Proc. 56th Southern Pasture and Forage Crop Improvement Conference, Springdale, AR April 21-22, 2001
The Conduct of Grazing Experiments: Measurements Explaining
Why Animal Response Differences Occur
J.C. Burns joe_burns@ncsu.edu and L.E. Sollenberger
USDA-ARS, North Carolina State University, Raleigh, NC,
and University of Florida, Gainesville, FL
In traditional grazing trials the results usually address the question “what might one expect in terms of animal and pasture production from some array of pasture treatments?” In such trials some basic pasture and animal measurements are obtained and presented. This area has been addressed in some detail by Dr. Sollenberger in this session (Sollenberger and Burns, this proceedings).
In traditional grazing trials, however, measurements are generally not taken that will explain why animal response differences resulted among the pasture treatments evaluated. For example, why animal response differences occurred among forage species being evaluated, or among herbage mass levels within forage species, or among different grazing methods, to mention only a few. For the purpose of this paper it is assumed that there is a need to determine why animal response differences occurred among treatments evaluated and that the costs (labor and sample intensive) are warranted. The objective of this paper is to address additional measurements that might be superimposed upon traditional grazing trials to expand the knowledge about each pasture treatment being evaluated. This information will contribute to answering the question “why did each pasture treatment result in the specific or relative animal response that was obtained?” A detailed literature review was not attempted, but some key references are included at the end for the interested reader.
Application of Intensive Measurements
The traditional, well conducted grazing trial provides the opportunity for the application of additional measurements that will provide insight and understanding into why animals respond as they do to each pasture treatment. This extension of the research is cost effective because the base trial is already in place and constitutes a major investment in its own right. Overlaying additional measurements will of course require further cost because these measurements are generally labor intensive and result in numerous samples that must be properly handled and analyzed. The judicious application of such intensive measurements is best achieved through a ‘Team’ effort with a scientist dedicated to each of the areas of interest (e.g., the plant, the animal, the soil, etc.). An essential member of the team is a person with bio-metric skills. These are needed to provide the pay-off which is the integration of the response data. Such skills may reside with one of the team members representing one of the biological areas or be contributed by a statistician.
Specific Measurements
Canopy Characterization
Morphology. Ruminant animals selectively graze and when possible generally consume green-leaf tissue. The separation of the pasture canopy into leaf, stem, and dead tissue provides insight into the potential diet that may be selected. Separation by some increment of canopy height, such as every 5 cm or perhaps an upper and lower layer, instead of the whole canopy provides more information about the canopies being compared and will aid in developing stronger relationships among plant and animal measurements because of the way most ruminants graze.
Sampling and Handling. In all experimentation, but especially when intensive methods are being applied, it is critically important that special care be given to the samples obtained. It is important that they are taken to be representative of what’s being measured. In grazing trials the relationships that are of interest are between 1) the animal and the fresh canopy as offered and consumed, 2) the plant and the soil, and 3) the animal and the soil. The intent is that the relationships reflect the natural state. Ideally, one would make all laboratory determinations on the fresh (wet) sample be it herbage, soil, or animal related. This is presently not feasible, especially when hundreds of samples are involved, and requires preservation of the fresh (wet) sample in a dry state until analyzed. While oven drying is the convention, heat applied to fresh (moist) tissue runs the risk of complexing the soluble constituents (especially nitrogen and carbohydrates), resulting in Maillard products, and can differentially alter the subsequent analytical data which may make it no longer useful for developing plant-animal relationships. The degree of complexing upon heating can vary with the concentration of water and soluble constituents in the sample so the actual or relative differences among treatments may have little to no meaning. The avoidance of artifact composition is best addressed by quick freezing fresh samples in liquid nitrogen, followed by freeze drying (96-98% DM), and subsequent storage in a freezer (-18 C) until analyzed. A critical point after drying is to keep the samples from reabsorbing moisture while in storage. This methodology adds a degree of assurance that analytical data reflect, as closely as possible, the fresh plant in situ as viewed by the animal.
Nutritive Value. Although the proportion of leaf, stem, and dead tissue that constitutes the whole canopy is important, the nutritive value of each fraction within and among treatments also has a major impact on the resulting animal response. To this extent an estimate of the digestibility (some form of in vitro bioassay) and crude protein and fiber fraction(s) concentrations from the freeze-dried tissue would be helpful in explaining differences in animal responses. Also, certain mineral concentrations of the various canopy fractions might be valuable in certain environments.
Diet Characterization
A ruminant meets its daily dry matter intake demand one bite at a time. It is this bite repeated over and over again during a 24-h period for each treatment evaluated that constitutes the dry matter which drives the animals’ daily response. An estimate of the diet consumed by the animal from each treatment and its nutritive value will provide insight regarding differences among pasture treatments in daily animal response.
Diet Collection. Several indirect and direct methods have been used to obtain an estimate of the animals diet. An indirect method that is practiced is hand plucking. In this case a person walks along side of a grazing animal and tries to hand pluck from the pasture the same material consumed by the animal.
A direct method that has been used is to remove the rumen contents from a rumen-fistulated steer, allow the animal to graze, then collect a sample of the ingested pasture. Another direct method, more frequently used, employs an esophageally fistulated animal that is assumed to be representative of the animals used in the formal grazing trial. The animal is permitted to graze and the ingested forage collected upon swallowing. Direct collection is complicated by the presence of saliva. Some investigators collect the ingested forage (masticate) in a net bag allowing the saliva to drain through the masticate and out of the sample onto the ground. Other investigators retain the saliva with the masticate. The collection of the masticate becomes an issue, however, because saliva is a very strong extracting media. Consequently, soluble constituents of the masticate can be readily lost from the collected diet if saliva is not retained. Following collection the residue masticate left after the saliva is drained or the total masticate (forage + saliva) constitutes an estimate of the animal’s diet and must be dried for subsequent analyses.
Masticate Handling. In the case of hand plucking as a means to obtain a sample of the animals diet, sample handling is straight forward. Each sample is best preserved by quick freezing as soon after sampling as possible followed by freeze drying and then holding in a freezer until analyzed.
When direct sampling is used the masticate sample is best preserved by avoiding oven drying, but rather freezing, followed by freeze drying and then holding in a freezer until analyzed. The methodology used in the collection of the masticate can result in two broadly different diets from the same pasture canopy. When the saliva is permitted to drain from the masticate, it will remove part of the soluble dry matter. This will likely vary among animals and among pasture treatments, thus can not be corrected for. When the saliva is retained with the masticate and freeze dried, some minute quantities of nutrients have been added by the saliva and retained in the masticate. This small degree of contamination will generally not be an issue and, if necessary, can be adjusted out by analyzing the saliva.
Particle Size. Although the ruminant meets its daily dry matter intake demand one bite at a time, the dry matter consumed can be viewed as being processed through the digestive system one particle at a time. Consequently, rate and extent of particle reduction is important relative to the animal response obtained and is both plant species and animal species dependent. Because particle size reduction (about 30 to 35%) begins with ingestive behavior, i.e., ingestive mastication, differences in diet particle sizes among treatments being compared may help explain differences in the resulting animal response. This is an aspect of the diet that cannot be examined when hand plucking is used.
Masticate particle size is generally determined through either wet sieving or dry sieving. If wet sieving is to be used a subsample of the freshly collected masticate needs to be obtained prior to freeze drying and then sieved using water and vibration to distribute the particles among the various sieve sizes (generally ranging from 0.125 to 5.6 mm).
In dry sieving, a subsample can be obtained from the fresh masticate, frozen, and then freeze dried, or a subsample can be obtained (carefully) from the freeze-dried masticate and separated into particle sizes through vibration only. One important advantage of dry sieving is that the nutritive value of each particle size can be reasonably determined. In wet sieving, the soluble nutrients may be differentially leached from the dry matter of the various particle sizes. Leaching may differ among parts of the same plant or among the same plant part of different forage species.
The various particle sizes obtained can be examined to determine mean and median particle size of the masticate and the data can be presented either by sieve size or several sieve sizes combined to give several particle size classes, i.e., large (>1.7 mm), medium (<1.7 mm > 0.5 mm), and small (> 0.5 mm) with associated nutritive value estimates. The presentation of the data will generally be dictated by how it is to be used.
Grazing Time
A ruminant’s day is spent in searching out, consuming, and ruminating its daily dry matter intake. Time is also spent resting, grooming, socializing, and in the setting of grazing trials, watching scientists trying to measure what residual is present and guessing at what the animals had already eaten. The time devoted to grazing is especially important because only dry matter that is ingested can influence the animal daily response. Ruminant species generally have periods of active grazing and rumination. In the case of cattle, they generally have a period of grazing after sunrise, a major period from mid-afternoon until dark and another grazing period about midnight. Under ideal conditions cattle may ingest their daily dry matter demand in 5 to 6 h but as pasture conditions depart from ideal (limited herbage mass, excessive pasture maturity, abundance of stems, or dead tissue etc.) the daily grazing time may be extended to 10 to 12 h. Extended and more frequent periods of grazing can compensate, up to a point, for less than ideal conditions. Consequently, grazing-time information among forage treatments being evaluated may provide valuable insight into subsequent animal responses.
Grazing time has been determined in various ways ranging from individual observers with hand-held watches, to motion sensors attached to the animal in such a manner that a pendulum inscribes a pattern on a recording chart during grazing (24 h d -1 for 4 to 5 d), to electron recorders that monitor the animals chewing behavior recording both grazing time and ruminating time (24 h d -1 for 4 to 5 d). The electronic versions are attractive because of their utility in down loading to a PC which greatly expedites data reduction and analyses.
Dry Matter Intake and Digesta Kinetics
Dry Matter Intake. Dry matter intake (DMI) and the digestibility of the dry matter consumed form the basis for nutritional studies in animal production systems. Generally DMI is considered to be more variable both among animals and among pasture treatments than is the associated digestibility of the diet selected. An accurate estimate of the daily DMI of the grazing animal would be extremely valuable in explaining differences in animal responses among pasture treatments. Even a method that yields precise estimates of DMI would be valuable in assessing relative differences among pasture treatments.
The methods used in measuring DMI of the grazing animal are categorized as either direct or indirect. The direct method generally consists of a ‘difference’ measurement. A pasture measurement would consist of determining the herbage mass before and after grazing. The difference is what was consumed. This measurement is best used in an intensive grazing method which involves a group of animals so differences are of sufficient magnitude to overcome inherent variation. An animal measurement would consist of an animal weight before and after grazing. The difference is what was consumed. This method is limited to a short-term intake estimate because the unit of weight, the animal, is a leaky system and losses that occur during grazing and weighing are difficult or impossible to adjust for. Another direct, short-term estimate of DMI has been attempted using rumen-fistulated animals. The rumen contents are first removed and the animal permitted to graze for a specific time. The resulting rumen contents are then removed and weighed and dry matter determined.
Generally, one of the indirect methods has been employed to estimate DMI in grazing trials. These consist of either a biological approach using the animal’s ingestive behavior or a physical approach using marker technology. Ingestive behavior measurements consist of estimating a short-term intake rate (g h -1) by multiplying bite rate by bite weight and scaling by daily grazing time to provide an estimate of daily DMI.
The more frequent and widely accepted approach involves the use of marker techniques. Markers are classified as either internal or external. The concept is to know the concentration (internal) or quantity (external) of an inert (not digested or if slightly digested that the degree is known) marker that moves through the digestive tract with the indigestible dry matter and can be measured in feces. An internal marker would be a constituent naturally present in the plant tissue that can be analyzed before ingested and in the feces. Examples of internal markers are lignin, indigestible fiber, silica, acid-insoluble ash and more recently, odd-numbered alkanes. An external marker is an inert substance fed in known quantity to an animal and recovered in the feces. Daily DMI can be estimated as follows:
DMI = Fecal DM output/(1.0 – DM digestibility)
This relationship shows that an estimate is needed of the animal’s daily fecal dry matter output and of the digestibility of the animal’s diet. The internal marker can be readily used to estimate digestibility of the forage consumed (marker ratio technique). The calculation is to divide the concentration of the marker in the forage by the concentration in the feces. This provides an estimate of the indigestibility of the forage dry matter. Another estimate can be obtained from an in vitro bioassay of the masticate, as discussed above. Although the bioassay of the animal diet is the easiest to obtain and frequently used, one would assume that an internal marker, which is a part of the natural composition of the plant tissue, would provide the best estimate.
Fecal dry matter output can be determined directly (fecal collection bags), but generally is determined using marker techniques. In this case an external marker is used. A known quantity of the marker is dosed daily, usually chromic oxide (chromium sesquioxide, Cr2O3) to establish a steady state of the marker. Fecal samples are then collected, generally two times a day, and analyzed for chromium concentration. More recently even-numbered alkanes have been used in place of chromic oxide. Fecal DM output is then estimated by dividing the daily marker dosed by the mean fecal concentrations of the marker.
Daily DMI (kg d -1) can now be estimated from the above equation using the diet dry matter digestibility and daily fecal dry matter output. It should be noted that errors in estimating either digestibility or fecal output can appreciably alter the DMI calculation. Further, care must be exercised in addressing all the requirements and assumptions of marker techniques.
Digesta Kinetics. Additional information that addresses something about how the animal handles the ingested dry matter can be obtained from the above technique. This requires that the external marker be given only once as a pulse dose, as opposed to daily as noted above. The material for the pulse dose is prepared first by collecting the animal’s diet, extracting using neutral detergent reagent to remove the soluble constituent, mordant (boil) the resulting fiber with chromium, then washing and drying the mordanted fiber.
This material is analyzed for chromium concentration, and a known quantity of chromium is stuffed into gelatin capsules and administered to the animal via balling gun. At initial dosing a fecal sample is obtained immediately and thereafter at 4-to 6-h increments until after the chromium concentration excreted in the feces has peaked. During the decay phase, sample frequency can be reduced but sampling generally continues through day 6 as chromium concentration in the feces returns to base-line. The chromium concentration is determined on each sample for each animal. The excretion curve is then fitted with a nonlinear equation to describe the relationship between marker concentration and time after dosing. The equation can be solved to obtain not only fecal dry matter output but also kinetic information as GI tract fill, mean retention time and passage rate.
Application of Specific Measurements
From the specific measurements described above information from each pasture is now available on the animal’s diet, its daily DMI and on aspects of digestive physiology. Differences in daily animal response among pasture treatments can now be examined to determine if they were related to the diet, DMI or to digestive physiology. If the diet appears limiting, then pasture morphology and associated plant-part characteristics can be examined. If daily DMI appear to be limiting, then measurements of grazing time, ingestive behavior and digesta kinetics can be examined. The weak link(s) in the pasture utilization process should be revealed. Grazing management strategies can now be adjusted to possibly circumvent the component to achieve a specific animal response. Further, this information will prove valuable in forage-genomics and-breeding programs where favorable pasture plant attributes can be emphasized and unfavorable ones de-emphasized.
In addition, with knowledge of the digestibility and crude protein concentrations of the animal’s diet and with an estimate of daily DMI one can now begin to access the nutrient requirement standards for ruminants, for example the “Nutrient Requirements of Beef Cattle” put out by the National Research Council. Assuming that the traditional in vitro dry matter disappearance value equates to total digestible nutrients (TDN) and using the relationship of 1.0 kg TDN = 4.4 M cal of digestible energy, the daily animal response can be determined. Information of this type from various forage species and management strategies should permit an estimate of daily animal performance that the producer might expect from a specific pasture-management system. Adjustments will be required in cases where daily DMI may be appreciably altered.
A component of the grazing system that can limit the practical application of these intensive measurements, however, is inadequate knowledge about short-term pasture daily growth rate and associated nutritive value during the growing season. These become critically important in developing intensively managed, e.g., daily allowance, grazing systems. Knowing the animal’s potential DMI and diet digestibility is not adequate to determine daily land allocation and associated stocking density and paddock number that must be anticipated in a production system. Stocking density and paddock number can interact to alter daily DMI. Extremely little agronomic data are published that present the daily regrowth rate and associated nutritive value during the spring, summer, fall, and winter growing season for any of our pasture species. Generally individual harvest yields are totaled for the season and the annual yields published along with associated mean annual nutritive value estimates weighted for individual harvests. Although these data are useful for making production comparisons, they are mainly useless in developing intensive grazing systems. The development of intensive grazing systems requires an estimate of the daily regrowth rate of the pasture to reasonably estimate paddock number and paddock size. Further, associated estimates of pasture nutritive value are needed, relative to the animal response desired by the producer, to assure adequate quantities of daily nutrient intake.
Summary
Formal grazing trials provide pasture and animal output data for a specific set of pasture treatments generated under reasonably controlled conditions. This base information is valuable in understanding what magnitude of pasture and animal output one might expect from each pasture treatment in an animal production system. The reason why these levels of performance occur may not be of much importance and, therefore, detailed measurements, other than herbage mass, botanical composition, and nutritive value of the diet are not warranted or even justified. If, however, there is a need to understand the more basic aspects of the plant-animal interface and to address why pasture and animal output responses differ among pasture treatments, grazing management practices, or methods of grazing, then intensive measurements are needed and can be readily justified.
Although a number of intensive pasture-animal measurements have been briefly discussed in this paper, no attempt has been made to assess the merit of one method over another in arriving at specific output data. This resides with the individual scientist, but clearly warrants considerable thought before a specific method is selected for use in experimentation. When alternative methods exist for the measurement of a specific plant or animal response, most have advantages and disadvantages that must be carefully considered while some are clearly superior to others.
The step into the arena of conducting intensive plant and animal measurements to address why different animal responses occur among pasture treatments takes a commitment of high labor input and analytical capacity to handle an extremely large number of plant and animal samples. If the planning process raises serious concern about adequate funding to make the desired measurements, the likelihood is that one should not become involved. Short-cuts may make the total effort of little value and render the data meaningless. When the need exists to justify intensive measurements and the project is adequately supported, then rewards are extremely beneficial. First is the benefit from understanding the dynamics of the plant-animal interface so one can begin to explain why results have occurred as they did. Second is the knowledge to identify plant and animal traits or characteristics that can be altered to positively change the outcome. Finally, is the wonderment of the total process after seeing how each of the biological systems operate not only in their own right but also as they respond to the influence of the other.
Suggested References
Pasture Canopy Attributes
Laca, E.A., and G. Lemaire. 2000. Measuring sward structure. p. 103-121. In L’t Mannetje and R.M. Jones (ed.) Field and Laboratory Methods and Grassland and Animal Production Research. CAB Int. Publishing, New York, NY.
Sollenberger, L.E., and J.C. Burns. 2001. Canopy characteristics, ingestive behaviour, and herbage intake in cultivated tropical grasslands. p. 321-327. In J.A. Gomide et al. (ed.)Proc. Int. Grassl. Cong., 19th, Piracicaba, Brazil, 10-21 Feb. 2001. Brazilian Society of Animal Husbandry, Piracicaba, Brazil.
Whalley, R.D.B., and M.B. Hardy. 2000. Measuring botanical composition of grasslands. p. 67-102. In L’t Mannetje and R.M. Jones (ed.) Field and Laboratory Methods and Grassland and Animal Production Research. CAB Intl. Publishing, New York, NY.
Sample Handling
Burns, J.C., C.H. Nolter, and C.L. Rhykerd. 1964. Influence of method of drying on soluble carbohydrates of Alfalfa. Agron. J. 56:364-365.
Fisher, D.S., and J.C. Burns. 1987. Quality analysis of summer annual forages. I. Sample preparation methods and chemical characterization of forages types and varieties. Agron. J. 79:236-242.
Diet Characterization
Pond, K.R., W.C. Ellis, and D.E. Akin. 1984. Ingestive mastication and fragmentation of forages. J. Anim. Sci. 58:1567-1574.
Pond, K.R., J.-M. Luginbuhl, J.C. Burns, and D.S. Fisher. 1990. Mastication of lignocellulose during ingestion and rumination. p. 23-32. In D.E. Akin et al. (ed.) Microbial and Plant Opportunities to Improve Lignocellulose Utilization by Ruminants. Elsevier Sci. Publ. Co., New York, NY.
Ingestive Behavior
Cosgrove, G.P. 1997. Grazing behaviour and forage intake. p. 59-80. In J.A. Gomide (ed.) Int. Symp. on Animal Production Under Grazing. 4-6 Nov. 1997, Vicosa, Brazil. Dep. de Zootec., Univ. Fed. de Vicosa, Vicosa, M.B. Brazil.
Hodgson, J., D.A. Clark, and R.J. Mitchell. 1994. Foraging behaviour in grazing animals and its impact on plant communities. p. 796-827. In G.C. Fahey, Jr. (ed.) Forage Quality, Evaluation and Utilization. ASA, CSSA, SSA, Madison, WI.
Ungar, E.D. 1998. Ingestive behavior. p. 185-218. In J. Hodgson and A.W. Illius (ed.) The Ecology and Management of Grazing Systems. CAB Int., New York.
Grazing Time
Hodgson, J. 1982. Ingestive behavior. p. 113-138. In J.D. Leaver (ed.) Herbage Intake Handbook. The British Grassl. Soc. Hurley, UK.
Marker Techniques and Application in Measuring Dry Matter Intake
Ellis, W.C., J.H. Matis, T.M. Hill, and M.R. Murphy. 1994. p. 682-756. In G.C. Fahey, Jr. (ed.) Forage Quality, Evaluation and Utilization. ASA, CSSA, SSA, Madison, WI.
Ellis, W.C., D.P. Poppi, J.H. Matis, H.Lippke, T.M. Hill, and F.M. Rouquette, Jr. 1999. Dietary-digestive-metabolic interactions determining the nutritive potential of ruminant diets. p. 423-481. In H.-J. G. Jung, and G.C. Fahey, Jr. (ed.) Nutritional Ecology of Herbivores: Proc. Vth Int. Symp. Nutr. Herbiv. Am. Soc. Anim. Sci., Savor, IL.
Illius, A.W., and M.S. Allen. 1994. Assessing forage quality using integrated models of intake and digestion by ruminants. p. 869-890. In G.C. Fahey Jr (ed.) Forage Quality, Evaluation and Utilization. ASA,SSA, SSA, Madison, WI.
Moore, J.E., and L.E. Sollenberger. 1997. Techniques to predict pasture intake. p. 81-96. In J.A. Gomide (ed.) Int. Symp. on Animal Production Under Grazing, 4-6 Nov. 1997. Vicosa, Brazil. Dep. de Zootec. Univ. Fed. de Vicosa, M.G., Brazil.
Measurements Applied in Experimentation
Burns, J.C., D.S. Fisher, K.R. Pond, and D.H. Timothy. 1992. Diet characteristics, digesta kinetics, and dry matter intake of steers grazing eastern Gamagrass. J. Anim. Sci. 70:1251-1261.
Burns, J.C., K.R. Pond, D.S. Fisher, and J.-M. Luginbuhl. 1997. Changes in forage quality, ingestive mastication, and digesta kinetics resulting from switchgrass maturity. J. Anim. Sci. 75:1368-1379.