Role of nitrergic nerves in the regulation of motility of the omasum and abomasum in healthy sheep (Ovis aries).

We examined the physiological role of nitrergic nerves in the regulation of omasal and abomasal motility in conscious healthy sheep and omasal muscle specimens. Nitric oxide (NO)-donor, S-nitroso-acethyl-dl-penicillamine (SNAP, 3-30 nmol/kg per min, i.v.) significantly inhibited omasal electromyographic (EMG) activity, whereas it did not alter EMG activity in the abomasal antrum. However, NO synthase inhibitor, Nomega-nitro-l-arginine-methyl ester (L-NAME, 0.3-3.0 micromol/kg per min, i.v.) did not alter EMG activity of the omasum and abomasum. In the in vitro experiments, SNAP application (6-200 micromol/l) significantly inhibited bethanechol (10 micromol/l)-induced contraction of longitudinal and circular muscles of the omasum. L-NAME application (0.03-3.0 mmol/l) enhanced electric field stimulation-induced contractions of the circular muscles. The results suggest that the omasal muscles are responsive to exogenous NO and that nitrergic nerves innervate the circular muscle layer of the omasum, however, nitrergic nerves are not or scarcely involved the physiological regulation of omasal and possibly abomasal motility in healthy sheep.

Morphometric and immunohistochemical study of the omasum of red deer during prenatal development.

The red deer is an important study species because of its value in the national economy and because it provides a wealth of genetic material. To date, there has been little research into the prenatal development of the stomach of ruminants, and none of the red deer. We therefore performed a histological evaluation of the ontogenesis of the omasum in the red deer. Histomorphometric and immunohistochemical analyses were carried out on 50 embryos and fetuses of deer from the initial stages of prenatal life until birth. For test purposes, the animals were divided into five experimental groups: Group I (1.4-3.6 cm crown-rump length, CRL; 30-60 days, 1-25% of gestation); Group II (4.5-7.2 cm CRL; 67-90 days, 25-35% of gestation); Group III (8-19 cm CRL; 97-135 days, 35-50% of gestation); Group IV (21-33 cm CRL; 142-191 days, 50-70% of gestation); and Group V (36-40 cm CRL; 205-235 days, 75-100% of gestation). At 67 embryonic days, the omasum wall was differentiated, and comprised three layers: the epithelial layer, pluripotential blastemic tissue and serosa. The stratification of the epithelial layer was accompanied by changes in its structure, with the appearance of four laminae of different sizes; in order of appearance these were: primary at 67 days, secondary at 90 days, tertiary at 97 days and quaternary at 135 days. At around mid-gestation, lateral evaginations were formed from the stratum basale of the primary and secondary smaller laminae. These were the primitive corneum papillae. From 205 days, the corneum papillae were present in all four sizes of laminae. The histodifferentiation of the lamina propia-submucosa, tunica muscularis and serosa showed patterns of development similar to those reported for the rumen and reticulum of red deer. The omasum of red deer during prenatal life, especially from 67 days of gestation, was shown to be an active structure with full secretory capacity. Its histological development, its secretory capacity (detected by the presence of neutral mucopolysaccharides) and its neuroendocrine nature (detected by the presence of positive non-neuronal enolase cells and the neuropeptides vasoactive intestinal peptide and neuropeptide Y) were parallel to the development of the rumen and the reticulum. However, its prenatal development was later than that of the omasum in sheep, goat and cow.

Structural differences of the enteric nervous system in the cattle forestomach revealed by whole mount immunohistochemistry.

The specific motility patterns of the forestomach of ruminants, composed of three structurally distinct compartments (rumen, reticulum, omasum), require an elaborate intramural innervation. To demonstrate the complex structure of the enteric nervous system (ENS), whole mount preparations obtained from different sites of the bovine forestomach were submitted to immunohistochemical procedures in which neuronal (protein gene product 9.5, neurofilament 200) and glial (protein S-100, glial fibrillary acid protein) markers were applied. Immunohistochemistry performed on whole mounts allowed a detailed two-dimensional assessment of the architecture of the intramural nerve networks. Generally, the myenteric and submucosal plexus layers were composed of ganglia and interconnecting nerve fiber strands, whereas the mucosal plexus consisted of an aganglionated nerve network. However, the texture of the ENS showed considerable regional differences concerning the ganglionic size, shape and density and the arrangement of nerve fiber strands. The myenteric plexus of the ruminal wall, showing a low ganglionic density and wide polygonal meshes, contrasted with the nerve network within the ruminal pillar which consisted of ropeladder-like nerve fiber strands and parallel orientated ganglia. The highest ganglionic density was observed at the reticular groove, the most prominent ganglia were found within the omasal wall. Branches of the vagal nerve frequently ramified within the myenteric plexus layers. The submucosal plexus of the rumen was divided into an external and internal layer; the reticular submucosal plexus followed the cristae and cellulae reticuli, the omasal submucosal (sublaminar) plexus showed intra- and parafascicular ganglia apart from ganglia located at the junctions of the nerve network. The mucosal plexus of the rumen consisted of thin nerve fascicles ramifying between the ruminal papillae, and reticular mucosal nerve fibers passed throughout the base of the cellulae reticuli. The highly specialised nerve network of the intralaminar omasal plexus showed radial and transverse trajectories reflecting the spatial arrangement of the intralaminar musculature. The demonstrated structural complexity of the ENS reflects the functional complexity of the ruminant forestomach and indicates the relatively high degree of autonomy in coordinating the different motility patterns required for the processing of the ingesta.

Morphological study of the submucosal and mucosal plexuses of the sheep forestomach.

Submucosal and mucosal nerve plexuses in the ovine forestomach were examined by immunohistochemical staining for protein gene-product 9.5 (PGP 9.5) and for S-100 protein (S-100), using whole-mount specimens that had been prepared by treatment with KOH. Nerve fibers of various sizes and glial cells (i.e., Schwann cells and satellite cells) were stained with antibodies against PGP 9.5 and S-100 respectively. The network of the submucosal plexus in the rumen is irregular and some nerve bundles in the plexus cross over other bundles. Some of the nerve bundles penetrate the ruminal papillae. The submucosal plexus in the reticulum consists of a network in the reticular wall and the reticular folds. The submucosal plexus in the omasum is also divided into two segments; namely, the sublaminar and the intralaminar plexuses. Most of the submucosal ganglion cells are unipolar and smooth-surfaced, being located singly or in small groups. A few perikarya were detected in the ruminal papillae. The number of perikarya per unit surface area is greater in the caudal portion of the omasal lamina (19.32 +/- 8.62 per cm2). In the mucosal plexuses, a well-developed network of beaded fibers with PGP 9.5-like immunoreactivity and a glial framework of S-100 like immunoreactivity was observed, in particular in the ruminal, reticular and omasal papillae. The intrapapillary nervous networks are interconnected by thin bundles of nerves in the interpapillary region. The present results suggest that some of the mucosal functions are intrinsically regulated by the submucosal and mucosal plexuses in the ovine forestomach.

Neuropeptides in the myenteric ganglia and nerve fibres of the forestomach and abomasum of grey, white and black Karakul lambs.

Previous studies indicated large, thin-walled, milk-filled rumens in lethal grey and white Karakul lambs. There was also a significant decrease in the number and size of the myenteric plexuses and the number of ganglion cells in these lambs. The purpose of this study was to determine whether the myenteric ganglia of the affected lambs are functional, by testing for the presence of vaso-active intestinal peptide, somatostatin, neurotensin, neuropeptide Y, met-enkephalin, calcitonin gene-related peptide and substance P in the myenteric ganglia and nerve fibres in the forestomach and abomasum of grey, white and black Karakul lambs. Four 1-cm2 samples were taken from analogous areas of the wall of the rumen, reticulum, omasum and abomasum of five grey, five white and five black newborn Karakul lambs. They were pinned to wax squares, fixed for 18 h in Zamboni's fixative, dehydrated and rehydrated through graded alcohols and stored in phosphate-buffered saline. The outer longitudinal muscle layer of each sample of the rumen, reticulum, omasum and abomasum was separated from the rest of the tissue layers, stained for each of the seven neuropeptides by employment of the immunofluorescence technique, and studied with a Leitz Orthoplan fluorescent microscope. All the material studied tested positive for all the neuropeptides. It is concluded that all the peptides tested for were present in all the lambs and that the myenteric ganglia are therefore functional in the lethal lambs.

Structure of the enteric nervous system in the sheep omasum as revealed by neurofilament protein-like immunoreactivity.

The general structure of the enteric nervous system in the omasum of sheep was investigated by immunohistochemical techniques using antineurofilament protein antibody. The myenteric plexus consisted of polygonal ganglia and interganglionic nerve bundles. The meshwork of the plexus in the oral region was denser than the middle and aboral regions. Intraganglionic laminar endings were present in the myenteric ganglia. The submucosal plexus was divided into sublaminar and intralaminar plexuses. The latter was denser near the free border of the omasal laminae than in the region near the omasal wall. There were no ganglion cells showing neurofilament protein-like immunoreactivity in the submucosal and mucosal layers. Probable mucosal receptors were found in the horny omasal papillae near the reticulo-omasal orifice. The findings demonstrate a complicated structure for the enteric nervous system in the omasum, presumably reflecting its regulatory activities.

The nerve cell bodies found in the ovine omasal laminae.

A histological study was made on the omasum of sheep, cows, sika deer, and Japanese serows in order to confirm the existence of the submucosal nerve cell bodies in the omasal laminae. In the present study, seventeen submucosal nerve cell bodies were found in seven sections from four out of twenty-two sheep.

Immunohistochemical study of the distributions of the peptide- and catecholamine-containing nerves in the omasum of the sheep.

The distribution in the sheep omasum of peptide- and catecholamine-containing neurons was investigated by an immunohistochemical technique using antibodies against six neuropeptides and two catecholamine-synthesizing enzymes. In the myenteric plexus, both nerve cell bodies and nerve fibers were positively immunostained for substance P (SP), methionine enkephalin (ENK), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and galanin (GAL). In contrast, immunoreactivity specific for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) was detected only in nerve fibers. In the omasal wall, nerve fibers with SP- and ENK-like immunoreactivity were prominent in the outer muscle layer, while numerous nerve fibers with VIP-, NPY- and GAL-like immunoreactivity were observed in the inner muscle layer. In the lamina propria mucosae, and in particular in the omasal papillae, a dense network of peptide-containing nerves was found. The distribution of nerves with CGRP-like immunoreactivity was different from that of other immunoreactive nerves. The networks of nerves with TH-, DBH- and NPY-like immunoreactivity were concentrated around the vascular components. The results of this study indicate the complexity of the enteric nervous system in the sheep omasum, reflecting the multiple functions of the nerves in the regulation of motility and/or absorption and secretion.

Substance P-immunoreactive neurons of the bovine forestomach mucosa: their presumptive role in a sensory mechanism.

Substance P-immunoreactive nerve fibers and cell bodies were examined in the forestomach mucosa of the calf and cow pretreated with colchicine, using thick (100 microns) floating sections. Intraepithelial nerve fibers were identified, appearing only rarely in the rumen and reticulum, and completely absent from the omasum. Nerve fibers were observed in the lamina propria of all the regions of the forestomach examined. A few thin nerve fibers were seen in the core of the ruminal papillae of the calf, whereas in the cow they appeared very coarse in nature. Flocculent and complicated nerve fiber networks were seen in the connective tissue of the reticular papillae. Mucosal nerve fibers formed a peculiar glomerulus-like architecture in the omasal papillae of the calf, while in the cow, the nerve fibers were largely restricted in distribution to the vicinity of the epithelium within the connective tissue pegs. Immunoreactive nerve cell bodies were found in the ruminal atrium, the dorsal sac and the ventral sac of the rumen of the calf and in the reticulum of both the calf and cow. Some of these neurons exhibited processes that appeared to course toward the papillae. In total, substance P-immunoreactive nerve fibers and cell bodies were more abundant in the calf than in the cow. These distributions demonstrate that the neural circuitry of the bovine forestomach contains substance P immunoreactivity in the mucosa as well as in the muscle layer, pointing to its possible importance in the regulation of the forestomach function. Substance P-immunoreactive nerve fibers were numerous in the reticular papillae of the calf and cow and in the omasal papillae of the calf. The positive fibers at these localities may act as mucosal receptors.

Ultrastructure of the myenteric ganglia in the rumen, reticulum, omasum and abomasum of grey, white and black Karakul lambs.

Homozygous grey and white Karakul lambs die after they have reached weaning age. This is due to a lethal gene causing digestive disturbances. Previous studies revealed that grey and white lambs have large, milk-filled rumens; the phenomenon was attributed to a significant decrease in the number of myenteric ganglia and neurons in the rumen wall. This study was undertaken to determine whether any morphological differences exist in the ultrastructure of the myenteric ganglia in the forestomach and abomasum of grey, white and black Karakul lambs. Samples of the forestomach and abomasum of grey, white and black Karakul lambs were prepared routinely for electron microscopy and studied with a Phillips electron microscope. No morphological differences could be detected in the structure of the components of the myenteric ganglia in the forestomachs and abomasums of grey, white and black Karakul lambs. It was therefore concluded that the lethal gene in grey and white Karakul lambs results in a paucity of the myenteric ganglia, but does not affect the ultrastructure of these structures.

A comparative histological study of the number and size of the myenteric ganglia and neurones in the fore-stomach and abomasum of grey, white and black Karakul lambs.

Homozygous grey Karakul lambs are born with a lethal genetic factor responsible for death at weaning age. When put on a high roughage diet under field conditions they develop distended, thin-walled rumens and sand impacted abomasa. Homozygous white Karakul lambs have a similar factor but survive for a longer period. Black Karakul lambs are not affected. The present study was undertaken to compare by image analysis the number and size of the myenteric ganglia, and the number of myenteric neurones in the walls of the fore-stomach and abomasum of 24-h-old grey, white and black Karakul lambs. One square centimetre samples were taken from analogous areas of the rumen, reticulum, omasum and abomasum of 38 embalmed Karakul lambs. Haematoxylin and eosin stained histological sections of each sample were studied with a Vids 2 Image Analyzer. One way analysis of variance indicated a significant difference between the groups regarding the number and size of the myenteric ganglia and in the number of myenteric neurones in the reticulum, rumen and abomasum. The number and size of the ganglia and the number of neurones was greatest in the black lambs and decreased progressively in the white and grey lambs. The omasum was not affected. It is suggested that the paucity of myenteric ganglia and neurones in the regions examined is instrumental in causing the lethal condition described above.

Distribution of methionine-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves in the forestomach of cattle.

The distribution of methionine-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves in the forestomach of calves and cows was studied, using immunohistochemical methods. Methionine-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves were found in all regions of the bovine forestomach. Cell bodies were found mainly in myenteric ganglia, and nerve fibers were found principally in muscular layers. Nerve fibers and ganglia were concentrated in the lips of the reticular groove, but were least numerous in ruminal and omasal walls. The reduced number of immunoreactive nerves in cows, compared with the number of those in calves, was striking, especially in the lips of the reticular groove and the omasal leaves. Results may indicate that the importance of methionine-enkephalin-Arg6-Gly7-Leu8-containing nerves in the physiologic regulation of the forestomach of the calf is different from that of the cow.

Immunohistochemical study on the distribution of neuron-specific enolase- and peptide-containing nerves in the omasum of cattle.

The distribution of nerves containing immunoreactivity for substance P (SP), vasoactive intestinal polypeptide (VIP), leucine-enkephalin (LENK), and gastrin-releasing polypeptide (GRP) in the margin of the reticulo-omasal orifice, omasum, and omasal pillar of calves and cows was studied by immunohistochemistry. The general distribution of nerves was determined by means of neuron-specific enolase (NSE) antiserum and then compared to the distribution of immunoreactive (IR) nerves stained for the four peptides. Marked differences in the distribution of immunoreactive nerves were associated with age and the segment examined. SP-IR fibers were abundant in the musculature of the ungulate papillae at the reticulo-omasal orifice and in the smooth muscle of the omasal leaves, moderately dense in the omasal pillar, and low in density in the inner muscle layer of the reticulo-omasal orifice and in the muscle of the omasal wall. In order of decreasing abundance, the cell bodies of SP-IR nerves were found at the reticulo-omasal orifice, in the omasal wall, and in the omasal pillar. LENK-IR fibers, though less abundant, showed a pattern of distribution that was similar to that of SP-IR fibers. Nerve cell bodies showing weak immunoreactivity for LENK were detected rarely. Abundant VIP-IR fibers were present in the inner muscle layer of the reticulo-omasal orifice and in the omasal wall, while moderate numbers were seen in the omasal pillar: they were low in density in the ungulate papillae and omasal leaves. Cell bodies of VIP-IR nerves decreased in number through the omasum, reticulo-omasal orifice, and omasal pillar. The distribution of GRP-IR nerve fibers was similar to that of VIP-IR fibers, although GRP-IR fibers were less abundant. Nerve cell bodies showing weak immunoreactivity for GRP were detected rarely. The individual distribution of peptide-IR nerves was similar in the calf and cow, but immunoreactive nerves were far more abundant in the calf. The present study provides valuable information for discussion of the possible role of nerves in the regulation of omasal function.

[Distribution of the vagus nerve of the stomach and certain lymph nodes of the sheep in the prenatal period (author's transl)]. / Die Versorgung des Magens und einiger Lymphknoten durch den Nervus vagus beim Schaf in der pränatalen Periode.

The goal of this study was the investigation into the vagus nerve structure in the ruminant stomach, and a description of the participation of this nerve in the stimulation of the atrial lymph nodes of the rumen and dorsal abomasal lymph nodes. This survey was carried out on 30 sheep fetuses, whose ages are designated according to Zietzschmann and Krölling (1955). The fetuses came from the third to fifth months of pregnancy. Observations and general morphological nature may be summarized as follows: 1. to the wall of the rumen reach atrial branches coming from both vagus trunks plus a dorsal ruminal branch, and a right ruminal branch coming from the dorsal vagus trunk, 2. the reticulum receives reticular branches, coming from both vagal trunks, 3. the omasum is innervated by omasal branches of the dorsal vagus trunk plus an omasoabomasal branch coming from the ventral vagus trunk, 4. to the wall of the abomasum reach the reticulo-abomasal and pyloric branches coming from the ventral vagus trunk plus a branch to the major abomasal curvature and a visceral abomasal branch coming from the dorsal vagus trunk, 5. arterial lymph nodes of the rumen receive innervation from the reticulo-abomasal branch or the pyloric branch, both leaving from the ventral vagus trunk, 6. dorsal abdominal lymph nodes innervated by nerve branches coming from a parietal abomasal branch, coming also from the ventral vagus trunk.

Regulation of motor activity of omasum after blockade of vagus nerves.

In 3 sheep with subcutaneously exposed vagus nerves and with chronic large fistula of the rumen pharmacological analysis of the motor activity of omasum was carried out after blockade of vagus nerves. The contractions of omasal canal, reticulum and rumen were recorded by the baloon method. After blockade of the vagus nerves with anesthetics the proper pharmacological agents were administered in intravenous injections. In the light of the obtained results it was found that contractions of the omsal canal, persisting after vagal blockade, are independent of the influence of the autonomic system and they are probably due to myogenic automatism. It has been also demonstrated that both alpha and beta adrenergic receptors participate in regulation of the tonous of omasum musclular layers.

The cyclic motility of the omasum and its control in sheep.

1. The mechanical activity of different parts of the omasum was recorded by means of small balloons and/or strain-gauges in conscious sheep. These events were related to the electrical activity of the omasal body, the omasal groove and the leaves and to the changes in diameter of the reticulo-omasal orifice. Humoral, behavioural and nervous influences were examined.2. Two patterns of contraction of the omasum were found. One, confined to the oral and middle thirds of the organ, originated at the omasal groove and proceeded to the right and then the left surfaces of the body and ceased at the onset of reticular contraction. The other was limited to the aboral third of the organ near the abomasum; in this part the contractions were prolonged regardless of the reticular contraction and some gave rise to a reversed propagation over the organ. The characteristic cyclic motility of the omasum was slightly reduced in sheep fasted for 48 hr and in those fed on pelleted food, and it persisted after vagotomy.3. The reticulo-omasal orifice always dilated strongly during the second phase of reticular contraction. It also dilated noticeably during the extra-contraction associated with regurgitation. In each case alternating opening and closing movements at a frequency of 5-7/min followed. Contractions of the omasal leaves, which were independent of those of the omasal body, occurred at a rate of approximately 2-3/min. They passed from the free border to the base and travelled in an aboral direction. After local application of fatty acids the frequency of their contractions increased to approximately the same rate as that of the orifice.4. It is concluded that the motility of the omasum in sheep involves a wave of contraction starting at a point on the omasal groove just posterior to the reticulo-omasal orifice and passing slowly over the omasal body. With the exception of the occasional reversed contractions this pattern is closely associated with rumino-reticular cycles even though the omasal contractions and the movements of the leaves are partially independent of vagal innervation.

Effects of stimulation of efferent fibres of the vagus on the reticulo-omasal orifice of the sheep.

1. The reticulo-omasal orifice has been observed and its activity recorded in decerebrate and spinal preparations of lambs and in lambs and sheep anaesthetized with chloralose.2. Spontaneous closure movements at about 6/min were seen, or recorded from balloons placed in the orifice in all types of preparation when both vagus nerves had been cut and after atropine, hexamethonium, phentolamine and propranolol had been given I.V.3. The effects on the reticulo-omasal orifice of stimulation in the neck of efferent fibres of the vagus nerves on either side differed according to the form of the stimulus applied.4. Threshold stimulation of the vagus nerve caused closing of the orifice which was blocked by atropine.5. Opening of the orifice and disappearance of spontaneous closing movements followed more intense stimulation of the vagus nerves.6. Vagal opening effects were obtained after atropine was given and after adrenergic opening and closure effects had been blocked with phentolamine and propranolol.