Evolution of intestinal microbiota and body compartments during spontaneous hyperphagia in the Greylag goose.

The aim of this work was to study the effects of spontaneous hyperphagia on the evolution of intestinal microbiota and body compartments in old goose. From October 25th to November 26th, 5-yr-old breeding Greylag Landaise geese (106 males and 106 females) were fed with grass during 1 mo (G period). From November 26th (0 d) the birds had ad libitum access to pellets (AMEn: 10.5 MJ/kg, CP: 18.9 g/kg; spontaneous fattening (SF) period). Some birds were killed at -31 d (n = 24; 50/50 sex ratio), 0 d (n = 48), 14 (n = 46), 22 d (n = 46), and 70 d (n = 48) after the start of G period to measure body traits. For microbial analysis, 10 of the samples per sex at 0 d, 14 d, and 70 d were selected to be representative of body traits. Between 0 and 22 d, liver weight increased from 98 g to 194 g in males and from 89 g to 199 g in females (P < 0.001). Liver weight decreased between 22 and 70 d from 194 to 174 g in males and from 199 to 163 g in females (P < 0.001). Irrespective of the diet (G or SF period) and the sex of the bird, the two major phyla were Proteobacteria (49%) and Firmicutes (48%). Bacteroidetes represented around 3.0% of the sequences. At order level, Firmicutes were dominated by Clostridiales (33% of total sequences) and Lactobacillales (13% of total sequences) and Proteobacteria were dominated by Campylobacteriales (34% of total sequences). Finally, Bacteroidetes were dominated by Bacteroidales. SF and sex did not change the microbial diversity but sparse partial least squares discriminant analysis allowed us to highlight discriminant operational taxonomic unit between experimental groups. In conclusion, our result showed that changes in the body compartments of old geese during spontaneous hyperphagia depend on the sex of the birds, but not so much in gut microbial composition. Further investigations are necessary to understand the functional microbiota and highlight the role of gut microbiota in hepatic steatosis induced with hyperphagia in geese.

Lactobacillus sakei modulates mule duck microbiota in ileum and ceca during overfeeding.

The supplementation with Lactobacillus sakei as probiotic on the ileal and cecal microbiota of mule ducks during overfeeding was investigated using high-throughput 16S rRNA gene-based pyrosequencing and real-time PCR. The ducks were overfed with or without L. sakei for 12 d with 56% ground corn and 42% whole corn. Samples were collected before the overfeeding period (at 12 wk), at 13 wk (meal 12 of overfeeding), and at 14 wk (meal 24), 3 h postfeeding. Whatever the digestive segment and the level of intake, Firmicutes, Bacteroidetes, and Proteobacteria were the dominant phyla in the bacterial community of mule ducks (at least 90%). Before overfeeding, ileal samples were dominated by Clostridia, Bacteroidia, and Gammaproteobacteria (80% and up), and cecal samples by Bacteroidia and Clostridia (around 85%). The richness and diversity decreased in the ileum and increased in the ceca after overfeeding. Overfeeding increased the relative abundance of Firmicutes and especially the Lactobacillus group in ileal samples. Nonmetric multidimensional scaling profiles separated the bacterial communities with respect to overfeeding only in cecal samples. Richness indicators decreased after L. sakei has been added at mid-overfeeding only in the ileum. In the ceca, the decrease of these indexes only occurred at the end of overfeeding. The addition of L. sakei triggers major changes in the ileum, whereas the ceca are not affected. Lactobacillus sakei decreased the relative abundance of Bacteroides at mid-overfeeding and the relative abundance of Enterobacteria at the end of overfeeding in the ileum.

Detection of QTL controlling metabolism, meat quality, and liver quality traits of the overfed interspecific hybrid mule duck.

The mule duck, an interspecific hybrid obtained by crossing common duck (Anas platyrhynchos) females with Muscovy (Cairina moschata) drakes, is widely used for fatty liver production. The purpose of the present study was to detect and map single and pleiotropic QTL that segregate in the common duck species, and influence the expression of traits in their overfed mule duck offspring. To this end, we generated a common duck backcross (BC) population by crossing Kaiya and heavy Pekin experimental lines, which differ notably in regard to the BW and overfeeding ability of their mule progeny. The BC females were mated to Muscovy drakes and, on average, 4 male mule ducks hatched per BC female (1600 in total) and were measured for growth, metabolism during growth and the overfeeding period, overfeeding ability, and the quality of their breast meat and fatty liver. The phenotypic value of BC females was estimated for each trait by assigning to each female the mean value of the phenotypes of her offspring. Estimations allowed for variance, which depended on the number of male offspring per BC and the heritability of the trait considered. The genetic map used for QTL detection consisted of 91 microsatellite markers aggregated into 16 linkage groups (LG) covering a total of 778 cM. Twenty-two QTL were found to be significant at the 1% chromosome-wide threshold level using the single-trait detection option of the QTLMap software. Most of the QTL detected were related to the quality of breast meat and fatty liver: QTL for meat pH 20 min post mortem were mapped to LG4 (at the 1% genome-wide significance level), and QTL for meat lipid content and cooking losses were mapped to LG2a. The QTL related to fatty liver weight and liver protein and lipid content were for the most part detected on LG2c and LG9. Multitrait analysis highlighted the pleiotropic effects of QTL in these chromosome regions. Apart from the strong QTL for plasma triglyceride content at the end of the overfeeding period mapped to chromosome Z using single-trait analysis, all metabolic trait QTL were detected with the multitrait approach: the QTL mapped to LG14 and LG21 affected the plasma cholesterol and triglyceride contents, whereas the QTL mapped to LG2a seemed to impact glycemia and the basal plasma corticosterone content. A greater density genetic map will be needed to further fine map the QTL.

Genetic parameters of product quality and hepatic metabolism in fattened mule ducks.

Genetic parameters of traits related to hepatic lipid metabolism, carcass composition, and product quality of overfed mule ducks were estimated on both parental lines of this hybrid: the common duck line for the maternal side and the Muscovy line for the paternal side. The originality of the statistical model was to include simultaneously the additive genetic effect of the common ducks and that of the Muscovy ducks, revealing a greater genetic determinism in common than in Muscovy. Plasma metabolic indicators (glucose, triglyceride, and cholesterol contents) were heritable, in particular at the end of the overfeeding period, and heritabilities increased with the overfeeding stage. Carcass composition traits were highly heritable in the common line, with values ranging from 0.15 for liver weight, 0.21 for carcass weight, and 0.25 for abdominal fat weight to 0.32 for breast muscle weight. Heritabilities of technological outputs were greater for the fatty liver (0.19 and 0.08, respectively, on common and Muscovy sides for liver melting rate) than for the pectoralis major muscle (between 0.02 and 0.05 on both parental sides for cooking losses). Fortunately, the processing industry is mainly facing problems in liver quality, such as too high of a melting rate, than in meat quality. The meat quality appraisal criteria (such as texture and cooking losses), usually dependent on pH and the rate of decline of pH, were also very lowly heritable. This study demonstrated that genetic determinism of meat quality and ability of overfeeding is not similar in the common population and in the Muscovy population; traits related to fattening, muscle development, and BW have heritability values from 2 to 4 times greater on the common line than on the Muscovy line, which is relevant for considering different selection strategies.

Is there a relationship between the kinetics of lipoprotein lipase activity after a meal and the susceptibility to hepatic steatosis development in ducks?

The difference in the ability of Pekin and Muscovy ducks to develop hepatic steatosis could result from a different peripheral lipoprotein lipase (LPL) activity, which hydrolyses triacylglycerol secreted by the liver. We studied the kinetics of plasma LPL activity in response to a meal at different ages in Pekin and Muscovy ducks. For that purpose, blood samples were taken at 5, 9, 12, 13, and 14 wk of age just before and 1, 2, 4, and 8 h after a meal. To release LPL into general circulation, an i.v. injection of heparin (400 IU/kg of BW) was administered 10 min before blood collection. For that reason, different ducks per genotype were used for each point of measurement (n = 6). Plasma LPL activity measured before the meal was negatively correlated with the weight of the fatty liver measured in the same ducks at 14 wk of age (r = -0.58, P < 0.001). Plasma triacylglycerol level measured before the meal was negatively correlated with plasma LPL activity measured in the same ducks (r = -0.31, P = 0.025) and was negatively correlated with plasma LPL activity measured in the same ducks for each age and each timing (r = -0.39, P < 0.001). At 14 wk of age for Muscovy and Pekin ducks, we observed that a high plasma LPL activity (>200 IU/L of plasma) corresponded to a relatively low development of fatty liver (190 g) induced by overfeeding, whereas a low plasma LPL activity (<150 IU/L of plasma) corresponded to a high propensity to develop fatty liver (470 g). In conclusion, plasma LPL activity measured just before the meal during the rearing period could be used as a marker of hepatic steatosis development during the overfeeding period.

Liver gene expression in relation to hepatic steatosis and lipid secretion in two duck species.

The susceptibility to development of hepatic steatosis is known to differ between Muscovy and Pekin ducks. Although some experiments were conducted to decipher these differences, few data have been produced to analyse the role of specific genes in this process. For this purpose, expression levels of genes involved in lipid (ATP citrate lyase, malic enzyme 1, fatty acid synthase, stearoyl-CoA desaturase 1, diacylglycerol O-acyl transferase 2, microsomal triglyceride transfer protein, apolipoprotein A1, apolipoprotein B, sterol regulatory element binding factor 1, hepatocyte nuclear factor 4, choline/ethanolamine phosphotransferase 1, carnitine palmitoyl transferase 1A, peroxisome proliferator-activated receptor alpha and sterol O-acyltransferase) and carbohydrate (activating transcription factor 4 or cAMP-response element binding protein, mitochondrial malate dehydrogenase 2 and carbohydrate responsive element binding protein) metabolism and in other functions were analysed in the liver of Pekin and Muscovy ducks fed ad libitum or overfed. A specific positive effect of feeding was observed on the expression of genes involved mainly in fatty acids and TG synthesis and glycolysis, and negative effect on genes involved in beta-oxidation. Interestingly, a strong species effect was also observed on stearoyl-CoA desaturase 1 and to a lesser extent on diacylglycerol O-acyl transferase 2 expression, leading to large differences in expression levels between Pekin and Muscovy overfed ducks, which could explain the difference in lipid metabolism and steatosis ability observed between the two duck species. These results should shed light on gene expression that might underlie susceptibility to hepatic steatosis in humans.

Gene expression and protein content in relation to intramuscular fat content in Muscovy and Pekin ducks.

Independent of their nutritional condition, Pekin ducks always exhibit higher i.m. fat content than Muscovy ducks. To understand this difference between species, the expression level of genes involved in lipid metabolism was analyzed in the pectoralis major muscle of Pekin and Muscovy ducks ad libitum-fed or overfed. The lipoprotein lipase (LPL) gene expression was not different between species and not influenced by overfeeding. The protein content for LPL was higher in Pekin ducks than in Muscovy ducks when birds were ad libitum-fed, whereas in overfed ducks, we found no difference between species. Adipocyte fatty acid-binding protein (A-FABP) gene expression and protein content were higher in Pekin ducks than in Muscovy ducks for each nutritional condition (suggesting a higher intracellular transport within i.m. adipocytes of fatty acids mainly provided by liver for this species). Overfeeding did not affect the expression of genes involved in oxidation [carnitine palmitoyl transferase 1A (CPT1A), cytochrome-c oxidase 4 (COX4), succinyl-coenzyme A:3-ketoacid coenzyme A transferase (SCOT)] but increased the expression of fatty acid synthase (FAS) involved in lipogenesis. For all nutritional conditions, Pekin duck exhibited higher expression levels of CPT1A, COX4, SCOT, and FAS than Muscovy ducks. Results for mRNA SCOT suggested that the muscles of Pekin ducks use ketone bodies as an energy source. In conclusion, i.m. lipogenesis could contribute to the i.m. fat, particularly in Pekin ducks.

Influence of lipoprotein-lipase activity on plasma triacylglycerol concentration and lipid storage in three genotypes of ducks.

The lipoprotein-lipase (LPL) hydrolyses the triacylglycerols (TG) secreted by the liver and, thus, allows the storage of lipids onto the extrahepatic tissues. The LPL activity has been studied by injection of LPL antibodies in three genotypes of ducks (Muscovy (Cairina moschat), Pekin (Anas plathyrhynchos) and Mule (hybrids of male Muscovy ducks and female Pekin ducks)) under overfeeding condition. The results show a similar weight gain between injected and control animals. A higher liver steatosis is observed in Mule ducks (616+/-18 g; 8.79% of body mass (BW)) and Muscovy ducks (514+/-13 g; 7.05% BW) compared to Pekin ducks (353+/-21 g; 5.89% BW, p<0.05). Pekin ducks showed a much marked extrahepatic fattening of abdominal and subcutaneous adipose tissues. The LPL activity was evaluated by comparing the evolution of the plasma TG concentrations after injections of saline (control animals) or injections of specific LPL-antibodies. Inhibition of LPL activity performed by intravenous injections of LPL-antibodies showed a spectacular increase in the plasma TG concentrations in the three genotypes. That increase was considerably higher in Pekin ducks (98+/-10 g/L) compared to Muscovy ducks (35+/-2 g/L, p<0.01) and Mule ducks (30+/-4 g/L, p<0.01). Those data suggest that a high export of lipids synthesized in liver and a high LPL activity occur in overfed Pekin ducks, which can favour the extrahepatic fattening to the detriment of the liver steatosis, and conversely in overfed Muscovy and Mule ducks.

Effects of dietary fructose on liver steatosis in overfed mule ducks.

Overfeeding of some waterfowl species results in obesity, which is mainly characterized by a dramatic hepatic steatosis induced by strong accumulation of lipids synthesized from dietary glucose in the liver. In mammals, fructose is known to be able to raise plasma triacylglycerol concentrations significantly; consequently, this may induce obesity. The aim of this study was to assess the effect of partial replacement of dietary glucose provided by corn starch with fructose on metabolism and fatty liver production in the Mule ducks. On the basis of 9.5 kg maize (132,920 kJ) given twice a day for 14 days, a supplementation of 9,800 kJ was provided in form of glucose, sucrose or high fructose corn syrup (HFCS: 50 % glucose, 42 % fructose and 8 % other saccharides). Fatty liver weight in ducks fed with glucose supplementation was 499 +/- 21 g. Sucrose or HFCS supplementation brought about a significant increase in liver weight (+ 18.7 % and + 16.3 % vs. glucose supplementation respectively, p < 0.05). These results suggest that the dietary fructose favors the liver steatosis by increasing hepatic lipogenesis. Postprandial plasma insulin concentrations were similar in ducks fed diets with or without fructose, suggesting that the effect of fructose on liver steatosis is not mediated by insulin.

Pancreatic hormonal and metabolic responses in overfed ducks.

When overfed at their maximum (intensive overfeeding) or at only 80% (moderate overfeeding) of food intake capacity, Mule ducks developed strong liver steatosis, whereas Pekin ducks showed very marked extrahepatic fattening. During overfeeding, evolution of plasma glucose and triacylglycerol concentrations suggested a very strong increase in the hepatic lipogenesis as well as genotype- and diet-independent lipoprotein secretion. In contrast, lipoprotein-lipase activity was dependent on alimentary status (the intensive overfeeding induces the highest activities), and Pekin ducks showed higher lipoprotein-lipase activity than Mule ducks, which could favor extrahepatic fattening to the detriment of hepatic steatosis. In Pekin ducks, plasma pancreatic hormone concentrations are related to diet levels and blood sugar. With similar food intake, Mule ducks (moderately overfed) showed global blood insulin lower than that of Pekin ducks (intensively overfed) despite similar blood sugar levels, suggesting a trend towards reduced pancreas response to glucose in Mule ducks. This may result from their lower lipoprotein-lipase activity as previously shown in these two ducks overfed at only 60% of their maximal food intake capacity (unpublished results). These results suggest that high plasma insulin concentrations may be necessary to induce an optimum lipoprotein-lipase activity in overfed ducks.

Metabolism in two breeds of geese with moderate or large overfeeding induced liver-steatosis.

Biochemical mechanisms which may control fat deposition in liver and/or peripheral tissues have been studied in Poland and Landes geese. Post-prandial plasma substrates and post-heparin lipoprotein-lipase (LPL) activity were measured in 10-week-old animals. At 23 weeks of age, geese were overfed for 14 days then slaughtered. Hepatic steatosis was more important in Landes geese, while muscle and subcutaneous adipose tissue were less developed. In this breed, fatty liver weight negatively scaled to LPL activity, suggesting that a low LPL activity is a limiting factor of peripheral fat deposition. Consequently, non-catabolized VLDL may return to liver and increase hepatic steatosis. In Poland geese, such a mechanism does not exist. On the other hand, fatty liver weight was positively correlated to very low density lipoproteins (VLDL) and triacylglycerols measured in overfed Poland geese, suggesting that lipids synthetized by liver are better transferred from liver to extrahepatic tissues. Kinetics of post-prandial plasma glucose, triacylglycerols, phospholipids and uric acid were similar in the two breeds. However, the marked decrease in post-prandial plasma glycerol in Poland geese suggests that an extrahepatic tissue lipolysis inhibition could contribute to the higher peripheral fattening in overfed Poland geese and could be a limiting factor of hepatic steatosis in this breed.

Cold adaptation of proteins. Purification, characterization, and sequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41.

The gene of subtilisin S41, an alkaline protease secreted by the psychrophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids residues. The nucleotide sequence and NH2- and COOH-terminal amino acid sequencing of the purified enzyme indicate that the mature subtilisin S41 is composed of 309 residues with a predicted M(r) = 31,224. Subtilisin S41 shares most of its properties with mesophilic subtilisins (structure of the precursor, 52% amino acid sequence identity, alkaline pH optimum, broad specificity, Ca2+ binding) but is characterized by a higher specific activity on macromolecular substrate, by a shift of the optimum of activity toward low temperatures, and by a low thermal stability. The enzyme also differs by an acidic pI (5.3) and the presence of one disulfide bond. It is proposed that the psychrophilic enzyme possesses a more flexible molecular structure when compared to mesophilic and thermophilic subtilases in order to compensate for the reduction of reaction rates at low temperatures. The model of subtilisin S41 indeed reveals several features able to induce a more flexible, heat-labile conformation: the occurrence of four extended surface loops, a very hydrophilic surface through 11 extra Asp residues, and the lack of several salt bridges and aromatic-aromatic interactions. The low affinity of the Ca1 calcium binding site (Kd(app) = 10(-6) M), resulting possibly from one chelating side chain substitution and the stacking of Gly residues, also reflect a less compact conformation. The difference of free energy of stabilization between subtilisin S41 and a mesophilic subtilisin suggests that the balance of exo- and endothermically formed weak bonds is critical for the enzyme flexibility.

Sequence of the subtilisin-encoding gene from an antarctic psychrotroph Bacillus TA41.

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph, Bacillus TA41, was determined. The primary structure of the subtilisin precursor corresponds to a preproenzyme of 419 amino acids. Asp144, His181 and Ser359 are the proposed catalytic residues of the protease active site.

Nucleotide and derived amino acid sequence of the subtilisin from the antarctic psychrotroph Bacillus TA39.

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph Bacillus TA39 was determined. The primary structure of the subtilisin precursor is composed of 420 amino acids giving rise to a mature enzyme of 309 amino acids. Asp-145, His-185 and Ser-361 are the proposed catalytic residues of the active site.