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Biomedical subjects

E M Green

Publications and source records attributed to E M Green.

At least 19 recordsLinked to original sources

Regulation of the sol locus genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 by a putative transcriptional repressor.

A gene (orf1, now designated solR) previously identified upstream of the aldehyde/alcohol dehydrogenase gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 176:871-885, 1994) was found to encode a repressor of the sol locus (aad, ctfA, ctfB and adc) genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824. Primer extension analysis identified a transcriptional start site 35 bp upstream of the solR start codon. Amino acid comparisons of SolR identified a potential helix-turn-helix DNA-binding motif in the C-terminal half towards the center of the protein, suggesting a regulatory role. Overexpression of SolR in strain ATCC 824(pCO1) resulted in a solvent-negative phenotype owing to its deleterious effect on the transcription of the sol locus genes. Inactivation of solR in C. acetobutylicum via homologous recombination yielded mutants B and H (ATCC 824 solR::pO1X) which exhibited deregulated solvent production characterized by increased flux towards butanol and acetone formation, earlier induction of aad, lower overall acid production, markedly improved yields of solvents on glucose, a prolonged solvent production phase, and increased biomass accumulation compared to those of the wild-type strain.

1-Butanol

Results of a combined dexamethasone suppression/thyrotropin-releasing hormone stimulation test in healthy horses and horses suspected to have a pars intermedia pituitary adenoma.

OBJECTIVE: To evaluate results of a combined dexamethasone suppression/thyrotropin-releasing hormone (TRH) stimulation test in horses suspected clinically to have a pars intermedia pituitary adenoma (PIPA). DESIGN: Case-control study. ANIMALS: 7 healthy adult horses and 5 horses suspected to have a PIPA. PROCEDURE: A baseline blood sample was collected, and dexamethasone (40 micrograms/kg [18 micrograms/lb] of body weight, IV) was administered; a second blood sample was collected 3 hours later, and TRH (1.1 mg, IV) was administered; serial blood samples were collected 15, 30, 45, 60, and 90 minutes and 21 hours after TRH administration (24 hours after dexamethasone injection). Cortisol concentration was determined for all blood samples. RESULTS: Baseline cortisol concentration was significantly lower in horses suspected to have a PIPA than in healthy horses. Cortisol concentration was suppressed by dexamethasone in both groups; however, after TRH administration, cortisol concentration returned to baseline values in horses suspected to have a PIPA, but not in healthy horses. Concentration was still less than the baseline value 24 hours after dexamethasone administration in healthy horses. CLINICAL IMPLICATIONS: The combined dexamethasone suppression/TRH stimulation test may be a useful diagnostic test in horses suspected to have a PIPA. For clinical application, collection of a blood sample 30 minutes after TRH administration is recommended.

Adenoma

Cell cycle analysis of proliferative zone chondrocytes in growth plates elongating at different rates.

Regulation of postnatal growth of long bones occurs in multiple levels of chondrocytic activity, including stem cell proliferation, proliferative zone cycling, and regulation of changes in chondrocytic shape during hypertrophy. The differentiation sequence of chondrocytes is the same in all growth plates, but rates of elongation at a single point in time and over a period of time differ widely among individual growth plates, which suggests that the rates of sequential gene activation and suppression in this phenotypic pattern can vary. The purpose of this study was to investigate, directly and in vivo, parameters of the cell cycle of proliferative chondrocytes in growth plates growing at widely different rates at a single point in time in order to analyze the relationship between cell cycle time, including the duration of each phase of the cell cycle (G1, S, G2, and M), and the rate of growth. The experimental design used repeated pulse labeling with bromodeoxyuridine and was analyzed using a regression model of time of pulse label with increasing labeling index. Total cell cycle time was calculated as the inverse of the slope of the relationship of the labeling index and the time between labels. The y intercept was the calculated labeling index at time zero. Multiple comparison contrasts were used to test for individual differences among four growth plates with growth rates ranging from approximately 50 to 400 microns per 24 hours from 28-day-old rats. The estimate of total cell cycle time for the proximal tibial growth plate was 30.9 hours. Cell cycle times for the other three growth plates were 34.0, 48.7, and 76.3 hours for the distal radius, distal tibia and proximal radius, respectively. Although the times for the proximal tibia and distal radius did not differ significantly, all other times were significantly different (p < 0.05). Almost all differences in total cell cycle time were attributable to significant differences in the length of the G1 phase. The S phase was estimated at 3.4-6.1 hours; the G2 phase, at 3.0 hours; and the M phase, at 0.5-0.6 hours. The current study suggests that regulation through cell cycle parameters, specifically in the G1 phase, may be involved in overall regulation of differential postnatal long bone growth. It has previously been established that increase and shape change of cellular volume during hypertrophy may be regulated at the level of individual growth plates and that both are significant in understanding differential growth of long bone at this level. By demonstrating that chondrocytes in the proliferating zone have different cell cycle times that are regulated primarily through differences in the duration of G1, this study suggests that, in addition to systemic controls of chondrocyte proliferation, local controls may modulate rates of proliferation of individual growth plates and thus may be another locally mediated regulator of differential growth.

Animals

Inactivation of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824.

A nonreplicative plasmid containing an internal aad gene fragment has been integrated into the chromosome of Clostridium acetobutylicum ATCC 824. Transformation was accomplished by electroporation with relatively high concentrations of methylated plasmid DNA. Southern hybridization experiments revealed that integration occurred by single crossover homologous recombination inactivating the aad gene. Integrants were relatively stable after 25 generations. Inactivation of the aad gene drastically reduced solvent production. This result suggests that aldehyde/alcohol dehydrogenase(AAD) plays a important role in butanol production.

Alcohol Dehydrogenase

Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824.

Integrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in Clostridium acetobutylicum ATCC 824. Non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome. Integration was assumed to occur by a Campbell-like mechanism, inactivating either pta or buk. Inactivation of the pta gene reduced phosphotransacetylase and acetate kinase activity and significantly decreased acetate production. Inactivation of the buk gene reduced butyrate kinase activity, significantly decreased butyrate production and increased butanol production.

Acetates

Induction of early-phase endotoxin tolerance in horses.

Six, clinically healthy horses, of mixed age and sex, were infused via a jugular venous catheter with 100 ml of pyrogenfree sterile saline (PFSS; 0.9% NaCl). Animals were infused with Escherichia coli O55:B5 endotoxin (total dose = 50 ng/kg bwt), 24 (LPS-1) and 48 h (LPS-2) after PFSS infusion. Blood was collected before, and every 15 min after, each infusion for the first 8 h and then every 2 h for the following 14 h. Clinical responses (rectal temperature, heart rate, respiration rate and blood pressure) were determined before and every 4 h after each infusion for 20 h. Geometric mean anti-endotoxin antibody titres in serum samples, harvested just before each infusion, were unchanged over the course of the experiment. Serum tumour necrosis factor-alpha (TNF alpha) activity was estimated using a cytotoxic bioassay and WEHI 164 clone 13 murine fibrosarcoma cells as targets. Mean clinical parameter values and geometric mean serum TNF alpha activity at given time points were compared across the 3 infusions. Both LPS-1 and LPS-2 resulted in elevated mean rectal temperature at 4 h after infusion. However, duration of mean rectal temperature elevation was greater (P < 0.05) after LPS-1 (through 12 h) than after LPS-2 (through 8 h). More substantial increases in systolic and diastolic blood pressure were observed after LPS-1 than LPS-2 and mean systolic blood pressure after LPS-1 was elevated at 4 h when compared to PFSS (P < 0.05). Decreased systolic and diastolic blood pressures were observed at 16 h after both LPS infusions, when compared to PFSS infusion. Heart rate was increased, compared to PFSS, after both LPS-1 (8-12 h) and LPS-2 (4-12 h) (P < 0.05). No significant elevations in mean respiratory rate were observed after either LPS-1 or LPS-2 when compared to PFSS. However, at 4 h post infusion, mean respiratory rate after LPS-2 was greater (P < 0.05) than that after LPS-1. Serum TNF alpha activity was not detected after infusion of PFSS, but was detected after both LPS-1 and LPS-2. Serum TNF alpha activity was elevated earlier, was present in higher concentrations and persisted longer after LPS-1 than after LPS-2 (P < 0.05). The decreased duration of fever and attenuated serum TNF alpha response subsequent to successive sublethal LPS challenge observed in this study support the conclusion that these horses developed early-phase endotoxin tolerance (EPET) and, therefore, contributes to the understanding of the role of endotoxaemia in a number of clinical conditions in horses.

Animals

Biochemical changes in three-day-event horses at the beginning, middle and end of Phase C and after Phase D.

Blood samples were collected 12-16 h before the Speed and Endurance test, immediately after steeplechase, midway through Phase C (4 km marker), at the end of Phase C and immediately after cross-country from 36 horses that completed a 3-day-event at the CCI* (n = 19) or CCI** (n = 17) level. Packed cell volume (PCV), plasma concentrations of sodium, potassium, chloride, ionized calcium, pH and lactate; and serum concentrations of total protein (TP), albumin, total calcium, alkaline phosphatase (AP), gamma glutamyl transferase (GGT), creatine kinase (CK), aspartate aminotransferase (AST), glucose and creatinine were measured. No differences were noted in any biochemical measurements between the CCI* and CCI** horses at any sampling time, despite differences in speed and length of various phases of the Speed and Endurance test. For all horses (n = 36), biochemical parameters changed significantly over time (P < 0.01). The PCV, calcium, potassium, lactate, total protein and albumin concentrations were significantly increased over pre-exercise concentrations immediately after Phase B. During Phase C, these parameters decreased towards pre-exercise concentrations, thereby supporting the concept that Phase C is a recovery phase. In contrast, chloride concentration decreased, and the creatinine concentration and CK increased compared to pre-exercise concentrations from the end of Phase B to the end of Phase C. These changes probably resulted from fluid and electrolyte losses in sweat, reduced renal blood flow and continued muscular activity. Many parameters did not change significantly between the 4 km marker (midway point on Phase C) and the end of Phase C. However, there were a few notable exceptions: potassium, chloride, lactate and glucose concentrations decreased, whereas pH and creatinine concentrations increased significantly from the 4 km marker to the end of Phase C. The most significant benefit of the extra distance from the 4 km marker to the end of Phase C was that it facilitated further dissipation of lactate concentrations prior to the start of Phase D.

Alkaline Phosphatase

An investigation into the development of early postural control.

Eighteen normal infants were studied longitudinally using video and photographic records of their levels of lying and sitting ability. A developmental sequence of postural control in lying and sitting was confirmed in the normal infants. All infants reached level 4 prone and supine lying ability before achieving level 3 sitting ability (maintaining independent sitting). 34 children with cerebral palsy in a cross-sectional study could be ascribed a level of sitting or lying ability. The relationship found in normal infants between lying and sitting was confirmed in all the children with cerebral palsy.

Adolescent

Haematological and biochemical changes in horses competing in a 3 Star horse trial and 3-day-event.

Haematological and biochemical changes in horses competing in the Endurance Test (Phase T and D) of an advanced Horse Trial (HT, n = 22) and the Endurance Test (Phases A-D) of an advanced (CCI) 3-day-event (TD, n = 11) over a similar course on the same day were studied. Environmental conditions during the event were cool (5.5-11.1 degrees C). Blood samples were collected from the horses in each group the evening prior to the Endurance Test, within 60 s after, and 10 min after, completion of Phase D (cross-country jumping). The following were determined in the blood samples and compared between the 2 groups of horses: packed cell volume (PCV), serum total protein [TP], serum albumin [ALB], plasma lactate [lactate], serum total calcium [TCa], plasma ionised calcium [Ca+2], serum inorganic phosphate [PO4], plasma pH, plasma sodium [Na], plasma potassium [K], serum chloride [Cl], serum urea nitrogen [SUN], serum creatinine [Cr] and serum glucose concentrations and aspartate aminotransferase (AST) and creatine kinase (CK) activities. The PCV and [Cr] were higher in the TD group and approached significance (P = 0.063 and P = 0.057, respectively). The [TP], [ALB], [Na], glucose concentration and CK, and AST were significantly higher and [Cl] and [PO4] were significantly lower in the TD group after exercise when compared to the HT group. It was deduced from these data that the horses competing in the 3-day-event experienced greater fluid and electrolyte losses, reduced glomerular filtration, higher glycogenolysis and had greater leakage of enzymes from working muscles during competition than horses competing in the horse trial.

Animals

Effects of shortening the steeplechase phase (phase B) of a 3-day-event.

Thirty-four horses competing in the Endurance Test of a 3-day-event were divided into 3 groups: horses in Group 1 (n = 15) competing in a 3.5 min steeplechase phase; horses in Group 2 (n = 13) in a 3 min steeplechase phase (Phase B) and horses in Group 3 (n = 6) in a 2.5 min steeplechase phase. The shortening of Phase B was associated with a lengthening of Phase C so that the total distance of the event for all horses was 14,940 m. Bodyweight (BW) was measured and total body water (TBW) and water loss estimated. Blood samples were collected from the horses prior to the Endurance Test, at the end of Phase B, the 4 km marker on Phase C (C4K), the end of Phase C, and 20 min after the completion of Phase D for measurement of packed cell volume (PCV), total plasma protein [TPP], lactate, ionised calcium, pH, sodium, potassium, chloride, total calcium and glucose concentrations, and aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities. Mean +/- s.d. ambient environmental temperature during the Endurance Test was 25.3 +/- 1 degrees C (range 20.3 degrees C-29.7 degrees C). Mean relative humidity was 43.8 +/- 2.4% (range 39%-48.6%) and the average 'comfort index' (CI) was 121. There were no significant differences between the groups competing in the Endurance Test, despite the shorter Phase B. However, there were significant decreases in BW, TBW, net exchangeable cations, chloride, ionised calcium, and pH. The sodium and total calcium concentrations remained at near pre-event values. The PCV, TPP, lactate, potassium, glucose, aspartate aminotransferase, and lactate dehydrogenase activity increased during the Endurance Test, when compared to pre-event values. Horses competing in this competition experienced significant fluid and electrolyte losses, reduced glomerular filtration, increased glycogenolysis and had significant leakage of enzymes from working muscles during competition. These changes could not be reduced by shortening Phase B and lengthening Phase C.

Animals

Aggregate risk study of exposure to endophyte-infected (Acremonium coenophialum) tall fescue as a risk factor for laminitis in horses.

Loline and ergot alkaloids found in endophyte-infected (Acremonium coenophialum) tall fescue (EITF) cause vasoconstriction of equine vessels in vitro. An aggregate risk study was used to evaluate the association between horses exposed to EITF and development of laminitis. Veterinary teaching hospitals participating in the Veterinary Medical Data Base were grouped by whether equine accessions were likely to have been at high, moderate, or low risk for exposure to EITF. From 1980-1990, there were 185,781 accessions, of which 5,536 had diagnosis of laminitis. Proportion of equine accessions with laminitis reported by veterinary teaching hospitals for high, moderate, and low risks, were 3.41, 3.04, and 2.00 cases/100 accessions, respectively (P < 0.0001). Comparison of the proportion of accessions with laminitis in the high- and moderate-risk groups with that in the low-risk group revealed significant differences between risk groups over all months (P = 0.063) and differences from month to month within risk groups (P = 0.0001). If the difference among risk groups is attributed entirely to exposure to EITF, the population-attributable risk is 7 cases/1,000 admissions, or 15% of all admissions for laminitis at veterinary teaching hospitals in our data base. Preliminary data support an association between horses exposed to EITF and increased risk of laminitis; however, studies at the individual animal level are indicated to confirm this hypothesis.

Acremonium

Blood ionized calcium concentrations in horses before and after the cross-country phase of three-day event competition.

Blood ionized calcium (Ca2+) and pH; plasma lactate concentrations; and total protein, total calcium (CaT), albumin, and phosphorus concentrations in serum were determined in 40 healthy horses before (T1), at the finish line (T2), and 10 minutes after the finish (T3) of the cross-country phase of a 3-day-event competition. Mean (+/- SEM) Ca2+ concentrations decreased from 6.22 +/- 0.04 mg/dl at T1 to 5.04 +/- 0.07 mg/dl at T2 (P < or = 0.05). This decrease was accompanied by a nonsignificant increase in CaT between T1 and T2. The mean (+/- SEM) percent ionization of calcium decreased significantly (P < or = 0.05), from 50.9 +/- 2.75% at T1 to 40.3 +/- 3.58% at T2. Significant increases in mean albumin, total protein, phosphorus, and lactate concentrations and a significant decrease in mean pH were observed at T2 (P < or = 0.05). At T3, mean Ca2+ and percent ionization had increased, but remained significantly less than resting values. Mean CaT was significantly decreased at T3, compared with values at T1 and T2. Correlation of mean Ca2+ concentration with all other measured variables at each time was evaluated; correlation coefficients between mean Ca2+ and all other variables were low (r2 < or = 0.38), indicating low biological significance.

Animals

Weight, water, and cation losses in horses competing in a three-day event.

Body weight of 48 horses competing in a 3-day event was measured the day before the event (baseline), following the dressage phase of the event (day 1), after the endurance phases of the event (day 2), and 18 to 24 hours after the endurance phases (day 3). Plasma sodium and potassium concentrations were measured the evening before, immediately after, and 10 minutes after the endurance phases. Total body water, water loss, and net exchangeable cation loss were then calculated. Body weight and total body water were significantly decreased, compared with baseline values, at all times during the event, and significant water loss was detected. The largest changes were recorded after the endurance phases of the event. Water deficits were still detected 18 to 24 hours after the endurance phases of the event. Mean plasma sodium concentration was significantly increased immediately after the endurance phases of the event, compared with concentration measured the evening before, and remained increased after the 10-minute recovery period, presumably because of dehydration. Mean plasma potassium concentration was significantly increased immediately after the endurance phases of the event, compared with concentration measured the evening before, but was not increased after the 10-minute recovery period.

Animals

Albumin quotient, IgG concentration, and IgG index determinations in cerebrospinal fluid of neonatal foals.

Total protein (TP), albumin, and IgG concentrations were measured in CSF from the atlanto-occipital (AO) and lumbosacral (LS) sites and in serum of 15 clinically normal neonatal foals < or = 10 days old (mean, 7.0 days). The albumin quotient (AQ; CSF albumin/serum albumin x 100) and IgG index ([CSF IgG/serum IgG] x [serum albumin/CSF albumin]), indicators of blood-brain barrier permeability and intrathecal IgG production, respectively, were then calculated. Mean +/- SD values obtained from the foals of this study were: serum albumin, 2,900 +/- 240 mg/dl; serum IgG, 1,325 +/- 686 mg/dl; AO CSF total protein (TP), 82.8 +/- 19.2 mg/dl; LS CSF TP, 83.6 +/- 16.1 mg/dl; AO CSF albumin, 52.0 +/- 8.6 mg/dl; LS CSF albumin, 53.8 +/- 15.7 mg/dl; AO CSF IgG, 10.2 +/- 5.5 mg/dl; LS CSF IgG, 9.9 +/- 5.7 mg/dl; AO AQ, 1.86 +/- 0.29; LS AQ, 1.85 +/- 0.51, AO IgG index, 0.52 +/- 0.28; and LS IgG index, 0.48 +/- 0.27. Significant difference between values for the AO and LS sites was not found. A CSF albumin concentration > 85.2 mg/dl or AQ > 2.4, as determined by mean +/- 2 SD, may indicate increased blood-brain barrier permeability. An IgG index value > 1.0 may indicate intrathecal IgG production. Values obtained for foals of this study should serve as baseline for comparison in the evaluation of blood-brain barrier permeability and intrathecal IgG production in neonatal foals with neurologic disease.

Albumins

A prospective study of septicaemia in colostrum-deprived foals.

Fourteen mares and their foals were attended at parturition. After mare-foal bonding, 8 colostrum-deprived (CD) foals were removed from their dams, deprived of colostrum, and provided with an alternative milk source for the first 24 h of life. The mares were milked out every 2-4 h during this period to remove colostrum, after which the CD foals were returned to their mares and allowed to nurse. Six colostrum-fed (CF) foals were allowed to suck colostrum in the normal manner. Foal serum IgG concentration was determined by single radial immunodiffusion (means, CD = 0 mg/dl; CF = 1,508 mg/dl). Accepted methods were used to minimise infections in the neonatal foals. Of the 8 CD foals, 7 demonstrated clinical signs of sepsis. Septicaemia was confirmed in 5 of the 7 septicaemic CD foals by ante-mortem blood culture or by culture of tissue at necropsy. Organisms isolated included: Actinobacillus equuli, Escherichia coli, undifferentiated coliforms, Pseudomonas spp., and Actinomyces pyogenes. Clinically ill foals were treated with antimicrobial drugs, intravenous fluid therapy, flunixin meglumine, and anti-endotoxin hyperimmune serum. Three septicaemic CD foals survived. Four of 7 septicaemic CD foals died or were destroyed. Post-mortem lesions included bacterial embolic pneumonia, glomerulonephritis/nephritis, lymphoid depletion/atrophy, splenic and lymphoid necrosis, hepatitis, septic arthritis, and systemic bacterial embolism. None of the CF foals became septicaemic. One CF foal had foal heat diarrhoea and 1 CF foal had a serum IgG concentration of 160 mg/dl (i.e. failure of passive transfer), but both foals were otherwise normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Serum tumor necrosis factor alpha concentrations and clinical abnormalities in colostrum-fed and colostrum-deprived neonatal foals given endotoxin.

We examined the effect of infusion of lipopolysaccharide (LPS) on serum tumor necrosis factor alpha (TNF alpha) concentration and clinical attitude in 2- 3-day-old colostrum-fed (CF) and colostrum-deprived (CD) foals. Eleven CF and 8 CD neonatal foals were given a bolus i.v. infusion of Escherichia coli O55:B5 lipopolysaccharide (0.5 microgram/kg of body weight) in sterile saline (0.9% NaCl) solution. Four CF and 2 CD foals were given saline solution alone. Serum IgG concentration and serum anti-LPS IgG(T) antibody titer were determined for each foal prior to infusion. A depression index was used to score clinical abnormalities. Serum TNF alpha concentration was estimated by use of an in vitro cytotoxicity bioassay that used WEHI 164 clone 13 cells as targets. The cytotoxic serum factor was identified as TNF alpha by immunoprecipitation with caprine antisera raised against the 15 NH2-terminal amino acids of human TNF alpha. Tumor necrosis factor alpha was not detected in any preinfusion serum samples nor in any samples from foals given saline solution alone. Serum TNF alpha concentration increased in all LPS-infused foals and peaked between 60 and 90 minutes after infusion. Serum TNF alpha concentrations, expressed as mean percentage of peak serum TNF alpha concentration, persisted longer in CD foals given LPS than in CF foals given LPS. All LPS-infused foals displayed clinical signs of endotoxemia, but mean depression index scores of the CF and CD foals given LPS were not significantly different at any time. Serum TNF alpha concentrations were correlated with depression index scores in both LPS-infused groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Serum interleukin-6 concentrations in endotoxin-infused neonatal foals.

Serum interleukin-6 (IL-6) concentration was measured in 11 colostrum-fed (CF) and 8 colostrum-deprived (CD) 2- to 3-day-old foals after foals were infused with lipopolysaccharide (LPS; Escherichia coli O55:B5 endotoxin, 0.5 microgram/kg of body weight in sterile saline [0.9% NaCl] solution). Four CF and 2 CD foals were given saline solution alone. Serum IL-6 concentration was estimated by use of an in vitro proliferative bioassay, using the IL-6 dependent B.13.29 clone 9 cells. Interleukin-6 concentration increased in all LPS-infused foals, and geometric mean serum IL-6 concentration was significantly higher in CF than CD foals 30 and 90 minutes after infusion. Both LPS-infused groups had multiple spikes of mean IL-6 concentration that peaked at 120 minutes in CF foals and 150 minutes in CD foals. Results indicated that IL-6 is produced in neonatal foals in response to LPS infusion. Furthermore, colostrum deprivation resulted in longer times to peak mean serum IL-6 concentration and tended to reduce serum IL-6 concentration in neonatal foals.

Animals