N.R. St-Pierre

Meta-analyses of experimental data in animal nutrition ⋆

Research in animal sciences, especially nutrition, increasingly requires processing and modeling of databases. In certain areas of research, the number of publications and results per publications is increasing, thus periodically requiring quantitative summarizations of literature data. In such instances, statistical methods dealing with the analysis of summary (literature) data, known as meta-analyses, must be used. The implementation of a meta-analysis is done in several phases. The first phase concerns the definition of the study objectives and the identification of the criteria to be used in the selection of prior publications to be used in the construction of the database. Publications must be scrupulously evaluated before being entered into the database. During this phase, it is important to carefully encode each record with pertinent descriptive attributes (experiments, treatments, etc.) to serve as important reference points for the rest of the analysis. Databases from literature data are inherently unbalanced statistically, leading to considerable analytical and interpretation difficulties; missing data are frequent, and data structures are not the outcomes of a classical experimental system. An initial graphical examination of the data is recommended to enhance a global view as well as to identify specific relationships to be investigated. This phase is followed by a study of the meta-system made up of the database to be interpreted. These steps condition the definition of the applied statistical model. Variance decomposition must account for inter- and intrastudy sources; dependent and independent variables must be identified either as discrete (qualitative) or continuous (quantitative). Effects must be defined as either fixed or random. Often, observations must be weighed to account for differences in the precision of the reported means. Once model parameters are estimated, extensive analyses of residual variations must be performed. The roles of the different treatments and studies in the results obtained must be identified. Often, this requires returning to an earlier step in the process. Thus, meta-analyses have inherent heuristic qualities.

A meta-analysis of the effects of high ambient temperature on growth performance of growing-finishing pigs

High ambient temperature (T) is one of the most important climatic factors influencing pig performance. Increased T occurs sporadically during summer heat waves in temperate climates and year round in tropical climates. Results of published experiments assessing the effects of high T on pig performance are surprisingly variable. Thus, a meta-analysis was performed to aggregate our knowledge and attempt to explain differences in the results across studies on the effect of increased T on ADFI and ADG in growing-finishing pigs. Data for ADFI and ADG were extracted from 86 and 80 trials, respectively, from articles published in scientific journals indexed in PubMed, Science Direct, and from proceedings of scientific meetings through November 2009. Data on ADFI and ADG were analyzed using a linear mixed model that included the linear and the quadratic effects of T and BW, and their interactions as continuous, fixed effects variables, and the trial as a random effect factor (i.e., block). In addition, the effects of housing type (2 levels: individual and group housing) and the year of publication (3 levels: 1970 to 1989, 1990 to 1999, and 2000 to 2009) on the intercept and the linear regression term for T (i.e., the slope) were also tested. Results showed that high T had a curvilinear effect on ADFI and ADG and that this effect was more pronounced in heavier pigs. Across T, ADFI was less when pigs were group-housed. The intercept and the regression coefficient (slope) for T were significantly affected by the year of publication. The effect of increased T was greater in more contemporary works, suggesting that modern genotypes could be more sensitive to heat stress than older genotypes of lesser growth potential. In conclusion, pig performance decreases at an accelerating rate as T is increased. The large between-study variability on the effects of high T on pig performance is partially explained by differences in pig BW and to a lesser extent by the year the study was published.

Myofibrillar 1-D fingerprints and myosin heavy chain MS analyses of beef loin at 36 h postmortem correlate with tenderness at 7 days

This study investigated the potential for relating changes in electrophoretic protein patterns derived from the longissimus dorsi of beef cattle 36 h postmortem with tenderness at 7 days. We report finding a significant correlation (R(2)=0.82) between electrophoretic l. dorsi myofibrillar fingerprints at 36 h postmortem and tenderness at 7 days, as determined by Warner-Bratzler shear analysis. In addition, we have used mass spectrometric analyses to identify fragments of bovine myosin heavy chain that are significantly correlated with tenderness. Furthermore, this method offers the potential to increase our understanding of the fundamental cellular mechanisms underlying the proteolytic breakdown of muscle proteins during the aging process.

Rumen ciliated protozoa decrease generation time and adjust 18S ribosomal DNA copies to adapt to decreased transfer interval, starvation, and monensin.

Defaunation studies have documented decreased ammonia concentrations associated with reduced microbial protein recycling and wastage of dietary protein, whereas many methods to suppress protozoa can reduce feed intake or depress ruminal organic matter or fiber digestibility. Therefore, more research is needed to optimize dietary conditions that improve protozoal growth and ruminal outflow relative to autolysis and recycling. Response in growth rate to ruminal outflow was simulated by abrupt changes in transfer interval of batch cultures, and substrate availability was evaluated by feeding without or with abrupt addition of monensin, which was postulated to inhibit digestive vacuole function. In experiment 1, Entodinium caudatum, a mix of Entodinium species, Epidinium caudatum, or Ophryoscolex caudatus cultures rapidly adjusted their generation times to approach respective changes in transfer interval from 3 to 2 or 1 d (cultures were always fed at 24-h intervals). Monensin (0.25 microM) consistently delayed this response. To evaluate a metabolic upshift associated with feeding or a downshift associated with substrate depletion, experiment 2 used real-time PCR to quantify protozoal 18S rRNA gene (rDNA) copies that were expressed relative to cell numbers or to the cellular constituents N and nucleic acids after feeding without or with monensin (0.5 microM). The 18S rDNA copies per milligram of nucleic acids were least for Ophryoscolex compared with the other cultures. When averaged over cultures (no culture x treatment interaction), 18S rDNA copies per unit of nucleic acids decreased at 16 h when cultures were starved but increased with feeding unless monensin uncoupled availability of consumed substrate. Rumen protozoal growth increased in response to decreased transfer interval in experiment 1. Substrate availability appeared to initiate metabolic responses preparing for cell growth, explaining how cultures could rapidly adjust to decreasing transfer interval in experiment 2. Because feeding was not coupled with transfer in experiment 2, however, a metabolic control probably arrested cell division to prevent overgrowth relative to substrate availability.

Isoleucine, leucine, methionine, and threonine effects on mammalian target of rapamycin signaling in mammary tissue

Improved representation of postabsorptive N metabolism in lactating dairy cows requires a better understanding of protein synthesis regulation in the mammary glands. This study aimed to determine the quantitative effects of Ile, Leu, Met, and Thr on the phosphorylation state of signaling proteins that regulate protein synthesis. The experiment used a composite design with a central point, 2 axial points per AA, and a complete 2(4) factorial. All of the other AA were provided at the concentrations in Dulbeccos modified Eagles medium. The experiment was replicated with tissues from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h. Total and site-specific phosphorylated mammalian target of rapamycin (mTOR; Ser2448), eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 2α (Ser51) were determined by western immunoblotting. Tissue concentrations of the 4 AA studied responded linearly to media supply. Addition of Ile, Leu, Met, or Thr had no effect on eukaryotic initiation factor 2α phosphorylation. Isoleucine and Thr positively affected mTOR phosphorylation. However, the 2 AA had an antagonistic relationship. Similarly, Ile linearly increased ribosomal protein S6 phosphorylation, and Thr inhibited the Ile effect. In addition, eEF2 phosphorylation was linearly decreased by Ile and Leu. Threonine curvilinearly decreased eEF2 phosphorylation, Ile and Leu negatively interacted on eEF2, and Thr tended to inhibit Leu effects on eEF2. This work demonstrated saturable responses and interactions between AA on activation of the mTOR pathway. Incorporation of these concepts into milk protein response models will help to improve milk and milk protein yield predictions and increase postabsorptive N efficiency and reduce N excretion by dairy cows.

Proteomic analysis of proteins associated with body mass and length in yellow perch, Perca flavescens.

The goal of commercial yellow perch aquaculture is to increase muscle mass which leads to increased profitability. The accumulation and degradation of muscle‐specific gene products underlies the variability in body mass (BM) and length observed in pond‐cultured yellow perch. Our objective was to apply a combination of statistical and proteomic technologies to identify intact and/or proteolytic fragments of muscle specific gene products involved in muscle growth in yellow perch. Seventy yellow perch randomly selected at 10, 12, 16, 20, and 26 wk of age were euthanized; BM and length were measured and a muscle sample taken. Muscle proteins were resolved using 5–20% gradient SDS‐PAGE, stained with SYPRO® Ruby and analyzed using TotalLab™ software. Data were analyzed using stepwise multiple regression with the dependent variables, BM and length and proportional OD of each band in a sample as a potential regressor. Eight bands associated with BM (R2 = 0.84) and nine bands with length (R2 = 0.85) were detected. Protein sequencing by nano‐LC/MS/MS identified 20 proteins/peptides associated with BM and length. These results contribute the identification of gene products and/or proteolytic fragments associated with muscle growth in yellow perch.

Technical note : Occurrence in fecal microbiota of genes conferring resistance to both macrolide-lincosamide-streptogramin B and tetracyclines concomitant with feeding of beef cattle with tylosin

Development of antimicrobial resistance in food animals receiving antimicrobials has been well documented among bacterial isolates, especially pathogens, but information on development of antimicrobial resistance at the microbial community level during long-term feeding of antimicrobials is lacking. The objective of this study was to examine the association between inclusion of tylosin in feed and occurrence of resistance to macrolide-lincosamide-streptogramin B (MLS(B)) in the entire fecal microbial communities of beef cattle over a feeding study of 168 d. A completely randomized design included 6 pens housed together in 1 barn, with each pen housing 10 to 11 steers. The control and tylosin groups each had 3 pens, with the former receiving no antimicrobial whereas the latter received both tylosin and monensin (11 and 29.9 mg/ kg of feed, respectively, DM) in feed. The abundance of genes conferring resistance to MLS(B) (erm genes) and tetracyclines (tet genes) were quantified using class-specific, real-time PCR assays. The abundances of erm and tet genes were analyzed with pens as experimental units using the MIXED procedure of SAS. Correlations between abundance of different resistance genes were calculated using the CORR procedure of SAS. We identified 4 classes (B, F, T, and X) of erm genes in fresh fecal samples collected at wk 2, 17, and 21 of feeding. From wk 2 to 17, the abundance of erm(T) and erm(X) increased (P < 0.05), whereas that of erm(B) and erm(F) did not. The abundance of the erm genes did not further change from wk 17 to 21. The tet(A/C), tet(G), and tet gene variants encoding ribosomal protection proteins (including classes M, O, P, Q, S, T, and W) appeared to be co-selected by tylosin feeding. Such co-selection of multiresistance at community level by one antimicrobial drug used in animals has the important implication that future studies should examine resistance to not only the antimicrobials used in animals, but also other antimicrobials, especially those used in human medicine, to fully assess the potential risk associated with antimicrobial use in animals. Both the erm and tet genes appeared to be disseminated among the microbial populations in all steers housed together.

Casein synthesis is independently and additively related to individual essential amino acid supply

Specific AA affect rates of milk protein synthesis in the mammary glands of lactating cows. The objective of this study was to quantify the rate of αS1-casein synthesis in response to Ile, Leu, Met, and Thr supplementation, and to test the single-limiting AA theory for milk protein synthesis by exploring interactions among these AA. Effects of Ile, Leu, Met, and Thr were studied in vitro with a composite design containing a central point repeated 4 times, with 2 axial points per AA and a complete 2(4) factorial. Other AA were at the concentration in Dulbeccos modified Eagle medium/F12 medium (DMEM). The experiment was replicated with mammary tissue from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h at 37°C in 5 mL of treatment medium containing (2)H5-Phe. Caseins were precipitated from cell homogenate supernatants. Enrichment with (2)H5-Phe of the N[34]LLRFFVAPFPE αS1 peptide was determined by matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF-TOF), which was used to determine enrichment of Phe in the transfer (t)RNA pool and αS1-casein fractional synthesis rates (CFSR). Data were analyzed with a polynomial mixed model containing linear, quadratic, and 2-factor interactions for Ile, Leu, Met, and Thr, and cow and residual as random factors. Interactions were not significant at P<0.1 and were removed from the model. Increasing concentrations of Ile, Leu, Met, and Thr simultaneously increased CFSR curvilinearly with a predicted maximum response of 4.32 ± 0.84%/h at 63% of DMEM concentrations. The maximum response to each of the 4 AA was at 71, 49, 60, and 32% of the concentration in DMEM, for Ile, Leu, Met, and Thr, respectively. These values correspond to 270, 120, 440, and 140% the plasma concentrations of Ile, Leu, Met, and Thr observed in lactating cows fed to meet National Research Council requirements, respectively. The CFSR estimated at those maxima were similar among AA (3.6 ± 0.6%/h). Individual AA effects on CFSR did not correlate with mammalian target of rapamycin (mTOR) signaling. Independent responses of CFSR to individual essential AA observed in this study contradict the single-limiting AA theory assumed in current requirement systems. The saturable responses in CFSR to these 4 AA also highlight the inadequacy of using a fixed postabsorptive AA efficiency approach for determining AA requirements for milk protein synthesis.

Intestinal digestibility of long-chain fatty acids in lactating dairy cows: A meta-analysis and meta regression

The objective of this analysis was to examine the intestinal digestibility of individual long-chain fatty acids (FA) in lactating dairy cows. Available data were collated from 15 publications containing 61 treatments, which reported total and individual FA duodenal flows and calculations of intestinal digestibility. All studies involved lactating dairy cows, and estimates of digestibility were based on measurements either between the duodenum and ileum (18 treatments) or between the duodenum and feces (43 treatments). Fatty acid digestibility was calculated for C16:0, C18:0, C18:1 (cis and trans isomers), C18:2, and C18:3. Digestibility of C18:0 was lower than for C18:1 and C18:3, with no difference in digestibility between saturated FA (C16:0 and C18:0). We weighted the studies by the reciprocal of the variance to generate best-fit equations to predict individual FA digestibility based on duodenal flow of FA and dietary independent variables. The flow of C18:0 negatively affected the digestibility of C18:0 and was also included in the best-fit equations for all other 18-carbon FA using duodenal flow characteristics. The type of fat supplemented had an effect on digestibility of individual FA, with whole seeds having reduced digestibility. Our meta-analysis results showed minimal differences in the digestibility of individual FA. However, C18:0 flow through the duodenum had a negative effect on the digestibility of several individual FA, with the largest negative effect on C18:0 digestibility. The mechanisms that reduce C18:0 absorption at high concentrations are unknown and warrant further investigation.

Effectiveness of potassium carbonate sesquihydrate to increase dietary cation-anion difference in early lactation cows

The effect of additional dietary potassium in early lactation dairy cows was evaluated with the addition of potassium carbonate sesquihydrate, which increased dietary K from 1.3 to 2.1% of dry matter (DM) from wk 3 to 12 of lactation. Cows fed potassium carbonate sesquihydrate in the form of DCAD Plus (Church & Dwight Co. Inc., Princeton, NJ) had increased DM intake, milk fat percentage and yield, energy-corrected milk, and efficiency of milk production per unit of DM intake. Milk fat of cows fed higher dietary K had a lower concentration of trans fatty acids, suggesting a role for potassium carbonate sesquihydrate in the rumen in the biohydrogenation processes converting linoleic to stearic acid. Cows fed the diet with 2.1% K had greater apparent balance of K, and no effects were noted on the concentration of blood Mg or amount of fecal Mg. The data support the feeding of greater amounts of K in the early lactation cow.