Exploring microbial digestive dynamics of ruminants in vitro

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  • Ruminants such as cattle possess a complex system of microorganisms in the gut which facilitate the breakdown of plant material. Pioneering studies in ruminant microbial ecology and health are being conducted by Dr Marshall D Stern and Dr Andres M Gomez from the University of Minnesota. Using a dual flow continuous culture system to mimic in vivo conditions, Dr Stern and Dr Gomez are investigating effects of various compounds on microbial fermentation and ecology using the in vitro system.

    The animal has a complex biological system comprised of a variety of chemical structures and a community of microorganisms that facilitate the breakdown of plant material. Because of the complexity of this system, it is essential to understand the biology of these microorganisms, the role they play in facilitating nutrient utilisation and their sensitivities to certain compounds that may have either deleterious or beneficial effects on nutrition and health. Investigating these issues using a non-invasive method are Dr Marshall D. Stern and colleagues from the University of Minnesota.

    Rumen biology
    Herbivorous animals such as cattle are ruminants which acquire nutrients from plant-based materials through a complex system of fermentation. This fermentation process, known as pre-gastric fermentation, is facilitated by microorganisms in the gut, or rumen, and allows ruminants to obtain energy from plants. Plant material such as cellulose is digested by the enzyme cellulase secreted by these microorganisms in the gut. Without this system, cellulose would not be digested by animals.

    Dr Marshall D Stern Examining his In Vitro Model of the Rumen.

    Besides digestion of cellulose, the system also allows microbes to synthesise protein from nitrogen, detoxify plant compounds, reduce the number of required B vitamins, and subsequently increase the efficiency of absorption of end-products in the lower gut. Therefore, studying this system in greater detail allows for a better understanding of the compounds and mechanisms involved in pre-gastric fermentation and thus developing methods for optimum nutrient absorption in cattle.

    The Dual Flow Continuous Culture Fermentation System “Artificial Rumens” in Dr Stern’s Laboratory.

    Non-invasive methods
    In vitro models provide a non-invasive way of studying the “the rumen” which is the first compartment of the stomach. Dr Stern and colleagues from the University of Minnesota utilise the continuous culture system to study various factors that affect rumen microbial fermentation and ecology. This in vitro study system is said to mimic in vivo conditions by maintaining microbes in an environment similar to that of the rumen. Conditions such as temperature, pH and digesta flow are maintained in this study system. Compared to using animal models, the continuous culture system is less harmful, less expensive, less time-consuming and more controlled.

    Recently, Dr Stern and his graduate student, Isaac Salfer, and other colleagues examined similarities and differences between bacterial and archaeal communities in the rumen of dairy cattle compared to the in vitro continuous culture system. Previous research using culture systems and oligonucleotide techniques established that some microbial populations in vitro may be maintained at abundances similar to the in vivo conditions in ruminants. By sequencing ribosomal bacterial and archaeal genes, Dr Stern’s laboratory investigated whether microbial communities and their abundances differed between the in vitro and in vivo conditions. Their results showed that while communities differed, the most abundant species were maintained across both study systems. This lends weight to the efficacy of in vitro culture systems for studying rumen conditions in a non-invasive way.

    The Rumen Fermentation and Microbial Ecology Laboratory
    in the Department of Animal Science at the University of Minnesota.

    In vitro in action
    This continuous culture system enables Dr Stern’s research group to study potentially negative effects of certain compounds on microbial growth and fermentation. One such compound is patulin, a secondary metabolite of toxigenic strains of Penicillium, Aspergillus and Byssochlaymis species which are common contaminants of fermented feeds. It is understood that patulin is toxic to many organisms and possesses an antimicrobial effect. In fact, Penicillium-contaminated silage has been found to be associated with hemorrhagic disorders in cattle. With this understanding, Dr Stern and colleagues evaluated the effects of varying concentrations of patulin on microorganisms in the in vitro continuous culture fermenters.

    In vitro models provide a non-invasive way of studying the rumen compartment of cattle and sheep.

    Results of this study confirmed that patulin can alter metabolic processes associated with nutrient absorption and disrupt the production of bacterial end-products. Dr Stern and colleagues have postulated that due to the interconnectedness of ruminal microbial populations, these changes could be have come about by direct inhibitory effects of patulin on bacterial growth or through a lack of essential nutrients from an altered food chain. Thus, these alterations may have negative consequences on the health and performance of ruminants such as cattle and sheep.

    Scanning electron micrograph showing the differential size of chains of rumen bacteria (Cocci) attached to the surface of a rumen Ciliated Protozoa (Entodiniomorph).

    Using the same fermenter system, Dr Stern and colleagues also investigated the effects of the sulphur binder bismuth subsalicylate (BSS) on the production of hydrogen sulphide gas, H2S.

    Hydrogen sulphide gas derived from sources of dietary sulphur is typically absorbed by the rumen and detoxified in the liver, however, eructation of this gas can result in it being inhaled by ruminants. This is problematic as hydrogen sulphide has previously been associated with several negative conditions affecting animal health. For instance, some animals have an increased risk of developing the neurological disease polioencephalomalacia. Bismuth subsalicylate has been previously used to decrease hydrogen sulphide accumulation in the gut of humans and thus may help alleviate the negative effects of this gas in ruminants.

    By administering varying concentrations of bismuth subsalicylate, Dr Stern’s group showed that there was a significant reduction in the amount of H2S in vitro. However, this reduction occurred at the expense of microbial fermentation and metabolism which was suppressed overall. Future studies in vitro and in vivo will need to determine whether BSS can be used in safe concentration to reduce H2S gas production whilst maintaining optimum microbial fermentation.

    Other methods of mitigating the negative effects of dietary sulphur have been considered. For instance, a common food for ruminants is dried distillers grains with solubles (DDGS) which is a source of dietary sulphur and subsequently H2S. Because H2S requires an acidic environment (low pH) to function, it has been postulated by Dr Stern and colleagues that a more alkaline condition, manipulated by diet, can mitigate the production of dietary sulphur and thus potentially alleviate the negative effects of hydrogen sulphide. Dietary roughage, which has previously been shown to increase ruminal fluid pH, was used to mitigate H2S production. However, the results of this study showed no beneficial effect of dietary roughage on reducing H2S production. Future studies may require researchers to investigate the effects of other sources of dietary roughage on H2S production.

    Compared to using animal models, this system is less harmful, less expensive, less time-consuming and more controlled.

    Future studies and implications
    Studies by Dr Stern and colleagues have examined the detrimental effects of microbial toxins and methods to mitigate harmful gas production. Future studies by Dr Stern and Dr Gomez aim to investigate key components of the rumen system by looking at the effects of enzymes on microbial ecology and nutrition. Using state-of-the-art DNA sequencing and biochemical fingerprinting methods, they are investigating the effect of three enzymes: a beta-glucanase, a protease and a cellulase, on rumen microbial composition and function. Results from their studies will contribute to a better understanding of how the in vitro system can be used to mimic in vivo conditions and thus may have implications for health and efficiency.

    This study along with future research will contribute to a wide body of data on rumen microbial communities and thus contribute to the understanding of in vitro and in vivo systems. Dr Stern’s and Dr Gomez’s collaborative research will hopefully inform others on the best ways to improve animal efficiency, performance and health.

    What are the biggest downfalls to using an in vitro system?
    The biggest downfall is that the microbial community in vitro is not identical to that found in vivo. While many bacterial species may be similar, ciliated protozoa found in the rumen cannot be well maintained in vitro. In spite of this downfall, research from Dr Stern’s laboratory demonstrated similarities in fermentation in vitro and in vivo, indicating that the dual flow continuous culture fermentation system
    is an excellent “rumen model”.

    References

    • Tapia, M.O. et al. (2001). ‘Effects of patulin on rumen microbial fermentation in continuous culture fermenters’. Animal Feed Science and Technology. 97:239-246.
    • Ruiz-Moreno, M. (2005). ‘Mitigation of in vitro hydrogen sulfide production using bismuth subsalicylate with and without monensin in beef feedlot diets’. Journal of Animal Science.,93:5346–5354.
    • Binversie, E.Y. (2016). ‘Effects of dietary roughage and sulfur in diets containing corn dried distillers grains with solubles on hydrogen sulfide production and fermentation by rumen microbes in vitro’. Journal of Animal Science. 94:3883-3893.
    • Fessenden, S.W. (2017). ‘Effects of bismuth subsalicylate and dietary sulfur level on fermentation by ruminal microbes in continuous culture’. Translational Animal Science. 1:1–11.
    • Salfer, I. J. 2018. ‘Comparisons of bacterial and archaeal communities in the rumen and a dual-flow continuous culture fermentation system using amplicon sequencing’. Journal of Animal Science, 96:1059-1072.
  • Research Objectives
    Dr Marshall D Stern and Dr Andres M Gomez from the University of Minnesota and their team are interested in how an in vitro dual flow continuous culture system may be used to investigate ruminant microbial ecology. By simulating in vivo conditions through controlled means, Dr Stern and Dr Gomez aim to better understand factors that affect rumen microbial ecology and how best to remedy performance and health issues that ruminants encounter.

    Funding
    National Institute of food and agriculture (NIFA)
    Title of Project: Impact of Factors that Affect Rumen Microbial Ecology and Fermentation on Protein Supply to the Small Intestine of Ruminant Animals.

    Collaborators

    • Dr Martin Ruiz-Moreno
    • Dr Ofelia Tapia
    • Dr Alex Bach
    • Dr Samuel Fessenden
    • Dr Abigail Carpenter
    • Elizabeth Binversie
    • Isaac Salfer
    • Dr Tom Jenkins
    • Dr Michael Sadowsky
    • Dr Christopher Staley
    • Dr Alfredo DiCostanzo
    • Haley Johnson


    Bio
    Morse Alumni Distinguished Professor of Animal Science with expertise in rumen fermentation and microbiology. Research emphasis has been to improve the efficiency of nutrient utilisation by ruminants. Recent research uses continuous culture fermenters to study mycotoxins, reduce gas emissions from the rumen and evaluate factors affecting ruminal microbial ecology using metagenomics.

    Contact
    Dr Marshall D. Stern

    E: stern002@umn.edu
    T: +1 612 624 9296
    W:
    W:

  • Exploring microbial digestive dynamics of ruminants in vitro

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