Ruminants, like sheep, goats, cows, deer, moose, etc.,  have a four-chambered stomach, the largest of which is called the rumen.  The rumen houses symbiotic microorganisms which break down plant fibers that the animal can’t digest on its own.  It’s estimated that up to 80% of a ruminant’s energy need is met from the volatile fatty acids (also called short-chain fatty acids) that bacteria produce from digesting fiber, and that up to 85% of a ruminant’s protein need is met from microbial proteins.

A lot of factors can be manipulated to help get the most out of one’s diet, including adjusting ingredients for water content, palatability, ease of chewing, and how easy the ingredients are to digest.  For example, highly fibrous foods with larger particles/pieces require more chewing, as well as a longer time spent in the rumen digesting so that microorganisms have plenty of time to break the chemical bonds of large molecules.  Smaller food particles can reduce the time and effort spent chewing, allow for more surface area on plant fibers for microorganisms to attach to and digest faster, and speed up the movement of food through the digestive tract.  On the other hand, moving food too quickly could reduce the amount of time microorganisms can spend digesting, or time the ruminant can absorb nutrients across their GI tract lumen, or cause slow-growing microbial species to wash out.

Surprisingly, almost no work has investigated the effect of diet particle size on the community, despite knowing that microbial digestion is contingent on the ability to attach to and process complex nutrient structures.  In this study, we observed the effect of particle size on rumen bacteria, by feeding long-stem (loose) alfalfa hay compared to a ground and pelleted version of the same alfalfa in yearling sheep wethers. 

The pelleted-hay diet group had a greater increase in bacterial richness, including common fibrolytic rumen inhabitants, which may explain the increase in average daily gain and feed efficiency in this group.

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Ishaq SL, Lachman MM, Wenner BA, Baeza A, Butler M, Gates E, et al. (2019) Pelleted-hay alfalfa feed increases sheep wether weight gain and rumen bacterial richness over loose-hay alfalfa feed. PLoS ONE 14(6): e0215797. Article.

Abstract

Diet composed of smaller particles can improve feed intake, digestibility, and animal growth or health, but in ruminant species can reduce rumination and buffering–the loss of which may inhibit fermentation and digestibility. However, the explicit effect of particle size on the rumen microbiota remains untested, despite their crucial role in digestion. We evaluated the effects of reduced particle size on rumen microbiota by feeding long-stem (loose) alfalfa hay compared to a ground and pelleted version of the same alfalfa in yearling sheep wethers during a two-week experimental period. In situ digestibility of the pelleted diet was greater at 48 h compared with loose hay; however, distribution of residual fecal particle sizes in sheep did not differ between the dietary treatments at any time point (day 7 or 14). Both average daily gain and feed efficiency were greater for the wethers consuming the pelleted diet. Observed bacterial richness was very low at the end of the adaptation period and increased over the course of the study, suggesting the rumen bacterial community was still in flux after two weeks of adaptation. The pelleted-hay diet group had a greater increase in bacterial richness, including common fibrolytic rumen inhabitants. The pelleted diet was positively associated with several Succiniclasticum, a Prevotella, and uncultured taxa in the Ruminococcaceae and Rickenellaceae families and Bacteroidales order. Pelleting an alfalfa hay diet for sheep does shift the rumen microbiome, though the interplay of diet particle size, retention and gastrointestinal transit time, microbial fermentative and hydrolytic activity, and host growth or health is still largely unexplored.

Feature image credit: Pellet Mill

Most studies that examine the microbial diversity of the gastrointestinal tract only look at one or two sample sites, usually the mouth, the rumen in ruminant animals, or the feces.  It can be difficult, expensive, invasive, or fatal to get samples from deep inside the intestinal tract; however many studies have pointed out that anatomical location and local environmental factors (like temperature, pH, host cells, nutrient availability, and exposure to UV light) can dramatically change a microbial community.  Thus, the microbes that we find in feces aren’t always what we would find in the stomach or along the intestines.

On top of that, certain microorganisms have been shown to closely associate with or attach to host cells lining the digestive tract, and the diversity of microbes next to host tissues can be different from what’s at the center of the intestines (the digesta).  This large, collaborative project took samples from nine different sites along the digestive tract of calves over the first 21 days of life to determine how body sites differed from each other, how sites changed over time as the calf matured, and how the lumen-associated bacteria would differ from the digesta-associated bacteria.

Samples from the mothers were also taken to understand how maternal microbial influence would affect body sites over time.  One of the most interesting finds of the study regarded colostrum, which is the special and highly-nutritious milk produced in the first 48 hours or so after parturition (birth).  Colostrum milk possessed a high diversity of bacteria, and is not sterile as was once assumed.

Not only that, but the bacterial community in colostrum had an impact on the bacterial community that developed along the calf digestive tract over time.  Calves received two doses of colostrum on the day of birth which had been aseptically collected from their dams and then fed to them, so that calves received milk but not the microbial influence of nursing and coming into contact with the dam.  After those two meals, calves were switched to milk replacer.  Surprisingly, the influence on the bacterial community wasn’t high on day one and then dropped off.  It increased over the first 21 days of life as bacterial communities from the digestive tract became more similar to bacterial communities found in colostrum (shown below).

In addition, we found that bacteria in the digestive tract became more similar to maternal samples moving from one end of the digestive tract to the other. We speculated that bacterial communities need time to develop, especially in a neonate ruminant which doesn’t have a functional rumen yet.  A flap of skin at the base of the esophagus (called the esophageal groove) shunts food into the omasum, bypassing the rumen and the reticulum where bacteria and other microorganisms would otherwise thrive.  After briefly passing through the omasum, milk would pass through the abomasum which is a glandular stomach (like the human stomach).  Both of those features are obstacles for ingested microorganisms to get past, and it would take time, and distance, to recover.

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Yeoman, C.J., Ishaq, S.L., Bichi , E., Olivo, S., Lowe, J., Aldridge, B.M. 2018. Biogeographical Differences in the Influence of Maternal Microbial Sources on the Early Successional Development of the Bovine Neonatal Gastrointestinal tract. Scientific Reports 8: 3197. Article.

Abstract

The impact of maternal microbial influences on the early choreography of the neonatal calf microbiome were investigated. Luminal content and mucosal scraping samples were collected from ten locations in the calf gastrointestinal tract (GIT) over the first 21 days of life, along with postpartum maternal colostrum, udder skin, and vaginal scrapings. Microbiota were found to vary by anatomical location, between the lumen and mucosa at each GIT location, and differentially enriched for maternal vaginal, skin, and colostral microbiota. Most calf sample sites exhibited a gradual increase in α-diversity over the 21 days beginning the first few days after birth. The relative abundance of Firmicutes was greater in the proximal GIT, while Bacteroidetes were greater in the distal GIT. Proteobacteria exhibited greater relative abundances in mucosal scrapings relative to luminal content. Forty-six percent of calf luminal microbes and 41% of mucosal microbes were observed in at-least one maternal source, with the majority being shared with microbes on the skin of the udder. The vaginal microbiota were found to harbor and uniquely share many common and well-described fibrolytic rumen bacteria, as well as methanogenic archaea, potentially indicating a role for the vagina in populating the developing rumen and reticulum with microbes important to the nutrition of the adult animal.

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Ishaq*, S.L., Bichi, E., Olivo, S.K., Lowe, J., Yeoman, C.J., Aldridge, B M. 2016. Influence of colostrum on the microbiological diversity of the developing bovine intestinal tract. Joint Annual Meeting, Salt Lake City, Utah, July 2016. (accepted talk)

In 2015, while working in the Yeoman Lab, I was invited to perform the sequence analysis on some samples from a previously-run diet study.  The study was part of ongoing research by Dr. Travis Whitney at Texas A & M on the use of juniper as a feed additive for sheep.  Coupled with the animal health and physiology aspect led by Travis, we published two companion papers in the Journal of Animal Science.  We had also previously presented these results at the Joint Annual Meeting of the American Society for Animal Science, the American Dairy Science Association, and the Canadian Society for Animal Science in Salt Lake City, UT in 2016.

The three main juniper species in Texas can pose a problem- while they are native, they have significantly increased the number of acres they occupy due to changes in climate, water availability, and human-related land use.  And, juniper can out-compete other rangeland species, which can make forage less palatable, less nutritious, or unhealthy for livestock.  Juniper contains essential oils and compounds which can affect some microorganisms living in their gut.  We wanted to know how the bacterial community in the rumen might restructure while on different concentrations of juniper and urea.

There was not a large effect of treatment on the rumen bacterial community in lambs (Figure 1B).  There was a change in animal production (feed intake and weight gain) (Whitney, 2017), yet, because bacterial diversity was largely unchanged by the diet, this was likely because the diet treatments reduced feed intake.  Plant secondary compounds, often called dietary toxins, can make it harder for animals to maintain a stable body temperature as they change fermentation in the rumen and how much heat is produced.  This increases the metabolic cost of thermoregulation as animals continuously have to adjust their rate of metabolism to keep their body temperature stable.  To avoid eating too many of these plant compounds, herbivores employ feeding strategies, such as reducing feed intake. It is possible that lambs ingesting high concentrations of juniper in Texas during the late summer simply consumed less supplemental diet in order to reduce toxin- and fermentation-related heat generation.

That’s not to say that there were no changes to the bacterial community at all; in fact, a number of important bacterial families were increased or decreased by increasing the amount of juniper, increasing the amount of urea, or both (Figure 2).

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Ishaq, S.L., Yeoman, C.J., Whitney, T.R. 2017. Ground Juniperus pinchotii and urea in supplements fed to Rambouillet ewe lambs. Part 2: Ewe lamb rumen microbial communities. Journal of Animal Science Oct; 95(10):4587-4599. Article.

Abstract

This study evaluated effects of ground redberry juniper (Juniperus pinchotii) and urea in dried distillers grains with solubles-based supplements fed to Rambouillet ewe lambs (n = 48) on rumen physiological parameters and bacterial diversity. In a randomized study (40 d), individually-penned lambs were fed ad libitum ground sorghum-sudangrass hay and of 1 of 8 supplements (6 lambs/treatment; 533 g/d; as-fed basis) in a 4 × 2 factorial design with 4 concentrations of ground juniper (15%, 30%, 45%, or 60% of DM) and 2 levels of urea (1% or 3% of DM). Increasing juniper resulted in minor changes in microbial β-diversity (PERMANOVA, pseudo F = 1.33, P = 0.04); however, concentrations of urea did not show detectable broad-scale differences at phylum, family, or genus levels according to ANOSIM (P > 0.05), AMOVA (P > 0.10), and PERMANOVA (P > 0.05). Linear discriminant analysis indicated some genera were specific to certain dietary treatments (P < 0.05), though none of these genera were present in high abundance; high concentrations of juniper were associated with Moraxella and Streptococcus, low concentrations of urea were associated with Fretibacterium, and high concentrations of urea were associated with Oribacterium and Pyramidobacter. Prevotella were decreased by juniper and urea. Ruminococcus, Butyrivibrio, and Succiniclasticumincreased with juniper and were positively correlated (Spearman’s, P < 0.05) with each other but not to rumen factors, suggesting a symbiotic interaction. Overall, there was not a juniper × urea interaction for total VFA, VFA by concentration or percent total, pH, or ammonia (P > 0.29). When considering only percent inclusion of juniper, ruminal pH and proportion of acetic acid linearly increased (P < 0.001) and percentage of butyric acid linearly decreased (P = 0.009). Lamb ADG and G:F were positively correlated with Prevotella(Spearman’s, P < 0.05) and negatively correlated with Synergistaceae, the BS5 group, and Lentisphaerae. Firmicutes were negatively correlated with serum urea nitrogen, ammonia, total VFA, total acetate, and total propionate. Overall, modest differences in bacterial diversity among treatments occurred in the abundance or evenness of several OTUs, but there was not a significant difference in OTU richness. As diversity was largely unchanged, the reduction in ADG and lower-end BW was likely due to reduced DMI rather than a reduction in microbial fermentative ability.

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Ishaq*, S.L., Yeoman, C.J., Whitney, T.R. 2016. Ground redberry juniper and urea in DDGS-based supplements do not adversely affect ewe lamb rumen microbial communities. Joint Annual Meeting, Salt Lake City, Utah, July 2016. (accepted talk). Travis Whitney’s companion presentation can be found here.

 

Featured Image Credit: National Park Service

 

What began as a simple data analysis project for me in the Yeoman lab turned into a publication, a conference presentation, a post-doc position, and a long-term, multi-project collaboration with the Menalled lab at Montana State University investigating soil microbial communities in agricultural settings and plant-soil feedbacks.

This study was part of a larger investigation on farming system (conventional or organic), and wheat-weed competition, as part of a master’s thesis by Stephen Johnson.  The publication on plant competition and crop performance can be found here.

The larger project involved soil collected from the fields of four farms around Montana which had both conventionally-managed and a USDA-certified organically-managed plots growing wheat.  Soil was brought back to Montana State University, where half of each field sample was sterilized to destroy living microorganisms.  A greenhouse study was performed using either the sterile or the living soil, and the soil was conditioned by growing either Amaranthus retroflexus L. (redroot pigweed) or Avena fatua L. (wild oat) for 16 weeks.  Following this plant growth phase, soil was collected and the bacterial community analyzed using Illumina MiSeq sequencing of the 16S rRNA gene.  The larger study then went on to study the performance of wheat crops in that preconditioned soil.

The strongest driving factor in soil bacterial communities was whether that soil had been sterile (purple) or living (green) in the greenhouse experiment, as seen below.  After that, farming system was the next strongest determinant of that community.  Interestingly, organically-sourced soil that had been sterilized was more similar to any living soil than conventionally-sourced sterile soil.  This indicates that organic soil was more favorable in recruiting a new soil community.

When comparing only the living soil samples, the samples reclustered by farming system; either organic or conventional.

Which weed species was growing was also an important factor, although much weaker.  A number of soil bacteria were more abundant in the soil around of the roots of one or the other plant.  Plants are known to associate with, and even recruit, different microbial communities, and this interaction can be plant-species-specific.

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Ishaq, S.L., Johnson, S.P., Miller, Z.J., Lehnhoff, E.A., Olivo, S.K., Yeoman, C.J., Menalled, F.D. 2017. A living soil inoculum increases soil microbial diversity, crop and weed growth using soil from organic and conventional farms in northeastern Montana. Microbial Ecology 73(2): 417-434. Impact 3.630. Article

Abstract

Farming practices affect the soil microbial community, which in turn impacts crop growth and crop-weed interactions. This study assessed the modification of soil bacterial community structure by organic or conventional cropping systems, weed species identity [Amaranthusretroflexus L. (redroot pigweed) or Avena fatua L. (wild oat)], and living or sterilized inoculum. Soil from eight paired USDA-certified organic and conventional farms in north-central Montana was used as living or autoclave-sterilized inoculant into steam-pasteurized potting soil, planted with Am. retroflexus or Av. fatua and grown for two consecutive 8-week periods to condition soil nutrients and biota. Subsequently, the V3-V4 regions of the microbial 16S rRNA gene were sequenced by Illumina MiSeq. Treatments clustered significantly, with living or sterilized inoculum being the strongest delineating factor, followed by organic or conventional cropping system, then individual farm. Living inoculum-treated soil had greater species richness and was more diverse than sterile inoculum-treated soil (observed OTUs, Chao, inverse Simpson, Shannon, P < 0.001) and had more discriminant taxa delineating groups (linear discriminant analysis). Living inoculum soil contained more Chloroflexi and Acidobacteria, while the sterile inoculum soil had more Bacteroidetes, Firmicutes, Gemmatimonadetes, and Verrucomicrobia. Organically farmed inoculum-treated soil had greater species richness, more diversity (observed OTUs, Chao, Shannon, P < 0.05), and more discriminant taxa than conventionally farmed inoculum-treated soil. Cyanobacteria were higher in pots growing Am. retroflexus, regardless of inoculum type, for three of the four organic farms. Results highlight the potential of cropping systems and species identity to modify soil bacterial communities, subsequently modifying plant growth and crop-weed competition.

 

Poster: Ishaq*, S.L., Johnson, S.P., Miller, Z.J., Lehnhoff, E.A., Olivo, S.K., Yeoman, C.J., Menalled, F.D. Farming Systems Modify The Impact Of Inoculum On Soil Microbial Diversity. American Society for Microbiology (ASM), Boston, MA, June 2016.