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. 2019Journal of Animal Science 97(2):687–697. Article.


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.

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An investigation into rumen fungal and protozoal diversity in three rumen fractions, during high-fiber or grain-induced sub-acute ruminal acidosis conditions, with or without active dry yeast supplementation. 

Ruminal acidosis is a condition in which the pH of the rumen is considerably lower than normal, and if severe enough can cause damage to the stomach and localized symptoms, or systemic illness in cows.  Often, these symptoms result from the low pH reducing the ability of microorganisms to ferment fiber, or by killing them outright.  Since the cow can’t break down most of its plant-based diet without these microorganisms, this disruption can cause all sorts of downstream health problems.  Negative health effects can also occur when the pH is somewhat lowered, or is lowered briefly but repeatedly, even if the cow isn’t showing outward clinical symptoms.  This is known as sub-acute ruminal acidosis(SARA), and can also cause serious side effects for cows and an economic loss for producers.

In livestock, acidosis usually occurs when ruminants are abruptly switched to a highly-fermentable diet- something with a lot of grain/starch that causes a dramatic increase in bacterial fermentation and a buildup of lactate in the rumen.  To prevent this, animals are transitioned incrementally from one diet to the next over a period of days or weeks.  Another strategy is to add something to the diet to help buffer rumen pH, such as a probiotic.  One of the most common species used to help treat or prevent acidosis is a yeast; Saccharomyces cerevisiae.

This paper was part of a larger study on S. cerevisiae use in cattle to treat SARA, the effects of which on animal production as well as bacterial diversity and functionality have already been published by an old friend and colleague of mine, Dr. Ousama AlZahal, and several others.

The main driver of fungal diversity was diet; moving from a high-fiber diet to a high-grain diet (Figure 1) triggered a change in available nutrients (more starch, less fiber), and decreased in rumen pH due to the byproducts related to microbial digestion of those nutrients.  Supplementation with active dry yeast only had minimal effect on fungal populations in the rumen, and did not help recover the fungal community found in healthy cows on a high-fiber diet.  Saccharomyces-related sequences all classified as S. cerevisiae, though to multiple strains, but were not found in >1% mean relative abundance in any treatment group or significantly more abundant in any group. Thus, it was unclear if the yeast supplement was actively part of the rumen fungal community.

PowerPoint Presentation
Figure 1. Relative abundance of rumen fungi genera for cows receiving a high fiber (HF) or high grain (HG) diet, with (Y) or without (C) yeast supplementation. Treatments include high-fiber control (HFC), high-fiber yeast (HFY), high-grain control (HGC), and high-grain yeast (HGY).

Similarly, diet was the major driver of protozoal diversity in the rumen (Figure 2), but there was also a small effect of the yeast supplementation.  Taxonomic diversity was also different between the high-fiber control (what the cows were before) and the high-grain yeast-supplemented group, indicating that yeast supplementation did not recover the initial protozoal community which healthy cows had.

PowerPoint Presentation
Figure 2. Relative abundance of rumen protozoal species for cows receiving a high fiber (HF) or high grain (HG) diet, with (Y) or without (C) yeast supplementation. Treatments include high-fiber control (HFC), high-fiber yeast (HFY), high-grain control (HGC), and high-grain yeast (HGY).

Another large difference was seen in the number and type of species found in three different locations within the rumen: those found in rumen fluid, those found attached to plant material (and presumably digesting it), and those found attached or associated with the rumen wall (epimural-associated).  In cows fed the high-grain diets, there were not enough fungi in the rumen fluid to generate enough sequences for comparison, and the high-grain diet tended to reduce the number of different species found in any location.  Fungal species richness was highest in plant-associated fractions, and there was surprisingly high species richness of fungi which were found along the rumen wall.  Protozoal species richness was likewise reduced by a switch to a high-grain diet, and was highest next to the rumen wall.

Ishaq, S.L., AlZahal, O., Walker, N., McBride, B. 2017. An investigation into rumen fungal and protozoal diversity in three rumen fractions, during high-fiber or grain-induced sub-acute ruminal acidosis conditions, with or without active dry yeast supplementation.  Frontiers in Microbiology 8:1943. Article.


Sub-acute ruminal acidosis (SARA) is a gastrointestinal functional disorder in livestock characterized by low rumen pH, which reduces rumen function, microbial diversity, host performance, and host immune function. Dietary management is used to prevent SARA, often with yeast supplementation as a pH buffer. Almost nothing is known about the effect of SARA or yeast supplementation on ruminal protozoal and fungal diversity, despite their roles in fiber degradation. Dairy cows were switched from a high-fiber to high-grain diet abruptly to induce SARA, with and without active dry yeast (ADY, Saccharomyces cerevisiae) supplementation, and sampled from the rumen fluid, solids, and epimural fractions to determine microbial diversity using the protozoal 18S rRNA and the fungal ITS1 genes via Illumina MiSeq sequencing. Diet-induced SARA dramatically increased the number and abundance of rare fungal taxa, even in fluid fractions where total reads were very low, and reduced protozoal diversity. SARA selected for more lactic-acid utilizing taxa, and fewer fiber-degrading taxa. ADY treatment increased fungal richness (OTUs) but not diversity (Inverse Simpson, Shannon), but increased protozoal richness and diversity in some fractions. ADY treatment itself significantly (P < 0.05) affected the abundance of numerous fungal genera as seen in the high-fiber diet: Lewia, Neocallimastix, and Phoma were increased, while Alternaria, Candida Orpinomyces, and Piromyces spp. were decreased. Likewise, for protozoa, ADY itself increased Isotricha intestinalis but decreased Entodinium furca spp. Multivariate analyses showed diet type was most significant in driving diversity, followed by yeast treatment, for AMOVA, ANOSIM, and weighted UniFrac. Diet, ADY, and location were all significant factors for fungi (PERMANOVA, P = 0.0001, P = 0.0452, P = 0.0068, Monte Carlo correction, respectively, and location was a significant factor (P = 0.001, Monte Carlo correction) for protozoa. Diet-induced SARA shifts diversity of rumen fungi and protozoa and selects against fiber-degrading species. Supplementation with ADY mitigated this reduction in protozoa, presumptively by triggering microbial diversity shifts (as seen even in the high-fiber diet) that resulted in pH stabilization. ADY did not recover the initial community structure that was seen in pre-SARA conditions.

Ishaq, S.L.*, O. AlZahal, N. Walker, B. McBride. 2017. Modulation of sub-acute ruminal acidosis by active-dry yeast supplementation and its effect on rumen fungal and protozoal populations in liquid, solid, and epimural fractions.  Congress on Gastrointestinal Function, Chicago, IL, April 2017. (accepted talk).


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