Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Difference in rates of net portal absorption between crystalline and protein-bound lysine and threonine in growing pigs fed once daily

Net portal absorption of AA during the 6-h postprandial period was measured in eight gilts (48.5 +/- 1.6 kg BW) in a crossover design. The pigs had chronic catheters placed in the portal vein, carotid artery, and ileal vein, and were trained to consume 1.2 kg of a standard grower diet once daily. Blood samples were taken every 30 min for 4 h and then hourly until 6 h after feeding. The first set of blood samples was taken after pigs were fed a meal of the test 16% CP corn-soybean meal diet (16% CP) or the test 12% CP corn-soybean meal diet supplemented with crystalline lysine, threonine, and tryptophan (12% CP + AA) to equal the three AA levels in the 16% CP diet. Pigs were then fed the standard diet for 2 d. Following that, blood samples were again taken after the pigs were fed a meal of the test diet that was not given to them at the first sampling period. Net portal AA absorption was calculated by multiplying porto-arterial plasma AA concentration difference by portal vein plasma flow rate (PVPF), estimated by an indicator-dilution technique employing p-aminohippuric acid as the indicator infused into the ileal vein. Plasma concentrations of lysine and threonine of pigs were affected by the diet x time interaction (P < 0.01). Portal and arterial plasma lysine and threonine concentrations in pigs attained the maximal level by 1 h postprandial when the 12% CP + AA diet was fed, but reached the peak level at 2.5 h postprandial when the 16% CP diet was given. The PVPF of pigs over the 6 h postprandial was less (P < 0.01) when the 12% CP + AA diet was given than when the 16% CP diet was fed. Net portal absorptions of lysine and threonine also were affected (P < 0.05) by time x diet interaction. The peak portal absorption of both lysine and threonine in pigs appeared at 0.5 h postprandial when the 12% CP + AA diet was given, but at 2.5 h postprandial with the feeding of the 16% CP diet. The early appearance of peak portal absorption of lysine and threonine from feeding the 12% CP + AA compared with the 16% CP diet indicates that crystalline lysine and threonine are absorbed more rapidly than protein-bound lysine and threonine in pigs fed once daily.     

Effects of dietary fat on growth performance and carcass characteristics of growing-finishing pigs reared in a commercial environment.

We conducted two experiments to evaluate the effects of added choice white grease on performance and carcass merit of barrows and gilts reared under commercial conditions. Pigs were housed either 20 (Exp. 1) or 25 (Exp. 2) per pen and were provided 0.67 m2 of pen space per pig. Diets were based on corn and soybean meal and fed in a meal form. The proportion of soybean meal was increased in diets with added fat to maintain the same calorie:lysine ratio in all diets within a weight phase. In Exp. 1, 480 pigs were fed diets with 0, 2, 4, or 6% fat. Total lysine contents of the control diets were 1.21, 0.88, and 0.66% during the weight phases 36 to 59, 59 to 93, and 93 to 120 kg, respectively. Gain:feed was increased linearly (P < 0.01) due to fat addition in all weight intervals and over the total experiment. The effect of added fat on ADG was not consistent among the weight phases; a linear (P < 0.01) improvement was found from 36 to 59 kg, but no effect was found during the heavier weight phases. Over the total experiment, however, ADG was improved (P < 0.01) linearly. Carcass traits were not affected by treatment. Experiment 2 used 900 pigs to evaluate possible carryover effects on performance and carcass merit from feeding 6% fat. The experiment was divided into four phases: 25 to 45, 45 to 70, 70 to 90, and 90 to 115 kg; lysine contents of the control diets fed in each phase were 1.23, 1.05, 0.81, and 0.63%, respectively. The six treatments consisted of no added fat throughout the experiment or 6% added fat fed from 25 to 45 kg, 25 to 70 kg, 25 to 90 kg, 25 to 115 kg, or 45 to 70 and 90 to 115 kg. Carryover effects for ADG and G:F (P < 0.07) were found for the 90- to 115-kg interval and for ADFI and ME intake (P < 0.05) for the 45- to 70- and 70-to 90-kg intervals. When fat was added in the previous weight interval, ADG and G:F were improved and ADFI and ME intake were decreased in the subsequent weight interval. Pigs fed fat from 25 to 115 kg had more (P < 0.05) backfat and lower (P < 0.05) carcass leanness than pigs on the other treatments. These data suggest that fat can be added or removed from diets of growing-finishing pigs without any detrimental carryover effects. In fact, the positive carryover effect on ADG and G:F from 95 to 115 kg suggests that feeding fat from 25 to 95 kg will maximize performance over the total growing-finishing period but minimize any detrimental effects of added fat on carcass leanness.     

Skin and coat traits in sheep in Brazil and their relation with heat tolerance

The objective of this study was to evaluate and compare physical parameters in groups of sheep with different phenotypic characteristics in the Centre-west region of Brazil. Five groups of sheep, with nine animals per group, were selected, three groups of Santa Inês animals with different coat colours (white, brown and black), one group with crossbred animals (Santa Inês × Bergamasca) and one group with animals of the Bergamasca breed. The following traits were evaluated: coat thickness, number and length of hair, pigmentation level in the coat and the skin as well as the percentage area of sweats glands in the skin tissue, carried out by histological analysis. The number of hairs and the area of sweats glands were not significantly different between the evaluated groups. The Bergamasca breed showed low pigmentation of the skin and long hairs. The levels of pigmentation of the hair and of the skin were highly correlated. Between the Santa Inês groups, the group with white hair showed the better parameters for heat adaptation, while the brown hair group showed the lower heat adaptation when compared with another hair breed groups.     

Effects of three-dimensional spheroid culture on equine mesenchymal stem cell plasticity

Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy fields. We optimize culture conditions of equine adipose tissue-derived MSCs (eAD-MSCs) for treatment of horse fractures. To investigate enhancing properties of three-dimensional (3D) culture system in eAD-MSCs, we performed various sized spheroid formation and determined changes in gene expression levels to obtain different sized spheroid for cell therapy. eAD-MSCs were successfully isolated from horse tailhead. Using hanging drop method, spheroid formation was generated for three days. Quantitative real-time PCR was performed to analyze gene expression. As results, expression levels of pluripotent markers were increased depending on spheroid size and the production of PGE₂ was increased in spheroid formation compared to that in monolayer. Ki-67 showed a remarkable increase in the spheroid formed with 2.0?×?10⁵ cells/drop as compared to that in the monolayer. Expression levels of angiogenesis-inducing factors such as VEGF, IL-6, IL-8, and IL-18 were significantly increased in spheroid formation compared to those in the monolayer. Expression levels of bone morphogenesis-inducing factors such as Cox-2 and TGF-β1 were also significantly increased in spheroid formation compared to those in the monolayer. Expression levels of osteocyte-specific markers such as RUNX2, osteocalcin, and differentiation potential were also significantly increased in spheroid formation compared to those in the monolayer. Therefore, spheroid formation of eAD-MSCs through the hanging drop method can increases the expression of angiogenesis-inducing and bone morphogenesis-inducing factors under optimal culture conditions.     

Soluble matrix of hen's eggshell extracts changes in vitro the rate of calcium carbonate precipitation and crystal morphology

1. Extra and intramineral eggshell matrix proteins were solubilised before and after demineralisation by sequential extractions using guanidine hydrochloride and EDTA.

2. The intramineral electrophoretic profile of SDS‐PAGE showed the presence of 80, 66, 43, 36 and 15 kDa bands with a predominance of a 17 kDa band. In the extramineral part, the major protein was the 15 kDa band.

3. The introduction of intramineral extract to a metastable solution of calcium carbonate delayed the rate of crystal growth. The delay in the rate of precipitation was elicited by a single fraction (MW 50–80 kDa), isolated by gel filtration chromatography, of eggshell extracts. Extramineral extracts had no effect.

4. Addition in vitro of intramineral eggshell extracts modified the morphology of calcite; the crystals aggregated and showed irregular surfaces.

5. These observations suggest that constituents of the eggshell matrix are involved in the control of calcite growth and crytallographic structure of the hen's eggshell.     

Analysis of the reactivity of anti-bovine CD8 monoclonal antibodies with cloned T cell lines and mouse L-cells transfected with bovine CD8

Mouse L-cells transfected with bovine CD8 and two Theileria parva-infected cloned T cell lines expressing bovine CD8 were used to screen the panel of ten monoclonal antibodies (mAbs) submitted to the workshop. Eight of the ten mAbs reacted with the transfectant and both the cloned T cell lines. However, two mAbs CC58 and BAT82A did not recognise the transfectant and only reacted with one of the T cell lines. Further biochemical studies indicated that the eight mAbs react with both homo- and heterodimeric forms of bovine CD8 whilst the two mAbs CC58 and BAT82A react with only heterodimeric forms. These data suggest that bovine DC8 is encoded by two genes as is the case in mouse and man.     

Uterine environment as a regulator of birth weight and body dimensions of newborn lambs

Pure-bred embryos were transferred within and reciprocally between large (Suffolk) and small (Cheviot) breeds of sheep to establish 4 treatment groups: SinS (Suffolk embryos in Suffolk dams), SinC (Suffolk embryos in Cheviot dams), CinS (Cheviot embryos in Suffolk dams), and CinC (Cheviot embryos in Cheviot dams). The recipient ewes carried single fetuses to term. The maternal plasma concentrations of ovine placental lactogen (oPL), progesterone, IGF-1, FFA, and glucose were measured on d 50, 90, 120, and 140 of pregnancy. Birth weight, body dimensions, and placental characteristics of lambs were recorded at birth. There was a recipient ewe breed × lamb breed × time interaction for the concentration of oPL (P = 0.03), but no such interaction was observed for progesterone (P = 0.42), IGF-1 (P = 0.57), glucose (P = 0.36), or FFA (P = 0.72). There were no differences in oPL (P = 0.28) and progesterone (P = 0.34) concentrations between SinC and SinS ewes. The concentrations of FFA on d 140 (P = 0.008), and those of glucose on d 50 (P = 0.02) and 120 (P = 0.01), were greater in SinC ewes than in SinS ewes. The ewes in CinS had less FFA concentration (P = 0.002) at all time points than CinC ewes. The concentrations of IGF-1 on d 90 were greater (P = 0.004) in CinS ewes than CinC ewes, but did not differ (P = 0.16) on d 50, 120, and 140. The concentrations of glucose on d 50 (P = 0.001), 90 (P = 0.03), and 140 (P = 0.03) were less in CinS ewes compared with CinC ewes. The birth weight of SinC lambs (5.04 ± 0.20 kg) was lighter (P = 0.001) than SinS lambs (5.94 ± 0.19 kg), and body dimensions of SinC lambs were smaller (P = 0.01) than SinS lambs. Neither birth weight nor the body dimensions of CinS lambs differed (P = 0.24) from CinC lambs. Cotyledon number was reduced (P = 0.04) in the CinS (57.5 ± 6.3) compared with the SinS group (74.2 ± 5.9), whereas mean cotyledon weight in CinS (2.42 ± 0.20 g) was greater (P = 0.02) than SinS (1.74 ± 0.21 g). It was concluded that the large genotype lambs were lighter and smaller when born to small genotype dams; however, the birth weight or body dimensions of small genotype lambs did not differ when born to large genotype dams. This study suggests that plasma oPL, progesterone, IGF-1, FFA, and glucose concentrations at different times throughout pregnancy reflect the regulatory effect of the uterine environment on the development of the fetus.     

Risk factors for metritis in Danish dairy cows

A retrospective longitudinal study of metritis was conducted in Denmark on data collected during 1993-1994. Data on herd size, breed, parity, and treatment of disease were obtained from the Danish Cattle Database. Management and production-facility data were collected using a questionnaire, conducted as a telephone interview in 1994. The study included 2144 herds from three regions in Denmark (102,060 cows). Herd-level variables included were: herd size, housing, flooring, grazing, calving measures, and calving supervision. Cow-level variables were: parity, breed, calving season and whether the cow had been treated by a veterinarian for dystocia or the diseases: retained placenta, reproductive disease, ketosis, milk fever, or dry cow mastitis.Marginal multivariable logistic-regression analyses were performed. The cow with highest odds of metritis was a first or greater than or equal to third parity cow, of large breed, that calved during November-April, in a zero-grazing herd. The cow had been treated for dystocia, retained placenta, and at least one other reproductive disease, but not for ketosis.     

Evaluation of protective immunity in pigs following oral administration of an avirulent live vaccine of Lawsonia intracellularis

OBJECTIVE: To evaluate the efficacy of an orally administered avirulent live vaccine to protect pigs against challenge exposure with virulent Lawsonia intracellularis. ANIMALS: 108 weaned 3-week-old pigs (35 in experiment 1 and 73 in experiment 2). PROCEDURE: 2 experiments were conducted. On day 0, vaccinates were orally administered vaccine via drench or in drinking water, whereas challenge-control pigs were administered cultured medium. On day 21, pigs were challenge exposed with a virulent heterologous isolate of L. intracellularis. Clinical observations, weights, seroconversion, and fecal excretion of L. intracellularis were measured until day 42. At study termination, pigs were euthanatized and examined for L. intracellularis-specific lesion development of the ileum and colon. RESULTS: Pigs receiving a single dose of vaccine were protected when challenge exposed with virulent L. intracellularis (at least 10(77) TCID50/dose). In experiment 1, vaccinates had significantly less fecal excretion (47% and 40% for days 35 and 42, respectively), compared with challenge-control pigs. In experiment 2, vaccinates had significantly less fecal excretion (50% and 58% for days 35 and 42, respectively), compared with challenge-control pigs. Significant reductions in lesion development were evident in the ileum of vaccinated pigs (70% and 56% at day 42 for experiments 1 and 2, respectively), compared with challenge-control pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration by drench or via drinking water of an avirulent live vaccine against L. intracellularis resulted in substantial protection against proliferative enteropathy among vaccinates and offers a better way to reduce stress of pigs during vaccine administration.