In vitro ruminal biotransformation of benzimidazole sulphoxide anthelmintics: enantioselective sulphoreduction in sheep and cattle

The comparative in vitro sulphoreduction of the (+) and (-) enantiomers of albendazole sulphoxide (ABZSO) and oxfendazole (OFZ) by ruminal fluid obtained from sheep and cattle, was investigated, under anaerobic conditions, in this study. Ruminal fluid samples were obtained from Holstein steers fitted with a permanent rumen fistula and from Corriedale lambs via an oesophageal tube. Albendazole sulphoxide, incubated as either the racemic (rac) mixture or as each individual enantiomeric form, was extensively sulphoreduced to form albendazole (ABZ) by ruminal fluid from both species. The concentrations of ABZ formed at different incubation times were between 55 and 158% greater after the incubation of cattle ruminal fluid with (+) ABZSO, compared with that produced when (-) ABZSO was the incubated substrate. Similarly, the concentrations of ABZ were 1.3--3.0-fold higher when (+) ABZSO was incubated with sheep ruminal fluid. Significantly higher rates of depletion were observed for the (+) enantiomeric form when ABZSO was incubated with ruminal fluid from both species. The rates of ABZ formation from both ABZSO enantiomeric forms were significantly higher in sheep compared with cattle ruminal fluid. Fenbendazole (FBZ) was the metabolite formed after the incubation of the racemic form of OFZ with ruminal fluid obtained from both species. The metabolic profile of both OFZ enantiomers followed a similar pattern to that observed for ABZSO enantiomers. A bi-directional chiral inversion of one enantiomer into its antipode was observed. The (+) enantiomer appeared in the incubation medium when (-) ABZSO was the incubated substrate, and also the (-) antipode was detected after (+) ABZSO incubation with ruminal fluid obtained from both species. The results reported here demonstrate an enantioselective ruminal sulphoreduction of ABZSO and OFZ (substrate enantioselectivity). These findings contribute to interpret the chiral behaviour of benzimidazole-sulphoxide anthelmintics.     

The role of calcium in the mechanical performance of cattle ruminal muscle

A viable preparation from the external muscle coat of cattle rumen for in vitro studies of contractility is described. The threshold Ca²⁺ concentration for contractile response in glycerol treated ruminal muscle was found to be pH dependent. At physiological pH it is situated between 10^-8 and 10^-7 M. Strips with intact membranes were studied in the depolarized state. In this preparation contraction to acetylcholine or histamine requires Ca²⁺ in the medium. However, contractility persists for several minutes in Ca²⁺-free solution at rest but disappears rapidly during stimulation. Recovery in a Ca²⁺-containing medium is much faster than the decline of responsiveness in a Ca²⁺-free medium. Tetracaine and D600 seem to inhibit contraction by blocking release of Ca²⁺ from and uptake of Ca²⁺ into hypothetical Ca stores inside the cell. The results are interpreted by assuming cellular Ca stores and two Ca pumps, one extruding Ca²⁺ into the medium and one accumulating Ca in the stores. Acetylcholine and histamine act by increasing Ca²⁺ permeability of both the membranes of the stores and the plasma membrane. The stimulator-induced and possibly the resting Ca²⁺ permeability in the depolarized state is reduced by tetracaine and D600 at both sites. The pumps are assumed not to be affected by stimulators and the mentioned drugs.     

Estimating ruminal microbial efficiencies in silage-fed cattle: comparison of an in vitro method with a combination of in situ and in vivo measurements

The achievement of maximum ruminal feed conversion into microbial biomass is a widely accepted concept of ruminant nutrition because high microbial efficiency improves microbial protein supply to the small intestine and, proportionally, reduces fermentative gaseous carbon losses (Beever 1993; Leng 1993). It has recently been demonstrated that differences between forages in in vitro microbial efficiencies, i.e. differences in the proportion of fermented substrate incorporated into microbial biomass, could be determined by a combination of in vitro gas volume measurements with a concomitant evaluation of the amount of substrate truly degraded during 24 h of incubation (for review see Blümmel et al. 1997a). It has been pointed out that these two measurements are not synonymous but complementary. The measurement of degradability is a modification by Goering and van Soest (1970) of the Tilley and Terry (1963) method to remove any residual microbial biomass from the undegraded substrate, thus allowing the calculation of the total amount of substrate dissimilated into all fermentation products, i.e. microbial biomass, short chain fatty acids (SCFA) and gases. In contrast, the gas volume measurement indicates how much of the fermented substrate was used for the formation of SCFA and gases since these two fermentation products are stoichiometrically very closely associated (Blümmel et al. 1997a). The measurements described above were performed after 24 h of incubation because the analytical approach used required all substrate solubles to already be fermented at the time of the residue determination; they would otherwise be removed by the treatment, leading to an overestimation of substrate degradability. On the other hand the determination should not be conducted too far beyond the microbial peak yield in order to minimize the possible contribution of microbial lysis to gas production. The ratio of truly degraded substrate to the gas volume thereby produced in 24 h was termed partitioning factor (PF). This factor denotes the substrate specific variation of in vitro microbial efficiency. The concept of the PF value was derived from, and applied to, crop residues of temperate and tropical origin (Blümmel et al. 1997b) and to Mediterranean grass and legume hays (Blümmel and Bullerdieck 1997). Forages with a high PF value, i.e. proportionally low gas production per unit of substrate degraded, were related to higher voluntary feed intakes than those with a low PF value. High in vitro PF values were also associated with high excretion of renal purine derivatives in Malawian goats fed maize stover leaves with different PF values but similar digestibilities (Mgomezulu and Blümmel 1996). For 61 straws and hays examined, the production of 1 ml of gas was associated with the concomitant in vitro true degradability of 2.74 to 4.65 mg of substrate i.e. PF values ranged from 2.74 to 4.65 mg/ml of which a minimum of 2.20 mg (Blümmel et al. 1997a) were required for the formation of acetate, propionate, butyrate and fermentative CO₂, CH₄ and H₂O, the latter produced upon the reduction of CO₂ to CH₄ as follows: CO₂+8H > CH₄+2H₂O. Carbon dioxide also arises from buffering the SCFA but this CO₂ is, contrary to the fermentative CO₂, not derived from the incubated substrate but from the buffer medium. Substrate degraded on top of these requirements (2.20 mg/ml) was available for microbial cell synthesis; proportionally more microbial biomass has been synthesized in fermentations having high PF values (Blümmel et al. 1997a). The objective of this work was to further investigate the relationship between in vitro microbial efficiencies as estimated by the PF value and in vivo microbial efficiencies using four perennial rye-grass silages harvested at different stages of maturity. These silages were fed to steers and the in vivo microbial protein synthesis was estimated from renal allantoin excretion, thus providing the opportunity for the comparison of in vitro and in vivo microbial efficiencies. Some aspects of the in vivo work have been reported by Philipczyk et al. (1996) and Schröder et al. (1997).     

In vitro bacterial growth and in vivo ruminal microbiota populations associated with bloat in steers grazing wheat forage

The role of ruminal bacteria in the frothy bloat complex common to cattle grazing winter wheat has not been previously determined. Two experiments, one in vitro and another in vivo, were designed to elucidate the effects of fresh wheat forage on bacterial growth, biofilm complexes, rumen fermentation end products, rumen bacterial diversity, and bloat potential. In Exp. 1, 6 strains of ruminal bacteria (Streptococcus bovis strain 26, Prevotella ruminicola strain 23, Eubacterium ruminantium B1C23, Ruminococcus albus SY3, Fibrobacter succinogenes ssp. S85, and Ruminococcus flavefaciens C94) were used in vitro to determine the effect of soluble plant protein from winter wheat forage on specific bacterial growth rate, biofilm complexes, VFA, and ruminal H2 and CH4 in mono or coculture with Methanobrevibacter smithii. The specific growth rate in plant protein medium containing soluble plant protein (3.27% nitrogen) was measured during a 24-h incubation at 39 degrees C in Hungate tubes under a CO2 gas phase. A monoculture of M. smithii was grown similarly, except under H2:CO2 (1:1), in a basal methanogen growth medium supplemented likewise with soluble plant protein. In Exp. 2, 6 ruminally cannulated steers grazing wheat forage were used to evaluate the influence of bloat on the production of biofilm complexes, ruminal microbial biodiversity patterns, and ruminal fluid protein fractions. In Exp. 1, cultures of R. albus (P < 0.01) and R. flavefaciens (P < 0.05) produced the most H2 among strains and resulted in greater (P < 0.01) CH4 production when cocultured with M. smithii than other coculture combinations. Cultures of S. bovis and E. ruminantium + M. smithii produced the most biofilm mass among strains. In Exp. 2, when diets changed from bermudagrass hay to wheat forage, biofilm production increased (P < 0.01). Biofilm production, concentrations of whole ruminal content (P < 0.01), and cheesecloth filtrate protein fractions (P < 0.05) in the ruminal fluid were greater on d 50 for bloated than for nonbloated steers when grazing wheat forage. The molecular analysis of the 16S rDNA showed that 2 different ruminal microbiota populations developed between bloated and nonbloated animals grazing wheat forage. Bloat in cattle grazing wheat pastures may be caused by increased production of biofilm, resulting from a diet-influenced switch in the rumen bacterial population.     

In vitro ruminal fermentation and in situ ruminal degradation of tamarind kernel powder extract residue in wethers

We examined the in vitro fermentation characteristics and in situ dry matter (DM) and crude protein (CP) degradability of tamarind kernel powder extract residue (TKPER), a by‐product of polysaccharides thickener processing. Two types of TKPER (I and II), of which the CP and neutral detergent fiber organic matter basis contents (%) were 41.4 and 42.0 and 1.4 and 0.5, respectively, were compared with dry heat soybean (SB), soybean meal (SBM) and dry soybean curd residue (SBCR). The TKPERs had significantly lower in vitro gas production compared to the other products at each observation time (P < 0.05). The in vitro DM and CP digestibility (%) of TKPER I and II were 67.7 and 64.9, and 64.5 and 58.0, respectively, significantly lower than those of SB and SBM (P < 0.05). We used four wethers (55.6 ± 4.6 kg) with ruminal cannulas to investigate the in situ degradability of TKPER I, SB, SBM and SBCR. At the ruminal outflow rate of 0.05/h, the effective degradability (%) values of the DM and CP of TKPER I were 64.1 and 49.7, respectively, which were similar to those of SBM. In conclusion, TKPER had high CP and exhibited in situ degradability similar to that of SBM, suggesting that TKPER could be used as a protein source feed.     

Responses of goat ruminal musculature to substance P in vitro and in vivo

A concentration-response curve obtained for contraction of goat rumen strips indicated that substance P (SP) is a very potent spasmogenic agent. In vivo, SP caused a dose-dependent inhibition of the extrinsic rumen contractions possibly due to an increase in ruminal smooth muscle tone, resulting in reflex inhibition. SP did not induce significant changes in heart rate. Further studies are necessary to elucidate a possible physiological role of SP in the modulation of forestomach motility.     

Responses of goat ruminal musculature to substance P in vitro and in vivo

A concentration-response curve obtained for contraction of goat rumen strips indicated that substance P (SP) is a very potent spasmogenic agent. In vivo, SP caused a dose-dependent inhibition of the extrinsic rumen contractions possibly due to an increase in ruminal smooth muscle tone, resulting in reflex inhibition. SP did not induce significant changes in heart rate. Further studies are necessary to elucidate a possible physiological role of SP in the modulation of forestomach motility.     

In vitro effects of individual fatty acids on protozoal numbers and on fermentation products in ruminal fluid from cattle fed a high-concentrate, barley-based diet

The objective of this study was to investigate the effects of sodium salts of individual fatty acids on protozoal numbers and ruminal fermentation variables in vitro. Ruminal inoculum was obtained from two heifers fed a finishing diet consisting of (DM basis) 90% rolled barley grain, 4% barley silage, 5% soybean meal, and 1% mineralized salt. Fatty acids (FA) were included individually in the inoculum as follows: C6:0, C8:0, and C10:0 at concentrations (wt/vol) of 0.0625, 0.125, and 0.25%; C14:0 and C18:0 at concentrations of 0.125, 0.25, and 0.5%; and C12:0, C16:0, C18:1, C18:2, and C18:3 at concentrations of 0.25, 0.5, and 1.0%. 15N-Labeled casein was included as a N tracer. In the presence of medium-chain saturated FA (particularly C10:0 and C12:0), no ciliate protozoa (99.8%Entodinium spp.) were recovered from the incubation medium. Long-chain unsaturated FA (C18:3, C18:2, C18:1) also decreased (P < 0.05) protozoal numbers. At all concentrations tested, C10:0 and C12:0 decreased (P < 0.05) ammonia and total VFA concentrations (by 29 and 22%, respectively) and increased (P < 0.05) concentrations of total free amino acids, reducing sugars, and soluble protein. At the greatest concentrations of these FA, xylanase and amylase activities of the incubation media were decreased (P < 0.05). The C18 unsaturated FA increased (P < 0.05) the polysaccharide-degrading activities of the media. These in vitro results suggest that long-chain unsaturated FA in combination with medium-chain saturated acids have the potential to decrease protozoal numbers and ruminal ammonia utilization in cattle fed high-grain diets.     

In vitro and in vivo acaricidal activity of a herbal extract

The anti-tick efficacy of combined aqueous herbal extracts of Azadirachta indica leaves, Nicotiana tabacum leaves, Calotropis procera flowers and Trachyspermum ammi seeds was evaluated using adult immersion test, larval packet test and ear bag method. The extract exhibited lethal effects on egg laying (index of egg laying=0.371404±0.00435), hatching (22.35%) and total larval mortality at 50 mg ml(-1) and reduced tick intensity on the infested calves (18 detached out of 35 at 45% (w/w) suspension, topically applied). The herbal extract exerted dose- and time-dependent response against all the developmental stages of Rhipicephalus (Boophilus) microplus considered in this study, thus justified their use in the traditional system of Pakistan.     

In vivo immunostimulatory effects of CpG oligodeoxynucleotide in cattle and sheep

Oligodeoxynucleotides (ODN) containing cytosine-phosphate-guanosine (CpG) motifs have been shown to activate the innate immune system and protect mice and chicken from bacterial and viral infections. Unfortunately, similar studies in other veterinary species are lacking. In this study we assessed the in vivo immunostimulatory effects of CpG ODN 2007, an ODN with previously demonstrated in vitro biological activity. The in vivo effects of ODN 2007 were compared in two closely related outbred species, sheep and cattle, to determine if there were common biological responses. We demonstrated that subcutaneous (s.c.) injection of the CpG ODN induces an acute phase response in the form of a transient fever, a mild transient increase in circulating neutrophils and elevated serum haptoglobin in both sheep and cattle. Sheep injected with CpG ODN also exhibited increased serum 2'5'-oligoadenylate (2'5'-A) synthetase activity, but no increase in serum 2'5'-A synthetase was detected in cattle. The ODN-induced responses were stronger in animals injected with CpG ODN formulated in 30% emulsigen than phosphate buffer saline (PBS) alone. These in vivo data demonstrate for the first time that a CpG ODN induces acute phase immunostimulatory responses in sheep and cattle. However, CpG ODN-induced antiviral effector molecule 2'5'-A synthetase was detected only in sheep but not in cattle.     

In vitro and in vivo temporal aspects of ACTH secretion: stimulatory actions of corticotropin-releasing hormone and vasopressin in cattle

The objective of the present study was to evaluate the temporal aspects associated with corticotropin-releasing hormone (CRH) and vasopressin (VP) stimulated bovine adrenocorticotropic hormone (ACTH) secretion in vitro and in vivo. For the in vitro studies, bovine anterior pituitary glands were enzymatically dispersed to establish primary cultures. On day 5 of culture, cells were challenged for 3 h with medium alone (Control) or various combinations and concentrations of bovine CRH (bCRH) and VP. Both CRH and VP each increased (P < 0.05) ACTH secretion. Maximal increases in ACTH secretion occurred in response to 0.1 microM CRH (5.5-fold) and 1 microM VP (3.7-fold), relative to Control cells. The in vivo portion of the study examined possible temporal differences in the activation of the pituitary-adrenal axis by CRH and VP. Jersey cows were randomly assigned to one of four groups (n = 8 cows/group): (i) Control (saline); (ii) bCRH (0.3 microg/kg BW); (iii) VP (1 microg/kg BW) and (iv) bCRH (0.3 microg/kg BW) + VP (1 microg/kg BW). Jugular blood samples were collected at 15-min intervals for 4 h pre- and for 6 h post-treatment; samples were also taken at 1, 5 and 10 min post-treatment. Plasma concentration of ACTH did not differ among treatment groups for the 4-h pre-treatment period. At 1 min post-treatment, bCRH + VP, VP and bCRH increased ACTH secretion by 22.4-, 9.6- and 2.2-fold, respectively, relative to Control (32.7 +/- 7.2 pg/ml). Maximal plasma concentration of ACTH occurred at 5, 10 and 15 min post-treatment for the VP (1017.7 +/- 219.9 pg/ml), bCRH + VP (1399.8 +/- 260.1 pg/ml) and bCRH (324.8 +/- 126.2 pg/ml) treatment groups respectively. Both the in vitro and in vivo data demonstrated that while VP acutely activates the bovine pituitary-adrenal axis, CRH-induced ACTH secretion is slower in onset but of longer duration. The present study also provides insight into the dynamics of ACTH and cortisol (CS) responsiveness to CRH and VP in cattle.     

Comparative measurements on ruminal pH-value in cattle

Subacute rumen acidosis (SARA) of ruminants is an important factor in terms of animal health, especially in high yielding cattle. In order to find an accurate method to determine the ruminal pH-value, three methods using eight rumen cannulated cattle were compared. An indwelling measuring unit (sensor) was used for continuous measurement of the ruminal pH-value. These results were compared to the pH-values of samplings via rumen fistula and to the results of samplings via oral stomach tube. Due to the different rations, mean pH-value of trial 1 (average of all methods) was 6.64 +/- 0.37 (hay ad lib., 2 kg concentrate/ animal) and 6.24 +/- 0.36 for trial 2 (75% maize silage, 1 kg hay, 2 kg soybean meal). In trial 1 no statistically significant differences between all methods could be observed. Under more acidic ruminal conditions of trial 2 all methods differed significantly (p < 0.05). In the lower pH-range of trial 2, a difference of 0.32 that data can be collected continuously. The sensor system was evaluated by a comparison with standardized pH-dilutions (pH 4, pH 7). The sensor system has proven to be an accurate and reliable instrument (r = 0.9984) and it represents an innovative system for answering scientific questions in terms of rumen physiology and rumen pathology.     

Effects of nonstructural carbohydrates and protein sources on intake, apparent total tract digestibility, and ruminal metabolism in vivo and in vitro with high-concentrate beef cattle diets

To investigate the effects of synchronizing nonstructural carbohydrate (NSC) and protein degradation on intake and rumen microbial fermentation, four ruminally fistulated Holstein heifers (BW = 132.3 +/- 1.61 kg) fed high-concentrate diets were assigned to a 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments studied in vivo and in vitro with a dual-flow continuous culture system. Two NSC sources (barley and corn) and 2 protein sources [soybean meal (SBM) and sunflower meal (SFM)] differing in their rate and extent of ruminal degradation were combined resulting in a synchronized rapid fermentation diet (barley-SFM), a synchronized slow fermentation diet (corn-SBM), and 2 unsynchronized diets with a rapidly and a slowly fermenting component (barley-SBM, and corn-SFM). In vitro, the fermentation profile was studied at a constant pH of 6.2, and at a variable pH with 12 h at pH 6.4 and 12 h at pH 5.8. Synchronization tended to result in greater true OM digestion (P = 0.072), VFA concentration (P = 0.067), and microbial N flow (P = 0.092) in vitro, but had no effects on in vivo fermentation pattern or on apparent total tract digestibility. The NSC source affected the efficiency of microbial protein synthesis in vitro, tending to be greater (P = 0.07) for barley-based diets, and in vivo, the NSC source tended to affect intake. Dry matter and OM intake tended to be greater (P > or = 0.06) for corn- than barley-based diets. Ammonia N concentration was lower in vitro (P = 0.006) and tended to be lower in vivo (P = 0.07) for corn- than barley-based diets. In vitro, pH could be reduced from 6.4 to 5.8 for 12 h/d without any effect on ruminal fermentation or microbial protein synthesis. In summary, ruminal synchronization seemed to have positive effects on in vitro fermentation, but in vivo recycling of endogenous N or intake differences could compensate for these effects.     

Blood and ruminal fluid profiles in carbohydrate-foundered cattle.

The relationships of acetylhistamine and histamine to the clinical signs of carbohydrate-induced acidosis were investigated in beef steers. Blood pH and plasma L-lactic acid decreased and serum sodium, serum potassium, ruminal fluid L-lactic acid, ruminal fluid histamine, and ruminal fluid and blood acetylhistamine increased in carbohydrate-engorged steers as compared with the changes in the steers while feeding on pasture (forage-fed steers). Twelve to 14 hours after the steers had become engorged, clinical signs of laminitis ("feedlot founder") were observed in three of six steers. These signs appeared 4 to 6 hours after blood acetylhistamine attained maximal concentration (2.9997 +/- 1.7054 microgram of histamine base/ml of blood) and blood pH decreased to 7.260 +/- 0.026 at 8 hours after engorgement. Blood histamine value reached 0.1298 +/- 0.1095 microgram of histamine base/ml 4 hours after engorgement (8 to 10 hours before the appearance of clinical illness), but had reached maximal concentration 32 hours after engorgement (0.3300 +/- 0.028 microgram of histamine base/ml of blood).     

In vitro effects of the ionophore lysocellin on ruminal fermentation and microbial populations.

Batch and continuous culture techniques were used to evaluate the effect of the ionophore lysocellin on ruminal fermentation and microbial populations. In batch culture, .5 and 1 ppm (of the fluid) lysocellin markedly decreased (P less than .01) the acetate:propionate ratio without affecting fiber digestion, ammonia concentration, or culture pH. Greater concentrations of lysocellin had negative effects (P less than .05) on fiber digestion and increased (P less than .05) culture pH. In continuous culture, a low level of lysocellin (33 ppm of the diet DM or about .7 ppm of the fluid) decreased pH (P less than .05) and methane (P less than .05) production but had no effect on fiber digestion. Lysocellin tended to increase (P less than .05) OM digestion in corn-based diets but decreased OM digestion in barley-based diets (starch source x lysocellin interaction, P less than .05). In addition, the molar proportion of propionate was increased more in barley- than in corn-based diets. Total anaerobes and amylolytic and lactate-utilizing microorganisms were not affected by the ionophore. In continuous culture, cellulolytic and lactate-producing organisms were insensitive to lysocellin, but, in lysocellin-treated media, cellulolytic organisms were resistant, whereas lactic acid producers were sensitive to lysocellin at 4 ppm. In summary, the ionophore lysocellin alters ruminal fermentation by decreasing ruminal methane production and increasing the molar proportion of propionate; however, effects varied depending on whether corn or barley served as the primary starch source.     

In vitro and in vivo glucose consumption in swine eperythrozoonosis

One complication of swine eperythrozoonosis is the hypoglycemia that occurs during parasitemia. To determine the cause of the hypoglycemia, we studied glucose consumption in splenectomized pigs infected with Eperythrozoon suis. With the rapid rise of erythroparasites, the in vitro glucose consumption of parasited whole blood increased dramatically, and hypoglycemia developed. Because mature porcine erythrocytes are impermeable to glucose, the increased glucose consumption is most logically the result of E. suis metabolism. Iodoacetamide and sodium fluoride (which inhibit glycolysis), but not sodium cyanide (which prevents cellular respiration), and tetracycline (which is used to treat eperythrozoonosis) inhibited glucose consumption. In vivo glucose turnover studies before infection and during peak parasitemia indicated an increased glucose production by infected pigs during parasitemia. The results suggest that hypoglycemia occurs during swine eperythrozoonosis because the parasite uses glucose faster than the gluconeogenic pathways can provide it.