Steroidogenic enzyme expression in estrogen production in the goat gastrointestinal (GI) tract and the effect of castration.

Extragonadal tissues are known to produce estrogens. At these sites, the C19 precursor is important for aromatase expression for the production of estrogen. Aromatase expression is tissue-specific and is controlled by hormones. Recent studies have shown that rat gastric parietal cells expressed aromatase. Our first objective was to investigate steroidogenic enzyme expression in estrogen biosynthesis; the second objective was to investigate which site(s) of the GI tract expressed steroidogenic enzymes; and the third objective was to assess the effects of castration on steroidogenic enzyme expression. CYP19A1, 17ß-HSD3, CYP17A1, 3ß-HSD and P450scc were quantified in the GI tract by real-time PCR. CYP19A1 was detected mainly in the body and pyloric regions of the abomasum, while we detected weak expression of CYP19A1 in other parts of GI tract. In addition, the expression of 17ß-HSD3 and CYP17A1 was detected in abomasum. 3ß-HSD expression was observed in duodenum and jejunum, while P450scc was not detectable in any part of GI tract. Immunohistochemical results showed immunolocalization of aromatase in parietal cells. Aromatase expression was observed to increase after castration. Furthermore, immunohistochemical results demonstrated that parietal cells also produced luteinizing hormone receptor (LHR). These results indicate steroidogenic enzymes required for the biosynthesis of estrogen were expressed, and the abomasum appeared to be the responsible organ for estrogen biosynthesis in the goat GI tract. In addition, parietal cells were responsible for estrogen production and the expression of LHR. Castration increased aromatase expression in abomasum through LH mediation.

Short communication: Sensory profile of raw goat milk cheeses made with artisan kid rennet pastes from commercial-weight animals: alternative to farmhouse goat cheeses.

The loss of traditional kid rennet pastes in the Canary Islands (Spain), as in many other regions, is most likely due to the custom of using abomasa from very young animals killed below desirable commercial weight. In addition, the reasonable price of commercial rennets (CR) has resulted in the loss of typical sensory characteristics for most farmhouse raw goat milk cheeses, placing them at a disadvantage when local and international markets are full of different cheeses, often with aggressive marketing strategies. This paper analyzes the sensory characteristics of raw goat milk cheeses made with rennet pastes prepared from commercial kid abomasa in 2 ways: dried while full of ingested milk [full, commercial, artisan kid rennet (FCKR)], or dried after being emptied of ingested milk and refilled with raw goat milk [empty, commercial, artisan kid rennet (ECKR)]. This latter practice allows the use of empty abomasa, or abomasa with grass, soil, and so on. Sensory profiles of cheeses made with FCKR and ECKR rennets were compared with those made with CR by an expert panel (n=7). The FCKR and ECKR cheeses had similar sensory profiles. Although scores for FCKR cheeses were somewhat higher than for ECKR cheeses, they were in the range found for traditional cheeses made with rennet prepared with abomasa from very young animals. The sensory profile of CR cheeses was very different. Almost 90% of consumer panelists (n=90) preferred cheeses made with the experimental rennet pastes. These results demonstrate the possibility to prepare artisan rennet pastes from commercial-weight kids in an easy way for farmhouse cheese makers using local resources that would otherwise be destroyed in abattoirs.

Fluorescein thiocarbamoyl-kappa-casein assay for the specific testing of milk-clotting proteases.

Milk-clotting proteases, which are widely used in the cheese-making industry, are enzymes that use soluble caseins as their preferential substrates. Here, we propose a modification to a method previously described for the specific determination of milk-clotting proteases by using kappa-casein labeled with fluorescein isothiocyanate as substrate. Validation of the modified method was confirmed using natural bacterial, fungal, plant, and animal milk-clotting proteases, as well as a milk-clotting enzyme of recombinant origin. The new modified method described here allowed specific quantification of the activity of milk-clotting proteases in a very sensitive way and permitted determination of the appropriate kinetic parameters of all the enzymes tested, consistent with their origin and degree of purity.

Purification and characterization of milk clotting enzyme from goat (Capra hircus).

Chymosin, the major component of rennet (milk clotting enzyme), is an acid protease produced in the fourth stomach of milk-fed ruminants including goat and sheep in the form of an inactive precursor prochymosin. It is responsible for hydrolysis of kappa-casein chain in casein micelles of milk and therefore, used as milk coagulant in cheese preparation. The present investigation was undertaken to purify and characterize goat (Capra hircus) chymosin for its suitability as milk coagulant. The enzyme was extracted from abomasal tissue of kid and purified nearly 30-fold using anion exchanger and gel filtration chromatography. Goat chymosin resolved into three major active peaks, indicating possible heterogeneity when passed through DEAE-cellulose ion exchange column. The purified enzyme had a molecular mass of 36 kDa on SDS-PAGE, which was further confirmed by Western blot analysis. The purified enzyme preparation was stable up to 55 degrees C with maximum activity at 30 degrees C. The milk clotting activity was decreased steadily as pH is increased and indicated maximum activity at pH 5.5. Proteolytic activity of goat chymosin increased with incubation time at 37 degrees C. Goat chymosin was found to be more thermostable than cattle chymosin and equally stable to buffalo chymosin.

Purification and characterization of chymosin and pepsin from kid.

The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3-6 zones at pH 4.6-5.1, while that of kid pepsin was at pH < or =3.0. Kid pepsin contained 0.37 molecules phosphorous per molecule and was totally inhibited by 5 muM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5.6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin

Resistance of rumen bacteria murein to bovine gastric lysozyme.

BACKGROUND: Lysozymes, enzymes mostly associated with defence against bacterial infections, are mureinolytic. Ruminants have evolved a gastric c type lysozyme as a digestive enzyme, and profit from digestion of foregut bacteria, after most dietary components, including protein, have been fermented in the rumen. In this work we characterized the biological activities of bovine gastric secretions against membranes, purified murein and bacteria. RESULTS: Bovine gastric extract (BGE) was active against both G+ and G- bacteria, but the effect against Gram- bacteria was not due to the lysozyme, since purified BGL had only activity against Gram+ bacteria. We were unable to find small pore forming peptides in the BGE, and found that the inhibition of Gram negative bacteria by BGE was due to an artefact caused by acetate. We report for first time the activity of bovine gastric lysozyme (BG lysozyme) against pure bacterial cultures, and the specific resistance of some rumen Gram positive strains to BGL. CONCLUSIONS: Some Gram+ rumen bacteria showed resistance to abomasum lysozyme. We discuss the implications of this finding in the light of possible practical applications of such a stable antimicrobial peptide.

Isolation, purification and characterization of chymosin from riverine buffalo (Bubalos bubalis).

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29-fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35.6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 degrees C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7.0. The ratio of milk clotting to proteolytic activity was 3.03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.

Effect of enhancing curd formation during the first colostrum feed on absorption of gamma glutamyl transferase by newborn calves.

OBJECTIVE: To examine the effects of curd formation within the abomasum, on the absorption of gamma glutamyl transferase (GGT) from colostrum in newborn calves. DESIGN: An in vivo physiological study with controls, and in vitro examination of calf abomasal fluid. PROCEDURES: Newborn calves were taken from cows without allowing them to suckle. They were fed either 1.5 kg colostrum or 1.5 kg colostrum plus rennet, with intervals between calving and colostrum feeding ranging from 0.4 to 12.7 h. Absorption of proteins from the whey component of colostrum was assessed from the rise in activity of serum GGT. In in vitro studies, colostrum was incubated with bovine amniotic fluid, newborn calf abomasal fluid or newborn calf forestomach contents, with or without rennet, to test the curd inhibiting effects of components in the abomasal fluid of newborn calves. RESULTS: In vivo: addition of rennet to the colostrum feed reduced the proportion of calves with serum GGT activity below 500 U/L by 60%. In vitro: 43% of newborn calves lacked curd forming activity in their abomasal fluid, and that deficiency was corrected by adding rennet to the incubation medium. CONCLUSIONS: Some calves are born with low amounts of curd forming enzyme activity in the abomasum. This may compromise their ability to absorb large whey proteins from the first feed of colostrum. Adding rennet to the first colostrum feed may improve passive immunity in those calves.

Effects of nutritional conditions around weaning on carbonic anhydrase activity in the parotid gland and ruminal and abomasal epithelia of Holstein calves.

Thirty-two male Holstein calves were used to investigate the effects of nutritional conditions around weaning and aging on carbonic anhydrase (CA) activity in the parotid gland and epithelium from the rumen and abomasum. We fed calf starter and lucerne hay as well as milk replacer (group N) or fed milk replacer either with (group S) or without (group M) administration of short-chain fatty acids (SCFA) through polypropylene tubing into the forestomach until 13 weeks of age. The diets were fed at 1000 hours and 1600 hours, and SCFA were administrated after milk replacer feeding at 1600 hours. Slaughter and tissue sampling were carried out between 1300 hours and 1430 hours at 1, 3, 7, 13, and 18 weeks of age. Tissue samples from five adult (1.5-2.0 years-old) Holstein steers were obtained from a local abattoir. In group N, CA activity in the parotid gland gradually and significantly increased toward the adult value, whilst in the epithelium from the rumen and abomasum, adult values were reached at 3 and 7 weeks of age, respectively. At 13 weeks, the activity for group N was significantly higher than that for the other two groups in the parotid gland, but there was no significant difference in the epithelium from the rumen and abomasum. The concentration of the carbonic isozyme VI in the parotid gland also changed with age but, in contrast to CA activity, had not reached adult levels by 13 weeks of age. In groups M and S, parotid saliva did not show any change toward an alkaline pH or toward a reciprocal change in the concentrations between Cl(-) and HCO(3)(-), even at 13 weeks of age. From these results we conclude that a concentrate-hay based diet around weaning has a crucial role in CA development in the parotid gland, but not in the epithelium of the rumen and abomasum.

Immunohistochemical study of the ontogeny of prochymosin--and pepsinogen-producing cells in the abomasum of sheep.

The appearance and development of prochymosin- and pepsinogen-producing cells were investigated in the ovine abomasum from fetus to adult using immunohistochemistry. Prochymosin immunoreactivity appeared first in the proper gastric glands of the 100-day-old fetus. The intensity and distribution of prochymosin-immunoreactive cells increased gradually with the progress of gestation, and their most intense immunoreactivities and widest distribution were observed in 3-day-old lambs. They were subsequently reduced throughout postnatal growth. A few prochymosin-immunoreactive cells were scattered in the glands of adult sheep. Pepsinogen immunoreactivity appeared at first in a small number of cells in the base of some proper gastric glands of 120-day-old fetuses. After 130 days, pepsinogen-immunoreactive cells increased their staining intensities and distribution. They reached a peak in area at 21 days, which is comparable to adult sheep. In the pyloric glands, prochymosin- and pepsinogen-immunoreactive cells appeared from 100 and 130 days, respectively. Numbers were reduced in comparison to gastric glands and their occurrence was capricious. The results demonstrated that the ontogeny of prochymosin- and pepsinogen-immunoreactive cells in the abomasum of sheep is more similar to that in cattle than to that in the goat. The present data will contribute to the overall understanding of the development of ruminant gastric proteases.

The effects of vagotomy on the abomasum in calves: radiography and protein gene product 9.5 immunohistochemistry.

Abomasal disorders of calves with total vagotomy, operated on at 1 week old, were investigated with radiography and protein gene product (PGP) 9.5 immunohistochemistry. Radiographic findings indicated abomasal atony with dilatation in all calves 2 weeks after vagotomy, while 4 weeks after vagotomy abomasal dilatation was detected in 2 calves and another 2 calves showed dilatation and impaction. The densities of PGP 9.5-immunoreactive nerves in the tunica muscularis decreased significantly in the corpus region of the greater curvature 2 weeks after vagotomy and in the pyloric region of the lesser curvature 4 weeks after vagotomy, and it was at its lowest 4 weeks after vagotomy in all regions examined. In conclusion, abomasal dilatation and/or impaction in vagotomized calves confirmed by radiography were related with a decreased frequency of nerves in the tunica muscularis of the abomasum.

Polyamines in the gastro-intestinal tract of goat kids and in the regenerating ruminal epithelium of sheep.

The aim of the present study was to investigate whether temporal changes in polyamine concentration and synthesis could be found in the luminal content and wall tissue of the rumen and abomasum, two organs which have entirely different growth patterns during the first month of life. In the abomasal mucosa there was a marked gradual decrease in the ornithine decarboxylase (ODC) activity during the first month of life, while the ODC activity in the ruminal mucosa was low during the whole experimental period. However, injury of the rumen wall was followed by increased ODC activity. The ODC activity in duodenal mucosa was about 10 times higher than in the ileal mucosa and the ruminal epithelium. In ruminal liquid a clear peak in ODC activity was observed during the period 51-70 days after birth. The polyamine concentration did not parallel the ODC activity, in either the ruminal epithelium or the ruminal liquid. Of the polyamines, the spermine concentration was always highest, and with the exception of duodenal mucosa, the putrescine concentration was lowest. In liver a clear decrease in spermidine concentration from day 1 to about day 60 after birth was observed. Otherwise no marked temporal changes in tissue polyamine concentrations were observed. Two and a half hours after oral administration of 14C-labelled spermine, nearly all of the radioactivity was found in the lumen of the gastrointestinal tract. On the other hand, 1 h after intravenous injection of polyamines the walls of the gastrointestinal tract were strongly labelled. In conclusion, the polyamines needed for ruminal epithelial development seem to come from sources other than the ruminal epithelium itself or the ruminal lumen.

Physicochemical, molecular and immunological characterization of camel calf rennet: a comparison with buffalo rennet.

Camel calf rennet (CCR) and buffalo calf rennet (BCR) were prepared from dried abomasa to study their physicochemical properties and electrophoretic behaviour and to carry out an immunological characterization of the rennet proteins. CCR was more thermostable than BCR. The milk clotting activity of both rennets increased as pH decreased. The optimum temperatures for CCR and BCR were 50 and 45 degrees C respectively. CCR was more sensitive to increased CaCl2 in milk than BCR. Addition of NaCl to milk in the range 0-100 g/l resulted in a marked decrease in the clotting activity of both rennets. When the rennets were treated with acetone, the activity of BCR was completely destroyed, but that of CCR was unaffected. The proteolytic activity of CCR was higher than that of BCR and pepsin towards both camel and cows' milk caseins at pH 6.0. SDS-PAGE electrophoretic patterns of CCR and BCR proteins gave two major bands with molecular masses estimated as 52 and 39 kDa for CCR and 50 and 35 kDa for BCR. Immunodiffusion and immunoelectrophoresis using anti-CCR serum demonstrated immunological cross reactivity between CCR and BCR.

In vitro motility disorders associated with displaced abomasum in dairy cows.

The objective of this study was to investigate in vitro abomasal motility in dairy cows diagnosed with displaced abomasum. Longitudinal muscle myenteric plexus preparations originating from the abomasal antrum of control cows, and cows diagnosed with left displaced abomasum (LDA), right displaced abomasum (RDA) or abomasal volvulus (AV) were used. In control preparations electrical field stimulation evoked an immediate cholinergic contractile response exceeding amplitude of basal contractions by 60%. In contrast, contractile activity was significantly inhibited during electrical stimulation in LDA, RDA and AV by 47%, 66% and 45%, respectively. This inhibition was reversed in the presence of L-NAME. The staining intensity of NADPH-positive myenteric neurones was significantly higher in displaced abomasa than in controls. Concentration-response curves indicated that preparations from displaced abomasa showed reduced sensitivity to acetylcholine. This study demonstrated motility disorders in displaced abomasa in vitro. The results suggested that abomasal displacement is associated with malfunctions at the level of the intrinsic nervous system combined with impaired cholinergic muscle responses. There appeared to be a predominance of nitrergic inhibitory mechanisms over excitatory mechanisms. These results might be of significance for diseases associated with gastric hypomotility and emptying disorders.

Changes in the zymogenic cell populations of the abomasa of sheep infected with Haemonchus contortus.

The effects of dietary urea supplementation and of a 10-week trickle infection regime, simulating chronic exposure to Haemonchus contortus, on the zymogenic population of the abomasa of Hampshire Down lambs was examined. At necropsy a variety of parameters including plasma pepsinogen concentration, the wet weights of abomasal fundic mucosal pieces and the amounts of pepsinogen contained in them, were assessed. Tissue pepsinogen concentration was measured as the total, acid-stable proteolytic activity present in mucosal homogenates, as well as immunohistochemically. The immunohistochemical findings were quantified using computer-aided image analysis. Elevation of plasma pepsinogen concentrations in infected animals was of borderline significance (P = 0.06). The fundic mucosae of infected animals were heavier (P < 0.02) than those of control animals, but there was no overall change in the pepsinogen content of tissues. Immunohistochemistry revealed that infected animals had increased numbers of zymogenic cells, due to mucous cell hyperplasia and the adaptation of cells to produce both mucins and pepsinogen. The pepsinogen content of chief cells, the major source of pepsinogen in uninfected animals, was reduced in infected lambs. Image analysis confirmed that at a mid-point of the mucosa of infected animals there was increased pepsinogen-specific immunoreactivity that corresponded with areas of mucosal hyperplasia. Mucous cell hyperplasia might therefore allow the maintenance of pepsinogen secretion in infected animals even if chief cell output is reduced.

The isolation and purification of a dual specific mast cell-derived protease from parasitised caprine jejunal tissue.

A mast cell granule protease has been isolated and purified from nematode-infected caprine jejunal homogenate by FPLC techniques and termed Goat Mast Cell Protease (GMCP). The purification steps were monitored for proteolytic activity against the synthetic substrate carboxybenzoyl-L-lysine thiobenzyl ester (BLT) and the presence of a homogenous protease preparation in the final sample was shown by SDS-PAGE electrophoresis. This protease was compared with enzymatic activity from isolated mucosal mast cells, which demonstrated the putative mast cell-derived source of the purified enzyme. Rabbit antiserum was raised against the protease and through the use of immunohistochemistry and Western blotting techniques the mast cell origin of the protease was confirmed. NH2-Terminal amino acid sequence analysis demonstrated a high degree of homology between GMCP and other previously isolated mast cell proteases including sheep mast cell protease (SMCP). Substrate analysis showed that GMCP also had an unusual dual chymotrypsin-like and trypsin-like activity similar to SMCP and bovine duodenase.

Ultrastructure of the zymogenic-cell lineage in abomasal mucosa of adult cattle.

The ultrastructural differentiation and maturation of the neck cells and the zymogenic cells during physiological cell renewal were investigated in the abomasal oxyntic-gland region of cattle. Immature neck cells of the distal isthmus and proximal neck exhibit transitional morphology to the predominantly mucous isthmus cells. Neck cells confined to the glandular neck are characterized by bipartite peptic-cored mucous secretory granules. In a proximal-distal gradient along the neck, a progressive increase in the peptic granular component and concomitant reduction in mucous components paralleled by proliferation of the rough endoplasmic reticulum creates pre-zymogenic cells in the proximal glandular base. These, in turn, give rise to mature zymogenic cells with pure peptic secretory granules and typical zymogenic cell morphology. In the depth of the gland, older degenerative zymogenic cells are found. Variations in size and number of the zymogenic granules point to different secretory activities of the mature zymogenic-cell population of the glandular base. These results favour the conception of a zymogenic-cell lineage arising within the isthmus and passing through different developmental stages, including neck cells, during their migration down the gland.

Heterogeneity and migration-related zonation of K(+)-ATPase activities in the oxyntic cell lineage of adult cattle.

Activities of the H+, K(+)-ATPase and the Na+, K+, ATPase have been localized in the morphologically heterogeneous oxyntic cell lineage of adult bovine abomasal mucosa, by means of K(+)-dependent paranitrophenylphosphatase (K(+)-pNPPase) histochemistry. At the light- and electron-microscopic level, only members of the mature oxyntic cell population within the oxyntic glandular base exhibit strong enzyme activity. Superficial oxyntic cells of the proximal isthmus and deep pit, arising from the upward migration of precursor cells and commonly supposed to have a high capacity of secreting acid, show weak or no enzyme activity. This is also true of the immature and pre-oxyntic cells of the generative zone. Global enzyme activity varies among the mature glandular oxyntic cell population. Ultracytochemically, strong H+, K(+)-ATPase (ouabain-insensitive K(+)-pNPPase) activity is associated with the apical plasmalemmal and expanded canalicular membrane in contrast to Na+, K(+)-ATPase (ouabain-sensitive K(+)-pNPPase) activity, which is localized on the basolateral plasmalemmal folds. In both cases, histochemical deposition is confined to the cytoplasmic aspect of the membranes. These results suggest a functional zonation and position-dependent heterogeneity of the oxyntic cell lineage related to the bidirectional mode of migration of pre-oxyntic cells during physiological cell renewal. Functional heterogeneity within the mature glandular oxyntic cell population is in accordance with the continuous mode of gastric acid secretion in cattle.

Prochymosin polymorphism in calves of black-and-white cattle and their crosses with Simental bulls.

Polymorphism of prochymosin was observed in individual calf abomasa, using agarose gel electrophoresis followed by detection of proteolytic activity. Abomasum samples were randomly collected during slaughtering from 239 and 146 calves (3-5 weeks old) of Black-and-White cattle and their crosses with Simental bulls, respectively. Four distinct prochymosins were found and, according to their decreasing electrophoretic mobility in alkaline agarose gel, termed as prochymosin A, D, B and C which occurred singly and in pairs (then with equal proteolytic activities of both components). Prochymosin A, B and C (designation according to FOLTMANN, 1966) activated at pH 4.7 was transformed into electrophoretically distinct chymosin. When prochymosin D was activated at this pH, chymosin D showed similar mobility as chymosin B both at alkaline and acidic pHs. Prochymosin variants occurred at genetical equilibrium in nine and ten phenotypes in the first and second genetic group. The distribution of phenotypes in the two groups differed significantly (P < 0.05). The gene frequencies of prochymosin A, D, B and C were 0.35, 0.11, 0.52 and 0.02 in Black-and-White calves, and 0.39, 0.08, 0.47 and 0.06 in crosses, respectively. These prochymosins were controlled by four pairs of codominant alleles. A possible correlation of the results obtained by FOLTMANN (1966) with ours and those of ASATO and RAND (1972, 1977) was discussed.