Ensiled Alfalfa Research Articles

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.

The effect of cultivar, wilting and storage period on fermentation and the clostridial community of alfalfa silage

The objective of this study was to gain deeper insights into chemical transformations and the clostridial community dynamics during the ensiling of alfalfa. A factorial experiment was conducted with four alfalfa cultivars [Sanditi (A1), AC Caribou (A2), WL319HQ (A3) and 4030 (A4)] × three wilting durations [0 h (direct-cut), 2 h and 4 h] × three storage periods (14, 28 and 56 days). The clostridial community was examined using denaturing gradient gel electrophoresis. High butyric acid and ammonia nitrogen (NH3–N) contents and clostridia numbers were observed in direct-cut silage, with higher values observed in A4 than in the other three cultivars. However, butyric acid and NH3–N contents and clostridia numbers decreased with wilting regardless of the cultivar. Although Clostridium ghonii and Clostridium sartagoforme were common to all direct-cut and wilted silages, bands for these species were faint. Differences in the appearing time of these species were observed, bands for C. ghonii were found after 14 and 28 days of ensiling, while those for C. sartagoforme were only found after 56 days. In addition, in direct-cut silage, distinct bands for Clostridium perfringens were detected in A1, A2 and A3, while those for Clostridium sporogenes were detected in A4. The inactive spores of clostridia could be observed in 4-h wilted silage as no butyric acid was detected. It was concluded that the enhanced clostridial fermentation of direct-cut silage was attributed mainly to C. perfringens and C. sporogenes, while C. ghonii and C. sartagoforme were involved in the restricted clostridial fermentation of 2-h wilted silage.

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

To gain deeper insights into the clostridial community dynamics and chemical transformations during the ensiling of alfalfa. Direct-cut alfalfa silage (with the dry matter content of 240gkg-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 56days 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. 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. 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.

Changes in carbohydrate and protein fractions during ensiling of alfalfa treated with previously fermented alfalfa juice or lactic acid bacteria inoculants

The effects of previously fermented juice (PFJ) prepared from alfalfa and lactic acid bacteria (LAB) inoculants on the dynamic changes of nutritive components in ensiled alfalfa after various ensiling periods were investigated by using the Cornell Net Carbohydrate and Protein System. The third-cut alfalfa was harvested at the budding stage, exposed to sunlight, weighed occasionally to estimate the dry matter (DM) content until the actual DM finally obtained was 347.8 g/kg fresh weight, and then chopped to 1–2-cm lengths. Chopped forages were treated with (1) distilled water (control), (2) alfalfa PFJ or (3) LAB at 1 mL/50 g fresh weight. The application amounts of PFJ and LAB to the fresh forage were 8.73 log (colony-forming units/mL) and 7.32 log (colony-forming units/mL) respectively. All silages were prepared in mini-silos of 100-mL polypropylene centrifuge tubes and kept in an incubator at 30°C, and triplicate silos from each treatment were opened after 1, 3, 7, 14 and 35 days of ensiling. Results suggested that silage treated with LAB and PFJ was of better quality than was the control silage, as evidenced by lower volatile fatty acid concentrations, as well as higher lactic acid, sugar, starch, soluble fibre and digestible natural detergent fibre production at various ensiling periods (P < 0.05), and a lower protein degradation as suggested by the low non-protein nitrogen production (P < 0.05). The effect of PFJ on alfalfa fermentation quality and protein degradation was greater than that of LAB, as evidenced by the lower pH value and volatile fatty acid content and the higher concentrations of lactic acid (P < 0.05). In addition, the cost of PFJ for 1 tonne of alfalfa silage is ~1/7–1/5 of that of LAB. In conclusion, adding PFJ to alfalfa forages before preservation as silage is a cost-effective way to improve the silage formation quality; in addition, its effect as a fermentation stimulant may be comparable to, or even better than, that of LAB inoculants at various ensiling periods.

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 21days including 16days for adaptation and 5days 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 45days. 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 5g waste date/95g DM, (iii) diet containing alfalfa silage with 10g waste date/90g DM, and (iv) diet containing alfalfa silage with 15g waste date/85g 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 15g waste date/85g DM and positive associative effects such as increased metabolizable energy (ME) and silage quality occurred.

Different aspects of Lactobacillus inoculants on the improvement of quality and safety of alfalfa silage

There is a significant range of bacterial inoculants for forage ensiling, but there is still a need for formulations to improve the safety of feed. The objective of the study was to evaluate the usefulness of three lactobacilli strains in alfalfa (Medicago sativa L.) The following bacterial inoculants were used: Lactocacillus plantarum K KKP 593p (LPK), L. plantarum C KKP 788p (LPC), L. buchneri KKP 907p (LB), and mix of all three strains (LPK+LPC+LB). The application of bacterial inoculants in alfalfa ensiling resulted in a reduction of the total number of molds, Clostridium perfringens and Listeria sp. (up to 5, 7, and 5 times respectively for LB inoculant in comparison to untreated silage). Total inhibition of Salmonella sp. and Escherichia coli growth was achieved in silages treated with all inoculants except for LPC. Aerobic stability in the control silage was the lowest (77 h) and doubled under the influence of bacterial inoculants. The most stable according to aerobic stability was silage treated with LB inoculant (175 h), where the highest concentrations of acetic acid (4.8 g kg-1), propionic acid (0.7 g kg-1) and 1,2-propanediol (526 mg kg-1) were reported. The study discussed that it is important to evaluate not only the effect of bacterial inoculants on physicochemical and microbiological silage properties, as the presence and expression of antibiotic resistance genes in lactic acid bacteria have been reported. The results of antimicrobial susceptibility testing of the strains showed that almost all minimum inhibitory concentrations values for eight antibiotics were equal to or below the corresponding breakpoints proposed by the European Food Safety Authority, Additives and Products or Substances used in Animal Feed Panel.

Fermentation characteristics, aerobic stability, proteolysis and lipid composition of alfalfa silage ensiled with apple or grape pomace

The effects of adding 100g of either dried apple pomace (AP), unground grape pomace (GP) or ground grape pomace (GGP) per kg wilted alfalfa (wet basis) on silage characteristics, aerobic stability, proteolysis and lipid composition were studied. The three combinations as well alfalfa without addition were ensiled in 1.8L- jars for 60d, with 4 jars per treatment. Addition of AP and GGP decreased pH (P<0.05). Pomace-treated silages contained more lactic acid than the control silage. Pomace treatments decreased aerobic stability of the ensiled alfalfa. The nonprotein nitrogen (NPN) concentrations in the ensiled alfalfa were decreased by 54%, 67% and 69% after being ensiled with AP, GP and GGP, respectively. Total fatty acid concentrations in the control silage and AP silage were comparable, but in GP or GGP-treated silages it was more than double that of the control silage. Application of pomace markedly increased (P<0.05) the proportions of oleic acid and linoleic acid in the ensiled alfalfa due to high proportions of these fatty acids in both pomaces. Silages treated by GP or GGP had a much greater proportion of linoleic acid than the control or AP treated silages (P<0.05); this fatty acid accounted for half of the fatty acids in GP or GGP-treated silages. The proportion of α-linolenic acid was lower in GP or GGP silages than in control silage or AP silage (P<0.05). In conclusion, application of apple or grape pomace at ensiling of alfalfa could not only employ this industrial waste as feed, but also inhibit proteolysis and alter the fatty acid composition of ensiled alfalfa.

Characterization of peptides in ensiled alfalfa treated with different chemical additives

Effects of different chemical additives on peptide composition in ensiled alfalfa (Medicago sativa L.) were investigated by using gel filtration and determination of N characteristics. The alfalfa silages were prepared untreated (control) or with formic acid, formaldehyde or tannic acid as additives at ensiling. All additives reduced non-protein N (NPN), ammonia nitrogen (NH3 -N) and amino acid N (AA-N) in the ensiled forage, and the most effective reduction of NPN and AA-N was observed in the formaldehyde-treated silages. Peptides in the control silage were mainly dipeptides to peptides with five amino acid residues. Most peptides in the formic acid-treated silage contained 4-12 amino acid residues. Although most peptides in the formaldehyde-treated silages contained 4-6 amino acid residues, there was a considerable proportion of peptides with 7-11 amino acid residues. Tannic acid had little effect on peptide size of ensiled alfalfa extract in which most peptides contained 5-6 amino acid residues. Peptide size in formic acid-treated alfalfa silage was greater than that in the other treatments. Addition of formic acid and formaldehyde not only increased the peptide concentration in alfalfa silage, but enlarged the peptide size.

Bacterial population dynamics during the ensiling of Medicago sativa (alfalfa) and subsequent exposure to air

To describe, at high resolution, the bacterial population dynamics and chemical transformations during the ensiling of alfalfa and subsequent exposure to air. Samples of alfalfa, ensiled alfalfa and silage exposed to air were collected and their bacterial population structures compared using 16S rRNA gene libraries containing approximately 1900 sequences each. Cultural and chemical analyses were also performed to complement the 16S gene sequence data. Sequence analysis revealed significant differences (P<0·05) in the bacterial populations at each time point. The alfalfa-derived library contained mostly sequences associated with the Gammaproteobacteria (including the genera: Enterobacter, Erwinia and Pantoea); the ensiled material contained mostly sequences associated with the lactic acid bacteria (LAB) (including the genera: Lactobacillus, Pediococcus and Lactococcus). Exposure to air resulted in even greater percentages of LAB, especially among the genus Lactobacillus, and a significant drop in bacterial diversity. In-depth 16S rRNA gene sequence analysis revealed significant bacterial population structure changes during ensiling and again during exposure to air. This in-depth description of the bacterial population dynamics that occurred during ensiling and simulated feed out expands our knowledge of these processes.

Effects of plant enzyme inactivation or sterilization on lipolysis and proteolysis in alfalfa silage

This experiment studied the contribution of plant enzymes and microbial activity on lipolysis and proteolysis in ensiled alfalfa. Before ensiling, the wilted alfalfa was treated with plant enzyme inactivation by autoclaving or with sterilization by γ-ray irradiation. The treated alfalfa was then inoculated with commercial lactic acid bacteria inoculants and ensiled for 40 d. Alfalfa without treatment was ensiled as the control. The content of total fatty acid (FA) after ensiling decreased 43% in the control silage and 28% in the γ-ray–treated silage, but did not change in the autoclave-treated silage. Among the major FA (C16:0, C18:2n-6, C18:3n-3), a considerable increase was observed in proportion of C16:0 in the control silage as compared with fresh alfalfa; conversely, decreases in proportions of C18:2n-6 and C18:3n-3 occurred during ensilage. Silage treated with γ-ray radiation at ensiling had a smaller proportion of C16:0 and greater proportions of C18:2n-6 and C18:3n-3 than control silage. Autoclave treatment further decreased proportions of C16:0 and most of the other FA, and increased C18:2n-6 and C18:3n-3 proportions in comparison with γ-ray treatment. Proportions of C16:0, C18:2n-6, C18:3n-3 and other detected FA (except for the proportion of C15:0) did not differ between fresh forage and autoclave-treated silage. Remarkably, smaller nonprotein nitrogen content was observed in the autoclave-treated silage compared with the γ ray-treated silage or the control silage. These results indicated that an extensive lipolysis occurred during ensiling of alfalfa, and plant enzymes played a major role in lipolysis and proteolysis.

Effect of Epiphytic Microorganisms and Exogenous Lactic Acid Bacteria on the Formation of Non-protein Nitrogen During the Ensiling of Alfalfa

Effect of Epiphytic Microorganisms and Exogenous Lactic Acid Bacteria on the Formation of Non-protein Nitrogen During the Ensiling of Alfalfa

Fecal pH and Microbial Populations in Thoroughbred Horses During Transition from Pasture to Concentrate Feeding

Abrupt dietary transitions and feeding of rapidly fermentable diets are common practices in the horse industry and have been associated with digestive and metabolic disorders that can impair the performance of horses. The present study investigated the effect of dietary transition from pasture grazing to confinement with concentrate feeding, and back, on fecal pH and bacterial populations of Streptococcus spp and Lactobacillus spp. Six Thoroughbred fillies, previously grazing perennial ryegrass and white clover-based pasture, were housed in individual stalls and fed an increasing ratio of concentrate to conserved forages for 13 days (days 1-13), followed by an abrupt transition back to only pasture-grazing for 3 days (days 14-16). The concentrate was initially offered at 0.83 kg dry matter (DM)/d and increased to 5 kg DM/d, whereas ensiled alfalfa was initially offered at 0.61 kg DM/d, increasing to 1.22 kg DM/d. Meadow hay was initially offered at 6.73 kg DM/d, decreasing to 1.6 kg DM/d. Fecal specimens were collected daily for determination of pH, and every 2 days for quantitative analysis of Streptococcus spp and Lactobacillus spp. Mean fecal pH increased significantly from pasture baseline values (pH 6.18) during the initial confinement and supplementation on day 1 (6.37), day 2 (6.52), day 3 (6.58), and day 4 (6.43) (standard error of mean [SEM]: 0.056; P < .001). By day 5, mean fecal pH values had decreased to, and remained at, baseline values until the horses returned to pasture, when another increase occurred at day 15 (6.45). Fecal colony forming units (cfu) of Streptococcus spp and Lactobacillus spp increased linearly (r = 0.94; P < .001) from 6.0 and 6.1 log10 cfu/g on day -4, to 7.8 log10 cfu/g on day 14 (SEM: 0.2 P < .001), respectively. Fecal cfu decreased on return to a pasture-only diet (P < .001). In this study, the increment of bacterial populations was associated with a relatively stable fecal pH and highlights the difficulty in identifying the effects of dietary transition on the equine hindgut health, without microbial culture.

Short communication: Characteristics of proteolytic activities of endo- and exopeptidases in alfalfa herbage and their implications for proteolysis in silage

The pH optimum and thermostability of both exopeptidases and endopeptidases were investigated in this study to elucidate the possible role of plant proteases in proteolysis during ensiling of alfalfa herbage. Proteolytic activities of 4 classes of endopeptidases (i.e., serine, metallo, aspartic, and cysteine peptidase) and 5 classes of exopeptidases (i.e., aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase) were examined within pH values of 3 to 9, and within temperatures from 20 to 90°C. Serine and metalloproteases, the principal endopeptidases that hydrolyzed most of the protein to nonprotein nitrogen in alfalfa silage, had optimum activities at pH 4. Among the major exopeptidases contributing protein degradation in ensiled alfalfa, dipeptidase and tripeptidyl-peptidase had stable activities between pH 4 and 6, and carboxypeptidase activity was optimal at pH 5. The optimum temperature for most peptidase activities was 40°C. Proteolytic activities of both endo- and exopeptidases increased with the elevation of incubating temperature from 20 to 40°C. The pH value in well-preserved alfalfa silage is often above 4.0, and the temperatures in the ensiled mass range from 25 to 40°C. Therefore, high proteolytic activities between pH 4 and 6 and the temperature range of ensiled alfalfa suggest that plant peptidases play a role in hydrolyzing protein during prolonged storage.

Contribution of exopeptidases to formation of nonprotein nitrogen during ensiling of alfalfa

The experiment was conducted to investigate the exopeptidase classes in alfalfa (Medicago sativa L.) leaves, and to determine their contribution to the formation of nonprotein nitrogen (NPN) components during ensiling. Six classes of inhibitors that included bestatin (aminopeptidase inhibitor), potato carboxypeptidase inhibitor (PCI, carboxypeptidase inhibitor), 1,10-phenanthroline (dipeptidase inhibitor), diprotin A (dipeptidyl-peptidase inhibitor), butabindide (tripeptidyl-peptidase inhibitor), and dipeptide Phe-Arg (peptidyl-dipeptidase inhibitor) were used. To determine the contribution of each exopeptidase to the formation of NPN products, aqueous extracts of fresh alfalfa were fermented to imitate the proteolytic process of ensiled alfalfa and to ensure that each class of exopeptidase inhibitor would have immediate contact with the proteases in the alfalfa extract. Five classes of exopeptidases; namely, aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase, were shown to be present in alfalfa leaves, each playing a different role in alfalfa protein degradation. Aminopeptidase, carboxypeptidase, and dipeptidase were the main exopeptidases contributing to the formation of NH3-N. Among the 5 exopeptidases, tripeptidyl-peptidase appeared to be the principal exopeptidase in hydrolyzing forage protein into peptides, whereas carboxypeptidase and dipeptidase appeared to be more important in contributing to the formation of amino acid-N. Dipeptidyl-peptidase and tripeptidyl-peptidase did not play a role in the formation of NH3-N or amino acid-N. Dipeptidase, carboxypeptidase, and tripeptidyl-peptidase were the principal exopeptidases for hydrolyzing forage protein into NPN during ensilage, and treatment with a mixture of the 5 inhibitors reduced the total NPN concentration in the fermented alfalfa extract to about 45% of that in the control after 21 d of fermentation.

Contribution of endopeptidases to the formation of nonprotein nitrogen during ensiling of alfalfa

The experiment was conducted to investigate endopeptidase classes in alfalfa ( Medicago sativa L.) leaves and to determine their contribution to the formation of nonprotein N (NPN) components during ensiling. Four classes of inhibitors ( i.e., phenylmethanesulfonyl fluoride (PMSF, serine peptidase inhibitor), 1,10-phenanthroline (metallo peptidase inhibitor), pepstatin A (aspirate peptidase inhibitor) and E-64 (cysteine peptidase inhibitor)) were examined. To determine the contribution of each endopeptidase to the formation of NPN products, green alfalfa extract was fermented to imitate the proteolytic process of ensiled alfalfa and to ensure that each class of endopeptidase inhibitor would have immediate contact with the alfalfa proteases in the alfalfa. Four classes of endopeptidases, namely serine, metallo, aspartic and cysteine peptidase, were shown to be in alfalfa leaves, each playing a different role in alfalfa protein degradation. Metallopeptidase inhibitor effectively inhibited the formation of NH 3-N during ensiling of alfalfa. Among the four classes of endopeptidases, the metallopeptidase was the principal peptidase in the formation of free amino acid N (AA-N) while aspartic peptidase made little contribution to AA-N. Serine and metallo peptidase contributed to the degradation of peptides into free AA, and degradation of protein into oligopeptides was mainly due to aspartic and cysteine peptidases. Metallo and cysteine peptidases were the principle peptidases for hydrolyzing forage protein into NPN during ensiling, and treatment with the mixture of four inhibitors reduced the total NPN content in the fermented alfalfa extract to about 44% of that in the control after 14 d of fermentation.

Modeling degradation ratios and nutrient availability of anthocyanidin-accumulating Lc-alfalfa populations in dairy cows

Dairy cattle eating fresh or ensiled alfalfa as the main portion of their diet often have low protein efficiency because of the rapid initial rate of ruminal protein degradation of fresh and ensiled alfalfa. Ruminal protein degradation of alfalfa might be reduced by introducing a gene that stimulates the accumulation of mono- or polymeric anthocyanidins in alfalfa. The objectives of this study were to fractionate protein and carbohydrates by in situ and chemical approaches, to evaluate in situ ruminal degradation characteristics and synchronization ratios, to determine protein availability to dairy cattle using the 2007 digestible intestinal protein/rumen-degraded protein balance (DVE/OEB) protein system, and to determine net energy for lactation using the Dutch net energy for lactation (VEM) system for 3 newly developed transgenic winter hardy anthocyanidin-accumulating T1 Lc-alfalfa populations. These T1 Lc-alfalfa populations, called T1BeavLc1, T1RambLc3, and T1RangLc4, had an average anthocyanidin accumulation of 163.4 μg/g of DM, whereas AC Grazeland (selected for a low initial rate of degradation) did not accumulate anthocyanidin. The basic chemical composition of the original samples, soluble and potentially degradable fractions, and degradation characteristics of crude protein and carbohydrates were similar in T1 Lc-alfalfa and AC Grazeland. The undegradable in situ crude protein and neutral detergent fiber fraction had 1.3% lower CP and 4.8% lower CHO, respectively, in T1 Lc-alfalfa compared with the amounts in AC Grazeland. The T1 Lc-alfalfa had a 0.34 MJ/kg of DM higher calculated net energy for lactation and 1.9% of CP higher buffer soluble protein compared with that in AC Grazeland. By the protein evaluation model, it was predicted that T1 Lc-alfalfa tended to have 11.9, 6.9, and 8.4 g/kg of DM higher rumen degradable protein, OEB, and intestinal available protein, respectively, compared with the amounts in AC Grazeland. The hourly OEB included an initial and substantial peak (oversupply) of protein relative to energy, which was highest in T1RangLc4 and lowest in T1RambLc3. The hourly OEB between 4 and 24h was similar and more balanced for all 4 alfalfa populations. In conclusion, T1 Lc-alfalfa accumulated anthocyanidin, tended to have higher predicted intestinal protein availability, and had higher predicted net energy of lactation availability for dairy cattle than did AC Grazeland.

A meta-analysis of passage rate estimated by rumen evacuation with cattle and evaluation of passage rate prediction models

A meta-analysis of studies using the flux/compartmental pool method with indigestible neutral detergent fiber (iNDF) as internal marker was conducted to study the effect of extrinsic characteristics and forage type on particle passage rate (kp) in cattle. Further, the kp prediction equations in the National Research Council (NRC) and the Cornell Net Carbohydrate and Protein System (CNCPS) were evaluated. Data comprised 172 treatment means from 49 studies conducted in Europe and the United States. In total, 145 diets were fed to dairy cows and 27 to growing cattle. A prerequisite for inclusion of an experiment was that dry matter intake, neutral detergent fiber (NDF), proportion of concentrate in the diet, body weight, and diet chemical composition were determined or could be estimated. Mixed model regression analysis including a random study effect was used to generate prediction equations of kp and to investigate the relationships between NRC and CNCPS predictions and observed kp of iNDF. Prediction equations were evaluated by regressing residual values on the predicted values. The best-fit model when forage type was not included was kp (%/h)=1.19+0.0879 × NDF intake (g/kg of body weight)+0.792 × proportion of concentrate NDF of total NDF+1.21 × diet iNDF:NDF ratio (adjusted residual mean square error=0.23%/h). The best general equation accounting for an effect of forage type was as follows: kp (%/h)=F+1.54+0.0866 × NDF intake (g/kg of body weight) (adjusted residual mean square error=0.21%/h), where F is the forage adjustment factor of the intercept. The value of F for grass silage, fresh grass, mixes of alfalfa and corn silage, and dry or ensiled alfalfa as sole forage component were 0.00, −0.91, +0.83, and +0.24, respectively. Relationships between predicted and observed kp were y=0.53(±0.187)+0.41(±0.0373) × predicted kp and y=0.58(±0.162)+0.46(±0.0377) × predicted kp for the NRC and CNCPS models, respectively. Residual analysis of the NRC and CNCPS models resulted in both significant mean biases (observed – predicted) of −2.40 and −1.70% and linear biases of −0.59 and −0.53, respectively. The results from this meta-analysis suggest that ruminal particulate matter kp is affected by forage type in the diet. Further, the evaluation of NRC and CNCPS models showed that passage rate equations developed from marker excretion curves markedly deviated from observed kp of iNDF derived using the rumen evacuation technique.

Characterization of protein fractions and amino acids in ensiled alfalfa treated with different chemical additives

Formic acid, formaldehyde, tannic acid or mixtures of two were studied on their effects on ensiled alfalfa ( Medicago sativa L.) amino acids and N fractions by the Cornell Net Carbohydrate and Protein System (CNCPS). The alfalfa forage was a second cut and was wilted to a mean over-dry dry matter (DM) content of 330 g/kg. All silages were prepared as mini-silos using 100 ml polypropylene centrifuge tubes (50 g) on a small laboratory-scale, with the additives added in 20 ml aliquots/kg herbage fresh weight (FW). After 35 d of ensiling, most of forage true protein was converted to fraction A and all of the added additives reduced fraction A content in the ensiled forages (P<0.05). The content of fraction B 1 in all of the additive-treated silages was higher (P<0.05) than that in control silage. Large proportions of true protein in the tannic acid/formaldehyde- and formic acid/formaldehyde-treated silages were fractions B 2 and B 3, respectively. No difference was observed on fraction C content between the control silage and silages treated with additives except for the formaldehyde or tannic acid-treated silages. Amino acids were well preserved in additive-treated silages compared with the control silage. Concentration of total amino acid was higher in formic acid-treated silages than that in the control and the other additive-treated silages (P<0.05). The pattern of changes in individual amino acid in all of the silages indicated that branched chain amino acids and methionine were relatively well preserved during fermentation but the basic and acidic amino acids were not.

The effect of enzyme treatment on the nutritive value of lucerne for equids

Two experiments were conducted to evaluate the effects of a fibrolytic enzyme preparation on the nutritive value of lucerne for equids. In both experiments four mature Welsh-cross pony geldings were used in a 4 × 4 Latin-square design to assess the effect of a fibrolytic enzyme treatment on the nutritive value of either high-temperature dried (HT) lucerne (experiment 1) or ensiled lucerne (experiment 2) for equids. Experiment 1 consisted of four levels of enzyme treatment (0 [WC], 2.3 [DE1], 4.7 [DE2] and 8.9 L tonne − 1 dry matter (DM) [DE3]), whilst in experiment 2 the four levels of enzyme treatment were: 0 (WS), 2.7 (WE1), 5.5 (WE2) and 10.2 L tonne − 1 DM (WE3). Each diet was fed to give a DM intake of 17.5 g kg − 1 of liveweight per day, offered in two equal meals at 12 h intervals. Digestibility was determined by total faecal collection. Feed and faecal samples were analysed for DM, OM, CP, ADFom, NDFom, starch, WSC, GE, Na, Ca, P, K, Mg and non-starch polysaccharide (NSP) content and composition. Silages were also analysed for pH, VFA, lactate, TN, ammonia–N and aerobic stability. In experiment 1, enzyme treatment significantly ( P < 0.001) reduced total NSP digestibility (TNSPD) at the lower levels of application in comparison to the control (551, 578 and 608 g kg − 1 for DE1, DE2 and WC, respectively), whilst in experiment 2, TNSPD was significantly ( P < 0.001) reduced at the higher application level compared to the control (550 and 421 g kg − 1 for WE3 and WS, respectively). The DM contents of the enzyme-treated silages were significantly lower (averaging 269 g kg − 1 ) compared to the control (302 g kg − 1 ) and butyrate content increased significantly ( P < 0.05) with increasing level of enzyme (2.6, 4.4, 7.8 and 7.8 g kg − 1 DM for WS, WE1, WE2 and WE3, respectively). Consequently, enzyme treatment does not appear to increase the nutritive value of high-temperature dried or ensiled lucerne for equids.

The nutritive value of sugar beet pulp-substituted lucerne for equids

Two experiments were conducted to evaluate the nutritive value of sugar-beet-substituted lucerne for equids. In both experiments four mature Welsh-cross pony geldings were used in a 4 × 4 Latin-square design to assess the effect of sugar beet pulp (SB) on the nutritive value of either high-temperature dried (HT) lucerne (Experiment 1) or ensiled lucerne (Experiment 2) for equids. Both experiments consisted of four levels of SB substitution: 0 [DC], 100 [SB10], 200 [SB20] and 300 [SB30] g/kg DM for Experiment 1, and 0 [WS], 100 [SB1], 200 [SB2] and 300 [SB3] g/kg DM for Experiment 2. Each diet was fed to give a total DM intake of 17.5 g/kg of liveweight per day, offered in two equal meals at 12-h intervals. Digestibility was determined by total faecal collection. SB substitution of HT and ensiled lucerne increased (P<0.01) total diet total non-starch polysaccharide (TNSP) digestibility compared to the control: 0.63, 0.65, 0.71 and 0.58 for SB10, SB20, SB30 and DC, respectively; and 0.62, 0.62, 0.69 and 0.55 for SB1, SB2, SB3 and WS, respectively. Furthermore, TNSP digestibility values for SB as a sole feed (calculated ‘by difference’ and predicted by regression) were greater than unity, indicating an increase in the digestibility of the cell wall fraction of the lucerne.