Parenteral administration of glutamine modulates acute phase response in postparturient dairy cows.

The objective of this study was to investigate whether administration of L-Gln would affect mediators of acute phase response in postparturient dairy cows. Twenty-four multiparous Holstein cows were blocked by the expected day of calving and randomly assigned to 1 of the 3 treatment groups (n = 8/group): 1) i.v. infusion of 10 L of 0.85% NaCl (control), 2) i.v. infusion of 106, or 3) 212 g/d of L-Gln mixed with 10 L of 0.85% NaCl solution; each treatment was given 8 h/d for each of 7 consecutive days starting on d 1 after calving. Blood samples were collected 1 wk before the expected day of parturition as well as on d 0, 7, 14, and 21 after parturition; plasma concentrations of serum amyloid A (SAA), haptoglobin, and lipopolysaccharide-binding protein were measured by ELISA, and alpha(1)-acid glycoprotein was assessed by radial immunodiffusion. Concentrations of SAA, haptoglobin, and alpha(1)-acid glycoprotein increased in control cows after parturition, reaching peak values on d 0 or 7 postpartum (60, 1,093, and 963 microg/mL, respectively). Cows infused with 106 g/d of L-Gln had greater concentrations of SAA in plasma on d 14 and 21 compared with controls (62.8 vs. 30.2 and 71.1 vs. 34.5 microg/mL, respectively). Cows infused with 212 g/d of L-Gln had greater concentrations of SAA on d 7 (82.5 vs. 53.9 microg/mL) and lower concentrations of haptoglobin on d 14 and 21 postpartum compared with controls (264 vs. 621 and 175 vs. 587 microg/mL, respectively). Cows treated with 106 and 212 g/d of L-Gln had greater plasma lipopolysaccharide-binding protein concentrations on d 7 compared with control group (50.0 and 35.6 vs. 10.8 microg/mL, respectively). There were no treatment differences with respect to milk yield and DM intake during the experimental period. In conclusion, our data indicate that i.v. administration of L-Gln modulated acute phase mediators in dairy cows after parturition and warrants further research into the mechanisms behind these effects.

Effects of fasting and aminophylline on norepinephrine-stimulated non-shivering thermogenesis.

In an attempt to further elucidate the mechanisms of fasting-depressed maximum thermogenesis and cold tolerance, norepinephrine (NE)-stimulated non-shivering thermogenesis (NST) in cold-acclimated rats was used as a functional index of possible alterations in adrenergic efficacy after fasting. Fasting decreased the magnitude of maximum NE-Stimulated NST by 18.2% [6.87 +/- 0.47 Kcal (Kg X 75 X min)-1 well-fed vs. 5.81 +/- 0.39 Kcal (Kg X 75 X min)-1 fasted], but the apparent adrenergic binding affinity was not affected [Ke = 0.43 micrograms NE min-1 well-fed vs. 0.55 micrograms NE min-1 fasted]. Pretreatment with aminophylline [15 mg Kg-1, i.p.], a phosphodiesterase inhibitor, restored the fasting-depressed NE-stimulated NST to the fed level. The results suggest that the depression of maximum thermogenesis after fasting is not due to changes in adrenergic binding characteristics but to alteration in cAMP production/degradation, resulting in decreased substrate mobilization for thermogenesis.

Treatment of gilts with leukocytes from the sire does not improve reproductive performance.

The effectiveness of pretreatment of gilts with leukocyte antigens on reproductive performance was studied. Two experiments were carried out that involved the treatment of gilts with leukocytes prior to artificial or natural insemination. Gilts were randomly assigned to one of three treatment groups. Group A gilts received two doses of 2 x 10(8) sire's leukocytes; one injected intra-peritoneal at first estrus and the other infused into the uterus immediately prior to insemination at the second estrus cycle. Group B gilts were similarly treated with the 'gilt's own' (autologous) leukocytes and Group C animals received phosphate buffered saline as controls. The same boars were used as sires across all three treatment groups. Gilts were slaughtered during the fifth week of pregnancy and their reproductive performance assessed. When data from the three groups were compared there were significant differences in embryo survival and placental weights between the sire's leukocyte treatment and PBS control groups. Embryo survival rate (No viable embryos/No corpora lutea x 100) at the fifth week of gestation was lower (p < 0.05) and placental weights were higher in the sire's leukocyte treatment group (p < 0.05). However, the latter may likely be due to fewer developing embryos. Embryo survival and placental weights did not vary significantly between the sire's and autologous leukocytes treatment groups. The treatment of gilts with leukocytes from the boars used as sires showed no improvement on subsequent reproductive outcome over that observed for the autologous leukocytes or PBS control treatments. Consequently, in a healthy well managed herd leukocytes treatment offers no advantage to pig reproductive function.

Effect of fasting on capillary blood flow in sheep.

Capillary blood flow rate was measured in 29 tissues in two hourly fed and 26 hour fasted Blackface and Clun lambs using the radiolabelled microsphere technique. Blood flow declined with fasting in alimentary tract, skin of the ear and some of the larger skeletal muscles. It was concluded that capillary perfusion in nerve tissue was not affected by fasting whereas tissues principally responsible for energy absorption, expenditure and retention can experience a fall in capillary blood flow during fasting.

Adipose tissue capillary blood flow in relation to fatness in sheep.

Capillary blood flow rate was measured in eight fat depots in eight Blackface and eight Clun lambs using the radiolabelled microsphere technique. Adipose tissue flow ranged from 3 to 47 ml 100 g-1 min-1 depending upon depot and degree of fatness. Blood flow declined with increasing fatness suggesting that perfusion was not an important constraint on the growth of fat. Blood flow rates were also measured in the fed and 26 hour fasted states but no effect from fasting was observed.

Acetyl-CoA carboxylase and fatty acid synthase activity and immunodetectable protein in adipose tissues of ruminants: effect of temperature and feeding level.

To gain insights into the regulation of fat synthesis, we have investigated the effect of cold environmental exposure and feed restriction of sheep on activity and immunodetectable protein content of acetyl-CoA carboxylase (ACC) and fatty acid synthase in adipose tissue. Subcutaneous and mesenteric adipose tissues were collected at slaughter from sheep exposed to either cold (0+/-2 degrees C) or warm (23+/-2 degrees C) environment, and given either ad libitum or restricted access to feed for three 5-wk periods. Acetyl-CoA carboxylase was isolated from frozen adipose tissue samples and activity determined as the rate of incorporation of H14CO3- into acid stable malonyl-CoA. Cold exposure and feed restriction reduced (P < .05) ACC activity in the two adipose tissue depots. Western blot analysis with peroxidase-conjugated streptavidin showed that both adipose tissue depots express a single isoform of ACC. In s.c. adipose tissue, cold exposure increased (P < .05) ACC protein abundance, which is opposite to the change in activity. However, feed restriction reduced immunodetectable ACC protein. There was no significant effect of environment or feeding level on ACC protein abundance in mesenteric tissue. Fatty acid synthase activity determined in ammonium sulfate extract by measuring the malonyl-CoA- and acetyl-CoA-dependent oxidation of NADPH was decreased (P < .05) by feed restriction in both s.c. and mesenteric tissues. Cold exposure reduced fatty acid synthase activity in s.c. but not in mesenteric tissue. There was no effect of environment on fatty acid synthase protein abundance in either adipose tissue depot. However, feed restriction significantly reduced fatty acid synthase protein abundance in the two depots. The data suggest that feed restriction and exposure of ruminants to cold environmental conditions may significantly down-regulate the activity of key lipogenic enzymes.

Effects of temperature and plane of nutrition on beta-adrenergic receptors in heart, kidney, and liver of lambs.

We determined the effects of temperature and feed intake on beta-adrenergic receptors (beta-adrenoceptors) in tissues of sheep. Twenty-four lambs were exposed during three 5-wk periods to either thermoneutral, control (W; 23+/-2 degrees C) or cold (C; 0+/-2 degrees C) temperatures and were fed either ad libitum (A) or restricted (R) levels of feed intake, resulting in four treatment groups: WA, WR, CA, and CR. Hearts, kidneys, and livers were harvested at slaughter and binding of [3H]dihydroalprenolol to plasma membrane extracts was used to determine densities (B(MAX)) and binding affinities (Kd) of beta1 and beta2 adrenoceptors. The B(MAX) values ranged from 12.10 to 201.26 and 3.38 to 12.30 fmol/mg protein for beta1 and beta2 adrenoceptors, respectively; heart and kidney had the highest and lowest values, respectively. Feed restriction reduced (P < .05) beta1 and beta2 receptor densities in heart but increased (P < .05) beta1 receptor density in kidney and liver. Cold temperature exposure reduced beta1 receptor density in heart tissue during feed restriction. The Kd values, ranging from 1.32 to 5.98 nM, were increased (P < .05) by cold exposure and feed restriction in kidney and liver. Because the effectiveness of hormones is a function of their concentrations, binding affinities, and their receptor densities, these results imply that cold temperature exposure and feed restriction could potentially reduce (in heart) and increase (in kidney and liver) metabolic responsiveness of tissues to catecholamines.

Blood flow distribution in hind limb bones and joint cartilage from young growing pigs.

Blood flow distribution was studied in hind limb bones and joint cartilage from four ten week old and four 20 week old pigs using 141Ce-microspheres. In general, blood flow rate was greater in the epiphyseal and metaphyseal cancellous bone than in the cortical bone and joint cartilage. In the epiphyseal cancellous bone, blood flow rate was three to tenfold greater (P less than 0.01) in the surface 2 mm layer than in the remaining deeper bone in all femoral and tibial samples examined. In the joint cartilage, blood flow rate in the femoral condyle was greater (P less than 0.05) than in the proximal femur, patella, central tarsus and metatarsus in ten week old pigs, and was greater (P less than 0.05) in the femoral condyle than in the patella and metatarsus in 20 week old pigs. A significant (P less than 0.05) age-associated decrease in the rate of blood flow was observed in the femoral, patellar and metatarsal cartilage. Within the femoral condyle, no differences in blood flow were found between areas associated with relatively high versus relatively low incidences of osteochondrosis.

Effects of adrenaline and noradrenaline on the heat production of warm- and cold-acclimated sheep.

The heat production (HP), heart rate (HR), respiration rate, rumen motility, and body temperature responses to 2.5-h adrenaline (A) and noradrenaline (NA) intrajugular infusions at 0.00, 0.15, 0.30, 0.60, and 0.90 micrograms.kg-1.min-1 were studied in 10 shorn wethers which had been chronically (3-8 weeks) exposed to warm (19-24 degrees C) or moderately cold (8-13 degrees C) temperatures. Heat production, as estimated from respired gas analysis, increased 40-45% with all doses of A and the effect was potentiated by chronic cold exposure. Only the higher dose rates of NA induced an increase in HP. The maximum HP increase due to NA was 30% and the effect was not influenced by chronic cold exposure. Thermoneutral HP was greater by 16-19% in cold-acclimated as compared with warm-acclimated sheep. Corresponding to the HP effects of A and NA, all doses of A and the highest dose of NA resulted in slight increases in rectal temperature. Respiration rate increased with increased dose rate of NA but only the highest dose of a resulted in an increase in respiration rate. HR, rectal temperature, and respiration rate responses to A and NA were not influenced by cold acclimation.

Changes during eating in oxygen consumption, cardiac function and body fluids of sheep.

1. A study was made of the changes taking place in O(2) consumption, cardiac function and the volume and composition of the body fluids of sheep while they consumed a meal of hay.2. During eating P(a, CO2) and P(v, CO2) both increased, pH decreased and free plasma [HCO(3) (-)] increased. Venous haematocrit increased sharply at the beginning of the meal, and declined slowly after feed was removed.3. Arterial P(O2) did not change significantly during eating. However P(v, O2) fell slightly but significantly. The O(2) saturation of venous blood fell due to the decline in pH. Estimated CO(2) in arterial blood increased as a consequence of increased haemoglobin content. The net effect was to increase arteriovenous difference in O(2) content from 4.4 ml./100 ml. before eating to 6.0 ml./100 ml. at the end of the meal.4. O(2) consumption increased about 60% during eating and fell rapidly thereafter. Heart rate followed a similar pattern. Cardiac output however increased only about 17%, from 6 to 7 l./min. Consequently stroke volume declined throughout the meal from 76 to 52 ml./beat.5. Plasma volume, estimated from measurements of T-1824, declined sharply by about 300 ml. at the beginning of the meal and recovered slowly after feed was removed. Blood volume declined less because of a rise in circulating erythrocytes.6. Extracellular fluid volume was estimated from measurements of thiocyanate and thiosulphate spaces. Thiocyanate space measurements were abandoned after thiocyanate was found to be concentrated in saliva. Considerable random variation occurred in measurements of changes in extracellular fluid from thiosulphate disappearance but the results did reveal a significant fall of 1000-1500 ml. in extracellular fluid volume during eating.7. The significance of these interrelated changes is discussed in relation to the maintenance of homoeostasis during eating in the sheep.

Effect of beta-adrenergic agonist L-644,969 on the impact of the thermal environment on in vitro fatty acid synthesis and lipogenic enzymes in sheep.

The effect of temperature and beta-adrenergic agonist (BAA) on in vitro rates of fatty acid synthesis and catalytic activity of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) was examined in wether lambs after 5 weeks at either 0 or 20 degrees C. Feeding BAA increased (P < 0.05) rate of fatty acid synthesis by 38% in subcutaneous adipose (SC) tissue from cold-acclimated animals but the rate decreased (P < 0.05) by 27% in SC tissue from warm-acclimated animals. In mesenteric fat (MS), BAA increased (P < 0.05) fatty acid synthesis in the cold environment. In perirenal (PR) fat, rate of fatty acid synthesis was reduced (P < 0.05) by 20% by BAA in the warm but had no effect in the cold. Activity of ACC in longissimus muscle was depressed (P < 0.05) when BAA was fed in the warm environment. In adipose tissues BAA reduced (P < 0.05) ACC activity in the warm, but reduced activity in the cold was limited to SC tissue. In PR tissue FAS activity was reduced (P < 0.05) in the cold environment, while BAA increased FAS activity in the warm environment. Western blot analysis showed two isoforms of ACC with MW of 280 000 and 265 000 Da in longissimus muscle whereas only one isoform was recognized in each of Biceps femoris (280 000 Da) and adipose tissues (265 000 Da). Feeding BAA in the cold environment reduced (P < 0.05) ACC and FAS immunoprotein expression in both MS and PR adipose tissues. The studies indicate that the effect of BAA on fatty acid synthesis and lipogenic enzymes is influenced by acclimation temperature.

Skeletal and cardiac muscle protein turnover during short-term cold exposure and rewarming in young rats.

Young animals exposed to cold environmental temperatures typically have decreased skeletal muscle accretion but increased heart masses. To explore these phenomena, we measured protein synthesis and degradation in vivo in cardiac and skeletal muscle in weanling rats during short-term cold exposure and rewarming. Control rats were housed at 25 degrees C throughout the experiment. Ad libitum-fed and pair-fed (to the intake of controls) rats were housed at 5 degrees C (cold) for 5 days and then at 25 degrees C (rewarmed) for another 5 days. Cold exposure decreased rates of protein accretion and synthesis in skeletal muscle, whereas degradation did not differ. The effects of cold exposure on skeletal muscle were similar in both pair-fed and ad libitum-fed rats, except growth was lower in pair-fed rats. In cardiac muscle, cold exposure increased rates of protein synthesis and degradation and resulted in increased cardiac mass. Results in pair-fed animals generally fell between those of control and ad libitum-fed cold rats. During rewarming, growth rates were not higher in skeletal muscle in ad libitum-fed re-warmed rats, although protein turnover returned toward control values; in pair-fed rats, it remained lower. In heart, growth rates of ad libitum-fed and pair-fed rewarmed rats decreased due to lower protein synthesis rates. These alterations appear to be consistent with a strategy designed to improve survival in cold environments.

Effects of cold exposure on feed protein degradation, microbial protein synthesis and transfer of plasma urea to the rumen of sheep.

1. Three diets of barley-canola-seed (Brassica campestris), lucerne (Medicago sativa) or chopped brome-grass (Bromus inermis) were given at intervals of 3 h to closely-shorn Suffolk wethers held at a temperature of 1-5 degree (cold) or 22-24 degree (warm). Apparent digestibility of organic matter (OM) and nitrogen was reduced by 0.08-0.05 and 0.04 units respectively for lucerne and brome-grass diets given to cold-exposed sheep, but no treatment effects on digestibility were observed for the barley-CSM diet. Measurements achieved using infusion of the digesta markers 58Co-EDTA and 103Ru-phenanthroline (103Ru-P) showed that cold exposure depressed apparent OM digestion in the stomach and intestines by 33 and 42 g/d for the lucerne diet, and 13 and 35 g/d for the brome-grass diet respectively. 2. The turnover time (h) of the 103Ru-P marker in the rumen of warm sheep was 38.9 for barley-CSM, 18.4 for lucerne, and 15.6 for brome-grass. In cold-exposed sheep, 103Ru-P turnover time (h) tended to be reduced to 32.3, 12.3 and 15.3 for the three diets, respectively. OM fermentation in the stomach was highly related to 103RU-P turnover time for lucerne and brome-grass diets. 3. Cold exposure increased the escape of dietary N from the abomasum by 0.04 and 0.09 of dietary N intake for sheep given lucerne and brome-grass diets respectively. Dietary N degradation was closely related to 103Ru-P turnover time for lucerne, and to the proportion of large particles in rumen digesta for the brome-grass diet. Estimates of feed N degradation made by use of information on the rate of fermentation of the diet in nylon bags and 103Ru-P turnover time were consistently lower than those observed in vivo for barley-CSM and lucerne diets. Intestinal digestibility of non-ammonia N was not significantly changed by cold exposure. 4. Transfer of urea from plasma to the rumen was 1.4-2.5 g N/d for the barley-CSM and lucerne diets, but the value for brome-grass was 4.5-4.9 g N/d. Cold exposure did not affect urea transfer. The production of ammonia from feed and endogenous protein was approximately 0.66 and 0.47 g N/g N intake of barley-CSM and lucerne diets, with no effect of cold exposure. Cold exposure reduced the value from 0.57 to 0.38 for brome-grass. 5. The results are compared with those obtained previously with pelleted hay, and the importance of large particle breakdown in the prediction of OM and N fermentation using nylon bags is discussed.

Apparatus for controlling temperature of catheter contents in cold environments.

An apparatus was developed to prevent the freezing of catheter contents during blood sampling or liquid infusion into animals maintained at sub-freezing temperatures. The apparatus consisted of a larger, outer set of plastic and copper tubes and a smaller, inner set of tubes. The catheter was passed inside the inner set. Water circulated through the outer set kept the contents of the catheter inside the inner set from freezing. The apparatus was also useful for the rapid cooling of blood to a predetermined temperature while a sample was being withdrawn from an animal.

The effect of cold exposure of sheep on digestion, rumen turnover time and efficiency of microbial synthesis.

1. Six closely shorn sheep were given brome grass (Bromus inermis) pellets at 1 h intervals and maintained at ambient temperatures of --I to Idegree and 18--21degrees for 28 d. Measurements of digestion were made during the last 10 d of temperature exposure. 2. Cold exposure resulted in a reduction in apparent dry matter (DM) digestibility from 0-482 to 0-450, and of apparent digestibility of organic matter (OM) from 0-511 to 0-477. Neither apparent digestibility nor retention of nitrogen was affected. 3. Apparent digestibility of OM in the rumen decreased from 0-300 to 0-242 with cold exposure, and was highly correlated with turnover time in the rumen of 103Ru, which was used as a particulate marker. 4. The efficiency of microbial synthesis (g N incorporated into microbial cells/kg OM apparently digested) was correlated with the dilution rate of the solute marker (51Cr) and with the turnover time of the particulate marker (103Ru) in the rumen. 5. Digestion in the intestine of DM and OM accounted for significantly more of apparent digestion in the whole gastrointestinal tract for sheep kept in the cold than for sheep kept in the warm. The apparent digestibilities of DM and OM entering the intestine were similar in sheep on both treatments, but significantly more non-ammonia-N was digested in the intestines of cold-exposed sheep. 6. The influence of dilution rate of rumen fluid on the efficiency of synthesis of microbial cells in the rumen is discussed.

The effect of a cold environment on protein and energy metabolism in calves.

Eleven Holstein bull calves 35 d of age were assigned to one of three treatment groups: (1) W72, warm environment (20 degrees), 72 g feed/kg body weight (BW)0.75 per d, (2) C72, cold environment (-5 degrees), 72 g feed/kg BW0.75 per d, or (3) C90, cold environment (-5 degrees), 90 g feed/kg BW0.75 per d. Fractional synthesis rates (FSR) of protein in the rumen wall, rumen papillae, omasum, duodenum, kidney, liver, heart, longissimus dorsi, biceps femoris and skin were determined following a continuous infusion of [3H]phenylalanine. Phenylalanine flux was elevated in both groups of cold-adapted calves. FSR of protein in the two muscles and skin were reduced along with N retention in the calves in the C72 group compared with the other two groups. Muscle protein degradation, estimated from urinary N tau-methylhistidine excretion, tended to be elevated in both groups of cold-adapted calves. Reduced protein synthesis and increased protein degradation in the C72 group contributed to reduced muscle protein gain. It appears that when feed intake is limited in cold-adapted animals, muscle and skin have a lower priority for nutrients than other organs and tissues, resulting in reduced protein synthesis. It seems unlikely that thermogenesis due to enhanced protein synthesis contributed to the increased heat production in the cold.

A porcine trophoblast cell line that secretes growth factors which stimulate porcine macrophages.

We have previously described experiments in both the mouse and the human indicating that cytokines capable of activating macrophages (colony-stimulating factor-1 [CSF-1], granulocyte-macrophage colony-stimulating factor [GM-CSF], and interleukin-3 [IL-3]) are produced by, and/or stimulatory of, trophoblast cells in these species. In contrast to the complex hemochorial placenta of the mouse and humans, the pig has a simple diffuse type of placenta, designated as epitheliochorial. To determine whether similar phenomena might not apply to the porcine pregnancy, we have isolated a cell line, designated Jag-1, from the trophoblastic tips of Day 14 porcine embryos. We report here that this cell line is cytokeratin-positive, vimentin-negative, and therefore of epidermal origin. It also shares various morphological characteristics with porcine trophoblast as demonstrated at both the light and electron microscopic levels. In addition, Jag-1 cells and primary trophoblast tissue from Day 14 blastocyst do not express classical major histocompatibility (MHC) class I and class II antigens, a unique feature of trophoblast in many species. To determine the ability of this cell line to produce cytokines, we have developed an assay for porcine macrophage growth factors that utilizes uptake of tritiated thymidine. This assay responds positively to recombinant bovine GM-CSF and, more importantly, detects a similar activity in supernatants of the porcine trophoblast cell line and of Day 14 blastocysts. Thus porcine trophoblast cells, like their murine and human counterparts, produce and potentially interact with lymphohematopoietic cytokines that are traditionally associated with macrophages.

Branched-chain alpha-keto acid analysis in biological fluids: preparative clean-up by anion-exchange and analysis by capillary gas chromatography.

A method is given for the quantitative analysis of the alpha-keto derivatives of the branched-chain amino acids in physiological fluids. A sample containing alpha-ketovalerate and alpha-ketocaproate as internal standards is passed through a weak anion-exchange resin at neutral pH. After washing the resin with distilled water, the alpha-keto acids are eluted with 4 M hydrochloric acid--ethanol (50:50). Quinoxalinol derivatives are prepared directly in the eluent, extracted with methylene chloride, and trimethylsilylated. Separation of the derivatives is by capillary gas chromatography on a 30 m fused-silica SE-30 column. Chromatographic separation is superior to that reported for packed column methods, thereby permitting the use of alpha-ketovalerate and alpha-ketocaproate as internal standards.

Capillary gas chromatographic analysis of amino acids in blood and protein hydrolysates as tert.-butyldimethylsilyl derivatives.

A method is given for a one-step derivatization and gas chromatography of amino acids in blood and protein hydrolysates. Blood samples are partially purified by solvent extraction. Protein hydrolysates are neutralized with a triethylamine solution. Then tert.-butyldimethylsilyl derivatives of the amino acids are prepared in a one-step procedure and separated on a 30-m fused-silica SE-30 capillary column. Except for tryptophan and cystine, amino acids are eluted within 30 min. Amino acids are derivatized more rapidly than their corresponding trimethylsilyl derivatives and do not degrade on the long fused-silica columns.