Food Sorting by Collared Lemmings (Dicrostonyx groenlandicus) and Prairie Voles (Microtus ochrogaster): A Cautionary Note for Digestibility Studies

Comp. Biochem. Physiol. Vol. 116A, No. 2, pp. 119–124, 1997 Copyright  1996 Elsevier Science Inc.

ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00161-2

Food Sorting by Collared Lemmings (Dicrostonyx groenlandicus) and Prairie Voles (Microtus ochrogaster): A Cautionary Note for Digestibility Studies Jan Peterson and Bruce A. Wunder Department of Biology, Colorado State University, Fort Collins, CO 80523, U.S.A. ABSTRACT. Small herbivores are often confronted with poor quality diets; in response to this situation, they may sort their food to increase diet quality. Neutral detergent fiber (NDF) levels were measured in three pelleted diets fed to collared lemmings (Dicrostonyx groenlandicus) and prairie voles (Microtus ochrogaster) and compared to levels in orts collected from the same diets and animals. Lemmings sorted and discarded the fibrous parts of food pellets on all three diets (17.3% to 25.9% higher NDF in orts), whereas voles sorted out fibrous particles on only one diet. Calculations of NDF digestibility were significantly reduced by particle sorting (up to 16.9% decrease in digestibility). In reviewing the literature, we found that in more than half of studies examined, orts may not have been analyzed for these differences. Researchers must carefully consider such effects, especially when conducting digestibility studies with herbivores on pelleted diets. Copyright  1996 Elsevier Science Inc. comp biochem physiol 116A;2:119–124, 1997 KEY WORDS. Orts, digestibility, fiber, voles, lemmings, selectivity, herbivores, digestion trial

INTRODUCTION Total balance digestion trials with captive animals are relatively straightforward and easy to carry out (27); however, several problems with this method have arisen. First, there is an underlying assumption that ‘‘the basic physiological processes of digestion are comparable between free-ranging and captive states’’ (27). This assumption is not easily testable because of the difficulty of conducting digestion trials on unrestrained animals. Secondly, it is often difficult to separate urine and feces in such a trial, and should feces become contaminated with urine, digestibility estimates of nitrogen and minerals may be reduced. This second problem may be of relatively minor importance, as Grodzinski and Wunder (12) estimated the daily loss of nitrogen in urine of small mammals to be about 2–3%. A third problem with total balance digestion trials can result from the ability of animals to selectively choose one forage over another from a mixture of foods. Domestic livestock, such as cows or sheep, are known to select preferred forage out of a mixture of different feeds, resulting in the consumption of a diet other than that which was intended. The refused portion of food is termed orts (36). Van Soest (36) suggests that one way to control or eliminate this potential problem with orts is to grind, chop, wafer or pellet Address reprint requests to: J. Peterson at: Department of Biology, Colorado State University, Fort Collins, CO 80523, U.S.A. Tel. (303) 491-2354 Fax (303) 491-0649. Received 20 December 1994; accepted 17 April 1996.

the diet. One may also limit the amount of food offered so that animals are certain to consume all of it, but then, of course, the condition of ad libitum feeding is jeopardized. In our experiment designed to examine the effects of different levels of dietary nitrogen and neutral detergent fiber (NDF) on digestibility and coprophagy (fecal reingestion) in prairie voles (Microtus ochrogaster) and collared lemmings (Dicrostonyx groenlandicus) (Peterson and Wunder, unpublished data), we had all of our diets ground and pelleted commercially to avoid the problem of sorting. However, an obvious dissimilarity in the physical appearance of food pellets and orts led us to hypothesize that these small animals were still able to sort food particles and discard the more fibrous portion of their pelleted food. Unlike livestock, voles and lemmings handle food pellets with their forepaws while feeding. To test the hypothesis that animals were sorting the diets, we compared dietary levels of NDF to levels of NDF in the orts. Additionally, we reviewed recent literature in order to ascertain whether or not it is standard practice in digestion trials to test for nutritional differences between orts and food, and to account for those differences in calculations of digestibility coefficients.

MATERIALS AND METHODS During a 5-day digestibility trial, groups of six animals of each species were randomly assigned to one of three treat-

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TABLE 1. Nutritional composition and ingredients in pel-

leted diets fed to collared lemmings (Dicrostonyx groenlandicus) and prairie voles (Microtus ochrogaster) Nutritional Composition Energy (KJ/g) Nitrogen (%) NDF (cell walls) (%) Dry matter (%) Ingredients Dehydrated alfalfa Wheat middlings Soybean hulls Cane molasses Corn Corn cobs Soybean meal Sorghum grain Rice mill feed Wheat bran Dicalcium phosphate Animal fat Vitamin mix

Diet #3 a Diet #1 Diet #2 Lemmings 18.1 2.3 42.2 94.0

Voles

17.6 2.2 38.2 92.0

17.1 1.1 47.1 92.0

17.9 0.9 57.9 92.0

Diet #1 b

Diet #2 %

Diet #3 %

27.5

8.35

5.0 3.9

5.0

X X X X X

46.3 X 25.9 20.4 17.3 X X X

32.1 7.4 Tr.

Tr.

Tr.

a

Diet #3 was identically formulated in two batches, but because of seasonal differences in ingredients, composition varied. b Diet formulation is patented by Purina Mills; thus, percentages are not included for ingredients.

ment diets and placed in metal metabolism cages (18 cm 3 20 cm 3 23 cm). Food and water were provided ad libitum. Feces and orts were collected daily and dried to a constant mass at 60°C to avoid formation of Maillard products (36). We measured NDF levels in food, orts, and feces using a modified Goering and Van Soest method (14). Because we were able to purchase only one commercial diet that contained low enough levels of nitrogen and high enough levels of fiber for our experiment (diet #2–Purina’s High Fiber Rabbit Chow), we formulated the other two diets ourselves (Table 1). Diet #2 had a pellet size of 0.39 cm diameter 3 0.64 cm length. Pellets for the other diets measured 0.95 cm diameter 3 1.91 cm length. Particle size of pellets was not measured. All diets were manufactured by the Test Diet Division of Purina Mills. Diet #3 was ordered in two separate batches and, although the formulation of ingredients was identical, the NDF levels were quite different because of seasonal variation in ingredients (Table 1). Thus, for diet #3, voles were given food that was 10% higher in NDF content than that of lemmings. Adult voles and lemmings (mean body mass 5 45.6 g 6 3.5 (SE) and 48.2 g 6 3.1, respectively), obtained from breeding colonies at Colorado State University, were acclimated to their respective diets for 18 days before the digestibility trials began. During the acclimation period and digestibility trials, lemmings were held at 16°C on a 24L

photoperiod, and voles were held at 23°C on a 14L:10D photoperiod. Although photoperiods were different, they represented ‘‘long days’’ for both species, and are the conditions under which they are normally held to simulate summer photoperiod for breeding purposes. Photoperiods that are much shorter than 24L generally cause lemmings to molt into winter pelage and gain body mass (26). Additionally, although lemmings were maintained at a lower ambient temperature than voles, Reynolds and Lavigne (26) reported the zone of thermoneutrality for Dicrostonyx to be between 15° and 20°C, about 10° lower than that for prairie voles (38). Digestibility of a diet is expressed as the proportion of matter or energy that does not appear in the feces. Digestibility is usually reported as apparent digestibility or apparent digestive efficiency, and can be calculated for any component portion of the diet, provided that component can be measured in the food, feces and orts. Apparent digestibilities are commonly calculated using the following equation: Digestibility (%) 5 Amount component consumed–Amount component excreted Amount component consumed

This equation is straightforward and digestibility coefficients are easily determined for animals on restricted intake diets in which no portion of the food is refused. However, for animals fed ad libitum, refused feed must be taken into account in the measurement of total consumption. In addition, if animals are able to practice selection and the orts differ in composition from the original diet, then this difference must also be factored into the equation. The above equation for apparent digestibility, revised to take into account the effects of nutrient selection, now appears as: Digestibility (%) 5 (Amount offered–Amount refused)–Amount in feces (Amount offered–Amount refused)

We compared differences in NDF concentration in orts and food, and differences between digestibilities with and without the correction factor using a paired Student’s t-test. All statistics were analyzed with a personal computer version of SAS (31). RESULTS Collared lemmings sorted and discarded more of the fibrous components of their food than did voles (Table 2). NDF concentration in orts was significantly higher (17.3% to 25.9%) than that in food for all lemming diet treatment groups, whereas, it was significantly higher (15.4%) for only one vole diet treatment group. Particle sorting had statistically significant effects on calculations of NDF digestibility that paralleled those for sorting (Table 3). Differences as great as 16.9% occurred, al-

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121

TABLE 2. NDF concentration (6SE in parentheses) of orts in comparison to that of food for collared lemmings (Dicrostonyx

groenlandicus) and prairie voles (Microtus ochrogaster) Species Treatment

n

Collared Lemmings Diet #1

6

Diet #2

6

Diet #3

6

Prairie Voles Diet #1

5

Diet #2

6

Diet #3

4

% NDF Food

% NDF Orts

Increase (%)

P-value

42.2 (0.66) 38.2 (1.19) 47.1 (0.41)

49.5 (1.53) 45.7 (2.49) 59.3 (1.37)

17.3

0.008

19.6

0.040

25.9

0.015

42.2 (0.66) 38.2 (1.19) 57.9 (0.71)

45.7 (0.86) 44.1 (0.53) 57.5 (0.90)

8.3

0.197

15.4

0.009

0

0.501

though much smaller differences significantly affected our calculations. In our review of the literature, we examined 30 recent studies reporting digestibility values (Table 4). Of these 30 articles, five authors reported that they had collected, dried, weighed and analyzed orts for nutrient composition. Although not explicitly stated, we assume that they then used their analyses to correct their estimates of digestibility. In one study, researchers fed animals at a level of restricted intake so as to insure that there would be no refused food remaining. In five of the papers, orts were collected, dried and weighed, and then used in calculating intake. Four investigators ground their diets to preclude selection by their experimental subjects. In 12 of these studies, however, no mention was made of orts or how the authors handled refused food. In one of the articles, the investigators collected and analyzed orts and then used the results of the analyses to estimate levels of nutrients in the diets that were fed. Levels of nutrients in the orts may have been vastly different from those in the diet, thus, this methodology appears to be inappropriate, at the least. Finally, in one paper authors

concluded that there was no selection, and in another, researchers conducted what appeared to be a partial analysis of orts. DISCUSSION There were no significant differences in body mass between voles and lemmings in this study; yet, lemmings sorted their food to a greater extent than voles did. Our results may be viewed as complementary to those of Justice and Smith (20), who reported a greater degree of sorting by smallerbodied woodrat (Neotoma spp.) species than by larger-bodied species. Those authors concluded that small animals likely sorted food to a greater extent because they are forced to moderate fiber intake. However, our use of two different species of approximately equal body mass revealed that although lemmings reduced their fiber intake this way, voles did not do so consistently. Perhaps if we had used only lemmings (or only voles) of large and small body mass, our results might agree more closely with the above authors (20) Our observed difference in sorting effort between the two

TABLE 3. Neutral detergent fiber (NDF) digestibility (6SE in parentheses) with and without correction for sorting of diets by

collared lemmings (Dicrostonyx groenlandicus) and prairie voles (Microtus ochrogaster) Species Treatment

n

NDF Digest. w/o correction

NDF Digest. w/ correction

Difference (%)

P-value

Lemmings Diet #1 Diet #2 Diet #3

6 6 6

35.4 (2.3) 42.9 (1.3) 35.7 (6.4)

30.4 (2.4) 36.4 (1.7) 18.8 (10.6)

5.0 6.5 16.9

0.000 0.011 0.055

Voles Diet #1 Diet #2 Diet #3

5 6 4

35.9 (1.9) 51.9 (3.3) 67.4 (3.6)

32.0 41.2 67.3

3.9 10.7 0.1

0.046 0.091 0.915

(2.8) (7.7) (3.0)

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TABLE 4. Comparison of orts analyses from total collection digestion trials

Digestibility

Diet Type

Intake

Species

DM, OM, F, FA

Ground mixture

Ad libitum

Dairy cows

DM

Ad libitum

DM, F DM, OM, P, F

Fresh forage & alfalfa pellets Ground pellets Twigs, rabbit chow Homogenized rabbit chow w/ additives Pellets & hay Semipurified pellets

DM, E, F

Ground alfalfa cubes

DM, E DM, N, F, NDS, E DM, OM, P, F

DM, E DM, P DM

OM, E, P, F, S DM, OM, N, CF, F, E DM, E OM, P, F, E, NFE DM, F, P DM, F DM, OM, P, F, NFE DM, E CHO, P DM, NDF, NDS DM, CHO DM, F, E DM, OM, E, N DM, OM DM, ME DM, NDF, E DM, P N, F DM, P

Orts

Source 32

Elk

Collected & analyzed Not mentioned

Premeasured Ad libitum Ad libitum

Voles Snowshoe hares Water voles

No selection Not mentioned No selection

6 33 37

Premeasured Ad libitum

Dried & weighed Collected & analyzed

23 29

Ad libitum

Pocket gophers Brushtail possums, ringtail possums, rabbits Woodrats

20

Fresh saltbush leaves

Premeasured

Fat sand rats

Pelleted mixture Cubed lucerne mixture Pelleted lucerne mixture Ground mixture Whole vegetables Pelleted alfalfa or pelleted mixture

Ad libitum Ad libitum

Prairie voles Rabbits, guinea pigs, rats, and hamsters Rabbits

Collected & analyzed Analyzed ‘‘leafscrapings,’’ not refused leaves Dried & weighed Not mentioned Not mentioned

11

Ground hay with supplements Ground forage w/ supplements Ground mixture Whole crickets Fresh forage Cut grass Cut leaves Pelleted grass and clover Sponges Oat hay & pelleted supplements Pasture vegetation Rabbit chow-plain and diluted w/cellulose Rabbit chow w/phenolic extracts Alfalfa-grain pellets and dog chow Fresh forage

Ad libitum

19

7 15 30

Restricted Ad libitum Ad libitum or maintenance Ad libitum and restricted Ad libitum

Rats and pigs Naked mole-rats Horses, ponies, rabbits and guinea pigs

No selection Not mentioned Not mentioned

9 5 34

Swine and rats

Not mentioned

22

Meadow voles

21

Ad Ad Ad Ad Ad Ad

Voles and mice Canyon lizards Butterflies Locusts Grasshoppers European hares

Collected & analyzed No selection Not mentioned Not mentioned Weighed Not mentioned Analyzed orts to determine diet composition Not mentioned Dried and weighed No selection Collected and analyzed Not mentioned

18

No orts remaining

28

Dried and weighed

16

libitum libitum libitum libitum libitum libitum

Ad libitum Ad libitum

Green turtles Sheep

Ad libitum Ad libitum

Sheep Collared lemmings

Ad libitum

European and mountain hares Mule deer, sheep and black bears Black-tailed deer

Restricted Ad libitum

8 3 2 17 10 24 4 35 1 25

DM 5 Dry Matter, OM 5 Organic Matter, F 5 Fiber, E 5 Energy, P 5 Protein, N 5 Nitrogen, S 5 Starch, FA 5 Fatty Acids, NFE 5 Nitrogen Free Extract, CHO 5 Soluble Carbohydrates, NDS 5 Neutral Detergent Solubles, CF 5 Crude Fat.

Food Sorting by Lemmings and Voles

similar-sized species suggests either that they have disparate strategies for dealing with high fiber food, or possibly that voles are less able to detect fiber in their food. Food sorting by voles and lemmings had significant effects on calculations of digestibility in our study. Although investigators trained specifically in animal nutrition are aware of the potential problems caused by not correcting for differences between orts and food, researchers in other areas often fail to take these differences into account. In our literature survey, we found that out of 30 recent studies reporting digestibility values, 12 made absolutely no mention of orts or refused food, even though animals were fed at an ad libitum level of intake. In five of the studies, authors stated that they did, indeed, collect, dry and weigh orts to calculate dry mass intake prior to calculation of digestibility or digestibility of components, but nowhere was it evident that there were any analyses to determine if orts were different from food in nutritional content. Additionally, none of these five studies reported using a correction factor of any kind for that presumed difference, when calculating digestibility coefficients. It is possible that all of these authors actually did conduct chemical analyses on the orts in their experiments, and then incorporated the differences into their calculations. Perhaps they just failed to report how they did this in the ‘‘Methods’’ section of their papers. If this is indeed the case, then investigators should be more conscientious in their reporting of experimental procedure. In our experiment, the sorting out of the more fibrous components in the diets resulted in our initially overestimating NDF digestibility. After making corrections for selection, our NDF digestibility coefficients decreased by as much as 16.9%. Although we did not conduct analyses on protein concentration differences in orts and food, we suspect that protein digestibilities would have been significantly higher because the orts most likely would have been lower in protein concentration. Dry matter digestibility also has the potential to be affected by selection. Depending on how the animal sorts its feed, dry matter digestibility could increase or decrease with the correction factor. In our review of the literature, we found that several researchers avoided the orts dilemma by feeding a ground diet that effectively prevented the animals from being selective, and another group avoided the problem by feeding at a restricted level of intake. These two options usually work well to alleviate the orts problem, but restricting intake level doesn’t always guarantee an absence of orts. When the amount of orts collected at the end of a trial is relatively large in comparison to what was offered and consumed, the investigator should check for a difference in composition between orts and the feed offered, especially if the diet was of heterogeneous composition. Grinding feed may present other problems when estimating digestibility of different feeds. The investigator must first be certain that particle size is actually small enough to prevent selection. Although smaller particle size may effec-

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tively preclude sorting in larger herbivores, such as ruminants, reduction in particle size has several other important consequences. Particle size reduction generally promotes a higher voluntary intake, at least in ruminants, which increases animal efficiency, while at the same time increasing rate of passage, which results in a depression of fiber digestibility and higher ratios of propionic acid to acetic acid in the rumen (36). In some instances, the depression in fiber digestibility due to the increased rate of passage may even offset the increased efficiency. Van Soest (36) described other potential problems associated with grinding and pelleting diets that are more difficult to control. He stated that the associated heat of friction often denatures proteins and gelatinizes starches. These effects can become important when feeding high-quality forages because they alter intake and digestibility. We urge investigators to exercise caution in analyzing, reporting and interpreting future digestibility studies, and not to assume that herbivores on a pelleted diet ingest exactly what they are fed. Our work was conducted with the approval of the Animal Care and Use Committee at Colorado State University in accordance with federal regulations governing humane treatment of research animals. We thank A. W. Alldredge, J. K. Detling, K. Castle and several anonymous reviewers for their helpful reviews of the manuscript. Financial support was provided in part by The American Museum of Natural History and The Program for Ecological Studies at Colorado State University.

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