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Stable carbon isotope analysis and maize-stalk beer diet in rats : implications for the origins of maize 2006

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S T A B L E C A R B O N I S O T O P E A N A L Y S I S A N D M A I Z E - S T A L K B E E R D I E T I N R A T S : I M P L I C A T I O N S F O R T H E O R I G I N S O F M A I Z E by M A R I A C E C I L I A C A N A L Licenciatura en Antropologia , Univers idad Nac iona l de L a Plata, 2000 A T H E S I S S U B M I T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F A R T S In T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Anthropology) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A October 2006 © Mar ia Cecil ia Canal, 2006 A B S T R A C T Maize is one of the world's most important staple crops but theories explaining the ancestry of maize are focused mostly on domestication as it relates to food for human consumption. Much research was conducted on the wild ancestor of maize; current trends support teosinte as the ancestor of maize; but the question that remains unexplained is why people initially cultivated teosinte considering the plant has so little yield? Smalley and Blake (2003), elaborating on a concept proposed by litis (2000), explored this question. litis argued that the ancestor of maize was first domesticated for its sugar content. Building on this idea, Smalley and Blake suggested also the possibility of making alcoholic beverages. This suggestion that the ancestor of maize was selected for its sugar content changes the focus of early maize research. Maize is found in the archaeological record at 5400 B.P. but was not yet a staple food crop. Researchers must consider alternate uses early Mesoamerican people had for this plant. The production of alcohol from the sugary maize stalk is an example of an alternate use for maize. In an attempt to understand what occurred between the initial appearance of maize as a crop and its use as a staple food source, researchers have been studying the plant's C 4 photosynthetic pathway and its impact on bone chemistry. The carbon isotope signature in human bone resulting from the consumption of maize is quite different depending on whether the maize is eaten direcdy as food or first converted to alcohol before being consumed. This study tests the hypothesis that C 4 carbon from maize-stalk beer leaves a signature in bone collagen. Rats were used in a feeding experiment to determine if a diet with a significant component of maize-stalk beer would elevate the stable carbon isotope ratios in the consumers. Results of this experiment showed that maize-stalk sugar converted to alcohol did not raise the stable carbon isotope ratio measured in the rats. This suggests that archaeologists must look for raised stable carbon isotope values in apatite, rather that collagen, if they are to detect maize alcohol use in ancient populations. i i T A B L E O F C O N T E N T S • Abstract » • Table of Contents «i • List of Tables v • List of Figures vi • Acknowledgements vii • Dedication viii • Introduction 1 • Background 5 Importance of Maize today The Ancestor of Maize - Teosinte 6 Spread and Timing of Maize 7 • Theory and Methods of Isotope Analysis 9 . • Consumption of Maize Products and Carbon Ratios 11 Making Maize-stalk Beer 12 • Experiment Design 14 • Experimenting with Rats 16 Animal Care During the Experiment 17 Methods of Alcohol administration 18 - The Liquid Diet 20 • Experimental Procedures 23 • Expectations of the Experiment 24 • Results 24 • Conclusion 29 iii Bibliography 32 Appendix 1. Rats' Weight Gain 36 Appendix 2. UBC Ethics Committee Approval Document 39 iv LIST O F T A B L E S Table 1. Composition and Diet of Experiment Groups 16 Table 2. Lieber-deCarli Liquid Diet 21 Table 3. Custom made Liquid Diet #710341 21 Table 4. Results of Isotopic Analysis from Group 1 (C 4 solid diet) 25 Table 5. Results of Isotopic Analysis from Group 2 (Maize-stalk beer diet) 26 Table 6. Results of Isotopic Analysis from Group 3 (C 3 solid diet) 27 Table 7. Results of the Diet Analysis 27 v LIST O F F I G U R E S Figure 1. Distribution of Carbon Ratios for Individual Rats vi A C K N O W L E D G E M E N T S I would like to thank a number of people who have helped me throughout this research. At UBC there are several archaeologists who deserve special mention. My thesis supervisor, Dr. Michael Blake has been generous with his assistance, guidance and kindness begmning with my application to UBC and continuing through the process of choosing the courses as well as assisting with this thesis research and experiment. Most importantly, I am indebted to him for suggesting this topic and for allowing me to be a part of this research. Dr. Brian Chisholm was an important supporter of this research with his scientific expertise but most importantly for showing me that there is a human part to grad school. Dr. Michael Blake and Dr. Brian Chisholm provided financial support for this research. I am grateful to Dr. R G. Matson and Dr. David Pokotylo for providing me with the opportunity and invaluable experience as their teaching assistant, as well as for the interesting talks we had about archaeology. I am also grateful to Dr. Zhichun Jing and Dr. Sue Rowley for the interest they generated in me while teaching me "other" archaeologies. Thanks to Patricia Ormerod and Mandy Adams for all your help during the MA and for the talks we have had that helped more than they know. I am indebted for life to Nadine Gray, who helped me do so many things during this MA, that it is impossible to mention them all. But most important, for the invaluable and uncountable hours of helping me edit my English, over and over. And for her friendship, that will last more than just the UBC period. During the research process I would like to thank the following: Dr. Joanne Weinberg for all the information she willingly provided about rats, diets and feeding methods. Thanks to Dyets Inc. and Test Labs Inc. for all the help with the development of the diets. Dr. John Pinel provided the lab space, and technicians Anne Cheng and Alice for their help. Thanks to Chris Ames for helping me with the sampling process. I am indebted to Lucille Hoover for her invaluable help and guidance during the whole experiment. Honestly, I don't know if I could have done this without your help. The same can be said for John Smalley and Carlo Baroccio for their expertise in beer brewing and their ability to answer endless questions, Cheers! On a more personal note I would like to thank Dr. Rita de Grandis and Dr. Blanca Muratorio at the beginning of this MA and for the good time we spent in Guatemala. Thanks to Dominion Tours Inc. for providing a job every summer. I also want to thank my brother Ernihano and all my friends and family, from Canada and Argentina, for being interested and encouraging during the whole process. Thanks to Vicky Kenny, who even though was pregnant with Olivia and always nauseous, went with me to the Lab to feed, clean and talk to the rats. Thanks to Dr. Maria Carbonetti, who made me believe I could do an MA at UBC and my grandmother Maria Rosa for all the encouragement over the years and to my in-laws who, even though they did not know much about what I was doing with the rats, were always there for me. Thanks to my brother Gabriel for long conversations deciding our futures, support while leaving Argentina and his ability to listen and encourage me. I cherish his wife Florencia, who never hesitates to help me and is now the sister I never had. My strongest supporters are my parents, Gloria and Jorge Canal, who always believed in me and encouraged me to be whatever I wanted to be. They provided financial but most importantly, emotional support over the years and for making life in Canada a possibility. Last but not least, to Tete, for always believing in the righteousness of my decisions, for his unconditional support and love over this long process. vii This thesis is dedicated to (Nora Zagorodny and 'Barbara (Baksta, for shouting me. that a future in archaeology was possible.. 9\ndto Tete, of course. Quiero dedicar esta tesis a (Nora Zagorodny y a (Barbara 'Baksta, por mostrarme que era posibte un futuro en !Arqueo(ogia. <y a Tete, por supuesto. vii i I N T R O D U C T I O N Maize is one of the world's most important staple crops but theories explaining the ancestry of maize are focused mosdy on domestication as it relates to food for human consumption. For more than 100 years, archaeologists, geneticists and botanists have studied the origin of maize and many theories have been proposed regarding how this crop evolved (Bennetzen et al. 2001; Gallinat et al 1984; Piperno and Pearsall 1998, Pope et al 2001). However, the most important aspects of this issue remain unexplained. As Mangelsdorf et al. (1964:538) explain, "a living wild form of corn has never been discovered, despite the extensive searches for it which have been carried on in various parts of the hemisphere." Much research has been conducted on the wild ancestor of maize and current trends support the teosinte hypothesis proposed by Beadle in 1939 (Beadle 1980), a geneticist (Doebley 2004:39-40). The teosinte hypothesis suggests that the wild annual grass, teosinte, is the sole progenitor of maize (Beadle 1980; Doebley 2004:40). Benz (2006:9) defines teosinte as an English term adapted from the Nahuat "tecintli" (good or evil grain) used widely to refer to the seven taxa of wild grasses that are closely related to maize. One of the seven taxa, Zea mays ssp. parvigumis, exhibits a close genetic relationship with maize and because of this evidence, teosinte is regarded as the ancestor of maize (Doebley 2004:39; Matsuoka et al. 2002). The physical differences between teosinte and maize are striking. Teosinte is a tall thin grass with many branches of tasselled spikes that produce small ears, approximately 10 cm long, with two rows of seeds (litis 2006:31-33). The yield from the teosinte plant is minimal compared to the maize plant that produces large ears containing as many as 16 rows of seeds (see Figure 3- 15A-F in litis 2006:40). If teosinte is in fact the wild ancestor of maize, the question that remains unexplained is why people initially cultivated teosinte considering the plant has so little yield? Another aspect to consider is that, based on ethnographic accounts and archaeological data, teosinte was known both as a "starvation" and an unpalatable food by early peoples of 1 Mesoamerica (Beadle 1980; Coe 1994:33; Flannery 1973:290). Consequendy, this leads to the important question, why were people cultivating teosinte i f it was so difficult to eat? This question was explored by J o h n Smalley and Michae l Blake (Smalley and Blake 2003) w h o elaborate o n a concept proposed by H u g h H . litis (2000). litis (2000:36) argued that the ancestor o f maize was first domesticated not for its grain but for its sugar content. Bu i ld ing on this idea, Smalley and Blake (2003:675) suggested, "that the social importance o f alcohol product ion was a precipitating factor i n Zea's early and rapid spread." This suggestion that the ancestor o f maize was selected not for its grain parts, but for its sugar content, changes the focus o f early maize research. U n t i l now, researchers focused on maize's evolved characteristics, and not the initial ones such as small, hard kernels, small cobs and sweet stalks (Smalley and Blake 2003:675). A s maize is currently an important staple crop, it is difficult for researchers today to focus on its origin as something different, a source o f sugar or alcohol. However , when considering the initial plant characteristics noted above, maize ears as a "staple c rop" become less important compared to the stalk, because it is the stalk that offers a more easily accessible source o f sugar. Consider ing this new perspective, the question then becomes: what types o f archaeological data w o u l d be most useful for illustrating the importance o f the sugary teosinte or maize stalk for early peoples? There is paleoenthnobotanical evidence showing that domesticated maize (Zea mays) w i th cobs was present in G u i l a Naqui tz Cave i n Oaxaca, M e x i c o at least 5400 carbon-14 years B . P . (Piperno and Flannery 2001:2102). However , isotopic data suggests that maize d id not become a dietary staple in many parts o f Mesoamerica unt i l 2500 years later (Smalley and Blake 2003:684). Since maize is found i n the archaeological record at 5400 B . P . but was not yet a staple food crop, researchers must consider the range o f alternate uses that early Mesoamerican people had for this plant. The product ion o f alcohol f rom the sugary maize stalk is an example o f an alternate use for maize. 2 There are archaeological residues and ethnographic evidence for the use o f alcohol, such as chicha (beer made from sprouted maize), i n communal drinking, feasting, as we l l as i n other polit ical and social activities (Dietler 1990:362; Has tor f 1999; Has to r f and Johannessen 1994; Jennings et al. 2005; Mandelbaum 1965; Marshal l 1979; M o o r e 1989; Ubelaker et al. 1995). Recent research by Jennings et al. (2005) discusses the archaeological evidence regarding the product ion and consumpt ion o f alcoholic beverages by ancient populations around the wor ld . Adams (2004) and Jennings et al. (2005) note that archaeological research o n feasting has tended to focus o n the poli t ical aspects o f feasting. This focus o n feasting as a poli t ical event " . . .can obscure the labour and resources commit ted to growing, harvesting, and processing the food and drink that were consumed o n these occasions" (Jennings et al. 2005:275). F o r Mesoamerica, Bruman (2000) describes the arious types o f alcoholic beverages that can be made f rom plants and fruits indigenous to Mex ico . O f particular interest for this thesis is the product ion o f "corn-stalk" wine 1 , wh ich he argues was one o f the beverages that people consumed prior to the introduction o f sugarcane to aboriginal A m e r i c a (Bruman 2000:57). A c c o r d i n g to B r u m a n (2000:57) many explorers and chroniclers attested to the economic importance o f maize-stalk syrup and that some tribes w h o made syrup, may have k n o w n the process o f diluting it wi th water and al lowing it to ferment to produce an alcoholic drink. In summary, the archaeological and ethnohistoric evidence indicates that maize was present by 5400 years B . P . and that at least by the time o f Columbus the plant was used to produce alcoholic beverages as maize-stalk beer (Piperno and Flannery 2001, Bruman 2000). In an attempt to understand what occurred between the initial appearance o f maize as a crop and its use as a staple food source, researchers have been turning their attention to the study o f the plant's C 4 photosynthetic pathway and its impact o n bone chemistry (Tykot 2006:132). The carbon isotope signature i n human bone resulting from the consumpt ion o f maize may be 1 Although Bruman (2000) uses the term "cornstalk, the more common term used today is maize, so I wi l l use maize throughout. Also, although technically fermented maize stalk juice is a "wine", I refer to it as beer, after Smalley and Blake (2003). 3 quite different depending on whether the maize is eaten direcdy as food or first converted to alcohol before being consumed. The basic hypothesis this thesis is trying to test is that C 4 carbon from maize-stalk beer does not leave a signature i n bone collagen. I f Mesoamerican people were cultivating maize and consuming it as an alcoholic beverage, it may not be reflected i n stable carbon isotope ratios present i n bone collagen (the preserved organic parts o f bone). The reason for this is the fact that the por t ion o f the diet that gets routed to the bone collagen comes from protein, not fat or carbohydrates (Ambrose and N o r r 1993:2; Ch i sho lm , personal communicat ion; Smalley and Blake 2003:684; Ubelaker et al. 1995). Bu i ld ing on the evidence provided by the experiments conducted by A m b r o s e and N o r r (1993:27-28) and the literature survey by C h i s h o l m et al (1982) I suggest that i n general, alcohol does not bui ld protein, because it contains mostly calories, vitamins and very little protein. Because this has never been tested experimentally, I designed an experiment to measure the effects o f a maize alcohol diet o n collagen product ion i n rats. Rats were chosen as a proxi for human consumers, because they breed rapidly, can be easily handled wi th some practice, and can be housed i n large numbers i n a relatively confined area. Moreover , being a small animal, it is both economical and practical to use large numbers in an experiment. M u c h is now k n o w n about their physiology, anatomy, genetics and behaviour which suggests that meaningful results can be obtained from rats that, i f interpreted wi th care, can be extrapolated to humans (Waynford and Flecknel l , 1992). In the following section, I present the theories and methods for stable isotope analysis and for alcohol administration i n rats, as wel l as the experimental design to determine i f C 4 f rom maize-stalk beer leaves any traces in bone collagen. In the third section I explain the procedures and methods used in this experiment. In the final section I discuss the results, the implications and conclusions o f this study and its archaeological importance. 4 B A C K G R O U N D Importance o f Maize today: Maize has become one o f the most important grain crops i n the wor ld , but it was not always a staple food crop. Tyko t and Staller (2002:667) argue that western scholars tend to overemphasize the importance that maize played i n early diets. Ma ize was an u n k n o w n product i n the O l d W o r l d before 1492 (Mangelsdorf et al. 1964:538; Mangelsdorf 1974:1). Spaniards made the first European historical reference to maize o n N o v e m b e r 5, 1492. They were exploring the island o f C u b a and then reported to Christopher Columbus that they had found "a sort o f grain they called mai^ wh ich was wel l tasted, bak'd, dry'd, and made into f lour" (Mangelsdorf 1974:1). The Nat ive Americans had, independendy from other areas o f the wor ld , developed food production. B y the time o f Columbus , Nat ive Americans had a vast knowledge o f many kinds o f plants, wh ich they used for subsistence, ritual activities, as wel l as medicinal purposes (Mangelsdorf 1974:1). Included among these many plants was maize. B y the time o f the Spanish conquest, maize was the most important staple crop i n Mesoamerica. Columbus brought maize back to Spain, and from there it quickly spread throughout Europe , to N o r t h Af r i ca , the Midd le East, India and China . Maize has remained an important staple crop in the Americas , and all over the wor ld . A s Mangelsdorf (1974:2) explains, a crop o f maize matures somewhere i n the w o r l d every month o f the year. It grows i n a diverse range o f environments from 58° N latitude, i n Canada and Russia, to 40° S latitude, in the southern hemisphere. Fields o f maize grow below sea level i n the Caspian plain and at altitudes o f more than 12,000 feet (more than 3600 meters) i n the Peruvian Andes . Maize is g rown i n regions wi th less than ten inches o f annual rainfall i n the semiarid plains o f Russia and regions o f more than 400 inches o f rainfall o n the Pacific Coast o f Co lombia . Today, maize is g rown i n every suitable agricultural region o f the globe (Mangelsdorf 1974:2). B y 1974, maize was grown on 119,770,684 hectares (Ha) and produced an annual grain crop o f nearly 5 306,287,347 metric tonnes. B y 2005, maize was grown on 147,017,069 hectares around the wor ld and produced 692,034,184 metric tonnes ( F A O 2005). Maize has m u c h higher yield per hectare (47,072 tonnes /Ha) compared wi th wheat (28980 tonnes /Ha) , rye (22670 tonnes /Ha) and barley (24484 tonnes /Ha) ( F A O 2005). B y current standards, maize is easy to grow and is inexpensive to purchase. Because o f this, it has become the dominant food and main source o f dietary energy and limited protein for underprivileged segments o f society around the wor ld . Cult ivat ing and consuming maize wi th beans and squashes, has long been recognized to provide an excellent diet. Maize supplies carbohydrates, small amounts o f protein, and fat while beans are the principal source o f protein, essential amino acids and vitamins. Squashes are important i n supplying additional calories as well as V i t a m i n A and fat (in the seeds) (Mangelsdorf 1974:1). The Ances tor o f Maize - Teosinte T h e most widely agreed-upon candidate for the wi ld ancestor o f maize is a perennial grass named teosinte (Doebley 2004:41). Teosinte is the name for a group o f large grasses o f the genus Zea that inhabits Central and South Amer ica . There are five species o f teosinte k n o w n today: Zea perennis, Zea luxuriant, Zea nicaraguensis, Zea diploperenni's, and Zea mays. T h e species Zea mays is divided into four subspecies: ssp. huehuetenangensis, ssp. mays, ssp. mexicana, and ssp. parviglumis. Zea mays ssp. mays, (maize or corn) is the only domesticated taxon i n the genus Zea , and is derived directly from Zea mays ssp./>tfrag/#tf?zj.(Matsuoka et al. 2002: 6080) However , the processes that led early people to interact wi th teosinte, harvest and utilize the seeds, leaves and stalks, and engage in the transportation or trade o f the plant into new areas require more research (Blake 2006:55). A s Tyko t and Staller (2002:667) note, "our understanding o f the phylogeny, origins, chronology, and routes o f dispersal o f maize remains incomplete." 6 Spread and T i m i n g o f Maize A new study on the genetics o f Zea mays by Matsuoka et al. (2002) indicates that all modern maize evolved from teosinte {Zea mays ssp. parviglumis) w h i c h originated i n the R i o Balsas drainage o f West Mex ico . F r o m there it spread out into new habitats, f rom eastern Canada to northern Chi le (Matsuoka et al. 2002: 6080). Independent domestications were proposed for maize, based o n morphological and genetic diversity (Gallinat 1988) however, this diversity can be explained by a single domestication and subsequent diversification (Matsuoka et al. 2002: 6080). F r o m an early diversification i n the Mex ican highlands, two lineages are proposed for the dispersal o f maize throughout the Americas (Blake 2006:55-59). O n e path leads f rom western and northern M e x i c o , into the southwestern U . S . and north to Canada. T h e second path can be traced from the Mex ican Highlands to the western and southern Lowlands o f M e x i c o , into Guatemala, the Caribbean Islands, the Lowlands o f South A m e r i c a and the Andes Mountains (Matsuoka et al. 2002: 6084). Researchers who study early maize have begun to focus their attention o n the direct dating o f maize macroremains using Accelerated Mass Spectrometry ( A M S ) radiocarbon dating (Blake 2006:56). Da t ing the early spread o f maize begins wi th the 5400 B . P . date from G u i l a Naqui tz Cave, Oaxaca (Piperno and Flannery 2001:2102) and San Marcos Cave i n the Tehuacan Valley (4700 ± 110 B.P.) (Benz and litis 1990; Benz and L o n g 2000). Blake (2006:55-59) summarizes the most recent data relating to the initial spread o f Zea mays from this region. The South to N o r t h progression o f maize dates suggests that maize spread slowly northward from its homeland i n the R i o Balsas drainage o f West M e x i c o to Tamaulipas (Romero's Cave 3903+ 50 B.P . ) , into Chihuahua (Cerro Juanaqueha 2980 ± 50 B.P.) and into the Amer i can Southwest (Fresnel Shelter, N e w M e x i c o 2945 ± 55 B.P.) where it then eventually spread into eastern N o r t h Amer ica . Matsuoka et al. (2002:6083) suggests that it is possible to estimate the date o f the origin o f maize as a single event using D N A microsatellite data. Microsatellite data utilizes a large number o f loc i 7 to construct the entire genome instead o f focussing on a single gene region (Benz 2006:13; Matsuoka et al. 2002:6080). A c c o r d i n g to Matsuoka et al. (2002:6083) teosinte, ssp. ^arviglumis and Mex ican maize have an average divergence date o f 9188 B . P (with a 9 5 % confidence interval between 5689 — 13093 B.P.) wh ich occurs more than 3500 years before maize was incorporated into the Tehuacan Cave deposits (Benz 2006:13) and is consistent wi th the archaeological estimates that crop domestication in Mesoamerica did not precede 10,000 B . P . (Matsuoka et al. 2002: 6083). Consider ing these early dates for maize manipulation in Mesoamerica, it is important to understand how people were uti l izing maize. O n e o f the ways to determine relationships between people and plants is to test stable isotope ratios i n bone collagen to determine h o w people were consuming maize. Smalley and Blake (2003:684) summarize published data o n stable carbon isotope analysis o f 622 individual human remains recovered from numerous South Amer i can and Mesoamerican sites. The pattern that emerges indicates that stable carbon isotope ratios increased, suggesting increasing reliance on maize in the diet, from the first appearance o f maize i n Central M e x i c o to the time o f the Spanish Conquest. In most regions, wi th few exceptions, the shift to higher stable carbon ratios, reflecting significant maize consumpt ion i n the diet, d id not occur unt i l after 3000 years ago. A s Smalley and Blake (2003:684) suggest, moderate to high stable carbon ratios are represented by values greater than —15.0%o. W h e n stable carbon ratios have values greater than -15.5%o i n non-marine environments, individuals are consuming a significant percentage o f their diet from C 4 plants, such as maize. I f maize has been present in the archaeological record (as seen i n micro and macro botanical remains) for 5000 years, yet not generally a dietary staple unti l after 3000 B . P . (Blake 2006:66) when higher stable carbon ratios are recorded, then it is logical to think o f an alternate explanation for the first use o f maize. 8 T H E O R Y A N D M E T H O D S O F I S O T O P E A N A L Y S I S Carbon, present i n the atmosphere as C O z , is incorporated into plant tissues by photosynthesis. Plants can be divided into three different categories. O n e category, plants that use the Calv in-Benson or C 3 photosynthesis pathway, generates a three-carbon molecule (phosphoglyceric acid) as the first photosynthesis intermediate. T h e second category is plants that use the C 4 , or Hatch-Slack pathway, wh ich incorporates the C 0 2 carbon into a molecule that contains four carbon atoms (oxaloacetate) as its first intermediate product (Chisholm 1989:12). The third group, "plants uti l izing the Crassulacean A c i d Metabol i sm ( C A M ) for carbon dioxide fixation have an isotopic content similar to C 4 plants" (Vogel and van der M e r w e 1977:239) when growing i n conditions that favour C 4 plants. Examples o f the Calv in-Benson or C 3 plants include most o f the flowering plants, trees and shrubs, and most o f the temperate zone grasses. O n l y ten plant families represent the Ha tch- Slack or C 4 plants. The most interesting for us, because they are part o f human diets, are maize, millet, sorghums, cane sugar and chenopods. The Crassulacean A c i d Metabo l i sm ( C A M ) plants include the pineapple and various cacti, some o f wh ich may be used as a food source (Chisholm 1989:12). A s the photosynthetic pathways diverge chemically they produce different degrees o f isotopic fractionation. This has been utilized to classify species as being C 3 , C 4 or C A M . Isotopic fractionation is . . . " the selection for or against one or more isotopes o f an element during the course o f a chemical or physical reaction. A s a result there is a change i n the relative concentration o f the isotopes involved i n the reaction" (Chisholm 1989:12). A s C h i s h o l m and K o i k e (1996:201) note, "...the values for meat are only slightly displaced from those o f the foods that the animals eat, wh ich may allows us to average meat and plants f rom the same food chains together to form human's alternative food groups." It is wor th ment ioning here that when marine 9 or terrestrial herbivores eat plants, their metabolism selects and recombines plant chemicals, resulting i n further fractionation o f the carbon isotopes (Chisholm 1989:13). Isotope ratio measurements are reported using the delta notation (for example 8 1 3 C , 8 1 5 N ) relative to international standards and are expressed i n parts per m i l (%o) (Tykot and Staller 2002: 669). M o d e r n C , plants have average values o f about -26.5 %o whereas modern C 4 plants have averages o f about -12.5 %o. This separation o f 14%o allows for discrimination between group averages. C A M plants, such as pineapples, agave and cacti, also produce high stable carbon ratios (Blake 2006:66) and according to Tyko t (2006:132), can switch between C 4 and C , pathways depending upon both their environment and geographic location. T h e analysis o f stable isotopes o f carbon from preserved bone has been used to study past diets and past environments (Ambrose and N o r r 1993; Burger and van der Merwe 1990; C h i s h o l m and K o i k e 1996; Tyko t and StaUer 2002; Ubelaker et al. 1995; van der M e r w e et al 1993; V o g e l and van der Merwe 1977). Stable carbon isotope analysis is particularly useful i n N e w W o r l d dietary studies (Tykot and Staller 2002; Ubelaker et al. 1995) because maize is often the only C 4 plant contributing significantly to past human diets (Tykot and Staller 2002: 670). The current trend for researchers interested in ancient diet involves both carbon and nitrogen isotope analysis ( D e N i r o and Epste in 1981; Tyko t 2006:134). Stable isotope analysis o f nitrogen is a clear indicator o f "...the effects o f climate and environment o n bo th plant and animal values and trophic level increases i n both terrestrial and marine ecosystems." A s Ubelaker et al. (1995:404) discuss, stable isotope analysis o f nitrogen is useful i n the study o f past human diet wi th regard to individuals who have more positive 5 1 5N values when compared to adults and older children. In general, nitrogen isotope ratios increase 2 to 3%o wi th each trophic level so terrestrial plants and animals have much lower values than fish and mammals from freshwater or marine environments. T h e nitrogen value o f maize (815N) is 2-3%o while freshwater and marine mammals and fish have a 8 1 5 N value o f 9-18%o (see Figure 10-2 i n Tyko t 2006:134). 1 0 U s i n g stable carbon and nitrogen isotopes for diet reconstruction is based on the assumption that you are what you eat. It is assumed that the carbon isotopic composi t ion in animal tissues is a direct and constant function o f the diet (Ambrose and N o r r 1993:1). A s previously mentioned, collagen i n consumers is made from the protein por t ion o f the diet, not f rom fats or carbohydrates (Ambrose and N o r r 1993:27-28). A m b r o s e and N o r r (1993:27-28) demonstrate that the value o f rat collagen largely reflects dietary protein, but it is poorly correlated wi th the whole diet when the isotopic composi t ion o f protein and non-protein components differ considerably. Therefore carbon i n dietary proteins is routed mainly to collagen (Chisholm et al 1982), rather than scrambled wi th that i n carbohydrates and lipids. An o t h e r source o f information comes from apatite (found mostly i n bone and tooth enamel) w h i c h is the inorganic por t ion o f bone and that reflects whole diet. Bone apatite carbonate provides the most accurate measure o f the energy por t ion o f the diet (Ambrose and N o r r 1993:27-8) while the protein o f the diet is routed to the bone collagen. F o r this thesis, I am interested i n the reconstruction o f diet by measuring the ratios o f carbon-13 to carbon-12 and nitrogen i n the collagen (protein portion) o f rat bone 2 . CONSUMPTION OF MAIZE PRODUCTS A N D CARBON RATIOS B o t h chicha (maize beer) and maize-stalk beer do not bu i ld up m u c h protein, but mosdy calories and vitamins (Chisholm, personal communicat ion; Smalley and Blake 2003:684). Thus, consumption o f maize beer or maize-stalk beer may not have produced higher stable carbon isotope ratios i n human bone collagen. It is important to distinguish the differences between Chicha and maize-stalk beer. Chicha has two c o m m o n recipes that produce a similar product (Jennings et al. 2005:278-279). O n e recipe involves the grinding o f maize kernels into flour, wh ich is then masticated wi th saliva. The second recipe is made wi th sprouted maize kernels, wh ich are 2 A later study will examine the 5 n C values in the apatite portion of the rat samples. 11 then ground into flour (Bruman 2000:40-41). The next step in both o f these recipes involves adding water and bringing the mixture to a low boi l . A t this stage the l iquid is transferred to jars to allow fermentation, (see Jennings et al. 2005 for a thorough description). In contrast, maize- stalk beer, is made wi th the sweet juice from the stalk. This process involves steaming maize stalks unti l they become soft and opaque green-brown i n colour. T h e stalk is then pressed or squeezed, much like sugar cane, to remove the juice out o f the stalk. The juice is then placed i n a container wi th a narrow neck and spout until the l iquid ferments (John Smalley, personal communicat ion, 2004). The key to both processes is the fermentation o f the maize sugar into alcohol . In order to test the idea that alcohol made from maize wi l l not leave a C 4 signature i n bone collagen, maize- stalk beer was brewed to conduct a feeding experiment wi th rats. Ch i cha was not used in this experiment because its isotopic reading wou ld be identical to maize-stalk beer. Future experiments, however, w i l l include chicha, particularly recipes that might have a higher protein component. M a k i n g Maize-stalk Beer The maize was harvested in late September 2004. O n l y the maize stalk is needed to make beer but because the modern emphasis is o n the maize ears, stalks were more difficult to purchase. Ralph's F a r m Market, on the Fraser Highway i n Langley, Br i t i sh C o l u m b i a had a small section o f their farm, wh ich they planned to turn into a maize maze for visitors. However , the area set aside for the maze was too small and the maize plants were left to grow. T h e maize plants were approximately 1.82 m (6ft) in height and were a mixture o f various types o f maize. U s i n g a small knife, 41 kg o f maize plant were harvested from the farm. T o prepare the stalks for storage, the other parts o f the plant were removed (leaves, cobs and seeds) and the stalks were cut into quarters to fit into plastic freezer bags. The stalks were kept i n two home freezers unti l the beer making process began i n mid - October. Freezing the stalks (and, later on , the beer) was done in 12 order to stop all bacterial growth and chemical reactions. Freezing w o u l d not have had any impact on the chemical composi t ion o f the stalks or beer (Chisholm, personal communicat ion) . J o h n Smalley had made maize-stalk beer i n the past and was able to explain the methods used to extract the l iquid from the maize stalk. F o r the first batch, ~21 kg o f maize stalk were defrosted overnight and then cut into small pieces (about 10 c m long). T h e maize stalks were then steamed i n 12 litre pots filled wi th 500 m l o f water, for about 20 minutes or unt i l the color o f the stalk changed from bright green to an opaque green/brown. T h e steamed stalks were placed i n a manual grape press i n order to remove the juice from the stalks. It took a lot o f arm strength to get all o f the juice out o f every batch. F r o m the 21 kg o f stalk, 12 kg o f juice were obtained and 9 kg o f pressed stalk were left. A beer and wine hydrometer was placed i n the juice to test the amount o f sugar present. The first test showed that wi th the amount o f sugar present, after the fermentation process, the beer w o u l d have an alcohol content o f 3%. T h e relatively l ow amount o f sugar recorded from this l iquid is likely a result o f harvesting the maize late i n the growing season when most o f the sugar had already migrated from the stalk into the cobs (John Smalley and Michae l Blake, personal communicat ion, 2005). The 12 kg o f juice were transported to the Department o f An th ropo logy and Sociology at U B C for fermentation. Commerc ia l yeast {Saccraromyces bayanus, 5 g) for wine /beer making was then added to the juice and left to ferment for about 4 days i n a clean plastic bucket covered with a sheet o f plastic as a l id . After 4 days the juice fermentation was complete and the alcoholic content o f the beer was measured at 3%. The beer was bottled into 12, 1 litre plastic bottles, the k ind used to store homemade beer. The 12 litres o f homemade maize-stalk beer were stored in a home freezer unt i l needed. A s the experiment progressed, more beer was needed but maize stalk was not available for harvest. The first batch was fermented a second time wi th the addition o f commercial maize sugar to increase the alcoholic percentage to a measurement o f 12%. The beer 13 was then left to ferment for another 10 days. A s the maize sugar is also a C 4 product, no potential impact was envisioned by this decision. A second batch o f beer was made wi th a different method i n late May , using the remaining ~21 kg o f frozen stalk. The stalks were defrosted overnight and cut i n 10 c m long pieces. Then , all the stalk pieces were placed i n a container (able to retain the heat), and boi l ing water was added unt i l all the stalks were covered. A s stalks tend to float, a weight was put on top o f the stalks to keep them submerged. This "tea" was left to infuse for 2 hours, then, 12 litres o f l iquid were extracted and deposited into a large glass botde for fermenting. T h e extraction process involved a long plastic tube, wh ich was connected to a copper pipe. Ice was packed around the copper pipe and served to coo l the hot "tea" as it was extracted. O n c e the "tea" reached the large glass botde it was left to cool . In order to reach the desired 12% alcohol content maize sugar was added to the juice. The juice was stored i n a large glass botde, and 5 grams o f yeast [Saccraromyces bayanus) was added to the juice. T h e botde was stored i n a r o o m at 24°C. T h e glass bottle was covered wi th a dark cloth i n order to keep the light out. In the spout o f the bottle, a pump was added, al lowing the gasses to escape and also preventing any contamination from the outside environment. The fermentation for an alcoholic content o f 12% took 10 days. E X P E R I M E N T D E S I G N The main purpose o f this experiment is to determine i f C 4 carbon f rom maize-stalk beer leaves any discernable traces i n rat bone collagen. T o do this, rats and not mice were used as consumers o f maize-stalk beer, because rats are larger and thus provide more bone collagen than mice. Bone collagen, tooth, hair and muscle samples were taken from second generation rats raised o n the same diets as their mothers. The second generation rats were selected because their diet had been controlled from the prenatal to postnatal stage. Consumers f rom the second 14 generation were alive for about 70 days or unti l they reached between 275-300 grams, i n order to even out irregularities in diet, the effects o f weaning and sexual d imorphism. The rats were divided in three groups: G r o u p 1, G r o u p 2 and G r o u p 3. Th is experiment analyzed 12 individuals i n G r o u p 1,10 individuals in G r o u p 2 and 12 individuals i n G r o u p 3. The special diet material for G r o u p 1 ( C 4 solid diet) and G r o u p 3 ( C 3 solid diet) was standard rat chow made by the company Test Diets Inc. ( P M I ® Nu t r i t i on International rodent diet #5012). T h e Test D ie t Inc. formula #5012 supplies complete life-cycle nutri t ion specifically designed to support reproduction, lactation, growth and maintenance o f rats. It is low in cholesterol content, wi th an increased level o f unsaturated fatty acids compared to other rodent diets. Some changes were required from the original formula because o f the C 4 ingredients that w o u l d have affected the carbon ratios. A s seen in Table 1, for G r o u p 1 ( C 4 sol id diet), all other sources wi th C 4 - l ike values, except maize were removed from the original formula and replaced wi th C 3 ingredients. The amount o f maize present in the formula was 70%. F o r G r o u p 3 ( C 3 solid diet), all sources o f C 4 were removed from the diet and exchanged for 100% C 3 ingredients. The Maize-stalk Beer diet o f G r o u p 2 was a custom made powdered diet (Dyets Inc. Formula #710341), wh ich was mixed wi th water and maize-stalk beer. F o r this powdered diet, maize o i l was replaced wi th soybean o i l , and the alcohol from the maize-stalk beer was the only source o f C 4 . It is important here to state that the U B C A n i m a l Care Centre d id not allow for a pure (100%) maize diet, or a pure maize-stalk beer diet because o f the nutritional concerns for the rats. Vi tamins and minerals had to be added to the diet in order to ensure a we l l balance diet. In order to confront to these requirements, the diets could not be pure C 4 (Group 1 diet) or only maize- stalk beer (Group 2), supplements o f vitamins and minerals were added to the diets o f all groups. 15 Table 1. Composition and Diet of Experiment Groups Group 1: Group 2: Group 3: (Ct solid diet) (Maize-stalk Beer diet) ( C 3 solid diet) Generation one: Pregnant Generation One: Pregnant Generation One: Pregnant mother mother mother Generation Two: 12 pups Generation Two: 10 pups Generation Two: 12 pups Diet: 70% C 4 plant based (pure Diet: Custom made diet Diet: C3 plant based + water maize) + 30% C 3 plant based + #710341 (Ci) + maize-stalk water beer (C4). N o other source of liquid. E X P E R I M E N T I N G WITH RATS Three pregnant rats3 arrived in the Department of Psychology4, on May 5th, 2005. The rats were housed individually and each was given a specific diet. The mother of Group 1 ( C 4 solid diet) was fed the C 4 diet (70% of maize + C3 + vitamins and minerals) and water. The mother of Group 2 (Maize-stalk Beer Diet) was fed the custom made liquid diet of C , + maize-stalk beer (the only C 4 source) with no other liquid. The mother of Group 3 (C , solid diet) was fed a 100% C 3 diet (no C 4 or maize) and water. The mother of Group 1 gave birth on May 6, 2005 to 10 males and 4 females. The pups remained with their mother until May 9lh, 2005, when 2 males were culled. The remaining Utter of 12 consisted of 8 males and 4 females. The mother of Group 2 gave birth on May 12th, 2005 to 14 pups, 8 males and 6 females. Five males and 5 females were kept, and were fed maize-stalk beer once they reached 25 days of age. The mother of Group 3 gave birth on May 12th, 2005 to 16 pups, 4 males, 11 females and one stillborn. Four males and 8 females were selected. Sprague-Dawley: A strain of albino rats developed by the Sprague-Dawley Animal Company, widely used in experimental work because of their calmness and ease of handling (NDI foundation 2006) 4 The feeding experiment took place in UBC Department of Psychology, Room #4310. Facilities in this department were ideal to conduct the experiment, as they are currently working in etho-experimental studies of rodent defensive behaviour and learning. 16 A n i m a l Care D u r i n g the Exper iment T h e cage o f G r o u p 1 was changed every 2 days and food and water were added when needed. T h e mother and pups were moved to a bigger cage when the pups were 10 days old. W h e n the pups were weaned they were pair-housed according to sex. T h e four females were divided into 2 cages while the 8 males were paired into 4 cages. T h e cage o f G r o u p 2 was also changed every 2 days but their feeding routine was different. The l iquid diet was administered every morning. The amount o f l iquid diet given changed f rom 100 m l per day o f the non-alcoholic diet (consumed by the mother during lactation) to 1200 m l per day just prior to the pups being weaned because the pups were now drinking. In accordance wi th the U B C A n i m a l Care Commit tee, the maize-stalk beer was kept out o f the diet unti l the pups were 25 days o ld and then alcohol was gradually added. O n c e the pups were weaned, they were housed according to sex. There were four cages; Cage 1 held 2 females, Cage 2 held 3 females, Cage 3 held 3 males and Cage 4 held 2 males. The cage o f G r o u p 3 was changed every 2 days wi th food and water added when needed. The mother and pups were moved to a bigger cage when the pups were 11 days old . O n c e the pups were weaned, they were pair-housed according to sex. There were 2 cages that held 2 males each and 4 cages that held 2 females each. O n c e the second generation rats had reached 275-300 grams, the experiment was completed. Fo l l owing the U B C Ethics Commit tee procedures for animal care and treatment, the second generation rats were euthanized (see Append ix 2 - U B C Ethics Commit tee A p p r o v a l Document) . There are many methods o f euthanasia. In selecting a method, the prime requisite is that it must be carried out humanely, causing only the absolute m i n i m u m amount o f anxiety and pain to the animal. The methods commonly used for rats include: an overdose o f anaesthetic, carbon dioxide, decapitation (guillotine), cervical dislocation and microwaves. Whi l e selecting a 17 method, it is important to take into account the purpose for wh ich the animal is being kil led (Waynford and Flecknel l , 1992). T h e method that was chosen was an overdose o f carbon dioxide ( C O _ . This method was selected i n part because the laboratory had the equipment i n place, because it was less intrusive and because it w o u l d not interfere wi th the carbon isotope analysis. A s the pups were born on different days, the euthanasia o f the second generation took place o n different dates. The euthanasia was carried out for G r o u p 1 on July 11* 2005, and o n July 19 t h , 2005 for G r o u p 2 and G r o u p 3. In the "Euthanasia r o o m , " C 0 2 was pumped into the cage for at least 5 minutes; the rat's heartbeat was moni tored to ensure they were not longer alive. After the euthanasia, G r o u p 1 remained i n a freezer i n the Department o f Psychology for 9 days while G r o u p 2 and G r o u p 3 were transported to the Department o f Anthropology and Sociology after only one day i n the Psychology Department's freezer. The mothers were donated to another project i n the Psychology Depar tment at U B C to be used as surrogate mothers. Before donating them, hair samples were taken for analysis (for a future experiment). Methods o f A l c o h o l Adminis t ra t ion i n Rat Populations Joanne Weinberg (1984:261-2; Weinberg, personal communicat ion, 2003) proposes four main methods to expose pregnant rodents to alcohol. These include; injection, intubation, a lcohol i n the dr inking water or, adding alcohol to a l iquid diet. B o t h injection and intubation have the advantage that a controlled dose o f alcohol can be administered and high b l o o d alcohol levels can be obtained. The major cri t icism o f these two methods is that both require a great deal o f handling o f the pregnant female and involve a fair amount o f stress for the rat. Since prenatal stress i n itself can affect hormonal , physiological and behavioural responses to rodent offspring, these may not be the best methods. A l s o , an increase i n fetal deaths a n d / o r resorptions, reduced 1 8 litter weights, and retarded postnatal growth has been observed using these methods (Gallo and Weinberg 1982; Wemberg 1984:261). Placing a lcohol i n the dr inking water and providing this as the only source o f fluid is regarded as the simplest method o f administrating alcohol. The majority o f rodents, however, w i l l not consume alcohol voluntarily, and i f it is placed pure i n the dr inking water, they w i l l reduce their fluid intake. W i t h water intake suppressed, the amount o f a lcohol ingested and therefore b lood alcohol levels are also reduced. Whi le using this technique, lower body weights, retarded growth, deficient skeletal and muscle development, and delayed eye opening have been observed (Weinberg 1984:261). The fourth opt ion involves adding alcohol to a l iquid diet. A l iquid diet is based on, casein, enriched wi th methionine and cystine sucrose, and o i l suspension mixture formulated to meet or to exceed the nutritional requirements needed (Lieber and D e C a r l i 1982: 523-26). This l iquid diet is then mixed wi th alcohol and water i n necessary quantities. Based o n the condi t ion that this is the only source o f nutrition, this form o f administering a lcohol to pregnant rats appears to be a more effective method. This method results i n greater and more consistent intake o f alcohol, and more consistently elevated b lood alcohol levels. Furthermore, it is less stressful to the pregnant rat than injection or intubation. Several concerns have been raised i n relation to this method. O n e is that animals consume greater amounts o f water wi th a l iquid diet than they w o u l d wi th a pelleted diet. It is possible that increased fluid intake could affect water balance and /o r kidney function, and thus contributed to fetal distress. The use o f a semi-purified diet formulated specifically for rodents, and tailored to meet the needs o f the particular experiment overcomes some o f the problems that may occur (Weinberg 1984:261-2). M a n y l iquid diets for rodents have been produced over the years; however, the most commonly used i n rodent labs throughout N o r t h A m e r i c a is the Lieber-deCarl i L i q u i d Diet . 19 The L i q u i d D i e t Pr io r to the Lieber-deCarl i diet, alcohol had been commonly administered to animals as part o f their dr inking water. W i t h this technique, however, a lcohol intake is insufficient to result i n sustained appreciable levels o f a lcohol i n the blood. This l ow intake results f rom a natural aversion by many animals for a lcohol wh ich was overcome by the feeding o f a lcohol through exclusive l iquid diets. (Lieber and deCarl i 1982). The composi t ion o f the Lieber-deCarl i l iquid diet formula is based o n amino acid, sucrose, and o i l suspension mixtures formulated to meet or to exceed the nutrit ional requirements needed. The adequacy o f the diet was illustrated by the fact that when fed ad libitum, this type o f diet promoted growth (in a way) comparable to that o f commonly acceptable commercia l diets. However , when alcohol was brought into the diet, spontaneous food consumpt ion decreased, but it was nevertheless sufficient to ensure continued growth o f the animals. T o avoid the need for expensive amino acids, the original formula was changed to substitute the amino acids wi th casein, enriched wi th methionine and cystine (Lieber and deCarli , 1982:523-26). The Lieber-deCarl i l iquid diet has three variants; The H i g h Prote in variant o f the diet is useful for conditions such as gestation and lactation that require increased protein consumption (Lieber and deCarl i 1982:529-30). The ingredients o f the H i g h Protein o f the Lieber-deCarl i l iquid diets are shown i n Table 2. D u e to the need for high nutrit ion during pregnancy and lactation, the H i g h Protein variant was used i n this experiment. The diet was custom made by Dyets Inc. i n the U S A . T o meet the requirements o f the experiment, the maize o i l was replaced by soy o i l and an increment o f the other oils already present i n the formula (olive and safflower) i n order to control the amount o f C 4 i n the l iquid diet shown in Table 3. B y doing this, the only C 4 the rats were exposed to was obtained from the maize-stalk beer, not f rom the l iquid diet. 20 Table 2. Lieber-deCarli Liquid Diet Table 3. Custom made Liquid diet Ingredients Grams/L Ingredients Grams/L Casein 57.6 Casein 57.6 L-cystine 0.65 L-cystine 0.65 D L - m e d i i o n i n e 0.4 DL-me th ion ine 0.4 Maize O i l 2.5 Soy bean O i l 2.5 O l ive O i l 8.4 O l ive O i l 8.4 Safflower o i l 2.7 Safflower o i l 2.7 Dextrin-maltose 155.6 ** Dextrin-maltose 66 V i t a m i n M i x 2.5 V i t a m i n M i x 2.75 Salt mix 8.75 Minera l mix 8.75 Chol ine bitartrate 0.53 Chol ine bitartrate 0.53 Sodium Carrageenate 2 Sod ium Carrageenate 2 Xan than G u m 3 Total Grams 241.63 Total Grams 163.03 ** in the alcohol formula, rc dextrin-maltose and 50 g of placed by 66.0 g of alcohol To make 1 Litre of Liquid Diet. Mix 163 grams of Dyets #710341 with 530 ml of maize stalk beer (12% alcohol) & complete with cold water to one litre and mix for 30 second in a blender. A l c o h o l interacts wi th nutrition i n many ways, and often, nutrit ion i n animal experiments is overlooked, even when there is a need o f nutritional control . W h e n alcohol is added to an animal diet, the animal may modify its nutritional intake because a lcohol has a high energy value (7.1 kcal/g) and may displace other foods in the diet. Calories i n a lcohol are not associated wi th vitamins, minerals, proteins or other essential nutrients. These calories are called "empty calories" and can result i n nutritional deficiencies. A l c o h o l also has an anorexigenic effect, and may compromise nutrient intake. It is wel l k n o w n that pregnant or lactating females, whose nutritional requirements are greater than non-lactating, can be i n a state o f nutritional deficiency when being fed alcohol i n large quantities (Weinberg 1984:262). 2 1 A s mentioned above, food intake and nutrient intake is invariably reduced i n animals and humans consuming alcohol i n large quantities. Investigators work ing wi th animal models have begun to include a control group, wh ich is "pair-fed" to the alcohol, i n order to control for this reduced intake. E a c h animal i n the control group receives control diet (with carbohydrates isocalorically substituted for alcohol) i n the amount consumed by its partner i n the alcoholic diet on the previous day. The inclusion o f a pair fed group enables the investigator to separate effects due to pharmacologic actions o f a lcohol from those related by a lcohol malnutr i t ion (Weinberg 1984:265). A l t h o u g h alcohol passes freely from the maternal circulation into the breast milk, and from there to the nursing infant, maternal under-nutrition can reduce the amount o f mi lk available and thus compromise offspring nutritional status. A l c o h o l can also directly affect mi lk secretion, and this could reduce offspring weight gain and harm nutritional status (Weinberg, 1984:266). A s the objective o f this experiment is to find out i f C 4 f rom maize stalk beer leaves any traces i n the bone collagen, and not the relationship between nutrit ion and alcohol , a "pair-fed" group was not used in this experiment. However , the nutritional status o f bo th mothers and pups was moni tored during the whole experiment to ensure that they were not under-nourished i n any way. Weight gain during the experiment was a very important issue to take i n consideration. In order to moni tor this, the rats were weighed every week to ensure weight gain. Append ix 1 illustrates the weight gain for individual rats i n every diet group compl ied wi th the standards outlined by the U B C A n i m a l Care Centre. 22 E X P E R I M E N T A L P R O C E D U R E S T h e euthanized rats were separated by diet and by cage, and were stored from m i d July unti l mid-September in a freezer i n the Anthropo logy Department. The 34 individuals were then transferred from the freezer to a fridge and stored for 24 hours. Unde r the supervision o f D r . Ch i sho lm, four samples were taken from each individual; a long bone sample f rom the hind leg, a por t ion o f muscle from the h ind leg, hair, and 2-4 teeth. In order to have samples for future study, a single h ind leg was removed for this experiment leaving the rest o f the body intact for long-term storage. T h e long bone samples were manually cleaned o f flesh, tendons, cartilage, and marrow. Soluble lipids (fat) were then removed by soaking the bone i n acetone ( C H 3 C O C H 3 ) until the bone was clean. The final step, to ensure collagen purification, involved a demineralization o f the bone using Hydrochlor ic A c i d (HCI). Collagen was then solubulized by heating the sample i n water at p H 3 ; it was then filtered, condensed by evaporation and freeze- dried (Ambrose and N o r r 1993:18-19; Ch i sho lm, personal communicat ion, 2005, C h i s h o l m and Blake 2006:162). The collagen samples where then sent to the Isotopic lab i n the Department o f Ear th and Ocean Science at U B C for stable carbon and nitrogen isotope analysis. The muscle material was manually removed from the bone samples, processed wi th acetone ( C H 3 C O C H 3 ) i n order to remove all the soluble lipids, and then transferred to a mortar and pesde for grinding. The crushing and grinding o f the muscle material involved adding more acetone when needed and then it was filtered through a thin paper filter and placed i n vials for long-term storage. A l s o placed i n long-term storage for future studies were 34 hair samples and 2-4 teeth taken from each o f the individuals. Ha i r samples were also taken from the 3 mothers pr ior to their transfer to another experiment. These samples i n long-term storage could be uti l ized, but not l imited to, experiments on bone apatite, tooth enamel, or collagen tests o f hair and muscle. I f necessary, the experiment on bone collagen discussed below could be replicated because the 34 individuals remain i n long-term storage. 23 E X P E C T A T I O N S O F T H E E X P E R I M E N T Consider ing the results o f the experiments conducted by A m b r o s e and N o r r (1993) and the Stalk-Sugar Hypothesis o f Smalley and Blake (2203), it was expected that the diet o f G r o u p 2 (maize-stalk beer diet) wou ld not fall i n the C 4 range o f the stable carbon isotope analysis because maize-stalk beer builds calories and vitamins, not protein. A s we know, collagen is formed from the protein por t ion o f the diet not from calories received from carbohydrates or fats. So, although the maize-stalk beer was brewed from parts (stalks) o f the maize plant (C 4 ) , when it is consumed i n the form o f an alcohol, it may not deposit in the collagen as an expected C 4 value. However , the G r o u p 1 diet was designed as a 70% C 4 diet wi th the expectation that when consumed it w o u l d produce a typical C 4 value (greater than -15%o) comparable wi th the studies by Blake (2006:66); C h i s h o l m (1989:13); Smalley and Blake (2003:684); Tyko t (2006:132); Tyko t and Staller (2002:669) and Ubelaker et al. (1995:404). A s a further test, the G r o u p 3 diet ( C 3 sol id diet) was expected to demonstrate values wi th in the typical range (-26.5%o to — 16%o) o f a diet that is derived from C , sources. Whi le the central goal o f this thesis focuses o n the consumpt ion o f maize-stalk beer and its stable carbon isotopic value, the diets created for G r o u p s 1 and 3 provide the necessary parameters to measure the values o f G r o u p 2. It was expected that G r o u p 2 (maize- stalk beer diet) w o u l d cluster closer to the individuals o f G r o u p 3 (C , solid diet) rather than the individuals i n G r o u p 1 ( C 4 solid diet) because the maize-stalk beer should not be recognized i n the collagen as a C 4 value. R E S U L T S Collagen was extracted from the bone samples using well-established laboratory procedures described above (see A m b r o s e and N o r r 1993:18-19; Br ian Ch i sho lm , personal communicat ion, 2005) and results from the stable isotope o f carbon and nitrogen analysis were received. A s the samples were divided in diet, cage number and gender (when applicable); so were 24 the results. Tables 4, 5 and 6 present the results o f the isotopic analysis o f stable carbon and nitrogen. Table 4, the results f rom G r o u p 1 ( C 4 sol id diet), shows a mean value o f 8 1 3 C -14.88%o, wh ich is consistent w i th the expectations for individuals consuming a moderate to high C 4 diet (i.e., is greater than -15%o). The mean for the 8 1 5 N value is 4.25%o, w h i c h is consistent wi th the expected values o f a individual consuming maize (C 4) or C 3 leguminous plants (see Figure 10-2 in Tyko t 2006:134; Ubelaker et al 1995:404). Table 4. Results of Isotopic Analysis from Group 1 (C4 solid diet) Sample C4 solid diet 6 1 5 N 6 1 3 C 1 Cage 2(F) Bone 4.56 -14.42 2 Cage 1 (F) Bone 4.47 -14.16 3 Cage 3 Bone 4.41 -14.62 4 Cage 1 Bone 4.25 -15.13 5 Cage 3 Bone 4.32 -14.96 6 Cage 1 (F) Bone 4.56 -14.76 7 Cage 4 Bone 4.14 -15.85 8 Cage 2(F) Bone 4.52 -14.50 9 Cage 1 Bone 4.24 -15.24 10 Cage 2 Bone 4.01 -14.92 11 Cage 4 Bone 4.15 -14.90 12 Cage 2 Bone 4.47 -15.11 M e a n 4.34 -14.88 Range 4.01 - 4.56 -14.16 to -15.85 The results from the G r o u p 3 diet can be seen in Table 5, and provide a mean o 1 3 C value o f —22.14%o, wh i ch falls wi th in the expected range o f individuals w h o consume C 3 plants wi th littie to no maize (-26.5%o to -16%o). The nitrogen values from this diet, w i th a mean o f 2.72 are again the expected values for individuals consuming either maize (C 4 ) or C 3 leguminous plants. Whi l e the carbon values from G r o u p 1 and G r o u p 3 diets indicate that the individuals are o n C 4 or C 3 diet respectively, the nitrogen values shows that both groups were consuming plants or terrestrial fauna, rather than fish or mammals. 25 Sample C 3 sol id diet 6 1 5 N 8 U C 16 Cage 4 B o n e 2.61 -22.49 17 Cage 5 Bone 2.54 -22.36 18 Cage 2 Bone 2.57 -23.62 19 Cage 2 Bone 2.45 -21.98 20 Cage 1 Bone 2.68 -21.95 21 Cage 6 Bone 2.91 -22.17 22 Cage 4 Bone 2.92 -22.35 23 Cage 6 Bone 2.73 -21.94 24 Cage 3 Bone 2.70 -21.76 25 Cage 5 Bone 2.85 -21.41 26 Cage 1 Bone 2.54 -22.08 27 Cage 3 Bone 3.08 -22.18 M e a n 2.74 -22.14 Range 2.45 - 3.08 -21.41 to -23.62 Finally, the results from G r o u p 2 (maize-stalk beer diet) can be seen i n Table 6, and produced interesting yet not unexpected results. The mean 8 1 3 C value o f -18.80%o wh ich falls wi th in the expected -20%o to -15%o range, but clearly when interpreting the -18%o value (8 1 3 C) the isotopic values for the powder por t ion o f the diet must be considered. T h e isotopic value (8 1 3 C) for the powder por t ion i f the diet was -18.17%o. It is important to note that although the C , por t ion o f the diet was min imal compared to the C 4 port ion, the results for 8 1 3 C show a stronger correlation to a C 3 value. The mean for the 8 1 5 N value o f 8.5l%o is consistent wi th the expected values o f individuals consuming C , based terrestrial fauna. The diets were also analysed to understand what influence the diets isotopic values may have had on the rat collagen, see Table 7. A s mentioned above, pure maize and a pure maize-stalk beer diet d id not fulfill the nutritional needs outlined by the U B C A n i m a l Care Centre. D u e to the fact that vitamins and minerals were added to all the G r o u p s ' diets, it was necessary to test the 8 1 3 C and 8 1 5 N values o f the diets i n order to incorporate these values into the bone collagen results (see Table 7 and Figure 1). It was important to test each diet to see i f the added vitarriins or minerals influenced the carbon or nitrogen values i n the food. A s the diets were served as the only source o f food for the rats, the carbon and nitrogen values i n each diet served as indicators o f the 26 diets the rats were consuming and what values could be expected for each group. A s mentioned before, modern C 3 plants have average values o f about —26.5%o whereas modern C 4 plants have averages o f about —12.5%o. This separation o f 14%o allows for discrirnination between group averages (Chisholm 1989:13). Table 6. Results of Isotopic Analysis from Group 2 (Maize-stalk beer diet) Sample Maize-stalk beer diet 6 1 5 N 6 1 3 C 29 Cage 2 Bone 8.80 -18.55 30 Cage 1 Bone 8.56 -18.69 31 Cage 4 Bone 8.34 -18.85 32 Cage 4 Bone 8.17 -19.66 33 Cage 3 Bone 8.20 -18.69 34 Cage 1 Bone 8.49 -18.51 35 Cage 2 Bone 8.72 -18.34 36 Cage 2 Bone 8.69 -19.20 37 Cage 3 Bone 8.50 -18.88 38 Cage 3 Bone 8M -18.60 M e a n 8.51 -18.80 Range 8.17 - 8 80 -18.34 to -19.66 Table 7. Results of the Isotopic Analysis of the Diets Sample 6 , 5 N 6 U C G r o u p 1 diet ( C 4 solid diet) 1.902 -15.74 G r o u p 2 Powder diet formula #710341 (CV) 5.546 -18.17 G r o u p 3 diet (C , solid diet) 1.606 -25.09 The C 4 solid diet consisted o f 70% C 4 and 30% C 3 wh i ch may account for why the diet returned a S13C value o f -15.74%o. This value clusters near the average value o f -12.5%o for C 4 plants; but the value has been affected by the C 3 component i n the diet. T h e C 3 sol id diet 8 1 3 C value o f -25.09%o is consistent wi th the average o f -26.5%o for modern C 3 plants. The powder diet formula #710341 has a 8 U C value o f -18.17%o, wh ich falls wi th in the 14%o separation between the C 3 and C 4 averages. This 8 1 3 C value was not expected because the diet was designed wi th only C 3 ingredients to wh ich the maize-stalk beer was added. This is o f interest because i n this experiment, the diets were controlled and the isotopic analysis o f this diet could be tested, but in an archaeological sample, the diets can only be postulated. 27 Figure 1 shows the distribution o f the stable carbon isotope ratios (8 C and 5 N) for all o f the individual rats sampled, as wel l as for the three diet samples tested. T h e three diets are grouped according to their isotopic ratio measurements. T h e values o f the food are also added to the graph. The figure shows that the isotopic reading for the rats is very similar to that o f the foods. The results for the individual rats in G r o u p 2 ( C 3 powder diet + C 4 f rom maize-stalk beer) who consumed the maize-stalk beer diet only reflect the C 3 value o f the original powder diet. A s expected, the maize-stalk beer (as a C 4 value) is virtually invisible i n the values measured for G r o u p 2, wh ich clusters closer to a C 3 reading than that o f a C 4 . • Rats on C3 Diet • Rats on C4 Diet • Rats on Maize-stalk beer Diet A C4 Solid Diet O Powder Diet O C3 Solid Diet -26 -21 -16 -11 6 1 3 C Figure 1. Distribution of Stable Carbon Isotope Ratios for Individual Rats The results o f this experiment support two important ideas. First, they support the hypothesis that maize consumed i n the form o f alcohol (beer) does not leave any measurable traces i n bone collagen. Second, this experiment supports the idea noted by A m b r o s e and N o r r (1993:27-28) and C h i s h o l m et al. (1982) that protein i n consumers is built f rom the protein 28 port ion o f the diet, not f rom fats or carbohydrates. Thus , the carbon isotopes that make up dietary protein are directly reflected in the carbon isotopes found i n the bone collagen. The content o f protein i n the maize-stalk beer is insignificant as it contains mostly sugars and water (Smalley and Blake 2003:684). This seems to be the reason why the maize-stalk beer did not show up i n the rats bone collagen analysis. C O N C L U S I O N Being able to demonstrate that consuming C 4 plants (maize) as an alcoholic beverage does not show in carbon isotope analysis o f bone collagen is a very important step i n archaeological research on the origin and spread o f early maize. F o r some years now, archaeologists have been trying to understand the reasons why the first Mesoamericans started to cultivate maize, and the focus has generally been o n the yield from the cobs rather than the other parts o f the plant. However , i f we can separate ourselves from the importance maize has as a cereal i n today's wor ld , and focus o n other uses maize may have had i n the past, this perspective can help us understand the reason for its first domestication and spread throughout the Americas . Research is begmning to consider other potential uses for the maize crop in the past. Iltis's (2000) suggestion that teosinte was first cultivated for its sweet stalk and tender ears; and Smalley and Blake's (2003) proposi t ion that the stalks were used to make alcoholic beverages are viable alternatives. A s mentioned previously, maize was first domesticated i n the R i o Balsas region o f southwestern M e x i c o before 6,000 B . P . (Benz 1999, 2006; Matsuoka et al, 2002, Smalley and Blake 2003:678). Paleoethnobotanical evidence shows the presence o f domesticated Zea at least as early as 5,400 B . P . in M e x i c o (Piperno and Flannery 2001:2102) and isotopic data suggests that maize did not become a dietary staple unti l 2500 years later. T o explain this "isotopic gap" i n maize use, Smalley and Blake (2003:678) proposed the "Stalk-Sugar Hypothesis" , i n w h i c h they argue that the extraction o f stalk juice - as a sweetener and possibly for making alcohol - may have been the key 29 factor i n the domestication o f Zea. I agree wi th Smalley, Blake and litis that this different way o f approaching the subject can yield some new light on the origin o f maize use. I also agree i n the advantages o f not focusing o n the tendency that Western scholars have i n overemphasizing maize's dietary importance. A s Tyko t and Staller (2002: 43) mentioned, i n South Amer ica , at least, maize was used more as a vegetable than as a cereal. W i t h regard to isotopic analysis, Smalley and Blake (2003) suggest the possibility that the practice o f convert ing maize to alcoholic beverages may explain l ow stable carbon isotope values during the "isotopic gap". They also explained "consumpt ion o f beer made f rom the juice wou ld not necessarily have produced higher stable carbon isotope ratios i n human bone col lagen" (Smalley and Blake 2003:686). The average 8 1 3 C value (of -18.80%o) obtained f rom G r o u p 2 offers validation o f the idea that beer made from maize products does not produce a strong C 4 reading i n bone collagen as predicted by A m b r o s e and N o r r (1993) and Smalley and Blake (2003). A s this experiment has shown, maize i n the form o f an alcoholic beverage does not exhibit a C 4 value i n stable carbon isotope analysis i n bone collagen. These results should serve as a caution for future investigations i n stable carbon isotopic analysis o n bone collagen. It appears that the importance o f maize present i n the diet o f ancient peoples o f Mesoamerica, is measurable through carbon isotope analysis o f collagen only i f people were eating maiz rather than "d r ink ing" it. (Ambrose and N o r r 1993; C h i s h o l m 1989; Farnsworth et al 1985; T y k o t and StaUer 2002; Uberlaker et al 1995; V o g e l and van der Merwe 1977 among others) T h e approach to overcoming the problem o f invisible maize beer consumption is to also analyze the stable carbon isotope values o f apatite i n bone and tooth enamel. These are proven to reflect the whole diet (Tykot 2006:131-2). Future analysis o f collagen and apatite from ancient peoples w h o were k n o w n to have consumed C 4 plant beverages such as chicha could be helpful i n better understanding this process and the necessary precautions to be taken when using stable carbon isotope analysis among (e.g., The W a r i brew master and Cerro Bau l i n Southern Peru, Moseley et al. 2006) 30 The possibility o f other uses o f early maize should be taken into consideration when searching for archaeological evidence. A s Smalley and Blake (2003:682) mentioned, maize stalk uses can be inferred both by direct and indirect archaeological evidence. T h e presence o f maize stalk and maize stalk quids 5 i n dry caves i n Archa ic Per iod and later deposits is so far the only direct evidence (see Smalley and Blake 2003:682-684 for a thorough description). Sources o f mdirect evidence are isotopic analysis in human bone samples (apatite i n bone and tooth enamel, as wel l as collagen and nitrogen); the presence o f pottery or any other type o f containers that may have worked as part o f the beverage preparat ion/storage/consumption process (large ceramic jars suitable for brewing as reported by Ubelaker et al. 1995:409) and artefacts that may have been use as presses to extract the juice o f the stalk. Chemica l studies o n ceramic vessels to identify function and uses o f the container can also be used as evidence. It is important to use as many tools as archaeologists have available today to help understand maize domestication and early spread. The results obtained i n this research, do not prove that the consumpt ion o f maize as an alcoholic beverage happened at the beginning o f maize domestication, but this research demonstrates that Smalley and Blake's and Iltis's hypotheses are viable and that this "alternate" use o f maize may have been one o f the original ones. The next step i n research that w i l l follow from this project is to analyze the stable carbon isotope values o f the remaining samples o f muscle, hair, specially the apatite i n both bone and teeth. O n c e these other values are known, we wi l l have a better understanding o f the differential pathways o f carbon from diet vs. carbon from alcohol consumpt ion i n ancient peoples. 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Universi ty o f Mich igan Press, A n n A r b o r Matsuoka, Y . , Y . Vigouroux , M . M . G o o d m a n , J . Sanchez, E . Buckler and J . Doebley 2002 A Single Domest ica t ion for Maize shown by Mul t i locus Microsatelli te Genotyping. Proceedings of the National Academy of Sciences 99(9):6080-6084. M o o r e , Jerry D . 1989 Pre-Hispanic Beer i n Coastal Peru: Technology and Social Context o f Prehistoric Product ion . American Anthropologist 91:682-695. Moseley, Michae l , D o n n a J . Nash , Patrick Ryan Wil l iams, Susan D . de France, A n a Miranda, and Mar io Ruales 2006 Burn ing D o w n the Brewery: EstabUshing and evacuating an ancient imperial economy at Cerro Bau l , Peru. Proceedings of the National Academy of Sciences 102(48):17264-17271. 34 N D I Foundat ion 2006 www.ndi f .org /Terms/Sprague-Dawley rats.html. Date Accessed: July 15, 2006. Piperno, D . R., and K . V . Flannery 2001 T h e Earliest Archaeological Maize (Zea mays L.) f rom High land M e x i c o : N e w Accelerator Mass Spectrometry Dates and their Implications. Proceedings of the National Academy of Science 98:2101-3. Piperno, D . R., and D . M . Pearsall 1998 The Origins of Agriculture in the Lowland Neotropics. Academic Press, San Diego . Pope K . O . , M . D . P o h l , J . G . Jones, J . S. Jacob, D . R. Piperno, S. D . D e France, D . L . Lentz , C . V o n Nagy, F . J . Vega , and I. R. Quitmeyer 2001 Or ig in and Envi ronmenta l Setting o f Ancien t Agricul ture i n the Lowlands o f Mesoamerica. Science 292:1370-1373. Smalley, J . and M . Blake 2003 Sweet Beginnings: Stalk Sugar and the Domest icat ion o f C o r n . Current Anthropology 44(5): 675-704. Tykot , Rober t H . 2006 Isotope Analysis and the Histories o f Maize . In Histories of Mai%e: Multidisciplinary Approaches to the Prehistory, Linguistics, Biogeography, Domestication and Evolution ofMai^e, edited by J . Staller, R. Tyko t and B . Benz , pp. 131-142. Academic Press, Bur l ington. Tykot , Rober t H . and J o h n E . Staller 2002 The Importance o f Ear ly Maize Agriculture i n Coastal Ecuador: N e w Data from L a Emerenciana. Current Anthropology 43(4):666-677. Ubelaker, D . H . , M . A . Katzenberg, and L . G . D o y o n 1995 Status and D i e t i n Precontact High land Ecuador. American Journal of Physical Anthropology 97:403-411. van der Merwe , N . J . , J . A . Lee-Thorp , and J . S. Raymond 1993 Light , Stable Isotopes, and the Subsistence Base o f Format ive Cultures i n Vald iv ia , Ecuador . In Prehistoric Human Bone: Archaeology at the Molecular Level, edited by J . B . Lamber t and G . Grupe , pp. 63-97. Springer-Verlag, Ber l in . V o g e l , J .C . and N . J . van der Merwe 1977 Isotopic Evidence for Ear ly Maize Cult ivat ion i n N e w Y o r k State. American Antiquity 42(2):238-242. Waynford , H . B . , P . A . Flecknel l 1992 Experimental and Surgical Technique in the Rat. Academic Press, L o n d o n . Weinberg, Joanne 1984 Nut r i t iona l Issues i n Perinatal A l c o h o l Exposure . Neurobehavioral Toxicology and Teraxology 6:261-269. 35 APPENDIX 1 - C.i solid diet Rats weight's gain Jun-08 Jun-15 Jun-22 Jun-29 Jul-06 124 184 236 296 340 Cage 1m 129 194 252 306 360 125 184 246 298 352 Cage 2 m 133 186 250 303 356 118 174 222 291 316 Cage 3 m 131 196 249 306 362 124 188 244 309 358 Cage 4 m 132 190 252 306 360 103 136 160 190 190 Cage 1 f 116 153 176 199 222 105 136 152 193 198 Cage 2 f 122 162 184 209 234 C4 solid diet 400 i 350 -\ 300 A 250 E s u ^ 200 £ 150 1(H) I 50 A - •—Cage lm/1 -A—Cage l m / 2 —A Cage 2 m/1 -A—Cage 2m/2 - A — Cage 3 m/1 - A — (-age 3m/2 -A—Cage 4m/1 -A—Cage 4m/2 - •—Cage lf/1 • Cage l f /2 —Cage 2 f / l Cage2f/2 3 Week 36 Maize-stalk beer diet Jun-15 Jun-22 Jun-29 Jul-06 Jul-13 78 122 145 170 180 Cage 1 2f 90 124 150 176 196 84 114 147 160 172 92 126 157 188 206 Cage 2 3f 94 136 164 192 208 99 152 209 258 286 103 160 230 300 294 Cage 3 3m 110 161 232 302 334 104 164 210 274 306 Cage 4 2m 110 172 229 286 326 L i q u i d Diet C3 + Corn Stalk Beet C4 40(1 Cage l-2f/l Cage l-2f/2 Cage2-3f/l Cage 2-3F/2 —•—Cage 2-3f/1 —jjfe—Cage 3-3m/l —A—Cage 3-3m/2 —A—Cage 3-3m/3 A Cage 4-2m/l —&— Cage 4-2m/2 3 Week 37 C3 solid diet Jun-15 Jun-22 Jun-29 Jul-06 Jul-13 128 188 227 300 344 Cage 1 m 132 194 254 314 372 122 180 240 294 338 Cage 2 m 128 188 248 308 366 104 140 169 186 214 Cage 3 f 120 154 179 204 234 114 150 183 190 220 Cage 4 f 128 170 199 228 256 108 140 167 190 216 Cage 5 f 118 150 174 198 236 116 154 184 196 236 Cage 6 f 119 162 191 220 260 C3 solid diet 400 -1 350 A A—Cage lm/1 £—Cage lm/2 3&— Cage 2 m/1 A—Cage 2m/2 • Cage 3f/l Cage 3f/2 •ii— Cage 4f/1 Cage4f/2 Cage5f/1 « - C a g e 5 f / 2 • - Cage 6f/l • • — Cage 6 f/2 Week 38 APPENDIX 2. U B C Ethics Commit tee A p p r o v a l Documen t https://rise.ubcxa/rise/Doc/0/BHG3IQUIOUE431KUM694TS7K74/... The University of British Columbia A n i m a l Care Certificate Application Number: ACM-1035 ; Investigator or Course Director: Michael T.M. Blake | Department: Anthropology & Sociology : Animals Approved: J Rals 39j i Start Date: 2005-1-15 Approval Date: 2005-2-22 Funding Sources: Funding Unfunded Research Agency: Title-''1'' Stable Isotope Analysis of Maize Beer Consumptic tttle- l n <' e <' Stable Isotope Analysis of Maize Beer Consumption The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility Office of Research Services and Administration 102, Agronomy Road, Vancouver, V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 l o t ! 2/22/2005 5:10 PM 39


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