An evaluation of the effectiveness of sumac and molasses as additives for alfalfa silage: Influence on nutrient composition, in vitro degradability and fermentation quality.

This study investigated the effects of sumac and molasses on nutrient composition, in vitro degradability and fermentation quality of alfalfa silage. Alfalfa was ensiled in quadruplicate in vacuum jars untreated group (A) or after the following treatments: sumac group at 10% (AS), molasses group at 5% (AM), and sumac (10%) and molasses (5%) group (ASM). Silos (n = 64) were stored for 0, 21, 45 or 60 days. The results showed that dry matter (DM) contents of the AS, AM and ASM groups were statistically higher than the control group (p < 0.001). Only on the 21st day of fermentation the crude ash content of the AS group was found to be significantly higher than the other groups (p < 0.05). In vitro, DM and organic matter degradation values of the AMS group increased significantly (p < 0.001). A significant decrease in alfalfa silage's pH values was determined with sumac and molasses additives (p < 0.001). The ammonia nitrogen (NH3-N) values of the control, AS, AM and ASM groups at Day 60 were determined as 9.08%, 7.22%, 7.00% and 6.81% respectively (p < 0.05). The water-soluble carbohydrate (WSC) values of all groups on the 60th day were significantly decreased compared to the 0th day (p < 0.001). When the groups were evaluated within themselves, there was a statistically significant difference between the 0th and 60th day lactic acid values. The acetic acid content of the A group on the 60th day was found to be significantly higher than the other groups (p < 0.01). There was a significant decrease in propionic acid levels on Days 21, 45 and 60 compared to Day 0 of fermentation (p < 0.001). The highest butyric acid (BA) level was determined in the A group on the 21st, 45th and 60th days of fermentation (p < 0.05). In conclusion, sumac prevents proteolysis depending on its tannin content. It improves silage fermentation positively thanks to its organic acid content, while the molasses additive is effective in silage fermentation, mainly depending on the WSC level. However, it was determined that neither additive could reduce the silage pH to the appropriate value ranges due to the low doses, and they could not mainly prevent the formation of BA.

Metagenomic insights into the microbiota involved in lactate and butyrate production and manipulating their synthesis in alfalfa silage.

AIMS: Lactate and butyrate are important indicators of silage quality. However, the microorganisms and mechanisms responsible for lactate and butyrate production in silage are not well documented. METHODS AND RESULTS: whole-metagenomic sequencing was used to analyse metabolic pathways, microbiota composition, functional genes, and their contributions to lactate and butyrate production in alfalfa silage with (SA) and without (CK) sucrose addition. Carbon metabolism was the most abundant metabolic pathway. We identified 11 and 2 functional genes associated with lactate and butyrate metabolism, respectively. Among them, D-lactate dehydrogenase (ldhA) and L-lactate dehydrogenase (ldhB) were most important for the transition between D/L-lactate and pyruvate and were primarily related to Lactobacillus in the SA group. The genes encoding L-lactate dehydrogenase (lldD), which decomposes lactate, were the most abundant and primarily associated with Enterobacter cloacae. Butyrate-related genes, mainly encoding butyryl-CoA: acetate CoA-transferase (but), were predominantly associated with Klebsiella oxytoca and Escherichia coli in the CK group. CONCLUSIONS: Enterobacteriaceae and Lactobacillaceae were mainly responsible for butyrate and lactate formation, respectively.

Exploring the rumen microbiota of Hu lambs in response to diet with paper mulberry.

Paper mulberry (Broussonetia papyrifera), as a new woody forage with high-protein characteristic, is being widely used in ruminant feeding. However, little is known about the comprehensive microbiota picture of whole ruminal niches (liquid, solid, and epithelium) under paper mulberry diet. To gain a better understanding of feeding paper mulberry on the rumen microbiota, the effects of fresh paper mulberry, paper mulberry silage, or a conventional high-protein alfalfa silage on rumen fermentation products and microbiota in rumen niches of Hu lambs were studied. Forty-five Hu lambs were randomly divided into 3 treatments with 15 replicates in each treatment. No significant difference was observed among treatments in the average daily gain (ADG). The fresh paper mulberry treatment had lower (P < 0.05) pH and higher (P < 0.05) total volatile fatty acids (TVFA) compared with silage treatments, but the fermentation parameters did not show significant differences between paper mulberry silage and alfalfa silage treatments. The Shannon index did not show a significant difference (P < 0.05) among treatments except between fresh paper mulberry and alfalfa silage treatment in rumen epithelial niches. Butyrivibrio and Treponema were the predominant genera in the rumen epithelial fraction, while Prevotella and Rikenellaceae_RC9 dominated in both rumen liquid and solid fractions. These results indicated the paper mulberry supplement did not have distinct impact on the microbial diversity and growth performance compared with alfalfa silage, especially for paper mulberry silage, which might help us develop an alternative animal feeding strategy of replacing alfalfa with paper mulberry. KEY POINTS: • Feeding paper mulberry silage did not show significant impact on the growth performance compared with alfalfa silage treatment. • Feeding fresh paper mulberry reduced rumen pH value and increased total volatile fatty acid. • The microbial diversity did not show significant difference among treatments.

Dry matter content and inoculant alter the metabolome and bacterial community of alfalfa ensiled at high temperature.

Alfalfa silage fermentation quality, metabolome, bacterial interactions, and successions as well as their predicted metabolic pathways were explored under different dry matter contents (DM) and lactic acid bacteria (LAB) inoculations. Silages were prepared from alfalfa with DM contents of 304 (LDM) and 433 (HDM) g/kg fresh weight and inoculated with Lactiplantibacillus plantarum (L. plantarum, LP), Pediococcus pentosaceus (P. pentosaceus, PP), or sterile water (control). The silages were stored at a simulated hot climate condition (35°C) and sampled at 0, 7, 14, 30, and 60 days of fermentation. The results revealed that HDM significantly improved the alfalfa silage quality and altered microbial community composition. The GC-TOF-MS analysis discovered 200 metabolites in both LDM and HDM alfalfa silage, mainly consisting of amino acids, carbohydrates, fatty acids, and alcohols. Compared with LP and control, PP-inoculated silages had increased concentrations of lactic acid (P < 0.05) and essential amino acids (threonine and tryptophan) as well as decreased pH, putrescine content, and amino acid metabolism. However, alfalfa silage inoculated with LP had higher proteolytic activities than control and PP-inoculated silage, as revealed by a higher concentration of ammonia nitrogen (NH3-N), and also upregulated amino acid and energy metabolism. HDM content and P. pentosaceus inoculation significantly altered the composition of alfalfa silage microbiota from 7 to 60 days of ensiling. Conclusively, these results indicated that inoculation with PP exhibited great potential in enhancing the fermentation of silage with LDM and HDM via altering the microbiome and metabolome of the ensiled alfalfa, which could help in understanding and improving the ensiling practices under hot climate conditions. KEY POINTS: • HDM improved fermentation quality and declined putrescine content of alfalfa silage • P. pentosaceus inoculation enhanced the fermentation quality of alfalfa silage • P. pentosaceus is an ideal inoculant for alfalfa silage under high temperature.

Occurrence and fate of antibiotic-resistance genes and their potential hosts in high-moisture alfalfa silage treated with or without formic acid bactericide.

Silage as the main forage for ruminants could be a reservoir for antibiotic resistance genes (ARGs) through which these genes got access into the animals' system causing a latent health risk. This study employed metagenomics and investigated the ARGs' fate and transmission mechanism in high-moisture alfalfa silage treated with formic acid bactericide. The results showed that there were 22 ARGs types, in which multidrug, macrolide-lincosamide-streptogramine, bacitracin, beta-lactam, fosmidomycin, kasugamycin, and polymycin resistance genes were the most prevalent ARGs types in the ensiled alfalfa. The natural ensiling process increased ARGs enrichment. Intriguingly, after 5 days of ensiling, formic acid-treated silage reduced ARGs abundances by inhibiting host bacterial and plasmids. Although formic acid bactericide enhanced the fermentation characteristics of the high-moisture alfalfa by lowering silage pH, butyric acid concentration, dry matter losses and proteolysis, it increased ARGs abundances in alfalfa silage owing to increases in abundances of ARGs carriers and transposase after 90 days of ensiling. Notably, several pathogens like Staphylococcus, Clostridium, and Pseudomonas were inferred as potential ARGs hosts in high-moisture alfalfa silage, and high-moisture alfalfa silage may harbor a portion of the clinical ARGs. Fundamentally, microbes were distinguished as the foremost driving factor of ARGs propagation in ensiling microecosystem. In conclusion, although formic acid bactericide improved the fermentation characteristics of high-moisture alfalfa during ensiling and reduced ARGs enrichment at the initial ensiling stage, it increased ARGs enrichment at the end of ensiling.

A novel species of lactic acid bacteria, Ligilactobacillus pabuli sp. nov., isolated from alfalfa silage.

In this study, we isolated a novel strain of lactic acid bacteria, AF129T, from alfalfa silage prepared locally in Morioka, Iwate, Japan. Polyphasic taxonomy was used to characterize the bacterial strain. The bacterium was rod-shaped, Gram-stain-positive, non-spore-forming and catalase-negative. The strain grew at various temperatures (15-40°C) and pH levels (4.0-8.0). The optimum growth conditions were a temperature of 30°C and a pH of 6.0. AF129T exhibited growth at salt (NaCl) concentrations of up to 6.5 % (w/v). The G+C content of the strain's genomic DNA was 41.5 %. The major fatty acids were C16 : 0, C18 : 1ω9c, C19 : 0cyclo ω8c and summed feature 8. 16S rRNA gene sequencing revealed that AF129T represents a member of the genus Ligilactobacillus and it has higher sequence similarities with Ligilactobacillus pobuzihii (98.4 %), Ligilactobacillus acidipiscis (97.5 %) and Ligilactobacillus salitolerans (97.4 %). The digital DNA-DNA hybridization values for AF129T and phylogenetically related species of the genus Ligilactobacillus ranged from 19.8% to 24.1%. The average nucleotide identity of the strain with its closely related taxa was lower than the threshold (95 %-96 %) used for species differentiation. In the light of the above-mentioned physiological, genotypic, chemotaxonomic and phylogenetic evidence, we confirm that AF129T represents a member of the genus Ligilactobacillus and constitutes a novel species; we propose the name Ligilactobacillus pabuli sp. nov. for this species. The type strain is AF129T =MAFF 518002T =JCM 34518T=BCRC 81335T.

Metagenomic analysis reveals the linkages between bacteria and the functional enzymes responsible for potential ammonia and biogenic amine production in alfalfa silage.

AIMS: To clarify the molecular mechanisms underlying ammonia (NH3 ) and biogenic amines (BAEs) formation in alfalfa silage, whole metagenomic sequencing analysis was performed to identify the linkages between functional bacteria and their responsible enzymes in alfalfa silage prepared with and without sucrose addition. METHODS AND RESULTS: Genes encoding nitrite reductase (nirB) resulting in NH3 formation were the most abundant and were mostly assigned to Enterobacter cloacae and Klebsiella oxytoca. Putrescine-related genes, classified mainly to encode ornithine decarboxylase (odcA), were predominantly carried by Escherichia coli, Ent. cloacae and Citrobacter sp. Escherichia coli and Kl. oxytoca were the important species responsible for cadaverine and tyramine formation. Ent. cloacae, E. coli, and Kl. oxytoca dominated the bacterial community in naturally fermented alfalfa silage, whilst sucrose-treated silages greatly inhibited the growth of these species by promoting the dominance of Lactobacillus plantarum, thus decreasing the concentrations of NH3 , cadaverine, putrescine and tyramine. CONCLUSIONS: Enterobacteriaceae bacteria are mainly responsible for the NH3 , putrescine, cadaverine and tyramine formations in alfalfa silage. SIGNIFICANCE AND IMPACT OF THE STUDY: Whole metagenomic sequencing analysis served as a useful tool to identify the linkages between functional bacteria and associated enzymes responsible for NH3 and BAEs formation.

Evaluation of gallnut tannin and Lactobacillus plantarum as natural modifiers for alfalfa silage: Ensiling characteristics, in vitro ruminal methane production, fermentation profile and microbiota.

AIMS: The potential of gallnut tannin (GT) and Lactobacillus plantarum (LP) on fermentation characteristics, in vitro ruminal methane (CH4 ) production and microbiota of alfalfa silage was investigated. METHODS AND RESULTS: Alfalfa was ensiled with GT (20 and 50 g kg-1 dry matter [DM]) and LP (3 × 108  CFU per gram fresh matter) alone or in combination for 60 days. The GT and LP alone or in combination decreased DM losses, pH and non-protein nitrogen contents of alfalfa silage. All additive treatments decreased ruminal CH4 production, and increased propionic acid molar proportions and Fibrobacter succinogenes numbers. The LP treatment increased nutrient degradation, cellobiase, pectinase and protease activities, and Prevotella ruminicola abundance, whereas high-dose GT treatment inhibited these variables. Importantly, LP together with GT alleviated the adverse effects of high-dose GT supply alone by enhancing pectinase and protease activities as well as Rumincoccus flavefaciens and P. ruminicola growth. CONCLUSIONS: Combination of GT and LP can be used as an efficient additive to improve silage quality and utilization by ruminants. SIGNIFICANCE AND IMPACT OF THE STUDY: Using GT-LP combination has practical implications, particularly concerning effects of tannins on ruminal CH4 mitigation, which may alleviate inhibitory effects of tannins on feed digestion through modulating ruminal microbiota.

Screening a Lactobacillus plantarum strain for good adaption in alfalfa ensiling and demonstrating its improvement of alfalfa silage quality.

AIM: To screen a lactic acid bacteria (LAB) strain for good adaption in alfalfa ensiling and to evaluate its effects on alfalfa ensiling. METHODS AND RESULTS: Lactobacillus plantarum (LP) strain ZZU203 was selected due to its superior ability to acidify structural carbohydrate metabolite and alfalfa powder. Alfalfa was ensiled with LP FG1 (from a commercial inoculant), LP ZZU203, or a no-additive control (CK) for 60 d. The two silages with LP additives significantly (P < 0·05) increased lactic acid (LA) and acetic acid (AA) concentrations compared to CK silage. The pH, ammonia nitrogen (NH3 -N) and cellulose concentrations, and the numbers of bacilli, coliform, aerobic bacteria and Clostridium decreased significantly (P < 0·05). The dry matter, LA, AA and water-soluble carbohydrate concentrations of ZZU203 silage were significantly (P < 0·05) higher, whereas the NH3 -N and the neutral detergent fibre concentrations, and the aerobic bacterial and Clostridium counts were significantly (P < 0·05) lower than in FG1 silage. CONCLUSION: ZZU203 obtained by the screening method of this study can improve the quality of alfalfa silage. SIGNIFICANCE AND IMPACT OF THE STUDY: The screening method designed for alfalfa silage conditions can effectively screen out LAB with potential application value in alfalfa silage.

Feeding diets varying in forage proportion and particle length to lactating dairy cows: I. Effects on ruminal pH and fermentation, microbial protein synthesis, digestibility, and milk production.

Physically effective neutral detergent fiber (peNDF) content of dairy cow diets was modified by varying the theoretical chop length of alfalfa silage and forage:concentrate (F:C) ratio, and effects on nutrient intakes, ruminal fermentation, site and extent of digestion, microbial protein synthesis, and milk production were evaluated. Estimates of dietary peNDF contents were compared with recommendations, and predictions of ruminal pH from peNDF and the recently developed physically adjusted neutral detergent fiber (paNDF) system were compared with observed pH. The experiment was designed as a triple 4 × 4 Latin square using 12 mid-lactating dairy cows with 4 intact, 4 ruminally cannulated, and 4 ruminally and duodenally cannulated cows. Site and extent of digestion and microbial protein synthesis were measured in a single 4 × 4 Latin square. Treatments were a 2 × 2 factorial arrangement; 2 forage particle lengths (FPL) of alfalfa silage (short and long) were combined with low (35:65) and high (60:40) F:C ratios [dry matter (DM) basis]. The peNDF contents were determined by multiplying the proportion (DM basis) of total mixed ration retained on 2 (8 and 19 mm; peNDF8.0) or 3 (1.18, 8, and 19 mm; peNDF1.18) sieves of the Penn State Particle Separator by the neutral detergent fiber content of the diet. The dietary peNDF contents ranged from 10.7 to 17.5% for peNDF8.0 or from 23.1 to 28.2% for peNDF1.18. Interactions between F:C ratio and FPL content were few. Increasing peNDF content of diets by increasing F:C ratio decreased DM intake, milk yield, and milk protein yield, whereas apparent total-tract DM digestibility and milk efficiency improved. Increasing F:C ratio improved ruminal pH status but decreased total volatile fatty acid concentration and microbial protein synthesis. Increasing peNDF content of diets via dietary FPL increased mean ruminal pH, but did not affect DM intake, total-tract digestibility, or milk production. The results indicate that feeding dairy cows a low F:C diet helps increase DM intake, milk production, and microbial protein synthesis, but may adversely affect feed digestibility and milk efficiency due to increased risk of subacute ruminal acidosis. Increased FPL improved ruminal pH status, but had minimal effects on feed intake, ruminal fermentation, nutrient digestibility, and milk production. The results indicate a trade-off between reducing the risk of subacute ruminal acidosis and maximizing ruminal fermentation, feed digestibility, and milk production of dairy cows. The paNDF model showed improvement in the predictability of ruminal pH over the peNDF model, but the accuracy of predictions varied depending upon the diet and ruminal fermentation variables considered in the equations.

Effects of lactic acid bacteria inoculation in pre-harvesting period on fermentation and feed quality properties of alfalfa silage.

OBJECTIVE: To develop the fermentation quality and chemical composition of alfalfa (Medicago sativa Lam.) silage, plants were inoculated with different lactic acid bacteria (LAB) strains at field 24 hours before harvest. METHODS: The treatment groups were as follow: silage without additive as a control and inoculated with each strains of Lactobacillus brevis (LS-55-2-2), Leuconostoc citerum (L-70-6-1), Lactobacillus bifermentans (LS-65-2-1), Lactobacillus plantarum (LS-3-3) and Lactobacillus plantarum (LS-72-2). All the silages were stored at 25 ºC. Parameters such as pH, microorganism and volatile fatty acid contents, crude protein, neutral detergent fiber, acid detergent fiber, net gas, metabolizable energy, organic matter digestibility, dry matter intake and relative feed value were measured to determine fermentation quality, chemical compositions and relative feed value of alfalfa silages. RESULTS: Significant differences were found among the control and treated groups in terms of pH and microorganism contents at all opening times and crude protein, net gas, metabolizable energy and organic matter digestibility of final silage. The pH values ranged from 4.70 to 5.52 for all treatments and control silage had the highest value of overall treatments at T75d silages. Volatile fatty acid of silages was not influenced significantly by inoculations. However, lactic acid content of L. bifermentans (LS-65-2-1) was higher than the other treatments. The highest metabolizable energy and organic matter digestibility were recorded from L. citerum (L-70-6-1) inoculation. In addition, no significant differences were found among treatments in terms of neutral detergent fiber, acid detergent fiber, dry matter intake and relative feed value. CONCLUSION: Among the treated lactic acid bacteria isolates, Lactobacillus bifermentans came into prominence especially in terms of organic acid composition and quality characters of silages.

Characterization and identification of ferulic acid esterase-producing Lactobacillus species isolated from Elymus nutans silage and their application in ensiled alfalfa.

AIMS: Ferulic acid esterase (FAE)-producing Lactobacillus species isolated from ensiled Elymus nutans growing on the Qinghai-Tibetan plateau were characterized, and effects of their application to the alfalfa ensiling process and the evidence to synergic effect between cellulase and FAE were investigated. METHODS AND RESULTS: The results of 16S rRNA gene sequence and species-specific polymerase chain reaction amplification showed that two screened strains with high FAE activity were Lactobacillus plantarum A1 (LP) and L. brevis A3 (LBr). The optimum temperature and pH for the LP and LBr was 37°C and 6·4 respectively. The FAE exhibited a good stability at temperatures between 25 and 50°C and at pH values of 5·0-7·0. The two strains and a commercial cellulase (CE) were applied as additives to alfalfa silage. After 60 days of ensiling, the lactic acid in the control and CE groups were significantly lower than those of the other treatment groups. The neutral detergent fibre and acid detergent fibre contents in the LP group were significantly lower than those observed in the other groups. At the same time, the combination of CE and FAE-producing lactic acid bacteria synergistically improved the fermentation quality of the silage. CONCLUSIONS: The addition of the FAE-producing strain of L. plantarum A1 to alfalfa silage improved its fermentation quality, and reduced the fibre content of the silage. SIGNIFICANCE AND IMPACT OF THE STUDY: The screened homo-fermentative and FAE-producing strain of L. plantarum A1 could be a candidate strain in improving fermentation quality and fibre digestibility of ensiled forages.

Effect of Lactobacillus plantarum expressing multifunctional glycoside hydrolases on the characteristics of alfalfa silage.

For the first time, Lactobacillus plantarum strains carrying heterologous genes encoding multifunctional glycoside hydrolases were constructed and used as additives for alfalfa silage. The chemical characteristics, nonstructural carbohydrate composition, and fermentation quality of alfalfa silage were examined. The supernatant of L. plantarum expressing CbXyn10C and Bgxg1 (LP11AG) showed activities on xylan, Avicel, and carboxymethylcellulose (CMC), while the supernatant of the wild-type L. plantarum showed no activity. When LP11AG was used as silage additive, the water-soluble carbohydrate content of alfalfa silage increased by 72%, 55%, and 155% compared with control when the silage was stored at 20 °C, 30 °C, and 40 °C, respectively. With LP11AG being used as an additive for the alfalfa silage stored at 20 °C, the hemicellulose, cellulose, and acid detergent ligninin (ADL) contents decreased by 17%, 6%, and 14% compared with the control (p < 0.05), respectively. Compared with the corresponding original contents, the contents of glucose, arabinose, galactose, and fructose detected in silage treated with LP11AG after 45 days of ensiling increased by 55%, 1494%, 68%, and 5% , respectively, when stored at 40 °C. Raffinose and stachyose, originally present in alfalfa, disappeared after ensiling. In conclusion, our results suggest that LP11AG provides a substantial benefit as a silage additive.

Lactic acid bacterial inoculant effects on the vitamin content of alfalfa and Chinese leymus silage.

OBJECTIVE: Information regarding the vitamin content of silage is limited. This study investigated the changes in the vitamin content of alfalfa and Chinese leymus silages with or without a lactic acid bacterial inoculant. METHODS: Alfalfa at the early flowering stage and Chinese leymus at the full-bloom stage were harvested. The treatments for each forage type were control (deionized water only) and 1×106 colony-forming units (cfu) Lactobacillus plantarum (LP) g-1 FM. After 45 d ensiling, all silages were sampled for evaluating the vitamin content, fermentation quality and chemical composition. RESULTS: The LP inoculant decreased the pH and ammonia nitrogen content of the alfalfa and Chinese leymus silages and significantly (p &lt; 0.05) increased the lactic acid, acetic acid, propionic acid content and Flieg`s points. Prior to ensiling, the levels of five B-group vitamins (thiamin, riboflavin, niacin, pantothenic acid, pyridoxine) and α-tocopherol in alfalfa were significantly higher than those in Chinese leymus (p &lt; 0.01). Ensiling decreased the levels of the five B-group vitamins in both alfalfa and Chinese leymus while increasing the α-tocopherol content of Chinese leymus. The thiamin, riboflavin, niacin and pantothenic acid levels in the LP-treated silage were significantly (p &lt; 0.05) lower than those in the untreated silage for both forages. The α-tocopherol content in the LP-treated alfalfa silage was significantly (p &lt; 0.05) higher than that in the untreated alfalfa silage. There was no significant (p &gt; 0.05) difference in pyridoxine content between the untreated and LP-treated silages for both forages. CONCLUSION: With or without LP inoculation, the levels of the five B-group vitamins (thiamin, riboflavin, niacin, pantothenic acid, pyridoxine) in alfalfa and Chinese leymus decreased after 45 days of ensiling, while the α-tocopherol content of Chinese leymus increased. The LP inoculant improved the fermentation quality of both the alfalfa and Chinese leymus silages but increased the thiamin, riboflavin, niacin and pantothenic acid loss in the two forages after fermentation.

Utilization of protein in red clover and alfalfa silages by lactating dairy cows and growing lambs.

Feeding trials were conducted with lactating cows and growing lambs to quantify effects of replacing dietary alfalfa silage (AS) with red clover silage (RCS) on nutrient utilization. The lactation trial had a 2 × 4 arrangement of treatments: AS or RCS fed with no supplement, rumen-protected Met (RPM), rumen-protected Lys (RPL), or RPM plus RPL. Grass silage was fed at 13% of dry matter (DM) with AS to equalize dietary neutral detergent fiber (NDF) and crude protein contents. All diets contained (DM basis) 5% corn silage and 16% crude protein. Thirty-two multiparous (4 ruminally cannulated) plus 16 primiparous Holstein cows were blocked by parity and days in milk and fed diets as total mixed rations in an incomplete 8 × 8 Latin square trial with four 28-d periods. Production data (over the last 14 d of each period) and digestibility and excretion data (at the end of each period) were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). Although DM intake was 1.2 kg/d greater on AS than RCS, milk yield and body weight gain were not different. However, yields of fat and energy-corrected milk as well as milk content of fat, true protein, and solids-not-fat were greater on AS. Relative to AS, feeding RCS increased milk and energy-corrected milk yield per unit of DM intake, milk lactose content, and apparent N efficiency and reduced milk urea. Relative to AS, apparent digestibility of DM, organic matter, NDF, and acid detergent fiber were greater on RCS, whereas apparent and estimated true N digestibility were lower. Urinary N excretion and ruminal concentrations of ammonia, total AA, and branched-chain volatile fatty acids were reduced on RCS, indicating reduced ruminal protein degradation. Supplementation of RPM increased intake, milk true protein, and solids-not-fat content and tended to increase milk fat content. There were no silage × RPM interactions, suggesting that RPM was equally limiting on both AS and RCS. Supplementation of RPL did not influence any production trait; however, a significant silage × RPL interaction was detected for intake: RPL reduced intake of AS diets but increased intake of RCS diets. Duplicated metabolism trials were conducted with lambs confined to metabolism crates and fed only silage. After adaptation, collections of silage refusals and excreta were made during ad libitum feeding followed by feeding DM restricted to 2% of body weight. Intake of DM was not different when silages were fed ad libitum. Apparent digestibility of DM, organic matter, NDF, and hemicellulose was greater in lambs fed RCS on both ad libitum and restricted intake; however, acid detergent fiber digestibility was only greater at restricted intake. Apparent and estimated true N digestibility was substantially lower, and N retention was reduced, on RCS. Results confirmed greater DM and fiber digestibility in ruminants and N efficiency in cows fed RCS. Specific loss of Lys bioavailability on RCS was not observed. Based on milk composition, Met was the first-limiting AA on both silages; however, Met was not limiting based on production and nutrient efficiency. Depressed true N digestibility suggested impaired intestinal digestibility of rumen-undegraded protein from RCS.

Dynamics of microbial community during ensiling direct-cut alfalfa with and without LAB inoculant and sugar.

AIM: To gain deeper insights into the clostridial community dynamics and chemical transformations during the ensiling of alfalfa. METHODS AND RESULTS: Direct-cut alfalfa silage (with the dry matter content of 240 g kg-1 ) was prepared with or without the addition of a lactic acid bacterial inoculant and sucrose. Silages were sampled at 0, 3, 7, 14, 28 and 56 days after ensiling and their bacterial community was determined using high-throughput sequencing with a special focus on the clostridial community. A clostridial fermentation occurred in the control silage, with high contents of acetic acid, butyric acid and ammonia nitrogen and Clostridia counts; while the inoculated silage was well preserved, with low pH and high lactic acid content. Lactic acid bacteria dominated the bacterial community during the ensiling process. In the control silage, Weissella confusa, Lactobacillus brevis, Enterococcus mundtii and Pediococcus acidilactici were identified at the beginning of the fermentation. Thereafter, W. confusa, Lactobacillus helsingborgensis and Bifidobacterium asteroides appeared and quickly prevailed. In the inoculated silage, Lactobacillus plantarum dominated the whole ensiling process. The genus Clostridium dominated the clostridial community, and was depressed with the inoculated treatment. Clostridium perfringens, Garciella sp. and Clostridium baratii were the main initiators of the clostridial fermentation of the control silage, while Clostridium tyrobutyricum became the most abundant Clostridia with prolonged ensiling. Overall in the inoculated silage, little changes in the clostridial community were observed throughout the ensiling period. Treating alfalfa silage with a homolactic acid-based bacterial inoculant prevented a clostridial fermentation resulting in more efficient fermentation. CONCLUSION: Distinct changes in the bacterial community with a special focus on the clostridial community were associated with the development of the clostridial fermentation during the ensiling of alfalfa. SIGNIFICANCE AND IMPACT OF THE STUDY: High-throughput sequencing based on a novel Clostridia-specific primer set proved a potentially useful tool to study the clostridial community dynamics, and could aid to elucidate the mechanism by which the clostridial fermentation develops during the ensiling of alfalfa.

Effects of addition of malic or citric acids on fermentation quality and chemical characteristics of alfalfa silage.

We studied the effects on alfalfa preservation and chemical composition of the addition of different levels of malic acid and citric acid at ensiling as well as the utilization efficiency of these 2 organic acids after fermentation. Alfalfa was harvested at early bloom stage. After wilting to a dry matter content of approximately 40%, the alfalfa was chopped into 1- to 2-cm pieces for ensiling. Four levels (0, 0.1, 0.5, and 1% of fresh weight) of malic acid or citric acid were applied to chopped alfalfa at ensiling with 4 replicates for each treatment, and the treated alfalfa forages were ensiled for 60 d in vacuum-sealed polyethylene bags (dimensions: 200 mm × 300 mm) packed with 200 to 230 g of fresh alfalfa per mini silo and an initial density of 0.534 g/cm3. The application of malic or citric acids at ensiling for 60 d led to lower silage pH than was observed in the control silage (0% of malic or citric acids). Application of the 2 organic acids led to higher lactic acid concentration in alfalfa silage than in the control silage except with the application rate of 1% of fresh weight. Silages treated with both organic acids had lower nonprotein nitrogen concentrations than the control silages, and the nonprotein nitrogen concentrations in ensiled forages decreased with the increase in malic or citric acid application rates. The application of the 2 organic acid additives led to lower saturated fatty acid proportions and higher polyunsaturated fatty acid proportions in ensiled alfalfa than in the control silage. The amount of malic and citric acids degraded during ensiling of alfalfa was 1.45 and 0.63 g, respectively. At the application rate of 0.5% of fresh weight, residues of malic acid and citric acid in alfalfa silage were 11.1 and 13.6 g/kg of dry matter. These results indicate that including malic or citric acids at the ensiling of alfalfa effectively improved silage fermentation quality, limited proteolysis, improved fatty acid composition of the ensiled forage, and could provide animals with additional feed additives proven to promote animal performance. However, when the application rate of both organic acids reached 1%, the concentration of lactic acid in silages decreased notably. Additionally, 0.5 and 1% application rates also increased the yeast count in ensiled alfalfa.

Replacing alfalfa silage with tannin-containing birdsfoot trefoil silage in total mixed rations for lactating dairy cows.

Two lactation trials were conducted comparing the feeding value of silages made from birdsfoot trefoil (BFT, Lotus corniculatus L.) that had been selected for low (BFTL), medium (BFTM), and high (BFTH) levels of condensed tannins (CT) to an alfalfa silage (AS) when fed as the principal forage in total mixed rations. Diets also included corn silage, high-moisture shelled corn, soybean meal, soy hulls, and supplemental fat. In trial 1, 32 lactating Holstein cows were blocked by days in milk, assigned to treatment sequences in 8 balanced 4 × 4 Latin squares, and fed 50% dietary dry matter from AS or 1 of 3 BFT silages containing 0.6, 1.2, or 1.7% CT. Diets averaged 17.5 to 19.5% crude protein and 26% neutral detergent fiber on a dry matter basis. Data were collected over the last 2 wk of each 4-wk period. Intakes were 1.3 to 2.8 kg of dry matter/d greater on BFT than on AS and cows gained 0.5 kg of body weight/d on BFT diets while losing 0.14 kg of body weight/d on the AS diet; this resulted in greater milk per dry matter intake (DMI) on AS. Linear effects indicated true protein yield and milk urea nitrogen declined with increasing CT concentration and quadratic effects indicated DMI, energy-corrected milk, and fat yield were increased at intermediate CT concentration. True protein yield and apparent N-efficiency were greater, and milk urea nitrogen lower, on all BFT diets than on AS. In trial 2, 50 lactating Holstein cows were fed a covariate AS diet for 2 wk and then blocked by parity and days in milk and randomly assigned to 1 of 5 diets that were fed continuously for 12 wk. Diets contained (dry matter basis) 48% AS, 16% AS plus 32% of 1 of 3 BFT silages with 0.5, 0.8, or 1.5% CT, or 48% of an equal mixture of each BFT silage. Diets averaged 16.5% crude protein and 30% neutral detergent fiber. Intake and milk yield tended to be lower on AS than BFT, but body weight gains averaged 0.6 kg/d on all diets. Cows fed any of the BFT silages had reduced milk urea nitrogen and ruminal ammonia and reduced urinary N excretion. Feeding the BFT mixture reduced concentrations of milk true protein and milk urea nitrogen and depressed apparent nutrient digestibility. Among diets containing the individual BFT silages, linear reductions in DMI and yield of milk, fat, true protein, lactose, and SNF were observed with increasing CT concentration. By contrast, a previous trial with the same BFT populations showed that substituting BFTH silage containing 1.6% CT for AS in rations containing 60% silage dry matter had no effect on intake, increased yield of milk, energy-corrected milk and milk components, elevated protein use-efficiency, but with a more modest reduction in milk urea nitrogen and urinary N excretion. Silage analyses suggested that the inconsistent responses among trials were related to growth environment or ensiling effects that altered tannin-protein interactions in BFT silage. Differences in diet formulation among trials may have also influenced responses. Results from the current and previous trials indicate further work is needed to identify optimum tannin levels in forages.

Effects of four short-chain fatty acids or salts on the dynamics of nitrogen transformations and intrinsic protease activity of alfalfa silage.

BACKGROUND: Short-chain fatty salts have been widely used as food and forage preservatives because of their antimicrobial properties. This study evaluated the effects of four chemical compounds with antimicrobial properties on nitrogen transformations and intrinsic protease activity of alfalfa silage. RESULTS: Potassium diformate (PD) and formic acid (FA) rapidly reduced silage pH. Silages treated with sodium diacetate (SD) and calcium propionate (CAP) had higher final peptide N concentrations than other silage. The free amino acid N contents in PD and FA treated silages were lower than other silages at all intervals of ensilage. The ammonia N concentrations in FA and PD silages were the lowest, followed by SD and CAP silages. As ensiling progressed, the aminopeptidase activity was completely lost by day 5 for FA and PD silages and inactive by day 7 for SD silage, while it remained active after day 7 for control and CAP silage. The carboxypeptidase activities in FA and PD silages were already reduced below 50% by day 1 of ensiling. CONCLUSION: Potassium diformate was as effective as formic acid in depressing the proteolysis, while sodium diacetate and calcium propionate were inferior to formic acid in protecting alfalfa proteins from being hydrolysed. © 2016 Society of Chemical Industry.

Chemical composition of alfalfa silage with waste date and its feeding effect on ruminal fermentation characteristics and microbial protein synthesis in sheep.

This study was conducted to evaluate the effect of feeding ensiled alfalfa with waste date on ruminal fermentation characteristics, microbial protein synthesis, protozoa population and blood parameters in sheep. Eight rams were used in a 2 × 2 change over design. Each experimental period consisted of 21 days including 16 days for adaptation and 5 days for sampling. For ensiling, fresh alfalfa (Medicago sativa L.) with different levels of waste date (Phoenix dactylifera L.) were mixed together and ensiled in 100-l containers for 45 days. Chemical composition of silages such as dry matter (DM), crude protein, NH3 -N, organic matter, NDF, ADF and pH were determined. Then, it was used as 30% (DM basis) in diets. The experimental diets were as follows: (i) control (diet containing alfalfa silage without waste date), (ii) diet containing alfalfa silage with 5 g waste date/95 g DM, (iii) diet containing alfalfa silage with 10 g waste date/90 g DM, and (iv) diet containing alfalfa silage with 15 g waste date/85 g DM. The results of this experiment showed that adding waste date to alfalfa during ensiling, improved silage quality, DM and energy level. Total protozoa population and all of holotrich, cellulolytic and entodinia in rumen fluid were increased linearly by increasing the level of waste date. Nitrogen (N) intake, urinary N excretion and nitrogen retention were affected by dietary treatments. Also, allantoin, uric acid, total purine derivatives, microbial protein synthesis, cholesterol level and blood urea nitrogen were significantly different. In conclusion, direct ensilage of alfalfa can be attained by mixing 15 g waste date/85 g DM and positive associative effects such as increased metabolizable energy (ME) and silage quality occurred.