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Methods in Primate Nutritional Ecology: A User's Guide

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2. amp Altmann S 1991 Resource base parity and reproductive condition affect females feeding time and nutrient intake within and between groups of baboon populations Oecologia 87 467 472 Nagy K A amp Milton K 1979 Energy metabolism and food consumption by wild howler monkeys Alouatta palliata Ecology 60 475 480 National Research Council 1998 Nutrient requirements of swine Washington DC National Academies Press National Research Council 2003 Nutrient requirements of nonhuman primates 2nd ed Washington DC National Academies Press g Springer 564 J M Rothman et al National Research Council 2007 Nutrient requirements of small ruminants Sheep goats cervids and New World camelids Washington DC National Academies Press Norconk M A amp Conklin N L 2004 Variation on frugivory The diet of Venezuelan white faced sakis International Journal of Primatology 25 1 26 Norconk M A Wright B W Conklin Brittain N L amp Vinyard C J 2009 Mechanical and nutritional properties of foods as factors in platyrrhine dietary adaptations In P A Garber A Estrada C Bicca Marques E Heymann amp K Strier Eds South American primates Testing new theories in the study of primate behavior ecology and conservation pp 279 319 New York Springer Oates J F 1978 Water plant and soil consumption by guereza monkeys Colobus guereza Relationship with minerals
3. Crowley B E Knott C D Blakely M D Larsen M D amp Dominy N J 2012 Non invasive method for quantifying nitrogen balance in free ranging primates International Journal of Primatology Waterman P G amp Mole S 1994 Analysis of phenolic plant metabolites Oxford Blackwell Watts D P 1984 Composition and variability of mountain gorilla diets in the central Virungas American Journal of Primatology 7 323 356 Worman C O amp Chapman C A 2005 Seasonal variation in the quality of tropical ripe fruit and the response of three frugivores Journal of Tropical Ecology 21 689 697 Wrangham R W amp Waterman P G 1981 Feeding behaviour of vervet monkeys on Acacia tortilis and Acacia xanthophloea with special reference to reproductive strategies and tannin production Journal of Animal Ecology 50 715 731 Wrangham R W Chapman C A amp Chapman L J 1994 Seed dispersal by forest chimpanzees in Uganda Journal of Tropical Ecology 10 355 368 Wrangham R W Conklin Brittain N L amp Hunt K D 1998 Dietary response of chimpanzees and cercopithecines to seasonal variation in fruit abundance I Antifeedants International Journal of Primatology 19 949 970 Zinner D 1999 Relationship between feeding time and food intake in hamadryas baboons Papio hamadryas and the value of feeding time as predictor of food intake Zoo Biology 18 495 505 AA Springer
4. 1217 Urquiza Haas T Serio Silva J C amp Hernandez Salazar L T 2008 Traditional nutritional analyses of figs overestimates intake of most nutrient fractions A study of Ficus perforata consumed by howler monkeys Alouatta palliata mexicana American Journal of Primatology 70 432 438 g Springer 566 J M Rothman et al van Schaik C P 1989 The ecology of social relationships amongst female primates In V Standen amp R A Foley Eds Comparative socioecology The behavioural ecology of humans and other mammals pp 195 218 Boston Blackwell Van Soest P 1963 Use of detergents in the analysis of fibrous feeds II A rapid method for the determination of fiber and lignin Journal of the Association of Official Agricultural Chemists 46 829 835 Van Soest P J 1994 Nutritional ecology of the ruminant Ithaca NY Cornell University Press Van Soest P J 1996 Allometry and ecology of feeding behavior and digestive capacity in herbivores A review Zoo Biology 15 455 479 Van Soest P J Robertson J B amp Lewis B A 1991 Methods for dietary fiber neutral detergent fiber and non starch polysaccharides in relation to animal nutrition Journal of Dairy Science 74 3583 3597 Vogel E R 2005 Rank differences in energy intake rates in white faced capuchin monkeys Cebus capucinus The effects of contest competition Behavioral Ecology and Sociobiology 58 333 344 Vogel E R
5. D amp Wrangham R W 2006 Energy intake by wild chimpanzees and orangutans Methodological considerations and a preliminary comparison In G Hohmann M M Robbins amp C Boesch Eds Feeding ecology in apes and other primates Ecological physical and behavioral aspects pp 445 571 Cambridge UK Cambridge University Press Cork S J amp Krockenberger A K 1991 Methods and pitfalls of extracting condensed tannins and other phenolics from plants Insights from investigations on Eucalyptus leaves Journal of Chemical Ecology 17 123 134 Curtis D J 2004 Diet and nutrition in wild mongoose lemurs Eulemur mongoz and their implications for the evolution of female dominance and small group size in lemurs American Journal of Physical Anthropology 124 234 247 Dammhahn M amp Kappeler P M 2008 Comparative feeding ecology of sympatric Microcebus berthae and M murinus International Journal of Primatology 29 1567 1589 Deblauwe I amp Janssens G P J 2008 New insights in insect prey choice by chimpanzees and gorillas in southeast Cameroon The role of nutritional value American Journal of Physical Anthropology 135 42 55 DeGabriel J L Wallis I R Moore B D amp Foley W J 2008 A simple integrative assay to quanity nutritional quality of browses for herbivores Oecologia 156 107 116 Deinum B amp Maassen A 1994 Effects of drying temperature on chemical composit
6. Researchers have used various ways to estimate the mass of food consumed the most popular are bites Oftedal 1992 Rode et al 2006 Watts 1984 and units Altmann 1998 Muruthi et al 1991 Rothman et al 2008b Vogel 2005 To estimate the number of bites the observer counts the number of times the subject takes a bite of food and estimates the mass of bite size Because bite sizes can be variable among individuals 1 e larger males may have bigger bites than females and juveniles Perry and Harstone Rose 2010 we recommend using unit counts instead of bites With unit counts the observer uses a predefined measurement a leaf fruit particular length of bark etc and notes each time the subject consumes the measured quantity When intake data are not available for every observation mean feeding rates can be used or a functional response curve can be used to estimate feeding rates Rode et al 2006 Shipley et al 1994 Spalinger and Hobbs 1992 Feeding rates differ not only among food items but also as primates deplete patches or become satiated or as a function of dominance thus counting the number of bites units over the duration of the feeding bout will provide more precise estimates Chivers 1998 Snaith and Chapman 2005 After consumption representative samples of the same unit are collected and weighed to the nearest 0 01 g For example if a primate eats a leaf a single leaf may be considered a unit Similarly a single fruit o
7. protein and any energetic returns from fiber National Research 2003 Most plant foods do not have appreciable quantities of fat aside from some fatty fruits and seeds such as palms Norconk and Conklin Brittain 2004 Norconk et al 2009 and fruits of Virola gt 30 Milton 2008 The best way to assess the fat contents in a sample is to estimate its fatty acid composition whereby fats are placed in a hydrophobic solvent purified esterified and then analyzed via gas chromatography Sukhija and Palmquist 1988 However few studies have analyzed the actual fatty acid composition of primate foods Chamberlain et al 1993 Reiner and Rothman 2011 probably because this analysis is time consuming and expensive Ether extract is a simple method that is commonly employed to estimate the crude fat in a sample Although ether extract gives a crude measure of fats and is appropriate for the measurement of triglycerides plants have nonfat components that are extracted by ether such as wax cutin galactose essential oils chlorophyll glycerol and other compounds that cannot be saponified and that are frequently indigestible Palmquist and Jenkins 2003 Forage leaves contained 5 3 fat as determined by ether extract but 57 of the ether extract was composed of non nutritive substances Palmquist and Jenkins 2003 Thus in the agricultural industry it is recommended that one is subtracted from the percentage of ether extract in forage leaves to a
8. while acid detergent fiber ADF is composed of cellulose and lignin Lignin is removed from cellulose and hemicellulose by treating the remaining fraction with 72 sulfuric acid The detergent analyses can be performed in a sequential fashion whereby NDF is analyzed first followed by ADF and lignin Alternatively if lignin or ADF is the only desired fraction then the NDF step can be omitted but the results will not necessarily be comparable with those for sequentially analyzed samples It should be noted that if ADF is completed directly and its value is higher than NDF of the same sample this is likely to be due to the confounding effects of tannins which are mostly removed during the NDF step High fat samples gt 10 should be preextracted in ether or acetone before NDF because lipids can interfere with the detergent All of the fiber fractions include minor components in their residues Neutral detergent residues include cutin portion of the waxy plant cuticle a minor portion of residual ash and hydrolyzable tannins that are not dissolved in the detergent solution Acid detergent residue includes cutin pectin total silica and tannin complexes Van Soest 1994 Lignin may contain a small amount of residual ash and cutin The potential impact of ash on fiber estimates should be considered high ash foods e g some exudates gt 25 ash Smith 2000 and nuts gt 40 Tsuji and Takatsuki 2008 may artificially inflate fiber estimates b
9. American spider monkey Ateles geoffroyi American Journal of Primatology 67 411 423 Robbins C T 1993 Wildlife feeding and nutrition San Diego CA Academic Press Robbins C T Hanley T A Hagerman A E Hjeljord O Baker D L Schwartz C C et al 1987a Role of tannins in defending plants against ruminants Reduction in protein availability Ecology 68 98 107 Robbins C T Mole S Hagerman A E amp Hanley T A 1987b Role of tannins in defending plants against ruminants Reduction in dry matter digestion Ecology 68 1606 1615 Rode K D Chapman C A Chapman L J amp McDowell L R 2003 Mineral resource availability and consumption by colobus in Kibale National Park Uganda International Journal of Primatology 24 541 573 Rode K D Chapman C A McDowell L R amp Stickler C 2006 Nutritional correlates of population density across habitats and logging intensities in redtail monkeys Cercopithecus ascanius Biotropica 38 625 634 Rothman J M Dierenfeld E S Molina D O Shaw A V Hintz H F amp Pell A N 2006a Nutritional chemistry of foods eaten by gorillas in Bwindi Impenetrable National Park Uganda American Journal of Primatology 68 675 691 AA Springer Methods in Primate Nutritional Ecology 565 Rothman J M Van Soest P J amp Pell A N 2006b Decaying wood is a sodium source for mountain gorillas Biology Letters 2 3
10. C H N H etc in a sample when it is irradiated with near infrared light These spectra are then statistically calibrated against reference values which are determined through traditional nutritional analysis to develop multivariate regression equations that can then be used to estimate nutritional values for samples similar to those in the calibration set With NIRS up to 150 plant samples per day can be analyzed for multiple nutrients stmultaneously after the spectrometer is calibrated with ca gt 100 samples similar to those that will be analyzed This offers a rigorous approach when sample sizes are numerous and many similar samples need to be analyzed Foley et al 1998 Rothman et al 2009a In a preliminary study NIRS was used to predict the N contents of bamboo eaten by lemurs Ortmann et al 2006 and in recent studies NIRS was used to investigate the nutritional ecology of spider monkeys Felton et al 2009b and mountain gorillas Rothman et al 2009a 2011 Conclusions Milton s 1979 1980 1981 1993 pioneering research on the differences in nutritional ecology of spider monkeys and howlers demonstrated that dietary adaptations had substantial implications for primate social behavior sociality and cognition However the field of primate nutritional ecology is still in its infancy Despite the theoretical importance of energy in primate socioecological models we know very little about primate energy intake Conklin Britta
11. Felton A M Felton A Lindenmayer D B amp Foley W J 2009a Nutritional goals of wild primates Functional Ecology 23 70 78 Felton A M Felton A Raubenheimer D Simpson S J Foley W J Wood J T et al 2009b Protein content of diets dictates the daily energy intake of a free ranging primate Behavioural Ecology 20 685 690 Felton A M Felton A Foley W J amp Lindenmayer D B 2010 The role of timber tree species in the nutritional ecology of spider monkeys in a certified logging concession Bolivia Forest Ecology and Management 259 1642 1649 Foley W J amp Moore B D 2005 Plant secondary metabolites and vertebrate herbivores from physiological regulation to ecosystem function Current Opinion in Plant Biology 8 430 435 Foley W J McIlwee A Lawler I Aragones L Woolnough A P amp Berding N 1998 Ecological applications of near infrared reflectance spectroscopy a tool for rapid cost effective prediction of the composition of plant and animal tissues and aspects of animal performance Oecologia 116 293 305 Food and Nutrition Board of the Institute of Medicine of the National Academy 2005 Dietary reference intakes for energy carbohydrate fiber fat fatty acids cholesterol protein and amino acids Washington DC National Academies Press Frey J C Rothman J M Pell A N Nizeyi J B Cranfield M R amp Angert E A 2006 Fecal bac
12. Matter 0 10 20 30 40 50 Sample Collection Number Fig 1 Intraspecific variability in the a crude protein and b neutral detergent fiber concentrations of 50 samples of Triumfetta tomentosa a staple food of Gorilla beringei We collected these samples in a variety of areas throughout the home range in June 2003 g Springer Methods in Primate Nutritional Ecology 547 variability implies that it will introduce considerable error when a single collection of a particular species is used to represent the nutritional parameters of multiple food items as subjects may discriminately select foods with specific nutrient qualities Thus to estimate nutrient intake we strongly advocate where possible collecting samples from the same plant from which the subject was feeding at the same time of day and within a few days of when it was consumed It is essential to collect the exact stage of food eaten because differences in e g fruit ripeness can have strong effects on nutritional composition For example ripe fruits eaten by chimpanzees Pan troglodytes and cercopithecines are higher in sugar and fat than unripe fruits Conklin Brittain et al 1998 Because accurate sampling requires numerous samples to be collected we suggest primatologists consider using near infrared reflectance spectroscopy to process a large number of samples effectively and reduce time and laboratory costs Rothman et al 2009a If time and finances are not ava
13. acid content of neotropical plant parts available to wild monkeys and bats Experentia 43 339 342 Milton K amp McBee R H 1983 Rates of fermentative digestion in the howler monkey Alouatta palliata Primates Ceboidea Comparative Biochemistry and Physiology Part A Molecular amp Integrative Physiology 74 29 31 Milton K Van Soest P J amp Robertson J B 1980 Digestive efficiences of wild howler monkeys Physiological Zoology 53 402 409 Mole S Butler L G amp Ianson G 1990 Defense against dietary tannin in herbivores A survey for proline rich salivary proteins in mammals Biochemistry Systematics and Ecology 18 287 293 Mould F L Kliem K E Morgan R amp Mauricio R M 2005 In vitro microbial inoculum A review of its function and properties Animal Feed Science and Technology 123 124 31 50 Mowry C B Decker B S amp Shure D J 1996 The role of phytochemistry in dietary choices of Tana River red colobus monkeys Procolobus badius rufomitratus International Journal of Primatology 17 63 84 Mueller Harvey I 2001 Analysis of hydrolysable tannins Animal Feed Science and Technology 91 3 20 Muetzel S amp Becker K 2006 Extractibility and biological activity of tannins from various tree leaves determined by chemical and biological assays as affected by drying procedure Animal Feed Science and Technology 125 139 149 Muruthi P Altmann J
14. amp Onderdonk D A 2002 Application of protein to fiber ratios to predict colobine abundance on different spatial scales International Journal of Primatology 23 283 310 Chapman C A Chapman L J Rode K D Hauck E M amp McDowell L R 2003 Variation in the nutritional value of primate foods Among trees time periods and areas International Journal of Primatology 24 317 333 Chapman C A Chapman L J Naughton Treves L Lawes M J amp McDowell L R 2004 Predicting folivorous primate abundance Validation of a nutritional model American Journal of Primatology 62 55 69 Chivers D J 1998 Measuring food intake in wild animals Primates Proceedings of the Nutrition Society 57 321 332 Conklin N L amp Wrangham R W 1994 The value of figs to a hind gut fermenting frugivore A nutritional analysis Biochemical Systematics and Ecology 22 137 151 Conklin Brittain N L Wrangham R W amp Hunt K D 1998 Dietary response of chimpanzees and cercopithecines to seasonal variation in fruit abundance II Macronutrients International Journal of Primatology 19 971 998 Conklin Brittain N L Dierenfeld E S Wrangham R W Norconk M amp Silver S C 1999 Chemical protein analysis A comparison of Kjeldahl crude protein and total ninhydrin protein from wild tropical vegetation Journal of Chemical Ecology 25 2601 2622 Conklin Brittain N L Knott C
15. conservation in Nigeria biodiversity hotspot pp 469 501 New York Springer Spalinger D E amp Hobbs N T 1992 Mechanisms of foraging in mammalian herbivores New models of functional response American Naturalist 140 325 348 Sterck E H M Watts D P amp van Schaik C P 1997 The evolution of female social relationships in nonhuman primates Behavioral Ecology and Sociobiology 41 291 309 Sterling E J Dierenfeld E S Ashbourne C J amp Feistner A T C 1994 Dietary intake food composition and nutrient intake in wild and captive populations of Daubentonia madagascariensis Folia Primatologica 62 115 124 Stewart J L Mould F amp Mueller Harvey I 2000 The effect of drying treatment on the fodder quality and tannin content of two provenances of Calliandra calothyrsus Meissner Journal of the Science of Food and Agriculture 80 1461 1468 Stork N E 1991 The composition of the arthropod fauna of Bornean lowland rain forest trees Journal of Tropical Ecology 7 161 180 Sukhija P S amp Palmquist D L 1988 Rapid method for determination of total fatty acid content and composition of feedstuffs and feces Journal of Agricultural and Food Chemistry 36 1202 1206 Tsuji Y amp Takatsuki S 2008 Effects of a typhoon on foraging behavior and foraging success of Macaca fuscata on Kinkazan Island Northern Japan International Journal of Primatology 29 1203
16. flavonoids tannins and salicylates Journal of Chemical Ecology 27 779 789 Karasov W H 1986 Energetics physiology and vertebrate ecology Trends in Ecology amp Evolution 1 101 104 Kay R F amp Davies A G 1994 Digestive physiology In A G Davies amp J F Oates Eds Colobine monkeys pp 229 250 Cambridge UK Cambridge University Press Kisidayova S Varadyova Z Pristas P Piknova M Nigutova K Petrzelkova K J Profousova I Schovancova K Kamler J amp Modry D 2009 Effects of high and low fiber diets on fecal fermentation and fecal microbial populations of captive chimpanzees American Journal of Primatology 71 548 557 Knott C D 1998 Changes in orangutan caloric intake energy balance and ketones in response to fluctuating fruit availability International Journal of Primatology 19 1061 1079 Koenig A 2000 Competitive regimes in forest dwelling Hanuman langur females Semnopithecus entellus Behavioral Ecology and Sociobiology 48 93 109 Lambert J E 1998 Primate digestion Interactions among anatomy physiology and feeding ecology Evolutionary Anthropology 7 8 20 Lambert J E in press In vitro fermentation of dietary carbohydrates consumed by African apes and monkeys Preliminary results for interpreting microbial and digestive strategy International Journal of Primatology in press Lee S C Prosky L amp DeVries J W 1992 Deter
17. total mass of the fruit and the masses of the pulp and seed separately for each fruit to differentiate the contribution of seed in the fruit Leighton 1993 Milton 2008 We also recommend that samples of the whole fruit pulp and seed are separated for analysis even when fruits are consumed whole However if is known that the seed is crushed and masticated along with the pulp the entire fruit should be analyzed It is important to note the processing procedure and the rationale for this separation should be discussed when presenting nutritional data Drying Samples It is rare to be able to analyze fresh samples because laboratories are typically unavailable at field sites and so drying the sample before transport to the laboratory is necessary The goals of drying primate foods are to inhibit enzymatic activity rapidly to prevent chemical and physical shifts and preserve the sample s nutritional attributes In the agricultural and food science industry freeze drying is thought to be the best way to preserve a sample in its current state In this process a sample is placed in liquid nitrogen where it is stored until it can be dehydrated with a freeze dryer However the procedure requires a supply of liquid nitrogen the ability to carry a liquid nitrogen tank to the field site storage facilities for the liquid nitrogen and a dry shipper which keeps samples frozen at cryotemperatures during transport The storage of large numbers of sample
18. vitro and in vivo Journal of Zoo and Wildlife Medicine 36 571 580 Shipley L A Gross J E Spalinger D E Hobbs N T amp Wunder B A 1994 The scaling intake rate in mammalian herbivores American Naturalist 143 1055 1082 Simmen B amp Sabatier D 1996 Diets of some French Guianan primates Food composition and food choices International Journal of Primatology 17 661 693 Simpson S J amp Raubenheimer D 1999 Assuaging nutritional complexity A geometrical approach Proceedings of the Nutrition Society 58 779 789 Smith A C 2000 Composition and proposed nutritional importance of exudates eaten by saddleback Sanguinus fuscicollis and mustached Saguinus mystax tamarins International Journal of Primatology 21 69 83 Snaith T V amp Chapman C A 2005 Towards an ecological solution to the folivore paradox Patch depletion as an indicator of within group scramble competition in red colobus monkeys Piliocolobus tephrosceles Behavioral Ecology and Sociobiology 59 185 190 Snaith T V amp Chapman C A 2007 Primate group size and interpreting socioecological models Do primates really play by different rules Evolutionary Anthropology 16 95 106 Sommer V Bauer J Fowler A amp Ortmann S 2011 Patriarchal chimpanzees matriarchal bonobos Potential ecological causes of a Pan dichotomy In V Sommer amp C Ross Eds Primates of Gashaka Sociology and
19. 21 324 Rothman J M Plumptre A J Dierenfeld E S amp Pell A N 2007 Nutritional composition of the diet of the gorilla Gorilla beringei A comparison between two mountain habitats Journal of Tropical Ecology 23 673 682 Rothman J M Chapman C A amp Pell A N 2008a Fiber bound protein in gorilla diets Implications for estimating the intake of dietary protein by primates American Journal of Primatology 70 690 694 Rothman J M Dierenfeld E S Hintz H F amp Pell A N 2008b Nutritional quality of gorilla diets Consequences of age sex and season Oecologia 155 111 122 Rothman J M Chapman C A Hansen J L Cherney D J amp Pell A N 2009a Rapid assessment of the nutritional value of foods eaten by mountain gorillas Applying near infrared reflectance spectroscopy to primatology International Journal of Primatology 30 729 742 Rothman J M Dusinberre K amp Pell A N 2009b Condensed tannins in the diets of primates A matter of methods American Journal of Primatology 71 10 76 Rothman J M Raubenheimer D amp Chapman C A 2011 Nutritional geometry Gorillas prioritize non protein energy while consuming surplus protein Biology Letters 7 847 849 Schmidt D A Kerley M S Dempsey J L Porton I J Porter J H Griffen M E Ellersieck M R amp Sadler W C 2005 Fiber digestibility by the orangutan Pongo abelii in
20. A Chapman C A amp Vickery W L 2007 Intratree variation in fruit production and implication for primate foraging International Journal of Primatology 28 1197 1271 Isbell L A 1991 Contest and scramble competition Patterns of female aggression and ranging behaviour among primates Behavioral Ecology 2 143 155 Isbell L A 1998 Diet for a small primate Insectivory and gummivory in the large patas monkey Erythrocebus patas pyrrhonotus American Journal of Primatology 45 381 398 Janson C H 1988 Intra specific food competition and primate social structure A synthesis Behaviour 105 1 17 Janson C H 2007 What wild primates know about resources Opening up the black box Animal Cognition 10 357 367 Janson C H amp Chapman C A 1999 Resources and primate community structure In J G Fleagle C Janson amp K E Reed Eds Primate communities pp 237 267 Cambridge UK Cambridge University Press Janson C H amp van Schaik C P 1988 Recognizing the many faces of primate food competition Methods Behaviour 105 165 186 Janson C H amp Vogel E R 2006 Estimating the effects of hunger on primate social ecology In G Hohmann M Robbins amp C Boesch Eds Feeding ecology in apes and other primates pp 285 312 Cambridge UK Cambridge University Press Julkunen Tiitto R amp Sorsa S 2002 Testing the effects of drying methods on willow
21. Int J Primatol 2012 33 542 566 DOI 10 1007 s10764 011 9568 x Methods in Primate Nutritional Ecology A User s Guide Jessica M Rothman Colin A Chapman Peter J Van Soest Received 26 May 2011 Accepted 26 September 2011 Published online 14 December 2011 Springer Science Business Media LLC 2011 Abstract An important goal of primatology is to identify the ecological factors that affect primate abundance diversity demography and social behavior Understanding the nutritional needs of primates is central to understanding primate ecology because adequate nutrition is a prerequisite for successful reproduction Here we review nutritional methods and provide practical guidelines to measure nutrient intake by primates in field settings We begin with an assessment of how to estimate food intake by primates using behavioral observations We then describe how to collect prepare and preserve food samples Finally we suggest appropriate nutritional assays for estimating diet nutritional quality and point to the merits and limitations of each We hope this review will inspire primatologists to use nutritional ecology to answer many unresolved questions in primatology Keywords Energy Feeding ecology Near infrared reflectance spectroscopy Protein J M Rothman lt Department of Anthropology Hunter College of the City University of New York New York NY USA e mail jessica rothman hunter cuny edu J M Rothman New Yo
22. alue of 6 25 is typically used to convert the amount of nitrogen in sample to its crude protein content however this value overestimates the amount of digestible protein Only 60 70 of the crude protein in domesticated plant species is true protein that is composed of amino acids Van Soest 1994 and in wild plants the true protein content is probably even less Milton and Dintzis 1981 Some of the nitrogen may be bound to the plant cell wall i e fiber bound nitrogen Rothman et al 2008a and it is resistant to digestion by animal and microbial enzymes Other nitrogen can AA Springer 552 J M Rothman et al be bound to secondary compounds particularly tannins and thus rendered indigestible Robbins et al 1987a Plants may also contain considerable amounts of nonprotein nitrogen in secondary compounds 1 e alkaloids glucosinolates and cyanides in nucleic acids or ammonia or as products of amino acid catabolism When crude protein is measured all of these different types of nitrogen are counted as protein even though they are not necessarily digestible Because the 6 25 protein conversion is inaccurate in accounting for nonamino forms of nitrogen studies have proposed adjustments to this factor to separate other N containing fractions from true protein Levey 2000 Milton and Dintzis 1981 However it is not known whether these other conversion factors are too restrictive in a study of tropical foliage Conklin Brittain
23. ample used for dry matter calculations should be used to calculate the organic matter or ash free portion of the sample This subsample is placed in a weighed labeled etched beaker It is then burned at 500 550 C and reweighed to provide an estimate of the minerals soil and dust contamination Nutrient values can be expressed as a percentage of the dry matter or organic matter DM or OM basis respectively Protein Milton 1979 observed that howlers Alouatta palliata selected leaves that were higher in protein and lower in fiber than those avoided suggesting that protein was an important criterion for leaf choice in folivores To estimate protein intake by primates it is important to consider not only crude protein but also its digestibility and quality Because animals do not require crude protein but amino acids the best approach to examine protein intake by primates is to estimate the amounts and types of amino acids in primate foods Amino acid composition is analyzed via high performance liquid chromatography HPLC which is based on separation of different amino acids based on their polarity Few studies have analyzed the amino acid composition of primate foods Curtis 2004 probably because amino acid analysis is quite expensive and requires a specialized technique Most primate studies measure the crude protein which is a measure of all the nitrogen in a sample Because pure protein is typically 16 nitrogen a conversion v
24. and toxins in the diet Biotropica 10 241 253 Oftedal O T 1992 The nutritional consequences of foraging in primates The relationship of nutrient intakes to nutrient requirements Philosophical Transactions of the Royal Society B Biological Sciences 334 161 170 Ortmann S Bradley B J Stolter C amp Ganzhorn J U 2006 Estimating the quality and composition of wild animal diets A critical survey of methods In G Hohmann M M Robbins amp C Boesch Eds Feeding ecology in apes and other primates Ecological physical and behavioral aspects pp 397 420 Cambridge UK Cambridge University Press Ozanne C M P Bell J R amp Weaver D G 2011 Collecting arthropods and arthropod remains for primate studies In J M Setchell amp D J Curtis Eds Field and laboratory methods in primatology 2nd ed pp 271 285 Cambridge UK Cambridge University Press Palmer B Jones R J Wina E amp Tangendjaja B 2000 The effect of sample drying conditions on estimates of condensed tannin and fibre content dry matter digestibility nitrogen digestibility and PEG binding of Calliandra calothyrsus Animal Feed Science and Technology 87 29 40 Palmquist D L amp Jenkins T C 2003 Challenges with fats and fatty acid methods Journal of Animal Science 81 3250 3254 Perry J M G amp Harstone Rose A 2010 Maximum ingested food size in captive strepsirrhine primates Scaling and the
25. aubenheimer 2011 will shed light on the nutritional priorities of animals coupled with good nutritional methods and an understanding of physiological capabilities these offer new approaches to understanding the patterns of nutrient intake by primates We hope that researchers will use nutritional ecology to tackle new and longstanding hypotheses in primatology Acknowledgments We thank Erin Vogel and Janine Chalk for inviting us to contribute this article We also thank Erin Vogel Janine Chalk and Peter Lucas for organizing a fruitful workshop on Innovative Methods in Feeding Ecology and we thank the participants of the workshop for inspiring discussions about nutritional methods We thank Deborah Cherney Ellen Dierenfeld Mary Beth Hall Skip Hintz Caley Johnson Joanna Lambert Alice Pell James Robertson Deborah Ross and Michael Van Amburgh for insights into the nutritional methods outlined here We appreciate the very helpful comments provided by Caley Johnson Erin Vogel and 3 anonymous reviewers We also thank our funding agencies including the National Science Foundation under grant 0922709 Hunter College Canada Research Chair program and the National Science and Engineering Research Council References Altmann J 1974 Observational study of behavior Sampling methods Behaviour 49 227 267 Altmann S A 1998 Foraging for survival Yearling baboons in Africa Chicago University of Chicago Press Altmann J amp Alb
26. bly can subsist on lower quality protein than simple stomached primates Their forestomach provides a rich source of microbial protein and microbes can use may many sources of protein that are unavailable to animals Van Soest 1994 For example in a study of protein utilization in hamsters and rats the foregut fermenting hamsters did not respond to amino acid supplementation while the rats did suggesting that hamsters are less dependent on dietary protein quality than rats Banta et al 1975 A common assumption in the literature is that colobines should select high protein diets Chapman and Chapman 2002 Mowry et al 1996 digestive physiology should factor more readily into these discussions Protein digestibility may be affected by tannins Robbins et al 1987a Robbins et al 1987b Both condensed tannins Glander 1982 Wrangham and Waterman 1981 and hydrolyzable tannins Marks et al 1988 have been shown to affect primate feeding behavior cf Barton and Whiten 1994 Chapman and Chapman 2002 However the role of tannins in primate nutrition is not clear Some primates including humans have adaptations to deal with tannins such as proline rich salivary proteins that bind tannins Mau et al 2009 2011 Mole et al 1990 and others host tannin degrading microbes in the gastrointestinal tract Frey et al 2006 Kay and Davies 1994 To complicate this discrepancies in analytical techniques make it A Springer Methods in Primate Nutri
27. ccount roughly for these compounds when fatty acid analysis is not available Palmquist and Jenkins 2003 and some have adopted this technique in primatology Rothman et al 2011 Using this method if the ether extract of a sample is below 1 it should be converted to 0 Carbohydrates and Lignin Carbohydrates are a major source of energy in primate diets They fall into 3 umbrella categories simple sugars which include glucose and fructose and their g Springer 554 J M Rothman et al conjugates storage reserve compounds which include starch sucrose and fructans and the structural polysaccharides which include the pectins hemicelluloses and celluloses Van Soest 1994 In the animal nutrition literature these plant compounds are divided into 2 categories nonstructural carbohydrates and structural carbohy drates plant cell wall It is important to recognize that the nonstructural carbohydrates include both the storage reserve compounds and the simple sugars but water soluble carbohydrates include only the rapidly digestible part of this fraction that are soluble in water including all of the simple sugars and a small fraction of the storage compounds Van Soest 1994 Of the nonstructural carbohydrates simple sugars are fully digested by animal enzymes and starches have high digestibility whereas structural carbohydrates are indigestible by animal enzymes but may be fermented with the aid of symbiotic gut microbes Dietar
28. ch is sodium rich Chancellor and Isbell 2009 Rode et al 2003 Knowledge of a species or a population s specific nutritional goals can be important in the construction of informed conservation or restoration plans for endangered species Chapman ef al 2004 Felton et al 2010 For example the protein to fiber ratio of mature leaves in a habitat is a good predictor of the biomass of colobines across forests Chapman et al 2004 Fashing et al 2007 and forest managers could use information about a species nutritional needs to pinpoint specific trees and areas to protect Chapman et al 2004 Felton et al 2010 We here review the methods for measuring nutrient intake by wild primates We begin with an assessment of how to estimate food intake by primates We then describe how to collect prepare and preserve food samples Finally we suggest appropriate nutritional assays for estimating diet nutritional quality This article is not intended to be a complete manual of primate nutritional ecology and associated laboratory methods but rather to point readers to potentially valuable approaches and methods We stress that it is important to outline the exact question that will be addressed so that methods can be tailored Felton et al 2009a For further information we suggest a few excellent textbooks Van Soest 1994 Barboza et al 2009 and Robbins 1993 Before embarking on research that involves collecting and processing primate f
29. e nutritional chemistry of the insects Estimating the amount of material to collect is dependent on the types of analyses needed We suggest that researchers collect gt 30 g of dry weight of material for analysis This provides 1 2 g for each macronutrient assay with some to spare in case of loss of sample during milling the need to run samples multiple times to obtain an accurate assessment or accidents during the analysis It is always better to have more sample than less To collect 30 g of dry weight may require collections of up to 500 g wet weight depending on the food item s moisture content Based on our experience pith stems contain the most moisture whereas bark wood contains the least Rothman et al 2006a Processing Samples Researchers should process the samples in the way they were processed by the consumer For example if the consumer eats the leaf lamina but not the leaf petiole the petiole should be removed before weighing and nutritional analysis If the consumer eats g Springer 548 J M Rothman et al only the pith of a stem and discards the outer herbaceous covering this covering should be removed This requires careful observation and meticulous and often very time consuming processing Some items are more difficult to process gorillas suck and spit out wood Rothman et al 2006b and chimpanzees make wadges or quids whereby they extract suction juices from fruits pith and bark and discard the fibro
30. ecause ash is only partially removed Energy Primates need energy for basal metabolism growth lactation and reproduction and energy intake has been related to fitness for some primate species Altmann 1998 Altmann and Alberts 2005 Karasov 1986 Nagy and Milton 1979 NRC 2003 The acquisition of energy is viewed as a fundamental facet of female competitive regimes among primates Janson 1988 Janson and van Schaik 1988 Snaith and Chapman 2007 Sterck et al 1997 van Schaik 1989 and higher ranking individuals are often AA Springer 556 J M Rothman et al able to obtain more energy than lower ranking individuals Koenig 2000 Vogel 2005 Seasonal energetic shortfalls may have large consequences for primates Knott 1998 An excellent step by step guide to estimating energetic contributions to primate diets is available Conklin Brittain et al 2006 Accordingly we briefly reiterate many of the points addressed in their article placing emphasis on what we view are a few key points There are 2 methods used to estimate energy intake Conklin Brittain et al 2006 NRC 2003 1 via actual energy measurements of foods and excreta via bomb calorimetry after carefully controlled feeding experiments and 2 via estimation of food intake and the energetic contributions of fat carbohydrates and protein There are 3 main classifications of energy Gross energy is the total energy content of a food which may be calculated as energy releas
31. ed after combustion in a bomb calorimeter The amount of energy released in a food is noted by a concomitant increase in water temperature within the bomb calorimeter The problem with using gross energy of food items as an indicator of what energy is available to the consumer is that not all energy within a food is digestible For example wood has a higher energy content than the same mass of sugar but it is far less digestible Many primate studies have used gross energy as a measure of energy available to primates Ganas et al 2008 Gould et al in press However this measure is misleading because without accompanying measures of fecal energy gross energy measures do not provide any indication of the energy received In addition a standard estimate of the digestibility of all foods is not adequate because foods differ in their fiber and lignin contents Digestible energy is the fecal energy subtracted from the gross energy In this case known quantities of each food ingested must be coupled with estimates of the energetic values of feces These studies have rarely been conducted in primates cf Edwards and Ullrey 1999a b Metabolizable energy is the urinary energy subtracted from the digestible energy which provides an even finer estimate of the energy consumed In cases where it is not possible to estimate digestible energy via bomb calorimetry on foods and excreta an approximation of energy gain is provided from physiological fuel values de
32. effects of diet American Journal of Physical Anthropology 142 625 635 Powzyk J A amp Mowry C B 2003 Dietary and feeding differences between sympatric Propithecus diadema diadema and Indri indri International Journal of Primatology 24 6 1143 1162 Raubenheimer D 2011 Towards a quantitative nutritional ecology The right angled mixture triangle Ecological Monographs 81 407 427 Rautio P Bergvall U A Karonen M amp Salminen J P 2007 Bitter problems in ecological feeding experiments Commercial tannin preparations and common methods for tannin quantifications Biochemical Systematics and Ecology 35 257 262 Reiner W B amp Rothman J M 2011 Fatty acids in gorilla diets Implications for primate nutrition and human health American Journal of Physical Anthropology 52 99 Remis M J Dierenfeld E S Mowry C B amp Carroll R W 2001 Nutritional aspects of western lowland gorilla Gorilla gorilla gorilla diet during seasons of fruit scarcity at Bai Hokou Central African Republic International Journal of Primatology 22 807 836 Reynolds V Plumptre A J Greenham J amp Harborne J 1998 Condensed tannins and sugars in the diet of chimpanzees Pan troglodytes schweinfurthii in the Budongo Forest Uganda Oecologia 115 331 336 Riba Hernandez P Stoner K E amp Lucas P W 2005 Sugar concentration of fruits and their detection via color in the central
33. ental feeding trials in which species are fed similar diet concentrations to that in the wild For example a feeding trial with chimpanzees on a diet of 34 NDF illustrated that they digested 54 3 of NDF Milton and Demment 1988 This fiber coefficient can be used to estimate the energetic contributions from the fermentation of fiber in wild chimpanzees Conklin Brittain et al 2006 When it is possible to estimate accurately estimate daily nutrient intake feces can be collected and dietary lignin is gt 5 feces can be used as an internal marker to determine diet and fiber digestibility Fahey and Jung 1983 Van Soest 1994 Using this approach the diet and feces are analyzed for lignin content and it is assumed that lignin is not digested Consequently NDF coefficients can be estimated and then incorporated into energy estimations Rothman ef al 2008b Another method is to culture and incubate fresh feces with food items as substrates to determine digestibility coefficients This method has been used in studies of captive lemurs Varecia variegata Eulemur fulvus Hapalemur griseus Campbell et al 2002 chimpanzees Kisidayova et al 2009 and orangutans Pongo abelii Schmidt et al 2005 Here fecal samples must be kept fresh so that microbial populations are preserved which is challenging in the field Instead of using physiological fuel values derived from human diets an alternative is to use the energetic equations developed for dom
34. ermination of glucose Journal of AOAC International 92 50 60 Hall M B amp Mertens D R 2008 Effect of sample processing procedures on measurement of starch in corn silage and corn grain Journal of Dairy Science 91 4830 4833 Hall M B Hoover W H Jennings J P amp Webster T K M 1999 A method for partitioning neutral detergent soluble carbohydrates Journal of the Science of Food and Agriculture 79 2079 2086 Harris T R amp Chapman C A 2007 Variation in the diet and ranging behavior of black and white colobus monkeys Implications for theory and conservation Primates 28 208 221 Harrison M E Vogel E R Morrogh Bernard H C amp van Noordwijk M A 2009 Methods for calculating activity budgets compared A case study using orangutans American Journal of Primatology 71 353 358 Hohmann G Fowler A Sommer V amp Ortmann S 2006 Frugivory and gregariousness of Salonga bonobos and Gashaka chimpanzees The influence of abundance and nutritional quality of fruit In G Hohmann M M Robbins amp C Boesch Eds Feeding ecology in apes and other primates Ecological physical and behavioural aspects pp 123 159 Cambridge UK Cambridge University Press Houle A Chapman C A amp Vickery W L 2004 Tree climbing strategies for primate ecological studies International Journal of Primatology 25 237 260 g Springer 562 J M Rothman et al Houle
35. erts S C 2005 Growth rates in a wild primate population Ecological influences and maternal effects Behavioral Ecology and Sociobiology 57 490 501 AOAC 1990 Official methods of analysis 15th ed Association of Analytical Chemists Banta C A Warner R G amp Robertson J B 1975 Protein nutrition of the golden hamster Journal of Nutrition 105 38 45 Barboza P S Parker K L amp Hume I D 2009 Integrative wildlife nutrition Berlin Springer Verlag Barton R A amp Whiten A 1994 Reducing complex diets to simple rules Food selection by olive baboons Behavioral Ecology and Sociobiology 35 283 293 Cameron J L 1996 Regulation of reproductive hormone secretion in primates Reproduction 1 117 126 Campbell J L 2000 Description of the gastrointestinal tract of five lemur species Propithecus tattersalli Propithecus verreauxi coquereli Varecia variecia Hapalemur grisus and Lemur catta American Journal of Primatology 52 133 142 Campbell J L Williams C V amp Eismann J H 2002 Fecal inoculum can be used to determine the rate and extent of in vitro fermentation of dietary fiber sources across three lemur species that differ in dietary profile Varecia variegata Eulemur fulvus and Hapalemur griseus Journal of Nutrition 132 3073 3080 Camperio Ciani A Martinoli L Capiluppi C Arahou M amp Mouna M 2001 Effects of water availability and habitat
36. ess the total water soluble carbohydrates using colorimetric assays with a sugar standard such as the phenol sulfuric acid assay Dubois et al 1956 which was developed for examining sea water In animal nutrition applications the protocol is typically modified we suggest the modifica tion of boiling a dry sample for 6 min in distilled water Protocols are available for partitioning of most nonstructural carbohydrates and soluble fiber such as starch and sugars Hall et al 1999 The current methods for estimating starch are outlined in Hall 2009 but researchers should also consider confounding compounds such as antioxidants and tannins that affect results Hall and Keuler 2009 To estimate the concentrations of soluble and insoluble fiber the TDF total dietary fiber TDF method that is used widely in human nutrition can be used it may be particularly helpful in identifying the portions of carbohydrates available to frugivores Conklin Brittain et al 2006 Food and Nutrition Board 2005 Lee et al 1992 Methods for particular soluble fibers such as pectins which are widespread in fruits and some tropical leaves are also available Milton 1991 In addition we recommend using a specific analysis for total nonstructural carbohydrates TNC Hall et al 1999 The TNC measure is frequently estimated by subtraction whereby the grams or g Springer Methods in Primate Nutritional Ecology 593 percentages of neutral detergent fiber p
37. estic animals For example for colobines which have pregastric fermentation Kay and Davies 1994 energetic equations based on small ruminants might be used NRC 2007 however without further research on colobine digestive ecology we do not know if they are analogous to small ruminants Milton 2006 For hindgut fermenters humans are good models for comparison Food and Nutrition Board 2005 Rothman et al 2011 and energetic equations of swine might be helpful NRC 1998 Rothman et al 2008b Minerals and Vitamins Little is known about vitamin and mineral requirements of wild primates but recent work highlights their significance in primate diets Fashing et al 2007 For example the soils of many tropical regions are sodium poor thus primates in the tropics often have difficulty obtaining required sodium Oates 1978 Rothman et al 2006b Sodium content of foods eaten by primates in Kibale National Park Uganda was extremely low no single food met the guidelines set by the NRC 2003 and sodium intake from the typical plant diet was well below suggested requirements throughout the year Rode et al 2003 Mineral analysis is typically preformed by ashing the sample and using atomic absorption spectroscopy The principle of this method is that an aqueous solution of the minerals sample ashed with acids is heated to vaporize and atomize the minerals Radiation beams are passed through the sample and the absorption of radiation is rela
38. et al 1999 noted that a 4 3 correction factor probably under estimates protein digestibility Thus they consider a fiber free protein an optimist s view of available protein estimation and a crude protein coefficient of 4 3 as a pessimist s view Without better measures of nitrogen balance through carefully controlled feeding experiments it is difficult to know how much protein is available Fortunately new methods are being developed to assess protein intake by primates and provide complementary approaches Vogel et al 2012 To make good approximations of protein availability and quality we recommend a few steps be employed First it is important to obtain an estimate of the crude protein in a sample through the Kjehdahl assay or combustion AOAC methods 984 13 and 990 03 AOAC 1990 Second researchers should estimate the portion of protein bound to fiber and indigestible Licitra et al 1996 Rothman et al 2008a This is accomplished by first following the steps to estimate acid detergent fiber ADF and then measuring the nitrogen that remains in the sample via Kjehdahl or combustion Third whenever possible other nonprotein nitrogenous compounds should be measured using stabilized tungstic acid Licitra et al 1996 Martinek 1964 However estimating the amino acids in a sample provides the most information about the quality of protein It is also worth mentioning that colobines which have foregut fermentation proba
39. he United States Department of Agriculture USDA for plants and insects and there are similar requirements for Canada Primate fecal material requires a separate permit from the Centers for Disease Control USA and Public Health Agency of Canada In our experience it can take 3 4 mo for such permits to be processed Similar regulations are probably applicable in other countries We found that the more physically processed the samples are the easier it is to obtain permits At present in the United States and Canada if plant samples are dried and milled before import a permit is not needed though a letter stating this from the appropriate agency is helpful for export and at customs Similarly if fecal samples are not infectious a permit is not necessarily required though a letter stating this is again helpful Thus we suggest drying and milling samples before export In addition this advance processing also helps to prevent mold and pathogens that can affect analyses In the Field Behavioral Methods The selection of methods to quantify a behavior represents a tradeoff between the amount and quality of the data that can be obtained There are many descriptions of the costs and benefits of different approaches Altmann 1974 Martin and Bateson 1986 and we do not attempt to describe these methods Full day continuous focal follows are ideal to obtain a robust sample of the daily nutrient intake Felton et al 2009a Knott 1998 However th
40. ilable to process individual samples we suggest numerous collections be combined to get a representative sample of foods in different seasons and microhabitats Taking subsamples and mixing them means that some sources of variance are not estimated but this will likely approximate the average nutritional composition of a food item The physical collection of primate food samples can be logistically difficult Terrestrial foods can be easily collected using a machete or plant pruners Tree saws and poles can be used to extract arboreal foods We also recommend opportunistic collection when primates drop branches from which food samples can be obtained There are also various tree climbing techniques that can be used to collect samples Houle et al 2004 Insects comprise a high portion of some primate diets Dammhahn and Kappeler 2008 Isbell 1998 but they are rarely analyzed for their nutritional composition because they are very difficult to collect cf Deblauwe and Janssens 2008 Insect collections are typically opportunistic but other methods can be employed Janson and Chapman 1999 Ozanne et al 2011 Primatologists should collaborate with entomologists to ensure proper identification of insects In addition entomologists often fog areas with insecticide and sort samples Stork 1991 primatologists could then sample insects used as primate food for nutritional analysis after the samples are sorted as the insecticide will likely not impact th
41. in et al 2006 Vogel 2005 and the digestive ecology of both frugivorous and folivorous primates cf Campbell 2000 Caton 1999 Caton et al 2000 Lambert 1998 Milton et al 1980 Although protein to fiber ratios of the most common trees in the forest predict colobine abundance at multiple scales Chapman et al 2004 it is unclear what nutritional mechanism is driving this relationship because the protein content of mature leaves in these forests is high Chapman et al 2002 and primates do not require high protein diets Oftedal 1992 we need to unravel this protein paradox in the primate literature Rothman et al 2011 Tests of cognitive maps of a group s home range are often based on simple assumptions about nutrition such as the g Springer Methods in Primate Nutritional Ecology 559 prediction that primates will choose the shortest distance between large high energy fruit resources Janson 2007 However it is possible that animals are making deviations from expected patterns to be able to obtain particular nutrients Harris and Chapman 2007 Link et al 2011 or have complex decision rules dependent on both mental abilities and nutritional needs Finally we need to understand the role of ecology and phylogeny in shaping the evolution of primate feeding adaptations Chapman and Rothman 2009 New frameworks of nutritional ecology such as nutritional geometry Simpson and Raubenheimer 1999 and right angle mixture triangles R
42. in plastic bags with desiccant in a dark area with low humidity and labeled with a chemical resistant marker Ensure that the desiccant is packed within a permeable cloth paper or cotton so that it does not directly mix with the samples We recommend that a small label be included inside the sample bag There is no particular recommended shelf life for dried plant samples and protein carbohydrates and minerals should be stable Unsaturated fatty acids and some secondary compounds may oxidize during storage However we analyzed the same samples using the same methods after 10 yr and they retained the same results for protein fiber fat and sugar Rothman unpubl data Laboratory Analysis Milling Samples Particle size is important in nutritional analysis because the size of a food particle will affect the reaction surface and thus the outcome of analyses Hall and Mertens g Springer 550 J M Rothman et al 2008 Mertens 1992 Samples can be ground by hand with gloves to avoid inflating fat and salt values to reduce bulk for storage but need to be milled to a uniform size for analysis Samples should not be ground via a mortar and pestle or coffee grinder because they will not produce uniform and known particle sizes Consideration should be taken in the type of mill used Mertens 1992 Cutting mills such as the Wiley mill control for particle size by allowing free fall of ground material through a uniform sieve Cyclone mills
43. ing at lt 45 C out of sunlight is preferable Julkunen Tiitto and Sorsa 2002 Mueller Harvey 2001 Palmer et al 2000 Stewart et al 2000 Light and heat can alter secondary compound composition Cork and Krockenberger 1991 Waterman and Mole 1994 though gt 1 study demonstrated no differences in the stability of polyphenols dried at 60 C Muetzel and Becker 2006 Qualitative tests presence absence of secondary compounds are not as sensitive to temperature adjustments When samples are dried at ambient temperature great care must be taken to avoid samples from molding as its inclusion will dramatically alter sample nutritional composition When there is no choice but to dry at ambient temperature a desiccant with a color moisture indicator should be included in a sample container This desiccant can be regenerated by heating in a cooking pan to restore function However this method has limited applicability for succulent fruits Another method is to place samples directly in a preserving solvent such as ethanol or methanol Although some primate studies have successfully preserved samples in solvents Powzyk and Mowry 2003 Remis et al 2001 we do not recommend it because a liquid extract containing ethanol soluble carbohydrates is produced and analyzing both the extract and sample is difficult After drying samples should be weighed to the nearest 0 01 g so that moisture can be estimated see section on Dry Matter and should be stored
44. ion and in vitro digestibility of forages Animal Feed Science and Technology 46 75 86 Dubois M Gilles K A Hamilton J K Rebers P A amp Smith F 1956 Colorimetric method for determination of sugars and related substances Analytical Chemistry 28 350 356 Edwards M S amp Ullrey D E 1999a Effect of dietary fiber concentration on apparent digestibility and digesta passage in non human primates I Ruffed lemurs Varecia variegata vauiegata and V v rubra Zoo Biology 18 529 536 Edwards M S amp Ullrey D E 1999b Effect of dietary fiber concentration on apparent digestibility and digesta passage in non human primates II Hindgut and foregut fermenting folivores Zoo Biology 18 537 549 Eitenmiller R R Landen W O amp Ye L 2007 Vitamin analysis for the health and food sciences 2nd ed Boca Raton FL CRC Press g Springer Methods in Primate Nutritional Ecology 561 Fahey G C amp Jung H G 1983 Lignin as a marker in digestion studies A review Journal of Animal Science 57 220 225 Fashing P J Dierenfeld E amp Mowry C B 2007 Influence of plant and soil chemistry on food selection ranging patterns and biomass of Colobus guereza in Kakamega Forest Kenya International Journal of Primatology 28 673 703 Fedigan L M 2010 Ethical issues faced by field primatologists Asking the relevant questions American Journal of Primatology 71 1 18
45. is method is dependent on the ability to obtain a large sample of complete days for each subject under study because if a day is not representative of the study period e g it is raining or the subject is sick it may be misleading and obtaining more samples during shorter periods of time might be more appropriate Frequently it is logistically unfeasible to conduct a full day follow because of poor visibility or government regulations Rothman et al 2008b In these instances it may be appropriate to conduct short focal follows with quantified food intake combined with scan samples of the group or individual or both to estimate the overall amount of time feeding Janson and Vogel 2006 Rothman et al 2008b Food Intake To measure nutrient intake it is essential to determine the amount of food 1 e in grams an individual consumes rather than the time it takes to consume a particular food item Time spent feeding is an excellent indicator of foraging effort but a poor indicator of nutrient intake Chivers 1998 Zinner 1999 Measures of feeding time can be misleading because an easily processed food item may take little time to prepare in relation to mass consumed or conversely a hard to chew item might take a longer time to consume with little mass intake Accordingly various field studies have demonstrated the discrepancy between feeding time and ingestion amounts g Springer Methods in Primate Nutritional Ecology 545 Chivers 1998
46. mination of total soluble and insoluble fiber in foods enzymatic gravimetric method MES TRIS buffer Collaborative study Journal of AOAC International 75 395 416 Leighton M 1993 Modeling dietary selectivity by Bornean orangutans Evidence for integration of multiple criteria in fruit selection International Journal of Primatology 14 257 313 Levey D J 2000 Conversion of nitrogen to protein and amino acids in wild fruits Journal of Chemical Ecology 26 1749 1763 Licitra G Hernandez T M amp VanSoest P J 1996 Standardization of procedures for nitrogen fractionation of ruminant feeds Animal Feed Science and Technology 57 341 358 Link A Galvis N Fleming E amp Di Fiore A 2011 Patterns of mineral lick visitation by spider monkeys and holwer monkeys in Amazonia Are licks perceived as risky areas American Journal of Primatology 73 286 396 Lucas P Osorio D Yamashita N Prinz J F Dominy N J amp Darvell B J 2011 Dietary analysis II Food chemistry In J M Setchell amp D J Curtis Eds Field and laboratory methods in primatology 2nd ed pp 255 270 Cambridge UK Cambridge University Press Malenky R K amp Wrangham R W 1994 A quantitative comparison of terrestrial herbaceous food consumption by Pan paniscus in the Lomako Forest Zaire and Pan troglodytes in the Kibale Forest Uganda American Journal of Primatology 32 1 12 Marks D L S
47. nt intake and provide equations for doing so Knott 1998 McCabe and Fedigan 2007 Rothman et al 2007 2008b and outlining the calculations employed in these estimations is essential Harrison et al 2009 Collecting Samples Whenever possible samples should be collected from the same plants eaten by the primates under study because the nutrient content of plants within a single species can vary over different spatial and temporal scales For example depending on location young leaves of the same species eaten by monkeys in Kibale National g Springer 546 J M Rothman et al Park Uganda varied in protein content from 22 to 47 Chapman et al 2003 and the fat content of a single species of ripe fruit varied seasonally from 0 3 to 30 0 Worman and Chapman 2005 Fruits in Kibale also varied in dry weight according to canopy height Houle et al 2007 and in Madagascar sun exposed leaves had more protein than shaded leaves Ganzhorn 1995 The nutritional components of terrestrial herbaceous vegetation eaten by mountain gorillas are highly variable Rothman ef al 2009a For example depending on its location samples of Triumfetta tomentosa collected in a single month over a range of locations varied in protein from 17 4 to 28 2 and neutral detergent fiber from 26 1 to 44 8 on a dry matter basis Rothman unpubl data Fig 1 This striking intraspecific a Crude Protein Dry Matter O Neutral Detergent Fiber Dry
48. nts thoroughly to ensure uniformity After the samples are milled they should be stored in tightly sealed plastic bags or glass jars to avoid moisture and insect consumption If a plant sample is particularly oily samples should be stored in brown jars because sunlight can affect the oxidation of some fatty acids Measuring Macronutrients and Energy Here we review some of the recommended measures for estimating macronutrients in primate foods We view our discussion as complementary to a review by Ortmann et al 2006 We also direct readers to the AOAC Official Methods of Analysis which provides detailed step by step protocols for most nutritional analyses and is currently available on the Internet Before conducting any nutritional analysis milled samples must be thoroughly mixed During storage heavier particles tend to settle toward the bottom of the container Stirring a sample is much more effective than shaking it Dry Matter and Organic Matter Moisture and the dry portion or dry matter of a food sample make up its weight However animals receive protein energy minerals and vitamins only from dry matter In addition because a variety of factors can affect the moisture of a food sample e g humidity sample characteristics it is necessary to account for any adsorbed moisture during nutritional analysis Sample moisture should be calculated g Springer Methods in Primate Nutritional Ecology 551 so that an estimate of we
49. oods for nutritional analysis it is important to realize that most countries require explicit permission to collect export and import wildlife samples including plants It is critical to obtain this proper documentation otherwise samples can be held up at country borders or confiscated and destroyed Unfortunately obtaining proper permits takes time For example we work in Uganda and import plant insect fecal and urine samples to the United States and Canada In Uganda in addition to obtaining permits to conduct research explicit written permission is required before collection of wildlife or their products When exporting samples a material transfer agreement is needed and fees are paid according to the amount of material removed After collection and before export the samples must be inspected After inspection it takes gt 6 wk for an application to be approved These are all reasonable requests made by the host country just as obtaining Animal Care approval is appropriate for the study of primates Fedigan 2010 Primatologists should inquire about the g Springer 544 J M Rothman et al procedures in their country of interest and budget their travel itineraries accordingly If researchers do not follow rules and export samples illegally it impacts all of the research community because countries will be reluctant to allow any samples to be exported in the future To import samples into the United States one needs permits from t
50. quality on bark stripping behavior in Barbary macaques Conservation Biology 15 259 265 Caton J M 1999 A preliminary report on the digestive strategy of the western lowland gorilla Australian Journal of Ecology 13 2 7 Caton J M Lawes M amp Cunningham C 2000 Digestive strategy of the south east African lesser bushbaby Galago moholi Comparative Biochemistry and Physiology Part A Molecular amp Integrative Physiology 127 39 48 AA Springer 560 J M Rothman et al Chamberlain J Nelson G amp Milton K 1993 Fatty acid profiles of major food sources of howler monkeys Alouatta palliata in the Neotropics Experientia 49 9 820 824 Chancellor R L amp Isbell L A 2009 Food site residence time and female competitive relationships in wild gray cheeked mangabeys Lophocebus albigena Behavioral Ecology and Sociobiology 63 1447 1458 Chapman C A 1995 Primate seed dispersal Coevolution and conservation implications Evolutionary Anthropology 4 74 82 Chapman C A amp Chapman L J 2002 Foraging challenges of red colobus monkeys Influence of nutrients and secondary compounds Comparative Biochemistry and Physiology Part A Physiology 133 861 875 Chapman C A amp Rothman J M 2009 Within species differences in primate social structure Evolution of plasticity and phylogenetic constraints Primates 50 12 22 Chapman C A Chapman L J Bjorndal K A
51. r a cluster of fruit can be considered a unit for analysis For bark stems and pith the approximate dimensions of the food eaten are estimated from food remains left behind e g the length and width of bark removed from a tree trunk Knott 1998 The mean unit weight provides an estimate of the mass of the food item and we suggest recording gt 30 individual unit weights for each food item to assess the variance of the mass To account for seasonal variation in unit mass and intraspecific variation in plant morphology we suggest this measurement be taken at least once a month for long term nutritional ecology studies particularly those assessing seasonal and spatial variability if the food item is a staple component of the diet These weights should be collected immediately after collection providing a wet mass so that nutrients can be determined on an as eaten basis and moisture can be calculated To measure nutrient intake the numbers of food units per unit of time are converted to dry weight intake and multiplied by the food s nutritional composition which is expressed as a percentage of dry or organic matter These values are summed for the day or scaled to the appropriate time frame If primates are feeding during travel or other activities this should be factored into estimates of nutrient intake e g mountain gorillas Gorilla beringei snack while traveling Rothman et al 2008b Several primate studies have estimated nutrie
52. rived from human diets which estimate metabolizable energy as additive amount of calories provided by 4 kcal g for carbohydrates 4 kcal g for protein and 9 kcal g fats Although this is an adequate approximation for human diets these values may not necessarily be appropriate for primates that eat very different foods and have different digestive physiology Lambert 1998 NRC 2003 In particular the energetic contributions from fiber fermentation need to be considered as in Conklin et al 2006 because depending on the digestive anatomy and physiology of primates these energy gains may be substantial Edwards and Ullrey 1999b Lambert in press Milton 1998 Milton and McBee 1983 To estimate the energetic contributions from fiber fermentation we need knowledge of the primates digestive anatomy and physiology fiber content of the diet and ability of the hosted microbes to ferment fiber These factors are considered in a study by Edwards and Ullrey 1999b in which they fed multiple monkey species different diets with varying fiber contents and noted their apparent digestibilities after a feeding trial On a high fiber diet the hindgut fermenters digested a mean of 47 of NDF and the foregut fermenters digested 77 of NDF g Springer Methods in Primate Nutritional Ecology 537 To estimate the digestive coefficients fraction of the ingested fiber that was fermented of wild primates we can use information gained from experim
53. rk Consortium in Evolutionary Primatology New York NY USA C A Chapman Department of Anthropology and McGill School of Environment McGill University Montreal Quebec H3A 2T7 Canada C A Chapman Wildlife Conservation Society Bronx NY 10460 USA P J Van Soest Department of Animal Science Cornell University Ithaca NY 14853 USA g Springer Methods in Primate Nutritional Ecology 543 Introduction An important goal of primatology is to decipher the ecological factors that influence primate abundance diversity life history and social behavior Nutritional ecology is at the core of these questions because primates must acquire adequate nutrition under a suite of environmental and social constraints to reproduce successfully Cameron 1996 For example insights into nutritional ecology not only provide understanding of determinants of diet selection or patch choice Leighton 1993 but also contribute to broader questions including hypotheses about the diversity of primates across continents Ganzhorn et al 2009 and primate niche separation Conklin Brittain et al 1998 Ganzhorn 1988 Milton 1981 Further investigation into nutritional ecology may lead to new insights with respect to competitive regimes and sociality Isbell 1991 Janson and van Schaik 1988 Sterck et al 1997 For example higher ranked female gray cheeked mangabeys Lophocebus albigena did not compete over fruit but did show agonism when eating bark whi
54. rotein fat and ash are subtracted from the total mass of the plant or 100 on a percentage basis This approach is adequate for a rough estimation of the nonstructural carbohydrates but does not consider the secondary compounds vitamins and other minor components of foodstuffs Another problem with this crude estimate is that any errors associated with the analysis of each portion protein fiber fats accumulate in the difference These errors may be major For example fiber bound nitrogen is measured twice once in the crude protein component and again in the portion of fiber which might result in substantial underestimation of TNC To remedy this researchers should subtract the fiber bound nitrogen from crude protein In addition in high fat foods if fatty samples are not preextracted before NDF analysis some of the fat might be measured twice once in the NDF and again in the ether extract portion These challenges highlight the importance of understanding the limits and merits of various analytical techniques To estimate structural carbohydrates and lignin we recommend the detergent system of analysis which is widely used to estimate the fiber contents of primate foods An excellent flow diagram of the procedure is found in Van Soest 1994 and details are outlined for the fiber procedure in Van Soest et al 1991 and lignin in Van Soest 1963 Neutral detergent fiber NDF is composed primarily of hemicellulose cellulose and lignin
55. s often makes freeze drying prohibitive Consequently few primate field studies have been able to freeze dry their samples cf Ganas et al 2008 Hohmann et al 2006 Sommer et al 2011 Because freeze drying facilities are not often available numerous studies have evaluated the analytical biases encountered when using various drying methods and g Springer Methods in Primate Nutritional Ecology 549 depending on the nutrient of interest these should be consulted before deciding on a drying method Samples should be quickly dried at or near 55 C to stop enzymatic activity and prevent the Maillard reaction in which amino groups react with sugar Deinum and Maassen 1994 This reaction responsible for the toasting of bread and roasted coffee will cause an overestimate of fiber and underestimate of soluble sugars Van Soest 1994 If electricity is available at the field site a drying oven may be used We also recommend the use of inexpensive food dehydrators such as Nesco models which have adjustable temperature control and multiple trays to hold and sort samples Where electricity is not available ovens can be made by storing samples in metal boxes over a charcoal stove where temperature is carefully controlled Conklin Brittain et al 1998 Powzyk and Mowry 2003 Wrangham et al 1998 When samples will be analyzed quantitatively for secondary compounds such as flavonoids tannins and silicates if freeze drying is not possible dry
56. some hypotheses of food selection by generalist herbivores American Naturalist 114 362 378 Milton K 1980 The foraging strategies of howler monkeys A study in primate economics New York Columbia University Press Milton K 1981 Food choice and digestive strategies of two sympatric primate species American Naturalist 117 496 505 Milton K 1991 Pectic substances in neotropical plant parts Biotropica 23 90 92 Milton K 1993 Diet and primate evolution Scientific American 269 86 93 Milton K 1998 Physiological ecology of howlers Alouatta Energetic and digestive considerations and comparison with the Colobinae International Journal of Primatology 19 513 548 Milton K 2006 Analyzing primate nutritional ecology In G Hohmann M M Robbins amp C Boesch Eds Feeding ecology in apes and other primates Ecological physical and behavioural aspects pp 381 396 Cambridge UK Cambridge University Press Milton K 2008 Macronutrient patterns of 19 species of Panamanian fruits from Barro Colorado Island Neotropical Primates 15 1 7 Milton K amp Demment M W 1988 Digestion and passage kinetics of chimpanzees fed high and low fiber diets and comparison with humans Journal of Nutrition 118 1082 1088 Milton K amp Dintzis F 1981 Nitrogen to protein conversion factors for tropical plant samples Biotropica 12 177 181 Milton K amp Jenness R 1987 Ascorbic
57. such as the Udy mill force grind plant material through a sieve at an angle For standardization most nutritional procedures suggest a Wiley mill fitted with a l1 mm screen or delivery tube It should be noted that the Wiley mini mill does not come standard with a 1 mm delivery tube it must be specially ordered no 20 screen is 0 85 mm and no 16 is 1 19 mm Because cyclone mills force material through a sieve they tend to produce an average particle size that is smaller than cutting mills thus when a cyclone mill is used samples should be milled to 2 mm to achieve a similar particle size to samples ground with a Wiley mill Mertens 1992 Sample sorting will occur as the sample is handled Sometimes very tough or fluffy material takes longer to go through the sieve All efforts should be taken to obtain a milled sample that is representative of the original sample so if some material does not go through the sieve it should still be mixed in with the rest of the milled sample and analyzed regardless of particle size Samples that are especially oily could be frozen in liquid nitrogen first so that they will easily go through the sieve If the samples are not mixed properly sub sampling can introduce very large errors in analysis When milling the sample as the material is passing through the sieve one should use a bag or jar that is large compared to the size of the resulting sample In this way there is room to stir sample conte
58. t weight intake can be attained this weight is the fresh weight as eaten or is often termed the as fed weight in agriculture In addition some primates may choose foods based on their water content particularly if they are faced with a water limited environment Camperio Ciani et al 2001 The moisture of food samples can vary greatly e g gorilla foods vary from 7 to 97 Rothman et al 2006a To calculate a sample s moisture and dry matter a 2 step process should be used including field and laboratory drying In the field the total sample should be weighed shortly after collection and before drying This initial or field moisture content should be calculated by subtracting the sample dry weight from the fresh weight obtained shortly after collection in the field Immediately before nutrient analysis a portion or subsample 0 5 1 0 g of the field dried samples should then be dried at 105 C for 16 h in a forced air oven to remove any adsorbed atmospheric water to calculate a coefficient for determining the 100 dry matter of a sample Goering and Van Soest 1970 This coefficient typically varies between 88 and 95 of the total sample weight It is important that the dry matter coefficient be calculated on each day that the sample is prepared for analysis because local weather varies which will affect adsorbed moisture Ash or inorganic matter represents the portion of the sample that does not contain organic matter The same subs
59. ted to the amounts of different minerals in the sample which can be differentiated by their absorption at different wavelengths This technique requires specialized equipment so primatologists usually send their samples to an outside laboratory The role of vitamins in primate diets is even less clear than that of minerals but because primates eat diets that are fresh and intentionally or accidentally consume g Springer 558 J M Rothman et al insects they are probably not susceptible to vitamin deficiencies Only a few studies have investigated a selection of vitamins in primate diets Milton and Jenness 1987 Sterling et al 1994 probably because vitamins are quite labile so samples need to be analyzed fresh Liquid chromatography mass spectrometry is now standard for vitamin analysis Eitenmiller et al 2007 Near Infrared Reflectance Spectroscopy Given the diversity of primate diets and the intraspecific variability of foods within primate diets methods used in agriculture and food sciences offer new options for primatologists for faster processing of primate foods Near infrared reflectance spectroscopy NIRS is a quick nondestructive and economical technology that can be used to assess the nutritional content of primate foods when large numbers of samples from the same habitat will be processed Rothman et al 2009a The general principle of NIRS is that spectra are reflected based on the number and type of chemical bonds
60. terial diversity in a wild gorilla Gorilla beringei Applied and Environmental Microbiology 72 3789 3792 Ganas J Ortmann S amp Robbins M M 2008 Food preferences of wild mountain gorillas American Journal of Primatology 70 927 938 Ganzhorn J U 1988 Food partitioning among Malagasy primates Oecologia 75 436 450 Ganzhorn J U 1995 Low level forest disturbance effects on primary production leaf chemistry and lemur populations Ecology 76 2084 2096 Ganzhorn J U Arrigo Nelson S Boinski S Bollen A Carrai V Derby A et al 2009 Possible fruit protein effects on primate communities in Madagascar and the Neotropics PloS One 4 e8253 Glander K E 1982 The impact of plant secondary compounds on primate feeding behavior Yearbook of Physical Anthropology 25 1 18 Goering H K amp Van Soest P J 1970 Forage fiber analysis United States Department of Agriculture Goodall J 1986 The chimpanzees of Gombe Boston Houghton Mifflin Gould L Power M L Ellwanger N amp Rambeloarivony H in press Feeding behavior and nutrient intake in spiny forest dwelling ring tailed lemurs Lemur catta during early gestation and early to mid lactation periods Compensating in a harsh environment American Journal of Physical Anthropology Hall M B amp Keuler N S 2009 Factors affecting accuracy and time requirements of a glucose oxidase peroxidates assay for det
61. tional Ecology 553 difficult to assess the quantities and biological activity of tannins Several recent reviews highlight these challenges and offer ways forward Foley and Moore 2005 Rautio et al 2007 Rothman et al 2009b A popular practice in primatology is to use the acid butanol assay Chapman and Chapman 2002 Powzyk and Mowry 2003 but the standard used Quebracho is not usually similar to the tannins within primate foods leading to erroneous results Rothman et al 2009b If this assay is employed we suggest that primatologists purify tannins from plant species of interest or report tannins in a qualitative fashion Rautio et al 2007 Rothman et al 2006a 2009b Such qualitative methods can apparently be performed in the field Lucas et al 2011 A newly developed assay uses fungal cellulases to simulate gut microbes and polyethyene glycol to account for the potential effects of tannins on digestibility DeGabriel et al 2008 Felton et al 2009b This measure may provide a useful estimate of food digestibility when tannins are present However it should also be viewed cautiously because it is still unclear what physiological adaptations primates have for coping with tannins such as salivary proteins and fungal cellulases do not necessarily have functions similar to those of gut microbes Mould et al 2005 Fat High fat foods are important energy sources for primates their caloric value exceeds that of carbohydrates
62. us component Goodall 1986 Malenky and Wrangham 1994 Reynolds et al 1998 Whether to remove seeds from fruit in nutritional analysis should be dependent on the subject s feeding behavior and nutritional questions asked If primates spit seeds then they should be removed before analysis If the primates chew the seeds and puncture the seed coat they are likely obtaining nutrients from the seeds and so the seed should be included in the assessment of the fruit s nutritional quality However if the primate swallows the seed the decision to include the seed is less clear Although there is evidence that a portion of the seed is altered as it is passed through primate guts Chapman 1995 Wrangham et al 1994 the majority of the seed is not Because seeds are typically high in protein and lipid and the seed coat high in fiber estimations of seeds in fruit samples will probably elevate estimations of fiber lipid and protein intake Conklin and Wrangham 1994 Milton 2008 Urquiza Haas et al 2008 In addition seeds fill the gut and reduce the amount of additional food the consumer can obtain Where studies of diet digestibility are of interest if seeds are included in the fecal sample output but not the diet intake input this will confound analysis Rothman et al 2008b As we have stressed repeatedly here again fruit samples should thus be processed according to the question posed In general we recommend that primatologists obtain the
63. wain T Goldstein S Richards A F amp Leighton M 1988 Chemical correlates of rhesus monkey food choice The influence of hydrolyzable tannins Journal of Chemical Ecology 14 213 235 Martin P amp Bateson P 1986 Measuring behaviour 2nd ed Cambridge UK Cambridge University Press g Springer Methods in Primate Nutritional Ecology 563 Martinek R G 1964 Stabilized tungstic acid reagent for blood deproteinization and glucose stabilization Chemist Analyst 53 108 109 Mau M Sudekum K H Johann A Sliwa A amp Kaiser T M 2009 Saliva of the graminivorous Theropithecus gelada lacks proline rich proteins and tannin binding capacity American Journal of Primatology 71 663 669 Mau M Martinho de Almeida A Coelho A V amp Sudekum K 2011 First identification of tannin binding proteins in saliva of Papio hamadryas using MS MS mass spectroscopy American Journal of Primatology 73 1 7 McCabe G M amp Fedigan L M 2007 Effects of reproductive status on energy intake ingestion rates and dietary composition of female Cebus capucinus at Santa Rosa Costa Rica International Journal of Primatology 28 837 851 Mertens D R 1992 Critical conditions in determining detergent fibers Paper presented at the Proceedings of the NFTA Forage Analysis Workshop September 16 17 Denver CO Milton K 1979 Factors influencing leaf choice by howler monkeys A test of
64. y fiber is typically considered the carbohydrate portion that animal enzymes cannot digest Van Soest 1996 This portion includes structural carbohydrates that occur in the plant cell wall and are either insoluble hemicellulose and cellulose or soluble gums pectin 8 glucans and various other carbohydrates such as fructans The soluble fibers are probably completely available to most primates through fermentation The fermentation of hemicellulose and cellulose varies with gut morphology and physiology and attributes of gut microbes hosted The insoluble fiber including hemicelluloses i e 5 carbon sugars is heterogeneous in its composition whereas celluloses 6 carbon sugars are fairly homogeneous Lignin is a structural component of the cell wall that is not well characterized chemically but can be considered the most significant factor limiting the availability of plant cell wall material to animal herbivores Van Soest 1994 A detailed overview of the carbohydrates and lignin is available elsewhere Van Soest 1994 as well as a glossary of nutritional terminology Conklin Brittain et al 2006 Measuring the quantities of specific simple sugars such as glucose and fructose is usually performed via HPLC Reynolds et al 1998 Riba Hernandez et al 2005 Simmen and Sabatier 1996 Colorimetric assays are not adequate to separate specific sugars but do provide a quantitative estimate of sugars in a sample A common approach is to ass

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