UC Agriculture & Natural Resources California Agriculture

Title In vitro gas production provides effective method for assessing ruminant feeds

Permalink https://escholarship.org/uc/item/2078m8m1

Journal California Agriculture, 58(1)

ISSN 0008-0845

Authors Getachew, Girma DePeters, Edward J. Robinson, Peter H.

Publication Date 2004

Peer reviewed

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RESEARCH ARTICLE

▲ ▲ In vitro gas production provides effective method for assessing ruminant feeds

Girma Getachew Edward J. DePeters To test the digestibility of Peter H. Robinson animal feeds, UC Davis graduate students Kelly McCaughey and ▼ Stefanie Cheng help postdoctoral associate Girma Getachew collect rumen fluid An animal’s feed intake, and how from “Tank.” The cow has been well that feed is digested, determine fitted with a permanent fistula, the feed’s production performance. allowing daily collection of rumen fluid. The in vitro gas production technique is a relatively simple method for can consume a wide variety of by- above). This method for forage evalua- evaluating feeds, as large numbers product feedstuffs derived from the tion was first reported in 1963, using of samples can be incubated and processing of plants for human food. ruminal fluid obtained from a sheep analyzed at the same time. This California livestock industries utilize with a rumen fistula (Tilley and Terry method has been applied success- the majority of these highly fibrous 1963). A rumen fistula is formed by sur- fully at UC Davis for a variety of pur- byproducts by including them in feeds gically transecting the skin and the ru- poses in feed evaluation, including for cattle, sheep and goats. The nutri- men, suturing the rumen to the skin calculating organic matter digestibil- tive value, or energy content, of an ani- and allowing the rumen to heal, creat- mal feed is determined predominately ing a permanent opening into the ru- ity, the metabolizable energy of by its digestibility, which affects intake, men. A soft cannula fits in the fistula to feeds and kinetics of their fermenta- or how much the animal will eat. close the rumen. The cannula can be tion; determining how feed value is Digestibility and intake, in turn, deter- opened to allow access to the rumen in affected by added fat, antinutritive mine the feed’s productive perfor- order to collect the bacteria-containing factors and rumen modifiers; quanti- mance, such as to support milk fluid needed for in vitro incubations. fying the energy value of feed mix- synthesis or muscle growth. However, Material not recovered in the residue tures (rations); monitoring microbial studies with live animals (in vivo) to de- following incubation is assumed to be change in the rumen; synchronizing termine the digestibility of feeds are fermented, providing estimates of the time-consuming, laborious, expensive extent of digestion for various feeds. nutrient digestion; and selecting for- and require large quantities of feed. More recent methods measure the age nutrient targets for agricultural Such experiments are not suited for the products of anaerobic fermentation. biotechnology. More than half of the rapid and routine feed evaluations un- Rumen fermentation by anaerobic mi- nutrients consumed by ruminant ani- dertaken by commercial laboratories crobes results in production of short- mals leave the animal unutilized and that provide feed information to live- chain fatty acids (SCFA), gases (carbon undigested, and are excreted in fe- stock producers and feed manufacturers. dioxide [CO2] and methane [CH4]) and ces, urine and gases. The in vitro gas The digestibility of feeds can also be microbial mass. The amount of gas pro- estimated by biological methods duced is proportional to acid produc- production method can be used to known as in vitro techniques, which are tion, thereby serving as an indicator of examine animal waste components conducted outside of the animal system acids produced by fermentation. The that impact the environment and de- but simulate the digestion process. amount of gas produced during incu- velop appropriate mitigations. Generally, in vitro techniques are those bation is measured to predict the extent based on measuring either fermenta- and rate of feed digestion. uminants have a four-compartment tion residues or products. The former In addition to quantifying the chemi- Rstomach. The rumen is the largest measures the unfermented residue re- cal composition of feeds, some com- compartment, where millions of bacte- maining after in vitro incubation of a mercial laboratories offer in vitro feed ria grow under anaerobic (low-oxygen) feed with rumen fluid. This approach digestibility as a component of their conditions. These bacteria are respon- involves collecting fluid by hand from feed analysis packages. This data can sible for the digestion of fiber (cellu- the rumen of a ruminant that has been be used in new ration-evaluation com- lose) and are the reason why ruminants fitted with a rumen fistula (see photo puter models with the goal of optimiz-

54 CALIFORNIA AGRICULTURE, VOLUME 58, NUMBER 1 Compared with laborious and expensive in vivo testing, the in vitro gas production method provides a quick and easy way to calculate organic matter digestibility, quantify the energy value of feed mixtures and monitor microbial change in the rumen. Left, Girma Getachew fills syringes with buffered rumen fluid. ing nutrient utilization and animal pro- cally controlled water bath (102°F). The Organic matter digestibility. The di- ductive performance, thereby minimiz- volume of gas produced in 24 hours gestibility of measured organic matter ing the environmental impacts of from incubating 200 milligrams (mg) of is closely correlated with that predicted nutrient excretion in the animal’s urine feed, together with the concentration of from gas production and the crude pro- and feces. crude protein and crude fat, is used to tein and ash contents of feeds. There- Gas-measuring techniques have predict ME. Large numbers of samples fore, the method can be used to predict been routine in feed evaluation since can be analyzed during a single 24-hour the extent of digestion for various the early 1980s, when a high correlation incubation run. feeds. was found between metabolizable en- At UC Davis, three nonlactating Energy contents of feeds. The gas ergy (ME) measured in live animals Holstein cows (about 1,450 pounds method has also been used successfully and that predicted from gas produc- each) are fitted with permanent rumen to predict the ME content of feeds. A tion. The in vitro gas technique has sev- fistulas. Twice daily, the cows are fed a regression equation has been devel- eral advantages over other in vitro diet of mostly oat hay with a small oped with data generated by in vivo methods that are based on measuring amount of alfalfa hay. This ensures that studies conducted with a variety of residues. Gas production reflects all nu- the ruminal microbial population feeds and in vitro gas production. The trients fermented, soluble as well as in- changes little from day to day. Rumen gas measurement provides a better esti- soluble; and fractions that are not fluid is collected after the morning mate of the ME level of feeds, when fermentable do not contribute to gas feeding using a manually operated combined with some chemical constitu- production. Furthermore, the kinetics vacuum pump. The fluid is placed into ents, compared with calculations based of fermentation can be obtained from a prewarmed thermos flasks, then mixed on chemical constituents only. Re- single incubation, allowing the rate of and filtered through four layers of cently, seven laboratories around the fermentation to be calculated. cheesecloth and flushed with carbon di- world that use a gas method — includ- Gas measurement is a direct mea- oxide in the laboratory. One part ru- ing UC Davis — carried out a compara- sure of microbial activity and can be a men fluid is mixed with two parts tive test to assess the repeatability of better index of forage ME content than buffered mineral solution (1:2 volume/ the technique in predicting the energy an indirect in vitro measure based on volume) and maintained at 102°F. Fi- value of feeds, and found that the gas nutrients fermented. The gas technique nally, this buffered rumen fluid (30 ml) is method was repeatable among labora- is relatively simple and does not re- pipetted into incubation syringes con- tories (Getachew et al. 2002). quire sophisticated equipment, making taining the ground test substrate and Kinetics of fermentation. In assess- it easy to conduct for research and com- placed in a 102°F water bath. Gas pro- ing nutritive value, the rate at which a mercial purposes. Rumen fluid is col- duction is measured by visually read- feed or its chemical constituents are di- lected from a cow with a rumen fistula. ing the scale on the side of each gested in the rumen is as important as Fermentations are conducted in large syringe. the extent of digestion. The pattern of (100 milliliter [ml]) calibrated glass sy- feed fermentation (kinetics of fermenta- ringes in an anaerobic medium inocu- Applicability of gas method tion) is one of several factors that influ- lated with rumen fluid. Incubations can The in vitro gas method has been ap- ence voluntary feed intake by ruminants. be carried out either in an incubator plied successfully at UC Davis for a va- The rate at which different chemical with a rotating disc or in a thermostati- riety of purposes in feed evaluation. constituents are fermented is a reflec-

http://CaliforniaAgriculture.ucop.edu • JANUARY-MARCH 2004 55 tion of microbial growth and accessibil- ity of the feed to microbial enzymes. By describing gas production mathemati- cally, kinetic data can be analyzed to evaluate substrate- and media-related differences as well as the fermentability of soluble and slowly fermentable com- ponents of feeds. The gas method is an ideal technique to generate kinetics of Left, dozens of syringes containing feed samples can be incubated at the same time during fermentation, as it allows recording of a single 24-hour incubation run. Right, gas production is measured by reading the scale on gas produced at several times in the in- the side of the syringe. cubation period, which is used to pre- dict the rate at which feed is digested. 2001)(fig. 3). Fatty acids in the form of Antinutritive factors. The gas The gas method has been used to triglyceride (YG) had no effect (when method can be used to measure how evaluate the effects of grain processing comprising up to 25% of the diet) on microbial activity lowers feed digest- on the rate and extent of gas produc- gas production, but fatty acids in the ibility. Some feeds, such as forage le- tion (DePeters et al. 2003)(fig. 1). Steam- form of potassium salts (YG soap) sig- gumes and cottonseed, contain flaked corn fermented faster than nificantly depressed gas production. In phenolics, alkaloids and saponins that whole grain. The first 16 hours of incu- the animal, however, there is a limit to have negative biological effects on mi- bation were important for measuring the amount of fatty acids that can be crobes and reduce microbial growth in starch availability. Different types of successfully fed, and this is lower than rumen. Tannins are naturally occurring forages, including cereal hays and si- in vitro. The fatty acids in potassium polyphenolic compounds found in lages, were also studied to determine salts are quickly available to microbes plants, which form complexes with their patterns of fermentation in the ru- as free fatty acids in ruminal fluid, and feed and microbial proteins and can men. Although alfalfa hay fermented have detrimental effects on microbial depress feed digestibility in the rumen. faster during the early stages, it was growth. In contrast, the fatty acids in The effect of tannins on the nutritive lower in extent of gas production com- the triglyceride form must be released value of feeds can be studied using pared to corn silage after 72 hours of through hydrolysis of the ester bond tannin-binding agents, such as polyeth- incubation (fig. 2). and therefore are available at a slower ylene glycol (PEG), which strongly Effects of added fat on feed degra- rate. Hydrolysis refers to breaking the binds to tannins and inhibits their bio- dation. Tallow and yellow grease (YG), chemical bond between the individual logical effects. The percent increase in both rendering byproducts, are typical fatty acid and the glycerol backbone of gas production when PEG is present in- fats used in the diets of lactating dairy the triglyceride. The effects of fatty ac- dicates the rate at which tannins de- cows. The gas technique was used at ids on rumen fermentation are impor- press rumen fermentation of feeds. UC Davis to examine the effect of tant because feeds with high levels of After adding PEG to limit tannin ef- sources and levels of added fat on gas residual fat, for example rice bran cre- fects, gas production increased by 22%, production and rumen fermentation ated in the production of white rice, are 71% and 211% in apple ring acacia of a total mixed ration (Getachew et al. commonly fed to ruminants. (Acacia albida), beach acacia (Acacia

Fig. 1. Kinetics of in vitro gas production Fig. 2. Pattern of in vitro gas production Fig. 3. Effect of yellow grease (YG) and during incubation of flaked and whole during incubation of alfalfa hay and yellow grease soap (YG soap) on in vitro corn grain. alfalfa, corn and wheat silages. gas production.

56 CALIFORNIA AGRICULTURE, VOLUME 58, NUMBER 1 cyanophylla) and red calliandra Jack Kelly Clark (Calliandra calothyrsus), respectively, which are browse plants (Getachew et al. 2000b)(fig. 4). Rumen modifiers. The gas method is also utilized to study feed additives and rumen fermentation modifiers, such as monensin sodium, by incubat- ing feeds in the presence or absence of these compounds. Rumen modifiers are compounds that are added to the diet to modify the populations of bacteria in the rumen. For example, some com- pounds are fed to reduce methanogenic bacteria to reduce methane production in the rumen. Previous studies have shown that the addition of saponins and tannins in an in vitro system in- creases microbial protein synthesis More than half of the nutrients consumed by ruminant animals such as cows are (Makkar et al. 1995). Yeast and yeast undigested and excreted into the environment. By using feed evaluations to improve fermentation products are routinely feed utilization, air and water pollution can be reduced. added to the diets of lactating dairy cows, although their mode of action has this is not always true. For example, tions in the rumen. This enables ma- not been clearly identified. By studying when poor-quality forage — such as nipulation of rumen microflora to in- the impact of various rumen modifiers wheat straw — is fed to a ruminant, its crease the utilization of feeds through on microbial fermentation, effects im- digestibility is low, but by adding nitro- degradation of fiber and lignin, increas- portant to milk production in commer- gen in the form of urea or protein, the ing the efficiency of nitrogen utilization cial dairy farms can be quantified. digestibility of the straw will be in- or allowing the degradation of anti- Feed associative effects. The in vitro creased and in turn, the energy derived nutritional and toxic components of gas production method is currently be- from straw organic matter in the diet feeds. Such controlled fermentation ing used to assess “associative” effects will be increased. Recent studies indi- systems could potentially be used with of feeds used in rations. Rations are cate that positive associative effects genetic engineering of plants to solve mixtures of individual feeds, with a on in vitro gas production occurred animal productivity problems. The multitude of possible combinations. when rice straw was incubated in technique is suitable for application The energy value of a ration is gener- mixtures with hay or mulberry leaves of molecular-based assays, such as ally calculated by adding up the energy (Liu et al. 2002). polymerase chain reaction (PCR) and values of the individual feeds in the ra- Monitoring rumen microbial change. ribonucleic acid (RNA)–targeted oligo- tion, on the assumption that the indi- In addition to rates and extents of di- nucleotide probes, to study and mea- vidual energy value of any particular gestion, the gas production method can sure rumen microbial growth, with the feed is the same in every possible com- be used to study substrate-related fac- goal of increasing the efficient utiliza- bination with other feeds. However, tors that influence microbial popula- tion of feeds and reducing environmen- tal impacts. Recently, Muetzel and Becker (2003) used the gas technique, in combination with ribosomal RNA- targeted probes, to measure the efficiency of microbial growth when barley straw was supplemented with legume leaves. Nutrient synchronization. Carbohy- drate and nitrogen sources must be available simultaneously in order to maximize microbial growth. Ruminal ammonia concentrations can be influ- enced by the degradation rates of car- bohydrates and nitrogen-containing compounds. For a given level of dietary Fig. 4. In vitro gas production of tannin-containing leaves in absence of PEG (0 PEG, protein, an increased rate of protein with tannins) and presence of PEG (+ PEG, with no tannins). degradation enhances the ruminal am-

http://CaliforniaAgriculture.ucop.edu • JANUARY-MARCH 2004 57 monia concentration while an increased Environmental degradation. More rate of carbohydrate degradation de- than half of the nutrients consumed by G. Getachew is Postdoctoral Associate, E.J. creases it. Increased carbohydrate avail- ruminants leave the animal unutilized DePeters is Professor, and P.H. Robinson ability for fermentation promotes and undigested, and are excreted in is Cooperative Extension Specialist, microbial growth and as a result less ni- feces, urine and gases. This increases Department of Animal Science, UC trogen is lost from the rumen in the animal production costs as well as envi- Davis. This research was supported by form of ammonia-nitrogen (Getachew ronmental impacts, by contaminating the California Agricultural Experiment et al. 2000a). The gas method offers an surface- and groundwater and contrib- Station, UC Davis. opportunity to study microbial require- uting to air pollution. The nitrogenous ments for nitrogen and carbohydrate to and organic compounds excreted are References enable efficient fermentative activity and further decomposed and can cause DePeters EJ, Getachew G, Fadel JG, et al. accumulation in the rumen. Using this odors in residential areas. Increasing 2003. In vitro rumen gas production as a technique, studies have been conducted the efficiency of feed utilization reduces method to compare fermentation character- istics of steam-flaked corn. Animal Feed Sci to assess rumen microbial requirements the amount of unutilized nutrients Technol 105:109–22. for nitrogen when different types of car- leaving the animal. Significant reduc- Getachew G, Crovetto GM, Fondevila M, bohydrate sources are incubated. tions in nitrogenous compounds et al. 2002. Laboratory variation of 24 h in Plant breeding, biotechnology. We (Kuelling et al. 2003) and in methane vitro gas production and estimated metabo- lizable energy value of ruminant feeds. Ani- believe that animal nutritionists and (Johnson and Johnson 1995) can be mal Feed Sci Technol 102:169–80. plant geneticists should collaborate to achieved by manipulating animal diets. Getachew G, DePeters EJ, Robinson PH, select genetic materials that have better The in vitro gas method can be used to Taylor SJ. 2001. In vitro rumen fermentation agronomic performance and superior study the efficiency of feed utilization and gas production: Influence of yellow grease, tallow, corn oil and their potassium nutritional qualities. Siaw et al. (1993) and to examine animal waste compo- soaps. Animal Feed Sci Technol 93:1–15. used the gas technique to evaluate nents that impact the environment in Getachew G, Makkar HPS, Becker K. large numbers of browse species in or- order to develop appropriate mitiga- 2000a. Effect of polyethylene glycol on in vitro degradability of nitrogen and micro- der to select those high in feeding tion strategies. bial protein synthesis from tannin-rich value. Browse is the edible parts of browse and herbaceous legumes. Br J Nutr woody vegetation such as leaves, stems Determining nutritive value 84:73–83. and twigs from bushes common on The nutrient composition of feeds is Getachew G, Makkar HPS, Becker K. 2000b. Tannins in tropical browses: Effects California foothills; they have been commonly determined primarily by on in vitro microbial fermentation and mi- identified as integral to the develop- chemical analyses. However, this does crobial protein synthesis in media contain- ment of fires that ravaged Southern not provide sufficient information to ing different amounts of nitrogen. J Agric California in the fall of 2003. Several determine the feed’s true nutritive Food Chem 48:3581–8. Johnson KA, Johnson DE. 1995. Methane forage and cereal crops have been ge- value. The efficiency by which an ani- emissions from cattle. J Animal Sci 73:2483–92. netically modified to increase yield, or mal utilizes feed nutrients has a signifi- Kuelling DR, Menzi H, Sutter F, et al. produce chemical constituents nor- cant impact on its productive 2003. Ammonia, nitrous oxide and methane emissions from differently stored dairy ma- mally deficient in a particular plant. performance and waste production. nure derived from grass- and hay-based ra- Forage plants are selected for rapid fi- The in vitro gas production system tions. Nutrient Cycling Agroecosystems ber digestibility. Plants have also been helps to better quantify nutrient utiliza- 65:13–22. genetically engineered to produce hu- tion, and its accuracy in describing di- Liu JX, Susenbeth A, Sudekum KH. 2002. In vitro gas production measurements to man lysozyme, but it is unclear what ef- gestibility in animals has been evaluate interactions between untreated fect lysozyme has on microbes in the validated in numerous experiments. Ani- and chemically treated rice straw, grass and rumen. Although many genetically engi- mal experiments will continue to add in- mulberry leaves. J Animal Sci 80:517–24. Makkar HPS, Blümmel M, Becker K. 1995. neered plants are intended for human formation to our understanding of In vitro effects of and interactions between consumption, their byproducts will be nutrient metabolism. However, where tannins and saponins and fate of tannins in fed to animals as a means of disposal. applicable the in vitro gas production sys- the rumen. J Sci Food Agric 69:481–93. The starch contained in cereals, including tem can be used to predict animal perfor- Muetzel S, Becker K. 2003. Supplementa- tion of barley straw with Sesbania corn and milo, is found in granules mance at a much lower cost. Based on the pachycarpa leaves in vitro: Effects of fermen- surrounded by a tough protein matrix strong relationship between measured tation variables and rumen microbial popula- that reduces enzymatic degradation. digestibility and that predicted from gas tion structure quantified by ribosomal There are new genetic varieties of production, regression equations have RNA-targeted probes. Br J Nutr 89:445–53. Siaw DEKA, Osuji PO, Nsahlai IV. 1993. these cereals with modified protein been developed and the method has been Evaluation of multipurpose tree germplasm: matrices. We are currently using the standardized. The use of gas production and rumen degra- gas production method to explore In addition, the method can evaluate dation characteristics. J Agric Sci (Cam- bridge) 120:319–30. whether these new varieties increase the impact of biotechnological changes in Tilley JM, Terry RA. 1963. A two-stage the extent and rate of starch digestion plants on their nutritive value and other technique for the in vitro digestion of for- (DePeters et al. 2003). factors that affect rumen fermentation. age crops. J Br Grassland Soc 18:104–11.

58 CALIFORNIA AGRICULTURE, VOLUME 58, NUMBER 1