Omasal Epithelium Research Articles

Canola meal or soybean meal as protein source and the effect of microencapsulated sodium butyrate supplementation in calf starter mixture. II. Development of the gastrointestinal tract

The aim of this study was to assess the effect of protein source, either soybean meal (SM) or canola meal (CM), and microencapsulated sodium butyrate (MSB) supplementation in a pelleted starter mixture on the development of the gastrointestinal tract (GIT) in dairy calves. Twenty-eight bull calves (8.7 ± 0.8 d of age and 43.0 ± 4.4 kg; mean ± SD) were assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement: CM as a main source of protein without or with MSB or SM without or with MSB. Calves were fed starters ad libitum and exposed to a gradual weaning program, with weaning taking place on 51.7 ± 0.8 d of age. Calves were observed for an additional 3 wk after weaning and slaughtered on d 72.1 ± 0.9 of age, after which the GIT was dissected. Morphometric measurements were recorded, and samples for determination of ruminal fermentation, histology, gene expression, and brush border enzyme activities were collected. Canola meal use in the starter mixture increased abomasal tissue weight, jejunal tissue weight and length, and mRNA expression of SLC16A4 (formerly known as MCT4) and FFAR2 (GPR43) in the ruminal epithelium, and decreased ruminal ammonia and mRNA expression of SLC15A2 (PEPT2) and SLC6A14 (ATB0+) in the proximal small intestine and ileum, respectively. However, MSB inclusion in the starter mixture decreased ruminal papillae length, ruminal epithelial surface, and ruminal epithelium dry weight, while increasing mRNA expression of SLC16A1 (MCT1) in ruminal epithelia. Reduced ruminal surface area associated with MSB supplementation was the most apparent when MSB was combined with CM in the starter mixture. Additionally, MSB supplementation decreased the thickness of omasal epithelium, omasal epithelium living strata, and stratum corneum, and increased duodenal and ileal aminopeptidase A enzymatic activity and ileal aminopeptidase N enzymatic activity. Overall, CM might increase growth of the GIT of calves, particularly of the small intestine, but may negatively affect intestinal epithelium function and peptide and AA absorption. Supplementation of MSB has a negative effect on the ruminal and omasal epithelium development, particularly when combined in a starter mixture with CM.

Carbonic anhydrase influences asymmetric sodium and acetate transport across omasum of sheep.

ObjectiveOmasum is an important site for the absorption of short chain fatty acids. The major route for the transport of acetate is via sodium hydrogen exchanger (NHE). However, a discrepancy in the symmetry of sodium and acetate transport has been previously reported, the mechanism of which is unclear. In this study, we investigated the possible role of carbonic anhydrase (CA) for this asymmetry.MethodsOmasal tissues were isolated from healthy sheep (N = 3) and divided into four groups; pH 7.4 and 6.4 alone and in combination with Ethoxzolamide. Electrophysiological measurements were made using Ussing chamber and the electrical measurements were made using computer controlled voltage clamp apparatus. Effect(s) of CA inhibitor on acetate and sodium transport flux rate of Na22 and 14C-acetate was measured in three different flux time periods. Data were presented as mean±standard deviation and level of significance was ascertained at p≤0.05.ResultsMucosal to serosal flux of Na (JmsNa) was greater than mucosal to serosal flux of acetate (JmsAc) when the pH was decreased from 7.4 to 6.4. However, the addition of CA inhibitor almost completely abolished this discrepancy (JmsNa ≈ JmsAc).ConclusionThe results of the present study suggest that the additional protons required to drive the NHE were provided by the CA enzyme in the isolated omasal epithelium. The findings of this study also suggest that the functions of CA may be exploited for better absorption in omasum.

Histomorphometric changes of the fore-stomach of lambs fed with diets containing spineless cactus genotypes resistant to Dactylopius sp.

The histomorphometric parameters of the fore-stomach epithelium of lambs fed with spineless cactus genotypes resistant to Dactylopius sp. were evaluated. Thirty-two lambs (23.0 ± 2.06 kg body weight) were distributed in a completely randomized design with four treatments and eight replications. The treatments consisted of the base diet and diets in which part of the elephant grass hay and all ground corn was replaced by the Miúda, IPA-Sertânia, or Orelha de Elefante Mexicana (OEM) spineless cacti. The keratin layers of the ruminal epithelium of the animals consuming the spineless cacti Miúda and OEM presented greater thickening relative to the control diet: 47.31, 41.30, and 25.81 μm, respectively. The heights of the ruminal papillae were higher in the animals that were fed with the OEM spineless cactus. The Miúda spineless cactus caused an enlargement in the keratinized layer of the reticulum epithelium, as well as a decrease in the thickness of the non-keratinized layers. The diet containing OEM spineless cactus resulted in greater thickening of the keratin layer of the omasal epithelium and decreased thickness of the non-keratinized layers. The spineless cacti Miúda and OEM changed the morphology of the fore-stomach epithelium and increased the dry matter intake and weight gain of lambs.

Effect of exogenous butyrate on the gastrointestinal tract of sheep. I. Structure and function of the rumen, omasum, and abomasum.

The aim of this study was to determine the effect of exogenous butyrate on the structure and selected functions of the stomach in sheep. Eighteen rams (30.8 ± 2.1 kg; 12 to 15 mo of age) were allocated to the study and fed a diet for 14 d without (CTRL) or with sodium butyrate (BUT; 36 g/kg of offered DM). Neither DMI nor initial BW differed between treatments (P ≥ 0.61), but final BW was greater for BUT compared with CTRL (P = 0.03). Butyrate concentration in the reticuloruminal fluid and abomasal digesta was greater for BUT compared with CTRL (P ≤ 0.01), but total short-chain fatty acids (SCFA) concentration, as well as concentration of other SCFA, did not differ between treatments (P ≥ 0.07). Relative to BW, reticuloruminal tissue mass tended (P = 0.09) to be greater and omasal digesta was less (P = 0.02) for BUT compared with CTRL. Dietary butyrate did not affect ruminal papillae length, width, and density nor did it affect ruminal epithelium thickness (P ≥ 0.12) in the ventral sac of the rumen. However, the DM of ruminal epithelium (mg/cm2) tended (P = 0.06) to be greater for BUT compared with CTRL. Omasal and abomasal epithelium thicknesses were greater (P ≤ 0.05) for BUT compared with CTRL. Mitosis-to-apoptosis ratio in the abomasal epithelium was less for BUT compared with CTRL (P = 0.04). Finally, the mRNA expression of peptide transporter 1 in the omasal epithelium was less (P = 0.02) and mRNA expression of monocarboxylate transporter 1 in the abomasal epithelium tended (P = 0.07) to be greater for BUT compared with CTRL. It can be concluded that exogenous butyrate supplementation affected not only the rumen but also omasum and abomasum in sheep.

Acidic pH and short-chain fatty acids activate Na+ transport but differentially modulate expression of Na+/H+ exchanger isoforms 1, 2, and 3 in omasal epithelium

Low sodium content in feed and large amounts of salivary sodium secretion are essential requirements to efficient sodium reabsorption in the dairy cow. It is already known that Na+/H+ exchange (NHE) of the ruminal epithelium plays a key role in Na+ absorption, and its function is influenced by the presence of short-chain fatty acids (SCFA) and mucosal pH. By contrast, the functional role and regulation of NHE in omasal epithelium have not been completely understood. In the present study, we used model studies in small ruminants (sheep and goats) to investigate NHE-mediated Na+ transport and the effects of pH and SCFA on NHE activity in omasal epithelium and on the expression of NHE isoform in omasal epithelial cells. Conventional Ussing chamber technique, primary cell culture, quantitative PCR, and Western blot were used. In native omasal epithelium of sheep, the Na+ transport was electroneutral, and it was inhibited by the specific NHE3 inhibitor 3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride, which decreased mucosal-to-serosal, serosal-to-mucosal, and net flux rates of Na+ by 80% each. The application of low mucosal pH (6.4 or 5.8) in the presence of SCFA activated the Na+ transport across omasal epithelium of sheep compared with that at pH 7.4. In cultured omasal epithelial cells of goats, mRNA and protein of NHE1, NHE2, and NHE3 were detected. The application of SCFA increased NHE1 mRNA and protein expression, which was most prominent when the culture medium pH decreased from 7.4 to 6.8. At variance, the mRNA and protein expression of NHE2 and NHE3 were decreased with low pH and SCFA, which was contrary to the published data from ruminal epithelial studies. In conclusion, this paper shows that (1) NHE1, NHE2, and NHE3 are expressed in omasal epithelium; (2) NHE3 mediates the major portion of transepithelial Na+ transport in omasal epithelium; and (3) SCFA and acidic pH acutely activate Na+ transport but suppress the expression of NHE2 and NHE3 in the longer term. By contrast, the expression of NHE1 is increased by SCFA and acidic pH, indicating a prominent role for NHE1 in the regulation of intracellular pH of omasal epithelium. Our results suggest a regulatable Na+ absorption in ruminal and omasal epithelium. It is of benefit for intracellular pH homeostasis and highly relevant to dairy cows fed on high-concentrate diets.

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.

Macroscopic and Microscopic Anatomy of the Omasum of the Baladi Goat

Scanning and transmission electron microscopy were used to study anatomical and histological features of the omasum in Baladi goats. Omasal laminae were arranged in four or five orders and varied by shape, distribution and direction of papillae. Spaces were present in the interpapillary and papillary surfaces of the omasal laminae as well as at the tips of some conical papillae. The omasal epithelium was keratinized stratified squamous consisting of four strata basalis; spinosum; granulose and cornum. Cells in the stratum basalis contained mitochondria and free ribosomes and had finger-like cytoplasmic processes that connected with neighboring cells via desmosomes. The stratum spinosum was rich in tonofilaments, free ribosomes, and cisternae of rough endoplasmic reticulum (rER). The cytoplasm of cells in the stratum granulosum was marked by keratohyalin granules of varying sizes with electron-dense contents. The stratum corneum consisted of several layers of flat, elongated cornified cells with varied cellular constituents. This study identified anatomical and histological features that may be responsible for increased absorption of water and nutrients from the leafy material ingested by Baladi goats,

Sodium transport across the isolated epithelium of sheep omasum is influenced by luminal ammonia.

Ammonia is a physiological fermentation product in the forestomachs and is absorbed from the rumen and omasum. Cellular uptake of ammonia affects the intracellular pH of polar and non-polar cells. The effect of the uptake on the pH of the cytosol depends on the predominant form of ammonia. NH(3) uptake and its intracellular protonation tend to alkalinize the cytoplasm, whereas the uptake of NH(4)(+) acidifies the cytoplasm by reversing this reaction. Consequently, the absorption of ammonia across the omasal epithelium could cause a change of the intracellular pH and pH-dependent transport mechanisms like Na/H exchange. Because no information is available about the form of ammonia absorbed in the omasum and, hence, possible modulation of Na transport by ammonia, the effect of increasing luminal ammonia concentrations (0, 5, 15 and 30 mmol/l) on Na transport were studied. In epithelia of hay-fed animals, ammonia linearly inhibited Na transport in a dose-dependent manner, at a luminal pH of 7.40, but not at a pH of 6.40. Ammonia did not influence Na transport in epithelia of concentrate-fed animals. Because luminal ammonia did not consistently change the short circuit current or tissue conductance absorption of ammonia as NH(4)(+) appears to be unlikely. The predominant form of ammonia absorbed in the omasum is probably NH(3), which is protonated in the cytosol. The reduced availability of protons may be the cause of inhibition of Na transport via Na/H exchange.

Absorption of 2-hydroxy-4-(methylthio)butanoic acid by isolated sheep ruminal and omasal epithelia.

Alimet (Novus Inter., Inc., St. Louis, MO) feed supplement (an 88% aqueous solution of 2-hydroxy-4-(methylthio) butanoic acid; HMB) is a source of L-Met commonly used in nonruminants and ruminants. The absorption of HMB across ovine omasal and ruminal epithelia was evaluated in this study. Ruminal and omasal epithelia were collected from eight lambs (BW = 67.6 kg +/- 9.1) and mounted in parabiotic chambers that were repeatedly sampled throughout a 60-min incubation. The appearance of HMB (using DL-[5-14C]-HMB as a radiolabeled marker) in serosal buffers increased quadratically (P < .004) with time in both tissues. More (P < .001) HMB appeared in the serosal buffers with omasal than with ruminal epithelia. Both tissues responded similarly, and, after 60 min of incubation, the accumulation of HMB within the tissues increased linearly (P < .001) as substrate concentration (.375, .75, 1.5, 3.0, 6.0, and 12.0 mM) increased in mucosal buffers. As the concentration of HMB in the mucosal buffers increased, there was a quadratic (P < .001) increase in the appearance of HMB in the serosal buffer of the omasal epithelium, indicating some saturation of the system. The increase in serosal appearance of HMB was linear (P < .001) with ruminal tissue. The results indicate that there are probably multiple mechanisms involved in the absorption of HMB. Because saturation was observed in the omasum, it is likely that mediated transport accounts for at least a portion of the absorption of HMB in the omasum. Other mechanisms (e.g., diffusion and(or) paracellular absorption) are responsible for the balance of the absorption. Omasal epithelium appears to have a greater capacity for HMB absorption than ruminal epithelium. The enzymes involved in the conversion of HMB to 2-keto-4-(methylthio)butanoic acid were found in ruminal and omasal epithelia, liver and kidney. These results indicate that HMB can be absorbed across ruminal and omasal epithelium and that HMB can be used as a source of L-methionine.

Glycyl-L-sarcosine absorption across ovine omasal epithelium during coincubation with other peptide substrates and volatile fatty acids.

To define the interactions between the absorption of glycyl-L-sarcosine (Gly-Sar; .1 mM) and glycine, L-methionylglycine, glycyl-L-leucine, L-carnosine, or L-methionylglycyl-L-methionyl-L-methionine (each at 5 mM), ovine omasal epithelium was collected from eight wethers (average BW=69+/-8.2 kg) and mounted in parabiotic chambers. [1,2]-[14C]Glycyl-L-sarcosine was used as a marker to monitor the presence of Gly-Sar. The Gly-Sar concentration in the omasal epithelium after 60 min of incubation was greatest (P < .05; .0055 nmol/mg dry tissue) when only Gly-Sar was present. Glycine inhibited (P < .05) Gly-Sar movement through the tissue by 20%, and peptide substrates inhibited (P < .05) Gly-Sar movement by 60 to 85%. The appearance of Gly-Sar in serosal buffers increased quadratically (P < .001) with time. Numerically, Gly-Sar appearance in serosal buffers was stimulated by the presence of glycine and peptide substrates. In a second experiment, ovine omasal epithelium was collected from four lambs (average BW=47+/-6.0 kg) to determine the interactions of Gly-Sar absorption (.1 mM) alone or when coincubated with either 10 mM butyric acid, or with a mixture of VFA (50 mM acetic acid, 40 mM propionic acid, and 10 mM butyric acid). The movement of Gly-Sar through the omasal epithelium was greatest (P < .05) when only Gly-Sar was present, and the VFA mixture inhibited (P < .05) Gly-Sar movement by 84%. Results from these studies support the idea that peptides can be absorbed across omasal epithelium and that the process involves mediated as well as nonmediated mechanisms, including possibly paracellular transport.

Poly(A)+ RNA from sheep omasal epithelium induces expression of a peptide transport protein(s) in Xenopus laevis oocytes.

To verify research from this laboratory indicating that sheep omasal epithelium contains mRNA encoding for a peptide transporter(s) and to determine di- to octapeptide transport capability, we injected poly(A)+ RNA isolated from sheep omasal epithelium into Xenopus laevis oocytes. Poly(A)+ RNA was functionally expressed in Xenopus oocytes 4 to 7 d after injection. Peptide (5 di-, 10 tri-, 6 tetra-, 2 penta-, 1 hexa-, 1 hepta-, and 1 octapeptide) transport capability was measured by impaling oocytes with a microelectrode to monitor membrane potential (Vm). Oocytes were maintained in pH 5.5 buffer. Peptide transport was identified as being expressed when, in the presence of a buffered peptide substrate (1 mM), the oocyte membrane showed persistent depolarization (a more positive Vm). In the absence of peptide transport, the membrane became depolarized with the addition of buffered substrate, but it rapidly repolarized to the resting potential. Peptide transport was expressed for some di-, tri-, and tetrapeptides. Measured depolarization ranged from 9.6 mV to 42.1 mV. Larger peptides were not transported by the oocytes. When transport expression was measured with the substrates in a pH 7.5 buffer, no transport occurred, indicating that transport was dependent on a proton gradient. Thus, sheep omasal epithelium contains mRNA that codes for a protein(s) capable of proton-dependent di-, tri-, and tetrapeptide transport. Results from the present study provide further evidence that absorption of peptides from the ruminant stomach is possible.

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.

Development of the omasum in sheep.

Data are presented on the histogenesis of the omasal mucosa in sheep from the 2.5 cm crown-rump (c-r) length fetus to the adult. 11 stages of fetal development, and 4 post-natal stages, were studies. The distribution of glycogen in the omasal epithelium was also studied. During fetal life the omasal epithelium was initially stratified cuboidal in type, but the superficial layers of cells became flattened in later stages of gestation. This epithelium became extremely thick by the late stages of fetal life, reaching a maximum of 358 micron, and consisting of greater than 20 layers of cells, in the 45 cm c-r fetus (approximatelay 140 days). After birth the epithelium became markedly reduced in thickness, being approximately 77 micron in the adult, and had differentiated into a cornified stratified squamous epithelium of the adult type by 12 weeks after birth. Glycogen was extremely abundant in the omasal epithelium of the 2.5 cm fetus, and declined gradually thereafter to be almost completely absent in post-natal specimens. 4 orders of laminae were present in the adult omasum, distributed in the seqeunce 1-4-3-4-2-4-3-4-1. The 1st order was already present in fetuses of 2.5 cm c-r length, with the 2nd, 3rd and 4th appearing by the 3.5, 5.5 and 11.0 cm stages, respectively. Initial stages in the development of conical papillae were first seen in 15.0 cm fetuses, but the development of these papillae was not completed until after birth.

RNA and DNA of gastric and duodenal mucosa in antrectomized and gastrin-treated rats.

RNA and DNA of gastric and duodenal mucosa in antrectomized and gastrin-treated rats.

Absorption of Organic Acids from the Omasum ,

Summary Absorption of volatile fatty acids (VFA) and lactic acid from the omasum of 4- or 5- mo.-old Holstein calves was studied by two methods. The first consisted of a study of the relative concentrations of these acids in the contents of the stomach compartments of six calves in relation to concentrations of chromic oxide remaining from previous oral adininistration. On a dry matter basis, levels of chromic oxide increased in order from the rumen, to the reticulum, to the omasum, to the abomasum; and, in the same order, VFA declined. The ratios of VFA to chromic oxide indicated a decrease in VFA of 51% from the reticulum to the omasum and a further decrease of 83% from the omasum to the abomasum. The second method involved injection of sodium-n-butyrate-1-C 14 into the exteriorized omasum of two calves. Organic acid levels and distribution of C 14 activity were determined on samples of blood drawn simultaneously from the omasal vein and the carotid artery at various time intervals during 1 hr. following injection. The animals then were sacrificed and concentrations of acids and distribution of label in the ingesta of the stomaeh compartments were studied. Butyrate-C 14 was absorbed rapidly from the omasum into the blood of the omasal vein, peak concentrations appearing at 5 to 10min. after injection. Labeled metabolite continued to be absorbed throughout the experiment. Conversion of butyrate to other VFA occurred in the omasum, as indicated by the presence of label in the acids isolated from ingesta in that organ. The appearance of 50% of the recovered label in omasal blood in the form of lactic acid suggests an unknown pathway for the conversion of butyrate to lactate by omasal epithelium. Average concentrations of butyric, propionic, and acetic acids were greater in the omasal vein blood than in the carotid artery blood; the reverse was true for lactic acid. It is concluded that absorption of organic acids does occur from the omasum and apparently in considerable magnitude.