Protein turnover in pigs: A review of interacting factors.

Protein turnover defines the balance between two continuous and complex processes of protein metabolism, synthesis and degradation, which determine their deposition in tissues. Although the liver and intestine have been studied extensively for their important roles in protein digestion, absorption and metabolism, the study of protein metabolism has focused mainly on skeletal muscle tissue to understand the basis for its growth. Due to the high adaptability of skeletal muscle, its protein turnover is greatly affected by different internal and external factors, contributing to carcass lean-yield and animal growth. Amino acid (AA) labelling and tracking using isotope tracer methodology, together with the study of myofiber type profiling, signal transduction pathways and gene expression, has allowed the analysis of these mechanisms from different perspectives. Positive stimuli such as increased nutrient availability in the diet (e.g., AA), physical activity, the presence of certain hormones (e.g., testosterone) or a more oxidative myofiber profile in certain muscles or pig genotypes promote increased upregulation of translation and transcription-related genes, activation of mTORC1 signalling mechanisms and increased abundance of satellite cells, allowing for more efficient protein synthesis. However, fasting, animal aging, inactivity and stress, inflammation or sepsis produce the opposite effect. Deepening the understanding of modifying factors and their possible interaction may contribute to the design of optimal strategies to better control tissue growth and nutrient use (i.e., protein and AA), and thus advance the precision feeding strategy.

Deciphering the complex interplay between pancreatic cancer, diabetes mellitus subtypes and obesity/BMI through causal inference and mediation analyses.

OBJECTIVES: To characterise the association between type 2 diabetes mellitus (T2DM) subtypes (new-onset T2DM (NODM) or long-standing T2DM (LSDM)) and pancreatic cancer (PC) risk, to explore the direction of causation through Mendelian randomisation (MR) analysis and to assess the mediation role of body mass index (BMI). DESIGN: Information about T2DM and related factors was collected from 2018 PC cases and 1540 controls from the PanGenEU (European Study into Digestive Illnesses and Genetics) study. A subset of PC cases and controls had glycated haemoglobin, C-peptide and genotype data. Multivariate logistic regression models were applied to derive ORs and 95% CIs. T2DM and PC-related single nucleotide polymorphism (SNP) were used as instrumental variables (IVs) in bidirectional MR analysis to test for two-way causal associations between PC, NODM and LSDM. Indirect and direct effects of the BMI-T2DM-PC association were further explored using mediation analysis. RESULTS: T2DM was associated with an increased PC risk when compared with non-T2DM (OR=2.50; 95% CI: 2.05 to 3.05), the risk being greater for NODM (OR=6.39; 95% CI: 4.18 to 9.78) and insulin users (OR=3.69; 95% CI: 2.80 to 4.86). The causal association between T2DM (57-SNP IV) and PC was not statistically significant (ORLSDM=1.08, 95% CI: 0.86 to 1.29, ORNODM=1.06, 95% CI: 0.95 to 1.17). In contrast, there was a causal association between PC (40-SNP IV) and NODM (OR=2.85; 95% CI: 2.04 to 3.98), although genetic pleiotropy was present (MR-Egger: p value=0.03). Potential mediating effects of BMI (125-SNPs as IV), particularly in terms of weight loss, were evidenced on the NODM-PC association (indirect effect for BMI in previous years=0.55). CONCLUSION: Findings of this study do not support a causal effect of LSDM on PC, but suggest that PC causes NODM. The interplay between obesity, PC and T2DM is complex.

Reduced risk of pancreatic cancer associated with asthma and nasal allergies.

OBJECTIVE: Studies indicate an inverse association between ductal adenocarcinoma of the pancreas (PDAC) and nasal allergies. However, controversial findings are reported for the association with asthma. Understanding PDAC risk factors will help us to implement appropriate strategies to prevent, treat and diagnose this cancer. This study assessed and characterised the association between PDAC and asthma and corroborated existing reports regarding the association between allergies and PDAC risk. DESIGN: Information about asthma and allergies was collated from 1297 PDAC cases and 1024 controls included in the PanGenEU case-control study. Associations between PDAC and atopic diseases were studied using multilevel logistic regression analysis. Meta-analyses of association studies on these diseases and PDAC risk were performed applying random-effects model. RESULTS: Asthma was associated with lower risk of PDAC (OR 0.64, 95% CI 0.47 to 0.88), particularly long-standing asthma (>=17 years, OR 0.39, 95% CI 0.24 to 0.65). Meta-analysis of 10 case-control studies sustained our results (metaOR 0.73, 95% CI 0.59 to 0.89). Nasal allergies and related symptoms were associated with lower risk of PDAC (OR 0.66, 95% CI 0.52 to 0.83 and OR 0.59, 95% CI 0.46 to 0.77, respectively). These results were supported by a meta-analysis of nasal allergy studies (metaOR 0.6, 95% CI 0.5 to 0.72). Skin allergies were not associated with PDAC risk. CONCLUSIONS: This study shows a consistent inverse association between PDAC and asthma and nasal allergies, supporting the notion that atopic diseases are associated with reduced cancer risk. These results point to the involvement of immune and/or inflammatory factors that may either foster or restrain pancreas carcinogenesis warranting further research to understand the molecular mechanisms driving this association.

Characterization of Microbial Populations in Two Distinct Dairy Manure Management Systems: Seasonal Effect and Implications for Pollutant Gases Emissions.

Following an increase of the demand of dairy products, higher quantities of manure are consequently produced, with the subsequent pollutant gas emission charge associated with its management. The two mostly used housing systems in the northeast of Spain, cubicles (CUB) and compost-bedded pack (CBP), entail different manure management techniques; thus, our main objective was to describe the microbiota present in manure of both systems during two distinct climatic situations (winter, mean temperature of 6.2ºC; and summer, mean temperature of 36.4ºC). The secondary aim was to correlate these microbiological profiles with literature findings on the emission of certain well-known pollutant gases from manure. CBP showed to have higher alpha biodiversity as well as presenting a remarkable clustering by season, but showed lower network complexity than CUB. Firmicutes/Bacteroidetes ratio was found superior in CUB, which also presented a significantly higher abundance of methanogenic genera belonging to Euryarchaeota phylum, such as Methanobrevibacter, Methanosaeta or Methanosarcina. On the other hand, CBP manure presented a significant presence of Corynebacterium, Pseudomonas or Truepera, among other genera, which activity has been linked to nitrogen (N) transformation pathways in manure. Season also had a relevant role to play on the fluctuation of these populations within each housing system under study. These results show how microbial populations change when manure is differently managed, and how these variations can be related to the synthesis of certain pollutant gases upon housing systems.

Measurement of Methane and Ammonia Emissions from Compost-Bedded Pack Systems in Dairy Barns: Tilling Effect and Seasonal Variations.

Dairy cattle contribute to environmental harm as a source of polluting gas emissions, mainly of enteric origin, but also from manure management, which varies among housing systems. Compost-bedded pack systems use manure as bedding material, which is composted in situ daily. As current literature referring to their impact on NH3 and CH4 emissions is scarce, this study aims to characterize the emissions of these two gases originating from three barns of this system, differentiating between two emission phases: static emission and dynamic emission. In addition, the experiment differentiated emissions between winter and summer. Dynamic emission, corresponding to the time of the day when the bed is being composted, increased over 3 and 60 times the static emission of NH3 and CH4, respectively. In terms of absolute emissions, both gases presented higher emissions during summer (1.86 to 4.08 g NH3 m-2 day-1 and 1.0 to 4.75 g CH4 m-2 day-1 for winter and summer, respectively). In this way, contaminant gases produced during the tilling process of the manure, especially during the warmer periods of the year, need to be taken into account as they work as a significant factor in emissions derived from compost-bedded pack systems.

First Steps into Ruminal Microbiota Robustness.

Despite its central role in ruminant nutrition, little is known about ruminal microbiota robustness, which is understood as the ability of the microbiota to cope with disturbances. The aim of the present review is to offer a comprehensive description of microbial robustness, as well as its potential drivers, with special focus on ruminal microbiota. First, we provide a briefing on the current knowledge about ruminal microbiota. Second, we define the concept of disturbance (any discrete event that disrupts the structure of a community and changes either the resource availability or the physical environment). Third, we discuss community resistance (the ability to remain unchanged in the face of a disturbance), resilience (the ability to return to the initial structure following a disturbance) and functional redundancy (the ability to maintain or recover initial function despite compositional changes), all of which are considered to be key properties of robust microbial communities. Then, we provide an overview of the currently available methodologies to assess community robustness, as well as its drivers (microbial diversity and network complexity) and its potential modulation through diet. Finally, we propose future lines of research on ruminal microbiota robustness.

Age Evolution of Lipid Accretion Rate in Boars Selected for Lean Meat and Duroc Barrows.

Fatty acid (FA) deposition in growing-fattening pigs is mainly based on endogenous lipid synthesis, but also direct FA incorporation from the diet. To evaluate the direct fat incorporation rates and the endogenous desaturation action of the stearoyl-CoA desaturase (SCD) enzyme, a deuterium (D)-labeled saturated FA (d35-C18:0) was added to the diet. Sixteen three-way (3W) crossbred boars, and thirty-two purebred Duroc barrows homozygous for the SCD single nucleotide polymorphism rs80912566 (16 CC/16 TT), were used. Half of the animals of each genotype belonged to the growing and fattening phases. The fractional incorporation rate (FIR) of dietary fat in growing pigs was generally higher in adipose tissues, whereas in fattening pigs it was higher in the liver. Duroc pigs exhibited lower FIRs than 3W pigs, suggesting lower rates of endogenous synthesis by 3W pigs. Real fractional unsaturation rates (FURs) increased with age by the higher FIRs in 3W pigs and the de novo synthesis pathway in Duroc genotypes. Moreover, pigs carrying the SCD_T allele showed more enhanced oleic acid biosynthesis than Duroc CC pigs. In conclusion, suitable feeding protocols should be designed for each pig type to optimize production traits, considering that the metabolic pathway of FA for its deposition may differ.

The Impact of Genetics on Gut Microbiota of Growing and Fattening Pigs under Moderate N Restriction.

Characterization of intestinal microbiota is of great interest due to its relevant impact on growth, feed efficiency and pig carcass quality. Microbial composition shifts along the gut, but it also depends on the host (i.e., age, genetic background), diet composition and environmental conditions. To simultaneously study the effects of producing type (PT), production phase (PP) and dietary crude protein (CP) content on microbial populations, 20 Duroc pigs and 16 crossbred pigs (F2), belonging to growing and fattening phases, were used. Half of the pigs of each PT were fed a moderate CP restriction (2%). After sacrifice, contents of ileum, cecum and distal colon were collected for sequencing procedure. Fattening pigs presented higher microbial richness than growing pigs because of higher maturity and stability of the community. The F2 pigs showed higher bacterial alpha diversity and microbial network complexity (cecum and colon), especially in the fattening phase, while Duroc pigs tended to have higher Firmicutes/Bacteroidetes ratio in cecum segment. Lactobacillus was the predominant genus, and along with Streptococcus and Clostridium, their relative abundance decreased throughout the intestine. Although low CP diet did not alter the microbial diversity, it increased interaction network complexity. These results have revealed that the moderate CP restriction had lower impact on intestinal microbiota than PP and PT of pigs.

The Impact of Producing Type and Dietary Crude Protein on Animal Performances and Microbiota Together with Greenhouse Gases Emissions in Growing Pigs.

In order to reduce dietary nitrogen and achieve an efficient protein deposition as well as decrease N wastage, we challenged the nutrient utilization efficiency of two different producing types in front of a dietary crude protein (CP) restriction and studied the role of the microbiota in such an adaptation process. Therefore, 32 pure castrated male Duroc (DU) and 32 entire male hybrid (F2) piglets were raised in a three-phase feeding regime. At each phase, two iso caloric diets differing in CP content, also known as normal protein (NP) and low protein (LP), were fed to the animals. LP diets had a fixed restriction (2%) in CP content in regards to NP ones throughout the phases of the experiment. At the end of third phase, fecal samples were collected for microbiota analysis purposes and greenhouse gases emissions, together with ammonia, were tested. No changes were found in average daily feed intake (ADFI) of animals of two producing types (Duroc vs. F2) or those consumed different experimental diets (NP vs. LP) throughout the course of study. However, at the end of each experimental phase the average body weight (BW) of hybrid animals were higher compared to Duroc pigs, whereas a reverse trend was observed for average daily gain (ADG), where Duroc pigs showed greater values with respect to hybrid ones. Despite, greater CH4 and ammonia emissions in Duroc pigs with respect to F2, no significant differences were found in contaminant gases emissions between diets. Moreover, LP diets did not alter the microbial community structure, in terms of diversity, although some genera were affected by the dietary challenge. Results suggest that the impact of reducing 2% of CP content was limited for reduction in contaminant gases emissions and highlight the hypothesis that moderate change in the dietary protein levels can be overcome by long-term adaptation of the gut microbiota. Overall, the influence of the producing type on performance and digestive microbiota composition was more pronounced than the dietary effect. However, both producing types responded differently to CP restriction. The use of fecal microbiota as biomarker for predicting feed efficiency has a great potential that should be completed with robust predictive models to achieve consistent and valid results.

Contribution of gut microbial lysine to liver and milk amino acids in lactating does.

The contribution of microbial amino acids through caecotrophy to tissue protein metabolism was investigated in lactating does. Attempts were made to vary microbial supply through a dietary antibiotic, Zn bacitracin, and to vary tissue demand through manipulation of litter size. Three groups of eight New Zealand does were fed different experimental diets from day 28 of pregnancy to day 26 of lactation. The control group received the basal diet formulated to meet requirements with grass hay, wheat, soybean meal and barley grain. The second (no antibiotic) group and the third (bacitracin; BAC) group ingested the basal diet supplemented with ammonium sulfate (5 g/kg), initially unlabelled (day 1 to day 8) then labelled with 15N (day 9 to day 30), while the BAC diet was also supplemented throughout with antibiotic (Zn bacitracin; 100 mg/kg). From just after birth each group of does was subdivided into two groups, each of four females, with the litter size either five (LS5) or nine (LS9) pups. The 15N enrichment in liver, milk and caecal bacteria amino acids was determined by GC-combustion-isotope ratio MS. All amino acids in bacterial protein were enriched with the (15 NH 4)2SO4 treatment, with lysine 15N enrichment significantly greater in caecal bacteria (0.23 (SE 0.0063) atom % excess (ape)) than in liver (0.04 (SE 0.0004) ape) or milk protein (0.05 (SE 0.0018) ape), confirming the double origin (bacterial and dietary) of tissue lysine. The contribution of microbes to tissue lysine was 0.23 (SE 0.006) when milk protein was used as reference.

Effect of antibiotics on the bacterial population of the rabbit caecum.

The effect feeding antibiotics has on the bacterial population of the rabbit caecum was investigated. No changes in total volatile fatty acid production or total bacterial counts were observed compared with nonantibiotic treated controls. However, treatment with chlortetracycline resulted in an increase of propionate at the apparent cost of butyrate (P<0.05). Denaturing gradient gel electrophoresis analysis indicated that the two antibiotics that inhibit protein synthesis (chlortetracycline and tiamulin) exerted the most similar changes on the bacterial population structure, decreasing the diversity of the profiles. Sequence analysis of DNA from excised denaturing gradient gel electrophoresis bands was carried out. The majority of the sequences observed were most similar to bacterial sequences previously described in other gut environments, with 11% being most similar to those previously reported from the rabbit, and 95% of the sequences having 95% or greater identity to sequences already in GenBank.

Molecular profiling of bacterial species in the rabbit caecum.

The diversity of bacteria present in the caecum of the rabbit was investigated. Partial bacterial 16S rRNA genes from a digested sample collected from the caecum of an adult rabbit were amplified by PCR. Sequence analysis of the amplified fragments indicated highest similarity was to bacterial sequences previously described from other gut environments. However, only one sequence showed significant identity (97% threshold) to any previously described bacterial 16S rRNA genes. Furthermore, most of the sequences clustered together in groups lacking representatives from sequences already described, suggesting that the rabbit caecal flora contains organisms not previously described.

Urinary excretion of purine derivatives in Bos indicus x Bos taurus crossbred cattle.

Four experiments were performed to study the kinetics of purine metabolism and urinary excretion in Zebu crossbred cattle. Fasting excretion was established in Expt 1, using eighteen male Bos indicus x Bos taurus crossbred cattle (261 (SE 9.1) kg body weight), six of each of the following genotypes: 5/8 Bos indicus, 1/2 Bos indicus and 3/8 Bos indicus. No significant differences were observed among genotypes in fasting purine derivative excretion (277.3 (SE 35.43) micromol/metabolic body weight). In a second experiment we measured the xanthine oxidase activity, which was higher in liver than in duodenal mucosa (0.64 and 0.06 (SE 0.12) units/g wet tissue per min respectively; P>0.05) being in plasma 0.60 (SE 0.36) units/l per min. The kinetics of uric acid were measured by intravenous pulse dose of [1,3-15N]uric acid (Expt 3). The cumulative recovery of the isotope in urine was 82 (SE 6.69) %, and uric acid plasma removal, pool size and mean retention time were 0.284 (SE 0.051) per h, 5.45 (SE 0.823) mmol and 3.52 (SE 0.521) h, respectively. Allantoin was removed from plasma at an estimated fractional rate of 0.273 (SE 0.081) per h and mean retention was 3.66 (SE 1.08) h. In Expt 4, the relationship between urinary purine derivative excretion (Y; mmol/d) and digestible organic matter intake (X, kg/d) was defined by the equation: Y=7.69 (SE 4.2)+5.69 (SE 1.68) X; n 16, Se 1.31, r 0.67.

Protein recycling in growing rabbits: contribution of microbial lysine to amino acid metabolism.

To study the absorption of microbial lysine in growing rabbits, a labelled diet (supplemented with (15)NH4Cl) was administered to six animals (group ISOT); a control group (CTRL, four rabbits) received a similar, but unlabelled, diet. Diets were administered for 30 d. An additional group of six animals were fed the unlabelled diet for 20 d and then the labelled diet for 10 d while wearing a neck collar to avoid caecotrophy (group COLL), in order to discriminate it from direct intestinal absorption. At day 30 animals were slaughtered and caecal bacteria and liver samples taken. The (15)N enrichment in amino acids of caecal bacteria and liver were determined by GC-combustion/isotope ratio MS. Lysine showed a higher enrichment in caecal microflora (0.925 atom% excess, APE) than liver (0.215 APE) in group ISOT animals, confirming the double origin of body lysine: microbial and dietary. The COLL group showed a much lower enrichment in tissue lysine (0.007 (se 0.0029) APE for liver). Any enrichment in the latter animals was due to direct absorption of microbial lysine along the digestive tract, since recycling of microbial protein (caecotrophy) was avoided. In such conditions liver enrichment was low, indicating a small direct intestinal absorption. From the ratio of [(15)N]lysine enrichment between liver and bacteria the contribution of microbes to body lysine was estimated at 23 %, with 97 % of this arising through caecotrophy. Absorption of microbial lysine through caecotrophy was 119 (se 4.0) mg/d, compared with 406 (se 1.8) mg/d available from the diet. This study confirms the importance of caecotrophy in rabbit nutrition (15 % of total protein intake).

Urinary excretion of purine derivatives as an index of microbial protein synthesis in the camel (Camelus dromedarius).

Five experiments were carried out to extend knowledge of purine metabolism in the camel (Camelus dromedarius) and to establish a model to enable microbial protein outflow from the forestomachs to be estimated from the urinary excretion of purine derivatives (PD; i.e. xanthine, hypoxanthine, uric acid, allantoin). In experiment 1, four camels were fasted for five consecutive days to enable endogenous PD excretion in urine to be determined. Total PD excretion decreased during the fasting period to 267 (SE 41.5) micromol/kg body weight (W)0.75 per d. Allantoin and xanthine + hypoxanthine were consistently 86 and 6.1 % of total urinary PD during this period but uric acid increased from 3.6 % to 7.4 %. Xanthine oxidase activity in tissues (experiment 2) was (micromol/min per g fresh tissue) 0.038 in liver and 0.005 in gut mucosa but was not detected in plasma. In experiment 3, the duodenal supply of yeast containing exogenous purines produced a linear increase in urinary PD excretion rate with the slope indicating that 0.63 was excreted in urine. After taking account of endogenous PD excretion, the relationship can be used to predict purine outflow from the rumen. From the latter prediction, and also the purine:protein ratio in bacteria determined in experiment 5, we predicted the net microbial outflow from the rumen. In experiment 4, with increasing food intake, the rate of PD excretion in the urine increased linearly by about 11.1 mmol PD/kg digestible organic matter intake (DOMI), equivalent to 95 g microbial protein/kg DOMI.