A. Palmonari

pH dynamics and bacterial community composition in the rumen of lactating dairy cows

The influence of pH dynamics on ruminal bacterial community composition was studied in 8 ruminally cannulated Holstein cows fitted with indwelling electrodes that recorded pH at 10-min intervals over a 54-h period. Cows were fed a silage-based total mixed ration supplemented with monensin. Ruminal samples were collected each day just before feeding and at 3 and 6h after feeding. Solid and liquid phases were separated at collection, and extracted DNA was subjected to PCR amplification followed by automated ribosomal intergenic spacer analysis (ARISA). Although cows displayed widely different pH profiles (mean pH=6.11 to 6.51, diurnal pH range=0.45 to 1.39), correspondence analysis of the ARISA profiles revealed that 6 of the 8 cows showed very similar bacterial community compositions. The 2 cows having substantially different community compositions had intermediate mean pH values (6.30 and 6.33) and intermediate diurnal pH ranges (averaging 0.89 and 0.81 pH units). Fortuitously, these 2 cows alone also displayed milk fat depression, along with markedly higher ruminal populations of 1 bacterial operational taxonomic unit (OTU) and reduced populations of another ARISA amplicon. Cloning and sequencing of the elevated OTU revealed phylogenetic similarity to Megasphaera elsdenii, a species reportedly associated with milk fat depression. The higher populations of both M. elsdenii and OTU246 in these 2 cows were confirmed using quantitative real-time PCR (qPCR) with species-specific primers, and the fraction of total bacterial rDNA copies contributed by these 2 taxa were very highly correlated within individual cows. By contrast, the fraction of total bacterial rDNA copies contributed by Streptococcus bovis and genus Ruminococcus, 2 taxa expected to respond to ruminal pH, did not differ among cows (mean= <0.01 and 10.6%, respectively, of rRNA gene copies, determined by qPCR). The data indicate that cows with widely differing pH profiles can have similar ruminal bacterial community compositions, and that milk fat depression can occur at intermediate ruminal pH. The results support recent reports that milk fat depression is associated with shifts in bacterial community composition in rumine and is specifically related to the relative abundance of Megasphaera elsdenii.

Influence of maturity on alfalfa hay nutritional fractions and indigestible fiber content

This study focused on changes in fibrous and protein fractions, changes in fiber digestibility and amount of indigestible neutral detergent fiber (NDF) as a consequence of increased maturity in alfalfa. A total area of 720 m(2) was divided in 18 blocks randomly assigned to 3 treatments, differing in cutting intervals. Treatment 1 was harvested with a 21-d cutting schedule, at a prebloom stage; treatment 2 with a 28-d schedule, at about first-bloom stage; whereas a full bloom was observed in treatment 3, harvested with a 35-d cutting schedule. Treatments were replicated 4 times through the spring-summer period for 2 subsequent years, 2011 and 2012. Statistical differences were observed for crude protein [treatment 1: 20.8%, treatment 2: 17.3%, and treatment 3: 17.0%; standard error of the mean (SEM)=0.83], soluble protein, and nonprotein nitrogen among treatments on a dry matter basis. Similar results were observed for acid detergent lignin (6.3, 6.9, and 7.3%, respectively; SEM=0.39), lower in treatment 1 compared with others, and in vitro NDF digestibility at 24 or 240 h. Indigestible NDF at 240 h resulted in lower values for treatment 1 compared with treatments 2 and 3 (15.5, 17.2, and 18.3%, respectively; SEM=1.54). Moreover, the indigestible NDF:acid detergent lignin ratio varied numerically but not statistically among treatments, being as much as 9% greater than the 2.4 fixed value applied for rate of digestion calculation and Cornell Net Carbohydrate Protein System (Cornell University, Ithaca, NY)-based model equations. Assuming the diet composition remained unchanged, treatment 3 (35-d cutting interval) would be expected to yield 1.4 kg less milk per day based on energy supply, and 2.8 kg less milk daily based on protein supply than treatment 1.

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.

Genome-Wide Association Mapping and Genomic Selection for Alfalfa ( Medicago sativa ) Forage Quality Traits

Genetic progress for forage quality has been poor in alfalfa (Medicago sativa L.), the most-grown forage legume worldwide. This study aimed at exploring opportunities for marker-assisted selection (MAS) and genomic selection of forage quality traits based on breeding values of parent plants. Some 154 genotypes from a broadly-based reference population were genotyped by genotyping-by-sequencing (GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein, neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib progenies in three growing conditions (summer harvest, full irrigation; summer harvest, suspended irrigation; autumn harvest). Trait-marker analyses were performed on progeny values averaged over conditions, owing to modest germplasm × condition interaction. Genomic selection exploited 11,450 polymorphic SNP markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a genome-wide association study (GWAS). GWAS confirmed the polygenic control of quality traits and, in agreement with phenotypic correlations, indicated substantially different genetic control of a given trait in stems and leaves. It detected several SNPs in different annotated genes that were highly linked to stem protein content. Also, it identified a small genomic region on chromosome 8 with high concentration of annotated genes associated with leaf ADL, including one gene probably involved in the lignin pathway. Three genomic selection models, i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were moderate (0.3–0.4) for stem NDFD and leaf protein content, modest for leaf ADL and NDFD, and low to very low for the other traits. Along with previous results for the same germplasm set, this study indicates that GBS data can be exploited to improve both quality traits (by genomic selection or MAS) and forage yield.

Effect of undigested neutral detergent fiber content of alfalfa hay on lactating dairy cows: Feeding behavior, fiber digestibility, and lactation performance

The objective of this study was to investigate the effects of 2 alfalfa hays differing in undigested neutral detergent fiber content and digestibility used as the main forage source in diets fed to high producing cows for Parmigiano-Reggiano cheese production. Diets were designed to have 2 different amounts of undigestible NDF [high (Hu) and low (Lu)], as determined by 240-h in vitro analysis (uNDF240). Alfalfa hay in vitro digestibility [% of amylase- and sodium sulfite-treated NDF with ash correction (aNDFom)] at 24 and 240 h was 40.2 and 31.2% and 53.6 and 45.7% for low- (LD) and high-digestibility (HD) hays, respectively. The 4 experimental diets (Hu-HD, Lu-HD, Hu-LD, and Lu-LD) contained 46.8, 36.8, 38.8, and 30.1% of alfalfa hay, respectively, 8.6% wheat straw, and 35.3% corn (50% flake and 50% meal; DM basis). Soy hulls and soybean meal were used to replace hay to balance protein and energy among diets. Eight multiparous Holstein cows (average milk production = 46.0 ± 5.2 kg/d, 101 ± 38 d in milk, and 662 ± 42 kg of average body weight) were assigned to a 4 × 4 Latin square design, with 2 wk of adaptation and a 1-wk collection period. Dry matter and water intake, rumination time, ruminal pH, and milk production and composition were measured. Diets and feces were analyzed for NDF on an organic matter basis (aNDFom), acid detergent fiber, acid detergent lignin, and uNDF240 to estimate total-tract fiber digestibility. Dry matter intake and rumination times were higher in HD diets compared with LD diets, regardless of forage amount. Rumination time was constant per unit of dry matter intake but differed when expressed as a function of uNDF240, aNDFom, or physically effective NDF intake. No differences were found among treatments on average ruminal pH, but the amount of time with pH <5.8 was lower in Hu-HD diets. Milk production and components were not different among diets. Total-tract aNDFom and potentially digestible neutral detergent fiber fraction digestibility was higher for the LD diets (88.3 versus 85.8% aNDFom in HD), for which lower feed intakes were also observed. The Hu-HD diet allowed greater dry matter intake, longer rumination time, and higher ruminal pH, suggesting that the limiting factor for dry matter intake is neutral detergent fiber digestibility and its relative rumen retention time.

Response on yield and nutritive value of two commercial maize hybrids as a consequence of a water irrigation reduction

The present study investigated in a practical farm condition the response of two commercial maize hybrids (maturity class FAO rating 700) grown for silage production on chemical composition and digestibility of whole maize plant as consequence of a severe water irrigation reduction. Based on different irrigation applications, water restricted (WR) and fully irrigated (FI) plots received 50 and 200 mm of irrigation water, respectively. A split-plot factorial arrangement in a randomised complete block design with two main plots (WR and FI) and two sub-plots (hybrid A and B) with 12 replications/treatment was performed. Studied parameters were dry matter (DM) yield, harvest index (HI), chemical composition, rumen in situ DM and neutral detergent fibre disappearance (DMD and NDFD, respectively), indigestible NDF (iNDF), 7h in vitro starch degradability (7hIVSD) and net energy (NE) for lactation content. Total DM and grain yields, HI and chemical composition differed (P<0.05) between FI and WR crops and only slight differences were recorded between hybrids. When compared to FI plants, WR had lower starch and higher fibre contents (P<0.05). Higher DMD (59.2 vs 56.4% DM) and NDFD (61.0 vs 58.4% NDFOM) were measured for FI with respect to WR crops, whereas iNDF was about 36% higher (P<0.05) in WR than FI. Lastly, WR plants had a lower NE content than FI plants (P<0.05). Our research showed that a drastic reduction in water irrigation negatively affected whole plant yield, chemical composition and nutrient availability of forage maize.

Effects of a completely pelleted diet on growth performance in Holstein heifers

Forage neutral detergent fiber (NDF) content and particle size are important factors that affect rumen function. The aim of the current study was to evaluate the effects on rumen health, NDF digestibility, and animal performance of pelleting a forage-based diet. Eight Holstein heifers (age 336±30d, body weight 346±35kg) were randomly assigned to a repeated crossover design. Animals were housed in tie-stalls and fed for ad libitum intake. The study included 4 periods of 3 wk, the first 2 wk for adaptation to the diet and the last wk for data collection. Diets had the same ingredients but had a different physical form: total mixed ration (TMR) and pellet (diameter=8mm). The physically effective NDF (peNDF) differed between the 2 treatments (39.8 and 11.8% of NDF in the TMR and pellet diets, respectively). During the trial, dry matter intake (DMI), water intake, rumination time, rumen temperature, and pH were evaluated daily. Fecal samples were collected in wk3 of each period to determine total-tract digestibility of the potential digestible (pd)NDF. Average daily gain and feed conversion ratio were calculated at the end of each period. With the pellet diet, DMI, DMI/body weight, and water consumption were higher. We observed no significant difference in average daily gain or feed conversion ratio. Rumination time was lower for the pellet diet than for the TMR diet (241 vs. 507min/d, respectively). Diet had no effect on rumen temperature or rumen pH. The total-tract digestibility of the pdNDF was greater with the TMR diet than with the pellet diet (90.25 vs. 86.82% pdNDF, respectively). The results of the current study suggest that a complete-feed pellet diet was well accepted by the animals, as demonstrated by higher DMI. Rumination time was reduced with the pellet diet, but rumen pH was not different. The pdNDF digestibility was high for both diets, but significantly higher for the TMR diet. Given that animal performance was similar between the 2 diets, although they differed with respect to DMI and fiber digestion, we hypothesize that the 2 diets had different retention times, related to their physical form. A complete-feed pellet diet formulated to provide a sufficient level of NDF from forages could be fed to growing ruminants without apparent negative effects on rumen health and animal productivity, at least for a short period. More research over a longer growing period is needed before recommending this feeding strategy for growing heifers.

Technical note: In vitro digestibility of amylase-treated, ash-corrected neutral detergent fiber, with addition of sodium sulfite, at 240 hours with or without rumen fluid reinoculation

Long-term in vitro fermentation (240 h) evaluating amylase-treated, ash-corrected neutral detergent fiber, with addition of sodium sulfite (aNDFom) digestibility is required to quantify the indigestible fiber fraction. It is commonly accepted to inoculate rumen fluid more than one time during such fermentations, every 96 h or at 120 h. However, no studies have been conducted to verify if the reinoculation is actually required to properly carry out the fermentation process. The current study aims to evaluate the effects of these procedures on aNDFom digestibility at 240 h. The study was conducted on a total of 24 forage samples (8 alfalfa hays, 8 grass hays, and 8 corn silages). Samples were digested in triplicate at 240 h in vitro. Rumen fluid was added twice (at 96 and 192 h) in treatment 1, after 120 h in treatment 2, whereas no addition was made in treatment 3. At the end of the fermentations, residual aNDFom was quantified to calculate digestibility. Among treatments, no difference was found in digestibility of aNDFom. Moreover, treatment 1 resulted in higher variability compared with other treatments. Results obtained in the current study show that subsequent addition of rumen fluid is not necessary for a proper estimation of aNDFom digestibility, and can be avoided.

Chewing and ruminating with various forage qualities in nonlactating dairy cows

This study investigated Italian typical dry forage diets used in the Parmigiano Reggiano PDO (protected designation of origin) area of Italy. The first cutting is typically a mixture of alfalfa and native wild grasses, resulting in a unique forage nutrient content. Alfalfa makes up less than 10% in the first cut because herbicides are not commonly used. Wild grasses are predominant in such conditions, whereas in the subsequent cuts alfalfa content increases as grasses decrease because of agronomical and climatic characteristics. Six multiparous, nonlactating Holstein cows were used in a replicated 3 × 3 Latin square to evaluate 3 different cuttings of alfalfa hay fed as the sole diet source. Eating and ruminating behavior were studied to investigate forage properties related to chewing activity. No differences were found in eating time; however, ruminating time per kilogram of physically effective NDF was greater when cows were fed firstcutting alfalfa than when they were fed the second or fifth cutting, despite similar digestibility and diet particle size of first- and fifth-cutting forage. In vivo digestibility of the diets revealed higher fecal NDF, ADF, and DM digestibility for first- and fifth-cutting hay. A similar trend was observed with the results of the in vitro data, with reduced digestibility for second-cutting forage.

Technical note: Near infrared reflectance spectroscopy to predict fecal indigestible neutral detergent fiber for dairy cows

In vitro and in situ procedures performed to estimate indigestible neutral detergent fiber (iNDF) in forage or fecal samples are time consuming, costly, and limited by intrinsic factors. In contrast, near infrared reflectance spectroscopy (NIRS) has become widely recognized as a valuable tool for accurately determining chemical composition and digestibility parameters of forages. The aim of this study was to build NIRS calibrations and equations for fecal iNDF. In total, 1,281 fecal samples were collected to build a calibration data set, but only 301 were used to develop equations. Once dried, samples were ground and chemically analyzed for crude protein, ash, amylase and sodium sulfite-treated NDF corrected for ash residue (aNDFom), acid detergent fiber, acid detergent lignin, and in vitro digestion at 240 h to estimate iNDF (uNDF240). Each fecal sample was scanned using a NIRSystem 6500 instrument (Perstorp Analytical Inc., Silver Spring, MD). Spectra selection was performed, resulting in 301 sample spectra used to develop regression equations with good accuracy and low standard error of prediction. The standard error of calibration (SEC), cross validation (SECV), and coefficients of determination for calibration (R2) and for cross validation (1 - VR, where VR = variance ratio) were used to evaluate calibration and validation results. Moreover, the ratio performance deviation (RPD) and ratio of the range of the original data to SECV (range/SECV; range error ratio, RER) were also used to evaluate calibration and equation performance. Calibration data obtained on fiber fractions aNDFom (R2 = 0.92, 1 - VR = 0.87, SEC = 1.48, SECV = 1.89, RPD = 2.80, and RER = 20.19), uNDF240 (R2 = 0.92, 1 - VR = 0.86, SEC = 1.65, SECV = 2.24, RPD = 2.57, and RER = 14.30), and in vitro rumen aNDFom digestibility at 240 h (R2 = 0.90, 1 - VR = 0.85, SEC = 2.68, SECV = 3.43, RPD = 2.53, and RER = 14.0) indicated the predictive equations had good predictive value.