MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS Dairy Year Book (2014-15) 122 Manipulation Of Ruminal Microbial Fermentations BY Shekhar Bhagwat Professor & Head, Department of Animal Nutrition, College of Veterinary Science & Animal Husbandry, SDAU, Sardarkrushinagar (BK)-385506 Introduction Overview Of Ruminal Microbial Fermentation Rumen is considered as a fermentation vat in which a complex and dense population of bacteria, protozoa and fungi convert feedstuff materials to VFA’s, CH 4 , CO 2, NH 3 and microbial cells. It is the dominant feature of the digestive tract of ruminants. It maintains a medium that supports a dense and varied population of microorganisms. These organisms ferment feed materials to produce mainly short chain organic acids or volatile fatty acids (VFAs), methane and carbon dioxide and the process provides substrate (the feed) and ATP (energy) for the growth of Micro-organisms. The microbial mix in the rumen is complex and highly dependent on diet. The main agents that break down fibre, sugars, starches and proteins in the rumen are all anaerobic and include bacteria, protozoa and fungi. The bacteria are the principal organisms that ferment plant cell-wall carbohydrates, but the anaerobic phycomycetous fungi may at times be extremely important. Protozoa are now recognised as having an overall negative effect in the rumen, particularly where ruminants are fed forage diets low in true- protein (Bird et al. 1990). Protozoa ingest and digest bacteria and reduce the bacterial biomass in the rumen (Coleman, 1980) and consequently the protein supply to the animal. Thus, they decrease the protein to energy ratio in the nutrients absorbed and increase the requirement of animals for true protein. The net result of the presence of protozoa is an increased requirement for dietary bypass protein and on low protein diets a decreased efficiency of utilisation of feed for growth and milk production (Bird et al. 1990). The presence of protozoa in the rumen may also reduce the rate at which bacteria colonise and degrade the ingested feed particles. Classification Of Rumen Microbes (Chiba, 2009) Types of bacteria (Special nutritional requirements) Cellulose Digesting Bacteria. Predominant: Ruminococcus flavefaciens Gram+ cocci, usually in chains Ferments cellulose, cellobiose & glucose Produces acetic, formic, succinic, some lactic Fibrobacter succinogenes Gram– rod Ferments cellulose, cellobiose & glucose Produces acetic, formic & succinic Ruminococcus albus Gram– cocci Ferments cellulose, cellobiose, usually not sugars Produces acetic, formic, lactic, ethanol & H 2 Strict anaerobes Tolerate narrow pH range (pH 6 to 7) Attach to feed particles Secondary: Eubacterium cellulosolvens Numbers usually low in rumen Gram– rod Ferments cellulose & soluble sugars Produces mostly lactic acid Butyrivibrio fibrisolvens Several strains in rumen Gram– curved rod Ferments cellulose (slow) & starch Produces formic, butyric & lactic acids, ethanol & H2 Strict anaerobes Tolerate narrow pH range (pH 6 to 7) Attach to feed particles. Hemicellulose Digesting Bacteria Butrivibrio fibrisolvens Prevotella ruminicola Gram– non motile rod Digests starch, cellulose not digested Produces succincic, formic, acetic and some strains propionic Eubacterium ruminantium Gram+ non motil rod Ferments cellobiose, dextrins, maltose, glucose, fructose, lactose, sucrose and 5-carbon sugars
Transcript
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 122
Manipulation Of Ruminal Microbial Fermentations
BY Shekhar Bhagwat
Professor & Head, Department of Animal Nutrition, College of Veterinary Science & Animal Husbandry,
SDAU, Sardarkrushinagar (BK)-385506
Introduction
Overview Of Ruminal Microbial Fermentation
Rumen is considered as a fermentation vat in which a complex and dense population of bacteria, protozoa
and fungi convert feedstuff materials to VFA’s, CH4
,
CO2, NH3
and microbial cells. It is the dominant
feature of the digestive tract of ruminants. It maintains a medium that supports a dense and varied population of microorganisms. These organisms ferment feed materials to produce mainly short chain organic acids or volatile fatty acids (VFAs), methane and carbon dioxide and the process provides substrate (the feed) and ATP (energy) for the growth of
Micro-organisms. The microbial mix in the rumen is complex and highly dependent on diet. The main agents that break down fibre, sugars, starches and proteins in the rumen are all anaerobic and include bacteria, protozoa and fungi.
The bacteria are the principal organisms that ferment plant cell-wall carbohydrates, but the anaerobic phycomycetous fungi may at times be extremely important. Protozoa are now recognised as having an overall negative effect in the rumen, particularly where ruminants are fed forage diets low in true-protein (Bird et al. 1990). Protozoa ingest and digest bacteria and reduce the bacterial biomass in the rumen (Coleman, 1980) and consequently the protein supply to the animal. Thus, they decrease the protein to energy ratio in the nutrients absorbed and increase the requirement of animals for true protein. The net result of the presence of protozoa is an increased requirement for dietary bypass protein and on low protein diets a decreased efficiency of utilisation of feed for growth and milk production (Bird et al. 1990). The presence of protozoa in the rumen may also reduce the rate at which bacteria colonise and degrade the ingested feed particles.
Classification Of Rumen Microbes (Chiba, 2009)
Types of bacteria (Special nutritional requirements) Cellulose Digesting Bacteria .
Predominant:
Ruminococcus flavefaciens
Gram+ cocci, usually in chains Ferments cellulose, cellobiose & glucose Produces acetic, formic, succinic, some lactic
Types of Rumen Fungi Initially thought to be a flagellated protozoa. Later showed to contain chitin – representative of fungi
Five genera have been found in the rumen:
Neocallimastix
Piromyces
Caecomyces
Orpinomyces
Anaeromyces
Anaerobic flagellated organisms
Life cycle includes motile zoospores and
non motile vegetative form
Zoospores attach to feed particles followed
by encystment and germination
Counts range from 1.5X103 to 1.5x10
6 per g rumen
contents
Pathways Of Ruminal Microbial Fermentation
Fermentation Of Starch
The degradation of the α-1linked starches (amylase and amylopectin) and the simple sugars (e.g., sucrose, maltose) is performed by several species of primary amylolytic bacteria. Some of these are capable of all four stages of the microbial processes, except for methane formation, whereas others carry out stages 1 and 2 but cease with the production of one of the metabolic acids, most commonly lactic acid. Unlike the cellulolytic bacteria, the amylolytic bacteria have faster fermentation rates, have much shorter doubling times (0.25 to 4 hours), and have a lower pH optimum to 5.5 to 6.6. This matches the lower ruminal pH values of ruminants on high concentrate (starch-rich) diets and is due to higher SCFA concentrations with an increase in the relative proportions of propionate, giving a typical acetate/propionate/butyrate ratio of 70:25:5, respectively. The increased proportion of propionate, as it produced less reducing equivalents (2H), means that there is not such a need for methane to be formed as a sink for reducing equivalents. In turn, this means that less dietary energy is lost as methane, and more is retained as propionate.
Amylolytic bacteria require not only a supply of NH3 but also some amino acids for protein synthesis. Secondary bacteria are required for methane formation (methanogenic bacteria) and for the conversion of the lactic acid and other metabolic acids to propionate (propionate bacteria). Both of these groups of secondary bacteria require amino acids for their protein synthesis, have a long doubling time (16 hours), and have an optimum ph of 6.2 to 6.8, which is higher than that required by amylolytic bacteria. Therefore when sudden changes are made from roughage to concentrate feeds, the amylolytic bacteria quickly increase both their numbers and the overall rate of fermentation, causing a rapid accumulation of SCFA among them lactic acid. This leads to a lowering of pH, which is within the pH optimum of the amylolytic bacteria but is too low for both kinds of secondary bacteria. Therefore lactic acid, a stronger organic acid than the volatile fatty acids (VFA: acetic-, propionic-, and butyric acids), increases still further while the potential for hydrogen disposal declines and may provide some degree of negative feed-back on further amylolytic activity.
The numbers of protozoa increase when concentrates are fed, probably owing to the greater availability of starch granules and of bacteria that feed on them. The protozoa thereby curb bacterial amylolysis, until the pH falls below 5.5 at which point protozoa are quickly inactivated and later die.
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 125
Fermentation of cellulose
The degradation of the β-1 linked compounds (cellulose, hemicelluloses,fructosans, pectin) is performed by several species of primary cellulolytic bacteria, which are capable of all stages of microbial activity, [(1)hydrolysis of polysaccharides, (2) Embden-Meyerhop pathway of anaerobic oxidation, (3) reactions producing the final metabolites of fermentation], except for methane formation (stage 4), which is carried out by methanogenic bacteria. The fermentation of cellulose is low, because cellulolytic bacteria have a low metabolic rate. As they take about 18 hours to double their numbers (the doubling time), population changes are also slow.
For protein synthesis, cellulolytic bacteria do not require a supply of amino acids but need NH3, the stages 2 and 3 intermediates, and small amounts of isoacids, which arise from the deamination of the branched amino acids in dietary plant proteins. The pH optimum is 6.2 to 6.8, which matches the typical ruminal pH of roughage-fed animal. The methanogenic bacteria have similar pH optimum, and they require a supply of formate, CO 2 and reducing equivalents (2H) to produce methane and a supply of amino acids to meet their protein requirements. The mixed population of cellulolytic and methanogenic microbes leads to the production of CO2, CH4 and the SCFA. The SCFA derived from the fermentation of cellulose – acetate, propionate, and butyrate – are generally in the ratio 75:15:10, respectively.
Fermentation of dietary protein
Proteolytic bacteria comprise only 12 to 38 percent of the total ruminal bacteria, and normally only about a half of the dietary protein is degraded in the rumen.
The original idea that soluble proteins but not insoluble proteins could be fermented is not tenable. Instead, dietary proteins are now classified as rumen degradable proteins (RDP) or as rumen undegradable proteins (RUP) Certain natural proteins (e.g., those in maize) and other processed (protected) proteins (e.g., those denatured by heat treatment or tanned by the application of formaldehyde) escape ruminal degradation but can be hydrolysed by the gastrointestinal enzymes. Bacterial proteolysis commences with extracellular protease activity to produce peptides that are actively absorbed and subjected to further hydrolysis within the bacterial cell. The end-products are amino acids, some of which are taken up by other microbes and the remainder deaminated to produce ammonia and various metabolic acids. These are formed to SCFA (the isoacids: isobutyrate and isovalerate), which arise from leucine, isoleucine, and valine and are required as minor nutrients by the cellulolytic bacteria.
Ammonia arises not only from the deamination of amino acids but also from the conversion of dietary and endogenous nonprotein nitrogen compounds (NPN). These include plant amides, nitrites, nitrates,
and endogenous urea. Urea both enters with saliva and readily diffuses across the rumen wall into the ruminal fluid. The diffusion is facilitated by the maintenance of a step concentration gradient across the rumen wall, due the high urease activity at the rumen wall – rumen fluid boundary, where urea is rapidly broken down to ammonia. Additional urease
activity is found in the fibrous raft in the dorsal ruminal sac. Ammonia is an important substrate for microbial protein synthesis, subject to the provision of (1) adequate amounts of α-ketoglutarate (for
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 126
amination to glutamate), (2) suitable SCFA (including isoacids) being available to provide the carbon skeletons onto which the amino acid groups can be added (by transamination from glutamate), (3) readily fermentable carbohydrates (e.g., starch) to
provide the energy (obtained from ATP generated by the Embden-Mayerhof pathway. needed for these synthetic reactions.
In practice, feeding regimens must, first, provide sufficient crude protein (true protein plus NPN) and readily fermentable carbohydrates. These ensure that the ruminal microbes have adequate amino acids, ammonia, carbon skeleton, and available
energy to meet the requirements of microbial protein synthesis for the maintenance of population numbers. Second, feeding regimens must ensure that excessive protein breakdown to SCFA and ammonia does not occur. Feeding protein in excess is a wasteful input of an expensive commodity, and it leads to the overproduction of ammonia, which takes
energy to convert it to urea (in the liver) and also creates a risk of ammonia toxicity.
In addition to the fermentation of dietary protein there is a continuous recycling of the protein of dead microbes, especially in the fibrous raft. Essentially none of the amino acids produced in the forestomach becomes immediately available to the ruminant. Instead, from the material that flows out of the forestomach into the abomasums and smack
intestine, the ruminant acquires unfermented dietary proteins and microbes. The microbial protein has a higher biological value (i.e., contains more essential amino acids) than the precursory plant proteins of the diet.
Fermentation Of Dietary Lipids
Dietary lipids occur as structural lipids in the leaves of forage plants and as storage lipids in oil seeds. The forage plant lipids are found mainly in cell
membranes and comprise 3 to 10 percent of the dry matter. Less than 50 percent of the total lipids are free fatty acids (FFA), and the majority are phospholipids, with palmitic,, linoleic, and linolenic acids being the predominant fatty acids.
In oil seeds, 65 to 85 percent of the lipids are triglycerides, with palmitic, oleic, and linoleic being
their predominant fatty acids. Ruminal microbes rapidly hydrolyze dietary lipids and, using the unsaturated fatty acids (oleic, linoleic, linolenic) as hydrogen acceptors, quickly convert most of them stearic acids. Most plant unsaturated fatty acids are in cis form. Ruminal microbes also synthesize
microbial lipids from SCFA, and many of these are in the trans form. Ruminal adipose tissue, intramuscular and milk lipids therefore contains fatty acids with both trans and cis forms.
Ruminant diets generally do not contain more than 5 percent dry matter (DM) as lipids. Higher values may have adverse effects on (1) food palatability; (2) cellulolytic activity; (3) food appetite and fore stomach motility, probably as a result of negative
feed-back by the cholecystokinin released when fat is present in the duodenum; (4) the physical consistency of concentrate pellets at high and low temperatures; and (5) the shelf-life of concentrate, lipids being prone to the development of rancid flavours.
Protozoa have an important role in ruminal lipid metabolism. They absorb some of the polyunsaturated fatty acids (PUFA), lock them away
in their own structure, and thereby protect them from hydrogenation. The protozoa that subsequently flow out of the rumen and undergo intestinal digestion release their PUFA content, this being probably the main source of PUFA for ruminants.
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 127
Compounds Used To Manipulate Riminal Microbial Fermentations
Ionophores
(A) Mode Of Action
Ionophores are used commercially throughout the world in the beef and poultry industries. Production efficiency of cattle is increased through alteration of rumen fermentation and control of protozoa that cause coccidiosis. Ionophores act by interrupting
transmembrane movement and intracellular equilibrium of ions in certain classes of bacteria and protozoa that inhabit the gastrointestinal tract. The actions of ionophores provide a competitive advantage for certain microbes at the expense of others. The basic function of ionophores is to create a flux of ion transport across cell membranes. They
bind to bacterial cell membranes and first cause an efflux of potassium from the cell and an influx of hydrogen into the cell. The increased hydrogen is exported out of the cell either by active transport involving adenosine triphosphate or passively via sodium entry into cells in exchange for hydrogen. In
order to maintain inner cell equilibrium, the bacterial cell expends energy and these results in death or reduced growth of the bacterium. Since gram-
negative bacteria have complex outer cell membranes, they are usually more resistant to the action of ionophores than are gram-positive bacteria. Ionophores, therefore, selectively inhibit gram-positive bacteria rather than gram-negative bacteria because of differences in bacterial cell wall structure.
In general, the metabolism of the selected
microorganisms favors the host animal. Major areas of animal metabolism include increased efficiency of energy metabolism, improved nitrogen metabolism, and general digestive effects, including reductions in both bloat and lactic acidosis. Monensin changes the ratio of volatile fatty acids in the rumen, increasing
propionic acid and reducing the molar percentages of butyric and acetic acids. Increased rumen propionic acid improves gluconeogenesis. Energy metabolism is enhanced through increased production of propionate among ruminal fatty acids with a concomitant reduction in methane. Ruminal degradation of peptides and amino acids is reduced,
thereby increasing the flow of protein of dietary origin to the small intestine. Total flow of protein to the lower tract is often increased with ionophore feeding. Risk of digestive disorders such as bloat and acidosis that result from abnormal rumen fermentation is reduced, as are certain conditions
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 128
caused by toxic products of fermentation, e.g., 3-methyl indole. Dry matter and nitrogen digestibilities are increased with ionophores, thereby providing environmental benefits.
Ionophores enhance the glucose status of dairy cows through increased production of propionate. Many of the demonstrated benefits of ionophores are associated with enhancement of the energy status of the cow in the transition period and during early lactation. The benefits include less mobilization of
body fat as evidenced by reduced blood nonesterified fatty acids and ketones and increased glucose. Animal manifestations include lower incidence of ketosis and displaced abomasum, reduced loss of body condition, increased milk production, and improved milk production efficiency. Improve feed efficiency. Benefits derived by cattle from the
biological actions of ionophores were classified into three areas of effects as follows (Bergen and Bates, 1984).
1. Increased efficiency of energy metabolism of rumen bacteria and (or) the animal.
2. Improved nitrogen metabolism of rumen bacteria
and (or) the animal.
3. Retardation of digestive disorders resulting from abnormal rumen fermentation.
Certain animal conditions, e.g., bloat, acidosis, and ketosis are caused by or related to disturbances in ruminal fermentation. These conditions are attenuated or reduced when ionophores are fed
because of a specific effect on a particular bacterial species, changes in eating behavior, or changes in end products of fermentation. Each action provides nutritional and metabolic advantages to the ionophore-supplemented animal over a nonsupplemented animal. The animal transforms
these into increased production or improved efficiency. Economic benefits derived from feeding ionophores include improved feed efficiency, increased weight gain, and a reduction in morbidity and mortality. Ionophores also help reduce the amount of excreta and gaseous emissions from animals. Thus, a positive effect on the environment
occurs when ionophores are fed.
The ionophores lasalocid and monensin are approved for use in dairy cows in many countries. Monensin is a carboxylic polyether ionophore produced by a naturally occurring strain of Streptomyces cinnamonensis and is provided to cattle orally, as a
sodium salt. Monensin exerts its many effects by shifting the microbial populations in the rumen. Claims are allowed for increased milk production, for improved feed efficiency, for control of subclinical and clinical ketosis, and for control of bloat. Findings from a nine-trial, dose-titration study in the United States and Canada designed for a production claim
for monensin in dairy cows were published recently (Symanowski et al., 1999).
B) Effects Of Ionophores On Rumen
Fermentations
Effects On Ruminal N And Energy Utilization
Because gram-positive bacteria mainly ferment dietary nutrients into ‘less desired’ products like acetate, H+, CH4, and NH3, ionophore treatment
improves rumen function and animal performance by reducing the production of these metabolites. Ionophore treatment typically reduces the acetate to propionate ratio in the rumen and hence improves the efficiency of ruminal energy utilization. In addition, ionophores like monensin can reduce
ruminal methane production by up to 30% by inhibiting bacteria providing precursors of methane (formate and H2) rather than directly inhibiting methanogens. For instance methanogenesis by Methanobacterium formicicum was inhibited when the bacteria were grown in a culture enriched with formate but it was largely unaffected when grown in
a H2 and CO2 enriched culture.Hydrogen-producing fungi and protozoa may also be inhibited. Ionophore treatment often reduces proteolysis slightly, but reduces peptidolysis and amino acid deamination markedly (Hobson and Stewart, 1997). This is often mediated by inhibition of gram-positive hyper
ammonia producers such as Peptostreptococcus anaerobius, Clostridium sticklandii, and C. aminopholum. Consequently, ionophore treatment often increases postruminal supply of proteins and peptides.
Effects on animal performance and health
Addition of ionophores to livestock diets has resulted
in both negative and positive effects on digestion due to differences in inclusion rate, diet composition, and level of feed intake (McGuffey et al., 2001). Nevertheless, a meta analysis involving 77dairy cow studies revealed that dry matter intake (DMI), milk fat and protein contents were reduced by 2, 3, and
1%, respectively, whereas milk yield, feed efficiency, and protein yield were each increased by about 2%. Ionophores like monensin are very effective at preventing coccidiosis in livestock. By inhibiting the growth of lactate-producing bacteria like Streptococcus bovis and Lactobacillus spp, ionophores can also reduce the risk of ruminal
acidosis and bloat.Furthermore, the increased glucose supply resulting from the stimulation of ruminal propionate production by ionophores can reduce the risk of ketosis and fatty liver syndrome in lactating dairy cows (Duffield et al., 2007a).
In summary, ionophores reduce the gram-positive
bacteria population in the rumen and this results in greater energetic efficiency (less CH4 & lower acetate to propionate ratio), better protein utilization (less peptidolysis and amino acid deamination, more bypass); less environmental pollution (less NH3 & CH4 production), reduced incidence of coccidiosis, acidosis, bloat, and ketosis; variable effects on fiber
digestion, increased efficiency of milk production and decreased milk fat concentration. Monensin has a benefit to cost ratio of 5 to 1 when added to dairy cow diets; it is recommended for increasing feed
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 129
efficiency in lactating cows and reducing metabolic disorders in dry cows (Hutjens, 2008).
Protease/Deaminase Inhibitors
Protein breakdown in the rumen is generally regarded
as detrimental to the efficiency of ruminant nutrition, certainly for animals on a relatively high plane of nutrition. Peptides and amino acids arising from proteolysis are potential nutrients for the growth of rumen microorganisms, but they are also liable to be degraded to ammonia and lost from the rumen.
Proteolytic activity occurs in all three main categories of rumen microorganisms. Bacteria are mainly responsible for dietary protein breakdown, while ciliate protozoa break down particulate feed protein of appropriate size and also bacterial protein. The key features of rumen proteolytic activity are that it varies greatly from animal to animal and from feed
to feed. The predominant mechanism of peptide degradation is biphasic, via dipeptidyl aminopeptidases which cleave dipeptides from larger peptides followed by dipeptidase.
Dipeptidyl aminopeptidase activity occurs only in Prevotella ruminicola among the common rumen
microbial species. In contrast, dipeptidase, which cleaves the dipeptide products from dipeptidyl aminopeptidase, is present in many species, including P. ruminicola, and is particularly high in rumen protozoa. Deamination of amino acids is carried out by a combination of numerous lowactivity bacteria and protozoa and a much smaller number of
high-activity species. Most ammonia production is probably carried out by the low-activity species, which again include P. ruminicola, but proliferation of the high-activity species may be a problem on certain diets.
The microbiology of protein breakdown in the rumen
is of interest because it deals with one of the major inefficiencies of ruminant nutrition, namely the too-rapid conversion of protein to ammonia in the rumen and the subsequent loss of that ammonia by absorption across the rumen wall and excretion as urea (Wallace,1994). This review describes the microorganisms involved at different stages of the
breakdown process and assesses the relative importance of different species in the light of population densities and the properties of the mixed rumen population.
Proteolytic ruminal microorganisms The mixed rumen microbial population has a proteolytic activity that is
only moderate compared with other proteolytic microorganisms and the host’s own gastric and pancreatic secretions, but the length of time that feed material is retained in the rumen means that this activity is able to break down a substantial proportion of most dietary proteins (Broderick et al, 1991). Many strains and species of rumen ciliate
protozoa, bacteria and anaerobic fungi have been found to be proteolytic, and they contain a variety of different types of proteolytic enzymes (Wallace, 1994).
The predominant species of proteolytic bacterium found in the rumen of most animals is Prevotella (formerly Bacteroides) ruminicola, which has been identified as proteolytic in many studies and which
can comprise more than 60% of the flora under some circumstances (Van Gylswyk, 1990). Its cell-associated, mainly cysteine protease activity, is fairly typical of the rumen bacterial population as a whole. Some animals possess Butyrivibrio fibrisolvens as the most prevalent proteolytic isolate (Wallace and
Brammall 1985).
Peptidolytic rumen microorganisms
Peptide breakdown to amino acids must occur for the amino acids to be incorporated into microbial protein, and when there is sufficient energy available to fuel biosynthesis, amino acids will be incorporated and
peptide breakdown would not be considered to be a major inefficiency in fermentation. However, when energy is unavailable, or when the rate of peptide breakdown exceeds the rate at which it can be assimilated, peptide catabolism leads to excessive ammonia production and poor N retention.
The great majority of peptidase activity in rumen fluid is aminopeptidase (Wallace et al, 1990 a). It is characterised by dipeptides rather than single amino acids being cleaved from the peptide chain (Wallace et al, 1990a, 1993). Enzymes of this nature are classified as dipeptidyl aminopeptidases (Webb, 1992). The main mechanism of hydrolysis in intact
rumen microorganisms appeared, from the hydrolysis of diagnostic synthetic substrates, to be dipeptidyl aminopeptidase type I (DAP-1 ) (Wallace and McKain, 1989), although other activities were also apparent. This pattern differs from that obtained with sonicated bacteria, which indicated among others a
strong X-Ala-p-nitroanilide arylamidase (Ala-DAP) activity (table II). The dipeptides released as a result of DAP activity are then broken down by separate dipeptidase activity. Ruminal peptide breakdown is therefore a two-stage process.
Rumen microorganisms forming ammonia from amino acids
Deamination is carried out by two distinct bacterial populations of either low-activity/high numbers or high-activity/low numbers characteristics. The former population is probably of greater significance under most circumstances. However, it is extremely important to suppress the proliferation of the high-activity species since the presence of only a small population of these organisms could have a major impact on the efficiency of N retention by the animal.
Methane Inhibitors
A) Introduction
Ruminal methane production, for instance, results in the inefficient conversion of potentially energy-
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 130
yielding substrates into a form that can not be conserved by the host. Methane emissions represent losses of up to 15% of gross energy intake for
forage-fed cattle and losses of 2–4% for cattle consuming diets rich in readily fermentable substates(Johnson and Johnson, 1995) and (Van Nevel and Demever, 1996). Methane is also a greenhouse gas implicated as a contributor to global warming. In the United States, approximately 21%
of the total methane production is from enteric fermentation and ruminants are major contributors (EPA, 2006).
One strategy for reducing ruminal methane production is to provide alternative electron acceptors that more effectively consume reducing equivalents produced during fermentation so as to redirect electron flow away from the reduction of carbon dioxide to methane (Sar et al., 2005b).
Other strategies involve supplementing ruminant diets with anti-methanogenic compounds that inhibit
methanogens directly or inhibit biochemical reactions involved in the production of methane. Compounds
such us Cl- and Br-methane analogues, Lauric acid, Lauricidin®, Marine algue, pyromec diimide Benzo 1,3 dioxins, Sulphate- sulphite, Nitrate-nitrite and
Bromoethane-sulphonic acid are used to inhibit methane production in ruminants.
B) Effects Of Methane Inhibitors On
Ruminal Microbial Fermentation
Effects of inhibitors on total gas and methane production and on residual hydrogen concentrations
A main effect of treatment was observed on total gas produced during the consecutive batch cultures, with 47% and 75% less total gas produced by Lauricidin
® and laurate-treated cultures, respectively,
than that produced by controls
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 131
Fermentation characteristicb Treatment
a
P value SEM
b
Control Sodium nitrate
Nitroethane Sodium laurate
Lauricidin® Marine algae
Headspace measurements
Total gas produced (ml) 7.2e 5.8
e,f 4.9
e,f,g 1.8
g 3.8
f,g 6.7
e,f 0.0003 0.8
Methane produced (μmol ml−1
) 21.24e 7.92
f 0.22
g 0.24
g 0.75
g 0.81
g <0.0001 0.92
Residual hydrogen (μmol ml−1
) 3.40 3.48 3.57 3.68 4.01 4.44 0.2250 0.33
Culture fluid measurements
Acetate (μmol ml−1
) 62.77e 71.40
e 40.75
f 29.64
f,g 20.39
g 41.33
f <0.0001 4.51
Propionate (μmol ml−1
) 34.42e 30.72
e,f 23.88
f,g 16.55
g 33.62
e 33.56
e <0.0001 1.73
Butyrate (μmol ml−1
) 10.63e,f
7.55f 11.27
e 3.31
g 4.09
g 11.85
e <0.0001 0.75
Valerate (μmol ml−1
) 2.54f,g
1.53h,i 3.13
e,f 0.87
i 2.14
g,h 4.07
e <0.0001 0.22
Isobutyrate (μmol ml−1
) 1.17f 1.17
f 1.17
f 0.90
g 0.97
f,g 1.61
e <0.0001 0.06
Isovalerate (μmol ml−1
) 0.70f 0.68
f 0.71
f 0.52
f 0.57
f 1.18
e <0.0001 0.05
Total volatile fatty acids (μmol ml
−1)
112.24e 113.06
e 81.10
f,g 51.80
g 61.79
g 93.61
e,f <0.0001 7.08
Acetate:Propionate ratio 1.81f 2.41
e 1.82
f 1.86
f 0.59
h 1.19
g <0.0001 0.10
Lactate (μmol ml−1
) 0.17 0.16 0.21 0.31 0.26 0.20 0.3519 0.05
Ammonia (μmol ml−1
) 5.57g 8.17
f 5.36
g 2.73
h 2.27
h 10.89
e <0.0001 0.28
Stoichiometric calculations
Hexose fermented (μmol ml−1
)c 61.85
e 60.22
e 47.07
e,f 27.44
g 33.37
f,g 53.74
e <0.0001 3.92
Reducing equiv. generated (μmol H2 equiv. ml
−1)
d
243.47e 244.16
e 193.41
e 122.06
f 126.73
f 213.50
e <0.0001 14.03
Reducing equiv. consumed (μmol H2 equiv. ml
−1)
d
185.40e 114.52
f 84.66
g 44.48
h 87.23
g 110.55
f,g <0.0001 6.25
Treatment by incubation series interaction observed on the production of propionate (Fig. A; P < 0.0001), butyrate (Fig. B; P = 0.0139), valerate (Fig. C; P = 0.0021) and on the ratio of acetate to propionate (Fig. D; P = 0.0004) during three consecutive batch cultures of ruminal microbes without (controls) or with addition of 1 mg sodium nitrate, 1 mg nitroethane; 5 mg sodium laurate; 5 mg Lauricidin
® and 10 mg marine algae per ml
incubation fluid.
Effects on lactate, volatile fatty acid and ammonia production
Effects on lactate, volatile fatty acid and ammonia Treatment by incubation series interaction observed on the production of propionate (Fig. A; P < 0.0001), butyrate (Fig. B; P = 0.0139), valerate (Fig. C; P = 0.0021) and on the ratio of acetate to propionate (Fig. D; P = 0.0004) during three consecutive batch cultures of ruminal microbes without (controls) or with addition of 1 mg sodium
Treatment by incubation series interaction observed on the accumulation of ammonia (P = 0.0002) during three consecutive batch cultures of ruminal microbes without (controls) or with addition of 1 mg sodium nitrate, 1 mg nitroethane; 5 mg sodium laurate; 5 mg Lauricidin
® and 10 mg marine algae per ml
incubation fluid.
Effects on estimates of hexose fermented and hydrogen balance
Treatment by incubation series interaction observed on estimates of amounts of reducing equivalents (P < 0.0001) during three consecutive batch cultures of ruminal microbes without (controls) or with addition of 1 mg sodium nitrate, 1 mg nitroethane; 5 mg sodium laurate; 5 mg Lauricidin
Five of the eight B vitamins are required as coenzymes in the complex series of steps to convert propionate to glucose in the liver of the goat. For the
average dairy owner and breeder to understand what this means and how it can relate to the health and wealth of his or her dairy goat herd, is important and requires delving into some background information as well as figuring out how to apply it to current feed and management systems.
Some time ago, when vitamins were first discovered, it was thought that there was only one B vitamin. Since then it has been established that there are eight, and although they are still classified as a B vitamin, they are very different from each other in structure and function. The eight recognized B vitamins are: B1 (thiamin); B2 (riboflavin); B3
(niacin); B5 (pantothenic acid); B6 (pyridoxine); B7 (biotin, sometimes called vitamin H); B9 (folic acid); and B12 (cobalamin).
Each B vitamin acts as a coenzyme. A coenzyme combines with another substance to form an enzyme. An enzyme is a type of protein produced by
living cells that activates chemical reactions. In one way or another, B vitamins play essential roles in hundreds of these biochemical processes. B vitamins often work together in some reactions and in some cases one B vitamin is required to make another B vitamin available to the cell.
Required only in tiny amounts, B vitamins are
involved in metabolic processes. Metabolism is defined as the creation of energy when one chemical is changed (broken down) into another. When protein, carbohydrates or fats are metabolized by enzymes, the result is a release of energy.
The interaction of B vitamins is well demonstrated in
an important process that occurs in all other ruminants. Rumen bacteria produce volatile fatty acids (VFA’s) as by-products of the fermentation of plant cellulose, starches and sugars. One of these VFA’s, called propionate, is converted to glucose in the ruminant’s liver by enzymes. This is the ruminant’s only source of glucose. Five of the eight B
vitamins play a role, as a coenzyme, in the chain of events where propionate is converted (metabolized) to glucose. Since glucose is included as part of the lactose molecule in the mammary cells, all five of these B vitamins are also essential to the production of milk.
The specialized bacteria that live only in the rumen synthesize (create) all eight B vitamins, as well as vitamin K, inside their cells. They in turn supply these vitamins to other rumen microbes and finally to the goat. Rumen bacteria have their own requirements for some of the B vitamins. Nearly all of the species require biotin (B7) for their own
growth and individual species have requirements for one or more of the other B vitamins. The ruminant obtains B vitamins when the rumen bacteria are passed to the abomasum (the fourth and true stomach) along with digested feed. Ruminants are
unique in that they produce an enzyme which digests bacteria releasing their B vitamin store.
In the rumen, hundreds to thousands times more B vitamins are found inside the bacterial cells than in the free fluid. The wall of the rumen absorbs only a
minimal amount of some B vitamins and in normal conditions is completely impermeable to others such as thiamin. B vitamins found in the feed or supplied as a supplement are either used by the microbes or changed by them into a different form, or may be absorbed across the rumen membrane. There is little information on just how much of the dietary B
vitamins are degraded in the rumen or manage to be available to the ruminant further on in the intestine. In studies where the entire rumen has been completely emptied, there is some indication that all B vitamins are absorbed through the rumen wall. And, when large quantities of B vitamins are
introduced to the rumen most of them, including thiamin, do appear to be absorbed across the rumen, although the process is slow. However, according to most research, normally in well-fed ruminants few B vitamins are absorbed across the rumen membrane.
Large, single-cell rumen protozoa have a requirement for many B vitamins and do not make their own.
They probably get most of what they require when they eat bacteria, which they enjoy in large numbers, but may use up some of those vitamins free in rumen fluid. This could account for some of the ruminal loss of supplemented vitamins. It is known that in normal conditions rumen protozoa use biotin
that is available in the diet and seem to have some negative effect on bacterial use or synthesis of biotin under certain conditions.
In order to understand the fate of B vitamins either synthesized by bacteria, present in feed or supplemented by keepers, researchers have placed sophisticated sampling tubes in the duodenum, (the
pouch located between the fourth stomach and the intestines) to sample the contents and look for levels of B vitamins that enter the intestines to be absorbed. The results seem to pose more questions than they answer. Depending on the study, either all of the B vitamins that exist free in the rumen
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 133
disappear before they reach the duodenum, or some, and not always the same ones, do and some do not. The explanations are as varied as the results. Some vitamins may pass through in different, yet still
bioavailable forms or attached to other molecules. Almost all researchers state that they do not completely understand whether B vitamins are absorbed in the rumen, used by the rumen microbes, destroyed or degraded by the rumen microbes, or possibly absorbed in the other stomachs or in the
duodenum before they reach the sampling apparatus. The most consistent thing known about the fate of B vitamins in the rumen is that the available data is quite inconsistent.
Attempting to study the vitamin requirements of rumen bacteria or the type of B vitamin that each synthesize is a challenge. Rumen bacteria do not like
living in laboratory conditions, much preferring the specific requirement of the rumen environment and company of other species which may provide them with necessities that scientists cannot duplicate. They resist being studied as an individual. The B vitamin requirements of some rumen bacteria are known,
and it is known that other bacteria produce B vitamins but which species produce or use what is still largely a mystery.
It has been accepted that ruminants do not require B vitamin supplements since the bacteria supply all that they need. However, some new studies have shown that supplementation of some of the B
vitamins have positive effects on hoof health, milk production, as well as other health benefits in ruminants.
Niacin has been extensively studied because supplementation in pregnant dairy cows has shown to decrease the incidence of ketosis. Even though
niacin has been determined to completely disappear in the rumen, suggesting that the microbes destroy it when supplemented, it seems to wind up getting into the system to help where needed.
Biotin is another B vitamin that has been supplemented with positive results. Biotin is important in helping to build the structure of the hoof
wall. Numerous studies have shown that supplementing biotin in the feed of cows improves the health of the hoof wall and decreases the occurrence of several common hoof problems especially those seen in wet conditions. At the same time it was noticed that there was also a positive
effect on milk production. In a significant number of trials the amount of milk produced by lactating dairy cows increased when supplemented with biotin. In some experiments there was also an increase in the amount of biotin present in the milk. There also appeared to be a positive effect on conception ra tes in some cows.
It is suspected that biotin may not be increasing milk production, but is helping to bring production up to what would be a normal level if the cow were not deficient in biotin. Modern dairy cows have been bred to produce more milk than nature intended. The
rumen bacteria may not be able to keep up with the high demand of heavy lactating dairy cows. Biotin is a coenzyme required in activating four different enzymes involved in milk production, and it is
essential for converting propionate into glucose. It is possible that if a lactating ruminant, such as a dairy goat, is slightly deficient in biotin this could mean that some of these important reactions may not be working efficiently. Adding biotin to the diet would then bring milk production up to full potential. This
idea is supported by the fact that biotin supplements help when given up to a certain amount. Supplementing beyond that limit ceases to have a positive effect on production.
Since biotin supplementation clearly improves the health of the hoof this may add to the positive effect on milk production and conception rates. Cows (and
goats) that can move around more comfortably produce more milk and are more efficient breeders.
With two exceptions, deficiencies of B vitamins are rare in ruminants. Thiamin, or vitamin B1, acts as a coenzyme in producing a neurotransmitter molecule which is critical to proper functioning of the nervous
system. A deficiency of thiamin causes the disease seen in goats called polioencephalomalacia (PEM) o r what is commonly called goat polio. If thiamin levels are low, this neurotransmitter cannot be formed and communication between nerves is interrupted. The result is a goat that shows signs of excitability, tremors, muscle spasms and convulsions.
The reason for this deficiency in ruminants is thought to happen due to the presence of an enzyme called thiaminase which destroys or alters thiamin. It is known that the production of thiaminase is related to rumen bacteria, but it is not known exactly under what condition or which species produces thiaminase.
It is suspected that diets high in concentrates which causes a more acidic environment in the rumen may either promote the growth of thiaminase-producing species or in some way increases the activity of the enzyme. Another possibility is that for some reason one or more species of rumen bacteria may produce a thiamin “look-a-like” molecule which competes for
the uptake of normal thiamin. There is also evidence that high levels of sulfur in the diet produces PEM in beef cattle. Sulfur in the rumen produces hydrogen sulfide gas which is toxic to the rumen bacteria. In addition, some plant species contain thiaminase which may cause a thiamin deficiency if ingested.
Thiamin is not stored in the body and is quickly depleted under high energy requirements. It is one B vitamin which must be continuously supplied. Therefore, if conditions in the rumen cause a destruction of thiamin the goat rapidly becomes deficient. When a ruminant develops PEM, any thiamin given orally will be degraded by the presence
or high activity of thiaminase in the rumen. To correct a thiamine deficiency, B1 must, therefore, be given as an injection.
B12 or cobalamin is the largest B vitamin molecule and has the most complex structure of all vitamins.
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 134
Unlike other B vitamins which can be synthesized in plants, it is only synthesized by bacteria and they require an atom of cobalt in order to make a molecule of B12. Along with assisting in the
metabolism of fats, carbohydrates, and proteins, B12 has the very important role in helping to produce all the red blood cells in the body. It also helps maintain the protective sheaths around nerves and to repair DNA. B12 works with folic acid in the production of an essential amino acid called methionine. In order
for folic acid to be used by cells it has to be altered by B12. A deficiency of B12 therefore, results in a deficiency of available folic acid.
Rumen bacteria use B12 during the fermentation process of forming propionate. B12 is used in the liver of the goat in the chain of chemical reactions which convert propionate to glucose. Obviously, B12
is an important vitamin, so fortunately it is very potent. In most animals only a small amount in the diet is required. In addition, unlike other B vitamins, B12 can be stored in the liver for a long period of time. Ruminants, however, have a higher requirement for B12 than monogastric animals
because the rumen microbes produce a large amount of propionate which requires B12 to convert it into usable molecules. Propionate can build up in the blood stream if there is a deficiency of B12.
Several factors can lead to a B12 deficiency. Since each molecule of B12 requires one atom of cobalt, a diet deficient in cobalt results in a B12 deficiency
since the rumen bacteria cannot synthesize it. Diets high in concentrates lead to a high level of propionate production and therefore a high demand for B12. These diets also promote the synthesis of molecules that are similar to B12 but are not beneficial. Therefore, a high concentrate diet causes
a higher demand for B12 but at the same time has a negative influence on the amount produced by rumen bacteria.
Severe parasite infections can damage the lining of the intestinal wall, reducing the amount of B12 that is absorbed. At the same time, chronic bleeding from the actions of these parasites causes an anemia
which requires B12 to help restore lost red blood cells.
Symptoms of B12 deficiency include depressed appetite, and poor growth in young ruminants. B12 supplied in the feed is not well absorbed through the wall of the rumen and most will be used by the
microbes since they have a high requirement. B12 is most useful as an injected supplement when required until the underlying cause of deficiency is corrected.
There are many ongoing studies related to understanding the role rumen microbes play in supplying our goats with their necessary B vitamins. Current focus is on producing B vitamin supplements
that are ruminally protected which may help improve the overall health and productivity of goats, cows, sheep and other ruminants.
Buffers Buffers are weak acids or alkalis that resist changes in H+ concentration or pH. They are added to diets to complement the buffering effect of saliva and
neutralize ruminal acidity. Consequently, buffer addition reduces the risk of acidosis in cattle fed starch-rich diets or acidic silages, and decreases the incidence of bloating in cattle fed spring grass/legume pastures. Examples include sodium bicarbonate, limestone, sodium bentonite, and
magnesium oxide. The main mode of action of buffers involves increasing pH or resisting a change in pH. Higher pH values facilitate fiber digestion, hence buffer addition has increased the acetate to propionate ratio in the rumen. In addition, certain buffers increase ruminal osmolality and thereby increase the ruminal fluid outflow rate, which is
associated with reduced ruminal propionate proportion and hence, increased milk fat synthesis (Hobson and Stewart, 1997).
In summary, buffers stabilize rumen pH thereby prevent acidosis, bloat, rumenitis, and laminitis. They also enhance water intake, ruminal fluid outflow,
fiber digestion and milk fat synthesis. Buffers are recommended for stabilizing ruminal conditions when acidic or bloat-inducing diets are fed.
Defaunation
A) Introduction
The process of making the rumen of animals free of
rumen protozoa is called defaunation and the animal is called defaunated animal. Rumen protozoa are the largest in size among rumen microbes and contribute 40-50% of the total microbial biomass and enzyme activities in the rumen (Agarwal et al., 1991).
Methods of defaunation
There are several ways to defaunate the animals and
to obtain a ruminant animal free from rumen ciliate protozoa.The different methods of defaunation are:
Isolation Of New Born Animals
One of the method of producing defaunated animals is the separation of newborn animals from their dams after birth and preventing them from any contact
with the adult ruminant animals. The newborn animals should be separated 2 to 3 days after birth. During this time the newborn animals gets contaminated with the native bacterial population but do not get rumen ciliate protozoa (Fonty et al., 1984). However, once the animal is separated, proper care should be taken so that the isola ted
animals do not come in contact with any adult animals as well as any contamination from the handlers who look after faunated and defaunated animals.
Chemical Treatment
Another method of defaunation is by use of chemicals and majority of researchers has used this method for obtaining animals free from rumen ciliate protozoa. The chemicals which have been widely used to
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 135
defaunate the animals are copper sulphate, manoxol and sodium lauryl sulphate, alkanates, teric GN9 and calcium peroxide . Chemicals which are used as defaunating agents are introduced in the rumen of
animals either orally by a stomach tube or through rumen fistula. However, these chemicals are not only toxic to the rumen protozoa but also kill the other rumen microorganism like bacteria. These chemicals are also toxic to the animals resulting in depressed feed intake, dehydration and some time mortality
also reported (Jouany et al., 1991).
Dietary Manipulation
The ciliate protozoa are very much sensitive to change in rumen pH. The activity of ciliate protozoa is adversely affected when the pH of the rumen fall below 5.8 and if the rumen pH fall below 5.0, the ciliate protozoa are be completely eliminated.
Therefore, offering high energy feed (especially cereal grains like barley, maize etc) to the starved (for 24 hours) animals creates acidic condition in the rumen and rumen pH fall below 5.0. This fall in rumen pH eliminate the ciliate protozoa completely and the animal become defaunated. However one
serious disadvantage of this method is that chances of developing acidosis in treated animal is more. Once rumen acidosis develops the animals will suffer form various secondary complications. The drenching of vegetable oils eliminate ciliate protozoa and hence can be used as a defaunating agent (Nhan et al., 2001).
(B) Effects Of Defaunation On Rumen Characteristics
Effect of defaunation on the rumen ecosystem rumen microbes
Defaunation causes both qualitative and quantitative
change in rumen bacterial population. After defaunation the bacterial population increased, since rumen protozoa feed on the rumen bacteria to meet their nitrogen requirement. A total of 4 to 45 g bacterial dry matter is engulfed by rumen protozoa per day per sheep. Defaunation increase the number
of amylolytic bacteria due to elimination of nutritional competition between bacteria and protozoa for using starch, whereas the cellulolytic bacterial population becomes decreased (Jouany et al., 1991). Fungal population in the rumen also increase due to defaunaton (Chaudhary et al., 1995).
Rumen pH
The buffering capacity of the rumen seems to be better in presence of protozoa on a wide variety of diets. The rumen pH starts falling immediately after ingestion of feed, both in faunated and defaunated
animals whereas, the drop in pH was much higher in defaunated than in faunated animals (Jouany et al, 1991; Nagaraja et al., 1992. Rumen protozoa engulf the readily fermentable carbohydrate (starch) which is stored in their body as amylopectin and thus decrease the rate of carbohydrate (starch degra-
dation) fermentation, resulting in a lower pH in the rumen of defaunated compared to faunated animals.
Volatile Fatty Acid (VFA) Production
The effect of defaunation on the production and composition of VFA is variable. The VFA production rate and its composition are greatly influenced by experimental diet. Increase in TVFA concentration in defaunating animals was reported by Santra et al.
(1996) and Santra and Karim (2002a) while non-significant effect was recorded by Ivan et al. (1992). Higher VFA conecntration in the rumen of faunated animals may be due to higher hydrolytic enzyme activity in the rumen protozoa because about 40-60% of hydrolytic enzyme activity is found in the rumen protozoa (Agarwal et al., 1991) and also due
to stimulatory effect of protozoa over bacteria.
Ammonia Nitrogen Concentration
Significant reduction in ammonia-N concentration in the rumen of defaunated animals was reported by
Chaudhary et al., 1995; Santra et al., 1996; Nhan et al., 2001; Santra and Karim, 2002a). Ammonia is utilized by bacteria to meet their nitrogen requirement for body protein synthesis while ciliate protozoa does not use it. In defaunated animals, the number as well as activity of rumen bacteria increase resulting in more uptake/utilization of ammonia by
bacteria and as a result, ruminal ammonia concentration is reduced.
Microbial Protein Synthesis
Microbial protein synthesis in the rumen of defaunated animals was higher than faunated animals. It is now generally accepted that in absence of rumen ciliate
protozoa, the efficiency of rumen bacterial growth is enhanced and more microbial protein flows from reticulo-rumen to duodenum. Although bacteria and protozoa are active in synthesis of microbial protein, outflow of microbial protein in to duodenum is primarily of rumen bacterial origin. About half of the microbial protein in the rumen can be of protozoal
origin while as a proportion of the microbial protein leaving the rumen, protozoal protein is usually under 10% because of higher rate of bacterial was out from reticulo-rumen (Bird et al., 1994).
Enzyme Profile:
Rumen ciliate protozoa secrete various hydrolytic enzymes which are responsible for break down of the plant cell wall poly saccharides (Agarwal et al.,
1991). The ciliate protozoa and fungi are most important microbial groups of the rumen organisms required for the ruminal digestion of plant fibre. Carboxymethyl cellulase enzyme activity was lower in the rumen of defaunated than faunated animals (Santra et al., 1996; Santra and Karim, 2002a). About 62% of the total rumen cellulase enzyme
activity is associated with rumen protozoal population. Hence elimination of ciliate protozoa decreases ruminal cellulase enzyme activity.
MANIPULATION OF RUMINAL MICROBIAL FERMENTATIONS
Dairy Year Book (2014-15) 136
Methane Production:
Defaunation is reported to considerably decrease the methane production compared with the normal faunated animals (Jouany et al., 1988; Santra et al., 1994b). The reduction in methane production in absence of rumen protozoa has been attributed to
various reasons. Rumen protozoa contribute hydrogen moiety for the production of methane by the methanogenic bacteria. Further, ectosymbiotic attachment methanogens have with ciliate protozoa and elimination of their symbiotic partner on defaunation results in reduced methane production.
Rumen Volume, Flow Rate Of Digesta And Physical Characteristics:
The effect of defaunation on rumen physiological
characteristics (e.g. motility, absorption) are lacking in literature. Contradictory reports have appeared on the effect of defaunation on rumen fluid volume and digesta flow rate. Chaudhary et al. (1995) reported higher rumen volume in defaunated buffaloes whereas liquid outflow rate remained unchanged
irrespective of presence or absence of ciliate protozoa. Kayouli et al. (1983/84) reported no difference in rumen volume and liquid fractional outflow rate in defaunated and faunated animals while, the particle outflow rate was significantly higher in the absence of ciliate protozoa.
(C) Future Direction
Results reported by different researchers on effect of defaunation are still contradicting each other. Contradictions on feed digestion and rumen metabolism are likely to happen because these two parameters are not as simple and easy as feed-intake and weight-gain parameters to measure
because rumen gastro-intestinal tracts are complex media. Therefore the contradictory results reported on feed digestion and rumen metabolism may arise from:
1. Variation between species of animals.
2. Variation in operator skills to apprehend small effects and analyze.
3. Variation in procedure and examination techniques.
4. Variation from technological level of apparatus or machine used to examine and analyze.
5. Variation in personal skill to interpret the analyzed
even smaller effects.
Contradictory results on easy-to-handle parameters like feed-intake and weight-gain can be cleared by conducting skilfully fine-tuning type of research on strictly homogenous animals. Whitelaw et al. (1972) noticed protozoa cannot convert NPN to protein because they have no ureases and so animal cannot
utilize urea and ammonia. Whitelaw et al. (1972) recommended further study on protozoa free animal for their maximum capacity on NPN utilization. Miresan et al. (2006) also noticed that defaunation may increase urea utilization and indicated the need for further testing of appropriate level of urea
utilization. Eryavuz et al. (2003) also pointed out this researchable gap saying the maximal dietary urea
level useful for defaunated animals remains to be determined.
Conclusions
Rumen is a natural fermentative anaerobic vat in which a complex and dense population of bacteria, protozoa and fungi convert feedstuff materials to VFA’s, CH
4
, CO2, NH3
and microbial cells and should
be manipulated essentially by altering the composition of ruminal microflora. There is ample scope to manipulate the ruminal microbial fermentations by using diverse chemical compounds in order to improve animal health (bloat, acidosis and ketones), productivity and environment.
The ionophores, non-ionophore antibiotics,
protease/deaminase and methane inhibitors, mineral salts, buffers and defaunating agents are capable of reducing ruminal microbial fermentations in the short-term, though further research must be conducted to find ways of extending the effects of these feed additives.
of reducing enteric methane production produced by ruminants, thereby decreasing the presence of methane in the atmosphere. Thus, supplementation of methane inhibitors is one strategy for mitigating the global warming. Future other strategies to reduce enteric methane production may be most successful
if they involve heterogeneous diets and combinations of some compounds which manipulate ruminal fermentations and methane inhibitor supplements.
References
1. Agarwal, N ., N . Kewalramani, D. N. Kamra, D. K.
Agrawal and K. Nath. 1991. Hydrolytic enzymes of
buffalo rumen: C omparison of cell free rumen fluid,
bac terial and protozoal fractions . Buffalo J. 7: 203-207.
2. Arse Gebeyehu and Yosef Mekasha
(2013).Defaunation: effec ts on feed intake, diges tion,
rumen metabolism and weight gain. Wudpecker Journal
of Agricultural Research, V ol. 2(5), pp. 134 – 141.
3. Bergen, W. G., and D. B. Bates (1984). Ionophores:
Their effec t on production efficiency and mode of ac tion.
J. A nim. Sci. 58:1465-1483.
4. Bird, S. H., and R. A . Leng. 1990. The effec ts of
defaunation of the rumen on the growth of cattle on low-
protein high-energy diets . Br. J. Nutr. 40:163-167.
5. Chaudhary L. C ., Srivastava A ., Singh K. K. (1995).
Rumen fermentation pattern and digestion of s tructural
carbohydrate in buffalo (Bubalus bubalis ) calves as
affected by c iliate protozoa. Anim. Feed Sc i. Technol., 56
(1995), pp. 111–117.
6. Chiba L.L.,2009. Rumen mic robiology and ferme-
27. Whitelaw, F. G., J. M . Eadie, S. O . Mann and R.
S. Reid. 1972. Some effec ts of rumen c iliate protozoa in
cattle given res tric ted amounts of a barley diet. Brit. J.
Nutr. 27:425.
28. William AG, Coleman GS(1988). The rumen
protozoa.In: Hobson, P .N. Ed.The rumen mic robial
ecosys tem. Elselvier Sci. P ublishers Ltd. London,
England, pp. 77 -128.
Feeding Of Forages For
Milk Production
Rations of lactating and non lactating animals have to be formulated with different types of feeds and fodders, so that their high level of requirements can be fulfilled within the limits of their intake. Along with maintenance ration, the ration of lactating animals should contain higher amount of minerals like Ca & P in addition to high protein. Fat percentage of the milk is also one of the deciding factors of nutrient requirements. During first lactation, aprox. 20% more protein and energy is required for medium producers while for high producers, the increase in the requirement can be up to 25%. Pregnant animals also require more nutrients during last 2 months of gestation. Requirement of energy increases from 235 Kcal in seventh month to 940 Kcal in ninth month. Depending upon the quantity of concentrate mixture in the ration total intake also varies. At 25% of conc. mix in the ration, total intake goes up to 2.4% of body weight while at 75% inclusion; total intake goes up to 3%. Animals producing 4 kg of milk can be maintained on green fodder, however, on leguminous fodders animals producing 6-8 kg can also be maintained without supplementing concentrate mixture. Exclusive feeding of green fodders can not fulfill nutritional requirements of high producing cows; therefore, supplementation of concentrate mixture is necessary to facilitate them to exhibit their full genetic potential.
