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.     

5-Hydroxytryptamine-4 receptor messenger ribonucleic acid levels and densities in gastrointestinal muscle layers from healthy dairy cows

Serotonin (5-hydroxytryptamine, 5-HT) is involved in gastrointestinal tract (GIT) motor functions through binding to specific receptors located in the GIT walls. The objectives of the current study were to compare mRNA levels and binding sites of 5-HT(4) receptors (5-HTR(4)) in smooth muscle layers from the fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows, and to verify whether mRNA and protein expression were correlated. Smooth muscle samples were prepared by scraping the mucosa and submucosa from full-thickness intestinal wall samples. The mRNA levels of 5-HTR(4) were measured by real-time PCR and expressed relative to those of the housekeeping gene glyceraldehyde phosphate dehydrogenase. Binding studies were performed using the 5-HTR(4) antagonist [(3)H]GR113808. The mRNA levels of 5-HTR(4) were affected (P < 0.05) by location along the GIT. The mRNA levels of 5-HTR(4) in the ELSC and the ileum were greater than in the PLAC (P = 0.05 and P = 0.07, respectively) but similar to those of all other locations. The competitive binding of [(3)H]GR113808 to suspended membranes from the fundus abomasi, pylorus, cecum, and ELSC was best fit by a 2-site receptor model, whereas it was best fit by a 1-site receptor model in the ileum and PLAC. The mRNA levels and numbers of 5-HTR(4) were not correlated (r = 0.14; P = 0.71). In conclusion, mRNA and binding sites for 5-HTR(4) are present in the smooth muscle layer of the entire GIT of dairy cows and may play a role with respect to motility. The effects of activation of this receptor subtype may be different among GIT locations due to differences in the amount of high- relative to low-affinity binding sites.     

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.     

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.     

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.     

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.     

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.     

Characterization of alpha-adrenoceptor subtypes in smooth muscle of equine ileum

OBJECTIVE: To determine the concentration and binding characteristics of alpha-adrenoceptor subtypes in smooth muscle cell membranes of equine ileum. SAMPLE POPULATION: Segments of longitudinal and circular smooth muscle from the ileum of 8 male and 8 female adult horses. PROCEDURE: Distribution of alpha-adrenoceptor subtypes was assessed by use of radioligand binding assays incorporating [3H]-prazosin and [3H]-rauwolscine, highly selective alpha1- and alpha2-adrenoceptor antagonists, respectively. Characterization of adrenoceptor subtypes was performed by use of binding inhibition assays. RESULTS: On the basis of binding affinity for specific radioligands, low- and high-affinity alpha1- and alpha2-adrenoceptors were detected. Concentration of low-affinity alpha2-adrenoceptors was significantly greater in male horses, compared with females. Competition studies confirmed the specificity of the radioligands used in the binding assays. Alpha1-adrenoceptors of both subtypes in male and female horses had a higher affinity for prazosin than phentolamine, whereas yohimbine did not compete with the radioligand for binding. For alpha2-adrenoceptors regardless of subtype, potency of inhibition elicited by each drug varied between sexes. In males, yohimbine was a more potent inhibitor than phentolamine, which was more potent than prazosin. In females, yohimbine was more potent than prazosin, which was more potent than phentolamine. CONCLUSIONS AND CLINICAL RELEVANCE: High- and low-affinity alpha1- and alpha2-adrenoceptors were detected in smooth muscle of equine ileum. Because alpha-adrenoceptor subtypes, particularly alpha2-adrenoceptors, are involved in the regulation of gastrointestinal tract function, characterization of these receptors may represent the basis for development of new therapeutic strategies for the control of gastrointestinal disturbances in horses.     

Effects of erythromycin on myoelectric activity of the spiral colon of dairy cows

The effects of erythromycin on myoelectric activity of the spiral colon of dairy cows were investigated in a prospective study. Four Simmental x Red-Holstein crossbred cows of similar body weight and condition with seven pairs of bipolar electrodes implanted in the intestine (one each in the distal ileum, caecum and proximal colon, and four in the spiral colon) were included. Erythromycin lactobionate (1 mg kg(-1)) and 0.9% sodium chloride solution (NaCl) were administered to each cow in a random order. Erythromycin was diluted with NaCl and both treatments were administered slowly intravenously over a period of 5 min 1 h after onset of a phase III of the bovine colonic migrating myoelectric complex (bcMMC). A 3-6-day washout period was scheduled between trials. Significant differences between the results of the treatments were observed for spike duration in phase I as well as for spike duration and duration of spiking activity during phase II of the second bcMMC, which were significantly higher after erythromycin treatment than after NaCl. These findings suggest an indirect effect of erythromycin on colonic motility in cattle.     

Hormonal interrelationships in postpartum suckled dairy cows.

Three cross-bred cows calved in March and April and were followed until day 62 after parturition. Each animal was suckled by 2 calves ad libitum. All calves were removed from the cows on day 55 after parturition. Blood was collected 3 times per day from the jugular vein by venipuncture. On 4 occasions after parturition--i.e. days 7-8, 21-22, 35-36 and 49-50, the cows were bled through a jugular venous catheter every 30 min during the 24 h. The plasma samples were analyzed for the content of 15-keto-13,14-dihydro-PGF2 alpha (main PGF2 alpha metabolite), LH, prolactin, cortisol and progesterone by radioimmunoassay methods. The concentration of PGF2 alpha increased from 280 to 730 pmol/l within the last 4 days before parturition. The highest geometric mean was 3106 pmol/l on the day of parturition. Thereafter a steady decrease of PGF2 alpha metabolite concentration was seen until day 21 when it reached plateau at 148 pmol/l. In all cows plasma LH concentrations increased significantly (P < 0.05) from about 1.6 micrograms/l on days 7-8 to 2.4 micrograms/l on days 21-22 post partum. The frequency of LH pulses showed no tendency to increase as the postpartum period progressed and averaged 6.5 pulses/24 h. Mean plasma LH concentrations increased from 2.1 micrograms/l 2 days before weaning to 3.2 micrograms/l 2 days after weaning (P < 0.05). LH peaks occurred less frequently in association with prolactin and cortisol peaks than in their absence. A partial positive correlation between PGF2 alpha metabolite and cortisol (r = 0.30) was found on days 7-8 post partum. Correlation between prolactin and cortisol on days 7-8 and 21-22 post partum was also positive (r = 0.20 and r = 0.27, respectively). There was a negative correlation between LH and cortisol on days 7-8 (r = -0.27) and days 49-50 (r = -0.21) post partum. The first and short progesterone increase observed after weaning was terminated in conjunction with PGF2 alpha metabolite peaks.     

Reproductive efficiency in grazing lactating dairy cows under a programmed reproductive management system

OBJECTIVE: To evaluate the effectiveness of a reproductive management program consisting of combinations of Ovsynch/TAI and prostaglandin (PG) F(2alpha) treatments in Holstein dairy cows under a pasture-based dairying system. DESIGN: Field trial. PROCEDURE: A total of 1177 cows in 8 commercial dairy farms were randomly allocated to control and treatment groups. Treatment group cows received one of two interventions depending upon the number of days postpartum (DPP) before the planned start of breeding. Cows more than 50 DPP by the planned start of breeding received the Ovsynch/TAI treatment, consisting of gonadotrophin releasing hormone (GnRH) - PGF(2alpha)- GnRH plus timed artificial insemination. Cows between 40 and 50 DPP received a PGF(2alpha) treatment followed by oestrus detection and, if the cow was not seen in oestrus, the cow received a second PGF(2alpha) 14 days later. Control cows were submitted to twice a day heat detection followed by artificial insemination. The experimental period was the start of the breeding season plus 21 days for cows over 50 DPP at the start of breeding, and was 40-61 DPP for cows that calved later and passed their voluntary waiting period after the start of the breeding season. RESULTS: Submission rate was higher for the treated group than for the control group (84.9% vs. 55.1%; P < 0.0001), as was the conception rate (51.0% vs. 46.1%; P < 0.03). Due to farm variations, pregnancy rate was similar in both groups (38.5% vs. 28.2%; P > 0.1). Within the treated group, conception rate and pregnancy rate of the cows inseminated after a PGF(2alpha) were higher than for timed artificial inseminated cows (51.4% vs. 32.6%; P < 0.001), and (37.8% vs. 32.6%; P < 0.001). CONCLUSION: A programmed reproductive management protocol may improve reproductive efficiency in dairy farms with seasonal breeding, by increasing submission and conception rates at the beginning of the breeding season and/or at the end of the voluntary waiting period. Fertility of cows bred after a PGF(2alpha) synchronised heat was greater than after an Ovsynch/TAI protocol.     

Distribution of muscarinic receptor subtypes and interstitial cells of Cajal in the gastrointestinal tract of healthy dairy cows

OBJECTIVE: To describe the distribution of muscarinic receptor subtypes M(1) to M(5) and interstitial cells of Cajal (ICCs) in the gastrointestinal tract of healthy dairy cows. SAMPLE POPULATION: Full-thickness samples were collected from the fundus, corpus, and pyloric part of the abomasum and from the duodenum, ileum, cecum, proximal loop of the ascending colon, and both external loops of the spiral colon of 5 healthy dairy cows after slaughter. PROCEDURES: Samples were fixed in paraformaldehyde and embedded in paraffin. Muscarinic receptor subtypes and ICCs were identified by immunohistochemical analysis. RESULTS: Staining for M(1) receptors was found in the submucosal plexus and myenteric plexus. Antibodies against M(2) receptors stained nuclei of smooth muscle cells only. Evidence of M(3) receptors was found in the lamina propria, in intramuscular neuronal terminals, on intermuscular nerve fibers, and on myocytes of microvessels. There was no staining for M(4) receptors. Staining for M(5) receptors was evident in the myocytes of microvessels and in smooth muscle cells. The ICCs were detected in the myenteric plexus and within smooth muscle layers. Distribution among locations of the bovine gastrointestinal tract did not differ for muscarinic receptor subtypes or ICCs. CONCLUSIONS AND CLINICAL RELEVANCE: The broad distribution of M(1), M(3), M(5), and ICCs in the bovine gastrointestinal tract indicated that these components are likely to play an important role in the regulation of gastrointestinal tract motility in healthy dairy cows. Muscarinic receptors and ICCs may be implicated in the pathogenesis of motility disorders, such as abomasal displacement and cecal dilatation-dislocation.     

Identification of plasma and hepatic parameters related to metabolic robustness in dairy cows

Blood plasma and hepatic parameters were identified that describe the differences between metabolically robust or vulnerable dairy cows grouped according to their past health status. Data from a field study on dairy cows were used from which metabolically challenged dairy cows were selected that had a milk fat percentage of >4.5 mg/g and a fat to protein ratio of >1.5 in their previous early lactation. The selected cows were either classified as metabolically robust or vulnerable based on the occurrence of various metabolic and (re)production disorders in their previous lactations. Blood and liver tissue samples were collected in week 3 ante partum (a.p.) (-3 wk), in week 4 (+4 wk) and in week 13 (+13 wk) post-partum (p.p.). Plasma concentrations of metabolites and hormones and mRNA expression of genes involved in metabolic pathways in the liver were used as variables for a two-group discriminant analysis (DA). Average discriminant scores (centroids) were different (p < 0.05) in -3 wk, +4 wk and in +13 wk. In -3 wk, significant variables that best explained the differences between metabolically robust and vulnerable cows were parity, plasma triglycerides, glucose and mRNA abundance of carnitine palmitoyltransferase 2 (CPT2). In addition, based on the classification matrix, 69% of the dairy cows were correctly classified. In +4 wk, identified significant parameters were parity, plasma glucose and urea, and 67% of the cows were correctly classified. In +13 wk, significant variables that explained the differences between the groups were parity, mRNA abundance of acyl-CoA synthetase long-chain 1 and CPT1, and 66% of the cows were correctly classified. In conclusion, the identified variables may distinguish from metabolically challenged cows, those cows that had a poorer health performance in their previous lactations.