Efficient Improvement of Silage Additives by Using Genetic Algorithms

The enormous variety of substances which may be added to forage in order to manipulate and improve the ensilage process presents an empirical, combinatorial optimization problem of great complexity. To investigate the utility of genetic algorithms for designing effective silage additive combinations, a series of small-scale proof of principle silage experiments were performed with fresh ryegrass. Having established that significant biochemical changes occur over an ensilage period as short as 2 days, we performed a series of experiments in which we used 50 silage additive combinations (prepared by using eight bacterial and other additives, each of which was added at six different levels, including zero [i.e., no additive]). The decrease in pH, the increase in lactate concentration, and the free amino acid concentration were measured after 2 days and used to calculate a “fitness” value that indicated the quality of the silage (compared to a control silage made without additives). This analysis also included a “cost” element to account for different total additive levels. In the initial experiment additive levels were selected randomly, but subsequently a genetic algorithm program was used to suggest new additive combinations based on the fitness values determined in the preceding experiments. The result was very efficient selection for silages in which large decreases in pH and high levels of lactate occurred along with low levels of free amino acids. During the series of five experiments, each of which comprised 50 treatments, there was a steady increase in the amount of lactate that accumulated; the best treatment combination was that used in the last experiment, which produced 4.6 times more lactate than the untreated silage. The additive combinations that were found to yield the highest fitness values in the final (fifth) experiment were assessed to determine a range of biochemical and microbiological quality parameters during full-term silage fermentation. We found that these combinations compared favorably both with uninoculated silage and with a commercial silage additive. The evolutionary computing methods described here are a convenient and efficient approach for designing silage additives.     

青贮对柳枝稷制取燃料乙醇转化过程的影响

青贮是一种传统的生物质原料保存方法,广泛应用于纤维素乙醇炼制领域尚需要考察其对原料品质和下游乙醇转化过程的影响。文中以秋季(初、中和末)收割的柳枝稷为原料,通过青贮、高温水热(LHW)预处理、纤维素酶水解和同步糖化与发酵(SSF)实验对上述问题予以回答。结果显示,秋季初收割的柳枝稷以不同湿度青贮后pH均小于4.0,干重损失小于2%,各主要成分与青贮前相比无明显变化;LHW预处理中青贮样品半纤维素水解率普遍高于未贮存样品,但青贮同样使原料获得了更高的发酵抑制物产生水平;青贮柳枝稷葡萄糖、木糖和半乳糖产量(预处理+酶水解)高于未贮存柳枝稷;经过168 h的SSF,青贮样品乙醇浓度为12.1 g/L,未贮存的秋季初、秋季中和秋季末柳枝稷为底物的浓度分别为10.3 g/L、9.7 g/L和10.6 g/L。综上,青贮有助于提高柳枝稷LHW预处理效率、酶水解率和乙醇产量。     

The effect of three commercial silage additives on numbers of lactobacilli entering the silo at the onset of fermentation

Abstract There is considerable debate regarding the effectiveness of currently available silage inoculants in the ensilage process. The large-scale farm experiment reported shows there to be no significant difference between 3 commercial inoculants and a control treatment with respect to numbers of lactobacilli at the onset of ensilage. Considerable variation in numbers of lactobacilli throughout both the silo and the bulk inoculant were also observed. Numbers of lactobacilli isolated from each of the inoculants were insufficient to produce a population density of 10⁶ organisms per g of grass.     

Influence of moisture content on microbial activity and silage quality during ensilage of food processing residues

Seasonally produced biomass such as sugar beet pulp (SBP) and tomato pomace (TP) needs to be stored properly to meet the demand of sustainable biofuel production industries. Ensilage was used to preserve the feedstock. The effect of moisture content (MC) on the performance of ensilage and the relationship between microorganism activities and MC were investigated. For SBP, MC levels investigated were 80, 55, 30, and 10% on a wet basis. For TP, MC levels investigated were 60, 45, 30, and 10%. Organic acids, ethanol, ammonia, pH and water soluble carbohydrates (WSC) were measured to evaluate the silage quality. Ensilage improved as the MC decreased from 80 to 55% for SBP and from 60 to 45% for TP. When the MC decreased to 30%, a little microbial activity was detected for both feedstocks. Storage at 10% MC prevented all the microbial activity. The naturally occurring microorganisms in TP were found to preserve TP during silage and were isolated and determined by polymerase chain reaction (PCR). The results suggest that partial drying followed by ensilage may be a good approach for stabilization of food processing residues for biofuels production.     

Silage studies IX biochemical changes in microbe-free silage

Summary If the aseptic plant material is regarded as a substrate for preservative microbial activity, it may be said that both the nitrogen and carbohydrate components undergo changes due to the influence of native enzymes in the plant material. These changes may very well be reflected in the microbial activity in a silage.It is interesting to compare the findings of this investigation, which show that during the days immediately after ensilage practically no changes take place when no bacteria are present. In a non-sterile ensilage, on the other hand, it is during this early stage that the briskest bacterial activity takes place.The chances of, say, an acid-forming microbial process actually lowering the pH in order that preservation of the material may take place are directly counteracted by the action of native enzymes of the vegetable material. During the days immediately after ensilage, however, if other conditions are favourable, microbial activity may clearly determine the whole course of events, and may possibly even inactivate the processes of the plant material itself.As is evident from the experiments described above, the activities inherent in the ensiled material itself are of no mean proportions. Further study of the mutual relationship of the ensilage and the various microbial fractions is therefore particularly necessary in order to be able to understand a number of processes of fundamental importance in biological preservation of plant material.     

Influence of Lactobacillus spp. from an Inoculant and of Weissella and Leuconostoc spp. from Forage Crops on Silage Fermentation

Lactobacillus spp. from an inoculant and Weissella and Leuconostoc spp. from forage crops were characterized, and their influence on silage fermentation was studied. Forty-two lactic acid-producing cocci were obtained from forage crops and grasses. All isolates were gram-positive, catalase-negative cocci that produced gas from glucose, and produced more than 90% of their lactate in the d-isomer form. These isolates were divided into groups A and B by sugar fermentation patterns. Two representative strains from the two groups, FG 5 and FG 13, were assigned to the species Weissella paramesenteroides and Leuconostoc pseudomesenteroides, respectively, on the basis of DNA-DNA relatedness. Strains FG 5, FG 13, and SL 1 (Lactobacillus casei), isolated from a commercial inoculant, were used as additives to alfalfa and Italian ryegrass silage preparations. Lactic acid bacterium counts were higher in all additive-treated silages than in the control silage at an early stage of ensiling. During silage fermentation, inoculation with SL 1 more effectively inhibited the growth of aerobic bacteria and clostridia than inoculation with strain FG 5 or FG 13. SL 1-treated silages stored well. However, the control and FG 5- and FG 13-treated silages had a significantly (P < 0.05) higher pH and butyric acid and ammonia nitrogen contents and significantly (P < 0.05) lower lactate content than SL 1-treated silage. Compared with the control silage, SL 1 treatments reduced the proportion of d-(−)-lactic acid, gas production, and dry matter loss in two kinds of silage, but the FG 5 and FG 13 treatments gave similar values in alfalfa silages and higher values (P < 0.05) in Italian ryegrass silage. The results confirmed that heterofermentative strains of W. paramesenteroides FG 5 and L. pseudomesenteroides FG 13 did not improve silage quality and may cause some fermentation loss.Silage is now the most common preserved cattle feed in many countries, including Japan. It is well established that lactic acid bacteria (LAB) play an important role in silage fermentation. Epiphytic microflora, the microorganisms naturally present on forage crops, are responsible for silage fermentation and also influence silage quality (3, 11, 15). Lactobacilli and lactic acid-producing cocci, e.g., leuconostocs, lactococci, streptococci, pediococci, and Weissella species, are major components of the microbial flora in various types of forage crops (3). Stirling and Whittenbury (21) reported that leuconostocs were the most numerous and widely distributed on forages and that lactobacilli occurred mostly on grasses. Cai et al. (3) examined a large number of forage crops and grasses and also found that the predominant LAB were lactic acid-producing cocci and that lactobacilli were the least numerous and mostly homofermentative. Ruser (17) found that although all LAB groups were present in chopped-maize samples, homofermentative lactobacilli and heterofermentative leuconostocs were present in the highest numbers.In order to improve silage quality, many LAB-containing biological additives have been developed and are currently available (13, 20, 25). These inoculants may inhibit the growth of harmful bacteria and enhance lactic acid fermentation during ensiling periods. The epiphytic LAB influence the effectiveness of silage inoculants because the introduced bacteria must compete with these LAB (12). Therefore, the LAB species and their characteristics in the silage environment require further study. However, while an increasing number of studies have reported positive benefits from using some bacterial inoculants as silage additives, relatively few have reported the effect of epiphytic LAB, especially Leuconostoc and Weissella species, on silage fermentation. In the present study, the characterization of Leuconostoc and Weissella species isolated from forage crops and their influence on silage fermentation were examined.     

The response of sheep to big bale grass silage ensiled with,or supplemented separately with,molassed sugar beet feed

Second cut big bale grass silage was prepared with grass ensiled alone or with added pelleted, molassed sugar beet feed (MSBF) at 32 or 64 g MSBF kg⁻¹ fresh weight of grass. Addition of MSBF at ensilage increased silage dry matter (DM), water soluble carbohydrate and lactic acid concentrations and decreased ammonia concentration. When fed to wether lambs, MSBF addition, whether at ensilage or given separately, variably improved DM intake, live-weight gain and feed conversion efficiency. Ruminal concentrations of ammonia were reduced and acetate molar proportion increased in diets supplemented with MSBF separately compared with the silage only control diet. Animal performance was generally improved by MSBF supplementation with responses to silages made with added MSBF comparable to those from silage supplemented with MSBF at feeding. There was little difference in animal performance between silages made with 32 or 64 g kg⁻¹ MSBF added at ensiling.     

玉米青贮过程中乳酸菌动态变化

为了揭示青贮玉米在发酵过程中乳酸菌数量及种群的动态变化,以及微生物添加剂对乳酸菌种群变化的影响,采用了培养方法计数发酵过程中乳酸菌数目的变化,利用16S rRNA基因序列比对方法分析青贮玉米中乳酸菌的多样性及种群变化趋势。经过对15d时间内青贮玉米中乳酸菌变化趋势的分析显示:一周后对照组乳酸菌数最高达到2.1×106CFU/g,两处理组中处理组Ⅱ乳酸菌数达到最高5.5×107CFU/g;利用MRS平板分离、培养出典型乳酸菌菌落152株,经16S rRNA基因序列比对分析为乳杆菌属和片球菌属,其中86%的菌株属于乳杆菌属。此研究表明微生物添加剂有利于青贮玉米发酵过程中乳酸菌的快速增殖,乳杆菌属和片球菌属都是青贮玉米发酵的启动菌之一,在发酵前期一直存在,但发酵后期乳杆菌属是玉米青贮过程中乳酸菌的主要菌群。     

Isolation of Salmonella spp. from liquid and solid excreta prior to and following ensilage in ten swine farms located in central Mexico

A study was carried out to define selected bacteriological characteristics of residues from 10 swine farms, 5 with or without prior clinical enteric disease (PCED) and to determine the effect of ensilage on the bacteria present in the solid fraction. At each farm, samples were taken from the sedimentation basin (SB), the solid fraction (SF), and the liquid fraction (LF). For each sample, CFU/g for enteric bacteria were quantified; Salmonella spp. were isolated and typified. Solid phase samples from each farm were used to prepare the ensilage, with a mixture of solids (80%), sorghum (12%) and molasses (8%). The quantity of enteric bacteria was significantly greater in farms without PCED (P < 0.05). Salmonella enterica were isolated from 8/10 of the farms with and without PCED; in 8 from SB; in 6 from LF; and in 5 from SF. Enteric bacteria were not isolated from silage, therefore, ensilage may be an alternative treatment for excreta that allows the elimination of pathogens such as Salmonella spp.     

Assessing the impact of various ensilage factors on the fermentation of grass silage using conventional culture and bacterial community analysis techniques

Aims: Grass silage is an important ruminant feedstuff on farms during winter. The ensilage of grass involves a natural lactic acid bacterial fermentation under anaerobic conditions, and numerous factors can influence the outcome of preservation. The aim of this study was to investigate the effect of dry matter concentration, ensiling system, compaction and air infiltration on silage bacterial community composition. Methods and Results: The impact of these factors was examined using conventional methods of microbial analysis and culture‐independent Terminal Restriction Fragment Length Polymorphism (T‐RFLP). Silage fermentation was restricted in herbage with a high dry matter concentration, and this was reflected in a shift in the bacterial population present. In contrast, ensiling system had little effect on bacterial community composition. Air infiltration, in the absence of compaction, altered silage bacterial community composition and silage pH. Conclusions: Dry matter concentration and the absence of compaction were the main factors affecting silage microbial community composition, and this was reflected in both the conventional culture‐based and T‐RFLP data. Significance and Impact of the Study: T‐RFLP proved a useful tool to study the factors affecting ensilage. Apart from monitoring the presence or absence of members of the population, shifts in the relative presence of members could be monitored.     

The effect of varying proportion and chop length of lucerne silage in a maize silage-based total mixed ration on diet digestibility and milk yield in dairy cattle

The objective was to assess the effects of inclusion rate and chop length of lucerne silage, when fed in a total mixed ration (TMR), on milk yield, dry matter (DM) intake (DMI) and digestion in dairy cows. Diets were formulated to contain a 50 : 50 ratio of forage : concentrate (DM basis) and to be isonitrogenous (170 g/kg CP). The forage portion of the offered diets was comprised of maize and lucerne silage in proportions (DM basis) of either 25 : 75 (high Lucerne (HL)) or 75 : 25 (low lucerne (LL)). Lucerne was harvested and conserved as silage at either a long (L) or short (S) chop length. These variables were combined in a 2×2 factorial arrangement to give four treatments (HLL, HLS, LLL, LLS) which were fed in a Latin square design study to Holstein dairy cows in two separate experiments. In total, 16 and 8 multiparous, mid-lactation cows were used in experiments 1 and 2, respectively. To ensure sufficient silage for both experiments, different cuts of lucerne silage (taken from the same sward) were used for each experiment: first cut for experiment 1 (which was of poorer quality) and second cut for experiment 2. Dry matter intake, milk yield and milk composition where measured in both experiments, and total tract digestibility and nitrogen (N) balance were assessed using four cows in experiment 2. In experiment 1, cows fed LL had increased DMI (+3.2 kg/day), compared with those fed HL. In contrast, there was no difference in DMI due to lucerne silage inclusion rate in experiment 2. A reduction in milk yield was observed with the HL treatment in both experiment 1 and 2 (−3.0 and −2.9 kg/day, respectively). The HL diet had reduced digestibility of DM and organic matter (OM) (−3% and −4%, respectively), and also reduced the efficiency of intake N conversion into milk N (−4%). The S chop length increased total tract digestibility of DM and OM (both +4%), regardless of inclusion rate. Inclusion of lucerne silage at 25% of forage DM increased milk yield relative to 75% inclusion, but a S chop length partially mitigated adverse effects of HL on DMI and milk yield in experiment 1 and on DM digestibility in experiment 2.     

Lack of nonadditive genetic effects on early fecundity in Drosophila melanogaster

Fecundity is usually considered as a trait closely connected to fitness and is expected to exhibit substantial nonadditive genetic variation and inbreeding depression. However, two independent experiments, using populations of different geographical origin, indicate that early fecundity in Drosophila melanogaster behaves as a typical additive trait of low heritability. The first experiment involved artificial selection in inbred and non-inbred lines, all of them started from a common base population previously maintained in the laboratory for about 35 generations. The realized heritability estimate was 0.151 +/- 0.075 and the inbreeding depression was very small and nonsignificant (0.09 +/- 0.09% of the non-inbred mean per 1% increase in inbreeding coefficient). With inbreeding, the observed decrease in the within-line additive genetic variance and the corresponding increase of the between-line variance were very close to their expected values for pure additive gene action. This result is at odds with previous studies showing inbreeding depression and, therefore, directional dominance for the same trait and species. All experiments, however, used laboratory populations, and it is possible that the original genetic architecture of the trait in nature was subsequently altered by the joint action of random drift and adaptation to captivity. Thus, we carried out a second experiment, involving inbreeding without artificial selection in a population recently collected from the wild. In this case we obtained, again, a maximum-likelihood heritability estimate of 0.210 +/- 0.027 and very little nonsignificant inbreeding depression (0.06 +/- 0.12%). The results suggest that, for fitness-component traits, low levels of additive genetic variance are not necessarily associated with large inbreeding depression or high levels of nonadditive genetic variance.     

Effects of lactic acid bacteria in inoculants on changes in amino acid composition during ensilage of sterile and non-sterile ryegrass

A study was carried out on the changes occurring in the amino acid fraction of a hybrid ryegrass during ensilage in laboratory-scale silos to help to establish the relative roles of plant and microbial proteases on protein degradation in the silo. Herbage treatments included (i) normal grass without treatment (ii) lambda-irradiated grass (sterile) without treatment (iii) sterile, inoculated with a strain of Lactobacillus plantarum and (iv) sterile, inoculated with a strain of Lactobacillus paracasei subsp. paracasei. These treatments had a significant effect on silage amino acid profiles. Concentrations of free amino acids and the extent of amino acid catabolism varied with treatment. However, levels were notably higher in control silages after 90 days (free amino acid nitrogen constituting 54% of total amino acid nitrogen compared with 37, 32 and 22% for treatments i, ii and iv, respectively). These results indicate that the extent of protein hydrolysis during ensilage is influenced by factors other than rate of pH decline and plant protease activity, and that microbial proteases play a role.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

Evaluation of the phytosanitary, fermentative and nutritive characteristics of the silage made from crude artichoke (Cynara scolymus L.) by-product feeding for ruminants

The crude by-product left after industrial processing of artichoke (Cynara scolymus L.) was ensiled in microsilos and sampled at different times. To evaluate its suitability as animal feed, various fermentative, chemical and phytosanitary parameters were determined. The by-product showed a good aptitude for ensilage, having a pleasant smell and good visual characteristics. The DM content was 297 g kg⁻¹, and no effluents were detected. It stabilized after 12 days of ensiling and showed a pH value of 4.1 at the end of the process. Only small losses were detected in its chemical value after the ensiling period. Prometryn was the only phytosanitary product found at day 0 (0.04 ppm a concentration below the maximum amount permitted by law), but was not detected after 12 days of ensilage. It is concluded that the silage by-product can be used as animal feed.     

Studies on fermentation of sorghum silage during storage — Effect of urea and molasses

Eighteen silages involving various treatments were prepared from Jowar (Sorghum vulgare) at the dough stage in 500 ml flasks. The treatment combinations were urea at 0% (U₀), 0.5% (U₁) and 1% (U₂), and molasses at 0% (M₀) and 1% (M₁). The flasks were opened for analysis at 30- (I₁), 60- (I₂) and 120- (I₃) day intervals. Urea levels increased the pH values irrespective of the presence of molasses. However, with increasing urea levels, the lactic acid increased in the absence of molasses while butyric acid increased in its presence. Fermentation quality was better for the silage prepared without additives and with molasses alone but was very poor when urea was added with molasses. Quality of silage with M₀U₀, M₀U₁ and M₁U₀ was maintained to the end of the experiment, whereas M₀U₂ silage deteriorated during the later part of the storage period.     

Chromosome Interactions in DROSOPHILA MELANOGASTER. II. Total Fitness

In a large experiment, using nearly 200 population cages, we have measured the fitness of Drosophila melanogaster homozygous (1) for the second chromosome, (2) for the third chromosome, and (3) for both chromosomes. Twentyfour second chromosomes and 24 third chromosomes sampled from a natural population were tested. The mean fitness of the homozygous flies is 0.081 ± 0.014 for the second chromosome, 0.080 ± 0.017 for the third chromosome, and 0.079 ± 0.024 for both chromosomes simultaneously. Assuming that fitnesses are multiplicative (the additive fitness model makes no sense in the present case because of the large selection coefficients involved), the expected mean fitness of the homozygotes for both chromosomes is 0.0066; their observed fitness is more than ten times greater. Thus, it appears that synergistic interactions between loci are considerable; and that, consequently, the fitness function substantially departs from linearity. Two models are tentatively suggested for the fitness function: a "threshold" model and a "synergistic" model.—The experiments reported here confirm previous results showing that the concealed genetic load present in natural populations of Drosophila is sufficient to account for the selective maintenance of numerous polymorphisms (of the order of 1000).     

Darwinian fitness and adaptedness in experimental populations of Drosophila willistoni

Fifteen second chromosomes were extracted from Drosophila willistoni flies collected in four natural populations. The adaptedness of populations homozygous for each chromosome was measured by average population size and productivity. Six control populations were established with mixtures of the wild second chromosomes. The Darwinian fitness of flies homozygous for each wild second ehromosome, and of flies carrying random combinations of these chromosomes, was measured relative to the fitness of flies heterozygous for a wild and a marker chromosome. The Darwinian fitness of homozygotes for each second chromosome relative to the fitness of flies carrying random combinations of the natural chromosomes was then inferred. The estimated loss of fitness on making the natural second chromosomes homozygous was substantial, ranging from 39 to 83 pereent, with an average reduction in fitness of 66 percent. These results with D. willistoni are consistent with those from similar experiments with other drosophila species, and they are compatible with a significant role for heterosis in the maintenance of genetic variability.Populations homozygous for wild chromosomes differ in their adaptedness to the experimental environment. Population size and productivity are correlated, although the correlation is far from complete. Some populations have high productivity and low population size, or vice versa. The control populations, with greater genetic variability, were superior in adaptedness to the average of the single-chromosome populations. The Darwinian fitness and the adaptedness of the genotypes in this experiment were not significantly correlated. It follows that certain measures used by population geneticists, such as genetic load and average Darwinian fitness, cannot be taken as general indices of how well adapted a population is to its environment.This work was supported by U.S. Public Health Service Grant RO1-HDO5055, NSF grant GB-20694 (International Biological Program). AEC contract AT-(30-1) 3096, and PHS Career Development Award K3 GM 37265. The collection of the flies was supported by the Fundacão de Amparo a Pesquisa do Estado de São Paulo, Brazil. The senior author's stay in New York, where the experiments were conducted, was financed in part by Research Fellowship 2-12861 from the Panamerican Union.     

Reconciling strong stabilizing selection with the maintenance of genetic variation in a natural population of black field crickets (Teleogryllus commodus)

Genetic variation in single traits, including those closely related to fitness, is pervasive and generally high. By contrast, theory predicts that several forms of selection, including stabilizing selection, will eliminate genetic variation. Stabilizing selection in natural populations tends to be stronger than that assumed in theoretical models of the maintenance of genetic variation. The widespread presence of genetic variation in the presence of strong stabilizing selection is a persistent problem in evolutionary genetics that currently has no compelling explanation. The recent insight that stabilizing selection often acts most strongly on trait combinations via correlational selection may reconcile this problem. Here we show that for a set of male call properties in the cricket Teleogryllus commodus, the pattern of multivariate stabilizing sexual selection is closely associated with the degree of additive genetic variance. The multivariate trait combinations experiencing the strongest stabilizing selection harbored very little genetic variation while combinations under weak selection contained most of the genetic variation. Our experiment provides empirical support for the prediction that a small number of trait combinations experiencing strong stabilizing selection will have reduced genetic variance and that genetically independent trait combinations experiencing weak selection can simultaneously harbor much higher levels of genetic variance.     

Survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in Wilted and Additive-Treated Grass Silage

Grass was field-dried to 3 different dry matter (DM) levels (200, 430 and 540 g/kg) and inoculated with 10⁶–10⁷ cfu/g of a Listeria monocytogenes strain sharing a phagovar occasionally involved in food-borne outbreaks of listeriosis. Formic acid (3 ml/kg) or lactic acid bacteria (8·10⁵/g) with cellulolytic enzymes were applied only to forages with low and intermediate DM levels. Forages were ensiled in laboratory silos (1700 ml) and were stored at 25°C for 30 or 90 days. After 90 days of storage, L. monocytogenes could not be detected in any silo, except one with the high dry matter grass without additive. After 30 days of storage, between 10² and 10⁶ cfu L. monocytogenes/g silage were isolated from the untreated silages. Increasing the DM content from 200 to 540 g/kg did not reduce listeria counts possibly because of the lower production of fermentation acids (higher pH). In silages treated with additives, counts of L. monocytogenes were always lower than in silages without additive. In wet silages (DM 200 g/kg) both additives were effective, but in the wilted silages (DM 430 g/kg) only the bacterial additive reduced listeria counts below detection level. Listeria counts were highly correlated to silage pH (r = 0.92), the concentration of lactic acid (r = -0.80) and the pooled amount of undissociated acids (r = -0.83).