Potential approaches and benefits of modulation of rumen ecosystem

V. R. Upadhyay1^*, Raju Kr. Dewry², Anupam Thakuria³ and Prashant Kumar⁴

¹PhD scholar, Animal Physiology Division,

²PhD scholar, Artificial Breeding Research Centre, ³MVSc scholar, Animal Nutrition Division,

⁴PhD scholar, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal

^*Email:vishwaranjanhzb@gmail.com

Introduction

Herbivores mostly rely on structural carbohydrates such as lignin, cellulose and hemicellulose as a source of energy, but the inability to produce respective hydrolytic enzymes make them to develop other means to thrive and utilize the available resources. Consequently, symbiotic microorganisms are established in their alimentary tracts that can hydrolyze and digest these structural plant sources to generate energy for themselves, as well as for the host animal. However, this microbial ecosystem is not fully efficient as there is significant portion of undigested feed in the feces with substantial loss of energy through CH₄ emission. Moreover, forages from tropical and subtropical area have some undesirable limitations like low energy value, deficiency of essential nutrients, imbalanced end productsand the presence of anti-nutritional compounds (Santra and Karim 2003 ) making it unsuitable for optimum growth and productivity. Thus to counteract the abovementioned limitations,this rumen microbial ecosystem should bestablized and optimized either as a result of natural selection or by artificial manipulation. Accordingly various traditional approaches through which overall performance of the rumen has been attempted to improve, like use of plant secondary metabolites, microbial feed additives, chemical feed additives, selective stimulation of beneficial rumen microbes and selective inhibition of harmful rumen microbes. Therefore, keeping these perspectives in mind this article attempts to provide the comprehensive idea about potential techniques and benefits of manipulation of rumen microflora and associated ruminal environment.

Rumen

The rumen is the largest compartment of the complex ruminant’s stomach occupying left half of the abdominal cavity and harbors an intricate bionetwork. Since, it has enormous space filled with digested and partially digested feed material, making the provision for regurgitation time and again. A combination of mechanical mastication and enzymatic action of the microbes on the hard fibre containing feeds results in breakdown into smaller pieces. For efficient action it has standardizedphysical and microbiological characteristics which are as follows(Castro- Montoya et al., 2011)–

Physical properties

• Dry matter (%) – 10–18
• Osmolality – 250–350 mOsmol/Kg ⁻¹
• pH – 5.5–6.9 (Mean 6.4)
• Redox potential -350 to −400 mV
• Temperature – 38–41 °C

Microbiological properties

• Anaerobic fungi – 10^3–5 g ⁻¹ (6 genera)
• Bacteria – 10^10–11g ⁻¹(>200 species)
• Bacteriophage – 10^7–9g ⁻¹particles ml ⁻¹
• Ciliate protozoa – 10^4–6g ⁻¹(25 genera)

Microbial Interactions

The biota of complex microflora inside the rumen is responsible for the complete degradation of organic matter ingested by the animals. There is no single organism that has the capacity for a complete degradation of complex substrates in the rumen.Therefore a complex process of cooperative catabolism undergoes for production and efficient utilization of fermentative end products. The complex feedstuffs are broken down into simple sugars by some group of microbes, transported, and fermented by other members of the microbial population that in turn produce branched-chain fatty acids, vitamins, or other cofactors for entirely other microorganisms.

Ruminal Manipulation

Dietary manipulation through selection of feed ingredients or by modulation in processing of feeds has been a traditional part of animal management, however direct modification of microbial populations and their activities through the use of natural or chemicals modifiers are latest favored approach to manipulate ruminal fermentation. Antibiotics were among the first compounds widely used to modify ruminal fermentation, but increased public health and consumer concern over residues in the products, and the potential for selection of antibiotic-resistant bacteria made nonantibiotic feed additives more popular. Selective stimulation of beneficial rumen microbes and competitive exclusion of harmful rumen microbes by the use of phytobiotics, probiotics, prebiotics, enzymes, other naturallyoccurring and chemical compound has enabled us to alter the fermentataion processin a desired manner. This whole manipulation process can be considered as an optimization process in which optimal condition are pursued by either altering the fermentation process or by modulating the substrate to be fermented. In simplistic terms, the objectives of ruminal manipulation are to enhance beneficial processes and to minimize, alter or delete processes harmful to the host which includes-

• Enhance fibrolytic activity
• Increase microbial protein synthesis
• Reduction in proteolysis
• Reduction in methanogenesis
• Prevention of acidosis
• Shifting acetate to propionate production
• Inoculation of novel microbes
• Metabolism of plant toxins
• Synthesis of useful enzymes and secondary metabolites

Methods of rumen manipulation can be classified in two broad groups i.e., genetic manipulation and non-genetic manipulation. In genetic manipulation, attempts were made to develop genetically engineered rumen microbes by gene transfer technique. Rumen manipulation using various‘omics’ technologies like genomics, proteomics and metabolomics is a more recent approach to understand and manipulate rumen function. Also exploring the diversity of rumen through variousfast and innovative molecular techniques like DNA profiling techniques, quantitativePCR, sequencing, fluorescent in situ hybridization(FISH), DNA microarrays and flow cytometry can be used in maintaining gooddensity and diversity whichsubsequently improves the performance of cattle rumen (Shrivastava et al., 2015). Although the introduction these approaches is a long-term goal of biotechnologists, the immediate benefitsof genetic engineering techniques are to gain better insight into the physiologyand genetics of ruminal microorganisms. Additionally, such techniques may lead to the productionof a wide variety ofready-made organic products that may be beneficial in further rumen fermentation, health and production.

Non genetic manipulation of the rumen can be done by physical methods (dietary manipulation) by using suitable organic and inorganic compounds (dietary modulation) and by microbial feed additives. The intention of thisdietary manipulation and nutritional interventions aims atmanipulating feeding strategy to promote stable ruminal fermentation, modulations in dietary compositions and controlling the metabolism of carbohydrate, nitrogen and lipid in the rumen (Hobson and Stewart, 1997).

• Manipulation of carbohydrate fermentation in the rumen-Optimizing starch utilization in the ruminant involves influencing the site of starch digestion, inhibition of high lactate production or enhancing its fermentation to VFAs in order to increase rate and extent of structural carbohydrate fermentation and maximize nutrient intake, availability and digestibility in the rumen.
• Manipulation of nitrogen metabolism– The rate and extent of proteolysis and ammonia formation affect not only the quantity of escape protein that reaches the abomasum but also the quantityand quality of microbial protein produced in the rumen. The logical strategy for manipulating nitrogen metabolism in the rumen is to enhance ruminaI escape of dietary protein by minimizing its degradationand optimizing microbial protein production from NPN sources of high biological value. Minimizationof protein degradation can be achieved by intervening at the proteolysis,peptidolysis or amino acid deamination stages which will reduce losses incurredin the conversion of dietary protein to microbial cell protein.
• Manipulation of lipid fermentation– Manipulation of ruminal lipid metabolismtargets tocontrol antimicrobial effects of fatty acids in order tominimize disruption of ruminal fermentation and to potentiate inclusion of higher levels of fat in the diet to overcome energy stress during transition and lactation period. Likewise it is also followed to control biohydrogenation process for altering theabsorption of selected fatty acids that may improve nutritional qualities ofanimal food products or even enhance animal performance via regulatoryeffects on cellular metabolism.Also the logic behind this manipulation is to regulate the flow of unsaturated fatty acids to the duodenum because of its various health promoting properties.

Possible approaches of rumen modulation-

Dietary interventions- This approachhave focused largely on increasing the nutrient digestibility ( particularly structural carbohydrate), protecting dietary protein against microbial attack in the rumen, increasing energy density of ration without altering normal rumen fermentation, reducing the incidence of metabolic disorders and minimizing energy loss in the form of methane.The establishment and maintenance of the stable population of microbes specially methanogens is affected by the type of diet and level and frequency of feeding which can be changed accordingly by choosing suitable dietary methodologies. Defaunation and probiotics supplementation are most potent technique to decrease energy losses through methanogenesis. In general dietary interventions involves supplementation of certain modifiers of rumen environment which have potential to alterassociated ruminal microbial activity, which includes–

• Phytobiotics– These arephytogenic feed additives or herbal products that have varying impact on the body (antimicrobial, antiviral, immunomodulatory, fungicidal or anti-inflammatory activity) and are used as animal feeds for increasing the productivity and the quality of food products of animal origin. The phytobiotics are divided into herbs (flowering, herbaceous and short-lived plants), spices (herbs with intensive odoror taste, usually added in food), ether oils (volatile lipophilic compounds obtained by cold pressing, steam or alcohol distillation) and resins (extracts obtained with the help of non-aqueous solvents). These phytobiotics are gaining popularity nowadays because of their high efficacy in rumen manipulation, in particular for defaunation and to reduce methane emission (Kamra et al. 2006 ).
• Plant secondary metabolites– Theypossess biological activity stimulating ruminal modulation and at the same time protecting the plants from the predation of insects or the grazing by herbivores. The microcidal or microstatic action of these compounds (phenolics, terpenes, steroids, alkaloids) mainly comes from the capacity of such molecules in intruding into the microorganism cell membrane and disintegrate its structures (Bodas et al. 2012 ).The inclusion of saponins in diet of animals changes the site of digestion of organic matter and fiber, from rumen to hindgut while tannins reduce ruminal protein digestibility and plant cell wall digestion by slowing their digestion rate. Saponins, tannins, and essential oils are also found to reduce the amount of NH₃produced in therumen, which improves assimilation of feed amino acid nitrogen by ruminants (Spanghero et al. 2008 ). Moreover, essential oils suppress colonization and digestion of readily degradable substrates by amylolytic and proteolytic bacteria without affecting fiber digestion by cellulolytic bacteria.
• Organic acids– The use of organic acids in ruminant feeding has focused on malate andfumarate, which are intermediates in the tricarboxylic acid cycle and in the pathway of propionate formation in the rumen. Stabilization of ruminal pH through a stimulation of lactate utilization by Selenomonas ruminantium by malate has been proposed as a beneficial effect on rumen fermentation. It has also been proposed that supplementation of organic acids can increase digestible energy content and propionate supply of low digestible diets to enhance lactate utilization and simultaneously diminish the risk of lactic acidosis and methane production in the rumen by competing for metabolic hydrogen.
• Buffering compounds(Alkalizers)– Excessive acids in the rumen in situations where natural buffering systems primarily salivary flow, may be inadequate due to metabolic disorders like lactic acidosis which necessitates the practical supplementation of NaHCO₃, CaCO₃, MgO, bentonite (aluminum silicate clay) and sodium sesquicarbonate. These chemical compounds neutralize the excessive acids and make the rumen environment conducive for cellulolytic bacteria. Also these rumen modifiers are somewhat found to shift the microbial population from amylolytic to cellulolytic ones.
• Ionophore antibiotics– These ion bearing compounds (monensin, lasalocid etc.) are members of a large and growing group possessing the ability to form lipid-soluble complexes with cations and mediate their transport across lipid barriers as growth promotants for ruminants.These antibiotics are also noticed to reduce protein degradation and deamination of amino acids, resulting in the improvement of nitrogen metabolism in the rumen and reduce lactic acid production and froth formation by inhibiting the growth of lactic acid bacteria, and consequently ruminal disorders. Ionophores, which have been used to enhance feed conversion efficiency and growth rate in cattle, have also been shown to increase production of propionate and decrease production of methane, resulting in increased efficiency of energy metabolism of the animal.
• Methane inhibitors– Approaches aiming at inhibition of enteric methane generation includes chemical inhibitors (ionophores), biological approaches (vaccine,bacteriophage, bacteriocins), hydrolyzed and condensed tannins, organic acids (fumarate) and unsaturated lipids (linoleic acid).Specific inhibitors of methane production are listed above, but methanogenesis can also be reduced by dietary fat supplementation and defaunationby means of diverting hydrogen away from methane formation (Wright and Klieve 2011 ).
• Growth factors, minerals and vitamins– Even when the diet is balanced for energy and protein, deficiency of growth factors can depress microbial growth, efficiency and yield. Therefore, supplementation of growth factors and limiting elements under these situations may have a positive impact on digestibility and intermediary metabolism in rumen.
• Microbial feed additives and enzymes– Numerous direct-fed microbials that are accessible today have been recognized for their positive effects on animal’s weight gain, rumen development, nutrient digestion, restoration of intestinal microflora (producing bacteriocins) and competitive exclusion of the opportunistic pathogens. Major groups of this type of additives include Lactobacilli, Bifidobacteria, Propionibacteria which can be fed in the form of prebiotic, probiotic or synbiotic. Yeast culture particularly of Saccharomyces cerevisiae stimulated the production of propionate at the expense of acetate (Yuan et al., 2015) and also found to improve ruminal fermentation, feed intake, and milk yield. Several microbial formulations are now commercially accessible, and their use serves as better alternative of antibiotics and growth promoters in livestock production. Similarly application of highly active enzymes (i.e. endoglucanase, xylanase, esterases, etc.) from the rumen source for commercial applications will provide a new dimension in agro-industries.

Physiological modification of the animal– This technique aims at achieving changes in ruminal fermentation which involve processes like increasing appetite, hormonal regulation and homeostasis, increasing salivary production, influencing the functional activity of the gut such as reticulo-ruminal motility and metabolic and absorptive function of the ruminal epithelium. Composition and activity of microbial populations through physiological processes, such as mastication, rumination, salivation, diffusion or secretion into the rumen, reticulo-ruminal motility, and the eructation and absorption of fermentation products can be altered which may have direct or indirect effect on fermentation process. Such improvements have been attributed to increase microbes and their beneficial by-products, reduction in pathogens, maintenance of favourable rumen pH, increase in ruminal fibrolytic enzymes, and improvement in ruminal fiber digestion and nutrient utilization,thereby resulting in increased feed digestibility and animal productivity.

Microbial intervention– Microbial feed additive (MFA) or direct-fed microbial (DFM) isformulation of viable naturally occurring bacteria, fungi, or yeastto exploit microorganismsand other feed additives in animal diets for improving animal’s healthiness and productivity. The benefits of supplementation may include stimulation of beneficial microbial growth in the rumen, decreased ruminal acidosis, stabilization of the rumen pH, positive alteration of ruminal fermentation and end-product production, increased ruminal propionate concentrations, increased plasma hormones concentration, increased nutrient flow, efficient nutrient digestibility, enhanced immune responses, alleviated metabolism, reduced rumen oxygen, detoxification and increased milk production.

It has been observed that several selected prebiotics could confer beneficial alterations in the gut microbiota, decrease rumen ammonia nitrogen and methane emission, and enhance microbial protein synthesis and weight gains in ruminants. Probiotics formulation in dairy cattle mostly comprise Lactobacilli, Bifidobacteria , Streptococci , Enterococci , and Propionibacteria (Walker, 2007 ). Yeast ( Saccharomyces cerevisiae ) or the aerobic fungus (Aspergillus oryzae ) are usually labelled as fungal feed additives or fungal probiotics and have reported to increase dry matter intake and milk production during negative energy balance. L.acidophilus stimulates fiber digestion and reduces protein degradation inrumen-simulating fermenters. Similarly inoculation with the rumenbacterium Megasphaera elsdenii helps to prevent the accumulation of lacticacid in rumen fluid during the transition from a low to a high concentratediet. Supplementing dairy cows with B. subtilis natto improves milk production and milk components yield, decreases SCC and promotes the growth of total ruminal bacteria, proteolytic and amylolytic bacteria, which indicate that B. subtilis natto has potential to be applied as a probiotic for dairy cows (Sun et al., 2011).

Additionally bypass nutritional interventions like bypass fat, bypass protein and rumen protected acids have immense potential to counter ill effects of NEB and optimize production without interfering the fermentation process in rumen. Bypass fat resist lipolysis and biohydrogenation by rumen microbes, but gets digested in lower digestive tract. Prilled fat andcalcium soaps of long chain fatty acid which are insoluble in rumen due to their temperature and pH sensitivity are soluble in the abomasum and lower digestive tract. Similarly formaldehyde treated meals is economically viable approach to optimise RDN/UDN at the site of metabolism preventing their wastage in rumen. Moreover modulation of the extent of protein degradation in the rumen is one method of influencing the amino acid supply to the lower gastrointestinal tract, so formulation of rations with low nitrogen solubility in rumen result in more protein escaping ruminal degradation and subsequent increases in production performance of animals. Thus adding these bypass nutrients to dairy rations can positively affect efficiency of dairy cows through combination of caloric and non-caloric effects and have been found effective to augment the overall productivity without affecting rumen microflora, metabolites and fermentation process.

Effect on productivity

The ultimate aim of manipulating ruminal fermentation is to maximize the efficiency of feed utilization and increase ruminant productivity, i.e. increase milk, meat and wool production. Administration of prebiotics to calves has been found to intensify nitrogen retention, increase microbial protein synthesis,weight gains, and improve feces consistency. Likewise, the inclusion of prebiotic oligosaccharides into the diet ofcalves has been shown to reduce the populations of E. coli and total anaerobes and concurrently increase the numbers of Bifidobacteria thus competitively exclude the harmful microorganism with increase in growth. Viable yeast cultures are also a good source to improve the performance with an increase in dry matter intake and milk production in dairy ruminants (Stella et al. 2007 ) and enhance the growthparameters, viz., average daily gain, final weight, feed intake, feed efficiency etc. in beef cattle. A recent transition cow study (Ramsing et al., 2009) reported that yeast culture supplementation at 57 g/d from approximately 21 d prepartum to 21 d postpartum improved dry matter intake and milk production significantly. Cattle with higher feed efficiencies are reported to produce 20–30 % less methane, clearly indicating that large amounts of ingested energy are usually discarded as methane in ruminant production. As a result dietary modulation of rumen metabolism mitigating enteric methane emission may improve animal production efficiency as well as contribute to alleviate the impact of ruminants on climate change (Khiaosa-Ard and Zebeli, 2012)

Conclusion

The rumen microbial ecosystem has a significant impact on productivity by improving feed utilizationthrough its complex bionetwork. Manipulating the dietary composition or altering the microbial ecosystemsis the key to lift growth, milk production and composition. Increasing the production of beneficial microbes in the rumen has strengthened awareness towards ruminal digestion and its potential application in industry for a cost-effective ruminant feed production and management of enteric fermentation. It can be easily speculated that prebiotics would soon become a regular part of animal diets as an additive for modulating the GI microflora and ultimately animal productivity. Due toits immense capacityto augment animal growth, production andperformance, this approachof using various microbial formulations undoubtedly going to play an important role in the future of livestock nutrition and production.Recent advances in molecular biology and genomics offer new opportunities to conduct a holistic examination of the structure and function of rumen microbial communities that can be further used to improve the rumen performance.

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