Evaluation of different ensiling methods for Saccharina latissima preservation: influence on chemical composition and in vitro ruminal fermentation.

Saccharina latissima is a brown seaweed that could be used in ruminant feeding, but its fast deteriorating and seasonal growth nature limit their utilisation in the practice. Ensiling could be used as a preservation method, but information of its effects on the nutritional value of the seaweed is limited. This study evaluated the in vitro ruminal fermentation of different S. latissima silages using ruminal inoculum either from goats fed a mixed diet (60:40 oat hay:concentrate) or from sheep fed a high-forage diet (90:10 alfalfa hay:concentrate) to simulate different small ruminant production systems. S. latissima was ensiled in vacuum bags without additives (Control), with formic acid (4 g/kg seaweed; FA), with lactic acid bacteria (LAB) or with LAB after a pre-wilting treatment to reach a seaweed dry matter (DM) content of 30% (30LAB). Ensiling S. latissima decreased (p < 0.05) the content in DM, neutral detergent fibre and total extractable polyphenols, but nitrogen and fat content were unaffected. For both ruminal inoculums, ensiling decreased (p < 0.05) the asymptotic gas production after 120 h of fermentation (excepting for FA silage with goats' inoculum), but the total volatile fatty acid (VFA) production was unaffected. The VFA profile shifted towards greater (p < 0.05) acetate and lower (p < 0.05) propionate proportions in all silages compared with the pre-ensiling S. latissima. When goats inoculum was used, greater (p < 0.05) CH4 production compared with pre-ensiling S. latissima was observed in all silages, except Control one, which led to greater (p < 0.05) CH4/total VFA ratio. In contrast, no differences among samples (p > 0.05) in either CH4 production or CH4/total VFA ratio were observed when sheep' inoculum was used. Fermentation of all samples started earlier with goats' inoculum than with sheep' inoculum, which was attributed to the different diet fed to the animals. These results suggest that ensiling S. latissima with either formic acid or lactic acid bacteria could be a viable conservation method to preserve the nutritive value.

Utilization of Avocado and Mango Fruit Wastes in Multi-Nutrient Blocks for Goats Feeding: In Vitro Evaluation.

This study was conducted to investigate the nutritive value of avocado and mango fruit wastes, and to assess the possibility of preserving the wastes into multi-nutrient blocks (MB). Both peels and a pulp:peels (PP) mixture of each fruit were analyzed for chemical composition and in vitro fermentation with goats' ruminal fluid. Wastes had low-dry matter (DM) content (<250 g/kg), with those from mango having high non-structural carbohydrates content (>800 g/kg DM) and those from avocado high fat levels (>580 g/kg DM). Mango wastes were fermented at a greater extent and faster rate than avocado ones. The PP mixture of each fruit was included into multi-nutrient blocks (MB) formulated to have similar chemical composition. There were only subtle differences in the fermentation of MB including wastes from either avocado or mango, but fermentation of avocado-MB resulted in significantly (p ≤ 0.032) greater acetate and lower propionate proportions than mango-MB. Including the PP mixture in the formulation of MB for goats feeding is a feasible option to reduce the environmental impact of avocado and mango fruit wastes, but studies on the acceptance of the MB by goats and their stability over long-time storage periods are needed.

Potential of Recycling Cauliflower and Romanesco Wastes in Ruminant Feeding: In Vitro Studies.

The nutritive values for ruminants of cauliflower (CAU) and Romanesco (ROM) wastes (leaves, stems and sprouts) were assessed by analyzing their chemical composition, in vitro ruminal fermentation, and in vitro intestinal digestibility. In addition, the in vitro ruminal fermentation of diets containing increasing amounts of CAU was studied. The dry matter (DM) content of leaves, stems and sprouts of both vegetables was lower than 10%, but they contained high crude protein (CP; 19.9 to 33.0%) and sugar (16.3 to 28.7%) levels, and low neutral detergent fiber (21.6 to 32.3%). Stems and sprouts were more rapidly and extensively fermented in the rumen than leaves, but there were only minor differences the fermentation profiles of both vegetables. The estimated metabolizable energy content ranged from 9.3 (leaves) to 10.8 (sprouts) MJ/kg DM. The CP rumen degradability (12-h in situ incubations) was greater than 80.0% for all fractions, and the in vitro intestinal digestibility of CP ranged from 85.7 to 93.2%. The inclusion of up to 24% of dried CAU in the concentrate of a mixed diet (40:60 alfalfa hay:concentrate) increased the in vitro rumen fermentation of the CAU diet, but did not affect methane (CH4) production, indicating the lack of antimethanogenic compounds in CAU.

Ruminal Fermentation, Growth Rate and Methane Production in Sheep Fed Diets Including White Clover, Soybean Meal or Porphyra sp.

The aim of the present work was to investigate the potential of Porphyra sp. as an alternative source of protein to soybean meal in diets for sheep. Our experimental treatments included a control diet (CON) based on grass silage and crushed oats and three diets containing protein supplements, clover silage (CLO), soybean meal (SOY) or Porphyra sp. (POR) to increase dietary crude protein concentrations. We studied its effects on rumen fermentation, growth rate and methane emissions. Ruminal fermentation characteristics, kinetics of gas production and methane production were studied in vitro by using batch cultures inoculated with rumen inoculum from sheep. There were no differences among diets in total volatile fatty acids (VFA) production or in the VFA profile in vitro. Across treatments, we measured no differences in methane production either in vitro or in vivo, and we saw no noticeable antimethanogenic effect of Porphyra sp. The present in vivo trial with lambs showed no differences in average daily weight gain when fed diets including Porphyra sp. or soybean meal diets (250 and 254 g/d, respectively). We conclude that Porphyra sp. has a protein value similar to high-quality protein sources like soybean meal.

Effects of Feeding Multinutrient Blocks Including Avocado Pulp and Peels to Dairy Goats on Feed Intake and Milk Yield and Composition.

Twelve Murciano-Granadina dairy goats were divided into two homogeneous groups, which were fed either a control diet composed of 40% alfalfa hay and 60% concentrate or a diet based on 40% alfalfa hay, 40% concentrate and 20% multinutrient blocks, including 14.8% avocado pulp and peels (APP). Total dry matter (DM) intake was similar (p = 0.709) for both diets, but APP-fed goats had lower (p = 0.024) concentrate intake and tended (p = 0.063) to have lower fat intake compared with those fed the control diet. The average intake of blocks was low (66.4 g DM/d), which was attributed to avocado lipids oxidation and rancidity. Neither milk yield (p = 0,921) nor the efficiency of energy and nitrogen use were affected (p = 0.909 and 0. 840, respectively) by the diet, but milk fat tended to be greater (p = 0.057) in the APP-fed goats compared with the animals fed the control diet. Other milk components were similar (p ≥ 0.110) for both diets, and only subtle changes in the milk fatty acid profile were observed. In summary, the intake of blocks containing avocado wastes by dairy goats was low probably due to avocado lipids oxidation causing off-flavors and reduced palatability.

Variability and Potential of Seaweeds as Ingredients of Ruminant Diets: An In Vitro Study.

This study was designed to analyze the chemical composition and in vitro rumen fermentation of eight seaweed species (Brown: Alaria esculenta, Laminaria digitata, Pelvetia canaliculata, Saccharina latissima; Red: Mastocarpus stellatus, Palmaria palmata and Porphyra sp.; Green: Cladophora rupestris) collected in Norway during spring and autumn. Moreover, the in vitro ruminal fermentation of seventeen diets composed of 1:1 oat hay:concentrate, without (control diet) or including seaweeds was studied. The ash and N contents were greater (p < 0.001) in seaweeds collected during spring than in autumn, but autumn-seaweeds had greater total extractable polyphenols. Nitrogen in red and green seaweeds was greater than 2.20 and in brown seaweeds, it was lower than 1.92 g/kg DM. Degradability after 24 h of fermentation was greater in spring seaweeds than in autumn, with Palmaria palmata showing the greatest value and Pelvetia canaliculata the lowest. Seaweeds differed in their fermentation pattern, and autumn Alaria esculenta, Laminaria digitata, Saccharina latissima and Palmaria palmata were similar to high-starch feeds. The inclusion of seaweeds in the concentrate of a diet up to 200 g/kg concentrate produced only subtle effects on in vitro ruminal fermentation.

Nutritive Value of Tomato Pomace for Ruminants and Its Influence on In Vitro Methane Production.

The objective of this study was to determine the variability in nutritive value for ruminants of tomato pomace (TP) samples and analyze its effect on in vitro fermentation when it was included in a high-concentrate diet. Twelve TP samples were obtained from two processing plants at weekly intervals and analyzed for chemical composition, in vitro rumen fermentation, and intestinal digestibility. The chemical composition of TP did not differ between processing plants and only slight variations were observed among sampling times. Tomato pomace had a low dry matter content (<300 g/kg), a high content of neutral detergent fiber, crude protein, and ether extract (572, 160, and 82.7 g/kg dry matter on average, respectively), and was rapidly fermented in the rumen. Protein degradability at 16 h in situ incubation was 510 g/kg and in vitro intestinal digestibility of protein was low (430-475 g/kg). Replacing soybean meal and barley straw by dried TP increased the in vitro fermentation rate and the production of volatile fatty acids and reduced NH3-N concentrations without affecting CH4. In summary, TP samples showed little variability in nutritive value over sampling time and TP of up to 180 g/kg could be included in high-concentrate diets without negatively affecting rumen fermentation.

Evaluation of the promiscuous component of several bacterial export pumps TolC as a biomarker for toxic pollutants in feedstuffs.

A significant risk to the food chain is the presence of noxious pollutants in the feeds of animals whose products are used in human nutrition. Consequently, analytical methods and biosensors have been developed to detect these types of contaminates in feeds. Here we have evaluated whether the expression of TolC, a promiscuous component of several ATP-dependent efflux pumps in E. coli, up-regulated in response to chemical stress, could be a useful biomarker for this aim. Changes in TolC expression in response to toxic compounds, with different abilities to induce DNA damage, were determined using two E. coli strains with (DH5α) and without (BL21(DE3)) inactivating mutation in RecA gene. Deoxycholic acid and potassium dichromate up-regulated TolC in both strains. In contrast, cisplatin-induced TolC up-regulation was abolished in the absence of a functional RecA. When the effect of several insecticides, herbicides, antibiotics and common soil pollutants on TolC expression was analyzed, a relationship between toxicity and their ability to up-regulate TolC was observed. However, this was not a general event because the insecticide α-cipermetrin induced a reduction in cell viability, which was not accompanied by TolC up-regulation. In contrast, the soil pollutant benzene was able to stimulate TolC expression at non-toxic concentrations. When this test was used to analyze aqueous extracts from different feedstuffs, up-regulation of TolC was found in the absence of cell toxicity and was even accompanied by enhanced cell viability. In conclusion, TolC expression is partly dependent on the integrity of the RecA/LexaA system. Although toxic compounds up-regulate TolC in a dose-dependent manner, this response is also activated by non-toxic agents. Thus, owing to its poor specificity regardless of its sensitivity, the use of TolC up-regulation in E. coli to detect the presence of toxic pollutants in conventional and unconventional sources of nutrients for ruminant feeding requires supplementary biomarkers.

Ruminal metagenomic libraries as a source of relevant hemicellulolytic enzymes for biofuel production.

The success of second-generation (2G) ethanol technology relies on the efficient transformation of hemicellulose into monosaccharides and, particularly, on the full conversion of xylans into xylose for over 18% of fermentable sugars. We sought new hemicellulases using ruminal liquid, after enrichment of microbes with industrial lignocellulosic substrates and preparation of metagenomic libraries. Among 150 000 fosmid clones tested, we identified 22 clones with endoxylanase activity and 125 with ß-xylosidase activity. These positive clones were sequenced en masse, and the analysis revealed open reading frames with a low degree of similarity with known glycosyl hydrolases families. Among them, we searched for enzymes that were thermostable (activity at > 50°C) and that operate at high rate at pH around 5. Upon a wide series of assays, the clones exhibiting the highest endoxylanase and ß-xylosidase activities were identified. The fosmids were sequenced, and the corresponding genes cloned, expressed and proteins purified. We found that the activity of the most active ß-xylosidase was at least 10-fold higher than that in commercial enzymatic fungal cocktails. Endoxylanase activity was in the range of fungal enzymes. Fungal enzymatic cocktails supplemented with the bacterial hemicellulases exhibited enhanced release of sugars from pretreated sugar cane straw, a relevant agricultural residue.

Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques for analysing the influence of diet on ruminal bacterial diversity.

The objective of this study was to compare the automated ribosomal intergenic spacer analysis (ARISA) and the denaturing gradient gel electrophoresis (DGGE) techniques for analysing the effects of diet on diversity in bacterial pellets isolated from the liquid (liquid-associated bacteria (LAB)) and solid (solid-associated bacteria (SAB)) phase of the rumen. The four experimental diets contained forage to concentrate ratios of 70:30 or 30:70 and had either alfalfa hay or grass hay as forage. Four rumen-fistulated animals (two sheep and two goats) received the diets in a Latin square design. Bacterial pellets (LAB and SAB) were isolated at 2 h post-feeding for DNA extraction and analysed by ARISA and DGGE. The number of peaks in individual samples ranged from 48 to 99 for LAB and from 41 to 95 for SAB with ARISA, and values of DGGE-bands ranged from 27 to 50 for LAB and from 18 to 45 for SAB. The LAB samples from high concentrate-fed animals tended (p < 0.10) to show greater peak numbers and Shannon index values than those isolated from high forage-fed animals with ARISA, but no differences were identified with DGGE. The SAB samples from high concentrate-fed animals had lower (p < 0.05) peak numbers and Shannon index values than those from animals fed high-forage diets with ARISA, but only a trend was noticed for these parameters with DGGE (p < 0.10). The ARISA detected that animals fed alfalfa hay diets showed lower (p < 0.05) SAB diversity than those fed grass hay diets, but no differences were observed with DGGE. No effect of forage type on LAB diversity was detected by any technique. In this study, ARISA detected some changes in ruminal bacterial communities that were not detected by DGGE, and therefore ARISA was considered more appropriate for assessing bacterial diversity of ruminal bacterial pellets. The results highlight the impact of the fingerprinting technique used to draw conclusions on dietary factors affecting bacterial diversity in ruminal bacterial pellets.

Biodetection of potential genotoxic pollutants entering the human food chain through ashes used in livestock diets.

Ash derived from energy generation is used as a source of minerals in livestock feeds. The microbial biosensor recApr-Luc2 was built to detect genotoxic hazard in recycled ash. Escherichia coli SOS gene (recA, lexA, dinI and umuC) expression in response to cisplatin-induced DNA damage led to the selection of the recA promoter. The biosensor required functional RecA expression to respond to genotoxic heavy metals (Cr>Cd≈Pb), and polluted ash induced a strong recApr-Luc2 response. In human liver and intestinal cells, heavy metals induced acute toxicity (Cr>Cd>Pb) at concentrations sufficient to activate recApr-Luc2. Cytostatic effects, including genotoxicity, were cell- and metal-dependent, apart from Cr. In agreement with the recApr-Luc2 bioassay, Cr had the strongest effect in all cells. In conclusion, recApr-Luc2 could be useful for evaluating the genotoxic risk of pollutants present in ash that might be concentrated in animal products and, thus, entering the human food chain.

Response of the rumen archaeal and bacterial populations to anti-methanogenic organosulphur compounds in continuous-culture fermenters.

Study of the efficacy of methanogenesis inhibitors in the rumen has given inconsistent results, mainly due to poorly understood effects on the key microbial groups involved in pathways for methane (CH4) synthesis. The experiment described in this report was designed to assess the effect of propyl propane thiosulfinate (PTS), diallyl disulfide (DDS) and bromochloromethane (BCM) on rumen fermentation, methane production and microbial populations in continuous culture fermenters. No effects on total volatile fatty acids (VFA) were observed with PTS or DDS, but VFA were decreased with BCM. Amylase activity increased with BCM as compared with the other treatments. A decrease in methane production was observed with PTS (48%) and BCM (94%) as compared with control values. The concentration of methanogenic archaea decreased with BCM from day 4 onward and with PTS on days 4 and 8. Pyrosequencing analysis revealed that PTS and BCM decreased the relative abundance of Methanomicrobiales and increased that of Methanobrevibacter and Methanosphaera. The total concentration of bacteria was not modified by any treatment, although treatment with BCM increased the relative abundance of Prevotella and decreased that of Ruminococcus. These results suggest that the inhibition of methane production in the rumen by PTS and BCM is associated with a shift in archaeal biodiversity and changes in the bacterial community with BCM.

A comparison of different legume seeds as protein supplement to optimise the use of low quality forages by ruminants.

The potential of different legume seeds species, including recently new developed varieties (Vicia faba: a commercial variety and varieties Alameda, Palacio and Baraka; Lupinus angustifolius; Pisum sativum and Cicer arietinum: varieties Fardon and Zegri) as protein supplements to low quality forages was evaluated. First, the chemical composition, in vitro digestibility, in situ degradability and in vitro/in situ intestinal digestibility of legume seeds were determined. The chemical composition was rather similar within genus. Vicia faba beans contained more condensed tannins (35.8-56.4 g/kg DM) and less ether extract (12.8-9.5 g/kg DM) than the other legumes. The rumen degradability and intestinal digestibility of the undegraded protein in the rumen was very similar among the seeds, with exception of lupins, having a much higher degradation rate than the rest. Second, the fermentation characteristics of diets, which were based on two low quality forages (olive leaves and barley straw) and feed blocks without or with supplementation of legume seeds or soybean meal, were investigated by using single-flow continuous culture fermenters. In this trial, the fermentation parameters (ammonia, pH and volatile fatty acids), the microbial protein synthesis and the degradation of olive leaves and barley straw promoted by the different diets were studied. Compared to soybean meal, beans and peas showed similar suitability as protein supplements for sustaining in vitro fermentation of low-quality forages. However, our results suggest a significant interaction between the type of legume used as supplement and the type of forage used, which need to be further studied in vivo.