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ABSTRACT: In modern breeding systems, cows are subjected to many stress factors. Animals fed a high-grain diet may have a decreased rumen pH, which would lead to subacute ruminal acidosis syndrome. The aim of this study was to investigate the evolution of microbial community composition in cows undergoing a dietary stress challenge. Twelve cows were subjected to a challenge period that consisted of a rapid change of ration, from a normal (45.4:54.6 forage:concentrate) to a high-grain content diet (24.8:75.2 forage:concentrate) to induce subacute ruminal acidosis. Individual rumen fluid content samples were collected before (T0) and during the challenge (T3, T14, T28). The DNA from rumen contents was extracted, purified, and sequenced to evaluate bacterial populations, and sequencing was performed on Illumina MiSeq. The effect of animal conditions on rumen microbial community was quantified through a linear mixed model. The acidogenic diet created 2 main clusters: ruminal hypomotility (RH) and milk fat depression (MFD). The microbial composition did not differ in T0 between the 2 groups, whereas during the challenge Ruminococcus spp., Treponema spp., Methanobrevibacter spp., and Methanosphaera spp. concentrations increased in RH cows; and Succinivibrio spp. and Butyrivibrio spp. concentrations increased in MFD cows. Prevotella spp. and Ruminococcus spp. were negatively correlated, whereas the Christenellaceae family was positively correlated with both Methanobrevibacter spp. and Methanosphaera spp. Moreover, the same diet affected cows' microbiota composition differently, underlying the impact of the host effect. Other studies are necessary to deepen the relationship between microbiota composition and host.

The aim of this study was to assess if molasses could modify VFA production and the rumen microbial community in vitro. Three beet (treatment Beet) and three cane (treatment Cane) molasses preparations were randomly selected from a variety of samples collected worldwide and incubated in vitro with rumen fluid along with a control sample (treatment CTR, in which no molasses was used). Flasks for VFA analysis were sampled at 0, 1, 2, 3, 4, 6, 8, and 24 h of each incubation. For microbiota analysis, samples from each fermentation flask after 12 and 24 h were subjected to microbial DNA extraction and V3–V4 16S rRNA gene sequencing on an Illumina MiSeq platform. Total net VFA production was higher in the beet and cane preparations than in the control (CTR) group at 24 h (33 mmol/L, 34 mmol/L, and 24.8 mmol/L, respectively), and the composition of VFAs was affected by the inclusion of molasses: acetic acid increased in the CTR group (73.5 mol%), while propionic acid increased in the beet and cane molasses (19.6 mol% and 18.6 mol%, respectively), and butyric acid increased, especially in the cane group (23.2 mol%). Molasses even influenced the composition of the rumen microbiota, and particularly the relative abundance of the most dominant family in the rumen, Prevotellaceae, which decreased compared to CTR (37.13%, 28.88%, and 49.6%, respectively). In contrast, Streptococcaceae (19.62% and 28.10% in molasses compared to 6.23% in CTR), Veillonellaceae (6.48% and 8.67% in molasses compared to 4.54% in CTR), and Fibrobacteraceae (0.90% and 0.88% in molasses compared to 0.62% in CTR) increased in the beet and cane groups compared to the CTR group. Another important finding is the lower proportion of Methanobacteriaceae following the addition of molasses compared to CTR (0.26%, 0.28%, and 0.43%, respectively). This study showed the impact of molasses in influencing VFA production and composition as a result of a modified rumen microbial composition.

Cows experience many physiological, nutritional and social changes during transition period. This study aims to evaluate the differences in nutrients total-tract digestibility (nTTD) between primiparous (PP) and multiparous (MP) cows through the transition period. From -23, -5, 0, 7, 14, and 30 days from calving a sample of feed and feces samples were collected from 25 Holstein cows (11 PP and 14 MP) as well as daily rumination time through accelerometers. The results show that average nTTD were different ( p  ≤ 0.02) for amylase-treated neutral detergent fiber organic matter (aNDFom) and potentially digestible neutral detergent fiber (pdNDF 240 ; 52.5 vs. 54.0 and 78.8 vs. 81.3, respectively in PP vs. MP), while no differences were found regarding pdNDF 24  and starch (88.5 vs. 88.6 and 95.1 vs. 96.1, respectively in PP vs. MP). Total-tract starch digestibility (TTstarchD) was different among timepoints ( p <  0.01), going from an average of 91.40 up to 97.39% of starch, on times -23 and 14, respectively. Differences in total-tract digestibility of aNDFom among timepoints ( p <  0.01) was expected because of differences in diet composition among lactating and non-lactating cows. No differences in daily rumination time ( p  = 0.92), TTstarchD and total-tract potentially digestible NDF digestibility at 24 h (TTpdNDF 24 D) were recorded. Our findings show that fiber digestibility during the transition period is higher in MP probably for a different ruminal retention time. These differences should be considered when formulating rations for groups with different parity number., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Beet and cane molasses are produced worldwide as a by-product of sugar extraction and are widely used in animal nutrition. Due to their composition, they are fed to ruminants as an energy source. However, molasses has not been properly characterized in the literature; its description has been limited to the type (sugarcane or beet) or to the amount of dry matter (DM), total or water-soluble sugars, crude protein, and ash. Our objective was to better characterize the composition of cane and beet molasses, examine possible differences, and obtain a proper definition of such feeds. For this purpose, 16 cane and 16 beet molasses samples were sourced worldwide and analyzed for chemical composition. The chemical analysis used in this trial characterized 97.4 and 98.3% of the compounds in the DM of cane and beet molasses, respectively. Cane molasses contained less DM compared with beet molasses (76.8 ± 1.02 vs. 78.3 ± 1.61%) as well as crude protein content (6.7 ± 1.8 vs. 13.5 ± 1.4% of DM), with a minimum value of 2.2% of DM in cane molasses and a maximum of 15.6% of DM in beet molasses. The amount of sucrose differed between beet and cane molasses (60.9 ± 4.4 vs. 48.8 ± 6.4% of DM), but variability was high even within cane molasses (39.2-67.3% of DM) and beet molasses. Glucose and fructose were detected in cane molasses (5.3 ± 2.7 and 8.1 ± 2.8% of DM, respectively), showing high variability. Organic acid composition differed as well. Lactic acid was more concentrated in cane molasses than in beet molasses (6.1 ± 2.8 vs. 4.5 ± 1.8% of DM), varying from 1.6 to 12.8% of DM in cane molasses. Dietary cation-anion difference showed numerical differences among cane and beet molasses (7 ± 53 vs. 66 ± 45 mEq/100 g of DM, on average). It varied from -76 to +155 mEq/100 g of DM in the cane group and from +0 to +162 mEq/100 g of DM in the beet group. Data obtained in this study detailed differences in composition between sources of molasses and suggested that a more complete characterization could improve the use of molasses in ration formulation., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 (R 2 ) 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 (R 2 = 0.92, 1 - VR = 0.87, SEC = 1.48, SECV = 1.89, RPD = 2.80, and RER = 20.19), uNDF240 (R 2 = 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 (R 2 = 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., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 (uNDF 240 ). 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 uNDF 240 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 uNDF 240 , 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., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In vitro methods requiring ruminal microorganisms to ferment and digest feeds have been used for decades. Though commonly accepted, collecting and pooling rumen fluid from different donor animals to avoid individual characteristics could affect in vitro fermentations. The current study evaluated the effects of individual or pooled liquors on in vitro digestibility of amylase- and sodium sulfite-treated NDF with ash correction (aNDFom). The study was conducted on 24 samples (8 alfalfa hays, 8 grass hays, and 8 corn silages). The 3 donor animals (treatment 1, 2, and 3) were selected based on similar body weights, parity, days in milk, milk production, and milk composition. Samples were digested in vitro via inoculation of different rumen fluid at different time points (12, 24, 72, and 120h). An equal amount of each liquor collected was sampled and equally mixed with the others to obtain treatment 4. For the alfalfa hay group, differences were observed at 12 (29.95, 27.07, 29.02, and 32.55% aNDFom for treatments 1, 2, 3, and 4, respectively) and 24h (37.35, 35.54, 36.44, and 40.56% aNDFom for treatments 1, 2, 3, and 4, respectively). The inoculum source did not affect in vitro digestibility over longer time periods (72 and 120 h). Similar results were observed in the grass hay group, in which the mixed inoculum had greater digestibility values at both 12 (28.86, 26.89, 27.88, and 30.92% aNDFom for treatments 1, 2, 3, and 4, respectively) and 24h (37.35, 35.54, 36.44, and 40.56% aNDFom for treatment 1, 2, 3, and 4, respectively), but not over longer time periods. For the corn silage group, we observed differences for treatment 4 only at 12h (35.78, 33.87, 34.83, and 37.80% aNDFom for treatment 1, 2, 3, and 4, respectively). These results underline the differences among donor animals, especially when evaluating short incubation time points, and that pooling rumen contents is not equal to averaging across individual animals. Reported data require a deeper investigation on whether or not the method of inoculating a pool of rumen contents represents the actual ability of the animal to digest fiber., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)