Establishment and identification of primary bovine omasal epithelial cells.

The establishment and culture of bovine omasal epithelial cells (BOECs) in vitro is a valuable tool for the study of the physiological function, nutrient absorption, and transport mechanisms of the omasum in dairy cows. This paper proposes a method for the culture of primary BOECs. Trypsin digestion was used to subculture the BOECs, which were passaged for 20 generations in vitro, and showed typical epithelial-like characteristics and a cobblestone morphology. The primary BOECs had a fast growth phase (between days 4 and 5) and were validated by their slight ß-galactosidase and visible cytokerat in 18 expression. In addition, RT-PCR results demonstrated that the monocarboxylate transporter 1 (MCT1), Na+/H+exchanger 1 (NHE1), and Na+/H+ exchanger 3 (NHE3) were expressed in the isolated primary BOECs. In conclusion, this primary BOEC isolation and culture model is a promising method for the study of nutrient absorption and regulation, as well as the immune regulation of epithelial cell transport in vitro.

Mechanism of peptide absorption in the isolated forestomach epithelial cells of dairy cows.

BACKGROUND: Peptide absorption from the forestomach plays a vital role in protein nutrition of dairy cows. This study was conducted to investigate the mechanism of dipeptide absorption in the forestomach of dairy cows using isolated omasal epithelial cells (OECs) and ruminal epithelial cells (RECs). RESULTS: Compared with RECs, the OECs formed a less tight monolayer, but had greater ability to transport glycylsarcosine (Gly-Sar) (P < 0.05). The OEC monolayers were immunopositive for the antibodies of anti-junction proteins. Gly-Sar transport was significantly greater at 37 °C than that at 4 °C, with an optimal pH of 6.0-6.5, and was decreased significantly by diethylpyrocarbonate and dipeptide Met-Gly (P < 0.05). The apical-to-basolateral transport was significantly greater than basolateral-to-apical transport (P < 0.05). Knockdown of peptide transporter 1 (PepT1) resulted in less Gly-Sar uptake in OECs, whereas overexpression of PepT1 in OECs resulted in higher Gly-Sar uptake (P < 0.05). Additionally, the expression of PepT1 was upregulated by the treatment with various dipeptides (P < 0.05). CONCLUSION: The OECs have a greater ability to transport Gly-Sar than RECs do. Both passive and active routes are involved in the process of Gly-Sar absorption in the isolated cultured forestomach epithelial cells from dairy cows. © 2018 Society of Chemical Industry.

A high-grain diet alters the omasal epithelial structure and expression of tight junction proteins in a goat model.

The omasal epithelial barrier plays important roles in maintaining nutrient absorption and immune homeostasis in ruminants. However, little information is currently available about the changes in omasal epithelial barrier function at the structural and molecular levels during feeding of a high-grain (HG) diet. Ten male goats were randomly assigned to two groups, fed either a hay diet (0% grain; n = 5) or HG diet (65% grain; n = 5). Changes in omasal epithelial structure and expression of tight junction (TJ) proteins were determined via electron microscopy and Western blot analysis. After 7 weeks on each diet, omasal contents in the HG group showed significantly lower pH (P <0.001) and significantly higher concentrations of free lipopolysaccharides (LPS; P = 0.001) than the hay group. The goats fed a HG diet showed profound alterations in omasal epithelial structure and TJ proteins, corresponding to depression of thickness of total epithelia, stratum granulosum, and the sum of the stratum spinosum and stratum basale, marked epithelial cellular damage, erosion of intercellular junctions and down-regulation in expression of the TJ proteins, claudin-4 and occludin. The study demonstrates that feeding a HG diet is associated with omasal epithelial cellular damage and changes in expression of TJ proteins. These research findings provide an insight into the possible significance of diet on the omasal epithelial barrier in ruminants.

High concentrate:forage ratio diet inhibiting omasal epithelium growth is associated with decreased cyclin D1 and CDK4 expression in growing goats.

The hypothesis that different concentrate : forage ratio diets alter omasal epithelium proliferation of growing goats via cyclins and regulation of the cell cycle was tested. Growing goats were fed with a high concentrate (HC, n = 8) or a low concentrate (LC, n = 8) diet for 42 days. The concentrate : forage ratio was 40:60 in the HC group and 0:100 in the LC group. In the HC group, the relative weight and DNA content of the omasal epithelium were lower, but the protein : DNA ratio was higher. Flow cytometry revealed that HC omasal cell numbers were smaller in S- and G2 /M-phases of the cell cycle and higher in the G0 /G1 -phases and were accompanied by reduced expression of cyclin D1 and CDK4 mRNA and protein. These data are consistent with morphologic observations in the HC that cell density decreased in the stratum spinosum (SS) plus stratum granulosum (SG) and stratum basale, and that cell density was lower in the SS plus SG. Thus, high-concentrate : forage ratio diet retards omasal epithelial growth by slowing the G1 to S phase transition of the cell cycle and is associated with decreased cyclin D1 and CDK4 expression in growing goats.

Sheep rumen and omasum primary cultures and source epithelia: barrier function aligns with expression of tight junction proteins.

The forestomachs of cows and sheep have historically served as important models for the study of epithelial transport. Thus, the ruminal epithelium was among the first tissues in which absorption of chloride against an electrochemical gradient was observed, requiring a tight paracellular barrier to prevent back-leakage. However, little is known about ruminal barrier function, despite the considerable implications for ruminant health. The tight junction proteins of the omasum have never been investigated, and no cell culture model exists. We present a new method for the isolation of cells from forestomach epithelia. Protein expression of cells and source tissues of sheep were studied using western blot, PCR and confocal laser scanning microscopy. Cultured cells were characterized by transepithelial resistance (TER) measurements and patch clamping. Cells developed TER values of 729±134 Ω cm(2) (rumen) and 1522±126 Ω cm(2) (omasum). Both primary cells and source epithelia of rumen and omasum expressed cytokeratin, occludin and claudins 1, 4 and 7 (but not claudins 2, 3, 5, 8 and 10), consistent with the observed paracellular sealing properties. Staining for claudin-1 reached the stratum basale. The full mRNA coding sequence of claudins 1, 4 and 7 (sheep) was obtained. Patch-clamp analyses of isolated cells proved expression of an anion conductance with a permeability sequence of gluconate

The surface morphology of the omasum of the African goat.

The microstructure of the omasum of the African goat was investigated macroscopically, as well as by scanning electron microscopy to determine how the omasum is anatomically specialised to enable the goat to graze and browse so successfully in the harsh environments of Africa. The omasum of the African goat was found to be more simple in structure than that of other domesticated ruminants. The omasal laminae were arranged in only 3 orders with the number of laminae varying between 27 and 33, considerably fewer than previously described for other goats, sheep or cattle. The large conical and unguiculiform papillae surrounding the reticulo-omasal orifice, as well as the hooked papillae present on the laminae, were more similar to those of wild ruminants that browsed than domestic grazing species. The papillae on the laminae were also found to be directionally orientated towards the orifices, which may suggest a "sieve mechanism' at the reticulo-omasal orifice and interlaminar spaces. In addition, it may suggest a directional movement of ingesta between the laminae. The stratified squamous epithelium lining the laminae and papillae was lightly keratinised which, along with the microplicae-like surface folds and pits of the superficial cells, indicated that it was functionally structured for absorption. These morphological specialisations of the omasal surfaces of the indigenous African goat demonstrated similarities with that of wild ruminants that are able to graze and browse.

Demonstration and characterization of dipeptide transport system activity in sheep omasal epithelium by expression of mRNA in Xenopus laevis oocytes.

Research from this laboratory has recently demonstrated that the omasal epithelium of sheep is capable of absorbing dipeptides. In order to express proteins potentially responsible for the mediated absorption of small peptides, size-fractionated poly(A)+RNA (RNA) isolated from omasal epithelial tissue of sheep (average BW 67.5 kg) were injected into defolliculated Xenopus laevis oocytes. The ability of oocytes injected with RNA or water to absorb [14C]glycyl-L-sarcosine (Gly-Sar) from media (usually pH 5.5) was compared. After 4 d (P < .02) of culture, specific RNA fractions induced an increased (P < .02) rate of Gly-Sar absorption, as compared with water-injected oocytes. The dependency of Gly-Sar uptake on the presence of a pH gradient was evaluated at pH 5.0, 5.5, 6.0, 6.5, and 7.5. Inducible uptake increased (P < .001) in the presence of increasing proton concentrations, whereas endogenous uptake of Gly-Sar decreased (P < .001). At pH 5.5, induced Gly-Sar uptake was saturable (Kt = .4 mM), but endogenous uptake was not. The specificity of Gly-Sar absorption was studied by the co-incubation of .1 mM Gly-Sar with 5 mM levels of competing substrates (pH 5.5). Induced uptake was inhibited (P < .05) 44% by carnosine, 94% by methionylglycine, and 91% by glycylleucine, but not by glycine. Incubation of RNA with DNA oligomers that were complementary to the rabbit intestinal transporter completely inhibited (P < .05) induced Gly-Sar uptake. These results indicate that sheep omasal epithelial cells express messenger RNA that encode for proteins that are capable of H(+)-dependent dipeptide transport activity.

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.

Histochemistry of the forestomach epithelium of the reindeer calf.

Samples from the rumen, reticulum and omasum of 26 reindeer calves were taken during the winter season. Non-specific alkaline and acid phosphatases, cytochrome and amine oxidases as well as succinate, lactate and 3-hydroxybutyrate dehydrogenases were demonstrated in the epithelium histochemically. The phosphatases were usually present in all the epithelial layers, whereas the activities of the other enzymes decreased in the outer layers and could not be demonstrated in the stratum corneum. The activity of alkaline phosphatase seemed to be highest in the reticulum and lowest in the omasum. The reason for the higher activity of this enzyme in epithelial taps in the rumen and omasum and in the reticular and omasal papillae may be the greater need for effective vertical transcellular transport in these regions. There was a tendency for enzymes other than phosphatases to be more active in the rumen than in the other forestomachs, which probably reflects the higher metabolic activity of the ruminal epithelium. No clear differences between early and late winter could be demonstrated.

Histomorphometric analysis of the omasum of sheep during development.

Histomorphometric and scanning electron microscopic analyses were performed on 74 embryos and fetuses and 20 sheep (early postnatal to adult age). Histologic differentiation of the omasum took place at 33 days of fetal life, with the appearance of first-order laminae. Second-, third-, and fourth-order laminae appeared at 39, 50, and 59 days, respectively. Neutral mucopolysaccharides first appeared in epithelial cells at 46 days of fetal life, decreasing quantitatively until birth, before subsequently stabilizing in postnatal life. Acid mucopolysaccharides, mucins, and mucoid compounds were not detected. Growth curves and formulas were constructed for each tissue layer. Initial tests involved multiplicative (y = axb), exponential (y = EXP [a + bx]), linear (y = a + bx), and polynomial models (y = a + bx + cx2 + dx3).

Do the cardiac glands exist? 7. The cow.

The glands distributed in the narrow region of the abomasum contiguous to the omasum of the cow have been described as cardiac glands. We doubted this assertion and therefore performed histological and histochemical investigations of the glands to clarify their characteristics. 1. All glandular cells except the parietal cells in a few glands contiguous to the omasum react strongly to PAS, AB(pH 2.5), and PAS-AB(pH 2.5) staining, and moderately to AB(pH 0.5) staining. 2. Glandular cells at the base of these glands contain fine pepsinogen granules and a few parietal cells are distributed in these glands, indicating that they are undifferentiated gastric glands and that the so-called cardiac glands do not exist in the cow stomach. 3. Glandular cells in undifferentiated gastric glands are filled with PAS, AB(pH 2.5 and 0.5) and PAS-AB(pH 2.5) positive substances. Which gradually decrease and finally disappear with differentiation, remaining only in the neck (mucous neck cells) and the cells in the upper part of the glandular body (immature chief cells), in mature gastric glands. 4. Mature chief cells in differentiated gastric glands are distributed in the middle and lower bodies and base of the glands and contain a number of PAS and PAS-AB(pH 2.5) positive granules and a large number of coarse pepsinogen granules, while pepsinogen granules in the mucous neck cells and immature chief cells are finer. 5. In the cow the region in which undifferentiated gastric glands are located is very narrow. 6. Parietal cells in the cow stomach are numerous.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of immunoglobulin-containing cells in calves inoculated orally with ruminal Bacteroides succinogenes and Selenomonas ruminantium.

Distribution of immunoglobulin(Ig)-containing cells was investigated in calves inoculated orally with live organisms of both Bacteroides succinogenes and Selenomonas ruminantium. Pathological changes and many Ig-containing cells were observed in calves which inoculated three times at 2, 3 and 26 days of age. Follicular germinal center was increased in number and size of the lymph nodes associated with the forestomach, suggesting activation of lymph apparatus. In the associated lymph nodes, IgG-containing cells were predominant and were located in both cortex and medulla, mainly in the medullary cord, B lymphocyte areas. Only a few IgA- and IgM-containing cells were observed in the lymph nodes. Accordingly, the inoculated bacteria may stimulate IgG-containing B lymphocyte populations. A few IgG-containing cells were detected in the mucosa of the forestomach. Ig-containing cells, predominantly IgG, were observed in the mucosa of the abomasum and intestine, and in the mesenteric lymph nodes. However, number of the cells in the mesenteric lymph nodes was smaller than that of the forestomach associated lymph nodes. The results suggest that the intraorally inoculated bacteria may stimulate the maturation of IgG positive lymphocytes in the lymph nodes associated with the forestomach.

Comparative distribution of immunoglobulin-containing cells in stomach, intestine and associated lymph nodes of cattle.

Immunoglobulin (Ig)-containing cells were investigated immunohistochemically in the stomach, intestine and associated lymph nodes of clinically normal calves, cows and fetuses to examine mucosal immune responses in the forestomach to rumen microbial flora. IgG-containing cells were observed in the mucosal propria of the forestomach of a 32 day-old, a 37 day-old, two 90 day-old calves, and of all 5 cases of cows. However, no IgA- and IgM-containing cells were observed. Further, no Ig-containing cells were detected in the associated lymph nodes of all calves and cows. Various numbers of Ig-containing cells, predominantly IgG, were observed in the mucosal propria of the abomasum, small intestine and cecum, and in the mesenteric lymph nodes of aged calves and all cows. No Ig-containing cells were observed in any tissues of fetuses. The results suggest that the development of mucosal immune responses in the forestomach may be incomplete as compared with that of the intestine.

Growth of foot-and-mouth disease virus in the different layers of bovine omasum in suspended cultures.

When suspended cultures of bovine omasum were cultured without agitation, the epithelium soon degenerated and foot-and-mouth disease virus multiplied mainly in the corium cells. Five days of preincubation were needed to reach a population of corium cells that could yield virus at a titer of 10(6.70) to 10(6.95) mean tissue culture infective doses per ml. The virus was freely released from the cells into the medium only when the degenerated epithelium was removed from the subepithelial tissue prior to virus inoculation. In agitated cultures, the viability of the epithelium was retained, the virus multiplied in all the layers of the epithelium and was freely released into the medium, and a virus titer of 10(6.95) mean tissue culture infective doses per ml was obtained without preincubation. The omasal laminae could be separated along the line of apposition of the two mucous membranes of the organ. The virus yield from these thin separated membranes was 0.5 to 1.0 log higher than that obtained from nonseparated laminae.