Microbial Fuel Cells and Microbial Ecology: Applications in Ruminant Health and Production Research

Microbial fuel cell (MFC) systems employ the catalytic activity of microbes to produce electricity from the oxidation of organic, and in some cases inorganic, substrates. MFC systems have been primarily explored for their use in bioremediation and bioenergy applications; however, these systems also offer a unique strategy for the cultivation of synergistic microbial communities. It has been hypothesized that the mechanism(s) of microbial electron transfer that enable electricity production in MFCs may be a cooperative strategy within mixed microbial consortia that is associated with, or is an alternative to, interspecies hydrogen (H₂) transfer. Microbial fermentation processes and methanogenesis in ruminant animals are highly dependent on the consumption and production of H₂in the rumen. Given the crucial role that H₂ plays in ruminant digestion, it is desirable to understand the microbial relationships that control H₂ partial pressures within the rumen; MFCs may serve as unique tools for studying this complex ecological system. Further, MFC systems offer a novel approach to studying biofilms that form under different redox conditions and may be applied to achieve a greater understanding of how microbial biofilms impact animal health. Here, we present a brief summary of the efforts made towards understanding rumen microbial ecology, microbial biofilms related to animal health, and how MFCs may be further applied in ruminant research.     

Dynamic role of single-celled fungi in ruminal microbial ecology and activities

In ruminants, high fermentation capacity is necessary to develop more efficient ruminant production systems. Greater level of production depends on the ability of the microbial ecosystem to convert organic matter into precursors of milk and meat. This has led to increased interest by animal nutritionists, biochemists and microbiologists in evaluating different strategies to manipulate the rumen biota to improve animal performance, production efficiency and animal health. One of such strategies is the use of natural feed additives such as single-celled fungi yeast. The main objectives of using yeasts as natural additives in ruminant diets include; (i) to prevent rumen microflora disorders, (ii) to improve and sustain higher production of milk and meat, (iii) to reduce rumen acidosis and bloat which adversely affect animal health and performance, (iv) to decrease the risk of ruminant-associated human pathogens and (v) to reduce the excretion of nitrogenous-based compounds, carbon dioxide and methane. Yeast, a natural feed additive, has the potential to enhance feed degradation by increasing the concentration of volatile fatty acids during fermentation processes. In addition, microbial growth in the rumen is enhanced in the presence of yeast leading to the delivery of a greater amount of microbial protein to the duodenum and high nitrogen retention. Single-celled fungi yeast has demonstrated its ability to increase fibre digestibility and lower faecal output of organic matter due to improved digestion of organic matter, which subsequently improves animal productivity. Yeast also has the ability to alter the fermentation process in the rumen in a way that reduces methane formation. Furthermore, yeast inclusion in ruminant diets has been reported to decrease toxins absorption such as mycotoxins and promote epithelial cell integrity. This review article provides information on the impact of single-celled fungi yeast as a feed supplement on ruminal microbiota and its function to improve the health and productive longevity of ruminants.     

Protozoa populations are ecosystem engineers that shape prokaryotic community structure and function of the rumen microbial ecosystem

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community—either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.Subject terms: Microbial ecology, Food webs     

Metagenomic analysis reveals a dynamic microbiome with diversified adaptive functions to utilize high lignocellulosic forages in the cattle rumen

Rumen microbiota play a key role in the digestion and utilization of plant materials by the ruminant species, which have important implications for greenhouse gas emission. Yet, little is known about the key taxa and potential gene functions involved in the digestion process. Here, we performed a genome-centric analysis of rumen microbiota attached to six different lignocellulosic biomasses in rumen-fistulated cattle. Our metagenome sequencing provided novel genomic insights into functional potential of 523 uncultured bacteria and 15 mostly uncultured archaea in the rumen. The assembled genomes belonged mainly to Bacteroidota, Firmicutes, Verrucomicrobiota, and Fibrobacterota and were enriched for genes related to the degradation of lignocellulosic polymers and the fermentation of degraded products into short chain volatile fatty acids. We also found a shift from copiotrophic to oligotrophic taxa during the course of rumen fermentation, potentially important for the digestion of recalcitrant lignocellulosic substrates in the physiochemically complex and varying environment of the rumen. Differential colonization of forages (the incubated lignocellulosic materials) by rumen microbiota suggests that taxonomic and metabolic diversification is an evolutionary adaptation to diverse lignocellulosic substrates constituting a major component of the cattle’s diet. Our data also provide novel insights into the key role of unique microbial diversity and associated gene functions in the degradation of recalcitrant lignocellulosic materials in the rumen.Subject terms: Bacterial genetics, Metagenomics     

奶牛瘤胃微生物研究进展和趋势

近年来在奶牛试验中,对瘤胃微生物的研究引起了人们越来越多的兴趣。这些研究的目的多是将微生物组成变化与日粮组成、宿主生产性能(如饲料效率,产奶量,乳脂等)、健康(如瘤胃酸中毒和亚急性酸中毒)以及环境(如甲烷排放)联系起来,另外还有一些研究则强调了微生物在多种反刍动物瘤胃发育中的作用。关于奶牛瘤胃微生物的大部分发现都是基于扩增子测序,可以揭示瘤胃微生物的分类组成,以及在不同处理条件下瘤胃菌群的变化。尽管新兴的宏基因组学和宏转录组学能够深入探索瘤胃微生物的功能,但在数据分析和解释方面也带来了更多的挑战,如目前大多数论文都严重依赖于相关性和推测分析。综述了奶牛瘤胃微生物研究的进展和局限,包括瘤胃微生物与产奶效率、甲烷排放以及瘤胃发育的关系,以及奶牛瘤胃微生物未来的研究趋势。     

Characterization of the rumen microbiota of pre-ruminant calves using metagenomic tools

The temporal sequence of microbial establishment in the rumen of the neonatal ruminant has important ecological and pathophysiological implications. In this study, we characterized the rumen microbiota of pre-ruminant calves fed milk replacer using two approaches, pyrosequencing of hypervariable V3-V5 regions of the 16S rRNA gene and whole-genome shotgun approach. Fifteen bacterial phyla were identified in the microbiota of pre-ruminant calves. Bacteroidetes was the predominant phylum in the rumen microbiota of 42-day-old calves, representing 74.8% of the 16S sequences, followed by Firmicutes (12.0%), Proteobacteria (10.4%), Verrucomicrobia (1.2%) and Synergistetes (1.1%). However, the phylum-level composition of 14-day-old calves was distinctly different. A total of 170 bacterial genera were identified while the core microbiome of pre-ruminant calves included 45 genera. Rumen development seemingly had a significant impact on microbial diversity. The dazzling functional diversity of the rumen microbiota was reflected by identification of 8298 Pfam and 3670 COG protein families. The rumen microbiota of pre-ruminant calves displayed a considerable compositional heterogeneity during early development. This is evidenced by a profound difference in rumen microbial composition between the two age groups. However, all functional classes between the two age groups had a remarkably similar assignment, suggesting that rumen microbial communities of pre-ruminant calves maintained a stable function and metabolic potentials while their phylogenetic composition fluctuated greatly. The presence of all major types of rumen microorganisms suggests that the rumen of pre-ruminant calves may not be rudimentary. Our results provide insight into rumen microbiota dynamics and will facilitate efforts in formulating optimal early-weaning strategies.     

Pantothenic acid supplementation to support rumen microbes?

Based on repeatedly reported extensive pantothenic acid disappearance in the rumen, the present study is aimed at examining if pantothenic acid is used for a more efficient ruminal fermentation and microbial growth in an artificial rumen (Rusitec). Three substrates differing in roughage/concentrate ratio were incubated with and without the addition of Ca-D-pantothenate. Pantothenic acid was extensively degraded without notably influencing fermentation, microbial protein synthesis and the status of other B-vitamins such as riboflavin, vitamin B6 and niacin. Therefore, pantothenic acid supplementation cannot be expected to contribute to microbial benefit for the ruminant animal.     

Xylan degradation, a metabolic property shared by rumen and human colonic Bacteroidetes

Microbial inhabitants of the bovine rumen fulfil the majority of the normal caloric requirements of the animal by fermenting lignocellulosic plant polysaccharides and releasing short chain fatty acids that are then metabolized by the host. This process also occurs within the human colon, although the fermentation products contribute less to the overall energy requirements of the host. Mounting evidence, however, indicates that the community structure of the distal gut microbiota is a critical factor that influences the inflammatory potential of the immune system thereby impacting the progression of inflammatory bowel diseases. Non-digestible dietary fibre derived from plant material is highly enriched in the lignocellulosic polysaccharides, cellulose and xylan. Members of the Bacteroidetes constitute a dominant phylum in both the human colonic microbiome and the rumen microbial ecosystem. In the current article, we review recent insights into the molecular mechanisms for xylan degradation by rumen and human commensal members of the Bacteroidetes phylum, and place this information in the context of the physiological and metabolic processes that occur within these complex microbial environments.     

Exploring the bovine rumen bacterial community from birth to adulthood

The mammalian gut microbiota is essential in shaping many of its host''s functional attributes. One such microbiota resides in the bovine digestive tract in a compartment termed as the rumen. The rumen microbiota is necessary for the proper physiological development of the rumen and for the animal''s ability to digest and convert plant mass into food products, making it highly significant to humans. The establishment of this microbial population and the changes occurring with the host''s age are important for understanding this key microbial community. Despite its importance, little information about colonization of the microbial populations in newborn animals, and the gradual changes occurring thereafter, exists. Here, we characterized the overall bovine ruminal bacterial populations of five age groups, from 1-day-old calves to 2-year-old cows. We describe the changes occurring in the rumen ecosystem after birth, reflected by a decline in aerobic and facultative anaerobic taxa and an increase in anaerobic ones. Some rumen bacteria that are essential for mature rumen function could be detected as early as 1 day after birth, long before the rumen is active or even before ingestion of plant material occurs. The diversity and within-group similarity increased with age, suggesting a more diverse but homogeneous and specific mature community, compared with the more heterogeneous and less diverse primary community. In addition, a convergence toward a mature bacterial arrangement with age was observed. These findings have also been reported for human gut microbiota, suggesting that similar forces drive the establishment of gut microbiotas in these two distinct mammalian digestive systems.     

Molecular analysis of the bacterial microbiome in the forestomach fluid from the dromedary camel (Camelus dromedarius)

Rumen microorganisms play an important role in ruminant digestion and absorption of nutrients and have great potential applications in the field of rumen adjusting, food fermentation and biomass utilization etc. In order to investigate the composition of microorganisms in the rumen of camel (Camelus dromedarius), this study delves in the microbial diversity by culture-independent approach. It includes comparison of rumen samples investigated in the present study to other currently available metagenomes to reveal potential differences in rumen microbial systems. Pyrosequencing based metagenomics was applied to analyze phylogenetic and metabolic profiles by MG-RAST, a web based tool. Pyrosequencing of camel rumen sample yielded 8,979,755 nucleotides assembled to 41,905 sequence reads with an average read length of 214 nucleotides. Taxonomic analysis of metagenomic reads indicated Bacteroidetes (55.5 %), Firmicutes (22.7 %) and Proteobacteria (9.2 %) phyla as predominant camel rumen taxa. At a finer phylogenetic resolution, Bacteroides species dominated the camel rumen metagenome. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism were the most abundant SEED subsystem representing 17 and 13 % of camel metagenome, respectively. A high taxonomic and functional similarity of camel rumen was found with the cow metagenome which is not surprising given the fact that both are mammalian herbivores with similar digestive tract structures and functions. Combined pyrosequencing approach and subsystems-based annotations available in the SEED database allowed us access to understand the metabolic potential of these microbiomes. Altogether, these data suggest that agricultural and animal husbandry practices can impose significant selective pressures on the rumen microbiota regardless of rumen type. The present study provides a baseline for understanding the complexity of camel rumen microbial ecology while also highlighting striking similarities and differences when compared to other animal gastrointestinal environments.     

Maternal versus artificial rearing shapes the rumen microbiome having minor long-term physiological implications

Increasing productivity is a key target in ruminant science which requires better understanding of the rumen microbiota. This study investigated how maternal versus artificial rearing shapes the rumen microbiota using 24 sets of triplet lambs. Lambs within each sibling set were randomly assigned to natural rearing on the ewe (NN); ewe colostrum for 24 h followed by artificial milk feeding (NA); and colostrum alternative and artificial milk feeding (AA). Maternal colostrum feeding enhanced VFA production at weaning but not thereafter. At weaning, lambs reared on milk replacer had no rumen protozoa and lower microbial diversity, whereas natural rearing accelerated the rumen microbial development and facilitated the transition to solid diet. Differences in the rumen prokaryotic communities disappear later in life when all lambs were grouped on the same pasture up to 23 weeks of age. However, NN animals retained higher fungal diversity and abundances of Piromyces, Feramyces and Diplodiniinae protozoa as well as higher feed digestibility (+4%) and animal growth (+6.5%) during the grazing period. Nevertheless, no correlations were found between rumen microbiota and productive outcomes. These findings suggest that the early life nutritional intervention determine the initial rumen microbial community, but the persistence of these effects later in life is weak.     

Dietary phytochemicals as rumen modifiers: a review of the effects on microbial populations

In the recent years, the exploration of bioactive phytochemicals as natural feed additives has been of great interest among nutritionists and rumen microbiologists to modify the rumen fermentation favorably such as defaunation, inhibition of methanogenesis, improvement in protein metabolism, and increasing conjugated linoleic acid content in ruminant derived foods. Many phytochemicals such as saponins, essential oils, tannins and flavonoids from a wide range of plants have been identified, which have potential values for rumen manipulation and enhancing animal productivity as alternatives to chemical feed additives. However, their effectiveness in ruminant production has not been proved to be consistent and conclusive. This review discusses the effects of phytochemicals such as saponins, tannins and essential oils on the rumen microbial populations, i.e., bacteria, protozoa, fungi and archaea with highlighting molecular diversity of microbial community in the rumen. There are contrasting reports of the effects of these phytoadditives on the rumen fermentation and rumen microbes probably depending upon the interactions among the chemical structures and levels of phytochemicals used, nutrient composition of diets and microbial components in the rumen. The study of chemical structure–activity relationships is required to exploit the phytochemicals for obtaining target responses without adversely affecting beneficial microbial populations. A greater understanding of the modulatory effects of phytochemicals on the rumen microbial populations together with fermentation will allow a better management of the rumen ecosystem and a practical application of this feed additive technology in livestock production.     

From rumen to industry

ABSTRACT: The rumen is one of the most complicated and most fascinating microbial ecosystems in nature. A wide variety of microbial species, including bacteria, fungi and protozoa act together to bioconvert (ligno)cellulosic plant material into compounds, which can be taken up and metabolized by the ruminant. Thus, the rumen perfectly resembles a solution to a current industrial problem: the biorefinery, which aims at the bioconversion of lignocellulosic material into fuels and chemicals. We suggest to intensify the studies of the ruminal microbial ecosystem from an industrial microbiologists point of view in order to make use of this rich source of organisms and enzymes.     

Effects of Dietary Linseed Oil and Propionate Precursors on Ruminal Microbial Community,Composition, and Diversity in Yanbian Yellow Cattle

The rumen microbial ecosystem is a complex system where rumen fermentation processes involve interactions among microorganisms. There are important relationships between diet and the ruminal bacterial composition. Thus, we investigated the ruminal fermentation characteristics and compared ruminal bacterial communities using tag amplicon pyrosequencing analysis in Yanbian yellow steers, which were fed linseed oil (LO) and propionate precursors. We used eight ruminally cannulated Yanbian yellow steers (510 ± 5.8 kg) in a replicated 4 × 4 Latin square design with four dietary treatments. Steers were fed a basal diet that comprised 80% concentrate and 20% rice straw (DM basis, CON). The CON diet was supplemented with LO at 4%. The LO diet was also supplemented with 2% dl-malate or 2% fumarate as ruminal precursors of propionate. Dietary supplementation with LO and propionate precursors increased ruminal pH, total volatile fatty acid concentrations, and the molar proportion of propionate. The most abundant bacterial operational taxonomic units in the rumen were related to dietary treatments. Bacteroidetes dominated the ruminal bacterial community and the genus Prevotella was highly represented when steers were fed LO plus propionate precursors. However, with the CON and LO diet plus malate or fumarate, Firmicutes was the most abundant phylum and the genus Ruminococcus was predominant. In summary, supplementing the diets of ruminants with a moderate level of LO plus propionate precursors modified the ruminal fermentation pattern. The most positive responses to LO and propionate precursors supplementation were in the phyla Bacteriodetes and Firmicutes, and in the genus Ruminococcus and Prevotella. Thus, diets containing LO plus malate or fumarate have significant effects on the composition of the rumen microbial community.     

Erosion reduces soil microbial diversity,network complexity and multifunctionality

While soil erosion drives land degradation, the impact of erosion on soil microbial communities and multiple soil functions remains unclear. This hinders our ability to assess the true impact of erosion on soil ecosystem services and our ability to restore eroded environments. Here we examined the effect of erosion on microbial communities at two sites with contrasting soil texture and climates. Eroded plots had lower microbial network complexity, fewer microbial taxa, and fewer associations among microbial taxa, relative to non-eroded plots. Soil erosion also shifted microbial community composition, with decreased relative abundances of dominant phyla such as Proteobacteria, Bacteroidetes, and Gemmatimonadetes. In contrast, erosion led to an increase in the relative abundances of some bacterial families involved in N cycling, such as Acetobacteraceae and Beijerinckiaceae. Changes in microbiota characteristics were strongly related with erosion-induced changes in soil multifunctionality. Together, these results demonstrate that soil erosion has a significant negative impact on soil microbial diversity and functionality.Subject terms: Soil microbiology, Microbial ecology     

Diet Alters Both the Structure and Taxonomy of the Ovine Gut Microbial Ecosystem

We surveyed the ruminal metagenomes of 16 sheep under two different diets using Illumina pair-end DNA sequencing of raw microbial DNA extracted from rumen samples. The resulting sequence data were bioinformatically mapped to known prokaryotic 16S rDNA sequences to identify the taxa present in the samples and then analysed for the presence of potentially new taxa. Strikingly, the majority of the microbial individuals found did not map to known taxa from 16S sequence databases. We used a novel statistical modelling approach to compare the taxonomic distributions between animals fed a forage-based diet and those fed concentrated grains. With this model, we found significant differences between the two groups both in the dominant taxa present in the rumen and in the overall shape of the taxa abundance curves. In general, forage-fed animals have a more diverse microbial ecosystem, whereas the concentrate-fed animals have ruminal systems more heavily dominated by a few taxa. As expected, organisms from methanogenic groups are more prevalent in forage-fed animals. Finally, all of these differences appear to be grounded in an underlying common input of new microbial individuals into the rumen environment, with common organisms from one feed group being present in the other, but at much lower abundance.     

The bacterial community and metabolome dynamics and their interactions modulate fermentation process of whole crop corn silage prepared with or without inoculants

Multi-omics approach was adopted to investigate the modulation of bacterial microbiota and metabolome as well as their interactions in whole crop corn ensiling systems by inoculating homofermentative Lactobacillus plantarum or heterofermentative Lactobacillus buchneri. Inoculations of the two different inoculants resulted in substantial differences in microbial community and metabolic composition as well as their dynamics in ensiled corn. Inoculants also altered the correlations of microbiota in different manners, and various keystone species were identified in corn silages with different treatments. Many metabolites with biofunctional activities like bacteriostatic, antioxidant, central nervous system inhibitory and anti-inflammatory were found in the present silage. A constitutive difference in microbiota dynamics was found for several pathways, which were upregulated by specific taxa in middle stage of fermentation, and widespread associations between metabolites with biofunctions and the species of lactic acid bacteria dominated in silage were observed. Multiple microbial and metabolic structures and dynamics were correlated and affected the fermentation process of the corn ensiling systems. Results of the current study improve our understanding of the complicated biological process underlying silage fermentation and provide a framework to re-evaluate silages with biofunctions, which may contribute to target-based regulation methods to produce functional silage for animal production.     

A commentary on methodological aspects of hydrolysable tannins metabolism in ruminant: a perspective view

Although, the application of tannic acid (TA), gallic acid (GA), natural hydrolysable tannins (HT)-rich ingredients, and HT-rich feeds in ruminant feeding have been explored in order to modify or manipulate microbial activities of digestive tract of animals, the interaction between HT and gastrointestinal microbiota and the fate of HT metabolites (GA, ellagic acid, pyrogallol, resorcinol, phloroglucinol, catechol and urolithin) derived from gastrointestinal microbial HT metabolism in the animal as a whole and animal products are missing. Incomplete biotransformation of HT and TA to GA, pyrogallol, resorcinol, phloroglucinol and other phenolic metabolites is a prevalent phenomenon discovered by researchers who examine the fate of HT metabolites in ruminant. While the rest of fellow researchers do not even examine the fate of HT metabolites and assume the complete biotransformation and fermentation of HT metabolites to volatile fatty acids (VFA). Only three studies have successfully identified the complete biotransformation and fermentation of HT metabolites to VFA in ruminant. The HT metabolites, mostly pyrogallol, produced through incomplete biotransformation of HT have adverse effects on gastrointestinal microbiota and host animal. Lack of awareness regarding the metabolism of HT metabolites and its consequences would compromise ruminant gastrointestinal microbiota, animal welfare, our environment and the power of research papers’ findings. In this perspective paper, I will bring to attention a new angle on the biotransformation and fermentation of HT metabolites in gastrointestinal tract, the role of gastrointestinal microbiota and deficiency of current approach in isolating tannin-degrading bacteria from rumen. Also, suggestions for better monitoring and understanding HT metabolisms in ruminant are presented.     

微生物介导反刍动物瘤胃氨生成及其对瘤胃功能的影响

反刍动物瘤胃中栖息着丰富多样的微生物,其在瘤胃内氨生成过程中发挥了重要的作用。微生物介导的氨基酸脱氨基作用和非蛋白氮水解作用是瘤胃内氨生成的主要途径。微生物介导了瘤胃内氨的生成,同时瘤胃内产生的氨也会反馈影响微生物菌群结构及瘤胃上皮功能,进而影响瘤胃发酵及宿主健康。本文主要综述了瘤胃微生物在介导氨生成中的作用和氨对瘤胃消化及瘤胃上皮功能的影响,以期对后续研究有所启发。     

Rumen Microbiome from Steers Differing in Feed Efficiency

The cattle rumen has a diverse microbial ecosystem that is essential for the host to digest plant material. Extremes in body weight (BW) gain in mice and humans have been associated with different intestinal microbial populations. The objective of this study was to characterize the microbiome of the cattle rumen among steers differing in feed efficiency. Two contemporary groups of steers (n=148 and n=197) were fed a ration (dry matter basis) of 57.35% dry-rolled corn, 30% wet distillers grain with solubles, 8% alfalfa hay, 4.25% supplement, and 0.4% urea for 63 days. Individual feed intake (FI) and BW gain were determined. Within contemporary group, the four steers within each Cartesian quadrant were sampled (n=16/group) from the bivariate distribution of average daily BW gain and average daily FI. Bacterial 16S rRNA gene amplicons were sequenced from the harvested bovine rumen fluid samples using next-generation sequencing technology. No significant changes in diversity or richness were indicated, and UniFrac principal coordinate analysis did not show any separation of microbial communities within the rumen. However, the abundances of relative microbial populations and operational taxonomic units did reveal significant differences with reference to feed efficiency groups. Bacteroidetes and Firmicutes were the dominant phyla in all ruminal groups, with significant population shifts in relevant ruminal taxa, including phyla Firmicutes and Lentisphaerae, as well as genera Succiniclasticum, Lactobacillus, Ruminococcus, and Prevotella. This study suggests the involvement of the rumen microbiome as a component influencing the efficiency of weight gain at the 16S level, which can be utilized to better understand variations in microbial ecology as well as host factors that will improve feed efficiency.