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 eScholarship.org Powered by the California Digital Library University of California 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.