Abomasal displacement in cattle is characterized by a gas-filled organ rising dorsally alongside the left abdominal wall. In North American Holstein cows, the mean incidence of this disease is 3% to 5% during lactation,1,2 although much higher incidences are observed in some herds.3,4 Obviously, there are considerable inter-individual differences concerning the predisposition for abomasal displacement within the Holstein breed itself. This indicates that genetic background is an important risk factor as has been determined in Holstein Friesians,2,5–10,a Simmental–Red-Holstein crossbreds,11 Brown Swiss, Ayrshires, Guernseys,6 and Jerseys.12 In some breeds, conversely, abomasal displacement occurs uncommonly (ie, German Fleckvieh,13 Swiss Browns,14 and Swedish Reds).15 Because of the aforementioned facts, a comparison of 2 breeds (ie, German Holsteins and German Fleckvieh) was performed in the present study.
It is assumed that dilatation of the abomasum is preceded by motility disorders, such as hypotonia or atonia.16 These disorders inhibit the active transport of gas out of the organ, which enters the abomasum with the chyme or is produced there. Normally, the gas escapes either back into the omasum via the omasal valve or into the small intestine via the pyloric channel. A previous studya revealed no significant differences concerning the innervation density of the abomasum between healthy cows and cows with abomasal displacement. Additionally, no differences have been detected concerning the spontaneous activity of smooth muscle specimens in vitro.17 Immunohistochemical studies18–25 on the gastrointestinal tract of ruminants are limited and basically deal with the distribution pattern of nervous structures, endocrine cells, and neuropeptides within the gastrointestinal wall.18–25 Until now, a comparison of breeds by means of immunohistochemistry has not been performed. Nevertheless, the physiologic importance of the neuropeptides VIP and substance P in different species is well accepted.11,26,27 Fahrenkrug27 gives a clear description of the role of VIP in the peripheral nervous system and suggests that an impairment of VIP nerves is involved in some autonomic dysfunctions. Vasoactive intestinal polypeptide is a 28 amino acid protein and is involved in the control of smooth muscle tone and motility because it is the nonadrenergic, noncholinergic transmitter for relaxation in the gastrointestinal tract.27 The undecapeptide substance P is a tachykinin and leads to contraction of gastrointestinal smooth muscle.11
Other than the mentioned neuropeptides substance P and VIP, NOS and ChAT are involved in the regulation of activity in the enteric motor neurons. Geishauser et al28 determined that acetylcholine leads to a dose-dependent enhancement of muscle tone in experimentally stimulated muscle strips from healthy cows, whereas higher doses of acetylcholine are needed for this effect in muscle strips from cows with displaced abomasum. Therefore, it would have also made sense to study the expression of ChAT. Nevertheless, NOS and ChAT have also been studied28 by means of immunohistochemistry. Smooth muscle preparations from the antrum pylori and the pylorus of the abomasum of cows were examined. Antibodies against NOS, ChAT, and PGP on tissue from healthy cows and cows with displaced abomasum were used. In doing so, no immunohistochemical differences could be found between the healthy cows and cows with abomasal displacement. Because of this, substance P and VIP were examined in the present study.
The aim of the study reported here was to examine possible factors that could explain the differences between breeds concerning the occurrence of abomasal displacement by means of immunohistochemical and histomorphometric analyses. For this purpose, the peptidergic innervation, overall nerve density, and layer thickness of the abomasal wall were taken into account. The study focused on the nerve fibers and neuropeptides of the myenteric plexus. As a secondary objective, 2 anatomic locations (antrum and corpus) in both breeds were compared. The hypothesis of the present study was that the predisposition for abomasal displacement in German Holstein cows might be associated with decreased content of motility-stimulating substance P or increased content of inhibiting VIP, compared with German Fleckvieh cows.
Materials and Methods
Sample population—Tissue specimens were collected from 20 German Holstein cows and 20 German Fleckvieh cows, 3 to 5 years of age, at a local slaughter-house within 20 minutes after stunning and euthanasia. The 2 biopsy specimens were taken from the region of the corpus abomasi and the antrum pylori, respectively. They were approximately 10 mm in diameter and included all layers of the abomasal wall. To ensure reproducible sampling sites, biopsy specimens were taken at the same area on an imaginary line between the major and minor curvatures of the abomasum on the parietal side of the organ (Figure 1).
Schematic illustration of the bovine abomasum and the locations of biopsy specimens taken from the corpus abomasi and antrum pylori between the minor and major curvature.
Citation: American Journal of Veterinary Research 69, 10; 10.2460/ajvr.69.10.1247
All biopsy specimens were immediately fixed in 4% paraformaldehyde for 24 hours, dehydrated in alcohol, and embedded in paraffin. Sections of 6 μm thickness were cut and mounted on 3 APES-coated slides. All sections were stained by use of the avidin-biotin complex/AEC technique as described.29,30
Immunohistochemical analysis—For immunohistochemical staining of substance P, the primary antibody AF 1021b was used in a dilution of 1:25. Specificity of the antibody has been reported.31 According to the symmetric expression pattern of substance P in the spinal cord first described by Cuello et al,31 sections of rat spinal cord served as positive controls. The antisera used in this study were tabulated (Table 1), and representative sections were prepared (Figure 2).
Antisera used for immunohistochemical analysis of evaluation of differences between breeds for substance R vaso-active intestinal polypeptide, and neurofilament 200 in the ab-omasal wall of cattle.
| Antibody | Host species | Dilution |
|---|---|---|
| Primary | ||
| Anti-SPAF 1021 [NC 1/34 HL] | Rat | 1:25 |
| Anti-VIPT4245 | Rabbit | 1:1,000 |
| Anti-NF200NFM002 | Mouse | 1:200 |
| Secondary | ||
| Anti-rat E 0467 | Rabbit | 1:300 |
| Anti-rabbit BA-1000 | Goat | 1:250 |
| Anti-mouse BA-2000 | Horse | 1:250 |
SP = Substance P. NF= Neurofilament.
Representative sections of abomasal samples collected from German Fleckvieh (GFV; A, C, and E) and those from German Holsteins (GH; B, D, and F) after staining for substance P (A and B), VIP (C and D), and neurofilament 200 (E and F). Positive signals are marked with a black arrowhead. A, B—Staining pattern for substance P (SP) in nerve fibers in the tunica muscularis of the antrum pylori. C, D—Staining pattern for VIP in nerve fibers in the tunica muscularis of the antrum pylori. E, F—Staining pattern for neurofilament (NF) 200 in nerve fibers in the tunica muscularis of the corpus abomasi. G—Negative control. H—Antigen preincubation of the polyclonal antibody for VIP resulted in a negative staining pattern. Bar = 50 μm.
Citation: American Journal of Veterinary Research 69, 10; 10.2460/ajvr.69.10.1247
For the immunohistochemical staining of VIP, the primary antibody T 4245c was used in a dilution of 1:1,000. Because this is a polyclonal antibody, preabsorption studies were performed to confirm specificity of the antibody. After preincubation with its specific antigen for 1 hour at 21°C (peptide concentration of 1 μg/ML vs IgG concentration of 2 μg/ML), no positive reaction was observed. According to the data sheet of the specific peptide H 3775,d 0.001 μg of antigen was neutralized by 2 mg of antibody IgG. Sections of rat duodenum served as positive controls.
As a common nerve fiber marker, neurofilament 200 was used to determine the overall nerve density. Neurofilaments are the intermediate filaments found specifically in neurons and nerve fibers of the central and peripheral nerve systems.26 For immunohistochemical staining of neurofilament 200, the primary antibody NFM 002e was used in a dilution of 1:200. Sections of rat brain served as positive controls.
Three 5-minute washing steps in PBS solution were performed after each incubation procedure. Antigen retrieval was performed by incubating the slides in boiling (100°C) citrate buffer for 20 minutes or, in the case of staining for neurofilament 200, incubating with proteinkinase K for 10 minutes. Endogenous peroxidase was inactivated by incubating the slides with 0.3% H2O2 in methanol for 30 minutes. Nonspecific tissue reactivity was blocked with 5% rabbit, goat, or horse normal serum dependent on the host species of the secondary antibody. When staining for VIP and substance P, primary antibodies were incubated overnight at 2° to 8°C, whereas staining for neurofilament 200 was performed for 3 hours at 21°C. Sections incubated in buffer without primary antibodies served as negative controls.
Primary antibodies were detected with biotin-conjugated secondary antibodies (Table 1) and HRP-conjugated streptavidin.f The colorimetric reaction was induced with the chromogen AECg and blocked by placing the slides in distilled water. The AEC signal development time varied between and within the different stainings (mean development times were as follows: substance P, 41.2 ± 4.6 minutes; VIP, 14.1 ± 4.3 minutes; and neurofilament 200, 13.4 ± 1.4 minutes). All slides were examined under a microscope,h and digital pictures were taken by use of a software program.i
Representative immunohistochemically stained sections for all 3 antigens examined were assessed by an experienced histologist,j who confirmed specific binding to the neurons and nerve fibers of the enteric nerve plexus of the abomasum.
Morphometric studies—For each animal, 1 slide/location was examined. For this purpose, 1 section of 3 consecutive sections was selected after the staining process. Selection was conducted on the basis of macroscopic characteristics; only completely preserved sections without lost portions were selected. On these selected slides, images of 5 randomly chosen visual fields were taken and analyzed.i The immunoreactive areas within the nerve fibers and ganglia of the myenteric plexus were outlined with a pen on a graphic tabletk and measured by use of the software program.i All areas on all 5 visual fields/location were summed for further statistical analysis. The visual fields were selected in a standardized manner by use of blinded adjustment of the microscope starting from a macroscopically and randomly located point.
Statistical analysis—Because the statistical distribution of the data (immunoreactive areas) was skewed to the right, a logarithmic transformation was done before statistical analysis to provide an approximately normal distribution. According to this, data are reported as geometric mean ± geometric SD or as box-and-whisker plots in the figures.
The differences of the immunoreactive areas between breeds were tested by use of the t test for independent samples. The differences between the 2 locations (antrum and corpus) within breeds were compared by use of the t test for 2 dependent samples. For all comparisons, P b 0.05 was considered significant. All analyses were performed with a statistical program package.32,l
Results
The expression pattern of the neuropeptides substance P and VIP was comparable to the expression pattern found in the intestine of pigs,29 although this study used whole-mount preparations instead of paraffin sections. This confirmed the specific binding of the antibodies used in the present study.
Substance P in the abomasal wall—The antral immunoreactive area for substance P was not significantly different between breeds (German Holsteins, 840 ± 1.75 μm2; German Fleckvieh cows, 910 ± 1.92 μm2)
Nonetheless, the corresponding area in the corpus abomasi was significantly smaller in the German Holsteins (679 ± 1.83 μm2) than in the German Fleckvieh cows (1,020 ± 1.65 μm2; Figure 3). No significant differences within breeds were detected between the 2 locations.
Box-and-whisker plots of substance P–immunoreactive areas in sections of the corpus abomasi and antrum pylori from 20 German Fleckvieh and 20 German Holstein cows. For each box, horizontal line represents the median value and the upper and lower boundaries represent the 75th and 25th percentiles respectively. Whiskers represent the minimum and maximum values, and black circles represent outlier values. *For the difference between the bracketed values, P = 0.025. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 69, 10; 10.2460/ajvr.69.10.1247
VIP in the abomasal wall—In biopsy specimens taken from the abomasum of German Holstein cows, VIP positive areas of 1,342 ± 1.62 μm2 were measured in the antrum pylori and 1,531 ± 1.51 μm2 in the corpus abomasi. In German Fleckvieh cows, values of 930 ± 1.95 μm2 and 1,444 ± 1.69 μm2 were measured, respectively. The difference between breeds did not reach significance (Figure 4). The VIP values differed between the antrum pylori and corpus abomasi in the German Fleckvieh cows, but not in the German Holsteins.
Box-and-whisker plots of VIP-immunoreactive areas in sections of the corpus abomasi and antrum pylori from 20 German Fleckvieh and 20 German Holstein cows. *For the difference between the bracketed values, P = 0.002. †For the differences in bracketed values, P = 0.054. See Figures 2 and 3 for remainder of key.
Citation: American Journal of Veterinary Research 69, 10; 10.2460/ajvr.69.10.1247
Overall nerve density in the abomasal wall—Innervation of the muscularis of the antral abomasal wall in German Holsteins, as measured by means of the immunoreactive areas for neurofilament 200, was denser than in German Fleckvieh cows (German Holsteins, 4,842 ± 1.29 μm2; German Fleckvieh, 3,333 ± 1.63 μm2). Similar results were observed in the corpus abomasi (German Holsteins, 4,800 ± 1.69 μm2; German Fleckvieh, 3,508 ± 1.62 μm2), but the difference between breeds did not reach significance (Figure 5).
Box-and-whisker plots of neurofilament 200-immunoreactive areas in sections of the corpus abomasi and antrum pylori from 20 German Fleckvieh and 20 German Holstein cows. *For the difference between the bracketed values, P = 0.056. †For the difference between bracketed values, P = 0.005. See Figures 2 and 3 for remainder of key.
Citation: American Journal of Veterinary Research 69, 10; 10.2460/ajvr.69.10.1247
Overall nerve density did not differ significantly between the 2 anatomic locations in either breed.
Thickness of abomasal wall layers—No significant differences between the 2 breeds examined were detected concerning the thickness of the different layers of the abomasal wall. In addition, the total thickness of the abomasal wall did not differ between the 2 breeds (German Holsteins: antrum, 11,992 ± 1.22 μm; corpus, 10,375 ± 1.13 μm vs German Fleckvieh: antrum, 12,803 ± 1.24 μm; corpus 10,673 ± 1.22 μm).
Discussion
During the past 50 years, a series of risk factors for the occurrence of abomasal displacement have been identified by means of epidemiologic and experimental studies. These include dietary effects (high-concentrate rations),33,34 stress conditions,33 metabolic disorders (insulin resistance in particular),23,34 neuronal dysfunctions,28 and genetic influences.10,35 The pathogenesis of the underlying motility disorders3,36 of the abomasum is still unknown. Recent studies8,28,37–39 have focused on possible functional disorders of the abomasal wall and its enteric nervous system. Nevertheless, the role of anatomic or functional properties of the abomasum in predisposition of some breeds and individuals for abomasal displacement is still unclear.
Various studies20,25,29,40–47 of the distribution pattern and the localization or colocalization of substance P and VIP in the intestinal wall have been reported. For example, NOS, the synthesizing enzyme for NO, is colocalized with VIP in the enteric neurons of the bovine gastrointestinal tract.48 Nitric oxide and VIP are involved in nonadrenergic, noncholinergic inhibitory neurotransmission in the mammalian gastrointestinal tract.11,48 In the guinea pig stomach, the action of NO and VIP is characterized by a functional synergism because NO increases the release of VIP and enhances its action on smooth muscle cells.49 Studiesm concerning the immunohistochemical expression pattern for NOS on bovine abomasal tissue reveal no differences between healthy animals and those with abomasal displacement. The present study therefore concentrated on the expression pattern of VIP as an inhibitory nonadrenergic noncholinergic transmitter.
To our knowledge, quantitative immunohistochemical studies have been performed primarily with the human intestine. Human intestinal diseases, such as ulcerative colitis, Crohn's disease, and slow transit constipation, are associated with changed expression patterns for neuropeptides.9,46,47,50 The expression pattern, importance, and motility disorders of the 2 neuropeptides VIP and substance P in the bovine abomasum have not been investigated. The authors presumed that a changed expression pattern of neuropeptides corresponding to that found in human motility disorders could also exist in bovine tissues. The hypothesis of the present study was, therefore, that the predisposition for abomasal displacement in German Holstein cows might be associated with decreased content of motility-stimulating substance P or increased content of inhibiting VIP, compared with German Fleckvieh cows.
The difference between the 2 breeds in this study—the significantly lower content of substance P in the corpus abomasi—suggests that in German Holsteins, the stimulating effects on the motility of the abomasum are smaller. Furthermore, considering the fact that the abomasal wall of German Fleckvieh cows in both locations examined had a lower number of nerve fibers than in German Holsteins, this finding is quite remarkable. Thus, the supply of neuropeptides to the musculature may be more efficient in German Fleckvieh cows than in German Holsteins. In addition to that, results of this study revealed a significantly lower content of VIP in the antrum pylori than in the fundic region of the abomasum in the German Fleckvieh breed. A diminished content of inhibiting VIP may be equated with a higher contractile motility. Because the antrum pylori is responsible for transporting the gastric contents (fluids and gas) toward the intestines like a pump, the efficiency of this pump seems to be higher than in German Holsteins. Consequently, the risk of distention and displacement of the abomasum is less than that in German Holsteins.
Conversely, there was wide variation within the German Holstein breed regarding these variables. This could explain the interindividual differences concerning the predisposition for occurrence of abomasal displacement of some cow families and therefore the known heritability of this disease. Another possible explanation for the variations could be that in this study, no differentiation between the immunoreactive areas of neurons and nerve fibers was performed. Thus, 1 ganglion in 1 slide could lead to an increase in the immunoreactive area. This fact certainly contributed to the considerable geometric SDs. A differentiation between somata of enteric neurons and nerve fibers could be the subject of future studies on this concern, but because the contribution of neurons (as 3-dimensional structures) to the immunoreactive areas depends on the layer examined, we have to be aware that a differentiation of neurons and nerve fibers could lead to false-negative results in the neuron fraction. This means that it is very unlikely that there are no fibers detectable in a section, but it is probable that there is no neuron to be found in the section.
Notwithstanding, results of the present study could be consistent with important differences between the 2 groups despite the considerable individual variability. Therefore, differentiation of neurons and nerve fibers could be reserved for future studies as well as a corresponding comparison of healthy cows and cows with abomasal displacement. The predisposition of German Holsteins for abomasal displacement could thus be explored more precisely.
Regarding the lower number of nerve fibers detected in the abomasal wall of German Fleckvieh cows in the present study, the chosen staining for neurofilament 200 as an overall nerve marker has to be challenged. Results of previous studies51,52 suggest that anti–Hu protein in human tissue or PGP 9.5 in cervine tissue are especially suitable overall nerve markers for examination of neurons in the human intestine and nerve fibers in the cervine testis, respectively. In addition to that, nonspecific enolase and neurofilament 200 do mark equal amounts of nerve fibers in cervine testis even though PGP 9.5 is better suited to depict the complete innervation pattern. Because the present study did not evaluate the total number of stained neurons and ruminant tissue was used, neurofilament 200 was chosen as an overall nerve marker. Staining for PGP 9.5 was not performed because of unpublished results from Herde et al,j which indicated that the use of neurofilament 200 antibody resulted in clearly specific staining in bovine tissue.
Results of the present study indicated a possible explanation for the more frequent occurrence of abomasal displacement in German Holstein cows. Results suggested that a genetically determined neuronal imbalance in German Holsteins could be predisposing for the development of abomasal displacement. Further investigations on this topic are needed to confirm this suggestion.
Abbreviations
| AEC | 3-amino-9-ethylcarbazol |
| APES | 3-aminopropyltriethoxysilan-3-triethoxy-silylpropylamin |
| ChAT | Choline-acetyltransferase |
| HRP | Horseradish peroxide |
| NO | Nitric oxide |
| NOS | Nitric oxide synthase |
| PGP | Protein-gene-product |
| VIP | Vasoactive intestinal polypeptide |
Clausen J. Untersuchungen zur Labmagengröße des Rindes in Beziehung zu Körpergröße, Alter, Futterzusammensetzung und Milchleistung. Doctoral thesis, Faculty of Veterinary Medicine, University of Giessen, Giessen, Germany, 1999.
AF 1021, Medicorp Inc, Montreal, QC, Canada.
T 4245, Bachem, St Helens, Merseyside, England.
H 3775, Bachem, St Helens, Merseyside, England.
NFM 002, Biologo, Kronshagen, Germany.
ABC kit, Vectastain ABC Kit Elite, PK-6100 Standard, Vector Laboratories Inc, Burlingame, Calif.
3-amino-9-ethylcarbazol (AEC); Peroxidase-Substratkit AE 002, Biologo, Kronshagen, Germany.
Axiophot Photomikroskop SIP 64614, Carl Zeiss AG, Oberkochen, Germany.
Analysis Pro, version 3.2, Olympus Soft Imaging Solutions GmbH, Münster, Germany.
Department of Veterinary Anatomy, Histology and Embryology, JLU Giessen, Germany.
Wacom intuos 3, Wacom Europe GmbH, Krefeld, Germany.
BMDP3D, Statistical Software Inc, Los Angeles, Calif.
Geishauser T. Untersuchungen zur Labmagenmotorik von Kühen mit Labmagenverlagerung. Habilitation thesis, Faculty of Veterinary Medicine, Univesity of Giessen, Giessen, Germany, 1995. of growth-associated protein-43 and substance P in ulcerative colitis. J Histochem Cytochem 2001;49:749–757.
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