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.     

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.     

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.     

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.     

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.     

Effect of lameness on the calving-to-conception interval in dairy cows

OBJECTIVE: To examine the relationship between lameness and the duration of the interval from calving to subsequent conception in lactating dairy cows. DESIGN: Cohort study. ANIMALS: 837 dairy cows. PROCEDURE: Cows affected with lameness were classified into 1 of 4 groups on the basis of types of disease or lesions observed, including foot rot, papillomatous digital dermatitis, claw lesions, or multiple lesions. Cows not affected with lameness were classified as healthy. Time from calving to conception was compared between lame cows and healthy cows. RESULTS: 254 (30%) cows were affected with lameness during lactation. Most lame cows (59%) had claw lesions. Lame cows with claw lesions were 0.52 times as likely to conceive as healthy cows. Median time to conception was 40 days longer in lame cows with claw lesions, compared with healthy cows. Number of breedings per conception for lame cows with claw lesions was significantly higher than that for healthy cows. CONCLUSION AND CLINICAL RELEVANCE: Claw lesions were the most important cause of lameness, impairing reproductive performance in dairy cows, as indicated by a higher incidence of affected cows and a greater time from calving to conception and a higher number of breedings required per conception, compared with healthy cows.     

Sequencing of canine 5-hydroxytriptamine receptor (5-HTR) 1B, 2A, 2C genes and identification of polymorphisms in the 5-HTR1B gene

Polymorphisms of human genes encoding 5-hydroxytriptamine (serotonin) receptors (5-HTRs) are thought to be associated with psychiatric disorders and behavioral traits. In the present study, we searched for corresponding polymorphisms in the dog and compared allelic frequencies for the canine 5-HTR1B, 5-HTR2A, and 5-HTR2C genes among five canine breeds. The canine genes consisted of the following: 5-HTR1B, 1170 bp; 5-HTR2A, 1413 bp; and 5-HTR2C, 1377 bp. All of these genes were highly homologous with the human genes. We found six single nucleotide polymorphisms (SNPs) in the 5-HTR1B gene (G57A, A157C, G246A, C660G, T955C, and G1146C). Genotyping of the respective SNPs revealed that there were inter-breed variations in the genotypes and allelic frequencies for four out of the six identified SNPs, suggesting that further analyses of the polymorphisms of the 5-HTR1B gene would be useful in order to gain an understanding of the genetic background underlying the diversified behavioral traits among canine species.     

Long-term fluctuations and effect of age on serum concentrations of certain fat-soluble vitamins in dairy cows

BACKGROUND: There is insufficient evidence in the literature concerning the fluctuations and correlations of serum fat-soluble vitamin concentrations during lactation in dairy cows and the potential effect of age of the animals on these values. OBJECTIVES: The objectives of this study were to determine fluctuations in mean serum concentrations of the fat-soluble vitamins A and E and b-carotene and the correlations among them in clinically healthy dairy cows, from the last month of the dry period until the end of lactation, and to investigate whether age was a factor that affects these serum concentrations. METHODS: Forty-five clinically healthy Holstein cows were assigned to 2 groups based on age. Group A consisted of 23 cows 4 years old (mean +/- SD, 3.24 +/- 0.44 years), and group B consisted of 22 cows >4 years old (6.68 +/- 1.66 years). Blood samples for determination of serum beta-carotene, vitamin A, and vitamin E concentrations were collected from each animal just before the start of study (1 month prior to expected parturition), at parturition, and thereafter, at monthly intervals until the end of lactation. RESULTS: There was a significant positive correlation between beta-carotene, vitamin A, and vitamin E values. Mean serum concentrations of beta-carotene, vitamin A, and vitamin E were significantly higher in younger animals. CONCLUSIONS: To our knowledge, this is the first evidence that age is a factor affecting serum concentrations of fat-soluble vitamins in dairy cows.     

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.     

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.     

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.     

Effect of lameness on milk yield in dairy cows

OBJECTIVE: To examine the relationship between lameness and milk yield in dairy cows. DESIGN: Cohort study. ANIMALS: 531 dairy cows. PROCEDURE: Cows affected with lameness were classified into 1 of 3 groups on the basis of type of diseases or lesions observed, including interdigital phlegmon (foot rot), papillomatous digital dermatitis (foot warts), or claw lesions. Cows not affected with lameness were classified as healthy. From Dairy Herd Improvement Association records, 305-day mature equivalent milk yield data were collected at the end of lactation or when the cow left the herd. Milk yield was compared between cows affected with lameness and healthy cows. RESULTS: 167 (31%) cows were affected with lameness during lactation. Lame cows had claw lesions (60%), papillomatous digital dermatitis (31%), or interdigital phlegmon (9%). Milk yield in lame cows with interdigital phlegmon (mean, 17,122 lb) was significantly less, compared with healthy cows (19,007 lb). CONCLUSIONS AND CLINICAL RELEVANCE: In this herd, interdigital phlegmon was associated with a 10% decrease in milk production. Lame cows with claw lesions or papillomatous digital dermatitis produced less milk than healthy cows, but the difference was not significant.     

Changes in the serum levels of primary bile acids in dairy cows

Serum cholic acid (SCA) and serum chenodeoxycholic acid (SCDCA) concentrations were determined in healthy dairy cows by radioimmunoassay (RIA). The levels of these two primary bile acids were correlated with the cows' reproductive status. The lowest concentrations were measured in dry cows (SCA: 7.8 +/- 3.3 mumol/l, SCDCA.: 1.5 +/- 1.0 mumol/l). In freshly calved cows SCA and SCDCA was 17.8 +/- 6.9 mumol/l and 2.3 +/- 1.0 mumol/l, respectively, while in milking cows SCA and SCDCA was 15.8 +/- 5.7 and 2.3 +/- 0.8 mumol/l, respectively. SCA concentration showed a characteristic change on the days immediately after calving: on calving day it was close to the mean SCA concentration found for dry cows, then it underwent a striking abrupt rise and reached the value typical of post-parturient cows by post-partum (PP) day 4-5. Cholic acid was found to be the major primary bile acid in the blood of dairy cows. In dry cows the SCA:SCDCA ratio is 5:1. If the serum bile acid concentration rises, the SCA:SCDCA ratio will increase further.     

Thiobarbituric acid reactive substances and plasma antioxidative capacity in dairy cows at different lactation stages

The aim of the present experiment was to investigate the level of the lipid peroxidation and plasma antioxidative capacity in plasma of dairy cows at different lactation stages. Twenty six healthy dairy cows were used in our experiment. The groups: I - dry cows (n = 7), II - dairy cows < 1 month after calving (n = 9), III - dairy cows 4 - 5 months after calving (n = 10). The highest level of lipid peroxidation (TBARS) was recognized in dairy cows in early lactation (II - 0.58+/-0.42 micromol/l) with significant differences between the groups (p < 0.05). In contrast, there was a tendency (p > 0.05) to the lowest plasma antioxidative activity in cows in the same group (158 +/- 44 micromol/l). The strongest trend to metabolic imbalance was found in the dairy cows in early lactation (group II). In conclusion, the metabolic stress of dairy cows associated with early lactation resulted in an increased level of plasma lipid peroxidation.     

Changes in peripheral leukocyte subsets in dairy cows with inflammatory diseases after calving

To clarify the cellular immune system in dairy cows with inflammatory diseases after calving, the leukocyte subsets were examined in Holstein dairy cows. Twenty Holstein cows reared in one herd, were used in this study. Nine cows (Group 1) experienced onset of mastitis or puerperal fever within 2 weeks after calving, and the other eleven cows remained healthy (Group 2) after calving. The numbers of CD3(+), CD4(+) and CD8(+) cells tended to be lower in Group 1 than in Group 2 from the day of calving through week 1. These results suggested that the cows with inflammatory diseases might have experienced a decline in T cells by the day of calving, before the onset of disease.