Nitrogen and fatty acid rumen metabolism in cattle offered high or low polyphenol oxidase red clover silage

Polyphenol oxidase (PPO) in red clover (RC) has been shown to reduce both lipolysis and proteolysis in silo and implicated (in vitro) in the rumen. However, all in vivo comparisons have compared RC with other forages, typically with lower levels of PPO, which brings in other confounding factors as to the cause for the greater protection of dietary nitrogen (N) and C18 polyunsaturated fatty acids (PUFA) on RC silage. This study compared two RC silages which when ensiled had contrasting PPO activities (RC+ and RC−) against a control of perennial ryegrass silage (PRG) to ascertain the effect of PPO activity on dietary N digestibility and PUFA biohydrogenation. Two studies were performed the first to investigate rumen and duodenal flow with six Hereford×Friesian steers, prepared with rumen and duodenal cannulae, and the second investigating whole tract N balance using six Holstein-Friesian non-lactating dairy cows. All diets were offered at a restricted level based on animal live weight with each experiment consisting of two 3×3 Latin squares using big bale silages ensiled in 2010 and 2011, respectively. For the first experiment digesta flow at the duodenum was estimated using a dual-phase marker system with ytterbium acetate and chromium ethylenediaminetetraacetic acid as particulate and liquid phase markers, respectively. Total N intake was higher on the RC silages in both experiments and higher on RC− than RC+. Rumen ammonia-N reflected intake with ammonia-N per unit of N intake lower on RC+ than RC−. Microbial N duodenal flow was comparable across all silage diets with non-microbial N higher on RC than the PRG with no difference between RC+ and RC−, even when reported on a N intake basis. C18 PUFA biohydrogenation was lower on RC silage diets than PRG but with no difference between RC+ and RC−. The N balance trial showed a greater retention of N on RC+ over RC−; however, this response is likely related to the difference in N intake over any PPO driven protection. The lack of difference between RC silages, despite contrasting levels of PPO, may reflect a similar level of protein-bound-phenol complexing determined in each RC silage. Previously this complexing has been associated with PPOs protection mechanism; however, this study has shown that protection is not related to total PPO activity.     

Influence of damaging and wilting red clover on lipid metabolism during ensiling and in vitro rumen incubation

This paper describes the relationship between protein-bound phenols in red clover, induced by different degrees of damaging before wilting and varying wilting duration, and in silo lipid metabolism. The ultimate effect of these changes on rumen biohydrogenation is the second focus of this paper. For this experiment, red clover, damaged to different degrees (not damaged (ND), crushing or frozen/thawing (FT)) before wilting (4 or 24 h) was ensiled. Different degrees of damaging and wilting duration lead to differences in polyphenol oxidase (PPO) activity, measured as increase in protein-bound phenols. Treatment effects on fatty acid (FA) content and composition, lipid fractions (free FAs, membrane lipids (ML) and neutral fraction) and lipolysis were further studied in the silage. In FT, red clover lipolysis was markedly lower in the first days after ensiling, but this largely disappeared after 60 days of ensiling, regardless of wilting duration. This suggests an inhibition of plant lipases in FT silages. After 60 days of ensiling no differences in lipid fractions could be found between any of the treatments and differences in lipolysis were caused by reduced FA proportions in ML of wilted FT red clover. Fresh, wilted (24 h) after damaging (ND or FT) and ensiled (4 or 60 days; wilted 24 h; ND or FT) red clover were also incubated in rumen fluid to study the biohydrogenation of C18:3n-3 and C18:2n-6 in vitro. Silages (both 60 days and to a lower degree 4 days) showed a lower biohydrogenation compared with fresh and wilted forages, regardless of damaging. This suggests that lipids in ensiled red clover were more protected, but this protection was not enhanced by a higher amount of protein-bound phenols in wilted FT compared with ND red clover. The reduction of rumen microbial biohydrogenation with duration of red clover ensiling seems in contrast to what is expected, namely a higher biohydrogenation when a higher amount of FFA is present. This merits further investigation in relation to strategies to activate PPO toward the embedding of lipids in phenol-protein complexes.     

Lipid metabolism in mixtures of red clover (Trifolium repens) and perennial ryegrass (Lolium perenne) in lab scale silages and in vitro rumen incubations

Most often, farmers consider red clover an unattractive forage because of its low ensilability. Nevertheless, several in vivo and in vitro experiments also showed advantages of red clover silages such as decreased rumen biohydrogenation of polyunsaturated fatty acids. This has been attributed to a possible protective role of protein-bound phenols, with polyphenol oxidase playing a key role in their formation. This enzyme is active in red clover, but not in other green forages, such as, for example, perennial ryegrass. Therefore, the aim was to study the lipid metabolism within red clover/ryegrass mixtures in lab scale silages and during in vitro rumen batch incubations. Ensilability of red clover increased with higher proportions of ryegrass in the silage mixture. However, the lipid-protecting mechanism of red clover does not seem to occur in the co-ensiled ryegrass as lipolysis of polar lipids linearly increased with increasing proportions of ryegrass (86.0%, 91.6%, 89.9%, 93.1% and 95.6% in 60-day-old silages with 100/0, 75/25, 50/50, 25/75 and 0/100 red clover/ryegrass, respectively). Rumen lipolysis and biohydrogenation of C18:3n-3 and C18:2n-6 were negatively related to red clover proportions in the silage mixtures. The lipid-protective mechanism in red clover silages is confirmed, but it seems not to be transferred to lipids in co-ensiled forages.     

Fermentative profile and lactic acid bacterial dynamics in non-wilted and wilted alfalfa silage in tropical conditions

This study was conducted to evaluate the fermentative profile and microbial populations of wilted and non-wilted alfalfa silages ensiled with or without inoculant and the population dynamics of lactic acid bacteria (LAB) of wilted alfalfa plant and theirs silage. A 2?×?2?×?6 factorial arrangement was used, with the absence or presence of wilting (W), with and without bacterial inoculant (I) and six fermentation periods (P) (1, 3, 7, 14, 28 and 56 days), in a completely randomized design, with three replicates. The alfalfa was slightly wilted for 6 h and increased the dry matter content from 133.9 to 233.4 g/kg. It was performed the cultivation, followed by the isolation of LAB from samples of alfalfa forage before ensiling and its silage only in non-inoculated silages, after different fermentation periods. DNA was extracted from the isolated strains of LAB; the 16S rRNA gene sequences were amplified by PCR and the sequences were compared to those available from the GenBank database. Wilting provided silages with lower pH, ammonia nitrogen and acetic acid concentrations. The wilting process did not alter the amount of LAB; however, it affected the LAB diversity of the silages. The Lactobacillus plantarum was the predominant species in non-wilted and wilted silages.

     

Red clover polyphenol oxidase and lipid metabolism

Increasing the polyunsaturated fatty acid (PUFA) composition of milk is acknowledged to be of benefit to consumer health. Despite the high PUFA content of forages, milk fat contains only about 3% of PUFA and only about 0.5% of n-3 fatty acids. This is mainly due to intensive lipid metabolism in the rumen (lipolysis and biohydrogenation) and during conservation (lipolysis and oxidation) such as drying (hay) and ensiling (silage). In red clover, polyphenol oxidase (PPO) has been suggested to protect lipids against degradation, both in the silage as well as in the rumen, leading to a higher output of PUFA in ruminant products (meat and milk). PPO mediates the oxidation of phenols and diphenols to quinones, which will readily react with nucleophilic binding sites. Such binding sites can be found on proteins, resulting in the formation of protein-bound phenols. This review summarizes the different methods that have been used to assess PPO activity in red clover, and an overview on the current understanding of PPO activity and activation in red clover. Knowledge on these aspects is of major importance to fully harness PPO's lipid-protecting role. Furthermore, we review the studies that evidence PPO-mediated lipid protection and discuss its possible importance in lab-scale silages and further in an in vitro rumen system. It is demonstrated that high (induction of) PPO activity can lead to lower lipolysis in the silage and lower biohydrogenation in the rumen. There are three hypotheses on its working mechanism: (i) protein-bound phenols could directly bind to enzymes (e.g. lipases) as such inhibiting them; (ii) binding of quinones in and between proteins embedded in a lipid membrane (e.g. in the chloroplast) could lead to encapsulation of the lipids; (iii) direct binding of quinones to nucleophilic sites in polar lipids also could lead to protection. There is no exclusive evidence on which mechanism is most important, although there are strong indications that only lipid encapsulation in protein-phenol complexes would lead to an effective protection of lipids against ruminal biohydrogenation. From several studies it has also become apparent that the degree of PPO activation could influence the mode and degree of protection. In conclusion, this review demonstrates that protein-bound phenols and encapsulation in protein-phenol complexes, induced by PPO-mediated diphenol oxidation, could be of interest when aiming to protect lipids against pre-ruminal and ruminal degradation.     

Ruminal large and small particle kinetics in dairy cows fed red clover and grass silages harvested at two stages of growth

Passage, comminution and digestion rates of large and small particles were estimated using a rumen evacuation technique and total faecal collection with five lactating dairy cows in a 5 × 5 Latin square experiment. Two grass and two red clover silages harvested at early and late primary growth stages and a 1:1 mixture of late harvest grass and early harvest red clover were the dietary treatments. Cows received 9.0 kg supplementary concentrate per day. Ruminal contents and faeces were divided into large (>1.25 mm) and small (1.25–0.038 mm) particles by wet sieving. Indigestible neutral detergent fibre (iNDF) was determined by 12 days ruminal in situ incubation followed by neutral detergent extraction. Plant species did not affect ruminal particle size distribution, whereas advancing forage maturity decreased the proportion of large particles for both grass and red clover silage diets. Ruminal pool size of iNDF was higher (P<0.001) with red clover compared to grass silage diets. Ruminal passage rates of iNDF and potentially digestible NDF (pdNDF) increased with decreasing particle size (P<0.01). Passage rate of iNDF for small particles was slower (P<0.01) when red clover compared to grass silage diets were fed. Particle comminution rate in the rumen was slower (P<0.001) with red clover compared to grass silage diets and it increased (P<0.01) with advancing forage maturity. The contribution of particle comminution to ruminal mean retention time of iNDF in the ruminal large particle pool was smaller (P<0.01) in red clover compared to grass silage diets and it increased (P<0.05) with the mixed silage compared to the separate silages. Passage rate of pdNDF for both large and small particles was not affected by dietary treatments. Digestion rate of pdNDF for large particles was faster (P<0.001) with red clover compared to grass silage diets. Differences in ruminal passage and digestion rates of the large and small particles, in addition to differences in the passage and digestion rates of red clover compared to grass silage diets, emphasize the need to consider particle size and forage type in metabolic models predicting feed intake and fibre digestibility in ruminants.     

Intake, rumen fermentation and nutrient flow to the omasum in beef cattle fed grass silage fortified with sucrose and/or supplemented with concentrate

The objective was to determine the relative effects of a specific increase in grass silage sucrose concentration, or a specific supplement of a starch-based concentrate, on rumen fermentation and nutrient supply to the omasum in beef cattle. Four ruminally cannulated Holstein–Friesian steers were fed grass silage only (G), G plus 3 kg concentrates/day (GC), G plus 90 g sucrose/kg dry matter (DM) (GS) and G plus 90 g sucrose/kg DM plus 3 kg concentrates/day (GCS) in a 4 × 4 Latin Square design experiment. Omasal flow was estimated using Co-EDTA, Yb-acetate and indigestible neutral detergent fibre (INDF) as digesta flow markers and purine bases as microbial markers. Concentrate supplementation reduced (P < 0.01) silage and increased (P < 0.001) total DM intake whereas sucrose had no effect. There was a sucrose × concentrate interaction (P < 0.05) for rumen pH whereby addition of sucrose to grass silage alone decreased pH and to grass silage plus concentrate had no effect. Rumen ammonia N (P < 0.01), total volatile fatty acid (VFA) concentration (P < 0.05) and the molar proportions of valerate (P < 0.05) and butyrate (P < 0.001) increased with concentrate supplementation whereas, sucrose supplementation had no effect on rumen fermentation parameters. Organic matter (OM) intake, omasal OM flow, the quantities of OM apparently (OMAD) and truly digested (OMTD) in the rumen (P < 0.001) and total tract OM digestibility (P < 0.01) increased, and apparent and true ruminal OM digestibility decreased (P < 0.05) with concentrate supplementation. Supplementation with concentrate decreased (P < 0.05) ruminal neutral detergent fibre (aNDFom) digestibility and increased (P < 0.05) aNDFom omasal flow. There was a tendency for addition of sucrose to increase (P < 0.1) ruminal OMAD and OMTD, while there was no effect of sucrose addition on intake or digestion of aNDFom. Concentrate supplementation increased (P < 0.001) N intake, flows of N, non-ammonia N (NAN), microbial N (MN) (P < 0.05) and non-ammonia non-microbial N (NANMN) (P < 0.01) and apparent total tract digestibility of N (P < 0.01), whereas sucrose reduced (P < 0.05) N intake and apparent ruminal N digestibility. There was no effect of concentrate or sucrose on N use efficiency or efficiency of microbial protein synthesis. Concentrate supplementation increased (P < 0.001) plasma β-hydroxybutyrate levels. In comparison to supplementing unwilted, well preserved grass silage of moderate digestibility with 3 kg starch-based concentrate per day, the limited response to the rate of sucrose supplementation employed suggests that increasing the water-soluble carbohydrate (WSC) concentration of grass silage through agronomic and/or ensiling practices will have relatively little effect on intake, rumen digestion or efficiency of microbial N synthesis.     

Effects of Bacillus subtilis and its metabolites on corn silage quality

Cellulolytic micro-organisms are potent silage inoculants that decrease the fibrous content in silage and increase the fibre digestibility and nutritional value of silage. This study aimed to evaluate the effects of Bacillus subtilis CCMA 0087 and its enzyme β-glucosidase on the nutritional value and aerobic stability of corn silage after 30 and 60 days of storage. We compared the results among silage without inoculant (SC) and silages inoculated with B. subtilis 8 log₁₀ CFU per kg forage (SB8), 9 log₁₀ CFU per kg forage (SB9) and 9·84 log₁₀ CFU per kg forage + β-glucosidase enzyme (SBE). No differences were observed in the levels of dry matter, crude protein and neutral detergent fibre due to the different treatments or storage times of the silos. Notably, the population of spore-forming bacteria increased in the SB9-treated silage. At 60 days of ensiling, the largest populations of lactic acid bacteria were found in silages treated with SB8 and SBE. Yeast populations were low for all silages, irrespective of the different treatments, and the presence of filamentous fungi was observed only in the SBE-treated silage. Among all silage treatments, SB9 treatment resulted in the highest aerobic stability.     

Characterization and Identification of Pediococcus Species Isolated from Forage Crops and Their Application for Silage Preparation

Pediococcus species isolated from forage crops were characterized, and their application to silage preparation was studied. Most isolates were distributed on forage crops at low frequency. These isolates could be divided into three (A, B, and C) groups by their sugar fermentation patterns. Strains LA 3, LA 35, and LS 5 are representative isolates from groups A, B, and C, respectively. Strains LA 3 and LA 35 had intragroup DNA homology values above 93.6%, showing that they belong to the species Pediococcus acidilactici. Strain LS 5 belonged to Pediococcus pentosaceus on the basis of DNA-DNA relatedness. All three of these strains and strain SL 1 (Lactobacillus casei, isolated from a commercial inoculant) were used as additives to alfalfa and Italian ryegrass silage preparation at two temperatures (25 and 48°C). When stored at 25°C, all of the inoculated silages were well preserved and exhibited significantly (P < 0.05) reduced fermentation losses compared to that of their control in alfalfa and Italian ryegrass silages. When stored at 48°C, silages inoculated with strains LA 3 and LA 35 were also well preserved, with a significantly (P < 0.05) lower pH, butyric acid and ammonia-nitrogen content, gas production, and dry matter loss and significantly (P < 0.05) higher lactate content than the control, but silages inoculated with LS 5 and SL 1 were of poor quality. P. acidilactici LA 3 and LA 35 are considered suitable as potential silage inoculants.     

Effects of formic acid,molasses and inoculant additives on corn silage composition,organic matter digestibility and microbial protein synthesis in sheep

This study examined the production of com silages with low or high lactic acid concentrations, provided by the addition of formic acid (0.5%), molasses (5%) or microbial inoculant (homofermentative lactic acid bacteria, 10 g/tonne). After the fermentation period, sheep were fed the silages to determine true and apparent digestibility of the organic matter and microbial protein synthesis. The experiment were carried out with four KıvırcıkxMorkaraman sheep, 1.5 years old, fixed with cannula in their rumen and duodenum.Lactic acid concentrations were significantly higher in silages treated with enzyme or molasses compared to other specific treatments. Acetic acid concentration was highest in silage treated with formic acid, and lowest in silage treated with molasses (P < 0.05). The by-pass of crude protein was highest in silage treated with formic acid.     

Both substrate availability and utilisation contribute to the defence of core temperature in response to acute cold

Acute cooling significantly increases energy demand in non-hibernators for the defence of core temperature but the contribution of the liver to thermogenesis is poorly understood. A two-tracer method to estimate lipid metabolism in cold-naïve control (CON) and cold-acclimated (CA) rats was employed to quantify hepatic rates of fat metabolism. Both fenofibrate, to increase liver mass and fat oxidation and dichloroacetate (DCA) to inhibit fat oxidation were used to alter lipid metabolism in CON animals. Following acute cooling, CA led to a doubling of the time to reach a core temperature 25 °C (P < 0.001), whereas DCA treatment decreased time of cooling (P < 0.01). DCA-treatment increased the gradient of Arrhenius-transformed rate–pressure product (P < 0.01). CA increased both palmitate uptake (P < 0.001) and β-oxidation (P < 0.01) whilst DCA treatment decreased uptake (P < 0.01) and β-oxidation (P < 0.05). Tissue-specific estimates of metabolism revealed that CA led to a 12-fold increase in β-oxidation for brown adipose tissue (P < 0.001) whilst fenofibrate halved β-oxidation in the liver (P < 0.01) despite doubling the liver mass (P < 0.001) and DCA decreased hepatic β-oxidation to 15% of control levels. Taken together, these results suggest that the liver has minimal contribution to thermogenesis in the rat, with brown adipose tissue significantly increasing both fat uptake and oxidation in response to CA.     

Effect of conjugated linoleic acid on dietary lipids utilization, liver morphology and selected immune parameters in sea bass juveniles (Dicentrarchus labrax)

Increased energy content in fish feeds has led to an enhanced fat deposition, particularly in European sea bass, concerning fish farmers. Inclusion of conjugated linoleic acid (CLA) could reduce fat deposition as in other vertebrates. To determine if dietary CLA affects fat deposition, lipid metabolism, lipid composition and morphology of different tissues, growth and selected immune parameters, European sea bass juveniles were fed 4 graded levels of CLA (0, 0.5, 1 and 2%). Growth and feed conversion were not affected by CLA, whereas feed intake was reduced (P < 0.05) by feeding 2% CLA. In these fish perivisceral fat was also reduced (P < 0.05), particularly reducing (P < 0.05) monounsaturated fatty acids. CLA has not affected tissue proximal composition, but reduced (P < 0.05) saturated and monounsaturated fatty acids and increased (P < 0.05) the n−3 and n−3 highly unsaturated fatty acids in muscle and increase (P < 0.05) CLA content in muscle, liver and perivisceral fat. A progressive reduction in lipid vacuolization of hepatocytes cytoplasm and regular-shaped morphology was found in fish fed increased CLA levels, together with a progressive increase in malic enzyme activity (only significant in fish fed 1% CLA). Finally, inclusion of CLA up to 1% increased (P < 0.05) plasma lysozyme activity and was positively correlated with alternative complement pathway.     

Positive effects of Taxol pretreatment on morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrification of in vitro matured porcine oocytes

This study was designed to determine the effects of Taxol pretreatment on the morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrified porcine oocytes matured in vitro. The result showed that: (1) the rate of normal mitochondria distribution in fresh group (92.85%) was significantly higher (P < 0.05) than that in other three groups (toxicity, 72.48%; vitrification, 50.83%; Taxol + vitrification, 69.98%) and Taxol pretreatment significantly (P < 0.05) increased the ratio of normal mitochondria distribution in vitrified oocytes; (2) lipid droplets in vitrified oocytes got cracked, resulting in a great number of smaller lipid droplets (diameter <5 μm). The number of lipid droplets (5–10 μm in diameter) in vitrified oocytes pretreated with Taxol was higher (P < 0.05) than that in the oocytes without Taxol pretreatment (81.87 ± 13.63 vs. 64.27 ± 13.72); (3) both toxicity and vitrification cause the difference in the ultrastructure of mitochondria and lipid droplets. Mitochondria were well maintained in the form of typical round and ellipse shape with smooth surface and clear outline and lipid droplets existed in the form of integrity in Taxol pretreatment group.In conclusion, Taxol pretreatment has positive effects on vitrified porcine oocytes matured in vitro in terms of morphology, distribution and ultrastructure of mitochondria and lipid droplets.     

Effect of harvest time of red and white clover silage on chewing activity and particle size distribution in boli,rumen content and faeces in cows

The study examined the effects of harvest time of red and white clover silage on eating and ruminating activity and particle size distribution in feed boli, rumen content and faeces in cows. The clover crops were harvested at two stages of growth and ensiled in bales. Red clover crops had 36% and 45% NDF in dry matter (DM) at early (ER) and late (LR) harvest, respectively, and the white clover crops had 19% and 29% NDF in DM at the early (EW) and late (LW) harvest, respectively. The silages were fed restrictively (80% of ad libitum intake) twice daily to four rumen cannulated non-lactating Jersey cows (588 ± 52 kg) in a 4 × 4 Latin square design. Jaw movements (JM) were recorded for 96 h continuously. Swallowed boli, rumen mat, rumen fluid and faeces samples were collected, washed in nylon bags (0.01 mm pore size) and freeze-dried before dry sieving through 4.750, 2.360, 1.000, 0.500, 0.212 and 0.106 mm into seven fractions. The length (PL) and width (PW) values of rumen and faeces particles within each fraction were measured by use of image analysis. The eating activity (min/kg DM intake; P < 0.05) was higher in LR compared with the other treatments. The eating activity (min/kg NDF intake; P < 0.05) was affected by clover type with highest values for white clover silage. The mean ruminating time (min/kg DM), daily ruminating cycles (P < 0.001) and JM during ruminating (P < 0.05) were affected by treatment with increasing values at later harvest time. The proportion of washed particle DM of total DM in boli (P < 0.001), rumen mat (P < 0.001), rumen fluid (P < 0.01) and faeces was (P < 0.001) highest by feeding LR. There were identified two peaks (modes 1 and 2) on the probability density distribution (PDF) of PW values of rumen mat and faeces, but only one peak (mode 1) for PL values. There was no difference in the mean and mode 1 PW and PL value in rumen mat between the four treatments. The mean PL, mode PL, mode 2 PW and mean PW in faeces were highest for LR (P < 0.05). The mean particle size in boli measured by sieving was higher at white clover compared with red clover treatments (P < 0.001) and the highest value in faeces was found in LR (P < 0.01). The two peaks on PDF for width values of rumen mat and faeces particles are most likely related to the leaves and the stems/petioles. In conclusion, the mean total chewing activity per kg DM was lowest for the white clover silage and increased for both silages due to later harvest time. The mean particle size in boli was smallest for LR, whereas the mean PL and PW in faeces were highest for the LR.     

Intake, digestibility, rumen fermentation and performance of beef cattle fed diets based on whole-crop wheat or barley harvested at two cutting heights relative to maize silage or ad libitum concentrates

This experiment aimed to quantify the relative intake, digestibility, rumen fermentation, performance and carcass characteristics of beef cattle fed diets based on good quality whole-crop wheat and barley silages, each harvested at two cutting heights, and to rank these relative to good quality maize silage and an ad libitum concentrates-based diet. Ninety beef steers, initial live-weight 438 ± 31.0 kg, were allocated to one of the following dietary treatments in a randomised complete block design: maize silage (MS), whole-crop wheat harvested at a normal cutting height (WCW) (stubble height 0.12 m) or an elevated cutting height (HCW) (stubble height 0.29 m), whole-crop barley harvested at a normal cutting height (WCB) (stubble height 0.13 m) or an elevated cutting height (HCB) (stubble height 0.30 m), each being supplemented with 3 kg concentrates/head/day, and ad libitum concentrates (ALC) supplemented with 5 kg grass silage/head/day for the duration of the 160-day study. Mean dry matter (DM) of the maize silage, whole-crop wheat, head-cut wheat, whole-crop barley and head-cut barley was 301, 488, 520, 491 and 499 g/kg, respectively. There were no differences in total DM intake among treatments, or in rumen fermentation characteristics (except ammonia), or in DM digestibility among the forage-based treatments. Neutral detergent fibre digestibility was lower (P<0.05) for whole-crop wheat than head-cut barley, and starch digestibility was lower (P<0.05) for whole-crop barley and head-cut barley than maize silage. Steers fed ALC had a higher carcass gain (P<0.001) and carcass weight (P<0.05) than all other treatments, but there were no differences between any of the forage-based treatments. Steers fed MS had a better feed conversion efficiency (FCE) than those on WCW or WCB (P<0.05) but were similar to HCW and HCB. The FCE was better for ALC versus any of the other treatments, particularly compared to WCW or WCB (P<0.001). Subcutaneous fat from steers fed ALC was more yellow (P<0.01) than that from steers fed the other treatments. Neither intake nor performance were altered by raising the cutting height of cereals or by replacing whole-crop wheat by barley. However, head-cut cereals numerically favoured DM intake, carcass gain and feed conversion efficiency values nearer to that of maize than whole-crop cereal silages. Ad libitum concentrates supported superior levels of growth by steers compared to all other treatments.     

Effect of combined ensiling of sorghum and soybean with or without molasses and lactobacilli on silage quality and in vitro rumen fermentation

Two sorghum (Sorghum bicolor (L.) Moench) varieties (SG₁ & SG₂), with the former showing higher grain and total DM yield, but also increased tannin contents compared to the latter, and one soybean (Glycine max (L.) Merr.) variety (INCASOY-35) were sown, harvested (at pasty grain state), chopped and ensiled (CIAP, Cuba). Silages were made in following combinations: either SG₁ or SG₂ combined with soybean in two proportions (0.4 and 0.6). All silages were prepared with or without molasses (3.5% of fresh material) and Lactobacillus sp. as inoculant (3 × 10⁵ colony forming units/g). Silage quality parameters included pH, ammonia, lactate, acetate, butyrate and water soluble carbohydrates content. Further, both fresh and ensiled materials were incubated in vitro with buffered rumen fluid to study the fermentation characteristics. Silage of a good quality could be produced with both sorghum varieties alone, but combined silages showed improved quality compared to soybean silage (p<0.05 for all quality characteristics). Addition of molasses and bacterial inoculant further improved silage quality (p<0.05 for all quality characteristics). In vitro incubation (24 h) of ensiled material resulted in lower acetate and higher propionate proportion compared to fresh forages. However, ensiling without molasses and inoculant reduced in vitro short chain fatty acid production and hence the apparent rumen degradability of organic mater as well as the fermentation rate. As expected, a higher proportion of sorghum increased the molar propionate proportion and the fractional fermentation rate, whereas ammonia (mmol/L) concentrations were reduced. SG₁ silages produced higher molar propionate proportions, lower acetate proportions and ammonia concentrations.     

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 B₁ 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 B₂ and B₃, 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.     

Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa

Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%-83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.     

Effects of <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> on fermentation,aerobic stability,bacteria diversity and ruminal degradability of alfalfa silage

Silages are important feedstuffs. Homofermentative lactic acid bacterial inoculants are often used to control silage fermentation. However, some research pointed out those homofermentative lactic acid bacteria (LAB) impaired the aerobic stability of wheat, sorghum, and corn silages. Adding heterofermentative LAB can produce more acetic acid, thereby stabilizing silages during aerobic exposure. Alfalfa is difficult to ensile. The present work was to study the effects of L. buchneri (heterofermentative LAB), alone or in combination with L. plantarum (homofermentative LAB) on the fermentation, aerobic stability, bacteria diversity and ruminal degradability of alfalfa silage. After 90 days ensiling, the pH, NH₃-N/TN, butyric acid content and molds counts of control were the highest. The inoculated silages had more lactic acid, acetic acid content and more lactic acid bacteria than the control. Inoculating LAB inhibited harmful microorganisms, such as Enterobacterium and Klebsiella pneumoniae. The L. buchneri + L. plantarum-inoculated silage had more acetic acid and less yeasts than other three treatments (P < 0.05), and lower NH₃-N/TN than control (P < 0.05). The CO₂ production of L. buchneri + L. plantarum-inoculated silage was less than that of L. plantarum-inoculated silage (P < 0.05). Inoculating LAB in alfalfa silages can decrease pH, increase the production of lactic and acetic acids, reduce the number of yeasts and molds, and inhibit Enterobacterium and K. pneumoniae. Inoculating with L. buchneri or L. buchneri + L. plantarum can improve aerobic stability of alfalfa silages. A combination of L. buchneri and L. plantarum is preferable because it enhanced alfalfa silage quality and aerobic stability.