Quantitative mRNA analysis of muscarinic acetylcholine receptors in the intestine of dairy cows with spontaneous caecal dilatation-dislocation

Muscarinic receptors mediate acetylcholine-induced muscular contractions. In this study, mRNA levels of muscarinic receptor subtypes 2 and 3 (M(2) and M(3)) in the ileum, caecum, proximal loop of the ascending colon (PLAC) and external loop of the spiral colon (ELSC) were determined by quantitative polymerase chain reaction in seven cows with caecal dilatation-dislocation (CDD) and seven healthy control cows. Levels of M(2) were significantly lower in the caecum, PLAC and ELSC and levels of M(3) were significantly lower in the ileum, caecum, PLAC and ELSC of cows with CDD compared to healthy cows (P<0.05). Down-regulation of M(3) may play a role in the pathogenesis of CDD.     

Effects of colostrum feeding and dexamethasone treatment on mRNA levels of insulin-like growth factors (IGF)-I and -II, IGF binding proteins-2 and -3, and on receptors for growth hormone, IGF-I, IGF-II, and insulin in the gastrointestinal tract of neonatal calves

The somatotropic axis regulates growth of the gastrointestinal tract (GIT). In addition, colostrum feeding and glucocorticoids affect maturation of the GIT around birth in mammals. We have measured mRNA levels of members of the somatotropic axis to test the hypothesis that colostrum intake and dexamethasone treatment affect respective gene expression in the GIT. Calves were fed either colostrum or an isoenergetic milk-based formula, and in each feeding group, half of the calves were treated with dexamethasone (DEXA; 30 microg/kg body weight per day). Individual parameters of the somatotropic axis differed (P < 0.05) among different GIT sections and formula feeding increased (P < 0.05) mRNA levels of individual parameters at various sites of the GIT. Effects of DEXA on the somatotropic axis in the GIT partly depended on different feeding. In colostrum-fed calves, DEXA decreased (P < 0.05) mRNA levels of IGF-I (esophagus, fundus, duodenum, and ileum), IGF-II (fundus), IGFBP-2 (fundus), IGFBP-3 (fundus), IGF1R (esophagus, ileum, and colon), IGF2R (fundus), GHR (fundus), and InsR (esophagus, fundus), but in formula-fed calves DEXA increased mRNA levels of IGF-I (esophagus, rumen, jejunum, and colon). Furthermore, DEXA increased (P < 0.05) mRNA levels of IGF-II (pylorus), IGFBP-3 (duodenum), IGF2R (pylorus), and GHR (ileum), but decreased mRNA levels of IGFBP-2 (ileum), and IGF1R (fundus). Whereas formula feeding had stimulating effects, effects of DEXA treatment on the gene expression of parameters of the somatotropic axis varied among GIT sites and partly depended on feeding.     

Expression of messenger RNA coding for 5-HT receptor, alpha and beta adrenoreceptor (subtypes) during oestrus and dioestrus in the bovine uterus

Serotoninergic and adrenergic receptors (5-HTR and AR) are involved in the regulation of uterine contractility. The objective of this study was to compare mRNA levels of 5-HTR(1A), 5-HTR(1B), 5-HTR(1D), 5-HTR(1F), 5-HTR(2A), 5-HTR(2B), 5-HTR(2C), 5-HTR(4) and alpha(1A), alpha(1B), alpha(1D), alpha(2AD), alpha(2B), alpha(2C), and beta(1), beta(2), beta(3)-AR in oestrus and dioestrus, and at three uterine locations (tip, middle and base) using quantitative real-time polymerase chain reaction. Uterine specimens consisting of endometrium and myometrium including vessels and serosa were collected from cows in oestrus (n = 10) and dioestrus (n = 15) respectively. Levels of 5-HTR and AR mRNA were expressed relative to the geometric mean of ribosomal RNA (18S), ubiquitin and glyceraldehyde phosphate dehydrogenase by the mean values of geNorm algorithm. 5-HTR(1A), 5-HTR(2C) and beta(3)-AR mRNA could not be detected in uterine tissues. The mRNA levels of 5-HTR(1F) and 5-HTR(2B) were lower (P < 0.05), but of 5-HTR(4) were higher (P < 0.05) in oestrus than in dioestrus. The mRNA levels of alpha(1A)-AR, alpha(2AD)-AR, alpha(2B)-AR were lower (P < 0.05), but of alpha(2C)-AR and beta(2)-AR were higher (P < 0.05) in oestrus than dioestrus. The mRNA levels of 5-HTR(1B) and 5-HTR(1D) (oestrus) and of alpha(2AD)-AR (dioestrus) differed among uterine locations (base > middle > tip; P < 0.05). The mRNA expression of 5-HTR and AR (subtypes) in bovine uterus was associated with cycle activity and varied according to uterine location. Additional studies on protein level will be carried out in order to elucidate the role of these receptor families on uterine contractility, which may then help to clarify clinical relevance.     

Regional and age dependent changes in gene expression of Toll-like receptors and key antimicrobial defence molecules throughout the gastrointestinal tract of dairy calves

The primary aim of this study was to determine regional and age-dependent expression patterns of Toll-like receptors (TLRs), peptidoglycan recognition protein 1 (PGLYRP1), and β-defensin in rumen, jejunum, ileum, cecum and colon of 3 week (n=8) and 6 month old (n=8) calves. The expression of most TLRs was significantly down-regulated throughout the gastrointestinal tract (GIT) with increasing age. TLR10 expression was significantly higher in ileum than all other gut regions, irrespective of age. TLR2 and TLR4 expression were significantly higher in the cecum and colon of 6 month old calves. Furthermore, expression of β-defensin, and PGLYRP1 was only detectable in 6 month old calves. The expression of TLRs was positively or negatively correlated with population of total bacteria and/or lactic acid bacteria depending on the GIT region. These observations indicate that innate immune responses to commensal microflora may vary significantly throughout the GIT and with age changes.     

Myoelectric activity in the intestines of healthy dairy cows during the recovery period after implantation of permanent electrodes

OBJECTIVE: To describe myoelectric patterns in the intestines of cows after electrode implantation. ANIMALS: 7 lactating Simmental-Red Holstein cows. PROCEDURE: Cows were implanted with 7 pairs of bipolar silver electrodes (1 each in the ileum, cecum, and proximal loop of the ascending colon (PLAC) and 4 in the spiral colon). Myoelectric activity was monitored during 10 periods within the first 3 weeks after surgery. Recordings from the first 2 weeks were compared with recordings from the third week, which was considered a steady-state condition. RESULTS: Significant changes over time were detected for 18 of 57 variables, including 3 variables describing myoelectric activity of the ileum, 6 variables of the cecum, 6 variables of the PLAC, and 3 variables of the spiral colon. Compared with values for the steady-state condition, 16 variables differed significantly for the 14-day period after surgery (7 variables until day 11, 2 variables until day 8, 4 variables until day 5, 1 variable until day 3, and 2 variables until day 2 after electrode implantation). None of the variables had significant changes that lasted only 1 day after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Significant changes were observed for several variables of myoelectric activity in all intestinal segments until as late as 11 days after electrode implantation, whereas a steady-state condition was reached 14 days after surgery. Effects of drugs, manipulations, or nutrition regimens on myoelectric activity of the bovine digestive tract should be evaluated no sooner than 2 weeks after electrode implantation.     

The potential role of myocardial serotonin receptor 2B expression in canine dilated cardiomyopathy

Serotonin signalling in the heart is mediated by receptor subtype 2B (5-HTR2B). A contribution of serotonin to valvular disease has been reported, but myocardial expression of 5-HTR2B and its role in canine dilated cardiomyopathy (DCM) is not known. The aim of the present study was to investigate myocardial 5-HTR2B mRNA expression in dogs with DCM and to correlate results with expression of markers for inflammation and remodelling. Myocardial samples from eight healthy dogs, four dogs with DCM, five with cardiac diseases other than DCM and six with systemic non-cardiac diseases were investigated for 5-HTR2B mRNA expression using quantitative PCR (qPCR). The results were compared to mRNA expression of selected cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP). Laser microdissection with subsequent qPCR and immunohistochemistry were employed to identify the cells expressing 5-HTR2B.The myocardium of control dogs showed constitutive 5-HTR2B mRNA expression. In dogs with DCM, 5-HTR2B mRNA values were significantly greater than in all other groups, with highest levels of expression in the left ventricle and right atrium. Myocytes were identified as the source of 5-HTR2B mRNA and protein. A significant positive correlation of 5-HTR2B mRNA with expression of several cytokines, MMPs and TIMPs was observed. The findings suggest that serotonin might play a role in normal cardiac structure and function and could contribute to myocardial remodelling and functional impairment in dogs with DCM.     

Myoelectric activity in the intestine of cows with strangulating obstruction of the distal small intestine

Myoelectric activity in 2 cows instrumented with permanent electrodes in the ileum, cecum, proximal loop of the ascending colon (PLAC), and spiral colon was analyzed after an obstruction developed in the distal small intestine. Results were compared with patterns from a group of 7 normal cows. Myoelectric activity in the ileum immediately orad to the occlusion was characterized by abolition of the migrating myoelectric complex (MMC) and a constant pattern of strong spike bursts of long duration. Cyclic activity was present in all parts of the large intestine, and propagation of phase III activity was evident from proximal to distal. A slight degree of disorganization in phase III propagation was restricted to the spiral colon. Activity cycles tended to be shorter in the cecum and PLAC of both cows with colic than in normal cows, and the intensity of spiking activity was generally lower. Changes in duration of the MMC in the spiral colon (bovine colonic MMC, bcMMC) were inconsistent, but the intensity of spiking activity tended to be lower in phases I and II of both cows compared to controls. The organization of phase III in several spindles typical of the bovine spiral colon was not disrupted, but phase IV of the bcMMC occurred only infrequently. Organized cyclic activity occurred in the large intestine of both cows despite complete disruption of the small intestinal MMC, indicating the presence of mechanisms able to initiate and regulate coordinated myoelectric patterns in the large intestine independent of the small intestine.     

Myoelectric activity of the spiral colon in dairy cows

OBJECTIVE: To describe myoelectric activity of the spiral colon in healthy cows. ANIMALS: 7 lactating Simmental X Red-Holstein crossbred cows. PROCEDURE: Cows were implanted with 7 pairs of bipolar silver electrodes (4 in the spiral colon and 1 each in the cecum, distal part of the ileum, and proximal loop of the ascending colon [PLAC]). Myo-electric activity was recorded during 4 days for each cow. Patterns were analyzed, using computer-based methods. RESULTS: Myoelectric activity of the spiral colon was closely associated with motility of the ileum and PLAC and showed the typical organization of migrating myoelectric complexes (MMC). The MMC in the bovine spiral colon (bcMMC) had a mean +/- SD duration of 188.6 +/- 30.8 minutes and was divided into 4 phases. Phases I and II lasted 11.3 +/- 1.4 and 159.4 +/- 33.3 minutes, respectively. Phase III (duration, 5.4 +/- 1.2 minutes) was characterized by 5.2 +/- 0.9 regular spindles (35.4 +/- 5.4 seconds) and 1 final elongated spindle (137.2 +/- 56.4 seconds). Phase III most commonly (73.8 +/- 16.1%) was followed by phase IV (duration, 173 +/- 3.6 minutes). Propagation velocity of phase III was 4.4 +/- 0.5 cm/min, and 13.6% of bcMMC were incompletely propagated through the spiral colon. CONCLUSIONS: Myoelectric activity of the bovine spiral colon is composed of a recurring cyclic pattern similar to MMC of the small intestine. Data of colonic myoelectric activity in healthy cows will serve as a basis for studies on cecal dilatation and dislocation in cattle.     

The response of gastrointestinal microbiota to avilamycin, butyrate, and plant extracts in early-weaned pigs

An experiment was designed to evaluate the effects of 3 different additives on the gastrointestinal microbiota of early-weaned pigs. Early-weaned (18 to 22 d; n = 32) pigs (6.0 +/- 0.10 kg of BW) from 8 litters were randomly distributed into 8 pens. Each pen was assigned 1 of 4 dietary treatments: a prestarter or control diet, the control diet with 0.04% avilamycin (AB), with 0.3% sodium butyrate, or with 0.03% plant extract mixture (XT; standardized mixture with 5% (wt/wt) carvacrol extracted from Origanum spp., 3% cinnamaldehyde extracted from Cinnamonum spp., and 2% capsicum oleoresin from Capsicum annum). At the end of the experimental period, 8 pigs per treatment were killed, and samples of their intestinal content were taken. The total bacterial load along the gastrointestinal tract (GIT; stomach, jejunum, cecum, and distal colon) and the lactobacilli and enterobacteria in the jejunum and cecum were measured by quantitative PCR. The total microbial counts along the GIT did not differ among the diets, but there was an increase in the lactobacilli:enterobacteria ratio in the cecum of the piglets on the XT diet (P = 0.003). Restriction fragment length polymorphism of the PCR-amplified V3, V4, and V5 regions of the 16S rDNA gene showed changes in the structure of the microbial community in the jejunum. Dendrograms grouped animals by diets; control with 0.3% sodium butyrate was the treatment that promoted the biggest changes in the microbial ecosystem, followed by AB and then XT. Biodiversity increased when using additives compared with the control diet (P = 0.002). Microbial metabolic activity along the hindgut was studied using the concentration of purine bases and carbohydrase activities. Different patterns for purine bases were observed between diets (diet x intestinal section, P = 0.01). The control diet reached a maximum purine base concentration at the end of the colon, whereas that of the AB diet was reached at the cecum. We could not detect any cellulase or xylanase activities in animals of this age. Appreciable amylase and amylopectinase activities were found, but they did not differ between diets. The results suggest that the effects of avilamycin, butyrate, or the plant extract would not be related to a reduction in the number of total bacteria inhabiting different sections of the GIT but rather to changes in the ecological structure and metabolic activity of the microbial community.     

An immunohistochemical study of gastrointestinal endocrine cells in the BALB/c mouse

The distributions and frequencies of some endocrine cells in the eight portions of the gastrointestinal tract (GIT) of BALB/c mouse were studied. Endocrine cells were stained using immunohistochemical method with seven types of antisera against bovine chromogranin (BCG), serotonin, gastrin, cholecystokinin (CCK)-8, somatostatin, glucagon and human pancreatic polypeptide (HPP), and the regional distributions and their relative frequencies were observed in the eight portions of the GIT of BALB/c mice. All seven types of immunoreactive (IR) cells were identified. Most of the IR cells in the intestinal portion were generally spherical or spindle in shape (open type cell) while round-shaped cells (closed type cell) were found in the intestinal gland and stomach regions occasionally. Their relative frequencies varied according to each portion of the GIT. BCG-IR cells were observed throughout the whole GIT except for the rectum and they were most predominant in the pylorus. Serotonin-IR cells were detected throughout the whole GIT and they showed the highest frequency in the fundus. Gastrin- and CCK-IR cells were restricted to the pylorus and duodenum with a majority in the pylorus and rare or a few frequencies in the duodenum. Compared with other mammals, somatostatin-IR cells were restricted to the fundus and pylorus with a few frequencies, respectively. In addition, glucagon- and HPP-IR cells were restricted to the fundus and duodenum, respectively, with relative low frequencies. Some species-dependent unique distributions and frequencies of endocrine cells were observed in the GIT of BALB/c mouse compared with other rodents.     

An immunohistochemical study of the gastrointestinal endocrine cells in the C57BL/6 mice

The regional distributions and relative frequencies of some gastrointestinal endocrine cells in the eight portions (fundus, pylorus, duodenum, jejunum, ileum, cecum, colon and rectum) of the gastrointestinal tract of C57BL/6 mouse was studied with immunohistochemical method using seven types of specific anti-sera against chromogranin A (CGA), serotonin, somatostatin, human pancreatic polypeptide (HPP), glucagon, gastrin and cholecystokinin (CCK)-8. In this study, all these seven types of immunoreactive (IR) cells were identified. Most of these IR cells in the intestinal portion were generally spherical or spindle in shape (open-type cell) while cells showing round in shape (closed-type cell) were found in the intestinal gland and stomach regions occasionally. Their relative frequencies were varied according to each portion of gastrointestinal tract. CGA-IR cells were demonstrated throughout the whole gastrointestinal tract and they showed most predominant in the pylorus and duodenum. Serotonin-IR cells were detected throughout whole gastrointestinal tract and they showed highest frequency in the stomach and colon. Somatostatin-IR cells were demonstrated throughout whole gastrointestinal tract except for large intestine and showed highest frequency in the fundus. HPP-IR cells were found in the fundus with rare frequency. Peculiarly, glucagon-IR cells were restricted to the fundus, ileum and colon with a few frequencies. Gastrin-IR cells were restricted to the pylorus with numerous frequency and CCK-8-IR cells were observed in the pylorus, duodenum and jejunum with numerous and/or a few frequencies, respectively. In conclusion, some peculiar distributional patterns of gastrointestinal endocrine cells were found in C57BL/6 mouse.     

不同产蛋水平肉种鸡繁殖性能、肠道组织形态、卵巢功能和盲肠微生物区系差异研究

本研究旨在比较不同产蛋水平肉种鸡繁殖性能、肠道组织形态、卵巢功能和盲肠微生物区系的差异。试验选用5000只同栋舍37周龄的爱拔益加父母代肉种鸡,记录2周内每只肉种鸡的产蛋率、受精率和孵化率,分别筛选出高和平均2种不同产蛋水平的肉种鸡各90只,设为2个组,分别为平均产蛋率组[AR组,产蛋率(79.34±0.49)%]和高产蛋率组[HR组,产蛋率(90.03±0.34)%],每组10个重复,每个重复9只鸡。预试期2周,正试期6周。结果表明:与AR组相比,HR组肉种鸡产蛋率和入孵蛋孵化率显著升高(P<0.05),血清谷丙转氨酶活性显著降低(P<0.05),空肠绒毛高度显著降低(P<0.05),回肠隐窝深度显著升高(P<0.05),卵巢细胞凋亡率极显著降低(P<0.01),卵巢促凋亡蛋白半胱氨酸天冬氨酸蛋白酶9(caspase 9)、卵泡发育相关蛋白骨形态发生蛋白受体1B(BMPR1B)、家鸡新型类催乳素(PRL?L)和转录因子GATA4表达水平显著升高(P<0.05),盲肠厚壁菌门(Firmicutes)和乳杆菌属(Lactobacillus)相对丰度显著升高(P<0.05),盲肠拟杆菌门(Bacteroidetes)和螺旋体门(Spirochaetes)相对丰度显著降低(P<0.05)。肉种鸡盲肠拟杆菌门相对丰度与卵巢BMPR1B和GATA4表达水平呈显著负相关(P<0.05),盲肠厚壁菌门相对丰度与卵巢BMPR1B、GATA4表达水平呈显著正相关(P<0.05),盲肠乳杆菌属相对丰度与卵巢caspase 9、BMPR1B、GATA4、PRL?L表达水平呈显著正相关(P<0.05),盲肠螺杆菌属(Helicobacter)相对丰度与卵巢GATA4表达水平呈极显著正相关(P<0.01)。综上所述,本试验结果表明,不同产蛋水平肉种鸡肠道组织形态、盲肠微生物区系和卵巢功能存在显著差异,盲肠厚壁菌门和乳杆菌属相对丰度与高繁殖性能密切相关。     

Concentration of volatile fatty acids in digesta samples obtained from healthy cows and cows with cecal dilatation or dislocation

OBJECTIVE: To compare concentrations of acetic, propionic, butyric, and i- and n-valerianic acids in digesta samples obtained from the rumen, cecum, proximal loop of the ascending colon (PLAC), and rectum of healthy cows and cows with cecal dilatation or dislocation (CDD). ANIMALS: 20 cows with CDD and 20 healthy cows. PROCEDURE: Samples were collected from all sites during surgical correction of CDD and also from the rectum 1, 2, and 3 days after surgery (group CDD). Samples from healthy (control) cows, matched on the basis of diet and milk yield, were obtained at a slaughterhouse. Concentrations of volatile fatty acids (VFA) were analyzed by use of gas chromatography. Absolute concentration of each VFA was additionally corrected for pH to allow calculation of the concentration of undissociated VFA. RESULTS: Absolute concentration and concentration of the undissociated form of all analyzed VFA were significantly increased in samples collected from the cecum and PLAC of cows in group CDD, compared with concentrations for control cows. Within 3 days after surgery, significant decreases of the absolute concentration of butyric, i- and n-valerianic acids, and undissociated i- and n-valerianic acids were evident in samples obtained from the rectum of group-CDD cows. Concentrations of VFA in samples obtained from the rectum during surgery correlated with corresponding VFA concentrations in samples obtained from the PLAC. CONCLUSIONS: Concentrations of VFA are increased in the cecum and PLAC of cows with CDD. However, the role of increased concentrations of VFA in the etiopathogenesis of CDD is unknown.     

Effects of dietary calcium and available phosphorus concentration on digesta pH and on the availability of calcium, iron, magnesium and zinc from the intestinal contents of meat chickens

1. The effects of high calcium intakes on the pH and availability of calcium, iron, magnesium and zinc in the gastrointestinal tract (GIT) contents of meat chickens were studied in two experiments. 2. A high dietary concentration of calcium (25.3 vs 10.7 g/kg) increased the pH of crop and ileum contents, but did not influence the pH of the contents of other segments of the GIT. 3. The solubilities of minerals in GIT contents were reduced and the size of dissolved mineral complexes were increased as the digesta moved from the duodenum and jejunum to the ileum. 4. After feeding diets with calcium and available phosphorus concentrations (15.3 and 4.3 g vs 21.8 and 4.3 g vs 22.6 and 8.3 g/kg), centrifugation of GIT contents showed that most (70-92%) of the calcium, iron, magnesium and zinc was in an insoluble form. High calcium diets reduced the proportion of soluble zinc associated with small complexes, and high calcium and available phosphorus diets reduced the proportions of soluble magnesium and zinc associated with small complexes. 5. These findings explain the mechanism of the reduced availability of zinc and magnesium in high calcium and high available phosphorus diets.     

Characterization of microbial populations and volatile fatty acid concentrations in the jejunum,ileum, and cecum of pigs weaned at 17 vs 24 days of age

In a series of five 17-d replicate trials, a total of 54 cannulated and 12 noncannulated pigs were used to determine the effects of weaning age (17 d or 24 d) on pH, dry matter percentage, aerobic and anaerobic microflora, lactate, and volatile fatty acid (VFA) concentrations in the jejunum, ileum, and cecum of weanling pigs. At -14 d of age, cannulated pigs were surgically fitted with T-cannulas in the jejunum (n = 20), ileum (n = 18), or cecum (n = 16). Upon weaning, cannulated pigs were individually caged in an environmentally controlled room with ad libitum access to a phase starter diet and water. Noncannulated pigs were killed at weaning and samples were collected from the jejunum, ileum, and cecum. Digesta and fecal swabs from cannulated pigs were collected twice weekly. The pH of cecal contents was lower (P < 0.05) and dry matter percentage was greater (P < 0.05) than those ofjejunal or ileal contents. Pigs weaned at 24 d of age had increased (P < 0.05) E. coli populations 3 d postweaning compared to preweaning populations, regardless of site of collection, whereas this increase was not observed in pigs weaned at 17 d of age. Unweaned pigs maintained higher (P < 0.05) lactobacilli populations compared to weaned pigs; however, populations declined (P < 0.05) in both groups by 3 d postweaning, with pigs weaned at 24 d of age having lactobacilli populations greater than pigs weaned at 17 d of age. Fecal populations of E. coli and lactobacilli declined (P < 0.05), whereas fecal bifidobacteria populations increased (P < 0.05) postweaning, regardless of weaning age. Concentrations of total fecal anaerobes declined (P < 0.05) in pigs weaned at 17 d of age but were maintained in pigs weaned at 24 d of age. Volatile fatty acid concentrations were greater (P < 0.05) in the cecum than in the jejunum or ileum, and acetic acid concentrations decreased (P < 0.05) postweaning regardless of weaning age. A tendency for L+ lactate concentrations to be greater (P < 0.07) in the ileum and jejunum vs the cecum was observed. Results indicate that weaning and weaning age have significant effects on microbial populations and VFA concentrations.