Siegfried Kuhla

Proteome analysis of fatty liver in feed-deprived dairy cows reveals interaction of fuel sensing, calcium, fatty acid, and glycogen metabolism

The liver of dairy cows is involved in signaling the current hepatic metabolic state to the brain via metabolites and nerval afferents to control and adjust feed intake. Feed deprivation may result in mobilization of body reserves favoring hepatic steatosis. While the overall metabolic changes are well characterized, specific regulatory mechanisms are not readily understood. To identify molecular events associated with metabolic adaptation and the control of energy homeostasis, liver specimens from six ad libitum-fed and six feed-deprived cows were analyzed for selected metabolites, for the activation of AMP kinase, and for regulatory/regulated proteins using two-dimensional gel electrophoresis and MALDI-TOF-MS. Feed deprivation increased total liver fat and the calcium content, as well as augmented AMPK phosphorylation, while it decreased the contents of protein, glucose, glycogen, and cholesterol when expressed as a percentage of dry matter. Among 34 differentially expressed proteins identified, we found downregulation of proteins associated with fatty acid oxidation, glycolysis, electron transfer, protein degradation, and antigen processing, as well as cytoskeletal rearrangement. Proteins upregulated after feed deprivation included enzymes of the urea cycle, fatty acid or cholesterol transport proteins, an inhibitor of glycolysis, and previously unknown changes in calcium signaling network. Direct correlation was found between expression of glycolytic enzymes and glucose/glycogen content, whereas inverse correlation exists between expression of beta-oxidative enzymes and total liver fat content. In conclusion, the regulatory response of identified proteins may help to explain development and consequences of hepatic lipidosis but also offers novel candidates potentially involved in signaling for maintaining energy homeostasis.

Effects of dietary energy intake during gestation and lactation on milk yield and composition of first, second and fourth parity sows

Abstract In order to determine the effects of a varied level of dietary energy intake during pregnancy and lactation on milk yield and composition, first, second and fourth parity sows (Large White × German Landrace) were provided with energy at a level of either: (i) 100% of ME requirement (MEreq) during pregnancy and lactation, (ii) 120% MEreq during pregnancy and 80% during lactation, and (iii) 80% MEreq during pregnancy and 120% during lactation. In spite of equal target levels feed analysis revealed that gestating first parity sows with 120/80 treatment combination and lactating sows of 80/120 treatment combination received 25, and 11 – 17% more digestible N than in the respective 100/100 treatment combination. Irrespective of this 120/80 sows responded with the highest milk DM, fat, and energy contents, and the lowest lactose concentrations whereas protein levels where not affected, irrespective of parity (p < 0.05). Milk yield of sows in 1st and 4th lactation was 85 and 106% of that in 2nd lactation, respectively. Average milk composition was 18.1% DM, 4.9% protein, 6.8% fat, 5.6% lactose, and 0.8% ash. Milk composition changes ceased at day 7 of lactation with a reduction of milk GE and protein, and an increase of lactose content. Concentrations of threonine, arginine, valine, leucine, tyrosine, phenylalanine, cystine, and tryptophan, as well as stearic, oleic, and linoleic acid were higher in colostrum than in milk at later lactation stages. In contrast, laurine, myristic, palmitic, and palmitoleic acids were lower concentrated in colostrum. In conclusion, these results illustrate the importance of body reserve mobilization for milk production in sows and indicate that low energy supply during gestation cannot be compensated by higher energy supply during lactation.

Effect of a high-protein diet on food intake and liver metabolism during pregnancy, lactation and after weaning in mice.

Major hepatic metabolic pathways are involved in the control of food intake but how dietary proteins affect global metabolism to adjust food intake is incompletely understood, particularly under physiological challenging conditions such as lactation. In order to identify these molecular events, mice were fed a high‐protein (HP) diet from pregnancy, during lactation until after weaning and compared with control fed counterparts. Liver specimens were analyzed for regulated proteins using 2‐DE and MALDI‐TOF‐MS and plasma samples for metabolites. Based on the 26 differentially expressed proteins associated with depleted liver glycogen content, elevated urea and citrulline plasma concentrations, we conclude that HP feeding during lactation leads to an activated amino acid, carbohydrate and fatty acid catabolism while it activates gluconeogenesis. From pregnancy to lactation, plasma arginine, tryptophan, serine, glutamine and cysteine decreased, whereas urea concentrations increased in both groups. Concomitantly, hepatic glycogen content decreased while total fat content remained unaltered in both groups. Consideration of 59 proteins differentially expressed between pregnancy and lactation highlights different strategies of HP and control fed mice to meet energy requirements for lactation by adjusting amino acid degradation, carbohydrate and fat metabolism, citrate cycle, but also ATP‐turnover, protein folding, secretion of proteins and (de)activation of transcription factors.

Effect of inulin supplementation on selected gastric, duodenal, and caecal microbiota and short chain fatty acid pattern in growing piglets

Abstract We explored whether bifidobacteria and lactobacilli numbers and other selected bacteria in the upper intestine and the caecum of growing pigs were affected by diet and intake of inulin. Starting at two weeks after weaning (28 d) 72 pigs were fed two types of diets (wheat/barley (WB) or maize/gluten (MG)), without or with 3% inulin (WB + I, MG + I) for three and six weeks. Intestinal bacteria were quantified by fluorescence-in-situ-hybridization (n = 8/group). Duration of feeding had no effect on the variables tested, so data for both periods were pooled. Gastric total bacteria amounted to log10 7.4/g digesta. Bifidobacteria were detected in stomach and duodenum two weeks after weaning and disappeared thereafter. In jejunum and caecum bifidobacteria were present at a level of log10 7.0/g digesta. Inulin did not alter numbers of lactobacilli, bifidobacteria, enterococci, enterobacteria and bacteria of the Clostridium coccoides/Eubacterium rectale-group. Inulin disappearance in stomach plus jejunum was higher with the MG diet (73.7 vs. 60.7%, p = 0.013). Caecal acetate was lower in inulin-supplemented diets (p < 0.05) whereas propionate and butyrate were higher in pigs fed the WB diets (p < 0.05). With the WB diet total caecal short chain fatty acids concentration was higher which resulted in a lower pH value (p < 0.05).

Influence of chlorocholinechloride-treated wheat on selected in vitro fertility parameters in male mice.

The aim of this study was to evaluate the influence of feeding with food and water containing chlorocholinechloride (CCC) on the fertility of male mice in a two-generation study. For this purpose the number of testicular spermatozoa and the relative proportion of primary and secondary spermatocytes involved in spermatogenesis were measured. Furthermore, the fertility of epididymal spermatozoa from tested male mice was investigated in a special in-vitro fertilization system. The experimental food was composed of CCC-treated wheat in the first experiment and CCC-free wheat and water mixed with pure CCC in the second experiment. The CCC residue content in the treated food and water was 0.21 mg/kg and 0.2 mg/L, respectively. Under the influence of feeding with CCC-treated wheat (Experiment 1) the fertilization and cleavage rates of oocytes incubated with spermatozoa from CCC-fed mice were reduced: the fertilization rate 65.1% vs. 21.1% and the cleavage rate 51.9% vs. 20.3%, p < 0.01 (control feeding vs. CCC feeding, respectively). Feeding of sperm donors with pure CCC mixed with untreated wheat pellets or water (Experiment 2) led to a reduction in the fertilization and cleavage rate (control: 60.8%, 32.4%; CCC-food: 29.8%, 12.1%; CCC-water: 30.1%, 10.2%; CCC-food/water: 36.6%, 12.5%; p < 0.01, respectively). The normal course of spermatogenesis was unchanged after the exposure to CCC. Testicular weight, the number of spermatozoa, and the proportion of haploid, diploid, and tetraploid testicular cells were not influenced. However, the functional competence of epididymal spermatozoa from CCC-fed donors was reduced, resulting in a significantly diminished fertilization and cleavage rate in vitro. The results suggest that CCC could interfere with epididymal protein secretion and the process of sperm maturation during passage through the epididymis.

Carbon and nitrogen content based estimation of the fat content of animal carcasses in various species

The objective of this study was to explore whether the C and N content can be used to estimate the fat content of animal carcasses. Considering the mean C and N contents of body fat and body protein, the fat content (EE) [%] can be predicted from C and N values [%] according to the generally valid equation EE=1.3038·C – 4.237·N. The application of this equation to estimate the total fat content of all animal carcasses results in significant differences in fat content between predicted and measured values. Therefore, we derived specific equations for rats, pigs, cattle, sheep, broilers and mice to predict the fat content by dual linear regression analysis (y=EE [% DM], x1=C [% DM], x2=N [% DM]) based on measured fat, C, and N contents of animal body samples. The specific equations for different animals showed residual standard deviations of 1.55, 1.63, 1.12, 1.35, 1.85 and 0.92% fat for rats, pigs, cattle, sheep, broilers and mice, respectively.

Alterations in the jejunum of young goats caused by feeding soy protein-based diets

This study was designed to investigate whether soy protein or soy protein supplemented with indispensable amino acids (AA) change the protein expression pattern and utilization of pre-cursors for RNA biosynthesis in jejunal mucosa in relation to casein and whether these changes affect mucosal cell growth. Kids were fed comparable diets based on cow;s milk, of which 50% of crude protein were replaced by either casein (CAS), soy protein (SP) or soy protein supplemented with indispensible AA (SPA) for 34 days (n = 4/group). Jejunal tissue was collected 5 h after adding a single dose of (15)N-RNA to the diet, in order to determine morphology, protein repertoire by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and RNA biosynthesis by isotope ratio-mass spectrometry. In mid-jejunum, morphological alterations induced by partial replacement of casein with soy protein were accompanied by changes in mucosal proteins related to generation of the cytoskeleton and in pathways for mucosal RNA biosynthesis, resulting in a smaller re-utilization of dietary RNA pre-cursors and in an increased activity of enzymes involved in nucleic acid breakdown. Soy protein supplemented with indispensible aminoacids tended to revise mucosal growth retardation with no impact on salvage of dietary RNA pre-cursors for mucosal RNA biosynthesis, but changes in cytoskeleton generation. Feeding soy protein with supplementation of indispensible AA does not ameliorate soy protein effects on mucosal morphology and RNA metabolism in the jejunum in a significant manner.

Effect of partial dehulling of two- and six-row barley varieties on precaecal digestibility of amino acids in pigs.

Abstract Five barrows (German Landrace; initial BW 58 kg, final BW 80 kg) fitted with an ileo-rectal anastomosis were used to determine the effect of partial dehulling and addition of barley hulls of two- and six-row barley varieties on the precaecal digestibility (pD) of CP and amino acids. The following diets were provided according to a standardized diet formulation and tested in seven consecutive periods (repeated group-period design): two-row barley (TRB) + casein (C), dehulled TRB + C, TRB + C + 10% hulls, six-row barley (SRB) + C, dehulled SRB + C, SRB + C + 1% hulls, and wheat starch + C. The diets were supplied at daily rates of 79 – 86 g DMI · kg BW−0.75 in barley containing diets and at 49 g DMI · kg BW−0.75 in the casein diet. The digestibility of amino acids in barley varieties was determined by the difference method (casein as basal diet) using quantitative digesta collection. In both varieties of barley the pD of CP and amino acids did not differ. The pD of CP was unchanged in regard to the treatments in both barley varieties. Due to dehulling in TRB the pD was improved significantly for most indispensable amino acids and in SRB for Met and Cys. Addition of 10% hulls to TRB led to equivalent pD of Arg, His, Leu, Tyr, and Trp compared to TRB, but the pD of Lys, Phe, Thr and Val was significantly decreased below the levels of TRB. Addition of even 1% hulls to SRB impaired the pD of Lys below the level in SRB. In conclusion, addition of barley hulls to pig diets impairs amino acids absorption in the small intestine. The pD values, measured under standardized experimental conditions (without a correction using basal endogenous amino acids), are similar to the values of true digestibility published by NRC (1998).

Phenotypic characterisation of extreme growth-selected mouse lines: An important prerequisite for future QTL analysis

A unique set of seven mouse lines, long-term selected for high growth, from different laboratories around the world has been comprehensively compared to evaluate these resources for future QTL and gene mapping for growth traits. The heaviest line (DUH) was 40% (males) to 44% (females) heavier than the smallest line (ROH) at birth, and 105% (males) to 114% (females) heavier at 98 d. Body conformation (body length and width, body areas), body composition (dry matter, fat, fatty acid composition, organ weights), and skeletal muscle cellularity also differed substantially. DUH was more than 20% longer (12.3 cm) compared to the shortest line ROH (9.7 cm). DAH (22.5%) had the highest percentage of gonadal fat and the leanest was BEH (7.7%). Line BEH (0.49 g) showed the highest weight for the left M. rectus femoris, which was 2.1 times higher, compared to ROH (0.23 g). These results suggest that different alleles, and possibly different physiological pathways, have contributed to the selection response in the different lines. Therefore these selection lines are an important tool with which to identify the genetic and physiological basis of growth as they may contain many, if not all, growth promoting alleles.