Effects of ensiling time on corn silage starch ruminal degradability evaluated in situ or in vitro.

Accurate measurements of concentration and ruminal degradability of corn silage starch is necessary for formulation of diets that meet the energy requirements of dairy cows. Five corn silage hybrids ensiled for 0 (unfermented), 30, 60, 120, and 150 d were used to determine the effects of ensiling time on starch degradability of corn silage. In addition, the effects of grind size of silage samples on 7-h in vitro starch degradability and the relationship between in vitro, in situ and near-infrared reflectance spectroscopy (NIRS) starch degradability were studied. In situ disappearance of corn silage starch increased from 0 to 150 d of ensiling, primarily as a result of an increase in the washout or rapidly degraded fraction of starch, particularly during the first 60 d of ensiling. When analyzed in vitro and by NIRS, ensiling time increased corn silage starch degradability either linearly or to a greater extent during the first 2 mo of ensiling. Differences in in situ starch disappearance among corn silage hybrids were apparent during the first 2 mo of ensiling but were attenuated as silages aged. No differences among hybrids were detected using a 7-h in vitro starch digestibility approach. Results from the in vitro subexperiment indicate that 7-h in vitro starch degradability was increased by reducing grind size of corn silage from 4 to 1 mm, regardless of ensiling duration. Fine grinding corn silages samples (i.e., 1-mm sieve) allowed distinguishing low- from medium- and high-starch degradability rated hybrids. Correlations among in situ, in vitro and NIRS measurements for starch degradability were medium to high (r ≥0.57); however, agreement among methods was low (concordance correlation coefficient ≤0.15). In conclusion, ensiling time linearly increased degradation rate of corn silage resulting in greater in situ starch disappearance after 150 d of ensiling. Reductions in grind size from 4 to 1 mm resulted in greater in vitro starch degradability, regardless of ensiling duration. Strong correlation but low agreement between starch degradability methods suggest that absolute estimations of corn silage starch degradability will vary, but all methods can be used to assess the effect of ensiling time on starch degradability.

Milk metabolome reveals pyrimidine and its degradation products as the discriminant markers of different corn silage-based nutritional strategies.

The purpose of this study was to evaluate the effect of 6 different feeding systems (based on corn silage as the main ingredient) on the chemical composition of milk and to highlight the potential of untargeted metabolomics to find discriminant marker compounds of different nutritional strategies. Interestingly, the multivariate statistical analysis discriminated milk samples mainly according to the high-moisture ear corn (HMC) included in the diet formulation. Overall, the most discriminant compounds, identified as a function of the HMC, belonged to AA (10 compounds), peptides (71 compounds), pyrimidines (38 compounds), purines (15 compounds), and pyridines (14 compounds). The discriminant milk metabolites were found to significantly explain the metabolic pathways of pyrimidines and vitamin B6. Interestingly, pathway analyses revealed that the inclusion of HMC in the diet formulation strongly affected the pyrimidine metabolism in milk, determining a significant up-accumulation of pyrimidine degradation products, such as 3-ureidopropionic acid, 3-ureidoisobutyric acid, and 3-aminoisobutyric acid. Also, some pyrimidine intermediates (such as l-aspartic acid, N-carbamoyl-l-aspartic acid, and orotic acid) were found to possess a high discrimination degree. Additionally, our findings suggested that the inclusion of alfalfa silage in the diet formulation was potentially correlated with the vitamin B6 metabolism in milk, being 4-pyridoxic acid (a pyridoxal phosphate degradation product) the most significant and up-accumulated compound. Taken together, the accumulation trends of different marker compounds revealed that both pyrimidine intermediates and degradation products are potential marker compounds of HMC-based diets, likely involving a complex metabolism of microbial nitrogen based on total splanchnic fluxes from the rumen to mammary gland in dairy cows. Also, our findings highlight the potential of untargeted metabolomics in both foodomics and foodomics-based studies involving dairy products.

Assessment of feed and economic efficiency of dairy farms based on multivariate aggregation of partial indicators measured on field.

Many of the metrics used to evaluate farm performance are only partial indicators of farm operations, which are assumed to be best predictors of the whole farm efficiency. The main objective of this work was to identify aggregated multiple indexes of profitability using common partial indicators that are routinely available from individual farms to better support the short-term decision-making processes of the cattle-feeding process. Data were collected from face-to-face interviews with farmers from 90 dairy farms in Italy and used to calculate 16 partial indicators that covered almost all indicators currently used to target feeding and economic efficiency in dairy farms. These partial indicators described feed efficiency, energy utilization, feed costs, milk-to-feed price ratio, income over feed costs, income equal feed cost, money-corrected milk, and bargaining power for feed costs. Calculations of feeding costs were based on lactating cows or the whole herd, and income from milk deliveries was determined with or without considering the milk quality payment. Multivariate factor analysis was then applied to the 16 partial indicators to determine simplified and latent structures. The results indicated that 5 factors explained 70% of the variability. Each of the original partial indicator was associated with all factors in different proportions, as indicated by loading scores from the multivariate factor analysis. Based on the loading scores, we labeled these 5 factors as "economic efficiency," "energy utilization," "break-even point," "milk-to-feed price," and "bargaining power of the farm," in decreasing order of explained communality. The first 3 factors shared 83% of the total communality. Feed efficiency was similarly associated with factor 1 (53% loading) and factor 2 (66% loading). Only factor 4 was significantly affected by farm location. Milk production and herd size had significant effects on factor 1 and factor 2. Our multivariate approach eliminated the problem of multicollinearity of partial indicators, providing simple and effective descriptions of farm feeding economics. The proposed method allowed the evaluation, benchmarking, and ranking of dairy herd performance at the level of single farms and at territorial level with high opportunity to be used or replicated in other areas.

Kinetics of gas production in the presence of Fusarium mycotoxins in rumen fluid of lactating dairy cows.

Little is known about the effects of Fusarium mycotoxins on the fermentation potential of rumen fluid sampled from lactating dairy cows ingesting diets contaminated at regular levels of these mycotoxins (i.e., contamination levels that can normally be found on dairy farms). In the current experiment, rumen donor animals received diets contaminated with both deoxynivalenol (DON) and fumonisins (FB) with or without a mycotoxin-deactivating product. The rumen fluid donor animals were 12 lactating Holstein dairy cows that received one of 3 experimental diets in agreement with a 3 × 3 Latin square design (3 periods and 3 treatments). The 3 diets were as follows: (1) a TMR contaminated with a regular level of Fusarium mycotoxins [340.5 ± 161.0 µg of DON/kg of dry matter (DM) and 127.9 ± 43.9 µg of FB/kg of DM; control diet, CTR], (2) a TMR contaminated with Fusarium mycotoxins at levels higher than CTR but below US and European Union guidelines (733.0 ± 213.6 µg of DON/kg of DM and 994.4 ± 323.2 µg of FB/kg of DM; MTX), and (3) the MTX diet (897.3 ± 230.4 µg of DON/kg of DM and 1,247.1 ± 370.2 µg of FB/kg of DM) supplemented with a mycotoxin-deactivator product (Mycofix, Biomin Holding GmbH; 35 g/animal per day; MDP). Each experimental period lasted 21 d, and rumen fluid was individually sampled from all cows on the last day of each intoxication period. Then, the 4 rumen fluids sampled from cows receiving the same experimental diets were pooled into a single rumen inoculum, which was used in the in vitro gas production test. For the gas production test, 3 different rumen inocula (i.e., CTR, MTX, and MDP) were buffered (buffer:rumen ratio of 2:1, vol/vol) and then used in 3 fermentation runs to evaluate gas production dynamics in the presence of 8 feeds (i.e., corn meal, barley meal, corn silage, sorghum silage, alfalfa hay, ryegrass hay, dry brewers barley grains, and dried distillers grains with solubles). The kinetic parameters of gas production and volatile fatty acid concentrations were evaluated at the end of fermentation. The block run (i.e., fermentation day) effect influenced all of the fermentative and kinetic parameters. Greater final volumes or rates of gas production over time were observed for MDP compared with MTX rumen inocula (i.e., 172.6 vs. 147.8 mL/g of organic matter or 0.078 vs. 0.063 h-1, respectively). However, the increase in rate of gas production was not consistent among tested feeds, meaning that a treatment by feed interaction was observed. Volatile fatty acid concentrations were not different among treatments, except for a slight increase of acetic acid in CTR compared with MTX (i.e., 71.0 vs. 67.9 mmol/L). This study showed that Fusarium-produced mycotoxins negatively affected the kinetics of gas production in feeds, whereas the presence of the mycotoxin-deactivator product in the diets of donor animals resulted in an increase in rumen fermentation potential, thus safeguarding the rumen environment.

Development of a linear programming model for the optimal allocation of nutritional resources in a dairy herd.

A linear programming model that selects the optimal cropping plan and feeds allocation for diets to minimize the whole dairy farm feed costs was developed. The model was virtually applied on 29 high-yielding Holstein-Friesian herds, confined, total mixed ration dairy farms. The average herd size was 313.2 ± 144.1 lactating cows and the average land size was 152.2 ± 92.5 ha. Farm characteristics such as herd structure, nutritional grouping strategies, feed consumption, cropping plan, intrinsic farm limitations (e.g., silage and hay storage availability, water for irrigation, manure storage) and on farm produced forage costs of production were collected from each farm for the year 2017. Actual feeding strategies, land availability, herd structure, crop production costs and yields, and milk and feed market prices for the year 2017 were used as model inputs. Through optimization, the feeding system was kept equal to the actual farm practice. The linear program formulated diets for each animal group to respect actual herd dry matter intake and fulfill actual consumption of crude protein, rumen-degradable and rumen-undegradable fractions of crude protein, net energy for lactation, neutral detergent fiber, acid detergent fiber, forage neutral detergent fiber, and nonfiber carbohydrate. Production levels and herd composition were considered to remain constant as the nutritional requirement would remain unchanged. The objective function was set to minimize the whole-farm feed costs including cash crop sales as income, and crop production costs and purchased feed costs as expenses. Optimization improved income over feed costs by reducing herd feed costs by 7.8 ± 6.4%, from baseline to optimized scenario, the improved was explained by lower feed costs per kilogram of milk produced due to a higher feed self-sufficiency and higher income from cash crop. In particular, the model suggested to maximize, starting from baseline to optimized scenario, the net energy for lactation (+8.5 ± 6.3%) and crude protein (+3.6 ± 3.1%) produced on farm, whereas total feed cost (€/100 kg of milk) was greater in the baseline (20.4 ± 2.3) than the optimized scenario (19.0 ± 1.9), resulting in a 6.7% feed cost reduction with a range between 0.49% and 21.6%. This meant €109 ± 96.9 greater net return per cow per year. The implementation of the proposed linear programming model for the optimal allocation of the nutritional resources and crops in a dairy herd has the potential to reduce feed cost of diets and improve the farm feed self-sufficiency.

Technical note: Evaluation of a novel enzymatic method to predict in situ undigested neutral detergent fiber of forages and nonforage fibrous feeds.

The purpose of this study was to optimize the conditions of a previously proposed enzymatic method used to estimate in situ undigested neutral detergent fiber (uNDF). We used a multi-step enzymatic approach, in which samples were first solubilized in NaOH solutions as a preincubation (PreInc) phase. After rinsing, samples were incubated (24 h at 39°C) in a buffered solution (pH 6) containing hemicellulase, cellulase, and Viscozyme L enzymes (Sigma-Aldrich s.r.l., Milan, Italy), followed by incubation (24 h at 39°C) in a buffered solution (pH 5) containing xylanase. Two sets of experiments were performed: a calibration trial (that tested different PreInc conditions on 9 selected forages) and a validation trial (that verified the results by testing multiple samples of 6 different forage types and a group of fibrous by-products). In the calibration trial, samples (300 mg in Ankom F57 filter bags; Ankom Technology Corp., Fairport, NY) were preincubated at 39°C in a 0.1 M NaOH solution for 90, 180, or 240 min, or in 0.2, 0.5, 1.0, or 2.0 M NaOH solution for 90 min. The results indicated that the best PreInc method, in terms of intra-laboratory repeatability and estimation of reference in situ values, was 90 min in a 0.2 M NaOH solution. Thus, we used this PreInc condition to determine enzymatic uNDF of 257 samples in the validation trial. Although the selected method generally had good accuracy in predicting in situ uNDF, inconsistencies were noted for certain forage types. Overall, when enzymatic uNDF was used to predict the in situ uNDF of all samples, the regression was satisfactory (intercept = 7.098, slope = 0.920, R2 = 0.73). The regression models developed for alfalfa hays, corn silages, and small grain silages had also acceptable regression performances and mean square error of prediction (MSEP) values, and the main sources of MSEP variation were error due to incomplete (co)variation and random error. Even when R2 values were >0.70, the MSEP value of the regression model for grass hays was 149.55, and that for nonforage fibrous feeds was 155.16. Although enzymatic uNDF partially overestimated the in situ uNDF, particularly in grass silages, the proposed procedure seems to be promising for accurately predicting in situ uNDF, because it generally had good repeatability and provided satisfactory estimates of in situ uNDF.

Short communication: In vitro rumen gas production and starch degradation of starch-based feeds depend on mean particle size.

Our objective was to model the effect of mean particle size (mPS) on in vitro rumen starch degradation (IVSD) and the kinetics of gas production for different starch-based feeds. For each feed, 2 batches of the same grains were separately processed through 2 different mills (cutter or rotor speed mills), with or without different screens to achieve a wide range of mPS (0.32 to 3.31 mm for corn meals; 0.19 to 2.81 mm for barley meals; 0.16 to 2.13 mm for wheat meals; 0.28 to 2.32 mm for oat meals; 0.21 to 2.36 mm for rye meals; 0.40 to 1.79 for sorghum meals; 0.26 to 4.71 mm for pea meals; and 0.25 to 4.53 mm for faba meals). The IVSD data and gas production kinetics, obtained by fitting to a single-pool exponential model, were analyzed using a completely randomized design, in which the main tested effect was mPS (n = 6 for all tested meals, except n = 7 for corn meals and n = 5 for sorghum meals). Rumen inocula were collected from 2 fistulated Holstein dairy cows that were fed a total mixed ration consisting of 16.2% crude protein, 28.5% starch, and 35.0% neutral detergent fiber on a dry matter basis. The IVSD, evaluated after 7 h of rumen incubation, decreased linearly with increasing mPS for corn, barley, wheat, rye, pea, and faba meals, and decreased quadratically with increasing mPS for the other meals. The y-axis intercept for 7-h IVSD was below 90% starch for corn, barley, and rye feeds and greater than 90% for the other tested feeds. The mPS adjustment factors for the rate of rumen starch degradation varied widely among the different tested feeds. We found a linear decrease in starch degradation with increasing mPS for barley, wheat, rye, and pea meals, whereas we noted a quadratic decrease in starch degradation for the other tested meals. Further, we observed a linear decrease in the rate of gas production with increasing mPS in each tested feed, except for pea meal, which had a quadratic relationship. For each 1 mm increase in mPS, the gas production was adjusted by -0.009 h-1 for corn, -0.011 h-1 for barley, -0.008 h-1 for wheat, and -0.006 h-1 for faba, whereas numerically greater adjustments were needed for oat (-0.022 h-1), rye (-0.017 h-1), and sorghum (-0.014 h-1). These mPS adjustment factors could be used to modify the starch-based feed energy values as a function of mean particle size, although in vivo validation is required.

Technical note: Relationship between in situ NDF degradability and enzymatic NDF hydrolysis in forages, nonforage fibrous feeds, and crop residues.

The study was performed on forages ( = 8), nonforage fibrous feeds ( = 10), and crop residues ( = 2). Samples were characterized for in situ NDF degradability (NDFD) at 6, 12, 18, 24, 30, 36, 48, 72, 96, 120, and 240 h of ruminal incubation. Then, samples were characterized for enzymatic NDFD by adopting a multistep enzymatic method consisting of a preincubation (PreInc) phase followed by enzymatic incubation (EnzInc) steps. In the PreInc phase, samples were incubated in a NaOH solution for 0, 30, 60, or 90 min. Then, in the EnzInc phase, samples were first incubated in a buffered enzymatic solution containing hemicellulase, cellulase, and Viscozyme L enzymes. Then, samples were incubated in a xylanase-buffered enzymatic solution. These 2-step EnzInc lasted for a total of 16 (8 h for the first enzymatic step + 8 h for the second enzymatic step), 32 (16 + 16 h), or 48 h (24 + 24 h). The enzymatic NDFD coefficients were increased by increasing both PreInc and EnzInc incubation times, and no PreInc × EnzInc interaction was observed, except for ryegrass hay. On average, enzymatic NDFD increased ( < 0.05) by 0.35, 0.54, or 0.68, respectively, for 30-, 60-, or 90-min PreInc compared with 0-min PreInc. The enzymatic NDFD increased ( < 0.05), on average, by 0.11 in 32-h EnzInc or 0.16 in 48-h EnzInc with respect to 16-h EnzInc. Enzymatic NDFD were used to predict in situ NDFD coefficients by adopting single regression equations. High coefficients of regression ( > 0.80, < 0.05) and low errors of prediction were measured when specific enzymatic conditions were performed to predict in situ NDFD at intermediate (from 24 to 48 h) ruminal incubation. Generally, worse regression performances were obtained when enzymatic NDFD were used to predict in situ NDFD evaluated after shorter or longer incubation times. The direct prediction of the rate of NDF degradation was not possible using enzymatic NDFD coefficients. Even if the proposed multistep enzymatic method appeared promising, further studies are required to improve enzymatic NDFD prediction ability within specific forage types or nonforage fibrous feeds.

Relationships among ensiling, nutritional, fermentative, microbiological traits and contamination in corn silages addressed with partial least squares regression.

The objective of this work was to reduce the predictor dimensionality and to develop a model able to forecast contamination in corn silages. A survey on 33 dairy farms was performed, and samples from core, lateral, and apical parts of the feed-out face of corn silage bunkers were analyzed for chemical, biological (digestible and indigestible NDF), fermentative (pH, ammonia nitrogen, lactic acid, VFA, and ethanol), and microbiological (yeasts and molds) traits. Corn silage samples were analyzed for cell and spore counts by adoption of a molecular DNA-based method. A partial least squares (PLS) regression with a leave-one-out cross-validation method was used to reduce the dimensionality of the original predictors ( = 30) by projecting the independent variables into latent constructs. In a first step of the model development, the importance of independent variables in predicting contamination was assessed by plotting factor loadings of both dependent and independent variables on the first 2 components and by verifying for each predictor the variable influence on projection values adopting the Wold's criterion as well as the entity of standardized regression coefficients. Three ensiling characteristics (bunker type, presence of lateral wrap plastic film, and penetration resistance as a measurement of the ensiled mass density), a chemical trait (DM), 9 characterizations of the fermentative profile (pH, ammonia nitrogen, acetic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, ethanol, and lactic acid), and 2 microbiological traits (yeasts and molds) were retained as important terms in the PLS model. Three reduced-variable PLS (rPLS) regressions-the first based on ensiling, chemical, fermentative, and microbiological retained important variables (rPLSecfm); the second based on chemical, fermentative, and microbiological retained important traits (rPLScfm); and the last based on only chemical and fermentative retained important variables (rPLScf)-were performed. The model that best fit the measurements was rPLSecfm. The rPLScfm and rPLScf models had similar regression performances but higher mean square errors of prediction than rPLSecfm. However, all tested models seemed adequate to rank corn silages for low, medium, and high risks of contamination. To avoid the visit on farm by trained people required to measure penetration resistance, the use of the rPLScf model is suggested as a useful tool to assess the risk of in corn silage.

Short communication: The effect of an exogenous enzyme with amylolytic activity on gas production and in vitro rumen starch degradability of small and large particles of corn or barley meals.

The objective of this study was to evaluate the effect of exogenous amylase supplementation on gas production and on in vitro rumen starch degradability (IVSD) of different sized particles of corn and barley meals (Cm and Bm, respectively). An aqueous liquid amylase formulation from Bacillus licheniformis was tested at 3 enzyme doses (EnzD; 0, 300 and 1,500 kilo novo units/kg of dry matter) on small (<750 µm) and large (≥750 µm) particle size (PS) of Cm and Bm. Data were analyzed according to a randomized complete block design with a factorial arrangement of treatments; the main tested effects were PS, EnzD, and their interaction. Fermentation run entered in the model as random effect. The mixed rumen fluid was collected from 2 rumen-fistulated Holstein dry dairy cows fed at maintenance (forage:concentrate ratio of 80:20; 12% crude protein; 55% amylase-treated neutral detergent fiber). Small particles of both Cm and Bm had a greater rate of fermentation and shorter lag time than large particles. The rate of starch degradation was greater for small than for large particles of Bm, being 0.187 and 0.125 1/h, respectively. Conversely, the rate of starch degradation of Cm averaged 0.063 1/h and was similar among treatments. Enzyme supplementation tended to reduce lag time and to increase rate of fermentation for both PS of Cm and Bm, with a more pronounced effect for small PS. A limited EnzD effect was measured for IVSD data and rate of starch degradation; PS influenced fermentation parameters and the magnitude of starch degradation more than EnzD. Supplementation with exogenous amylase influenced the rumen fermentation pattern of small and large PS of Cm and Bm, even if the effect of the enzyme supplementation differed according to the PS of cereal meals.

Gas production and starch degradability of corn and barley meals differing in mean particle size.

The objective of this study was to verify the effect of mean particle size (mPS) on both gas production and in vitro rumen starch degradability (IVSD) of corn and barley meals (Cm and Bm, respectively). Batches of the same Cm or Bm were separately processed through 2 different mills (i.e., a cutter mill or a rotor speed mill) equipped with or without different screens to achieve different mPS for each tested meal. Samples were analyzed accordingly to a completely randomized design and the main tested effect of model was mPS (n=11, from 0.46 to 3.50mm mPS for Cm or n=10, from 0.11 to 2.98mm mPS for Bm). For both in vitro assays, the rumen inocula were collected from 2 rumen-fistulated Holstein lactating dairy cows fed a total mixed ration with 16.2% crude protein, 28.5% starch, and 35.0% neutral detergent fiber on a dry matter basis. To fit gas production data, 1-pool exponential model and 1-pool or 2-pool Gompertz models were adopted. The rate of gas production decreased and lag increased by increasing mPS of both Cm and Bm, irrespective of adopted 1-pool models. When the 2-pool Gompertz model was used to fit gas production data, a shift of particles from fast to slow fermentable pools was measured by increasing mPS. In particular, the ratio between fast and slow final volumes ranged from 0.90 at 0.11mm mPS to 0.10 at 2.98mm mPS for Bm. For Cm, the ratio between fast and slow final volumes decreased quadratically by increasing mPS, with the highest value (i.e., 0.58) measured at the lowest tested mPS. Values lower than 0.10 were measured for mPS greater than 1.93mm for Cm. Concerning IVSD data, linear decreases in rate of starch degradation equal to -0.049 or -0.092h(-1) for each 1-mm increase in mPS were achieved for Cm and Bm, respectively. The 7-h IVSD decreased by 6.3 or 6.5% starch for each 1-mm increase in mPS of Cm or Bm, respectively. Present findings supported the hypothesis that different particle sizes within the same starch source represent an important factor influencing both fermentation kinetic parameters and IVSD.

Estimation of the indigestible fiber in different forage types.

The role of indigestible NDF is essential in relation to OM digestibility prediction, total tract digestibility, rumen fill, passage rate, and digestion kinetics. Moreover, the truly indigestible NDF (iNDF) represents a core point in dynamic models used for diet formulations. However, despite its wide possible applications, few trials have been conducted to quantify iNDF and even fewer to investigate whether or not it is consistent among different forage sources. The objective of this study was to predict the iNDF by measuring the residual NDF after 240-h in vitro fermentation to determine the unavailable NDF (uNDF) within and among various forage types. Finally, a mathematical approach was investigated for the estimation of the uNDF fraction. In all, 688 forages were analyzed in this study. This pool included 122 alfalfa hays, 282 corn silages, and 284 grass hays. Values of uNDF varied among different forages and within the same type (22.7% ± 4.48%, 20.1% ± 4.23%, and 11.8% ± 3.5% DM for grass hay, alfalfa hay, and corn silages, respectively). The relationship among uNDF and ADL was not constant and, for grass hay and corn silage, was different ( 0.05) from the 2.4 × lignin value applied by the traditional Chandler equation. The observed uNDF:ADL ratio was 3.22 for grass hay and 3.11 for corn silage. Relationships among chemical and biological parameters and uNDF were investigated via simple and multiple regression equations. The greatest correlation with a single variable was obtained by ADL and ADF when applied to the whole data set ( = 0.63). Greater coefficients of determination resulted from a multiple regression equation for the whole data set ( = 0.80) and within each forage type ( = 0.65, 0.77, and 0.54 for grass hay, alfalfa hay, and corn silage, respectively). In conclusion, a regression approach requires specific equations and different regression coefficients for each forage type. The direct measurement of uNDF represented the best approach to obtain an accurate prediction of the iNDF and to optimize its specific purpose in dynamic nutrition models.

Effects of two different blends of naturally mycotoxin-contaminated maize meal on growth and metabolic profile in replacement heifers.

The aim of this trial was to assess the effects of the administration of different combinations of mycotoxins in naturally contaminated maize grains on dairy heifer growth, blood measurements and puberty onset. A total of 35 Friesian female heifers were randomly allotted to three experimental groups from 18-21 to 42-45 weeks of age. During the 24-week experimental period (EP), heifers were fed the same diet, but with maize meal derived from three differently contaminated lots: very low contamination, as control (C); medium-low aflatoxin-contaminated (A); and mixed aflatoxin-fumonisin contaminated (A-F). At the end of the EP, they returned to a common diet without contaminated maize, and they were monitored for an additional period of 12 weeks (post-experimental period, PEP). BW, wither height, hip height, body length and heart girth were measured every 4 weeks from the beginning of EP to the end of PEP. At the same time, body condition score was evaluated and blood samples were taken from the jugular vein to be analysed for haematological, serum protein and metabolic profiles. Age at puberty was assessed by measuring weekly plasma progesterone levels from 40 to 52 weeks of age. Body growth measurements were processed both by ANOVA of average daily gain of EP and PEP separately, and by the analysis of growth curve parameters. Haematological, serum protein and metabolic profile were evaluated using a mixed model, taking into account the repeated measurements in time on each animal. Heifers' growth was delayed both in A and A-F groups during EP, as evidenced by the different linear coefficients of the BW growth curve in the three groups. Differently contaminated diets did not affect the haematological profile, so that it can be concluded that these levels of mycotoxin contamination do not determine any specific effect on haematopoiesis and immunity in growing heifers. The main blood marker of mycotoxin chronic toxicity was the γ-glutamyl transferase activity level in plasma, which appeared to be altered even after the removal of mycotoxins. During EP, plasma glucose was lower in the groups fed contaminated diet compared with C. The joint actions of an altered nutritional status and a long-lasting liver damage were probably the causes of the delay in puberty attainment in A and, particularly, in the A-F group. The results from this trial evidenced that a chronic aflatoxin-fumonisin contamination in diets of dairy heifers can determine an important delay in the reproductive career of these animals.

In vitro production of short-chain fatty acids from resistant starch by pig faecal inoculum.

The need to improve the knowledge of fermentation processes within the digestive tract in pigs is growing, particularly for ingredients that may act as potential prebiotic sources, such as resistant starch (RS). A study (based on enzymatic digestion followed by in vitro fermentation) was conducted to investigate whether various sources of RS, obtained from eight native starches characterized by inherent heterogeneous starch chemistry and structure, can influence short-chain fatty acid (SCFA) concentrations and relative production kinetics. Total and individual SCFA productions were evaluated over time and up to 72 h of incubation. The in vitro hydrolysis of native starches allowed a classification from very high [≥ 650 g/kg dry matter (DM)] to low (<50 g/kg DM) RS amount. The total SCFA production was similar between ingredients, whereas acetate and butyrate molar ratios in the SCFA profile differed (from 0.48 to 0.56 and from 0.17 to 0.25, respectively; P < 0.05). Differences in fermentation kinetic parameters for total and individual SCFA productions were observed (P < 0.05). Considering the total SCFA production after 72 h of incubation, the time at which half of the maximum production has been reached (T 1/2), the maximum rate of production (R max) and its time of occurrence (T max) differed between ingredients (P < 0.05), with values ranging from 6.1 to 11.9 h, from 0.459 to 1.300 mmol/g DM incubated per hour and from 5.1 to 9.8 h, respectively. Overall, a similar trend was observed considering individual SCFA productions. In particular, T 1/2 ranged from 6.4 to 12.5 h, from 5.5 to 12.5 h and from 6.7 to 11.3 h for acetate, propionate and butyrate, respectively (P < 0.05). For R max, differences were obtained for propionate and butyrate productions (P < 0.05), whereas no difference was recorded for acetate. In summary, our findings indicated that both quantitative and qualitative production of SCFA and related kinetics were influenced by fermentation of RS obtained from native starches characterized by heterogeneous starch characteristics. Current findings are based on an in vitro approach, and thus require further in vivo validations.

Use of principal component analysis to classify forages and predict their calculated energy content.

A set of 180 forages (47 alfalfa hays, 26 grass hays, 52 corn silages, 35 small grain silages and 20 sorghum silages) were randomly collected from different locations of the Po Valley (Northern Italy) from 2009 to 2010. The forages were characterised for chemical composition (11 parameters), NDF digestibility (five parameters) and net energy for lactation (NEL). The latter was calculated according to the two approaches adopted by the 2001 Nutrient Research Council and based on chemical parameters either alone (NEL3x-Lig) or in combination with 48 h NDF degradability in the rumen (NEL3x-48h). Thereafter, a principal component analysis (PCA) was used to define forage populations and limit the number of variables to those useful for obtaining a rapid forage quality evaluation on the basis of the calculated NEL content of forages. The PCA identified three forage populations: corn silage, alfalfa hay and a generic population of so-called 'grasses', consisting of grass hays, small grain and sorghum silages. This differentiation was also confirmed by a cluster analysis. The first three principal components (PC) together explained 79.9% of the total variation. PC1 was mainly associated with protein fractions, ether extract and lignin, PC2 with ash, starch, NDF and indigestible NDF (iNDF) and PC3 with NDF digestibility. Moreover, PC2 was highly correlated to both NEL3x-Lig (r = -0.84) and NEL3x-48h (r = -0.94). Subsequently, forage-based scores (FS) were calculated by multiplying the original standardised variables of ash, starch, NDF and iNDF with the scoring factors obtained from PCA (0.112, -0.141, 0.227 and 0.170, respectively). The FS showed a high determination coefficient for both NEL3x-Lig (R 2 = 0.86) and NEL3x-48h (R 2 = 0.73). These results indicate that PCA enables the distinction of different forage classes and appropriate prediction of the energy value on the basis of a reduced number of parameters. With respect to the rumen in situ parameters, iNDF was found to be more powerful at discriminating forage quality compared with NDF digestibility at different rumen incubation times or rates of NDF digestion.

Plasma glucose response and glycemic indices in pigs fed diets differing in in vitro hydrolysis indices.

Different dietary starch sources can have a great impact in determining starch digestion potential, thus influencing the postprandial blood glucose response. Our objectives were to define: (i) the incremental plasma glucose response in pigs fed diets containing various sources of starch differing in in vitro digestion patterns, (ii) the in vivo glycemic index (GI) values for the same diets, (iii) the possible relationship between in vitro and in vivo data. Diets, formulated with 70% of starch from five heterogeneous sources, were characterized in depth by using two distinct in vitro evaluations. The first one was based on the Englyst-assay for nutritional classification of starch fractions, whereas the second one was based on a time-course multi-enzymatic assay up to 180 min from which the hydrolysis indices (HIs) were calculated and used as a link between the physicochemical properties of starch from diets and the in vivo responses. For the in vivo study, five jugular-catheterized pigs (35.3 ± 1.1 kg body weight) were fed one of the five diets for 6-day periods in a 5 × 5 Latin square design. On day 5, blood was collected for 8 h postprandially for evaluating glucose appearance. On day 6, blood was collected for 3 h postprandially for the estimation of the GI. Starchy diets differed for rapidly digestible starch (from 8.6% to 79.8% of total starch (TS)) and resistant starch contents (from 72.5% to 4.5% of TS). Wide between-diets variations were recorded for all the kinetic parameters and for the HI calculated from the in vitro digestion curves (P < 0.05). On the basis of the obtained HI, diets contained starch with a very low to a very high in vitro digestion potential (ranging from 26.7% to 100.0%; P < 0.05). The glucose response differed among diets (P < 0.05), with marked differences between 15 and 120 min postprandial. Overall, the ranking of incremental glucose appearance among diets agreed with their in vitro HI classification: high HI diets increased plasma glucose response more (P < 0.05) than low HI diets. Lastly, different in vivo GIs were measured (ranging from 30.9% to 100.0%; P < 0.05). The relationship between HI and GI showed a high coefficient of determination (R2 = 0.95; root mean square error (RMSE) = 15.8; P < 0.05). In conclusion, diets formulated with starches with a wide range in HI potential can strongly affect the postprandial glucose response in pigs.

Melamine in eggs, plasma and tissues of hens fed contaminated diets.

A study was conducted to evaluate the excretion pattern of melamine from feed into eggs, plasma, kidney, liver and muscle of laying hens. In particular, 90 laying hens were randomly allocated to three dietary treatments and fed diets contaminated with melamine at a level of 2.5, 25 and 250 mg of melamine/kg of diet for T1, T2 and T3 groups, respectively. The diets were offered in six replicate boxes (five hens each) for 13 days. Eggs were collected from each group for melamine quantification on days 0, 1, 3, 6, 9 and 13. At the end of the experimental period, one hen per box was randomly selected and slaughtered to collect plasma, liver, kidney and muscle samples. During the experiment, feeding diets with increasing levels of melamine had no effect (P > 0.05) on weight gain, feed intake, egg production, egg weight and mortality of laying hens. The melamine in eggs increased from day 1 after melamine ingestion and reached a plateau between days 6 and 13 of melamine ingestion. At steady-state condition, the melamine egg concentrations increased (P < 0.01) with treatments, being 0.026, 0.352 and 4.631 mg/kg for T1, T2 and T3, respectively. Similarly, the carryover of melamine from feed to egg increased (P < 0.05) with the levels of melamine in the diets, varying from 0.50 to 0.70 and 0.84 for T1, T2 and T3, respectively. The melamine was detected in plasma of all tested groups, increasing (P < 0.01) with levels of melamine in the diets (0.030, 0.266 and 4.102 mg/l in T1, T2 and T3, respectively). Melamine was not detected in kidney, liver and muscle of hens fed T1. Except for kidney sampled in the T3, no melamine concentration higher than 2.5 mg/kg, representing the maximum allowable limit set by the US Food and Drug Administration and European Union for food and feeds, was measured. The melamine resulted higher in plasma and kidneys than in the liver and muscle both in T2 and T3. The results confirmed the presence of an excretion pattern of melamine from feed to eggs and tissues in laying hens.

Technical note: quantification of zeins from corn, high-moisture corn, and corn silage using a turbidimetric method: comparative efficiencies of isopropyl and tert-butyl alcohols.

Zeins are corn endosperm storage proteins that encapsulate starch granules into a protein matrix, which can act as a barrier to starch accessibility and digestion. Laboratory methods to quantify zein are seldom used because they are considered arduous and time-consuming. A recently published rapid turbidimetric method (mTM) was reinvestigated by changing the solution originally used for the zein solubilization step. In particular, the aim was to explore whether, and to what extent, the use of tert-butyl alcohol (t-BuOH-mTM) in lieu of isopropyl alcohol (i-PrOH-mTM) was able to improve the quantification of zeins from dry corn, high-moisture corn, and corn silage samples. The nature of the alcohol influenced the zein extraction values, and t-BuOH-mTM gave higher zein values in corn (3.6 vs. 3.3 g/100 g of dry matter) and corn silage samples (1.2 vs. 0.9 g/100 g of dry matter) compared with i-PrOH-mTM. In contrast, similar zein extraction values were obtained for high-moisture corn (2.1 vs. 1.9 g/100 g of dry matter, respectively). Sodium dodecyl sulfate-PAGE analysis revealed no contamination by nonzein proteins with the use of tert-butyl alcohol. Overall, these findings indicated that tert-butyl alcohol has a greater ability to solubilize zein compared with isopropyl alcohol and thus the t-BuOH-mTM allowed greater extraction of zeins. Considering the growing interest of animal nutritionists in zein proteins, such results should provide useful information for routine laboratory analysis.

Transfer of melamine from feed to milk and from milk to cheese and whey in lactating dairy cows fed single oral doses.

A study was conducted to evaluate the excretion pattern, after a single oral dose, of melamine from feed into milk, and the subsequent transfer to cheese and whey. The transfer of cyanuric acid was also investigated. Twenty-four lactating Holstein cows were randomly allocated to 4 treatments and received single doses of melamine as follows: 0.05, 0.50, 5.00, and 50.00 g/cow for groups D1, D2, D3, and D4, respectively. Individual milk samples were collected for melamine and cyanuric acid analyses on d 1, 2, 3, 4, 5, and 7. Milk collected individually from the second milking after melamine ingestion was used to make cheese on a laboratory scale. Melamine and cyanuric acid were extracted using a solid-phase extraction cartridge, and analyses were carried out by liquid chromatography-mass spectrometry/mass spectrometry. Maximal melamine concentrations occurred between 6 and 18 h after treatment and increased with log dose (linear and quadratic), ranging from 0.019 to 35.105 mg/kg. More than 60% of the melamine that was transferred to the milk was observed within 30 h after melamine ingestion. Melamine was not detected (limit of detection was 0.002 mg/kg) in milk 5 d after treatment in group D1, and 7 d after treatment in groups D2, D3, and D4. Blood urea nitrogen was not influenced by melamine ingestion. During cheese making, melamine was transferred mainly to the whey fraction. Cyanuric acid was not detected in any of the samples (milk, cheese, or whey). The excretion pattern of melamine in milk and whey may represent a health concern when cows ingest more than 0.50 g of melamine/d. However, only at intake levels of 5 and 50 g/d did cheese exceed the limits as set forth by the European Union. The results confirmed that melamine contamination of milk and milk products may be related not only to direct contamination, but also to adulteration of animal feeds.

Technical note: precision and accuracy of in vitro digestion of neutral detergent fiber and predicted net energy of lactation content of fibrous feeds.

The objective of this study was to test the precision and agreement with in situ data (accuracy) of neutral detergent fiber degradability (NDFD) obtained with the rotating jar in vitro system (Daisy(II) incubator, Ankom Technology, Fairport, NY). Moreover, the precision of the chemical assays requested by the National Research Council (2001) for feed energy calculations and the estimated net energy of lactation contents were evaluated. Precision was measured as standard deviation (SD) of reproducibility (S(R)) and repeatability (S(r)) (between- and within-laboratory variability, respectively), which were expressed as coefficients of variation (SD/mean × 100, S(R) and S(r), respectively). Ten fibrous feed samples (alfalfa dehydrated, alfalfa hay, corn cob, corn silage, distillers grains, meadow hay, ryegrass hay, soy hulls, wheat bran, and wheat straw) were analyzed by 5 laboratories. Analyses of dry matter (DM), ash, crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) had satisfactory S(r), from 0.4 to 2.9%, and S(R), from 0.7 to 6.2%, with the exception of ether extract (EE) and CP bound to NDF or ADF. Extending the fermentation time from 30 to 48 h increased the NDFD values (from 42 to 54% on average across all tested feeds) and improved the NDFD precision, in terms of both S(r) (12 and 7% for 30 and 48 h, respectively) and S(R) (17 and 10% for 30 and 48 h, respectively). The net energy for lactation (NE(L)) predicted from 48-h incubation NDFD data approximated well the tabulated National Research Council (2001) values for several feeds, and the improvement in NDFD precision given by longer incubations (48 vs. 30 h) also improved precision of the NE(L) estimates from 11 to 8%. Data obtained from the rotating jar in vitro technique compared well with in situ data. In conclusion, the adoption of a 48-h period of incubation improves repeatability and reproducibility of NDFD and accuracy and reproducibility of the associated calculated NE(L). Because the in vitro rotating jar technique is a simple apparatus, further improvement would probably be obtained by reducing the laboratory differences in rumen collection procedures and type of animal donors, which, however, reflect practical conditions.