Localisation and expression of TRPV6 in all intestinal segments and kidney of laying hens

1. The aim of this study was to investigate the localisation and expression of the epithelial Ca2+ channel TRPV6 (transient receptor potential vanilloid channel type 6) in different intestinal segments and kidney of laying hens during peak lay. 2. Immunohistochemical analysis of the intestine indicated that TRPV6 was localised to the brush-border membranes of the duodenum, jejunum, ileum, caecum, and rectum. Expression was weaker in the rectum, and little or no expression was found in crypt and goblet cells. In addition, TRPV6 mRNA was quantified amongst different intestinal segments, and expression was highest in the duodenum and jejunum. Furthermore, Western blotting indicated that the duodenum expressed the greatest amount of TRPV6 and the rectum the least with the other segments expressing intermediate levels. 3. In the kidney, distinct immunopositive staining for TRPV6 was detected at the apical domain of the distal convoluted tubules (DCT) and medullary connecting tubules (CNT). Interestingly, distribution of TRPV6 extended to the proximal convoluted tubules (PCT). Furthermore, the kidney expressed lower TRPV6 mRNA and protein levels compared with that in the duodenum. 4. In conclusion, the epithelial Ca2+ channel TRPV6 is strongly expressed in the apical cells of the entire intestine and the renal tubules, suggesting that active Ca2+ transcellular transport plays a crucial role in dietary calcium (re)absorption in laying hens.     

Expression of TRPV6 and CaBP-D28k in the egg shell gland (uterus) during the oviposition cycle of the laying hen

1. The aim of this study was to investigate the localisation of the transient receptor potential vanilloid channel type 6 (TRPV6) in egg shell gland (ESG) and examine the dynamic expression of TRPV6 and Calbindin-d28k (CaBP-D28k), as well as the changes in concentration of total calcium (Ca), total inorganic phosphorus (P), alkaline phosphatase (ALP), parathyroid hormone (PTH) and calcitonin (CT) in plasma during the oviposition cycle.

2. The plasma ALP activity was notably increased at 8 h. In addition, plasma CT was highest at 0 h and significantly lower at 8 h. The change of plasma PTH concentration increased slightly post-oviposition and reached a maximum at 16 h.

3. Immunohistochemical analysis indicated that TRPV6 was strongly localised to the apical luminal epithelium of the mucosa. The mRNA levels of TRPV6 and CaBP-D28k in the ESG remained very low from 0 to 4.5 h, but were significantly increased at 16 h. Furthermore, Western blotting analysis showed that the expression of TRPV6 and CaBP-D28k also reached a maximum at 16 h and was different from the concentration of CaBP-D28k.

4. In conclusion, the epithelial Ca²⁺ channel TRPV6 is strongly expressed in the epithelial cells of the eggshell gland, and the increase of TRPV6 and CaBP-D28k mRNA and protein expression during eggshell formation suggests that active Ca²⁺ transcellular transport exerts significant effects in delivering active calcium in the ESG.     

亚急性瘤胃酸中毒耐受性不同的奶牛瘤胃上皮形态及功能差异研究

本试验旨在探究SARA（亚急性瘤胃酸中毒）耐受性不同奶牛的瘤胃上皮形态及其功能差异。选取12头装有永久性瘤胃瘘管的泌乳中期荷斯坦奶牛,饲喂精粗比为4∶6的日粮,并根据瘤胃pH值的高低,分为SARA易感组（SUS,n=4）和SARA耐受组（TOL,n=4）。瘤胃上皮形态及功能分析结果显示,与TOL组比较,SUS组奶牛瘤胃上皮的棘突层和基底层厚度明显增厚（P<0.05）,SUS组奶牛瘤胃上皮组织中参与挥发性脂肪酸（VFA）吸收的PAT1、MCT4和DRA基因表达量较TOL组显著下调（P<0.05）,而H+转运载体NHE1、NHE2、NHE3和调节胞内pH的vH+ATPase和Na+/K+ATPase的表达量显著升高（P<0.05）;对参与调控瘤胃VFA代谢的基因定量结果表明,SUS组PDHA1和SREBP2的表达量显著高于TOL组（P<0.05）,而HMGCL-2的表达量显著降低（P<0.05）;此外,SUS组CDK2、CDK6和Cyclin D1、Bad及Caspase-9等参与瘤胃上皮细胞增殖与凋亡的基因表达量显著高于TOL组（P<0.05）。结果说...     

TRPV5与TRPV6在哺乳动物妊娠期钙代谢的适应性调节作用

在哺乳动物妊娠期,母体的钙代谢通过适应性调节满足胎儿对钙的需求,包括肠钙吸收的增强、肾钙排泄的减少。妊娠钙代谢适应性需要钙离子通路蛋白参与,TRPV5/TRPV6是实现钙代谢调节的重要蛋白。TRPV6高表达于小肠,主要介导肠钙转运,TRPV5与肾脏钙离子重吸收相关,此外TRPV6介导胎盘组织的母体一胎儿钙转运。     

蛋鸡TRPV6基因的原核表达、纯化及抗体制备

为制备蛋鸡TRPV6蛋白多克隆抗体,根据其基因序列,设计1对特异性引物,以卵巢组织中提取的总RNA为模板,扩增蛋鸡TRPV6基因1 801～2 176nt的375bp序列,构建原核表达质粒pET-32a（＋）-TRPV6;将重组质粒转化BL21（DE3）,经IPTG诱导表达TRPV6融合蛋白,通过镍离子螯合柱纯化后免疫新西兰大白兔,获得兔抗鸡TRPV6多克隆抗体,分别通过酶联免疫吸附试验（ELISA）法和Western blot检测抗体的效价和抗体特异性。结果表明,试验成功构建原核表达载体pET-32a（＋）-TRPV6,SDS-PAGE蛋白电泳检测发现目的蛋白大小约35 000;Western blot分析显示,表达的TRPV6融合蛋白具有良好的免疫原性,其抗体可与大肠杆菌表达的产物特异性结合;ELISA显示其抗体效价达1∶100 000。获得纯化的融合蛋白和多克隆抗体对研究TRPV6钙离子通道在蛋鸡髓质骨形成的作用机理具有重要作用。     

The absorption of phosphate ions from the ovine reticulorumen

The reticulorumen is now recognised to be an important site of net absorption of phosphate ions from ruminal fluid containing phosphate concentrations appropriate to those found in normal farming practice. These rates of absorption were measured in vivo from solutions placed in the washed reticulorumen, isolated in situ, in conscious, trained sheep. Reducing the ruminal sodium concentration led to reduced absorption of phosphate, suggestive that phosphate and sodium fluxes across the apical wall of the ruminal epithelial cell are linked, as they are in the kidney. Increased absorption of short chain fatty acids led to enhanced absorption of phosphate ions. Conversely, inhibition of carbonic anhydrase activity, by the addition of 1 mM acetazolamide to the ruminal fluid, led to a reduction in phosphate absorption. An increase in the acidity of the ruminal fluid also increased the absorption of phosphate, as did an increase in the ruminal Ca(2+) concentration over the range 1-4 mmol per litre. It is suggested that these effects can be accounted for by a Na(+)/H(+) antiporter coupled with a phosphate/proton symporter in the apical membrane of the ruminal epithelial cell.     

Effect and absorption of histamine in sheep rumen: significance of acidotic epithelial damage

The significance of ruminal histamine for the induction of epithelial damage and systemic histaminosis during the ruminal lactic acidosis syndrome was investigated using the Ussing chamber technique. Histamine did not affect the electrophysiological characteristics of ovine ruminal epithelia under shortcircuit conditions. In contrast, mucosal acidification to pH 5.1 induced pronounced effects on tissue conductance (Gt) and short-circuit current (Isc). Using [3H]histamine for flux determination (hist-rad fluxes), significant net absorption of hist-rad (.40+/-.07 nmol x cm(-2) x h(-1); n = 6) was evident under short-circuit conditions in the presence of a mucosal-to-serosal (ms) histamine gradient (80 microM:12 microM). In comparison to hist-rad, absorption of native histamine (ms histamine gradient 80 microM:0 microM) measured with HPLC under open circuit conditions was smaller (.010+/-.003 nmol x cm(-2) x h(-1); n = 10). Mucosal acidification to pH 5.1 led to an increase (P<.05) in net absorption of hist-rad (to .67+/-.06 nmol x cm(-2) x h(-1); n = 6) and a dramatic increase (P<.01) in the absorption of native histamine (to .27+/-.04 nmol x cm(-2) x h(-1); n = 10). Absorption of ruminal histamine should be considered an important cause of systemic histaminosis in acidotic ruminants. Histamine absorption is linked to ruminal epithelial damage, which is primarily induced by luminal acidity and not by histamine.     

Role of TRPV1 channels during the acquisition of fertilizing ability in boar spermatozoa

Recently, the transient receptor potential vanilloid type 1 (TRPV1) channel was shown to be involved in capacitation, the process that allows mammalian spermatozoa to acquire their fertilizing ability within the female genital tract. Unfortunately, the role of TRPV1 in this process is still unclear. Thus, the aims of the present work were to 1) investigate the function of TRPV1 in the male gamete signaling system and 2) modulate TRPV1 activity by administering a specific activator, capsaicin, or a specific inhibitor, capsazepin, to spermatozoa during in vitro capacitation. Using confocal microscopy, cellular responses were assessed in terms of changes in 1) cell membrane resting potential, 2) intracellular calcium concentrations, and 3) actin polymerization dynamics. As a result, TRPV1 channels were shown to act as specific cationic channels: their activation led to membrane depolarization and, consequently, the opening of voltage-gated calcium channels and an increase in intracellular calcium concentrations. These ionic events promote actin cytoskeletal depolymerization and a loss of acrosome structure integrity. In contrast, TRPV1 inhibition caused a slowing of the capacitation-dependent increase in intracellular calcium concentrations, a reduction in actin polymerization, and acrosome rupture. In conclusion, these results suggest that TRPV1 channels modulate the major pathways involved in capacitation.     

Ruminant Nutrition Symposium: Molecular adaptation of ruminal epithelia to highly fermentable diets

Feeding highly fermentable diets to ruminants is one strategy to increase energy intake. The increase in short-chain fatty acid (SCFA) production and reduced ruminal pH associated with highly fermentable diets imposes a challenge to the metabolism and the regulation of intracellular pH homeostasis of ruminal epithelia. The ruminal epithelia respond to these challenges in a coordinated manner. Whereas the enlargement of absorptive surface area is well documented, emerging evidence at the mRNA and transporter and enzyme activity levels indicate that changes in epithelial cell function may be the initial response. It is not surprising that gene expression analysis has identified pathways involved in fatty acid metabolism, ion transport, and intracellular homeostasis to be the pathways dominantly affected during adaptation and after adaptation to a highly fermentable diet. These findings are important because the intraepithelial metabolism of SCFA, particularly butyrate, helps to maintain the concentration gradient between the cytosol and lumen, thereby facilitating absorption. Butyrate metabolism also controls the intracellular availability of butyrate, which is widely regarded as a signaling molecule. Current data indicate that for butyrate metabolism, 3-hydroxy-3-methylglutaryl-CoA synthase and acetyl-CoA acetyltransferase are potential regulatory points with transient up- and downregulation during diet adaptation. In addition to nutrient transport and utilization, genes involved in the maintenance of cellular tight junction integrity and induction of inflammation have been identified as differentially expressed genes during adaptation to highly fermentable diets. This may have important implications on ruminal epithelial barrier function and the inflammatory response often associated with subacute ruminal acidosis. The objective of this review is to summarize ruminal epithelial adaptation to highly fermentable diets focusing on the changes at the enzyme and transporter activity levels, as well as the underlying molecular changes at the mRNA and protein expression levels.     

产蛋循环过程中TRPV6、CaBP-D_（28K）和PMCA 1b在蛋壳腺的动态表达

将40羽220日龄ISA蛋鸡分为5组,于产蛋后0、2、4.5、8、16h断头处死,采集蛋壳腺组织,运用Real-time PCR和Western-blot方法检测产蛋循环过程中瞬时性受体单位香草精受体6（Transient receptor potential vanilloid receptor 6,TRPV6）、钙结合蛋白（calbindin D28K,CaBP-D28K）、质膜钙离子ATP酶1b（plasma membrane Ca2＋-ATPase 1b,PMCA 1b）mRNA和蛋白浓度的动态变化。结果显示,卵子未进入蛋壳腺前（产蛋后0~4.5h）,蛋壳腺内TRPV6、CaBP-D28K和PMCA 1bmRNA表达水平较低,随后表达量逐渐升高,在蛋壳钙化过程中达到最大值（产蛋后16h）,与0h相比,TRPV6和CaBP-D28KmRNA表达差异均极显著（P〈0.01）;另外,产蛋循环过程中,TRPV6、CaBP-D28K和PMCA 1b蛋白浓度变化与mRNA变化一致,产蛋后16h到达最大值,其中CaBP-D28K蛋白浓度与0h相比,差异显著（P〈0.05）。结果表明,产蛋循环可调控蛋壳腺内TRPV6、CaBP-D28K和PMCA 1b的表达,并提示钙离子跨细胞转运途径在钙离子进入蛋壳腺形成蛋壳过程中发挥重要作用。     

Effects of calcium soaps of long-chain fatty acids on feedlot performance, carcass characteristics and ruminal metabolism of steers

Two trials were conducted to determine the effects of calcium soaps of long-chain fatty acids (calcium soap) on feedlot performance, diet digestibility, carcass characteristics and ruminal metabolism of steers fed diets (85% concentrate:15% corn silage) containing 0, 2, 4 or 6% calcium soap. In Trial 1, increasing calcium soap decreased (P less than .05) DM, CP and gross energy intake but increased total fatty acid intake. Feed to gain ratio tended to improve with increased calcium soap; gross energy conversion was not affected (P greater than .05) by diet. Average daily gain and hot carcass weight decreased (P less than .05) with addition of calcium soap; other carcass characteristics were not affected (P greater than .05). Apparent digestibilities of DM, N, energy and ash were not affected (P greater than .05) by calcium soap. Neutral detergent fiber digestibility increased linearly (P less than .08) with increasing calcium soap, whereas digestibility of total fatty acids was affected quadratically (P less than .05); fatty acid digestibility was similar among 0, 2 and 4% calcium soap diets but decreased for the 6% calcium soap diet. In Trial 2, increased calcium soap did not affect (P greater than .05) ruminal VFA concentrations, pH or in sacco NDF disappearance of orchardgrass following 12, 24 and 48 h of incubation. Calcium soap increased (P less than .07) ruminal concentrations of calcium soap fatty acids at 1, 2, 4 and 8 h postfeeding. Calcium soap did not improve performance of feedlot cattle fed high-concentrate diets. Further, calcium soap did not affect ruminal fermentation and did not dissociate significantly even when ruminal pH was below 6 for extended periods of time.     

日粮添加尿素对瘤胃上皮细胞增殖、凋亡以及吸收转运能力的影响

本研究探讨了在日粮中添加尿素替代部分豆粕对山羊瘤胃发酵、上皮细胞增殖、凋亡和吸收转运能力的影响。将18 只波杂山羊随机分为3 组（n=6）,分别饲喂3 种日粮：LC组（纯粗料）、MC组（30%精料）以及Urea组（1%DM缓释尿素替代部分豆粕+30%精料）。饲喂Urea组和MC组的山羊瘤胃中短链脂肪酸（SCFA）浓度、pH值均显著高于LC组,而该两组之间无显著差异;但Urea组在MC组的基础上进一步显著提高了瘤胃NH₃和血浆尿素氮（BUN）浓度。因此日粮添加尿素对于瘤胃上皮中受瘤胃SCFA浓度、pH值调节的上皮生长、细胞周期、增殖凋亡相关基因和SCFA转运载体mRNA表达的影响与MC组相似,但对受瘤胃NH₃调节的尿素转运、细胞内pH（pHi）调节相关蛋白mRNA表达则有显著的抑制效果,即显著高于LC组,但低于MC组。     

Ruminant Nutrition Symposium: Role of fermentation acid absorption in the regulation of ruminal pH

Highly fermentable diets are rapidly converted to organic acids [i.e., short-chain fatty acids (SCFA) and lactic acid] within the rumen. The resulting release of protons can constitute a challenge to the ruminal ecosystem and animal health. Health disturbances, resulting from acidogenic diets, are classified as subacute and acute acidosis based on the degree of ruminal pH depression. Although increased acid production is a nutritionally desired effect of increased concentrate feeding, the accumulation of protons in the rumen is not. Consequently, mechanisms of proton removal and their quantitative importance are of major interest. Saliva buffers (i.e., bicarbonate, phosphate) have long been identified as important mechanisms for ruminal proton removal. An even larger proportion of protons appears to be removed from the rumen by SCFA absorption across the ruminal epithelium, making efficiency of SCFA absorption a key determinant for the individual susceptibility to subacute ruminal acidosis. Proceeding initially from a model of exclusively diffusional absorption of fermentation acids, several protein-dependent mechanisms have been discovered over the last 2 decades. Although the molecular identity of these proteins is mostly uncertain, apical acetate absorption is mediated, to a major degree, via acetate-bicarbonate exchange in addition to another nitrate-sensitive, bicarbonate-independent transport mechanism and lipophilic diffusion. Propionate and butyrate also show partially bicarbonate-dependent transport modes. Basolateral efflux of SCFA and their metabolites has to be mediated primarily by proteins and probably involves the monocarboxylate transporter (MCT1) and anion channels. Although the ruminal epithelium removes a large fraction of protons from the rumen, it also recycles protons to the rumen via apical sodium-proton exchanger, NHE. The latter is stimulated by ruminal SCFA absorption and salivary Na(+) secretion and protects epithelial integrity. Finally, SCFA absorption also accelerates urea transport into the rumen, which via ammonium recycling, may remove protons from rumen to the blood. Ammonium absorption into the blood is also stimulated by luminal SCFA. It is suggested that the interacting transport processes for SCFA, urea, and ammonia represent evolutionary adaptations of ruminants to actively coordinate energy fermentation, protein assimilation, and pH regulation in the rumen.     

奶牛亚急性瘤胃酸中毒（SARA）的易感因素及其对瘤胃功能的影响

在奶牛养殖中,亚急性瘤胃酸中毒（subacute ruminal acidosis,SARA）是一种高发的营养代谢病。随着我国奶牛养殖集约化程度的提高,为提升奶牛生产性能饲喂大量高能谷物饲料极易诱发SARA,导致瘤胃液pH值降低,瘤胃菌群发生改变,瘤胃上皮黏膜层受损,进而影响瘤胃代谢功能。分析了奶牛不同生理阶段、择食行为以及个体差异等SARA的易感因素,探讨了SARA对奶牛瘤胃菌群变化、瘤胃上皮细胞基因表达、瘤胃屏障功能的影响,以期为阐明SARA造成瘤胃功能损伤的机制、降低奶牛发生SARA的风险提供参考。     

Performance, methanogenesis and nitrogen metabolism of finishing steers fed monensin and nickel

Sixty-four Angus steers initially averaging 354 kg were allotted to a 2 X 2 factorial arrangement of treatments to determine the effects of dietary Ni (0 or 5 mg/kg supplemental), monensin (0 or 33 mg/kg) and their possible interaction on performance, methane production and N metabolism. The basal diet was a high energy, corn-cottonseed hull based diet containing 10.2% crude protein and .30 mg/kg Ni on a dry matter basis. Monensin reduced (P less than .05) feed intake, did not affect average daily gain and improved (P less than .05) feed conversion over the 102-d study. Nickel supplementation did not significantly alter or interact with monensin to affect steer performance. However, steers fed Ni tended to have higher average daily gains and improved feed conversions. Monensin decreased (P less than .05) in vitro methane production, altered several carcass traits, increased (P less than .05) molar proportion of ruminal propionate and decreased (P less than .05) molar proportion of ruminal acetate. Nickel did not alter methane production, carcass characteristics or ruminal volatile fatty acid proportions. Both monensin and Ni increased (P less than .05) ruminal fluid urease activity when samples were obtained before feeding. A significant monensin X Ni interaction was found to affect ruminal epithelial urease activity. Monensin increased ruminal epithelial urease in steers not receiving supplemental Ni, but had no effect on ruminal epithelial urease activity in steers fed supplemental Ni. Ruminal fluid protein and ammonia-N were decreased (P less than .05) by monensin. Results of this study indicate that Ni may interact with monensin to affect ruminal epithelial urease activity but not performance or methane production in finishing steers.     

Characterization of bovine ruminal epithelial bacterial communities using 16S rRNA sequencing, PCR-DGGE, and qRT-PCR analysis

Currently, knowledge regarding the ecology and function of bacteria attached to the epithelial tissue of the rumen wall is limited. In this study, the diversity of the bacterial community attached to the rumen epithelial tissue was compared to the rumen content bacterial community using 16S rRNA gene sequencing, PCR-DGGE, and qRT-PCR analysis. Sequence analysis of 2785 randomly selected clones from six 16S rDNA (～1.4kb) libraries showed that the community structures of three rumen content libraries clustered together and were separated from the rumen tissue libraries. The diversity index of each library revealed that ruminal content bacterial communities (4.12/4.42/4.88) were higher than ruminal tissue communities (2.90/2.73/3.23), based on 97% similarity. The phylum Firmicutes was predominant in the ruminal tissue communities, while the phylum Bacteroidetes was predominant in the ruminal content communities. The phyla Fibrobacteres, Planctomycetes, and Verrucomicrobia were only detected in the ruminal content communities. PCR-DGGE analysis of the bacterial profiles of the rumen content and ruminal epithelial tissue samples from 22 steers further confirmed that there is a distinct bacterial community that inhibits the rumen epithelium. The distinctive epimural bacterial communities suggest that Firmicutes, together with other epithelial-specific species, may have additional functions other than food digestion.     

Influence of lysocellin and monensin on mineral metabolism of steers fed forage-based diets

Studies were conducted to determine the effects of lysocellin and monensin on mineral metabolism of steers fed forage-based diets. In each study treatments consisted of 1) control, 2) 100 mg lysocellin/d, 3) 200 mg lysocellin/d and 4) 200 mg monensin/d. Twenty-four growing Hereford steers were used in each of two experiments to evaluate the effects of ionophore feeding on plasma and ruminal soluble mineral concentrations. Steers were fed individually greenchop (tall fescue and bermudagrass) ad libitum and .91 kg/d of a corn-trace mineral salt-ionophore supplement. Plasma and ruminal fluid samples were obtained on d 28 and 84 in both studies. Ruminal concentrations of soluble phosphorus (P) and iron (Fe) were higher (P less than .05), whereas soluble manganese (Mn) was lower (P less than .01), in steers fed lysocellin than in controls. Steers fed lysocellin had higher (P less than .05) plasma magnesium (Mg) concentrations than control steers. Plasma and ruminal soluble mineral concentrations generally were similar for the monensin and 200 mg lysocellin treatments. Two additional studies were conducted to determine the effects of lysocellin and monensin on macromineral apparent absorption and retention in steers fed tall fescue greenchop. Steers were adjusted to their diets for 28 d and then placed in metabolism crates for a 6-d acclimation followed by a 5-d collection of urine and feces. Percent apparent absorption of calcium (Ca), potassium (K), Mg and P was higher (P less than .05), whereas sodium (Na) absorption was lower (P less than .05), in steers fed lysocellin than in controls. Mineral absorption was similar in steers fed 200 mg lysocellin or monensin. Calcium (P less than .05) and K (P less than .10) retention (percent of intake) was increased by ionophore feeding. Results indicate that lysocellin and monensin alter apparent absorption and retention of certain minerals in steers fed forage-based diets.