,浓度为0、5、10、50μg/mL的LPS作用6、12、24、48 h后,细胞活力无显著变化（P>0.05）;浓度为100μg/mL的LPS作用24 h时,细胞存活率显著低于对照组（P<0.05）,细胞培养上清中的IL-1β、TNF-α、IL-6、IL-8含量均显著高于对照组及低浓度组（P<0.05）,引起细胞的炎症反应。与对照组相比,模型组MUC-1 mRNA及蛋白的相对表达量均极显著增加（P<0.01）;Wnt-7a mRNA及蛋白的相对表达量均极显著下降（P<0.01）;Integrin αvβ3、IFNAR1和IFNAR2 mRNA相对表达量均极显著下降（P<0.01）,蛋白相对表达量均显著下降（P<0.05）。结果表明,浓度为100μg/mL的LPS作用24 h可成功构建子宫内膜炎体外感染模型,子宫内膜炎对奶牛子宫容受性因子MUC-1、Wnt-7a、IFNAR1、IFNAR2、Integrin αvβ3 mRNA及蛋白表达的影响可能是引起奶牛屡配不孕和繁殖率低下的重要原因。     

白藜芦醇对体外培养的水牛卵丘细胞增殖活力及其相关基因mRNA表达的影响

【目的】研究白藜芦醇(resveratrol, RES)对水牛卵丘细胞体外培养过程中细胞增殖活力、激素分泌、卵丘扩展及抗凋亡和抗氧化能力的影响。【方法】用不同浓度(0(对照组)、1、10、20、30、40、50和60μmol/L)RES培养卵丘细胞,用CCK-8试剂盒测定细胞增殖活力,筛选最佳RES处理浓度及时间用于后续试验。用ELISA法测定培养液中卵丘细胞分泌的雌二醇(estradiol, E₂)和孕酮(progesterone, P₄)的含量,实时荧光定量PCR法测定卵丘细胞扩展、凋亡和抗氧化相关基因的相对表达量。【结果】与对照组相比,在体外培养24～36 h内,1、10和20μmol/L RES组水牛卵丘细胞的增殖活力均有升高趋势,10μmol/L RES处理36 h细胞活力最强,因此用于后续试验中细胞的处理。体外培养36 h时,10μmol/L RES组细胞增殖能力和E₂、P₄的分泌显著增加(P<0.05);10μmol/L RES组卵丘细胞扩展相关基因穿透素3(PTX3)、前列腺素...     

Effect of TGF‐β1 Superfamily Members on Survival of Buffalo (Bubalus bubalis) Embryonic Stem‐like Cells

This study examined the effects of supplementation of ES‐like cell culture medium with bone morphogenetic protein (BMP)‐4 (0, 10, 20 or 100 ng/ml) or Noggin (250, 500 or 750 ng/ml) or TGF‐β1 (0, 0.1, 1 or 10 ng/ml) or SB431542 (0, 10, 25 or 50 μm ), an inhibitor of TGF‐β1 signalling, on survival, colony area and expression level of pluripotency genes in buffalo ES‐like cells at passage 40–80, under different culture conditions. BMP‐4 supplementation significantly reduced (p < 0.05) colony survival rate, percentage increase in colony area and relative mRNA abundance of OCT4, whereas that of NANOG and SOX‐2 was increased significantly (p < 0.05). Noggin supplementation did not affect the colony survival rate and percentage increase in colony area in the presence of FGF‐2 and LIF. In the presence of FGF‐2 alone, it significantly reduced (p < 0.05) the relative mRNA abundance of OCT4 and SOX‐2 and increased (p < 0.05) that of NANOG. Supplementation with TGF‐β1 at 1.0 ng/ml but not at other concentrations increased colony survival rate but had no effect on percentage increase in colony area at any concentration. Supplementation with SB‐431542 decreased (p < 0.05) colony survival rate at 50 μm but not at other concentrations. The percentage increase in colony area was lower (p < 0.05) with 10 μm SB‐431542 than that in the controls, whereas at higher concentrations of 25 or 50 μm , SB‐431542 decreased (p < 0.05) the colony size instead of increasing it. In conclusion, these results suggest that BMP‐4 induces differentiation in buffalo ES‐like cells, whereas TGF‐β/activin/nodal pathway may not be playing a crucial role in maintaining pluripotency in these cells.     

前列腺素E2和F2α对LPS刺激后奶牛子宫内膜上皮细胞COX-1和COX-2表达的影响

试验旨在阐明前列腺素E₂（prostaglandin E₂,PGE₂）和F_2α（prostaglandin F_2α,PGF_2α）对体外培养的奶牛子宫内膜上皮细胞中环氧合酶-1（cyclooxygenase-1,COX-1））与环氧合酶-2（cyclooxygenase-2,COX-2）表达的影响。培养奶牛子宫内膜上皮原代细胞和传代细胞,第4代细胞以1×10⁶个/孔接种于6孔板,以10^-7mol/L PGE₂和PGF_2α分别预处理细胞24 h,以100 ng/mL细菌脂多糖（lipopolysaccharides,LPS）刺激细胞4、8和12 h后分别提取RNA和总蛋白质,采用实时荧光定量PCR与Western blotting等技术检测COX-1与COX-2 mRNA和蛋白质的表达量。结果表明,与对照组相比,COX-1 mRNA表达量在PGE₂单独作用4、8和12 h后显著上调（P<0.05）;COX-2 mRNA表达量在PGE₂单独作用4和12 h后显著上调（P<0.05）,PGE₂单独处理使COX-1、COX-2蛋白表达量均显著上调（P<0.05）。与对照组相比,LPS刺激8和12 h时COX-1 mRNA表达量显著下调（P<0.05）,LPS刺激后COX-1蛋白表达量无显著变化（P>0.05）;LPS刺激后4、8和12 h时COX-2 mRNA表达量显著上调（P<0.05）,LPS刺激后COX-2蛋白表达量显著上调（P<0.05）。与LPS单独处理组相比,LPS+PGE₂处理组在8和12 h时COX-1和COX-2 mRNA表达量均显著上调（P<0.05）,同时COX-1和COX-2蛋白表达量也显著上调（P<0.05）。PGF_2α在LPS未刺激和刺激后对COX-1和COX-2 mRNA的表达无显著影响（P>0.05）,仅在PGF_2α单独处理8和12 h后COX-1 mRNA表达量上调（P<0.05）。两种激素联合处理与各自单独处理及LPS单独刺激相比,对COX-1和COX-2 mRNA表达具有一定的协同诱导作用。     

Antibody‐Dependent Killing of Virus‐Infected Targets by NK‐Like Cells in Bovine Blood

Natural killer (NK) cell lysis of target cells by an Fc receptor‐mediated mechanism has not been conclusively demonstrated in cattle (Campos and Rossi, Vet. Immunol. Immunopathol. 8, 351–362, 1985), although it is well recognized in other species (Sulica et al., Nat. Immun. 14, 123–133, 1995). To resolve this problem, bovine peripheral blood mononuclear cells were completely depleted of adherent monocyte/macrophage type cells. The resulting enriched population of lymphocytes, was totally devoid of adherent monocytes, but contained up to 2 % NK‐like cells. On their own, this population had very low background levels of cytotoxicity for virus‐infected target cells in ⁵¹chromium release assays, but following the addition of virus‐specific antibodies, high levels of lysis were observed. This enhanced level of antibody‐dependent cytotoxicity demonstrated that bovine NK‐like cells can mediate killing of targets by an Fc receptor‐mediated mechanism as has been demonstrated for NK cells from other species.     

Prostaglandin F2α Stimulates Endothelial Nitric Oxide Synthase Depending on the Existence of Bovine Granulosa Cells: Analysis by Co‐culture System of Endothelial Cells,Smooth Muscle Cells and Granulosa Cells

Prostaglandin F_2α (PGF_2α) induces luteolysis in the mid but not in the early luteal phase; despite this, both the early and the mid corpus luteum (CL) have PGF_2α receptor (FPr). We previously indicated that the luteal blood flow surrounding the CL drastically increases prior to a decrease of progesterone (P) in the cows, suggesting that an acute increase of luteal blood flow may be an early sign of luteolysis in response to PGF_2α and that this may be induced by a vasorelaxant nitric oxide (NO). The aim of this study was to investigate the luteal stage‐dependent and the site‐restricted effect of PGF_2α and NO on the mRNA expressions and P secretion. To mimic the local luteal region both of peripheral and central areas of the CL, we utilized co‐cultures using bovine aorta endothelial cells (EC), smooth muscle cells (SMC) and luteinizing granulosa cells (GC) or fully‐luteinized GC. PGF_2α stimulated the expression of endothelial NO synthase (eNOS) mRNA at 0.5 h in mix‐cultures of EC and SMC with fully‐luteinized GC but not with luteinizing GC. The expression of eNOS mRNA in EC was increased by PGF_2α at 1 h only when EC was cultured together with fully‐luteinized GC but not with luteinizing GC. In all co‐cultures, PGF_2α did not affect the mRNA expression of FPr. Treatment of NO donor inhibited P secretion at 0.5 h. In conclusion, the present study suggests that the coexistence of the mature luteal cells (fully‐luteinized GC) with EC/SMC may be crucial for acquiring functional NO synthesis induced by PGF_2α.     

Measurement of C‐reactive protein and Prostaglandin F2α Metabolite Concentrations in Differentiation of Canine Pyometra and Cystic Endometrial Hyperplasia/Mucometra

Canine pyometra is a dioestrus period disease in which systemic inflammatory response syndrome (SIRS) is a common outcome due to the response of the body to the bacterial infection. The purpose of this study was i) to differentiate canine pyometra and cystic endometrial hyperplasia (CEH)/mucometra by measuring serum C‐reactive protein (CRP) and prostaglandin F_2α metabolite (PGFM) concentrations in blood and ii) to compare serum concentrations of CRP and PGFM in bitches with a pathological uterus (pyometra or CEH/mucometra) to concentrations in bitches with a healthy uterus. Mean CRP concentrations were found significantly higher (p < 0.001) in dogs with pyometra compared to those with CEH/mucometra or healthy uterus. However, no statistical difference could be detected between the groups for mean PGFM concentrations. Mean white blood cell count (WBC), alkaline phosphatase (ALP) and total protein concentrations were found significantly higher (p < 0.001) in dogs with pyometra. Escherichia coli was the most frequently isolated microorganism from dogs with pyometra (64.3%). Edwardsiella spp. was detected in a single case of pyometra for the first time. In conclusion, our results demonstrate that serum CRP concentrations were increased in dogs with pyometra and thus we conclude that serum CRP concentration but not PGFM might be useful as a marker to differentiate a case of CEH/mucometra from pyometra in female dogs. To the authors' knowledge, this is the first report in which Edwardsiella spp. has been isolated in the canine uterus.     

Dietary supplementation of β‐hydroxy‐β‐methylbutyrate in animals – a review

The leucine metabolite β‐hydroxy‐β‐methylbutyrate (HMB) has been studied by many researchers over the last two decades. In particular, the utility of HMB supplementation in animals has been shown in numerous studies, which have demonstrated enhanced body weight gain and carcass yield in slaughter animals; positive immunostimulatory effect; decreased mortality; attenuation of sarcopenia in elderly animals; and potential use in pathological conditions such as glucocorticoid‐induced muscle loss. The aim of this study was to summarize the body of research on HMB supplementation in animals and to examine possible mechanisms of HMB action. Furthermore, while the safety of HMB supplementation in animals is well documented, studies demonstrating efficacy are less clear. The possible reasons for differences in these findings will also be examined.     

α‐6 Integrin Expression in Bovine Spermatogonial Cells Purified by Discontinuous Percoll Density Gradient

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α‐6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self‐renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two‐step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α‐6 integrin by flow cytometry and real‐time RT‐PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two‐step enzymatic digestion. An average of 1 × 10⁵ viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α‐6 integrin expression. Flow cytometry analysis demonstrated no differences in the α‐6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real‐time PCR analysis (p > 0.05). In addition to α‐6 integrin, the expression of GFRa‐1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α‐6 integrin expression.     

应用生物反应器和微载体培养Vero细胞生产猪流行性腹泻病毒的研究

对应用生物反应器系统和微载体培养Vero细胞生产PEDV进行了实验研究,通过分析和比较批培养及换液培养过程中细胞生长、代谢和病毒增殖的相互关系,发现PEDV随着细胞的生长而不断在胞内增殖和积累,72h后Vero细胞需经过一个短暂的停止生长阶段,再继续呈指数生长。反应器换液培养的单位培养基细胞产量比批培养提高了28%,比方瓶培养提高了138%,疫苗生产效率显著提高。当接种密度为1 23×105cells/ml时,换液培养至96h时获得了1 74×106cells/ml的高细胞密度,细胞扩增了14 3倍,且单位细胞的病毒产量并不低于静态培养。     

Antiviral Effect of Saccharomyces cerevisiaeβ‐glucan to Swine Influenza Virus by Increased Production of Interferon‐γ and Nitric Oxide

The aim of these experiments was to investigate the potential antiviral effect of Saccharomyces cerevisiaeβ‐glucan on the pneumonia induced by swine influenza virus (SIV). Forty colostrum‐deprived 5‐day‐old piglets were randomly divided into four groups of 10. The 20 pigs in groups 1 and 2 were administered Saccharomyces cerevisiaeβ‐glucan orally (50 mg/day/pig; En‐Bio Technology Co., Ltd) for 3 days before SIV infection and those in groups 3 and 4 were given culture medium/diluent alone. Groups 1 and 3 were inoculated intranasally with 3 ml of tissue culture fluid containing 2 × 10⁶ tissue culture infective doses 50% (TCID₅₀)/ml of SIV and those in groups 2 and 4 were exposed in the same manner to uninfected cell culture supernatant. The microscopic lung lesions induced by SIV infection (group 1 pigs) were significantly more severe than those induced by infection in animals pre‐administered β‐glucan (group 3) (P < 0.05). Significantly more SIV nucleic acid was detected in the lungs of pigs experimentally infected with SIV only (group 1) at 5, 7 and 10 days post‐inoculation (dpi) compared with lungs from pigs pre‐administered β‐glucan and infected with SIV (group 3) (P < 0.05). The concentrations of interferon‐γ (IFN‐γ) and nitric oxide (NO) in bronchoalveolar lavage fluid from pigs pre‐administered β‐glucan and infected with SIV (group 3) were significantly higher than for any other group at 7 and 10 dpi for IFN‐γ, and at 5, 7 and 10 dpi for NO (P < 0.05). Saccharomyces cerevisiaeβ‐glucan reduced the pulmonary lesion score and viral replication rate in SIV‐infected pigs. These findings support the potential application of β‐glucan as prophylactic/treatment agent in influenza virus infection.     

Effects of β‐carotene‐enriched dry carrots on β‐carotene status and colostral immunoglobulin in β‐carotene‐deficient Japanese Black cows

Data from 18 β‐carotene‐deficient Japanese Black cows were collected to clarify the effects of feeding β‐carotene‐enriched dry carrots on β‐carotene status and colostral immunoglobulin (Ig) in cows. Cows were assigned to control or carrot groups from 3 weeks before the expected calving date to parturition, and supplemental β‐carotene from dry carrots was 138 mg/day in the carrot group. Plasma β‐carotene concentrations in the control and carrot groups at parturition were 95 and 120 μg/dL, and feeding dry carrots slightly improved plasma β‐carotene at parturition. Feeding dry carrots increased colostral IgA concentrations in cows and tended to increase colostral IgG₁, but colostral IgM, IgG₂, β‐carotene and vitamin A were not affected by the treatment. Feeding dry carrots had no effects on plasma IgG₁, IgA and IgM concentrations in cows, but plasma IgG₁ concentrations decreased rapidly from 3 weeks before the expected calving date to parturition. These results indicate that feeding β‐carotene‐enriched dry carrots is effective to enhance colostral IgA and IgG₁ concentrations in β‐carotene‐deficient cows.     

Plasma Concentrations of 13, 14‐Dihydro‐15‐keto‐Prostaglandin F2α, Progesterone and Cortisol During Periparturient Period in Yaks (Poephagus grunniens L.)

An attempt was made to determine plasma concentrations of, 13, 14‐dihydro‐15‐keto‐prostaglandin F_2α (PGFM), cortisol and progesterone during periparturient period in yak. Plasma PGFM level showed an increasing trend beginning day 5 prior to parturition (0.48 ± 0.14 ng/ml) and increased steeply thereafter to reach a peak level (17.16 ± 1.31 ng/ml) on the day of parturition. The levels, then, declined sharply on day 1 postpartum to reach 1.20 ± 0.40 ng/ml and thereafter declined gradually over the days to reach 0.28 ± 0.09 ng/ml on day 20 postpartum and this level was maintained with fluctuation within narrow limits thereafter till conclusion of the blood sampling on day 90 post‐calving. The plasma progesterone concentration on days 7 and 6 before parturition was high (2.10 ± 0.10 and 2.12 ± 0.10 ng/ml, respectively). The level then decreased gradually and abrupt fall was observed 1–2 days before parturition and became basal on day of parturition (0.24 ± 0.04 ng/ml). This basal level was maintained till the end of the blood sampling on day 90 postpartum. Plasma cortisol level showed an increasing trend beginning day 2 prior to parturition (2.36 ± 0.65 ng/ml) and increased steeply thereafter to reach a peak level (26.65 ± 5.28 ng/ml) on the day of parturition. The levels, then, declined gradually over the days and touched 2.36 ± 0.25 ng/ml on day 3 postpartum and this level was maintained with fluctuation within narrow limits thereafter till day 7 post‐calving.