Use of an in vitro gas production method to investigate interactions between veld hay and Napier hay or groundnut hay supplements

Measurement of gas produced during in vitro fermentation was used to investigate the fermentability of poor quality natural pasture (veld) hay mixed with different amounts of Napier hay or groundnut hay. In vitro fermentations were conducted in nitrogen-rich (NR) and nitrogen-free (NF) media. Groundnut hay was more rapidly fermented than Napier hay, the nitrogen content of the medium making little difference to the fermentation characteristics of either hay. Veld hay was the least fermentable substrate, particularly when NF medium was used. Statistically significant positive interactive effects were observed between both supplements and veld hay fermented in both media as gauged by gas production and dry matter disappearance. Evidence of significant interactions had not been obtained from earlier measurements of in vivo digestibility using the same feeds, but they may have been obscured by increased rates of passage with increased supplementation. Differences between gas productions in NR and NF media were explored as possible indicators of nitrogen deficiency in feed mixtures. Both Napier hay and groundnut hay appeared to be balanced in terms of energy and nitrogen fermentable by rumen microbes.     

Production of aflatoxin by an Aspergillus flavus isolate cultured under a limited oxygen supply

In a previous experiment on the preservation of hay of high moisture content with formic acid, among other agents, aflatoxin was formed in the hay, and aflatoxin-forming strains of Aspergillus flavus were isolated from this hay after incubation in air as well as in an anaerobic jar. One isolate from the anaerobic jar was cultivated in a chemostat (Bioflo model C 30; New Brunswick Scientific Co.) in a defined medium with added B vitamins, yeast extract, or formic acid, with or without gas flow (air or nitrogen). In all cases where spore germination occurred, aflatoxin was formed in the cultures with gas flow, and small quantities of aflatoxins B1 and B2 occurred even in an atmosphere of nitrogen. Addition of B vitamins and supply of traces of air gave an approximately 15-fold increase in the amount of aflatoxin in 2 days. Carbon dioxide enrichment hindered aflatoxin formation on the defined medium even in the presence of B vitamins, but when formic acid was added, small quantities (5 to 15 micrograms/liter) were formed, and this low level remained constant until the gas flow was started.     

Production of aflatoxin by an Aspergillus flavus isolate cultured under a limited oxygen supply.

In a previous experiment on the preservation of hay of high moisture content with formic acid, among other agents, aflatoxin was formed in the hay, and aflatoxin-forming strains of Aspergillus flavus were isolated from this hay after incubation in air as well as in an anaerobic jar. One isolate from the anaerobic jar was cultivated in a chemostat (Bioflo model C 30; New Brunswick Scientific Co.) in a defined medium with added B vitamins, yeast extract, or formic acid, with or without gas flow (air or nitrogen). In all cases where spore germination occurred, aflatoxin was formed in the cultures with gas flow, and small quantities of aflatoxins B1 and B2 occurred even in an atmosphere of nitrogen. Addition of B vitamins and supply of traces of air gave an approximately 15-fold increase in the amount of aflatoxin in 2 days. Carbon dioxide enrichment hindered aflatoxin formation on the defined medium even in the presence of B vitamins, but when formic acid was added, small quantities (5 to 15 micrograms/liter) were formed, and this low level remained constant until the gas flow was started.     

Impaired semen quality of AI bulls fed with moldy hay: a case report

The daily quality control of semen at a Finnish artificial insemination (AI) bull station is based on subjective motility and sperm morphology of young bulls entering the semen collection program. Semen quality dropped suddenly in autumn 1998. During 5 consecutive months, the number of rejected ejaculates and discarded frozen semen batches due to poor motility increased, and the number of all forms of abnormal spermatozoa increased. However, for the accepted ejaculates, a 60 day nonretum rate was normal. The summer of 1998 in Finland was rainy, and the hay used in the AI station was visibly moldy. Immunoassay and gas chromatography-mass spectrometry (GC-MS) detected Fusarium mycotoxins HT-2 and T-2, but no zearalenone in the hay. Occurrence of mycotoxins such as T-2 and HT-2 in the moldy hay coincided with, and may have been responsible for the impaired semen quality in AI bulls. This case report will draw the attention to the possible hazards when feeding moldy hay.     

Evaluation of a Method of Measuring Fermentation Rates and Net Growth of Rumen Microorganisms

The method of el-Shazly and Hungate for measuring gas production in rumen contents was slightly modified and used throughout this investigation. The variation in the fermentation rates due to samples collected separately from a sheep fed on hay was less than 2%. When samples obtained through a stomach tube were compared with samples collected through the rumen fistula, the variation was about 3%. The rates of gas and acid production were approximately similar in samples obtained from the rumen at the same time when no sodium bicarbonate was added. During in vitro incubation of whole ruminal contents, there was a highly significant correlation between the net growth rate values (obtained by using fermentation capacity as an index) and the change in concentration of viable rumen bacteria or total ciliate protozoa.     

16S rRNA基因高通量测序方法检测奶牛场常用干草表面微生物群落结构及多样性

【目的】随着中国奶牛业的发展,干草需求量与日俱增。作为天然牧草,干草可以成为家畜传播病原体的载体。以干草表面附着物为研究对象,了解干草中细菌群落结构以及致病菌属特征。【方法】对来自6个不同奶牛场饲草舍的干草样本,应用Illumina Mi Seq高通量测序技术测定干草表面附着物细菌的16S r RNA基因V3-V4变异区序列,分析不同干草样本细菌群落组成。【结果】干草样本中的细菌在97%的相似水平下共得到OTU个数为15 416,涵盖了29门87纲144目219科323属的细菌。微生物多样性分析表明,干草样本具有很高的细菌多样性,不同样本多样性存在差异。对干草样本菌群中丰度较高的14种病原菌属进行分析,发现相较于人工种植牧草制备的干草,天然牧草制备的干草中病原菌属丰度较高。【结论】研究解析了干草样本中微生物的多样性、丰度及主要病原菌属的特征,对奶牛场疾病防控有一定指导意义。     

The effects of management on vegetation trajectories during the early‐stage restoration of previously arable land after hay transfer

The restoration of floodplain grasslands has benefited from many studies of the underlying mechanisms. Among the operational tools that resulted, hay transfer is now used increasingly to alleviate the effects of limited seed dispersal and recruitment. To improve this method, we still need to understand how it can affect restoration trajectories, and particularly their direction and magnitude during the early stages of restoration. Based on concepts from the field of community ecology theory, we investigated the effects of early‐stage management through grazing or mowing on restoration trajectories after soil harrowing and hay transfer. We established a randomized block design experiment and quantified several community‐related metrics to formalize restoration trajectories for 3 years after hay transfer on a previously arable alluvial island in southwestern France. Whatever the management treatment, the species richness and evenness were significantly higher in hay‐inoculated than in control plots. This effect was linked to the recruitment of species originating not only from the reference grassland through hay transfer, but also from the seed bank, a well‐known effect of soil harrowing. Although generally oriented toward the reference grassland, the origin, direction, and magnitude of the trajectory of hay‐inoculated plots all depended on the management applied. Sheep grazing applied at the same time as hay transfer enhanced the recruitment of reference species as from the first experimental year, because it controlled aboveground competition and maintained the window of opportunity open for a sufficiently longer period of time. Our findings show that the type of management applied simultaneously to hay transfer influences the origin of a grassland trajectory, while its direction and magnitude are dependent on the management applied in subsequent years. Grazing immediately after hay transfer may be appropriate to accelerate the recruitment of species from the reference grassland.     

Passive dispersal of <Emphasis Type="Italic">Metrioptera bicolor</Emphasis> (Phillipi 1830) (Orthopteroidea: Ensifera: Tettigoniidae) by transfer of hay

The transfer of hay from a donor site onto a receptor site is currently a widely accepted restoration procedure to establish grasslands with high biodiversity and rich in species. The impact of this procedure on vegetation has been well studied. However, its influence on the insect population has not been investigated. This study verifies that individuals of Metrioptera bicolor (Orthopteroidea: Tettigoniidae) were successfully transferred with hay. Three individuals transferred with the hay were still able to reproduce after the hay transfer. This result corresponds to 4.6% of the 65 marked individuals that were mapped on the donor site on the day of hay transfer. Of the estimated 1220–3013 individuals on the donor site (8300 m²), 56–139 reproductively able individuals were transferred with the hay. Suitable habitat structures presupposed, this number is sufficient to initiate a persistent colonisation of the species on the receptor site. The loss rate of M. bicolor due to grass cutting totalled 42%, which can be considered a very high mortality rate. Comparable studies on bush crickets show a medium loss rate of 21%, in relation to body size.     

Identification of swainsonine as a probable contributory mycotoxin in moldy forage mycotoxicoses

When infested with the fungus Rhizoctonia leguminicola, certain forages, e.g., red clover hay, can cause a "slobber syndrome" of varying severity when consumed by ruminants. The causative agent has been presumed to be slaframine [(1S,6S,8aS)-1-acetoxy-6-aminooctahydroindolizine], which is produced by R. leguminicola. In one serious outbreak of the slobber syndrome in horses, the red clover forage involved was carefully examined and found to contain R. leguminicola and slaframine. An identical hay sample is shown here by ion-exchange chromatographic and gas chromatographic-mass spectrometric analysis of appropriate hay extracts to also contain swainsonine [(1S,2R,8R,8aR)-1,2,8-trihydroxyoctahydroindolizine], a potent alpha-mannosidase inhibitor. Swainsonine has previously been isolated from pure cultures of R. leguminicola and from higher plants, namely the Darling pea (Swainsona canescens) and spotted locoweed (Astragalus lentiginosus). Consumption of Darling pea and spotted locoweed by livestock results in a severe neurological condition resembling that observed in hereditary mannosidosis in cattle and humans. Our findings indicate that swainsonine may be viewed as a mycotoxin when present in moldy forages consumed by livestock. The extent to which slaframine and swainsonine mycotoxicosis pose threats to animal husbandry and, indeed, to humans, if these alkaloids were to enter the human food chain, deserves serious consideration.     

Evaluation of Initial Restoration Measures during the Restoration of Calcareous Grasslands on Former Arable Fields

In 1993, experiments on the restoration of calcareous grasslands on ex‐arable fields were started in order to provide new habitats for species of a small nature reserve with ancient grasslands north of Munich (Germany). The effects of diaspore transfer by the application of seed‐containing hay on vegetation establishment were studied on restoration fields with and without topsoil removal for 5 years. The aim of the study was to assess plant diversity for the evaluation of restoration success by different methods including determination of species with viable seeds in the hay by germination tests, phenological investigations on hay‐transfer source sites at the time of harvest, and vegetation analyses on the restoration sites. Total seed content of the hay and the number and composition of plant species with viable seeds were affected by the time of harvesting and differed between a site which had been used as arable field until 1959 and ancient grassland sites. Nevertheless, the number of established hay‐transfer species showed only few differences between restoration fields. The proportion of species transferred to restoration fields in relation to the number of species with viable seeds in the hay was between 69 and 89%. Five years after the hay transfer, the proportion of the established species was still between 58 and 76%. Up to now, topsoil removal had no significant effect on the number of established hay‐transfer species. After triple hay application the absolute number of transferred grassland species was higher than on sites with single hay application, but restoration efficiency was lower because many of the species with viable seeds in the hay did not establish. In general, our results showed that the transfer of autochthonous hay is a successful method to overcome dispersal limitation in restoration projects.     

Identification of swainsonine as a probable contributory mycotoxin in moldy forage mycotoxicoses.

When infested with the fungus Rhizoctonia leguminicola, certain forages, e.g., red clover hay, can cause a "slobber syndrome" of varying severity when consumed by ruminants. The causative agent has been presumed to be slaframine [(1S,6S,8aS)-1-acetoxy-6-aminooctahydroindolizine], which is produced by R. leguminicola. In one serious outbreak of the slobber syndrome in horses, the red clover forage involved was carefully examined and found to contain R. leguminicola and slaframine. An identical hay sample is shown here by ion-exchange chromatographic and gas chromatographic-mass spectrometric analysis of appropriate hay extracts to also contain swainsonine [(1S,2R,8R,8aR)-1,2,8-trihydroxyoctahydroindolizine], a potent alpha-mannosidase inhibitor. Swainsonine has previously been isolated from pure cultures of R. leguminicola and from higher plants, namely the Darling pea (Swainsona canescens) and spotted locoweed (Astragalus lentiginosus). Consumption of Darling pea and spotted locoweed by livestock results in a severe neurological condition resembling that observed in hereditary mannosidosis in cattle and humans. Our findings indicate that swainsonine may be viewed as a mycotoxin when present in moldy forages consumed by livestock. The extent to which slaframine and swainsonine mycotoxicosis pose threats to animal husbandry and, indeed, to humans, if these alkaloids were to enter the human food chain, deserves serious consideration.     

Fate of inoculated Escherichia coli in hay

AIMS: To monitor the fate of inoculated Escherichia coli in dry and moist hay of various types, under laboratory conditions. METHODS AND RESULTS: Wheat, vetch and clover hay were used as received or wetted to 250-300 g kg(-1) moisture. The hay was inoculated at about 10(6) CFU g(-1) with a kanamycin-resistant E. coli strain that expresses the green fluorescence protein, and was stored in small open glass jars that were covered with aluminium foil. Three jars per treatment were sampled on days 1 and 3, or 4 and 7, or 8, 20 and 50, respectively, after the initiation of the experiments, and the numbers of E. coli in the hay were determined. The results indicated that E. coli disappeared from both dry and moist hay by 7-8 days after inoculation. However, in a few cases colonies that were presumed to be E. coli developed after incubation in Luria broth medium. CONCLUSIONS: The tagged E. coli strain usually disappeared rapidly from both the dry and the moist hay, in spite of the high level of inocula used. However, in some cases a few, possibly injured E. coli might have persisted, and could be detected after incubation in a rich growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is part of a risk assessment associated with sewage irrigation of forage crops in Israel. The results indicate that E. coli added to the hay is not likely to pose a health risk to cattle or to humans. Nevertheless, more research with natural strains of E. coli and other enteric pathogens that might be more adapted to forage conditions is warranted in order to ensure the safety of sewage-irrigated crops.     

Morphological and physiological aspects of digestive processes in the graminivorous primate Theropithecus gelada-a preliminary study

Hindgut fermentation has been suggested to contribute significantly to the digestive process in the gelada (Theropithecus gelada). We therefore hypothesized that in an in vitro fermentation test (Hohenheim gas test, using gas production as measure of microbial digestion) inoculum based on fresh gelada feces would degrade grass to a similar degree as zebra (Equus burchelli chapmani) feces and to a higher degree than that of hamadryas baboons (Papio hamadryas). Additionally, morphology of gelada tongue, salivary glands, stomach, and intestine were examined in this study. Gas production was measured between 4 and 96 hr using animal feces incubated with 200 mg of air-dry hay or mixed concentrate sample. For grass hay, 12-hr gas production was as follows: T. gelada (19.9 ml)>Papio (18.4 ml)>Equus (15.7 ml). After 24 hr, gas production changed: Papio (35.1 ml)>T. gelada (31.9 ml)>Equus (27.9 ml). At 96 hr, Papio was unexpectedly the most effective species with the highest gas production (53.1 ml)>zebra (51.2 ml)>gelada (49.4 ml). With a concentrate standard, 12-hr gas production was as follows: T. gelada (38.5 ml)>Equus (36.8 ml) = Papio (36.4 ml). At 24 hr, gas production differed: Papio (51.7 ml)>Equus (47.0 ml) = T. gelada (46.8 ml). At 96 hr, zebra was the most effective species with the highest gas production (63.9 ml)>Papio (60 ml) = T. gelada (59.9 ml). In conclusion, the results show that the microbial population present in gelada feces is able to ferment forage and concentrate substrates in vitro, although this fermentation did not occur with the expected effectiveness. Future studies should therefore focus also on the bacteria species involved.     

Impact of subacute ruminal acidosis (SARA) adaptation and recovery on the density and diversity of bacteria in the rumen of dairy cows

Subacute ruminal acidosis (SARA) is characterized by ruminal pH depression and microbial perturbation. The impact of SARA adaptation and recovery on rumen bacterial density and diversity was investigated following high-grain feeding. Four ruminally cannulated dairy cows were fed a hay diet, transitioned to a 65% grain diet for 3 weeks, and returned to the hay diet for 3 weeks. Rumen fluid, rumen solids, and feces were sampled during weeks 0 (hay), 1 and 3 (high grain), and 4 and 6 (hay). SARA was diagnosed during week 1, with a pH below 5.6 for 4.6±1.4 h. Bacterial density was significantly lower in the rumen solids with high grain (P=0.047). Rumen fluid clone libraries from weeks 0, 3, and 6 were assessed at the 98% level and 154 operational taxonomic units were resolved. Week 3 diversity significantly differed from week 0, and community structure differed from weeks 0 and 6 (P<0.0001). Clones belonging to the phylum Firmicutes predominated. Compared with the hay diet, the high-grain diet contained clones from Selenomonas ruminantium and Succiniclasticum ruminis, but lacked Eubacterium spp. SARA adaptation was found to significantly alter bacterial density, diversity, and community structure, warranting further investigation into the role bacteria play in SARA adaptation.     

The feeding biology of Hydrobia ventrosa (Montagu). II. Allocation of the components of the carbon-budget and the significance of the secretion of dissolved organic material

The deposit-feeding prosobranch Hydrobia ventrosa (Montagu) was fed ¹⁴C-labelled food for a short period. As food, sterile detritus (homogenously ¹⁴C-labelled, dried barley hay), detritus with attached bacteria, and pure bacteria were used. The distribution of the ingested ¹⁴C was followed for a 24-h period. It was found that the assimilation efficiencies of sterile hay, hay with bacteria, and pure bacteria were 34, 56, and 70 %, respectively. This indicates a significance of bacteria for deposit-feeders. There is a considerable loss of dissolved organic material, in part due to leakage from faecal pellets (13 % of the ingested C in the case of a pure bacterial meal and 7 % of the ingested C in the case of sterile hay). The animals also excrete about 30 % of the assimilated carbon. Excretion of mucus constitutes about 9 % of the assimilated carbon. The fraction of assimilated carbon respired depends on the nature of the food. For sterile hay, hay with bacteria, and pure bacteria the percentage respired was 53, 30, and 38 %, respectively. Growth efficiency is, therefore, higher when protein-rich bacteria are included in the diet.     

Expression of conditioned preference for low-quality food in sheep is modulated by foraging costs

Past positive experiences can increase herbivores’ motivation to eat low-quality foods. However, this is not always translated into a higher preference for low-quality foods in choice tests among foods of higher nutritional quality. Foraging behavior is also affected by properties of the feeding context because the quality and abundance of foods in nature change in time and space. We hypothesized that in a choice situation, the expression of a past positive experience with a low-quality food is modulated by the costs associated with selecting a high-quality food option. A total of 24 sheep were randomly assigned into two groups (n=12). During conditioning phase, one group (CS+; i.e., conditioned group) was fed with oat hay (a low-quality food) for 20 min and immediately after a ration of soybean meal (a nutritious food), whereas the other group was also fed with oat hay but the offer of soybean meal was delayed 5 h (CS−; i.e., control group). After conditioning, we assessed sheep motivation to eat the oat hay in an experimental arena in which accessibility to alfalfa hay (a high-quality food) was increasingly restricted. When alfalfa hay was readily accessible, CS+ and CS− sheep almost exclusively selected this food, showing a small and similar preference for oat hay. However, when accessibility to alfalfa hay decreased, intake and selection of oat hay was greater in the CS+ sheep than in the CS− sheep. The latter was a consequence of differential changes in behavior between groups; for example, sheep in CS+ spent more time foraging oat hay and were more likely to switch to oat hay if they had previously been eating alfalfa hay than sheep in CS−. Our results show that behavioral expression of the conditioned preference for a low-quality food depends on parameters of the feeding context (e.g., availability). We suggest that this can be the link between learning models and optimal foraging models of diet selection.     

Effects of a Bacterial Hay Preservative (Pediococcus pentosaceus) on Hay under Experimental Storage Conditions

The abundant growth of molds and thermophilic actinomycetes in stored hay decreases its quality and can be hazardous for the producer who inhales these contaminants when the moldy hay is fed in closed barns. These microbes are responsible for a respiratory disease called farmer's lung. Products, including bacterial cultures that can be inoculated in hay, are available to prevent hay deterioration by molds and bacteria. The aim of this study was to verify the effectiveness of Pediococcus pentosaceus (a bacterial inoculant) in preventing hay deterioration at different humidity levels in a laboratory experiment. Mixtures of grasses (mostly alfalfa, timothy, and clover) placed in plastic bags were treated with the commercially available product (live culture of P. pentosaceus) at 500,000 and 5,000,000 CFU/g of hay and humidified at different levels (20, 25, 30, and 35%). Control batches of hay (untreated) were prepared at the same humidity levels. The growth of inoculated bacteria in hay, pH level, and hay deterioration were evaluated. Under these experimental conditions, the growth of P. pentosaceus was abundant only when it was inoculated in very moist hay (35% moisture), resulting in bacterium levels of 6.3 x 10(sup8) CFU/g after 30 days. This abundant growth did not prevent the pH from increasing (final pH of about 9.0), nor did it prevent molding. At lower humidity levels (20, 25, and 30%), the bacterial inoculant used did not grow and did not prevent hay deterioration.     

Effect of Hay Transfer on Long-Term Establishment of Vegetation and Grasshoppers on Former Arable Fields

The transfer of seed‐containing hay is a restoration measure for the introduction of plant species of local provenance. We investigated the effect of hay transfer on species richness and on long‐term establishment of target plant and grasshopper species on former arable fields with and without topsoil removal in comparison to reference sites in a nature reserve. Plant species richness, the number of target plant species, and Red List plant species were significantly positively affected by hay transfer, both on the scale of whole restoration fields and on permanent plots of 4 m². Eight years after the start of the restoration, only few of the transferred plant species had disappeared and some target species were newly found. Grasshoppers were affected not by hay transfer but by topsoil removal. The proportion of target grasshopper and plant species and Red List grasshopper species was higher on topsoil removal sites with low standing crop and high cover of bare soil than on sites without soil removal. On topsoil removal sites without hay, however, plant species richness was very low because of the slow natural dispersal of the target species. Vegetation and grasshopper communities still differed between restoration fields and the nature reserve. Nevertheless, our results indicate that the transfer of autochthonous seed‐containing hay is a successful method to establish species‐rich grasslands with a high proportion of target species.     

Soil Contamination of Grass Biomass Hay: Measurements and Implications

A large area of unutilized or underutilized marginal land in the northeastern USA, some of which currently produce a mature hay crop for use as mulch, is potentially suitable for bioenergy crop production. Mature-mixed grass hay bales (n?=?1980) were sampled across New York in late summer and fall of 2011 and 2012 from 65 farms. Chemical analysis of 19 parameters, including gross calorific value, fiber, and elemental analyses, was conducted on the samples. In addition, 156 soil samples, representing 67 soil types, were analyzed for 12 to 14 parameters, using two different extraction procedures. Results indicate that hay composition is extremely variable among bales, and that much of the variation is due to soil contamination. Soil contamination reduced energy content of hay, a reduction best estimated from ash content of the hay. Standard plant analyses of contaminated hay samples determine total elemental content, but the same analyses only partially extract soil elements. A subset of samples showed that fiber analysis of soil-contaminated hay is problematic, with results impacted greatly by soil type and gravimetric filtration method. Aluminum, because of its low plant uptake potential, its high concentration in most soils, and its relatively moderate range in concentration across soil types compared to other soil elemental predictors, is the best indicator of soil contamination of biomass when the soil type is unknown. Evaluation of herbaceous plants for bioenergy parameters should include ash and Al analysis to assess soil contamination, which could significantly bias other compositional analyses.     

Incidence of allergic rhinitis in general practice, 1981-92.

OBJECTIVES--To determine the epidemiology of hay fever and to consider the role of pollution. DESIGN--Examination of data on weekly incidence of allergic rhinitis and hay fever by age, sex, region, and location. SETTING--Royal College of General Practitioners Weekly Returns Service. Practice data were based on registered populations of 220,000 in 1981, rising to 700,000 in 1992 from England and Wales. MAIN OUTCOME MEASURES--Numbers of new cases of hay fever and allergic rhinitis. Data on pollen counts for Darlington, Derby, and London. RESULTS--The incidence of allergic rhinitis fluctuated greatly from year to year but showed no trend. Peaks in hay fever coincided with peak pollen counts. No important differences were found between urban and rural locations or different parts of the country with respect to both size and timing of the peaks. Incidence was highest in children (5-14 years). CONCLUSIONS--The similarity of the results throughout England and Wales does not support an important role for local pollutants in hay fever. However, the possibility that levels of pollutants are high enough to act as an adjuvant in hay fever across the whole study area has not been excluded.