Case study: manipulating the rumen ciliates

The current data on function of the rumen protozoa suggest that lowering their densities or removing them entirely from the rumen microbiome may be an effective strategy to ensure high production and lower environmental impact of ruminants. The rumen protozoa are passed between animals by direct transfer through saliva (Becker and Hsiung, 1929; Yanez-Ruiz et al., 2015). Ciliate protozoa can normally be seen in the rumen of young ruminants within 2 weeks of birth with small Entodinia established before the large protozoa (Eadie, 1962). As such, protozoal-free rumen populations may be established in calves or lambs by rearing them in an environment devoid of other ruminants with a normal protozoal population from an early age (typically around 24-48 hours after birth, following initial suckling to expose them to maternal colostrum). Likewise, chemical treatment of the foregut can be used to remove the protozoal population later in life. While both of these approaches can be achieved in a research environment, neither is particularly practical in an agricultural setting. Instead manipulation, including either reduction or eradication, of the rumen ciliate population must be achieved by alternative means to make it practical.

One of the most successfully implemented examples of the use of supplements to reduce protozoal numbers and improve ruminant production efficiency, while minimising environmental impact.aretheionophores(including monensin) (Nagaraja et al., 1997). lonophores have growth-promoting effect on ruminants. Their mode of action is, to date, not fully understood although monensin has been shown to inhibit proteolysis within storage vesicles in the non-rumen ciliate Paramecium (Fok and Ueno, 1987) and inhibits lysosomal fusion of the food vacuole (Gautier et al., 1994). Asa consequence, it is assumed that monensin works in a similar way against the related rumen protozoa, albeit this has not been confirmed. Nonetheless, the rumen protozoa have also been shown to adapt to the addition of ionophores, thus reducing their ability to reduce protozoal density (Nagaraja et al., 1997). This protozoal adaptation coupled with a ban in 2006 for ionophore use in livestock production in the European Union due to concerns on the development of antimicrobial resistance means that alternatives need to be found.

Following the ban on ionophores, a substantial focus was placed onto evaluating phytochemicals as replacements. In particular, much emphasis has been given to the plant saponins, which consist of an aglycone or sapogenin linked to one or more sugar moieties through a glycosidic bond (Francis et al.,

2002). Different sources of saponins have been shown to have different levels of effect (Wallace et al„ 2002), for example, saponins from alfalfa have been shown to alter fermentation in a continuous culture (Lu et al., 1987) and reduce both protozoal numbers and methane production (Patra and Saxena, 2009). However, the results of application of saponins in the ruminant diet are mixed, with many studies reporting no effects (Sliwinski et al„ 2002; Holtshausen et al., 2009) but some reporting reductions in methane of 6.7% (Santoso et al., 2004). It is suggested that reductions in methane production are indirect and are a result of an increase in propionate production and reduction in protozoal numbers (Hristov et al., 1999). It is hypothesised that this variation in results is due to the differing sources and concentrations used in studies, for example, extracts from the fruit Sapindus saponaria were used in a study by Hess et al., (2004) which resulted in an inclusion rate of 0.75% which are much higher than the levels occurring in Yucca extracts (Sliwinski et al., 2002; Goel and Makkar, 2012). There is also the added complication of the accompanying diets in these studies, which vary from grazed pasture to forage/concentrate diets (Goel and Makkar, 2012).

Tannins work in a similar way to saponins as naturally occurring plant secondary metabolites with antimicrobial action and effects on rumen fermentation pathways. Two types of tannins exist - condensed and hydro- lysable, both of which may have adverse and beneficial effects on the animal simultaneously. To that end, condensed tannin-rich plants or their extracts are mostly used to reduce the risk of toxicity (Beauchemin et al., 2008). It is suggested that the methane-reducing effects of tannins may be a result of one of two actions: by direct effects on the rumen methanogens or by reducing feed degradation which lowers hydrogen production (Tavendale et al„ 2005). The negative effects of tannin application on ruminal fibre degradation are likely due to a reduction in cellulolytic bacteria and the formation of recalcitrant tannin-cellulose complexes (McSweeney et al., 2001; Makkar et al., 1995). However, as with studies investigating the effects of saponins, the research surrounding tannins can be inconsistent and contradictory, with conflicting evidence for effects on the rumen protozoa and the magnitude of effects observed (Benchaar et al., 2008; Goel and Makkar, 2012).

Other dietary supplements have also been used with some degree of success in reducing protozoal numbers (Benchaar et al„ 2008). This has included essential oils which have previously been shown to be effective against certain populations of rumen bacteria, and also tannins. The main issue in applying essential oils to the diet of animals is a lack of clarity about their exact mechanism and the contradictory research that has been published. While some studies are able to demonstrate significant effects, others are unable to replicate these results (Patra, 2011). There are also regulatory and safety concerns associated with the use of essential oils, as often high concentrations are required to produce a significant result which may lead to toxic effects as well as feeding into poor profitability.

Despite the research conducted on phytochemical supplementation of ruminant diets, these supplements are currently not used extensively in practice, mainly due to regulatory challenges, practical and financial concerns and limited feed industry uptake, although a company has been established (Delacon), which sell phytogenic supplements for enhancing ruminant production and health. It should also be noted that future strategies should aim to target removal of the order Vestibuliferida, due to their association with methanogens. Conversely, the current data suggest that due to their fibrolytic capacity, the order Entodiniomorphida should be retained.

Future trends and conclusions

With the global human population predicted to reach around 9.8 billion in 2050 one of the primary concerns facing society is the provision of adequate and nutritional food (UN, 2017). As a result, the agricultural industry will likely see a major surge in demand for food of animal origin, while concurrently experiencing pressure to increase sustainability and minimise environmental impact. One method of addressing this issue is to maximise rumen function and efficiency, however, to achieve improvements it is essential that we first have a complete understanding of the rumen microbiome. As previously highlighted, our knowledge of the rumen protozoa is far from complete, as such, further research using the latest sequencing and molecular techniques is vital. These methods offer the greatest potential to help truly elucidate the role of the protozoa in the rumen and therefore how best to manipulate the microbial population in our questto improve sustainability. Meta-omictechnologies such as metagenomics, metabolomics, metaproteomics and metatranscriptomics offer the opportunity to examine whole populations as opposed to the more limited methods that use pure cultures. Used in conjunction, these methods could provide an in-depth meta-analysis detailing interactions within the microbiome as well as between the microbiome and the host or ingested plant material. The biggest advantage of these technologies, in terms of the rumen protozoa, is their ability to bypass the roadblock of developing and maintaining a protozoal culture (Newbold et al„ 2015). Despite the development of advanced, culture-free techniques, the current representation of the rumen protozoa in sequence libraries is poor and limited to one macronuclear genome sequence of Entodinium caudatum alongside a handful of fibrolytic proteins (Park et al., 2018). As such, during bioinformatic analyses it is feasible that the protozoal fraction of a sample is significantly mis- or under-represented leading to unrepresentative results and a smaller perceived role in many metabolic processes (Comtet-Marre et al.,

2017). It is therefore important that research utilises NGS and omictechnologies to their fullest to deliver further functional annotation and genome sequencing of the rumen inhabitants - specifically the protozoa. The collection of more, in-depth and robust data seems to be the key to many potential interventions, including the development of microbiome biomarkers, protozoal-methanogen relationships and host-microbiome interaction, all of which affect the efficiency and environmental impact of the animal.

There is also a gap in the literature for more in-depth characterisation of the interactions between protozoa and the other microbial groups in the rumen, in particular the relationship of the methanogen populations with protozoa. It is currently established that abundance and structure of methanogen colonisation of the rumen protozoa differ depending on the genera, for example, smaller Entodinium spp. have been associated with higher levels of methane production than larger species, such as P. multivesiculatum (Ranilla et al., 2007).

The relationship between the host genotype and microbiome also remains to be fully elucidated, particularly in relation to methane production. When examining the effect of host genetics and the microbiome on methane production, a study by Difford et al. (2018) attributed 13% of variation to the rumen microbiome and 21% to host genetics, further suggesting that the two may not be linked. If this were the case then these two factors could be targeted independently. In summary, working with rumen protozoa is challenging, leading to difficulties for enhancing our understanding of their exact role in the rumen. Therefore, research on these unique protozloa still needs to be enhanced in order to fully understand their function and allow development of innovative strategies to target specific rumen protozoa to improve host phenotype and environmental impact.

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