Review: Methods and biomarkers to investigate intestinal function and health in pigs.

Society is becoming increasingly critical of animal husbandry due to its environmental impact and issues involving animal health and welfare including scientific experiments conducted on farm animals. This opens up two new fields of scientific research, the development of non- or minimally invasive (1) methods and techniques using faeces, urine, breath or saliva sampling to replace existing invasive models, and (2) biomarkers reflecting a disease or malfunction of an organ that may predict the future outcome of a pig's health, performance or sustainability. To date, there is a paucity of non- or minimally invasive methods and biomarkers investigating gastrointestinal function and health in pigs. This review describes recent literature pertaining to parameters that assess gastrointestinal functionality and health, tools currently used to investigate them, and the development or the potential to develop new non- and minimally invasive methods and/or biomarkers in pigs. Methods described within this review are those that characterise gastrointestinal mass such as the citrulline generation test, intestinal protein synthesis rate, first pass splanchnic nutrient uptake and techniques describing intestinal proliferation, barrier function and transit rate, and microbial composition and metabolism. An important consideration is gut health, and several molecules with the potential to act as biomarkers of compromised gut health in pigs are reported. Many of these methods to investigate gut functionality and health are considered 'gold standards' but are invasive. Thus, in pigs, there is a need to develop and validate non-invasive methods and biomarkers that meet the principles of the 3 R guidelines, which aim to reduce and refine animal experimentation and replace animals where possible.

Effects of increasing levels of whole Black Soldier Fly (Hermetia illucens) larvae in broiler rations on acceptance, nutrient and energy intakes and utilization, and growth performance of broilers.

Meal of black soldier fly larvae (BSFL), which requires extraction of protein and fat, is a novel protein source for poultry, while unprocessed whole BSFL could even directly be fed to chickens. Newly hatched Ross-308 chicks (n = 252) received whole BSFL at 10% (L10), 20% (L20), or 30% (L30) of voluntary feed intake (FI) of control chickens (CON) that received no BSFL but only age-specific diets (n = 63 birds / group) for 42 days (d). Acceptance and nutrient and energy intake of birds by BSFL and FI were calculated. Plasma metabolites were measured using an automatic enzymatic analyzer and immunoglobulins with ELISA. Depending on the variable, data were analyzed using ANOVA or repeated measures ANOVA to address treatment, time and interaction effects. Birds consumed all offered larvae. With the exception of d1, time spent by birds eating their daily portion of larvae (TSL, min/pen) did not differ among the larvae supply groups (P = 0.982). The L10 had a higher larvae eating rate (LER) that is, speed of larvae intake than did L20 and L30 (P < 0.05), implying increased competition for less available BSFL. The ratio of LER to feed eating rate (FER) was greater than 50 fold change difference (FCD), indicating a strong interest of chickens in BSFL over regular feed. Whole BSFL intake up to 30% of voluntary FI did not adversely affect broiler growth (P > 0.05). The L30 had lower total dry matter and metabolizable energy intakes (P < 0.05), although total fat intake was higher in L30 than in CON (P < 0.05). Compared with CON, 30% whole BSFL increased dietary protein-to-energy ratios, plasma uric acid and serum alkaline phosphatase concentrations (P < 0.05). We conclude that whole BSFL can be included in broiler rations up to 20% without negatively affecting growth performance and nutrient conversion efficiency, whereas a higher proportion is associated with lower protein utilization efficiency, possibly due to lower total energy intake.

Dietary fiber and its role in performance, welfare, and health of pigs.

Dietary fiber (DF) is receiving increasing attention, and its importance in pig nutrition is now acknowledged. Although DF for pigs was frowned upon for a long time because of reductions in energy intake and digestibility of other nutrients, it has become clear that feeding DF to pigs can affect their well-being and health. This review aims to summarize the state of knowledge of studies on DF in pigs, with an emphasis on the underlying mode of action, by considering research using DF in sows as well as suckling and weaned piglets, and fattening pigs. These studies indicate that DF can benefit the digestive tracts and the health of pigs, if certain conditions or restrictions are considered, such as concentration in the feed and fermentability. Besides the chemical composition and the impact on energy and nutrient digestibility, it is also necessary to evaluate the possible physical and physiologic effects on intestinal function and intestinal microbiota, to better understand the relation of DF to animal health and welfare. Future research should be designed to provide a better mechanistic understanding of the physiologic effects of DF in pigs.

Proteasome activity and expression of mammalian target of rapamycin signaling factors in skeletal muscle of dairy cows supplemented with conjugated linoleic acids during early lactation.

The mammalian target of rapamycin (mTOR) is a major regulator of protein synthesis via its main downstream effectors, ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E binding protein (4EBP1). The ubiquitin-proteasome system (UPS) is the main proteolytic pathway in muscle, and the muscle-specific ligases tripartite motif containing 63 (TRIM63; also called muscle-specific ring-finger protein 1, MuRF-1) and F-box only protein 32 (FBXO32; also called atrogin-1) are important components of the UPS. We investigated 20S proteasome activity and mRNA expression of key components of mTOR signaling and UPS in skeletal muscle of dairy cows during late gestation and early lactation and tested the effects of dietary supplementation (from d 1 in milk) with conjugated linoleic acids (sCLA; 100 g/d; n = 11) compared with control fat-supplemented cows (CTR; n = 10). Blood and muscle tissue (semitendinosus) samples were collected on d -21, 1, 21, and 70 relative to parturition. Dry matter intake increased with time of lactation in both groups. It was lower in sCLA than in CTR on d 21, which resulted in a reduced calculated metabolizable protein balance. Most serum and muscle concentrations of AA followed time-related changes but were unaffected by CLA supplementation. In both groups, serum and muscle 3-methylhistidine (3-MH) concentrations and the ratio of 3-MH:creatinine increased from d -21 to d 1, followed by a decline on d 21. The mRNA abundance of MTOR on d 21 and 70 was greater in sCLA than in CTR. The abundance of 4EBP1 mRNA did not differ between groups but was upregulated in both on d 1. The mRNA abundance of S6K1 on d 70 was greater in CTR than in sCLA, but remained unchanged over time in both groups. The mRNA abundance of FBXO32 (encoding atrogin-1) on d 21 was greater in sCLA than in CTR. The mRNA abundance of TRIM63 (also known as MuRF1) showed a similar pattern as FBXO32 in both groups: an increase from d -21 to d 1, followed by a decline. The mRNA for the α (BCKDHA) and ß (BCKDHB) polypeptide of branched-chain α-keto acid dehydrogenase was elevated in sCLA and CTR cows on d 21, respectively, suggesting a role of CLA in determining the metabolic fate of branched-chain AA. For the mTOR protein, no group differences were observed. The abundance of S6K1 protein was greater across all time points in sCLA versus CTR. The antepartum 20S proteasome activity in muscle was elevated in both groups compared with postpartum, probably reflecting the start of protein mobilization before parturition. Plasma insulin concentrations decreased in both groups postpartum but to a greater extent in CTR than in sCLA, resulting in greater insulin concentrations in sCLA than in CTR. Thus, the greater abundance of MTOR mRNA and S6K1 protein in sCLA compared with CTR might be mediated by the greater plasma insulin postpartum. The upregulation of MTOR mRNA in sCLA cows on d 21, despite greater FBXO32 mRNA abundance, may reflect a simultaneous activation of both anabolic and catabolic signaling pathways, likely resulting in greater protein turnover.

Evaluation of electrical broad bandwidth impedance spectroscopy as a tool for body composition measurement in cows in comparison with body measurements and the deuterium oxide dilution method.

Body fatness and degree of body fat mobilization in cows vary enormously during their reproduction cycle and influence energy partitioning and metabolic adaptation. The objective of the study was to test bioelectrical impedance spectroscopy (BIS) as a method for predicting fat depot mass (FDM), in living cows. The FDM is defined as the sum of subcutaneous, omental, mesenteric, retroperitoneal, and carcass fat mass. Bioelectrical impedance spectroscopy is compared with the prediction of FDM from the deuterium oxide (DO) dilution method and from body conformation measurements. Charolais × Holstein Friesian (HF; = 18; 30 d in milk) crossbred cows and 2 HF (lactating and nonlactating) cows were assessed by body conformation measurements, BIS, and the DO dilution method. The BCS of cows was a mean of 3.68 (SE 0.64). For the DO dilution method, a bolus of 0.23 g/kg BW DO (60 atom%) was intravenously injected and deuterium (D) enrichment was analyzed in plasma and whey by stabile isotope mass spectrometry, and total body water content was calculated. Impedance measurement was performed using a 4-electrode interface and time domain-based measurement system consisting of a voltage/current converter for applying current stimulus and an amplifier for monitoring voltage across the sensor electrodes. For the BIS, we used complex impedances over three frequency decades that delivers information on intra- and extracellular water and capacity of cell membranes. Impedance data (resistance of extra- and intracellular space, cell membrane capacity, and phase angle) were extracted 1) by simple curve fit to extract the resistance at direct current and high frequency and 2) by using an electrical equivalent circuit. Cows were slaughtered 7 d after BIS and D enrichment measurements and dissected for the measurement of FDM. Multiple linear regression analyses were performed to predict FDM based on data obtained from body conformation measurements, BIS, and D enrichment, and applied methods were evaluated by cross-validation. The FDM varied widely between cows and was correlated to D enrichment in plasma ( = 0.91, < 0.05). Prediction of FDM by body size measurements was less precise ( = 0.84), but FDM prediction was more accurate using D enrichment in plasma ( = 0.90) and BIS ( = 0.99) data. Therefore, both BIS and D enrichment analysis resulted in similarly good predictions of FDM in cows, and we conclude that BIS could have the potential to predict FDM in dairy cows from 40 to 380 kg.

Moderate high or low maternal protein diets change gene expression but not the phenotype of skeletal muscle from porcine fetuses.

The aim of our study was to characterize the immediate phenotypic and adaptive regulatory responses of fetuses to different in utero conditions reflecting inadequate maternal protein supply during gestation. The gilts fed high- (250% above control) or low- (50% under control) protein diets isoenergetically adjusted at the expense of carbohydrates from the day of insemination until the fetuses were collected at day 64 or 94 of gestation. We analyzed body composition, histomorphology, biochemistry, and messenger RNA (mRNA) expression of fetal skeletal muscle. Both diets had only marginal effects on body composition and muscular cellularity of fetuses including an unchanged total number of myofibers. However, mRNA expression of myogenic regulatory factors (MYOG, MRF4, P ≤ 0.1), IGF system (IGF1, IGF1R, P ≤ 0.05) and myostatin antagonist FST (P = 0.6, in males only) was reduced in the fetal muscle exposed to a maternal low-protein diet. As a result of excess protein, MYOD, MYOG, IGF1R, and IGFBP5 mRNA expression (P ≤ 0.05) was upregulated in fetal muscle. Differences in muscular mRNA expression indicate in utero regulatory adaptive responses to maternal diet. Modulation of gene expression immediately contributes to the maintenance of an appropriate fetal phenotype that would be similar to that observed in the control fetuses. Moreover, we suggest that the modified gene expression in fetal skeletal muscle can be viewed as the origin of developmental muscular plasticity involved in the concept of fetal programming.

Effects of general anesthesia with ketamine in combination with the neuroleptic sedatives xylazine or azaperone on plasma metabolites and hormones in pigs.

Physiological research with swine often includes sedation or general anesthesia (GA), which may influence the basal physiological responses of experimental animals and may have the potential to confound or interfere with the effects of experimental factors of interest. Using 6 adult female pigs, we investigated whether selected plasma metabolites and hormones are influenced by GA induced with ketamine (K) and 2 neuroleptic sedatives, namely azaperone (A) and xylazine (X). Fasted pigs rotationally received either no drug, a single intravenous administration of A or X, or A or X combined with ketamine (AK or XK, respectively), and plasma concentrations of glucose, lactate, non-esterified fatty acids (NEFA), triglycerides (TG), glucagon, insulin, and cortisol were determined for a 5-h period following administration. Azaperone and X induced deep sedation, whereas AK and XK induced GA. Overall, the average plasma glucose concentrations were increased by A and X, with the latter exerting a stronger effect that was also associated with hypoinsulinemia ( < 0.05). Time-dependent effects indicated a more rapid increase in glucose concentration due to X or XK than AK. Plasma NEFA concentrations were elevated by A and AK and to a lesser extent by X and XK ( < 0.05). Plasma lactate and TG levels were elevated by A and AK and remained unaffected by X or XK. Plasma cortisol concentrations were elevated ( < 0.05) by X and XK and even more so with a single administration of A ( < 0.05), while the combined effect of A with ketamine resulted in the highest cortisol concentrations ( < 0.05). Our data suggest that the effects of azaperone are mediated by cortisol but less so for xylazine, which also indicates that azaperone elicits a stronger stress response in pigs. Xylazine probably induces long-lasting, fasting-state hyperglycemia through the stimulation of hepatic glucose production associated with hypoinsulinemia. A discriminant analysis based on the variation in all of the measured metabolites and hormones, collectively, indicated that ketamine induced no additional effect on the overall physiological response patterns than that of the individual sedatives. In conclusion, the neuroleptic sedatives azaperone, and to a lesser extent, xylazine, acutely affect the metabolism of pigs, so primary metabolic readouts obtained under these drugs may be confounded.

Technical note: Analytical refinements of the methane indicator archaeol in bovine feces, rumen fluid, and feedstuffs.

Archaeol (1,2-di-O-phytanyl-sn-glycerol) is a cell membrane lipid component of methanogens that has the potential to be used as a biomarker for methane production in ruminants. However, its analysis via gas chromatography-mass spectrometry (GC-MS) is challenging because of its molecular size and structure. In this study, 2 different sample preparation methods were tested, Soxhlet and sonication-aided extraction, and the methods were compared for extraction efficiency using the internal standard (IS; 1,2-di-o-hexadecyl-rac-glycerol). The extraction efficiency of the Soxhlet method for fecal archaeol was twice that of sonication. With the use of a high-temperature GC column, the retention times of IS and archaeol were 17.6 and 19.4 min, respectively, with a total run time of only 25 min. The molecule ions m/z 611.4 (IS) and m/z 725.8 (archaeol), or alternatively the fragment ion of the glycerol moiety m/z 130.0, were used for identification and quantification via GC-MS in positive chemical ionization mode. The intra-assay coefficients of variation for fecal archaeol measurements were 1.3% (m/z 725.8) and 2.1% (m/z 130.0) (n=3), respectively. Fecal archaeol quantifications did not differ between the use of the molecule or glycerol moiety ions (paired t-test, n=156). Archaeol concentrations tended to be 3.3% greater in samples stored at -20°C before drying compared with samples that were immediately dried after collection (paired t-test, n=5). The detection limit of archaeol was 0.5 µg/g of fecal dry matter (DM); no archaeol could be detected in feed samples. In different fractions of rumen fluid, archaeol levels ranged from 1.9 to 24.0 µg/g of DM. In 10 cows fed the same grass and corn silage/hay-based ration, diurnal variations of fecal archaeol levels (5 time points over 2 d) were cow dependent and ranged from 26.2 to 77.2 µg/g of DM (mean 48.4 µg/g of DM). Thus, within-animal variation in cows on the same diet was between 4 and 27%. We suggest that this finding is related to the amount and time of the latest feed intake event before the fecal sampling. Feeding pattern can determine the passage rate of digesta through the alimentary tract and thus the duration of contact time of archaea with their substrate.

Effects of a six-week intraduodenal supplementation with quercetin on liver lipid metabolism and oxidative stress in peripartal dairy cows.

The purpose of this study was to evaluate possible effects of quercetin (Q) on liver lipid metabolism and antioxidative status in periparturient dairy cows. The periparturient period is associated with enormous metabolic changes for dairy cows. Energy needs for incipient lactation are too high to be balanced by feed intake, leading to negative energy balance and body fat mobilization. It has been estimated that this leads to the development of fatty liver in about 50% of cows, which are at high risk for disease. Furthermore, the antioxidative status of these cows may be impaired. Quercetin is a plant flavonoid having hepatoprotective and antioxidative potential and the ability to reduce liver lipid accumulation in monogastric animals. Little information is available in regard to these effects in ruminants. To prevent microbial Q degradation in the rumen, Q was administered via a duodenal fistula to improve systemic availability. Five cows of the Q-treated group received, daily, 100 mg of quercetin dehydrate/kg BW in a 0.9% sodium chloride solution from d -20 until d 20 relative to calving, whereas 5 control (CTR) cows received only a sodium chloride solution. Blood samples were taken weekly and liver biopsies were performed in wk -4, -2, and 3 relative to calving. Cows treated with Q showed a tendency ( = 0.082) for lower liver fat content compared with CTR cows. Liver glycogen, glutathione concentrations, and relative mRNA abundance of genes related to hepatic lipid metabolism and antioxidative status as well as parameters of antioxidative status in plasma were not affected ( > 0.1) by Q supplementation. In conclusion, liver fat content in dairy cows tended to be reduced by Q supplementation, but potential underlying mechanisms remain unclear because analyzed parameters related to hepatic lipid metabolism and antioxidative defense were not altered by Q supplementation.

Endogenous and dietary lipids influencing feed intake and energy metabolism of periparturient dairy cows.

The high metabolic priority of the mammary gland for milk production, accompanied by limited feed intake around parturition results in a high propensity to mobilize body fat reserves. Under these conditions, fuel selection of many peripheral organs is switched, for example, from carbohydrate to fat utilization to spare glucose for milk production and to ensure partitioning of tissue- and dietary-derived nutrients toward the mammary gland. For example, muscle tissue uses nonesterified fatty acids (NEFA) but releases lactate and amino acids in a coordinated order, thereby providing precursors for milk synthesis or hepatic gluconeogenesis. Tissue metabolism and in concert, nutrient partitioning are controlled by the endocrine system involving a reduction in insulin secretion and systemic insulin sensitivity and orchestrated changes in plasma hormones such as insulin, adiponectin, insulin growth factor-I, growth hormone, glucagon, leptin, glucocorticoids, and catecholamines. However, the endocrine system is highly sensitive and responsive to an overload of fatty acids no matter if excessive NEFA supply originates from exogenous or endogenous sources. Feeding a diet containing rumen-protected fat from late lactation to calving and beyond exerts similar negative effects on energy intake, glucose and insulin concentrations as does a high extent of body fat mobilization around parturition in regard to the risk for ketosis and fatty liver development. High plasma NEFA concentrations are thought not to act directly at the brain level, but they increase the energy charge of the liver which is, signaled to the brain to diminish feed intake. Cows differing in fat mobilization during the transition phase differ in their hepatic energy charge, whole body fat oxidation, glucose metabolism, plasma ghrelin, and leptin concentrations and in feed intake several week before parturition. Hence, a high lipid load, no matter if stored, mobilized or fed, affects the endocrine system, metabolism, and feed intake, and increases the risk for metabolic disorders. Future research should focus on a timely parallel increase in feed intake and milk yield during early lactation to reduce the impact of body fat on feed intake, metabolic health, and negative energy balance.

Insulin-dependent glucose metabolism in dairy cows with variable fat mobilization around calving.

Dairy cows undergo significant metabolic and endocrine changes during the transition from pregnancy to lactation, and impaired insulin action influences nutrient partitioning toward the fetus and the mammary gland. Because impaired insulin action during transition is thought to be related to elevated body condition and body fat mobilization, we hypothesized that over-conditioned cows with excessive body fat mobilization around calving may have impaired insulin metabolism compared with cows with low fat mobilization. Nineteen dairy cows were grouped according to their average concentration of total liver fat (LFC) after calving in low [LLFC; LFC <24% total fat/dry matter (DM); n=9] and high (HLFC; LFC >24.4% total fat/DM; n=10) fat-mobilizing cows. Blood samples were taken from wk 7 antepartum (ap) to wk 5 postpartum (pp) to determine plasma concentrations of glucose, insulin, glucagon, and adiponectin. We applied euglycemic-hyperinsulinemic (EGHIC) and hyperglycemic clamps (HGC) in wk 5 ap and wk 3 pp to measure insulin responsiveness in peripheral tissue and pancreatic insulin secretion during the transition period. Before and during the pp EGHIC, [(13)C6] glucose was infused to determine the rate of glucose appearance (GlucRa) and glucose oxidation (GOx). Body condition, back fat thickness, and energy-corrected milk were greater, but energy balance was lower in HLFC than in LLFC. Plasma concentrations of glucose, insulin, glucagon, and adiponectin decreased at calving, and this was followed by an immediate increase of glucagon and adiponectin after calving. Insulin concentrations ap were higher in HLFC than in LLFC cows, but the EGHIC indicated no differences in peripheral insulin responsiveness among cows ap and pp. However, GlucRa and GOx:GlucRa during the pp EGHIC were greater in HLFC than in LLFC cows. During HGC, pancreatic insulin secretion was lower, but the glucose infusion rate was higher pp than ap in both groups. Plasma concentrations of nonesterified fatty acids decreased during HGC and EGHIC, but in both clamps, pp nonesterified fatty acid concentrations did not reach the ap levels. The study demonstrated a minor influence of different degrees of body fat mobilization on insulin metabolism in cows during the transition period. The distinct decrease in the glucose-dependent release of insulin pp is the most striking finding that explains the impaired insulin action after calving, but does not explain differences in body fat mobilization between HLFC and LLFC cows.

Metabolic and clinical response to Escherichia coli lipopolysaccharide in layer pullets of different genetic backgrounds supplied with graded dietary L-arginine.

L-arginine (Arg) is an essential amino acid in birds that plays a decisive role in avian protein synthesis and immune response. Effects of graded dietary Arg supply on metabolic and clinical response to Escherichia coli lipopolysaccharide (LPS) were studied over 48 hours after a single intramuscular LPS injection in 18-week-old genetically diverse purebred pullets. LPS induced a genotype-specific fever response within 4 hours post injectionem. Whereas brown genotypes showed an initial hypothermia followed by longer-lasting moderate hyperthermia, white genotypes exhibited a biphasic hyperthermia without initial hypothermia. Furthermore, within 2 hours after LPS injection, sickness behavior characterized by lethargy, anorexia, intensified respiration, and ruffled feathers appeared, persisted for 3 to 5 hours and recovered 12 hours post injectionem. The varying grades of Arg did not alter the examined traits named above, whereas insufficient Arg reduced body growth and increased relative weights of liver and pancreas significantly. At 48 hours post injectionem, increased relative weights of liver and spleen were also found in LPS treated pullets, whereas LPS decreased those of pancreas, bursa, thymus, and cecal tonsils. Moreover, LPS lowered the sum of plasma amino acids and decreased plasma concentrations of Arg, citrulline, glutamate, methionine, ornithine, phenylalanine, proline, tryptophan, and tyrosine, and increased those of aspartate, glutamine, lysine, 1- and 3-methyl-histidine. Elevating concentrations of dietary Arg led to increasing plasma concentrations of Arg, citrulline, ornithine, and 3-methyl-histidine subsequently. As quantitative expression of LPS-induced anorexia, proteolysis, and the following changes in plasma amino acids, pullets showed a significant decrease of feed and nitrogen intake and catabolic metabolism characterized by negative nitrogen balance and body weight loss in the first 24 hours post injectionem. Pullets recovered from the challenge within the second 24 hours post injectionem and changed to anabolism with re-increased feed and nitrogen intake, positive nitrogen retention, and weight gain. To conclude, present results confirmed that LPS induced numerous metabolic and physiological changes in pullet's genotypes, whereas dietary Arg affected the examined traits only slightly.

Acute effects of general anesthesia with propofol, pentobarbital or isoflurane plus propofol on plasma metabolites and hormones in adult pigs.

Experimental setups for physiological research, in which acute operative interventions need to be performed, can require inclusion of general anesthesia (GA), which may interfere or confound with the effects of the experimental factors of interest on measured variables. It was recently shown that the most commonly used sedatives/anesthetics in pigs (e.g., ketamine, xylazine, azaperone) affect physiological responses and thus the primary metabolic readouts have the potential to be confounded. To extend the search for a physiologically-friendly anesthesia regime for such studies, we investigated effects of GA induced by propofol (Prop) or pentobarbital (Pent) or propofol plus isoflurane (Prop + Isof) on plasma concentrations of commonly measured metabolites and hormones. In 2 experimental runs, 6 female pigs fitted with jugular vein catheters were used. Fasted pigs received either no drug (CON) or anesthetized rotationally either with Prop, Pent or Prop + Isof on different days, separated with washout periods of sufficient length (2 to 3 d). Six-h profiles of glucose, lactate, non-esterified fatty acids (NEFA), triglycerides (TG), cholesterol, urea as well as hormones including glucagon, insulin and cortisol were determined. Concentrations of cholesterol, urea and glucagon remained unaffected by any of the treatments ( > 0.05). Pent tended to increase cortisol from 30 to 90 min after drug administration. Glucose and lactate concentrations were increased ( < 0.05) by Prop and Pent within the first hour of GA ( < 0.05). Propofol and Pent reduced NEFA concentrations, which were more pronounced during the last 2 h of the studied period. Triglyceride concentrations were increased by all 3 agents within the first 45 min with Prop containing treatments exerting a stronger effect than Pent. Our data suggest that GA with Prop and particularly with Pent adulterate plasma metabolite and hormone profiles of pigs acutely, and thus has the potential to confound the effects of experimental factors of interest. Although Prop + Isof anesthesia did not differ from the controls, providing a physiologically-friendly GA, both single and the isoflurane-combined treatment of Prop induced hypertriglyceridemia due to the lipid adjuvant of the Prop drugs. It is concluded that readouts obtained under GA may be influenced both by physiological adulterations as response to anesthesia as well as by artifacts due to accompanying ingredients of the drug formulations.

Ruminal degradation of quercetin and its influence on fermentation in ruminants.

The aim of the present study was to investigate the ruminal degradation of the flavonol quercetin and to determine its potential antimicrobial effects on ruminal fermentation in cows. Ruminal degradation of quercetin (0 or 100µmol/L, respectively) as well as its influence on ruminal gas production (0, 50, or 100µmol of quercetin equivalents/L, respectively, either applied as aglycone or as its glucorhamnoside rutin) using concentrate, grass hay, and straw as substrates were investigated in vitro using the Hohenheim gas test. Additionally, the influence of quercetin on ruminal concentrations of volatile fatty acids and their molar ratio in rumen-fistulated, nonlactating cows (n=5) after intraruminal application of quercetin as aglycone or as rutin (0, 10, or 50mg of quercetin equivalents/kg of BW, respectively) was evaluated. Quercetin was rapidly and extensively degraded, whereby the disappearance of quercetin was accompanied by the simultaneous appearance of 2metabolites 3,4-dihydroxyphenylacetic acid and 4-methylcatechol. In vitro total gas and methane production were not reduced by the addition of quercetin aglycone or rutin, respectively, using concentrate, grass hay, and straw as substrates. As expected, however, effects of the substrates used were detected on total gas and methane production. Highest gas production was found with concentrate, whereas values obtained with grass hay and straw were lower. Relative methane production was highest with grass hay compared with concentrate and straw (27.1 vs. 25.0 and 25.5%). After intraruminal application of the quercetin aglycone or rutin, respectively, neither total concentration nor the molar ratio of volatile fatty acids in the rumen fluid were influenced. Results of the present study show that quercetin underlies rapid ruminal degradation, whereby 3,4-dihydroxyphenylacetic acid and 4-methylcatechol are the main metabolites, whereas the latter one most likely is formed by dehydroxylation from 3,4-dihydroxyphenylacetic acid. Regarding antimicrobial effects of quercetin, results obtained indicate that fermentation processes in the forestomachs are not substantially influenced by quercetin or rutin, respectively. With regard to potential health-promoting effects of quercetin, its application in cows, especially in the form of the better available rutin, might not be accompanied by negative effects on ruminal fermentation.

Body composition and plasma lipid and stress hormone levels during 3 weeks of feed restriction and refeeding in low birth weight female pigs.

Compensatory growth in response to feed restriction (FR) affects deposition rates of lean and adipose tissues. It is, however, unclear whether pigs with low birth weight differ from their counterparts with normal birth weight with regard to compensatory growth. Female littermate pigs with low (UW; 1.1 kg) and normal (NW; 1.5 kg) birth weight were fed to appetite (control, CON) or feed restricted (RES) at 60% of DMI of the CON group between 78 and 98 d of age and subsequently refed at the level of the CON group until 131 d of age. Subgroups of pigs were slaughtered at 75, 98, 104, and 131 d of age to compare BW and body composition. Blood samples were taken at 98 and 119 d of age to analyze plasma metabolites and hormones. At birth UW pigs were shorter and had lower BW until 131 d of age than NW pigs ( < 0.05). Feed intake per kilogram of BW was greater in UW than in NW pigs ( < 0.01). The UW and NW pigs differed in carcass composition as indicated by greater relative subcutaneous fat at 75 d ( < 0.1), greater shoulder back fat ( < 0.05) at 98 d, and lower carcass weight at 131 d with greater abdominal and subcutaneous neck back fat in UW compared with NW pigs ( < 0.05). During FR, BW gain of RES pigs was lower than in NW pigs. The RES pigs showed greater feed intake after termination of FR until 131 d than CON pigs ( < 0.01). At 98 d RES pigs were leaner than CON pigs ( < 0.05). After 6 d of refeeding (104 d) relative fat depot weights were still smaller ( < 0.03) in RES pigs than in CON pigs. After 5 wk of refeeding, RES pigs had lower abdominal fat weights and greater plasma cortisol levels than CON pigs ( < 0.05). Regarding the plasma metabolite and hormone response, at 98-d fasting levels of plasma NEFA and glycerol were greater in RES than in CON pigs ( < 0.05), and after the drop in their levels after morning feeding ( < 0.001), plasma NEFA and glycerol and also triacylglycerol increased until the next meal in RES vs. CON pigs ( < 0.01). Plasma cortisol was greater in RES pigs after 3-wk FR ( < 0.05), whereas only a trend for increased plasma adrenalin concentrations in RES pigs at the end of the FR period and after 5 wk of refeeding was found ( < 0.1). In conclusion, UW pigs at 75 d of age (20 to 23 kg BW) had greater subcutaneous fat, whereas at 131 d (61 to 68 kg BW) they showed greater abdominal fat than NW pigs, suggesting that subcutaneous fat is deposited earlier than abdominal fat. The FR caused similar changes in body composition, plasma lipids, and stress hormones in UW and NW pigs.

Methyl-coenzyme M reductase A as an indicator to estimate methane production from dairy cows.

The evaluation of greenhouse gas mitigation strategies requires the quantitative assessment of individual methane production. Because methane measurement in respiration chambers is highly accurate, but also comprises various disadvantages such as limited capacity and high costs, the establishment of an indicator for estimating methane production of individual ruminants would provide an alternative to direct methane measurement. Methyl-coenzyme M reductase is involved in methanogenesis and the subunit α of methyl-coenzyme M reductase is encoded by the mcrA gene of rumen archaea. We therefore examined the relationship between methane emissions of Holstein dairy cows measured in respiration chambers with 2 different diets (high- and medium-concentrate diet) and the mcrA DNA and mcrA cDNA abundance determined from corresponding rumen fluid samples. Whole-body methane production per kilogram of dry matter intake and mcrA DNA normalized to the abundance of the rrs gene coding for 16S rRNA correlated significantly when using qmcrA primers. Use of qmcrA primers also revealed linear correlation between mcrA DNA copy number and methane yield. Regression analyses based on normalized mcrA cDNA abundances revealed no significant linear correlation with methane production per kilogram of dry matter intake. Furthermore, the correlations between normalized mcrA DNA abundance and the rumen fluid concentration of acetic and isobutyric acid were positive, whereas the correlations with propionic and lactic acid were negative. These data suggest that the mcrA DNA approach based on qmcrA primers could potentially be a molecular proxy for methane yield after further refinement.

Maternal perinatal undernutrition modifies lactose and serotranferrin in milk: relevance to the programming of metabolic diseases?

A close link between intrauterine growth restriction and development of chronic adult diseases such as obesity, diabetes, and hypertension has been established both in humans and animals. Modification of growth velocity during the early postnatal period (i.e., lactation) may also sensitize to the development of metabolic syndrome in adulthood. This suggests that milk composition may have long-lasting programming/deprogramming metabolic effects in the offspring. We therefore assess the effects of maternal perinatal denutrition on breast milk composition in a food-restricted 50% (FR50) rat model. Monosaccharides and fatty acids were characterized by gas chromatography, and proteins were profiled by surface-enhanced laser desorption/ionization-time-of-flight analysis in milk samples from FR50 and control rat dams. Milk analysis of FR50 rats demonstrated that maternal undernutrition decreases lactose concentration and modulates lipid profile at postnatal day 10 by increasing the unsaturated fatty acids/saturated fatty acids and diminishes serotransferrin levels at postnatal day 21. Our data indicate that maternal perinatal undernutrition modifies milk composition both quantitatively and qualitatively. These modifications by maternal nutrition open new perspectives to identify molecules that could be used in artificial milk to protect from the subsequent development of metabolic diseases.

The energy expenditure of 2 Holstein cow strains in an organic grazing system.

Until recently, measurements of energy expenditure (EE; herein defined as heat production) in respiration chambers did not account for the extra energy requirements of grazing dairy cows on pasture. As energy is first limiting in most pasture-based milk production systems, its efficient use is important. Therefore, the aim of the present study was to compare EE, which can be affected by differences in body weight (BW), body composition, grazing behavior, physical activity, and milk production level, in 2 Holstein cow strains. Twelve Swiss Holstein-Friesian (HCH; 616 kg of BW) and 12 New Zealand Holstein-Friesian (HNZ; 570 kg of BW) cows in the third stage of lactation were paired according to their stage of lactation and kept in a rotational, full-time grazing system without concentrate supplementation. After adaption, the daily milk yield, grass intake using the alkane double-indicator technique, nutrient digestibility, physical activity, and grazing behavior recorded by an automatic jaw movement recorder were investigated over 7d. Using the (13)C bicarbonate dilution technique in combination with an automatic blood sampling system, EE based on measured carbon dioxide production was determined in 1 cow pair per day between 0800 to 1400 h. The HCH were heavier and had a lower body condition score compared with HNZ, but the difference in BW was smaller compared with former studies. Milk production, grass intake, and nutrient digestibility did not differ between the 2 cow strains, but HCH grazed for a longer time during the 6-h measurement period and performed more grazing mastication compared with the HNZ. No difference was found between the 2 cow strains with regard to EE (291 ± 15.6 kJ) per kilogram of metabolic BW, mainly due to a high between-animal variation in EE. As efficiency and energy use are important in sustainable, pasture-based, organic milk production systems, the determining factors for EE, such as methodology, genetics, physical activity, grazing behavior, and pasture quality, should be investigated and quantified in more detail in future studies.

Effects of parturition and feed restriction on concentrations and distribution of the insulin-like growth factor-binding proteins in plasma and cerebrospinal fluid of dairy cows.

Hormones and metabolites act as satiety signals in the brain and play an important role in the control of feed intake (FI). These signals can reach the hypothalamus and brainstem, 2 major centers of FI regulation, via the blood stream or the cerebrospinal fluid (CSF). During the early lactation period of high-yielding dairy cows, the increase of FI is often insufficient. Recently, it has been demonstrated that insulin-like growth factors (IGF) may control FI. Thus, we asked in the present study if IGF-binding proteins (IGFBP) are regulated during the periparturient period and in response to feed restriction and therefore might affect FI as well. In addition, we specifically addressed conditional distribution of IGFBP in plasma and CSF. In one experiment, 10 multiparous German Holstein dairy cows were fed ad libitum and samples of CSF and plasma were obtained before morning feeding on d -20, -10, +1, +10, +20, and +40 relative to calving. In a second experiment, 7 cows in second mid-lactation were sampled for CSF and plasma after ad libitum feeding and again after feeding 50% of the previous ad libitum intake for 4 d. Intact IGFBP-2, IGFBP-3, and IGFBP-4 were detected in plasma by quantitative Western ligand blot analysis. In CSF, we were able to predominantly identify intact IGFBP-2 and a specific IGFBP-2 fragment containing detectable binding affinities for biotinylated IGF-II. Whereas plasma concentrations of IGFBP-2 and IGFBP-4 increased during the periparturient period, IGFBP-3 was unaffected over time. In CSF, concentrations of IGFBP-2, both intact and fragmented, were not affected during the periparturient period. Plasma IGF-I continuously decreased until calving but remained at a lower concentration in early lactation than in late pregnancy. Food restriction did not affect concentrations of IGF components present in plasma or CSF. We could show that the IGFBP profiles in plasma and CSF are clearly distinct and that changes in IGFBP in plasma do not simply correspond in the brain. We thus assume independent control of IGFBP distribution between plasma and CSF. Due to the known anorexic effect of IGF-I, elevated plasma concentrations of IGFBP-2 and IGFBP-4 during the postpartum period in conjunction with reduced plasma IGF-I concentrations may be interpreted as an endocrine response against negative energy balance in early lactation in dairy cows.