Intrinsic ruminal innervation in ruminants of different feeding types

Abstract

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.

Fig. 1

Ganglion size (A) and ganglion density (B) in the rumen of different species. *Asterisks in (A) and (B) indicate significant (P < 0.05) differences between species (one-way anova with subsequent Student–Newman–Keuls test). Data were obtained from 12–24 preparations from 8–15 animals. Columns in (C) are calculated from the respective mean values of columns in (A) and (B).

Fig. 3

NADPH-diaphorase reaction (A) and immunohistochemical staining against NOS (B) marked the same neurons.

Fig. 2

Cholinergic and nitrergic neurons in the rumen. Immunohistochemical detection of ChAT (A) revealed a subpopulation of all myenteric neurons in goat rumen stained against HuC/D. Neurons that were ChAT negative and HuC/D positive were labelled by NADPH-diaphorase reaction [dark stained neurons in (C), indicated by arrows]. NADPH/D-positive neurons were clustered at the margins of ganglia in goat (C) and cattle (D) or were equally distributed in the ganglia of fallow deer (E) and sheep (F).

Fig. 5

Proportion of neurochemically defined subpopulations in the rumen of different species. Within all four species, the proportion of the subpopulation was in descending order: ChAT/SP > NOS/NPY/VIP > ChAT/– = NOS/NPY (the significant differences between the relative sizes of the subpopulations are not indicated in the figure). *Asterisks indicate significant (P < 0.05) differences within one subpopulation between species (two-way anova with subsequent Student–Newman–Keuls test). Data were obtained from 5–11 preparations from 4–10 animals.

Fig. 4

Complete colocalization of NOS and NPY in the rumen is shown in (A) and (B). The majority of NPY (C) and NOS (E) immunoreactive neurons colocalized with VIP (D,F). Only a small proportion of neurons [indicated by arrows in (C–F)] expressed immunoreactivity for NPY (C) or NOS (E), respectively, but not for VIP (D,F).

Fig. 6

Neurons immunoreactive for NOS (A) did not show immunoreactivity for SP (B) and vice versa. Consequently, SP immunoreactive neurons had a cholinergic phenotype. In cattle, the majority of CALB immunoreactive neurons [arrows in (C)] expressed SP [arrows in (D)]. One neuron immunoreactive for CALB but not for SP is marked by an arrowhead.

Fig. 7

Immunoreactivity for SOM in the ruminal myenteric plexus of fallow deer (A) and goat (B). In fallow deer not only fibres but also neuronal somata were immunoreactive for SOM [arrows in (A)]. In goats, no SOM-positive neuronal somata but various fibres [marked by arrows in (B)] could be detected.