Pelleted-hay alfalfa feed increases sheep wether weight gain and rumen bacterial richness over loose-hay alfalfa feed.

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.

Fig 2. Observed bacterial richness (A) and Shannon diversity (B) in the rumen of wethers on loose-hay or pelleted-hay alfalfa diets. Significance was determined at p < 0.05, by linear mixed model for observed SVs and Conover test for Shannon diversity, with sheep ID as a fixed effect.
Fig 5. Discriminatory rumen bacterial sequence variance by treatment group for wethers receiving loose-hay or pelleted-hay alfalfa diet treatments.Significance (p < 0.05) determined by binomial test. 

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

Zinc amino acid supplementation alters yearling ram rumen bacterial communities but zinc sulfate supplementation does not.

Zinc is an important mineral in your diet; it’s required by many of your enzymes and having too much or too little can cause health problems. We know quite a bit about how important zinc is to sheep, in particular for their growth, immune system, and fertility.  We also know that organically- versus inorganically-sourced zinc differs in its bio-availability, or how easy it is for cells to access and use it.  Surprisingly, we know nothing about how different zinc formulations might affect gut microbiota, despite the knowledge that microorganisms may also need zinc.

This collaborative study was led by Dr. Whit Stewart and his then-graduate student, Chad Page, while they were at Montana State University (they are now both at the University of Wyoming).   Chad’s work focused on how different sources of zinc affected sheep growth and performance (previously presented, publication forthcoming), and I put together this  companion paper examining the effects on rumen bacteria. Unfortunately, the article is not currently open-access.


Ishaq, S.L., Page, C.M., Yeoman, C.J., Murphy, T.W., Van Emon, M.L., Stewart, W.C. 2019. Zinc amino acid supplementation alters yearling ram rumen bacterial communities but zinc sulfate supplementation does not. Journal of Animal Science 97(2):687–697. Article.

Abstract

Despite the body of research into Zn for human and animal health and productivity, very little work has been done to discern whether this benefit is exerted solely on the host organism, or whether there is some effect of dietary Zn upon the gastrointestinal microbiota, particularly in ruminants. We hypothesized that 1) supplementation with Zn would alter the rumen bacterial community in yearling rams, but that 2) supplementation with either inorganically-sourced ZnSO4, or a chelated Zn amino acid complex, which was more bioavailable, would affect the rumen bacterial community differently. Sixteen purebred Targhee yearling rams were utilized in an 84 d completely-randomized design, and allocated to one of three pelleted dietary treatments: control diet without fortified Zn (~1 x NRC), a diet fortified with a Zn amino acid complex (~2 x NRC), and a diet fortified with ZnSO4 (~2 x NRC). Rumen bacterial community was assessed using Illumina MiSeq of the V4-V6 region of the 16S rRNA gene. One hundred and eleven OTUs were found with > 1% abundance across all samples. The genera PrevotellaSolobacteriumRuminococcusButyrivibrioOlsenellaAtopobium, and the candidate genus Saccharimonas were abundant in all samples. Total rumen bacterial evenness and diversity in rams were reduced by supplementation with a Zn-amino-acid complex, but not in rams supplemented with an equal concentration of ZnSO4, likely due to differences in bioavailability between organic and inorganically-sourced supplement formulations. A number of bacterial genera were altered by Zn supplementation, but only the phylum Tenericutes was significantly reduced by ZnSO4 supplementation, suggesting that either Zn supplementation formulation could be utilized without causing a high-level shift in the rumen bacterial community which could have negative consequences for digestion and animal health.

Featured Image Source: Wikimedia Commons

Biogeographical Differences in the Influence of Maternal Microbial Sources on the Early Successional Development of the Bovine Neonatal Gastrointestinal tract.

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.

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Figure 1 Mean bacterial diversity at the phylum level for maternal and calf lumen (A) and mucosal (B) samples.

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.

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Figure 1 (partial) Mean bacterial diversity at the phylum level for maternal and calf lumen (left) and mucosal (right) samples.

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).

colostrum
A poor-quality GIF, showing bacterial communities from the calf digestive tract (each other the colored shaped) becoming more similar to maternal colostrum (milk) samples (grey asterisk) over the first 21 days of life.

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.

GI tract
An equally poor-quality GIF, showing bacterial communities (colored shapes) from the calf digestive tract samples becoming more similar to maternal samples (grey asterisks) as one moves along the digestive tract.


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: 3197Article.

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.


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)

 

 

A living soil inoculum increases soil microbial diversity, crop and weed growth using soil from organic and conventional farms in northeastern Montana.

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.

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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.


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.

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Poster presentation at ASM 2016.

Ishaq et al ASM 2016 poster