In vitro effects of glucosamine and acetylsalicylate on canine chondrocytes in three-dimensional culture

OBJECTIVE: To determine effects of glucosamine and acetylsalicylate on canine chondrocytes in 3-dimensional culture. SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of 2 adult female dogs recently euthanatized for reasons unrelated to orthopedic abnormalities. PROCEDURE: Chondrocytes were cultured in a 3-dimensional agarose-based medium alone (control), with glucosamine (100 microg/ml; GL), or with acetylsalicylate (18 microg/ml; AS). Supernatant and agarose plugs from 4 wells/group/d were collected on days 3, 6, and 12 of culture. Agarose plugs were evaluated for percentage of viable cells, percentage of cells producing pericellular or territorial matrix, glycosaminoglycan (GAG) concentration, and type-II collagen production. Prostaglandin E2 concentration in supernatants was determined. RESULTS: Chondrocytes in all groups had characteristics indicative of viability and differentiation; however, on day 12, a lower percentage of viable cells was detected in the AS group, compared with the other 2 groups. On day 6, GAG concentration in the AS group was significantly greater than concentrations in the other 2 groups. On day 12, GAG concentrations in the GL and AS groups were significantly less than in the control group. Within the GL and AS groups, cell viability was significantly less on day 12, compared with day 3. Significant differences in PGE2 concentration among or within groups and evidence of type II collagen production were not detected. CONCLUSIONS: 3-dimensional culture of canine chondrocytes allows for production of hyaline cartilage matrix constituents and growth of cells with morphologic characteristics similar to those of articular cartilage. Acetylsalicylate and glucosamine, at the single concentration evaluated, had detrimental effects on chondrocyte viability, GAG production, or both.     

In vitro effects of tachykinins on the smooth musculature of horse gut

The contractile effects of the tachykinins eledoisin, substance P and neurokinin A and B were investigated in vitro on circular and longitudinal muscle strips from horse duodenum, ileum and colon. Circular smooth muscle of the small intestine was highly responsive, large intestine circular smooth muscle less so, while longitudinal muscle from all gut segments was much less sensitive. pD₂ values and intrinsic activities on small intestine circular muscle indicated differences in receptor distribution between the duodenum and ileum: NK₃ and a smaller number of NK₂ receptors being present in the duodenum, and NK₂ receptors predominating in the ileum. Notwithstanding this, eledoisin and neurokinin B were the most active substances on duodenum and ileum, respectively. These findings suggest that tachykinins may play a role in equine gastrointestinal pathophysiology.     

In vivo and in vitro effects of three glucocorticoids on blood leukocyte chemotaxis in the dog

The effects of three glucocorticoids on random migration (RM) and oriented migration (OM) of dog blood leukocytes either from dogs treated in vivo (at therapeutic dosage regimen) or leukocytes treated in vitro (at pharmacological concentrations), were investigated using an agarose gel technique. After in vivo treatment, methylprednisolone sodium succinate (MPSS) produced a stimulation of both RM and OM in the three treated dogs. Dexamethasone produced a stimulation of both RM and OM in the three treated dogs. Dexamethasone produced a stimulation of these parameters in all but one of the three treated dogs, but the difference was not statistically significant. After in vitro treatment of leukocytes from eight dogs, MPSS significantly stimulated OM. Dexamethasone was without significant effect except at a higher than therapeutic concentration (0.5 micrograms/ml), for which RM was stimulated. Hydrocortisone sodium succinate was without statistically significant effect. Under no conditions, except after suprapharmacological concentrations, did glucocorticoids inhibit RM or OM.     

In vitro evidence for a bacterial pathogenesis of equine laminitis

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation.These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis.     

In vitro assessment of movements of the sacroiliac joint in the horse

REASONS FOR PERFORMING STUDY: Sacroiliac joint (SIJ) disease is associated with poor hindlimb action, lameness and poor performance in horses. However, little is known about the biomechanics of this low-motion joint. OBJECTIVES: To determine in vitro the capacities of movement of the SIJ in the sagittal plane, and to test the effect of a sacrosciatic and sacrotuberal desmotomy on its stabilisation. METHODS: Six anatomical specimens underwent cycles of flexion-extension of the lumbosacral joint (LSJ) before and after desmotomy. Kinematic triads were linked rigidly to the sacrum, spinous process of vertebra L5 and iliac wing. Angles were measured using a joint coordinate system based on anatomical frames. RESULTS: The LSJ underwent regular movements of flexion and extension (overall mean +/- s.d. range 23.4 +/- 1.6 degrees). The only recorded movement of the SIJ was a nutation during LSJ flexion (overall mean +/- s.d. 0.8 +/- 0.5 degrees). Desmotomy induced an increase of that nutation (overall mean +/- s.d. 1.7 +/- 0.2 degrees). CONCLUSIONS AND POTENTIAL RELEVANCE: Movements of the SIJ were small and coupled only with the flexion of the LSJ. The ligaments surrounding the SIJ have a strong effect on the stabilisation of this joint. Due to the limited amount of movement, its biomechanical study in vivo seems to be difficult. Further in vitro studies would be useful to determine the role of each ligament, to better understand the clinical consequences of the tears frequently observed during necropsy.     

In vitro effects of lipopolysaccharides on bovine uterine contractility

Metritis is an important disorder in dairy cows during the early postpartum period. Myometrial contractility is a prerequisite for uterine involution; however, very scanty literature is available about the effect of metritis on this process and endocrine responsiveness. This study was aimed to evaluate the effect of inflammation on uterine contractility in vitro, and the inflammation was induced by incubating myometrial strips with lipopolysaccharides (LPS). Myometrial samples were collected from 17 healthy Holstein Friesian cows during caesarean section. Eight longitudinal strips from each cow were incubated in organ baths with LPS concentrations of 0 (LPS₀), 0.1 (LPS_0.1), 1 (LPS₁) and 10 µg/ml (LPS₁₀). Spontaneous contractility and contractility induced by increasing concentrations of oxytocin (10^–10 – 10^–7 mol/L) were recorded during nine 30-min intervals (T1 to T9). The minimum amplitude (minA), maximum amplitude (maxA), mean amplitude (meanA) and area under the curve (AUC) were calculated for each time interval. LPS had an effect (p ≤ .05) on maxA, meanA and AUC. In T1, myometrial strips incubated with LPS_0.1 and LPS₁ had higher (p ≤ .05) maxA, meanA and AUC than the strips incubated with LPS₀. In T9 without oxytocin, LPS₀ led to higher (p ≤ .05) maxA, meanA and AUC than LPS_0.1 and LPS₁. In T8 and T9 with oxytocin, LPS₁ had lower (p ≤ .05) maxA, meanA and AUC than the other LPS concentrations. Interestingly, the results show that LPS has a transient positive effect on myometrial contractility in vitro and that this effect is dependent on LPS concentration and duration of incubation.     

Relaxant effects of theophylline and clenbuterol on tracheal smooth muscle from horse and rat in vitro

A comparison between the relaxant effects of clenbuterol and theophylline on horse tracheal smooth muscle has been made in vitro. Rat tracheal smooth muscle was also investigated as a reference. The tracheal preparations were initially contracted with carbachol since the smooth muscle did not spontaneously develop tone. The response of the carbachol-contracted preparations to theophylline was the same in the two species. The response to clenbuterol varied. In only five out of eleven horses were the tracheal smooth muscles sensitive to clenbuterol (mean pD2 = 7.92 M). In the remaining six horses the tracheal smooth muscles were insensitive to clenbuterol (mean pD2 = 3.59 M), yet the preparations responded well to theophylline with complete relaxation. All rat tracheal preparations were insensitive to clenbuterol.     

In vitro evaluation of a closed-bowel technique for one-layer hand-sewn inverting end-to-end jejunojejunosotomy in the horse

OBJECTIVE: To report a technique for closed-bowel 1-layer inverting end-to-end jejunojejunal anastomosis in horses. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Fresh cadaveric jejunal segments from 12 horses. METHODS: For each bowel segment a 1-layer closed and a 2-layer inverting end-to-end jejunojejunosotomy was created. Anastomosis construction time and anastomotic bursting pressure were measured and compared. RESULTS: Closed-bowel anastomosis was significantly faster to create than a 2-layer technique. Luminal narrowing (<30%) was similar with both techniques and comparable with other inverting techniques. Bursting pressure was significantly higher for the 2-layer technique, although all anastomoses resisted pressures higher than those reported for other jejunojejunal anastomosis techniques. CONCLUSIONS: A 1-layer hand-sewn, closed, inverting jejunojejunosotomy using a modified Doyen clamp was easy and faster to perform, and resulted in functional characteristics similar to, a 2-layer hand-sewn inverting technique. CLINICAL RELEVANCE: A closed, 1-layer inverting technique could be considered for equine jejunal anastomosis but requires in vivo evaluation before recommendation for clinical use.     

不同精粗比和菜粕水平对饲粮组合效应的影响简

 为了探讨在反刍动物中等或较低水平粗饲料中需要添加能量和蛋白质饲料的量和比例,试验采用体外产气法测定玉米、菜粕和小麦秸或氨化小麦秸在不同精粗比（７０∶３０,６０∶４０,５０∶５０,４０∶６０,３０∶７０）和菜粕水平（６％,１２％,１８％,２４％）下各组合及４种饲料分别培养２,４,６,９,１２,２４,３６,４８,７２,９６ｈ内的产气量,并通过２４ｈ产气量和各组合的加权估算值计算出各组合的组合效应值。结果表明：１）小麦秸基础料中,１５个饲粮组合为负组合效应,而高精粗比７０∶３０高菜粕水平１８％和２４％两组合和低精粗比４０∶６０低菜粕水平６％和１２％两组合均表现出正组合效应,且高精粗比７０∶３０的组合效应值极显著高于低精粗比４０∶６０和３０∶７０组（P＜０．０１）。２）氨化小麦秸基础料中,１９个饲粮组合均不同程度地表现出正组合效应值。各精粗比间组合效应值,３０∶７０组极显著高于７０∶３０组（P＜０．０１）。各菜粕水平间组合效应值,２４％组显著高于１２％组（P＜０．０５）。低精粗比和高菜粕水平组合时组合效应值最大。使用氨化小麦秸要比使用小麦秸更能充分利用粗饲料,节省精饲料。     

In vitro gastrointestinal digestion study of two wheat cultivars and evaluation of xylanase supplementation

Background

The filamentous fungus Talaromyces versatilis is known to improve the metabolizable energy of wheat-based poultry diets thanks to its ability to produce a pool of CAZymes and particularly endo-β(1,4)-xylanases. In order to appreciate their in vivo mode of action, the supplementation effect of two of its xylanases, XynD and XynB from families GH10 and GH11 respectively, have been evaluated on two different wheat cultivars Caphorn and Isengrain, which were chosen amongst 6 varieties for their difference in non starch polysaccharides content and arabinoxylan composition.

Results

Polysaccharides digestion was followed during 6 h along the digestive tract using the TNO gastrointestinal model-1, to mimic monogastric metabolism. Polysaccharide degradation appeared to occur mainly at the jejunal level and was higher with Isengrain than with Caphorn. For both cultivars, XynD and XynB supplementation increased notably the amount of reducing end sugars into the jejuno-ileal dialysates, which has been confirmed by a valuable increase of the soluble glucose into the jejunal dialysates.

Conclusions

The amounts of arabinose and xylose into the dialysates and ileal deliveries increased consequently mainly for Caphorn, suggesting that XynD and XynB supplementation in wheat-based diet could alleviate the anti-nutritional effects of arabinoxylans by limiting the physical entrapment of starch and could increase the available metabolizable energy.     

In vitro gastrointestinal digestion study of two wheat cultivars and evaluation of xylanase supplementation

Background:The filamentous fungus Talaromyces versatilis is known to improve the metabolizable energy of wheatbased poultry diets thanks to its ability to produce a pool of CAZymes and particularly endo-β(1,4)-xylanases.In order to appreciate their in vivo mode of action,the supplementation effect of two of its xylanases,XynD and XynB from families GH10 and GH11 respectively,have been evaluated on two different wheat cultivars Caphorn and Isengrain,which were chosen amongst 6 varieties for their difference in non starch polysaccharides content and arabinoxylan composition.Results:Polysaccharides digestion was followed during 6 h along the digestive tract using the TNO gastrointestinal model-1,to mimic monogastric metabolism.Polysaccharide degradation appeared to occur mainly at the jejunal level and was higher with Isengrain than with Caphorn.For both cultivars,XynD and XynB supplementation increased notably the amount of reducing end sugars into the jejuno-ileal dialysates,which has been confirmed by a valuable increase of the soluble glucose into the jejunal dialysates.Conclusions:The amounts of arabinose and xylose into the dialysates and ileal deliveries increased consequently mainly for Caphorn,suggesting that XynD and XynB supplementation in wheat-based diet could alleviate the anti-nutritional effects of arabinoxylans by limiting the physical entrapment of starch and could increase the available metabolizable energy.     

In vitro antiprotozoal effects of artemisinin on Neospora caninum.

Neospora caninum is an intracellular apicomplexan parasite that infects a wide range of mammals and has been associated with abortion in cattle worldwide. Artemisinin is an effective antimalarial compound derived from a traditional Chinese herbal remedy, qinghao or Artemisia annua L. In the study reported, the cultured host cells (vero cells or mouse peritoneal macrophages) infected with N. caninum tachyzoites were incubated with alpha-MEM (minimal essential medium) 10%HS supplemented with various concentration or artemisinin (20, 10, 1, 0.1 and 0.01 microg/ml) to examine the efficacy of artemisinin against N. caninum tachyzoites intracellular multiplication. In long-term studies, at 20 or 10 microg/ml for 11 days, artemisinin reduced N. caninum and completely eliminated all microscopic foci of N. caninum. At 1 microg/ml for 14 days, artemisinin reduced N. caninum and completely achieved elimination of all microscopic foci of N. caninum. There was no apparent toxicity to host cells in long-term studies. In short-term studies, at > or = 0.1microg/ml, artemisinin reduced N. caninum tachyzoites intracellular multiplication, significantly (P < 0.05) and appeared to depend on the artemisinin concentrations. Pretreatment of host cells or N. caninum tachyzoites with artemisinin had no effect on N. caninum tachyzoites intracellular multiplication. These results demonstrate that artemisinin inhibited N. caninum tachyzoites intracellular multiplication.     

In vitro effects of 2-methoxyestradiol on canine tumour cells

The in vitro antiproliferative, apoptotic and cell‐cycle effects of 2‐methoxyestradiol (2ME₂), an endogenous oestrogen metabolite, were investigated using a variety of canine tumour cell lines. The cells were cultured under standard conditions and incubated with varying concentrations of 2ME₂. Inhibition of tumour cell proliferation was evaluated using a tetrazolium‐based colorimetric assay. DNA content analysis was performed using propidium iodide staining and flow cytometry. Cytologic analysis with Leukostat staining solution and Hoechst 33342 staining and Annexin V‐fluorescein isothiocyanate (FITC) fluorescence were used to quantify cell‐cycle distribution and apoptosis induction. Tumour cell proliferation was inhibited by 50% at concentrations of 2ME₂ ranging from 0.88 to 7.67 µM, depending on the cell line tested. Profound G₂/M phase arrest, an increase in binucleate cells and induction of apoptosis were observed in all cell lines tested, in a dose‐dependent manner. Based on these results, this compound has potential as an agent for the treatment of canine cancer and warrants further investigation. The canine lymphoma cell line, 1771, was inhibited at concentrations that may be achievable in vivo.     

Fructooligosaccharide supplementation in the yearling horse: effects on fecal pH, microbial content, and volatile fatty acid concentrations

Short-chain fructooligosaccharides (FOS) were supplemented to the diets of nine quarter horses ranging in age from 489 to 539 d with initial BW averaging 400.6 +/- 21.2 kg. The objectives of this study were to determine the effects of dietary FOS on the fecal responses in terms of pH, the microbial population, and VFA concentrations. The horses were used in a 3 x 3 replicated Latin square design, fed according to NRC requirements, and their individual diets were supplemented with no FOS (CON), 8 g of FOS/d (LOW), or 24 g of FOS/d (HIGH) over three 10-d feeding periods. On the last 3 d of each 10-d feeding period, a single fecal sample was collected between 0730 and 0930. Fecal pH decreased linearly (P = 0.01) from 6.48 with the CON diet to 6.38 with the HIGH diet, but there was no change (P = 0.19 for linear effect) in fecal consistency among treatments. A quadratic effect (P < 0.01) was observed for fecal Escherichia coli population, but no difference (P = 0.88 for linear effect) was found in fecal Lactobacilli enumeration among treatments. The presence of fecal Bifidobacteria was unable to be confirmed and was therefore not reported. Fecal acetate concentrations increased linearly (P = 0.03), with means of 2.13, 2.18, and 2.52 mg/g of wet feces for CON, LOW, and HIGH treatments, respectively. Similarly, fecal propionate concentrations increased linearly (P = 0.01), with means of 0.58, 0.64, and 0.73 mg/g for CON, LOW, and HIGH treatments, respectively. Fecal butyrate concentrations also increased linearly (P = 0.02), with means of 0.40, 0.46, and 0.54 mg/g for CON, LOW, and HIGH treatments, respectively. Total VFA (P = 0.01) and lactate (P = 0.02) concentrations increased linearly, with total VFA means of 3.47, 3.69, and 4.25 mg/g for CON, LOW, and HIGH treatments, respectively, and lactate means of 0.36, 0.41, and 0.47 mg/g for CON, LOW, and HIGH treatments, respectively. Supplementing FOS in diets fed to yearling horses altered fecal microbial populations, fecal VFA concentrations, and pH.     

In vitro evidence for the importance of cultivation conditions on the effects of Calliandra tannins on ruminal escape of soybean protein and its post‐ruminal degradability

Purified condensed tannins (CT) extracted from the legume Calliandra calothyrsus (var. San Ramón CIAT 22310), harvested in the dry and the rainy season and cultivated with low or high level of fertilization were added to soybean meal in a ratio of 600 mg/g of the incubated crude protein (CP). Effects on degradation either in ruminal fluid only, or in ruminal fluid followed by incubation in HCl/pepsin, were evaluated using a modified two‐step in vitro method. Season was found to have larger effects on in vitro ruminal and post‐ruminal CP degradation than fertilization. Condensed tannins from the rainy season harvest reduced ruminal CP degradation less than that from the dry season harvest. They had also less negative effects on the degradability of rumen escape protein and enhanced the proportion of post‐ruminally degraded CP more than CT from the dry season harvest. An increase in level of fertilization reduced ruminal CP degradation in CT from the rainy season plants but this was not associated with effects on post‐ruminal degradation. The study demonstrated the importance of environmental factors for the efficiency of CT in modifying ruminal and post‐ruminal CP degradation.