Age-associated changes of the intrinsic nervous system in relation with interstitial cells in the pre-weaning goat rumen.

In this study, we investigated the neural changes and their relationships with interstitial cells (ICs) in the rumen of pre-weaning goats by transmission electron microscopy, western blot and immunofluorescence (antibody: general neuronal marker-Protein Gene Product (PGP9.5)/ IC marker-vimentin). The immunofluorescence results showed that PGP9.5-positive reaction was widely distributed in neuronal soma (NS) and nerve fibre (NF). The NSs were observed in the ganglia of the myenteric plexus (MP) but not in the submucosal plexus. The mean optical density (MOD) of the whole of PGP9.5-positive nerves and the protein expression level of PGP.5 in the rumen wall both decreased significantly with age. However an obvious increase MOD of PGP.5-positive NFs within the rumen epithelium were observed. In the MP, the nerves and ICs were interwoven to form two complex networks that gradually tightened with age. Furthermore, NSs and nerve trunks were surrounded by a ring-boundary layer consisting of several ICs that became physically closer with aging. Moreover, ICs were located nearby NFs within the ML, forming connections between ICs, smooth muscle cells and axons. This study describes the pattern of neural distribution and its association with ICs in the developing rumen which shed light on the postpartum development of ruminants.

Disorders of Rumen Distension and Dysmotility.

Rumen distension and hypomotility are common clinical findings in ruminants. A thorough physical examination to assess the rumen shape and consistency of rumen contents are critical to determining the underlying pathology. Most cases can be classified into 1 of the 4 types of vagal indigestion. Type 1 is characterized by gas distension of the rumen dorsally on the left side. Types 2, 3, and 4 will often appear similar on physical examination with fluid distension of the rumen on the left and ventrally on the right. Serum chloride and bicarbonate measurement and assessment of rumen chloride allow for differentiation of type 2 versus types 3 and 4 vagal indigestion. This is critical, as type 2 vagal indigestion will commonly require a rumenotomy, whereas types 3 and 4 typically are addressed through a right flank exploratory.

Intrinsic ruminal innervation in ruminants of different feeding types.

According to their feeding habits, ruminants can be classified as grazers, concentrate selectors and those of intermediate type. The different feeding types are reflected in distinct anatomical properties of the forestomachs. The present study was designed to investigate whether the intrinsic innervation patterns of the rumen (the main part of the forestomach) differ between intermediate types and grazers. Myenteric plexus preparations from the rumen of goats (intermediate type), fallow deer (intermediate type), cattle (grazer) and sheep (grazer) were analysed by immunohistochemical detection of the following antigens: Hu-protein (HuC/D), choline acetyltransferase (ChAT), nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), substance P (SP), calbindin (CALB) and somatostatin (SOM). Myenteric ganglia of cattle contained 73 +/- 6 neurons per ganglion, whereas the ganglia of sheep were significantly smaller (45 +/- 18 neurons per ganglion). The ganglion density of the myenteric plexus was highest in fallow deer (15 +/- 3 ganglia per cm(2)) and lowest in cattle (6 +/- 1 ganglia per cm(2)). All myenteric neurons were either ChAT or NOS positive. The proportion of NOS-positive neurons was significantly lower in sheep (29.5 +/- 8.2% of all neurons) than in goats (44.2 +/- 9.8%). In all species, additional analysis of the different neuropeptides revealed the following subpopulations in descending order of percentile appearance: ChAT/SP > NOS/VIP/NPY > ChAT/- > NOS/NPY. Expression of CALB was detected in a minority of the ChAT-positive neurons in all species. Somatostatin immunoreactive somata were found only in preparations obtained from fallow deer and sheep. These data suggest that the rumen of grazers is under stronger cholinergic control than the rumen of species belonging to the intermediate type, although most subpopulations of neurons are present in all species. However, whether the strong mixing patterns of low quality roughage during digestion are enabled by the prominent excitatory input of the rumen of grazers requires elucidation in further studies.

Intrinsic innervation patterns of the smooth muscle in the rumen and reticulum of lambs.

The rumen and reticulum of sheep serve as a fermentation chamber. Both compartments exhibit specific motility patterns. With developmental changes, the size of the reticulorumen dramatically increases when newborn lambs mature to adult sheep. This makes it possible to investigate the intrinsic innervation of the reticuloruminal muscles in lambs by taking the entire reticulum and rumen into account. The aim of the study was to analyse the projections and neurochemistry of myenteric neurons in the rumen and reticulum, which project to the inner or outer muscle layer, respectively. Therefore, we applied retrograde tracing with the fluorescent dye 1,1'-didodecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) and subsequent immunohistochemical detection of choline acetyltransferase (ChAT), substance P (SP) and vasoactive intestinal peptide (VIP). In both compartments innervation of both the inner and the outer muscle layer consisted mainly of cholinergic neurons (65-70%). The majority of them co-localized SP. The non-cholinergic neurons projecting to the muscle expressed immunoreactivity for VIP. Polarized innervation of the muscle layers was found neither in the rumen nor in the reticulum. Consequently, intrinsic innervation patterns for the smooth muscle layers in the rumen and reticulum differ from all gastrointestinal regions examined thus far.

Age-associated plasticity in the intrinsic innervation of the ovine rumen.

The rumen of adult sheep functions as a large fermentation chamber. In the newborn suckling ruminant, the rumen is bypassed and milk enters the abomasum directly. It was the aim of our study to investigate whether the transmitter content of intrinsic nerves changes with the developmental stage. The neurochemical code of myenteric neurons in the rumen from suckling lambs, fattened lambs and adult sheep was determined by using quadruple immunohistochemistry against choline-acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). Three neurochemically distinct subpopulations were identified within the rumen. They expressed the code ChAT/-, ChAT/SP and NOS/VIP. The number of ChAT/SP neurons did not change during development. It was 62% in the newborn lamb and remained stable in fattened lambs (63%) and adult sheep (63%). By contrast, the number of ChAT/- neurons decreased significantly from 20% in suckling lambs to 11% and 7% in fattened lambs and adult sheep, respectively. Simultaneously, the proportion of NOS/VIP neurons increased from 16% in suckling lambs to 29% in adult sheep. The increase in the proportion of NOS/VIP immunoreactive neurons indicates an adaptation to large volumes of ingesta at the beginning of roughage intake and rumination. We conclude that the age-associated changes in neurochemical code of myenteric neurons in the forestomach are related to the adaption of the rumen to different functional properties during development.

Ruminal muscle of sheep is innervated by non-polarized pathways of cholinergic and nitrergic myenteric neurones.

The motility patterns of the reticulorumen evoke mainly mixing of the ingesta. So far unknown, intrinsic neural circuits of the enteric nervous system are involved in the control of these motility patterns. The aim of the study was to characterize neurochemically sheep ruminal myenteric neurones, in particular the neural pathways innervating the ruminal muscle layers. Cell bodies within the myenteric plexus projecting to the longitudinal or circular muscle layer were retrogradely labelled by direct application of the fluorescent tracer 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) onto the circular or longitudinal muscle. The neurochemical code of myenteric neurones was identified by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). According to their neurochemical code, ruminal myenteric neurones were divided into three populations: ChAT/SP (68% of all myenteric neurones), NOS/VIP (26% of all myenteric neurones) and ChAT/- (5% of all myenteric neurones). Application of DiI onto the circular or longitudinal muscle revealed on average 64 or 44 labelled cell bodies in the myenteric plexus, respectively. DiI-labelled neurones expressed the code ChAT/SP or NOS/VIP. In the pathways to circular or longitudinal muscle, ChAT/SP-positive neurones outnumbered NOS/VIP-immunoreactive neurones by 5:1 and 2:1. Pathways to the circular or longitudinal muscle did not exhibit any pronounced polarized innervation patterns. This study demonstrated specific projections of myenteric neurones to the ruminal muscle. Neurones expressing the code ChAT/SP might function as excitatory muscle motor neurones, whereas NOS/VIP neurones are likely to act as inhibitory muscle motor neurones.

Tremorgenic mycotoxins paxilline, penitrem and lolitrem B, the non-tremorgenic 31-epilolitrem B and electromyographic activity of the reticulum and rumen of sheep.

The mycotoxic tremorgens penitrem, paxilline and lolitrem B had profound effects on electromyographic (EMG) activity of smooth muscle of the reticulorumen in conscious sheep, with a similar time course of action to their respective characteristic effects on the induction (1 to 2, 15 to 20 and 20 to 30 minutes) and the duration (1 to 2, 1 to 2 and 8 to 12 hours) of tremoring. Responses to penitrem revealed a greater sensitivity of smooth muscle than skeletal muscle. Effects included an inhibition of the vagally-dependent cyclical A and B sequences of contraction of the reticulorumen, an increase in their amplitude and an excitation of local intrinsic activity contributing to elevated baselines and the occurrence of chaotic activity of the reticulum. The excitatory local effects were partially blocked by atropine, indicating that stimulation of muscarinic cholinoceptors was involved. Increased local activity may mediate a reflex inhibition of cyclical contractions. A non-tremorgenic isomer of lolitrem B (31-epilolitrem B) had no effect on the reticulorumen. The intensity and duration of the effects of lolitrem B, up to 12 hours, indicate that severe disruption of digestion may occur in animals grazing endophyte-infected pasture.

Antagonist of nitric oxide synthesis inhibits nerve-mediated relaxation of isolated strips of rumen and reticulum.

The present study investigated the possibility that nitric oxide is a nonadrenergic, noncholinergic neurotransmitter of nerves that are intrinsic to the forestomach. Tunica muscularis, myenteric plexus preparations of bovine reticulum and rumen were maintained in vitro in a physiological solution of buffer that contained scopolamine. Trains of electric field stimulation transiently reduced (relaxed) the tone induced by BaCl2. NG-Nitro-L-arginine methyl ester, a nitric oxide synthase competitive antagonist, inhibited relaxation of the rumen and reticulum preparations that had been induced by the electrical field. The actions of NG-nitro-L-arginine methyl ester were partially reversed by L-arginine. These data suggest that nitric oxide, or a related substance, is an inhibitory neurotransmitter of nerves that are intrinsic to tunica muscularis, myenteric plexus preparations of the bovine forestomach.

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.

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.

Beta-endorphin-induced inhibition of rumen contractions in sheep. The effect of hypothalamic de-efferentiation.

In conscious sheep, beta-endorphin (1 and 2 micrograms/kg) administered into the third cerebral ventricle caused psychomotor excitability and a significant inhibition of the frequency of rumen contractions. The amplitude of the first rumen contractions, following immediately after the end of endorphin infusion, and the average amplitude of primary rumen contractions were also inhibited. De-efferentiation at the level of the hypothalamus prevented both the inhibitory effect of beta-endorphin on the frequency of rumen contractions and the drug-induced psychomotor excitability. However, de-efferentiation did not prevent beta-endorphin-induced inhibition of the mean amplitude of rumen contractions. The character of pathohistological changes induced by de-efferentiation showed descending degenerative changes of the nerve tracts connecting the hypothalamus with the pons and the medulla oblongata. These results, together with previously published evidence, do suggest that de-efferentiation at the level of the hypothalamus causes degeneration of inhibitory descending opioid-noradrenergic pathways connecting the hypothalamus with the gastric centres in the medulla oblongata.

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.

Ruminal lactic acidosis: forestomach epithelial receptor activation by undissociated volatile fatty acids and rumen fluids collected during loss of reticuloruminal motility.

Forty-three reticuloruminal epithelial receptors, with excitatory receptive fields within the reticulum, were isolated in 19 anaesthetised sheep. The responsiveness of these receptors to acetic, propionic, butyric and DL-lactic acids were assessed as well as their ability to be activated by rumen fluids obtained from sheep with induced ruminal lactic acidosis. Eighteen (41.9 per cent) receptors were excited by all three volatile fatty acids (VFAs) and 17 (39.5 per cent) responded to two VFAs. Butyric acid was the most potent volatile fatty acid eliciting responses in 41 (95.3 per cent) receptors. Acetic acid activated 33 (76.7 per cent) receptors and propionic acid excited 22 (53.5 per cent) receptors. DL-lactic acid only activated 13 (30.2 per cent) receptors, six of which required concentrations of 200 mM to be activated. Rumen fluids obtained when forestomach motility was impaired, activated 37 (81.1 per cent) of the 43 receptors, whereas rumen fluids obtained when forestomach motility was normal did not activate any receptors. Despite the high levels of lactic acid in rumen fluids which activated epithelial receptors, this acid was not responsible for receptor excitation.

[Masticatory cycles of the rumination type in vagotomized lambs]. / Cycles masticatoires de type mérycique chez la brebis vagotomisée.

Contractions of the fluid-filled RR dependent upon the activity of the myenteric plexus were observed within 2 weeks after vagotomy in sheep maintained on a liquid diet. Spontaneous but not adrenergic evoked rumination was recorded beyond the 5th week after vagotomy. The observation of ruminating-like activity during 120 to 200 min per day in chronically-vagotomized sheep indicates that the onset of rumination does not require a gastric afferent pathway.

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.

Hexamethonium: a probe to assess autonomic nervous system involvement in upper gastrointestinal functions in conscious sheep.

Hexamethonium, which inhibits cholinergic transmission by preventing acetylcholine release, has been considered an ideal reference drug for the blockade of autonomic ganglia, Auerbach plexus and reflex gastrointestinal secretions. The degree of inhibition of ruminant gastrointestinal functions with this reference drug were as follows: cyclical contractions of the reticulo-rumen and abomasal motility greater than gastric acid secretion and duodenal migrating myoelectrical complexes. Although reduced at high dosages, the initiation of migrating myoelectric complexes was enhanced at clinically used dosages. The duration of the inhibition of reticular contractions was dose-related varying from 0.5 to 5 h for 1.25 to 20 mg/kg subcutaneously. Abomasal motility and acid secretion were similarly reduced but exhibited strong and long-lasting rebound effects. Inhibition of the reticulum by the blockade of muscarinic receptors by atropine was also dose-related lasting from 0.5 to 3 h for 0.5 to 2 mg/kg, whereas inhibition of the abomasal motor and secretory functions lasted from 1 to 6 h. These results suggest a higher degree of impingement of the parasympathetic pathways on abomasal acid secretion and motility than on the cyclical activity of the reticulum and only a modulatory role of the extrinsic neural activity on the cyclical motor events of the duodenum.