Dietary Modulation of Intestinal Fermentation in Raptors

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Dietary modulation of intestinal fermentation in Raptors.

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Anika Daneel Student number: 01110677

Supervisor: Prof. dr. Geert Janssens Supervisor: Dr. Katherine Whitehouse-Tedd

A dissertation submitted to Ghent University in partial fulfillment of the requirements for the degree of Master of Veterinary Medicine

Academic year: 2017 - 2018

Ghent University, its employees and/or students, give no warranty that the information provided in this thesis is accurate or exhaustive, nor that the content of this thesis will not constitute or result in any infringement of third- party rights. Ghent University, its employees and/or students do not accept any liability or responsibility for any use which may be made of the content or information given in the thesis, nor for any reliance which may be placed on any advice or information provided in this thesis.

Foreword

I would like to thank Professor Geert Janssens and Dr Katherine Whitehouse-Tedd for their excellent guidance, countless revisions and support throughout my thesis. I also wish to thank Herman De Rycke, Katja Van Nieuland and all the people that assisted with the measurements, Donna Vanhauteghem and Gerry Whitehouse-Tedd, without whose cooperation I would not have been able to collect and analyse all the data. I would also like to thank my friends and family for their moral support.

To my mother who deserves a particular note of thanks: thank you for assisting with the revision and your wise counsel.

I hope you enjoy your reading.

Index

1. Abstract and Summary…………………………………………………………………………………………………………………………………………………. 1 2. Literature study…………………………………………………………………………………………………………………….…...... 2 2.1. Introduction………………………………………………………………………………………………………………….…………………………………….. 2 2.2. Anatomy and physiology of the raptor digestive system………………………………………….…………………………………..…… 3 2.2.1. Esophagus………………………………………………………………………………………………………………………………………………… 4 2.2.2. Ingluvies………………………………………………………………………………………………………………..…………………………………..….4 2.2.3. Proventriculus………………………………………………………………….…………………………………………………………………….... 5 2.2.4. Ventriculus………………………………………………………………………………...…………………………………………………………….....5 2.2.5. Small intestine (duodenum loop, jejunum and ileum)………………………………………………………………………….….. 7 2.2.6. Large intestine (colon)………………………………………………………………….……………………….………………………………….. 7 2.2.7. Caeca………………………………………………………………………………………………………………………………….…………………..…..8. 2.2.8. Cloaca…………………………………………………………………………………………………………………………..……………………………. 9 2.3. Intestinal separation mechanism (SM)………………………………………………………………………….…...... 9 2.4. Microbiota of the digestive tract………………………………………………………………………………………………………………………….10 2.4.1. Bacteria in the different parts of the digestive tract………………………………..……………….……………………………….. 12 2.4.2. Bacterial phyla found in the digestive tract of birds……………………………………………….…...... 12 2.5. Nutrient content of prey…………………………………………………………………..……………………………..…………………………………. 16 2.6. Protein catabolism………………………………………………………………………………...... ……………………………….. 19 2.7. Microbial fermentation………………………………………………………………………….…………………………...... 22 2.7.1. Short chain fatty acids (SCFA) ……………………………………………………….…………………………...... 23 2.7.2. Microbial fermentation……………………………………………………………….…………………………...... 25 2.8. Fermentation of uric acid………………………………………………………………………………………………….……………………………….. 25 2.9. Digesta passage rate…………………………………………………………………..………………………………………..……………………………. 26 2.10. Mean retention time (MRT)…………………………………………..………………………………………..………………………………………… 26 2.10.1. Influence of quantity and type of diet……………………………………………………………………………………………………….26 2.10.2. Influence of body temperature…………………………………………………………………..………………………………………..…..26 2.10.3. Influence by length of digestive tract………………………………………………………………………………………………………….27 2.11. Inert markers………………………………………………………………..……….…….…………………………………………………………………….. 27 2.11.1. C isotopes…………………………………………………………………..……………………………………………………………………….. 28 2.11.2. N isotopes………………………………………………………………...……………………………………………………………………………..28 2.11.3. S isotopes………………………………………………………………..……...... 29 2.11.4. H isotopes………………………………………………………………………………………………………….…………………………………….29 3. Hypothesis…………………………………………………………………………………………………………….……………………………………………………… 29 3.1. Function of vestigial caeca………………………………………………………………………………………………………….……….….……………29 3.2. Efficiency of digestion…………………………………………………………………………………………………………….………………………………29 3.3. Speed of excretion………………………………………………………………….………………………………………………………………………………30 4. Aim…………………………………………………………………………………………….………………………………………………………………………………… 30 4.1. Objective 1: Food intake and excretion pattern………………………………………………………………………………………………………30 4.2. Objective 2: Efficiency of diet utilization……………………………………………………………………………………………………………. 30 4.3. Objective 3: Passage rate and mean retention time (MRT)…………………………………………………………………………………….31 5. Material and methods………………………………………………………………………….………………………………..………………………………………31 5.1. Food intake and excretion pattern…………………………………………………….……………………………………………………………….. 31 5.2. Efficiency of diet utilization…………………………………………..…………………………………………...... 33 5.3. Passage rate…………………………………………………………………………………..……………………………………...... 34 6. Results………………………………………………………………………………………………..………………………………………………………………………… 36 6.1. Food intake and excretion pattern……………………………………………………………………………………………………………………….36 6.2. Efficiency diet utilization……………………………………………………………….……...... 39 6.3. Passage rate…………………………………………………………………………………..……………………………………………………………………….41 7. Discussion…………………………………………………………………………………………………………….………………………………………………………. 43 7.1. Food intake and excretion pattern…………………………………………………………………………………………………………….…………43 7.2. Efficiency of diet utilization…………………………………………………………………………………………………………….…………………… 45 7.3. Passage rate……………………………………………………………………………………………………………………………………………………….. 46 8. Conclusion……………………………………………………………………………………………………………………………………………………………………..46 9. Reference list…………………………………………………………………………………………………………………………………………………………………..47 10. Attachments…………………………………………………………………………………………………………………………………………………………………. 56

1. Abstract

Birds of prey also known as raptors are carnivore birds that feed on other animals, which can be categorized into 7 families. Strigidae, Tytonidae and Falconidae are the only families that do not belong to Accipitriformes. When considering their digestive tracts Strigiformes is the only order that has a different digestive tract: they have appendicular caeca, while the rest of the raptors have vestigial caeca. This is of importance since all raptors live on the same high proportion of animal tissues. In this study two vulture species were used to determine how they accommodate these differences, how efficient they digest food and what are their passage rates. The actual collection period lasted four days. During the first three days the efficiency of digestion was studied by measuring the change in stable isotopes from food to excreta.

The last day a bolus of titanium dioxide (TiO2) was given to each vulture, which was used to determine the passage rate. Variance analysis was applied to evaluate effects of vulture species. Lappet-faced vultures (Torgos tracheliotos) ingested significantly more than griffon vultures (Gyps fulvus) despite similar body weights, produced significantly more brown fecal samples than green fecal samples compared to griffon vultures. Although the stable isotope analysis showed no difference in digestive efficiency, the excreta C and N analysis still suggested that the lappet-faced vultures digested and absorbed protein more efficiently than griffon vultures. The high amount of leftover pieces collected from griffon vultures indicate that nutritional values based on whole prey is not entirely adequate for raptors and can lead to shifted dietary nutrient profiles. Although only numerical, griffon vultures appeared to have a faster passage rate than lappet-faced vultures. The consistency of fecal samples and mean retention time (MRT) were not different between vulture species. The results demonstrate that the differences in digestive anatomy between lappet-faced vultures and griffon vultures is associated with higher food intake and a higher digestive capacity, in particular for diets with high amounts of animal fiber.

1. Samenvatting

Roofvogels zijn vogels wiens dieet voornamelijk bestaat uit andere dieren. Ze kunnen in 7 verschillende families ingedeeld worden. Strigidae, Tytonidae en Falconidae zijn de enige families die niet tot Accipitriformes behoren. Wanneer wordt gekeken naar het spijsverteringsstelsel van Strigiformes, zijn ze de enige groep roofvogels die een appendiculair caeca heeft, terwijl de andere roofvogels een rudimentair caeca hebben. Dit is van belang aangezien alle roofvogels van dezelfde hoge proportie dierlijk weefsels afhangen. Tijdens deze studie zijn twee type gieren gebruikt om te evalueren hoe ze met deze verschillen omgaan, hoe efficiënt ze hun eten verteren en wat de passagesnelheid is voor de twee type gieren. De eigenlijke collectieperiode duurde vier dagen. De eerste drie dagen werd de verteringsefficiëntie door middel de verandering in stabiele isotopen profiel van voer tot excreta. Tijdens de laatste dag kregen de gieren elk een bolus van titanium dioxide (TiO2) die gebruikt werd om de passagesnelheid te evalueren. Variantieanalyse werd gebruikt om verschillen tussen de giersoorten aan te tonen. De oorgier (Torgos tracheliotos) at meer dan de vale gier (Gyps fulvus), had meer bruine mest dan groene mest in vergelijking met de vale gier. Hoewel de stabiele isotopenanalyse geen verschil toonde in verteringsefficiëntie, suggereert de analyse van C en N in de excreta dat de oorgier het eiwit beter verteerde en benutte dan de vale gier. De hoge hoeveelheid etensresten gecollecteerd van de vale gieren toont aan dat de selectieve opname van bepaalde delen kan leiden tot een afwijkend nutriëntenprofiel in de uiteindelijke voeding. Hoewel enkel numeriek, lijkt het of vale gieren een snellere passagesnelheid hadden dan oorgieren. De consistentie van de mest en de gemiddelde passagesnelheid was niet significant verschillend tussen de twee type gieren. De resultaten tonen aan dat de verschillen in spijsverteringsanatomie tussen oorgieren en vale gieren geassocieerd is met hogere voeropname en een hogere verteringscapaciteit, in het bijzonder voor diëten met grote hoeveelheden dierlijke vezel.

2. Literature study

2.1 Introduction

What is the definition of a raptor or ‘bird of prey’? The question is not so easily answered. First of all, raptor or ‘bird of prey’ is not an order, family or a classification, rather it is used for the category of birds which feeds primarily on other animals. These include mammals, other birds, fish or insects. Raptors include a diverse group of species and because of this, it is still open for discussion which species belong to this category.

Falconiformes is more often used as the order which include Falconidae and Accipitridae (Brown and Mindell, 2009; Lacasse, 2015). In this study, Falconiformes will be used as the order name for the family falcons and Accipitriformes will be used as the order name for family of buzzards, eagles, harrier, hawks, kites and Old World vultures. This is done because a recent study, using retinal transcription sequencing (Wu et al., 2016), classified the two orders as two different categories containing different species.

Birds of prey are classified from the ancestral group of Telluraves (Fig. 1), where it then divides into two groups namely the Afroaves and Australaves (Wu et al., 2016). The Afroaves evolved into the different orders namely Accipitriformes, Strigiformes and Coraciimorphae. The Australaves evolved into the Falconiformes, family Falconidae (Murray, 2014; Wu et al., 2016).

Accipitriformes consist of Accipitridae, Pandionidae and Cathartidae. Accipitridae is comprised of eagles, buzzards, hawks, harriers, kites and Old World vultures. The osprey (Pandion haliaetus) belongs to the family Pandionidae and Cathartidae includes the condors and the New World vultures. Strigiformes includes the families Strigidae (true owls) and the Tytonidae (barn owls). Falconiformes makes up the family Falconidae. Since Falconidae belongs to Australaves it is genetically the furthest removed from the other birds of prey (Wu et al., 2016). However, when using evolution, Strigiformes contains the oldest raptors species; dating back 71 million years (Brown and Mindell, 2009).

The difference between true owls and barn owls is that true owls are arboreal, they evolved to live and move in trees, while barn owls live primarily in open country. True owls come in different colors and have large heads, while barn owls have white hart-shaped faces with a white abdomen. Both types of owls have round facial discs around their eyes. These facial discs are arranged in such a position to deliver the maximum amount of sound to the ears.

The evolution of Old and New World vultures is very extraordinary due to the “convergent” evolution they underwent. Genetically the two families are different but the integument looks similar. Even though there was an ecological separation between the two families, due to competition for food sources, the body size and integument developed in the same way, meaning that the two different families have either a nude head or a head with soft, fine feathers, a hooked beak and a well-developed crop (Hertel, 1994). The New World vultures are genetically more closely related to storks than to the Old World vultures (Mundy, 1993; Wink, 1995). The main difference between the two families is that the New World vultures rely on smell to find prey, while the Old World vultures rely on sight to find their prey.

Figure 1. The taxonomy of Birds of Prey (modified after Brown and Mindell, 2009 and Wu et al., 2016)

2.2 Anatomy and physiology of the raptor digestive system

The majority of raptors are diurnal birds of prey. The exception to this rule are Strigiformes which are either nocturnal or crepuscular birds of prey (Ponder and Willette, 2015). Diurnal birds primarily hunt during day time, while nocturnal birds only hunt during night time and crepuscular only hunt during dusk or dawn. Raptors have a sharp, hooked tomia (Ford, 2010; Murray, 2014; Lacasse, 2015) which is the cutting edge of the raptors’ beak. The beak is adapted to the type of food that they consume. A sharp, hooked beak makes cutting into or tearing up the prey easier. The maxilla is a few centimeters longer than the mandible and has a crooked tip pointed downwards. Falcons are known to have a tomial tooth and notched edges for cutting prey more easily (Stevens and Hume, 1998). The Eurasian kestrel (Falco tinnunculus) uses the tomial tooth to severe the vertebrae of their prey (Klaphake and Clancy, 2005; Lacasse, 2015). Vultures uses three different feeding strategies namely 1) ripping, 2) gulping and 3) scraping their prey (Hertel, 1994). Since vultures prefer rotten food and very rarely catch their own prey, there was no evolutional process to develop a tomial tooth; they are also known to feed on carrions in large groups.

Food digestion and nutrient absorption is the primary function of the digestive tract. The digestive tract of birds contains more organs than that of mammals, therefore the inter-organ communication system is more complex than in mammals. This is due to the additional caecum, the presence of ingluvies (crop), two stomachs and the cloaca. The digestive tract is adapted to accommodate flying and hunting behaviors of raptors, resulting in the length and weight of the digestive tract becoming small in relation to their body mass (Klaphake and Clancy, 2005). The digestive tract of raptors has some similarities to the digestive tract of carnivorous mammals. For example, in mammals the length of the digestive tract of carnivores is small relative to their body mass, compared to herbivores and omnivores (Stevens and Hume, 1998). The carnivorous digestive tract is also less complex since the small intestine and the colon have become relatively small compared to the herbivores (Stevens and Hume, 1998) and the caecum is vestigial. A big difference between raptors and carnivorous mammals

is that fermentation only occurs in the caeca in raptors, whereas in carnivorous mammals it primarily occurs in the colon.

The digestive tract of birds (Fig. 2) consists of the 1) esophagus, 2) ingluvies (crop), 3) proventriculus (pars glandularis), 4) ventriculus (gizzard, pars muscularis), 5) small intestine, 6) caeca, 7) large intestine (colon) and the 8) cloaca (Klaphake and Clancy, 2005; Pollock, 20161).

Figure 2. The digestive tract of Accipitriformes and Falconiformes (A) and Strigiformes (B). Figure A has a vestigial caeca and Figure B has an absent ingluvies and well-developed caeca. (Taken out of Raptor Gastroenterology written by Klaphake and Clancy, 2005)

2.2.1. Esophagus

The esophagus lies along the right side of the neck and has longitudinal folds made up of incomplete keratinized stratified squamous epithelial cells, which provide elasticity and aid in passage of food or water. The elasticity in Accipitriformes and Falconiformes is more pronounced than in Strigiformes. The esophagus of Strigiformes has no dilation capacity and does not possess a crop, but instead they have a fusiform enlargement of the distal esophagus that is used for storage of ingested food (Duke, 1997; Klaphake and Clancy, 2005).

2.2.2. Ingluvies

The ingluvies is the storage unit for ingested food and can soften the ingested food. It is found cranial to the thoracic inlet. The distal dilation of the esophagus forms the ingluvies (Klaphake and Clancy,

1 Pollock: https://lafeber.com/vet/raptor-gastrointestinal-anatomy-physiology/ (20/05/2018)

2005). The ingluvies of most raptors are well-developed and are spindle-shaped (Murray, 2014); a true ingluvies, as in doves, has a functional sphincter under conscious control while in raptors the lower sphincter is poorly formed (Ford, 2010). In the evolution of the ingluvies, it has taken on different shapes and sizes in different species.

Accipitriformes and Falconiformes have a well-developed ingluvies, and in the Old World and New World vultures it has become particularly well-developed (Klasing, 1998). The bearded vulture (Gypaetus barbatus) is an exception in Accipitriformes since the well-developed ingluvies has become extinct; it has no crop but instead relies on the esophagus for storage of food (Houston and Copsey, 1994; Klaphake and Clancy, 2005). The black-winged kite (Elanus caeruleus) also forms an exception, since it has been found to have a wide esophagus and a poorly developed ingluvies (Hamdi et al., 2013). In Strigiformes the ingluvies is none existent.

2.2.3. Proventriculus

When food is passed from the ingluvies into the proventriculus, the food will be “put over”. This is done by extending the head upwards and then pushing it down in a flattening movement (Klaphake and Clancy, 2005).

The proventriculus is well-developed in all raptors. It can be distinguished from the ventriculus through the longitudinal folds and by the secretion of gastric juices: pepsinogen and HCl-. The gastric juices cross to the ventriculus to make the ventriculus more acidic. Duke et al. (1975) and Ford (2010) have found that the pH prior to digestion can be as low as 1.7 for Accipitriformes and Falconiformes and 2.4 for Strigiformes. The conversion of the proventriculus and the ventriculus gives the two organs a pear shape with a mean length of 16 cm (Houston and Cooper, 1975).

The absence of ingluvies in the bearded vulture have resulted in a high concentration of acid-secreting cells in the proventriculus, which are used to digest food (Klaphake and Clancy, 2005).

Bearded vultures have been found to primarily eat left over bones when eating from a carrion. After the bones have been stripped of the majority of meat, by predators and other scavengers, like the griffon vulture (Gyps fulvus), bearded vultures come and eat the leftover bones. Small bones can be swallowed whole while larger bones will be broken into multiple pieces before being swallowed. The advantage of having no crop and a very low pH in the proventriculus and ventriculus, is that bones can be swallowed in its entirety with no possibility of penetrating the crop and then be digested in the ventriculus. The high concentration of bones provides the vultures with almost as much energy (387 kJ/100g of bones), as when eating meat (440 kJ/100g of meat) (Brown and Plug, 1990; Houston and Copsey, 1994; Margalida and Villalba, 2017). This is due to the high fat content in mammalian bones.

2.2.4. Ventriculus

The primary function of the ventriculus is mechanically massaging food and crushing it into smaller pieces, increasing the surface area. Compared to other animals, the ventriculus in raptors is thin-walled and reduced in size. The ventriculus is lined with koilen (a horny material) that is secreted by mucus glands to protect the ventriculus against a low pH, received from the acid produced by the proventriculus. In the white-backed griffon vulture (Gyps africanus), the koilen seems to be absent which means the lumen is separated from the mucosa through a thin coat of cellular debris (Houston and Cooper, 1975).

Murray (2014) found that pH in the ventriculus of Accipitriformes and Falconiformes was around 1.6 and between 2.3-2.5 in Strigiformes. In studies done by Duke et al. (1975) and Houston and Cooper (1975), pH of gastric contents range from 1.0-2.0 in vultures and 2.5-5.0 in barn owls (Tyto alba) and European kestrels. Due to the difference in pH, it would seem that the digestion of bones is probably more efficient in Accipitriformes and Falconiformes (Murray, 2014; Ponder and Willette, 2015).

An acid pH in vultures contributes to digestion of food and pathogenic organisms. Since vultures primarily feed on carrions, which include animals that have died from illnesses, starvation or fell prey to other animals, these carrions are more likely to carry pathogens. Because of the acid pH throughout the digestive tract of vultures, pathogens are systematically digested, and vultures therefore contribute to the prevention of transmission of pathogenic organism like anthrax (Houston and Cooper, 1975).

The ventriculus has another special function, it produces pellets or casts. Pellets are egested by owls while casts are casted by the rest of the raptors. Pellets and casts are the undigested food that does not pass through the small intestine and the colon; rather it is egested through the beak. Egestion of pellets or casts follows three phases, 1) mechanical-digestion, 2) chemical-digestion and 3) fluid evacuation, development and egestion of the pellet or cast (Kostuch and Duke, 1975; Fuller and Duke, 1978). Birds have a very interesting mechanism to transport food from the ventriculus through the ingluvies and out of the beak. They make use of anti-peristaltic muscular waves to transfer the content in a retrograde direction. At the time of egestion, the anti-peristaltic waves move the pellet from the ventriculus through the esophagus and out of the beak. The equivalent of egestion is regurgitation in mammals. The difference with regurgitation is that abdominal muscles are used rather than anti- peristaltic waves in the esophagus (Duke et al., 1976a).

Studies have found that Strigiformes produce a pellet after each meal period, while Accipitriformes and Falconiformes usually eat more than one item before egesting a cast (Duke et al., 1975; Duke et al., 1976b; Cooper, 2002). Even though the egestion of casts in Accipitriformes and Falconiformes is correlated with dawn, before the first meal of the day, the frequency of casting is still unclear. They will egest to ensure the crop is empty and use the rest of the day to find their prey (Balgooyen, 1971; Duke et al., 1976b). Birds can eat with a half full crop, but when fresh food is mixed with a cast or a pellet, chances of getting crop rot increases.

In Accipitriformes and Falconiformes, the cast consist mostly of fur and feathers. Studies done on hawks have shown that the cast of a hawk contains hair, fur and feathers but no bones; this is due to the lower pH in the ventriculus which digest bones completely. Vultures also have a lower pH resulting in pellets containing hair, vegetable matters and minimal bone (Murray, 2014).

Strigiformes have a higher pH, which results in incomplete bone corrosion and pellets that contain fur, feathers, bones or a full skeleton (Cummings et al., 1976; Klaphake and Clancy, 2005; Ponder and Willette, 2015).

Studies have shown that the pellet or cast can also contain insect chitin. In the American kestrel (Falco sparverius) 23.8 % of the pellet contents consists of insect chitin while the pellet in the eastern screech owl (Strix varia) contains 29.6 % insect chitin (Klaphake and Clancy, 2005).

The size of the pellet or cast varies in and between species and has no connection with the total amount of food eaten (Erkinaro, 1973). Larger raptors have been found to egest larger pellets when compared to smaller raptors although the size of the pellet varies between the same size raptors.

Ponder and Willette (2015) have found that the meal-to-pellet interval (MPI) varies with the quantity of food eaten; in owls it can take up to 10 to 13 hours. The meal-to-pellet interval of other raptors is longer and differs between captive and wild raptors. A study done by Balgooyen (1971) suggest that the photoperiod plays a role in the time of egestion and on the MPI. Hawks fed late in the day have shorter MPI than hawks fed at dawn, even though they did not egest at dawn.

2.2.5. Small intestine (duodenum loop, jejunum and ileum)

In raptors, length of the intestine is relatively short in contrast to other birds like granivores and herbivores (Duke, 1997). The relatively short digestive tract is compensated for with well-developed, finger-like villi (Houston and Cooper, 1975; Murray, 2014). However, the length differs according to the type of food that the birds consume, even within the raptors, and is linked to the digestive efficiency. When relying on highly digestible food sources, the digestive tract can be 20-40% shorter (Klasing, 1998); as in the case of the sparrow-hawk (Accipiter nisus), kestrel (Falco tinnunculus), peregrine falcon (Falco peregrinus) and honey buzzard (Pernis apivorus). The shorter tract allows the raptors to be lighter and more maneuverable for catching prey. It however, decreases the efficiency of digestion, resulting in the need of more prey per day and a reduced choice of prey. Due to this, raptors with a short digestive tract have found to be largely feeding on small passerines (high body fat). The Eurasian buzzard (Buteo buteo), marsh-harrier (Circus aeruginosus) and red-kite (Milvus milvus) have a relatively long digestive tract; this prolongs the mean retention time (MRT) and increases the efficiency of digestion. So although eating less digestible food, like carrions or prey with low-energy levels, all the available nutrients can be absorbed. These raptors are also known as opportunistic birds, they will feed when the opportunity presents itself; they are known to soar over vast amounts of landscapes searching for prey or pounce when the opportunity presents itself; they rely less on speed and agility to catch their prey and proportionally need less food than raptors with short digestive tracts.

The small intestine is very similar between bird species in contrast to other organs. Exceptions are the American kestrel which has an enlarged duodenum that efficiently stirs the ingesta and speeds up digestion, and the osprey which has a lengthy digestive tract formulated in small loops (Klaphake and Clancy, 2005; Pollock, 20161). In the majority of raptors, the duodenum loops around the ventriculus, however, in the red kite, kestrel and peregrine falcon, the duodenum is coiled up like a snail coil.

The pH of the small intestine is higher than in the ventriculus due to the secretion of bicarbonate by the pancreas. Bicarbonate will counterbalance the pH of the digesta, neutralizing the pH and aiding in the function of bile salt and intestinal enzymes.

Enzymatic digestion and nutrient absorption occurs in the small intestine. The end products are absorbed by the epithelial cells (enterocytes) and transported into the blood stream. Mammals use mechanical and enzymatic digestion to digest food. However, in raptors mechanical digestion does not exist, since raptors have no teeth to mechanically digest their food (King and McLelland, 1984).

2.2.6. Large intestine (colon)

The colon is short, straight and is found to be relatively large in all raptors, with an abnormally large colon in the kestrel (Ford, 2010). The colon is predominantly used for reabsorption of water. After one

of the pacemakers, located in the intestine, produces anti-peristaltic muscular waves, the colon contracts and the digesta refluxes back up the digestive tract.

The digestive tract contains four different pacemaker regions (Stevens and Hume, 1998; Klasing, 1999), all located in the tunica muscularis of the intestine and is made up of interstitial cells of Cajal (ICC), meshed in between nerve fibers and muscular cells. The different types of ICC have their own function and are located in different segments of the digestive tract. ICC mediate enteric motor neurotransmission and produce electric pulses, resulting in contraction of the smooth muscle. The generated pulses move retrograde (Lecoin et al., 1996; Ward and Sanders, 2001).

Due to the presence of pacemakers, the digestive passage rate is prolonged. By prolonging time spend in the colon and caeca, the efficiency of absorbing nutrients and water increases. Due to this process, the size of the prey does not play a significant role in the absorption of nutrients. When the content is moved back up the digestive tract and into the caeca there must be a safety system to ensure the content does not move into the small intestine, this is ensured though the ileocecal sphincter at the end of the small intestine. At the entrance of the caeca, the villi forms a meshwork to filter out large particles, only permitting fluids and small particles through (Clench and Mathias, 1995).

2.2.7. Caeca

Birds have paired caeca. The insertion site of the caeca starts at the junction where the ileum ends, the distal segment of the small intestine, and the colon begins (Clench and Mathias, 1995). The caeca are blind-ended cannulae, the length of both caeca are approximately the same and the caeca have two different insertion sites, while the pH is roughly between 6.0-6.5 (Hill, 1971).

The length of the caeca was found by DeGolier et al. (1999) to be 6.9 cm in barn owls and 9.9 cm in true owls (Strigidae). In a study done by Gussekloo (2006) and Klaphake and Clancy (2005) the caeca in owls was found to be 4 – 11 cm while that of Accipitriformes and Falconiformes was found to be on average only 0,04 cm. Houston and Cooper (1975) found the mean caeca length in white-backed griffon vultures to be 0,85 cm. Through multiple studies done on the caeca of different bird species, the length of the caeca was found to be related to the diet (Naik and Dominic, 1962; DeGolier et al., 1999). In raptors, this finding is peculiar since the diet of owls and the rest of the raptors are very similar still the length of the caeca differs greatly. According to DeGolier et al. (1999), the difference can be due to the necessary absorption of water and nitrogen in the caeca in owls, which would not seem to be necessary in the other raptors since their caeca is thought to be nonfunctional.

When in the caeca, the digesta is fermented by local microbiota and is afterwards excreted, this product is called a caecal dropping. Caecal droppings of owls are known to be dark brown, have a softer texture and are homogeneous (Murray, 2014; Ponder and Willette, 2015). Caecal production frequency has been observed to be less frequent that the normal fecal production. Also, owls are known to release a caecal dropping in response to stress.

It is important to differentiate a caecal dropping from diarrhea. Since caecal droppings have never been observed in other raptors, it is unclear what they look like.

Through evolution, the caeca has evolved differently in the different bird species. There are four types of caeca namely 1) intestinal, 2) glandular, 3) vestigial and 4) lymphoid type. Strigiformes have well- developed glandular caeca, which contain goblet cells and secretory glands. A study done on the great horned owl (Bubo virginianus) has found that the glandular caeca plays a very important role in water

absorption. The same study found that caeca plays an important role in water absorption in other birds, and have a major role in the urinary water reabsorption (Duke et al., 1981). Accipitriformes and Falconiformes have a lymphoid caeca that has become vestigial (Murray, 2014).

The caeca have a variety of important functions 1) intestinal and urinary fluid absorption, 2) absorption of solid particles and molecules in the fluid by microbiota, fungi and other micro-organisms, 3) fermentation of food, 4) digestion of food, 5) absorption of nitrogenous components, 6) activity of microbiota, consisting of pathogens and non-pathogens and 7) production of antibodies and immunoglobulins (McNab, 1973; Clench and Mathias, 1995).

2.2.8. Cloaca

The cloaca is located at the end of the colon, therefore, at the end of the digestive tract. It is distinguishable from the colon by a much larger diameter. The cloaca consist of three compartments 1) the coprodeum, 2) urodeum and 3) proctodeum. The coprodeum functions as the storage unit for excreta and urine, while the ureters extending from the kidneys and the oviduct or deferent ducts end in the urodeum (Klasing, 1999). Urine is excreted in the urodeum since birds do not contain bladders and holds the endogenous produced uric acid. Uric acid is produced when nitrogen is metabolized. It enters the cloaca and can be excreted directly or it can be refluxed with the digesta. When refluxing the uric acid, it will be converted into amino acid that is absorbed by the host (Vispo and Karasov, 1997). The kidney-cloaca system balances the water and sodium losses. By refluxing uric acid, the available water can be absorbed. The nasal salt glands have the same function (Cade and Greenwald, 1966), but owls are not known to have these glands and only use the kidney-cloaca system for water and sodium absorption.

Excreta consist of two substances; the first is the fecal matter, which has to be dry and dark, and the second content is uric acid. Uric acid is white and a fluid, it can be accommodated with fecal matter or it can be excreted on its own while fecal matter will always be accommodated with uric acid. Fecal matter is a lighter brown than a caecal dropping. This is known for owls, since it is still unclear whether the other raptors produce a caecal dropping. Green fecal matter can mean that the bird is anorexic or has fasted. The green coloring is due to the presence of unused bile from the gallbladder (Klaphake and Clancy, 2005).

2.3 Intestinal separation mechanism (SM)

In herbivorous, such as lagomorphs, ruminants, rodents and some birds (Stevens and Hume, 1998; Björnhag, 1981; Dittman et al., 2015; Frei et al., 2015; 2017), and recently in dogs (De Cuyper et al., 2018), it has been suggested that they have an intestinal separation mechanism (SM) that allows certain nutritional particles to enter the caecum while other particles will pass into the colon where it will be excreted. The SM is thought to be able to distinguish between particles that have more nutritional value, such as smaller or liquid particles and exclude particles that have little nutritional value, such as larger particles (Björnhag, 1981; Björnhag et al., 1984).

The smaller and liquid particles that enter the caeca can be fermented more easily, while the larger particles which are more difficult to ferment will be excreted (Björnhag, 1981; Björnhag et al., 1984). This results in fluids and smaller particles having a longer MRT while the larger particles having shorter MRT (Gasaway et al., 1975; Björnhag, 1981; Frei et al., 2017).

When the particles enter the caeca, they are fermented by the local microbiota, producing short-chain- fatty-acids (SCFA) and other molecules, which will ultimately result in energy production. After fermentation, the caecal content is released into the proximal colon, where fluids and smaller particles are captured in the haustra of the colon and moved back into the caeca through the retro peristaltic contractions produced by the local pacemakers (Björnhag, 1981). The rest of the digesta will be excreted, producing a soft defecation sample, namely a caecal dropping.

It is a well-known fact that lagomorphs consume some of their fecal samples, the part that is formed in the caecum a behavior called caecotrophy. In rodents, the same behavior can occasionally be seen but is called coprohagy. Since the caecal dropping was produced in the caecum it contains a different molecular composition than a normal fecal sample, such as high concentrations of SCFA, B vitamins, microbial proteins, Na, K and water and is highly nutritional, while a normal fecal sample contains very little SCFA, microbial proteins and water. Caecotrophy provides rabbits with a third of their nitrogen intake, made up of microbial proteins that have high concentrations of essential amino acids (Stevens and Hume, 1998).

Due to the SM, fecal samples are presented in two different forms, dry and soft. Dry samples occur due to larger particles passing through the digestive tract and contains very little SFCA, other molecules and water (Stevens and Hume, 1998), while soft samples are made up of smaller particles that entered the caecum were it was fermented and contain high concentrations of SCFA, other molecules and water (Stevens and Hume, 1998). Due to the difference in consistency, rabbits are able to distinguish between the two samples and will only consume the nutritionally valuable fecal samples.

Recently the SM has been observed in dogs (De Cuyper et al., 2018), which were thought to have a non-functional caecum. In the study done by De Cuyper et al. (2018), dogs were observed excreting two different types of samples, dry and soft, which could mean that there is some sort of mechanism that distinguish between small, liquid and larger particles.

Since studies have suggested the existence of a SM in birds (Frei et al., 2017), and the recent study done on dogs (De Cuyper et al., 2018), which are carnivorous and have short caeca this could mean that the SM could potentially be present in raptors.

2.4 Microbiota of the digestive tract

Microbiota are capable of fermenting unused energy substrates (Waite and Taylor, 2014), regulating the immune system by producing SCFA, preventing the growth of pathogens, regulating the development of the digestive tract, producing vitamins for the host (Coates et al., 1968), and producing hormones to direct the host in storing fats (Józefiak et al., 2004; Kohl, 2012). When bacteria fail to fulfill any of their functions, it can result in an impaired host, meaning the host can experience immune deficiencies, enteritis, inflammation of the intestines and disruption of the normal development of the host (Jacob and Pescatore, 20132; Kierończyk et al., 2016). The local microbiota is broadly seen as an endocrine-organ (Clarke et al., 2014).

The normal microbiota of the digestive tract consist primarily of beneficial bacteria and trace amounts of pathogenic bacteria. In raptors the local microflora are largely unclear. It is however clear that the digestive tract is host to a large variety of bacteria, protozoa and fungi. There have been very little

2 Jacob and Pescatore: http://www2.ca.uky.edu/agcomm/pubs/ASC/ASC203/ASC203.pdf (20/05/2018)

studies done on raptors describing local microflora and the majority of the research was done on domestic birds and was then extrapolated to wild birds.

Through recent DNA (16S rRNA sequencing) sequencing methods, it has become possible to effectively identify bacteria in raptors. The technique is based on extraction and sequencing of nucleic acid from fecal samples (Amann et al., 1994; 1995; Kohl, 2012; Waite and Taylor, 2014; Mackenzie et al., 2015; Meng et al., 2017). This revolution makes it possible to collect digestive content from different parts of the digestive tract and to determine what the different functions of bacteria are in the different segments of the digestive tract based on bacteria identified (Godoy-Vitorino et al., 2012a; 2012b).

It is widely known that animals are hosts to a diverse range of bacteria (Mackie, 2002). The local bacteria in each individual is specific and varies in quantity and type of bacteria present. Microbiota are influenced by different factors, such as type of food eaten by the specific animal (carnivores, herbivores, granivores and insectivores), morphology of the digestive tract, environment mostly whether birds are in captivity or not (Waite and Taylor, 2014), length of each segment, pH of the segment, time digesta spend in the segment and age of the individual.

Since each segment must fulfill different functions, it is understandable that each segment contains different types of bacteria and in different quantities. For example, the small intestine enzymatically digests food particles and therefore less bacteria are required, while the caeca ferment these particles into SCFA and a great deal of bacteria are necessary to fulfill the function. The hoatzin (Opisthocomus hoazin), a herbivorous bird, is the only bird known to use their ingluvies to ferment food particles, resulting in foregut fermentation. As a result the ingluvies are dominated by bacteria of the phyla Bacteroidetes, Firmicutes and Proteobacteria while they also host Aquificae, Coprothermobacteria, Thermodesulfobacteria and Caldithrix bacteria (Grajal et al., 1989; Godoy-Vitorino et al., 2010; 2012a; 2012b), phyla that are more closely related to those found in the rumen of ruminant animals compared to those found in the digestive tract of other birds.

The amount of bacteria is positively correlated to the health of the digestive tract in mammals. When bacteria colonize the lumen, it increases the quality of the epithelium wall; this only occurs when bacteria colonizing the lumen are not pathogenic. When pathogenic bacteria colonize the lumen, the epithelium wall can be disrupted, resulting in less water and nutrient absorption, causing diarrhea. Under healthy conditions, the quality of the epithelium wall is maintained through an increase in cross- bridging proteins. These proteins protect the epithelium wall against invasion of pathogenic bacteria and the absorption of endotoxins (Hooper et al., 2001). Birds do not rely on increasing bacteria to keep the epithelium healthy, instead they rely on decreasing the concentration of bacteria and increasing paracellular transportation. The evolutional pressures of flying have decreased the intestinal surface area, and the digestive tract of birds primarily uses paracellular absorption, which transfers water- soluble nutrients between the enterocytes. If the amount of bacteria were to increase, less nutrients and vitamins would be absorbed. The balance between a too high concentration of bacteria, too dense a population, and the correct concentration of bacteria is very narrow (Ford and Coates, 1971; Caviedes-Vidal et al, 2007). Not only is the density important but also the correct composition of bacteria. When both factors are respected, the host will contain a healthy microbiota.

By maintaining a healthy digestive microbiota, the host is unlikely to fall ill to diseases related to the digestive tract (Jacob and Pescatore, 20132). It is therefore important to know how microbiota works and what functions they fulfil. It has been proven that the local microbiota plays a very important role in the health of the host (Kohl, 2012), and is therefore important to maintain a good and healthy colonialization of bacteria. The health of the host entails normal growth of the digestive tract, fulfilling

the normal functions of the digestive tract namely, digestion and absorption of food particles, absorption of water and normal development of the immune system (Leser and Mølbak, 2009; Kohl, 2012). The functions of microbiota are diverse and complex and since very little is known of bacteria and their functions in raptors, more research is needed to fully understand the roles that microbiota play.

2.4.1 Bacteria in the different parts of the digestive tract

2.4.1.1 Small intestine

Bacteria in the small intestine consist of anaerobic and aerobic bacteria while microbiota in the colon and the caeca primarily consist of anaerobic bacteria. This is due to the lack of oxygen in the lower digestive tract. Aerobic bacteria rely on oxygen to produce ATP (adenosine triphosphate), to regulate their metabolism and to multiply, without oxygen these bacteria will not survive; while anaerobe bacteria do not require oxygen to survive. These bacteria thrive when there is no oxygen present. Facultative anaerobe bacteria can also be found. These bacteria are capable of producing ATP from oxygen, in an oxygen rich environment while they are capable of switching to fermentation when there is no oxygen present (Thauer et al., 1977).

2.4.1.2 Caeca

Over time, there have been countless studies done on the caeca of chickens. The studies were done on different stages of development and different ages of chickens, from hatchlings to adults. The studies all resulted in the same findings, the caeca consist mainly of anaerobic and facultative anaerobic bacteria, all belonging to one of these four most commonly identified phyla, namely Actinobacteria, Bacteroidetes, Firmicutes or Proteobacteria (Barnes et al., 1972; Salanitro et al., 1975; Mead, 1989; Clench and Mathias, 1995; Van der Wielen et al., 2001; Józefiak et al., 2004). The caeca is also host to yeast and protozoa but in lower quantities. Studies have shown that local bacteria evolve and increase with increasing age of the host (Rehman et al., 2007).

2.4.1.3 Crop

Bacteria isolated in the crop of broiler chickens were mainly gram-positive facultative anaerobic bacteria (Rehman et al., 2007). The most common bacteria isolated were Lactobacillus species, while other common bacteria isolated where Escherichia coli, Klebsiella species, Staphylococcus lentus, Micrococcus luteus, Enterobacter aerogenes, Pseudomonas aeruginosa, Eubacterium species and Sarcina genus (Rehman et al., 2007).

2.4.2 Bacterial phyla found in the digestive tract of birds

Local microbiota can be divided into three categories namely the dominant, sub-dominant and temporary categories (Barnes, 1979; Józefiak et al., 2004). The most dominant bacteria found in herbivorous and carnivorous birds belongs to the phylum Firmicutes while Actinobacteria, Bacteroidetes and Proteobacteria were commonly found in the different categories (Waite and Taylor, 2014; 2015).

A study done by Hird et al. (2014), found that local microbiota between bird species (herbivores, granivores and carnivores) were more similar then compared to microbiota of mammals or insects while reptile microbiota showed more similarities to that of birds. The study demonstrates that extrapolating information known about microbiota of mammals or insects or even reptiles to birds can result in erroneous conclusions and should be done with care.

This is in contrast to the study done by Kohl (2012) who found that the higher up the taxonomic ladder, the less diversity there seemed to be between local microbiota in birds and mammals, and he found that the two most dominant phyla, out of 75 bacterial phyla, in birds and mammals were Firmicutes and Bacteroidetes. This finding corresponds with the study done by Waite and Taylor (2014).

When combining both studies it can be concluded that there are some similarities between birds and mammals when comparing the phyla but when extrapolating information between the two groups, it must be done with care since there seems to be a high percentage of bacterial genera differences between the two groups.

The most common aerobic bacteria that were identified in the majority of the studies belonged to the phyla Proteobacteria, namely Escherichia coli, Proteus sp., Enterobacter sp., Salmonella sp., Klebsiella sp. and Citrobacter sp., while the most common anaerobic bacteria identified belonged to the phyla Bacteroidetes, Proteobacteria and Firmicutes, which are gram-negative cocci, facultative aerobic cocci and Eubacterium.

Local microbiota exists in a very delicate environment. When the microflora is disrupted or overwhelmed by parasites, pathogenic bacteria or toxins, competing bacteria or parasites are presented with the opportunity to grow (Jacob and Pescatore, 20132). Competing bacteria can either be exogenous-pathogenic or endogenous-pathogenic bacteria (local microbiota). When a species of endogenous bacteria overgrows in the digestive tract it most likely will result in becoming pathogenic. Under normal conditions the local bacteria, parasites and yeast are not pathogenic. However, they can become pathogenic when the immunity is inefficient, for instance, in chronically ill raptors. In these cases, the bacterial infection is secondary (Willette et al., 2009).

Examples of endogenous-pathogenic bacteria are Salmonella sp. (Jacob and Pescatore, 20132), E. coli, Proteus sp., Pasteurella multocida, Klebsiella sp., Pseudomonas aeruginosa, Shigella sp., and Clostridium botulinum (Naldo and Samour, 2004; Samour and Naldo, 2005; Jones, 2006). A study done in the United Arab Emirates on falcons found the most identified endogenous pathogenic bacteria to be E. coli, Chlamydia psittaci, Pseudomonas sp., and Clostridium perfringens (Muller et al., 2006). These endogenous-pathogenic bacteria have been identified in clinically sick and clinically healthy birds.

2.4.2.1 Hawks

Lamberski et al. (2003) found, through cloacal swabs, that the most common aerobic microbiota in red-tailed hawks (Buteo jamaicensis) consisted of coagulase-negative Staphylococcus sp., Micrococcus sp. and Streptococcus sp., and in wild Cooper’s hawks (Accipiter cooperii) coagulase-negative Staphylococcus sp., Micrococcus sp., and Escherichia sp., while in the captive Cooper’s hawks the most common bacteria were Escherichia sp., coagulase-positive Staphylococcus sp. and Streptococcus sp.

While Salmonella sp., Bacillus sp. and Corynebacterium sp. were commonly observed in all the wild hawks but not in the captive hawks, Pasteurella sp. was not observed in any of the hawks involved. Cooper (2002) observed the most common bacteria in the lower digestive tract in raptors to be E. coli

and Proteus sp. The difference in microbiota in the captive and wild hawks and between the two hawk species can be due to different environments, diet and age of the raptors. The environment has been suggested to play a bigger role in the presence of different microbiota than originally thought (Hird et al., 2014; Waite and Taylor, 2014; 2015).

2.4.2.2 Vultures

Vultures are a very unique group of raptors, since they primarily feed on carrions or decaying animals which means that the animals either fell prey to other animals, or died of diseases, illnesses or old age, which ultimately means the dead animals can contain pathogenic bacteria. Vultures are able to ingest these pathogenic bacteria without falling ill like other animals would. Several studies have been conducted on different vulture species to determine what technique they use to overcome this obstacle. The digestive tract was found to mainly host gram-negative rods belonging to the class Gammaproteobacteria namely the family Enterobacteriaceae (Klaphake, 2006; Battisti, 1998; Meng et al., 2017).

This finding is supported by the study done on wild turkey vultures (Cathartes aura), where the vultures were shot, dissected, 1g of the intestine and its content collected and cultivated (Winsor et al., 1981). The gram-negative aerobic bacteria that where isolated, in decreasing percentage, were E. coli, Proteus mirabilis, Plesiomonas shigelloides, Enterobacter cloacae, Salmonella sp., E. aerogenes, Klebsiella pneumoniae, Citrobacter freundii and other Proteus sp. Another study done on American black vultures (Coragyps atratus), where the digestive tract was split into three segments (proximal, medial, distal segment) and the stomach and then cultivated (de Carvalho et al., 2003), showed that the stomach had a low density of bacteria, which is thought to be due to the very low pH (1.0-2.0). The three segments were found to contain a similar bacterial complex, but in different quantities. The most abundant bacteria in the proximal segment was found to be Clostridium bifermentans, and in the medial and distal segment Actinomyces bovis. Quantitatively, the distal segment compared to the proximal segment contained the highest amount of bacteria. The difference in the proximal segment can be due to the lower pH, shorter mean retention time and presence of pancreatic enzymes and bile acids. In the distal segments, pancreatic enzymes decrease and bile acids are deconjugated, creating an environment rich for bacterial inhabitance and growth (Rehman et al., 2007). When combining these studies, it can be concluded that bacteria found in the digestive tract of vultures are primarily of the gram-positive type, they are able to survive the ventriculus where the environment is very acid, the dominant genera differ along the digestive tract and percentage bacteria present increases along the digestive tract.

Roggenbuck et al. (2014) studied two types of New World vultures, the black and the turkey vulture, where both species were swabbed on the face and in the cloaca. The DNA collected was analyzed using deep sequencing and PCR amplification. Findings showed that facial microbiota and microbiota in the digestive tract of both species were largely the same. The study also indicated that microbiota on the face contained a wider range of bacteria than that of the digestive tract. And that the majority of the digestive microbiota isolated were from the same sources as the facial microbiota. Since both species feed in the same environments and on the same carrion, it can be expected that bacteria found were of the same phyla. The fact that the digestive tract contained less diversity of bacteria than the face can be due to the digestion of bacteria before they reach the small and lower intestine. The most dominant bacteria found cultivating in the digestive tract in both the black and turkey vulture were Clostridia and Fusobacteria, both fecal anaerobic bacteria. These bacteria were also found on the faces of both vulture species.

Since both groups are anaerobic bacteria, it was unexpected to find these bacteria on the face were percentage oxygen is high, however, the finding is not particularly significant since these vultures feed on carrions, going through the anus to get inside the carrion. Percentage Clostridia found on the face and in the digestive tract of both vulture species was approximately the same, while percentage Fusobacteria was lower in the digestive tract of black vultures compared to turkey vultures, but percentage Fusobacteria on the faces was comparable for both species. This study indicates the uniqueness of microbiota in vultures. Vultures fall under the rare group of birds that are able to tolerate Fusobacteria without becoming ill. One other bird that can withstand Fusobacteria is the American flamingo (Phoenicopterus ruber) (Roggenbuck et al., 2014).

Meng et al. (2017) used metataxonomics on fecal samples to identify the microbiome of nine vultures from three different species. The most commonly isolated bacteria belonged to two phyla, namely Firmicutes and Proteobacteria and four classes, namely Clostridia, Gammaproteobacteria, Bacillus and Alphaproteobacteria. The four most commonly detected orders were Clostridiales, Enterobacteriales, Lactobacillales and Sphinogomonadales and the six most common families were Clostridiaceae, Peptostreptococcaceae, Eubacteriaceae, Sphingomonadaceae, Peptoniphilaceae and Lactobacillaceae. Meng et al. (2017) also identified the seven most most commonly detected genera out of 98 belonging to Clostridium, Peptostreptococcus, Peptoniphilus, Eubacterium, Sporacetigenium, Sphingomonas and Lactobacillus.

The operational phylogenetic units (OPU) is when a grouping takes place after a phylogenetic interference. This is done to decrease sequence errors and indels influencing the endresult (Meng et al., 2017). According to the study done by Meng et al. (2017) only six OPU were commonly identified in all nine vultures. They were C. perfringens, P. russellii, E. moniliiforme/E. multiforme, Sporacetigenium sp., S. melonis/S. aquatilis and Peptoniphilus sp.

Meng et al. (2017) compared the OPU between Old and New World vultures and found that 40 out of the 105 OPUs were mutual in both families (Roggenbuck et al., 2014). Fecal matter of New World vultures identified 65 of the 105 OPUs. And the five most commonly identified OPUs in all the vultures were C. perfringens, P. russellii, E. coli/Shigella, Sporacetigenium sp. and Fusobacterium sp.2. Fusobacterium sp.1, E. moniliiforme/E. multiforme, Enterococcus clade 7, uncultured Firmicutes and uncultured Veillonellaceae were the most commonly identified species only isolated in the Old World vultures.

2.4.2.3 Variety of raptors

Roggenbuck et al. (2014) also identified the local microbiota in five different bird species in Copenhagen zoo all from different classes and they were also able to compare zoo turkey vultures with wild turkey vultures. In the African spotted owl (Bubo africanus), the most abundant bacteria found were in decreasing percentage Gammaproteobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Verrucomicrobia, Clostridia, Shingobacteria and Flavobacterium. In the red-tailed hawk, the most common bacteria were Gammaproteobacteria, Actinobacteria, Clostridia, Bacilli and Flavobacterium. The captive turkey vulture had in decreasing percentage Clostridia, Fusobacteria and Bacteroidetes.

The study found that even though red-tailed hawk is from a different order than African spotted owl, there were more similarities found in the local microbiota between these raptors, than compared to the turkey vulture whom is more closely related to the red-tailed hawk. The most common bacteria in

red-tailed hawk and African spotted owl were Gammaproteobacteria, Actinobacteria and Clostridia. The most obvious similarity of the captive turkey vulture with the other two raptors was the abundance of Clostridia, however in turkey vulture Clostridia was the most commonly identified bacteria, while in the two other raptors it was Gammaproteobacteria.

Captive and wild turkey vultures had the same microbiota except for Bacteroidia which was not identified in wild turkey vultures. This suggest that food is probably not the only factor important for determining local microbiota, but genes and environment also play an important part (Kierończyk et al., 2016). Roggenbuck et al. (2014) came to the same conclusion, however they did not describe the diet of captive raptors and therefore the implication of diet on microbiota could not be determined.

According to the study done by Sala et al. (2016) on mountain caracaras (Phalcoboenus megalopterus), Egyptian vultures (Neophron percnopterus), red-headed vultures (Sarcogyps calvus), Eurasian eagle- owls (Bubo bubo) and snowy owls (Bubo scandiacus), the most common bacteria identified in the ingluvies were Staphylococcus sp., while only Accipitriformes tested negative. In the cloaca E. coli was identified as the most dominant bacteria, then Klebsiella sp. followed by Proteus sp. and Staphylococcus sp. in all species studied.

2.5 Nutrient content of prey

Raptors are known to ingest a high protein diet. This entails that raptors primarily feed on animal proteins; which they acquire by hunting or feeding on other animals including mammals, birds, fish, reptiles, amphibians and insects (Palmer, 1988; Willette et al., 2009). Animal proteins are found in muscles, bones, feathers, hairs, skin, intestine and other organs and due to the volume of skeletal muscle, it contains the highest concentration of peptide-bound and free amino acids in the body (Davis and Fiorotto, 2009), resulting in the highest concentration of endogenous nitrogen.

While hunting, most raptors will consume the whole prey, which ensures the intake of essential amino acids as well as appropriate amounts of amino acids (Klasing, 1998) while hair and feathers are used to aid in the egestion of pellets and casts (Klasing, 1998). Since raptors rely on such a high protein they have evolved efficient pathways for converting amino acids into energy (Ford, 2010).

Due to their high protein intake, which entails highly digestible animal tissues and low concentration of carbohydrates, the digestive tract did not evolve into a more complex system, in contrast to birds living on high carbohydrate diets (Barton and Houston, 1994; Duke, 1997; Ford, 2010; Hamdi et al., 2013). Owls present the exception since they also have a high protein diet but have a more complex digestive tract than the rest of the raptors, since they have an appendicular caeca of 4.4-9.9 cm long (DeGolier et al., 1999). One possible reason for this difference could be the variance in acidity. The pH (2.2-2.5) (Murray, 2014), in the digestive tract of owls (higher than for the other raptors), could result in a decreased enzymatic digestion of proteins. With the presence of functional caeca, owls are able to ferment undigested proteins and absorb available nutrients.

Studies have shown that all raptors feed on other animals, resulting in similar diets. The main difference between feeding on mammals, birds, fish, reptiles or amphibians is the relative fat proportion in the prey item. Proportion of fat in the prey is not constant and varies between seasons, and between and within species (i.e. according to age or habitat). When prey is capable of storing high amounts of fat, they are considered as superior prey and they are more likely to be hunted by raptors, especially by raptors with a relatively short digestive tract, since a high percentage fat means relatively more energy (Duke and Houston, 2007).

As explained earlier, length of the digestive tract correlates with the efficiency of digestion. A longer digestive tract is capable of digesting food more thoroughly then a shorter digestive tract resulting in raptors with a shorter digestive needing to hunt more frequently and they have resorted to hunting prey with a high percentage body fat (Duke and Houston, 2007). There are several ways to look at this theory. For instance, the high fat content of prey means that a shorter tract is also capable of efficiently digesting fat, resulting in enough energy to hunt frequently. Or when hunting frequently, raptors with a shorter tract are able to digest food faster and can hunt more regularly due to the high energy intake.

A number of studies (Bird and Ho, 1976; Bird et al., 1982; Clum et al., 1997) analyzed nutrient content of commonly fed or commonly hunted domestic prey species. The studies mainly focused on percentage protein, fat, ash and fiber in the different prey items.

The most common prey items analyzed were rats (Rattus norvegicus), mice (Mus musculus), chickens (Gallus domesticus), day old chicks, house sparrows (Passer domesticus), meadow voles (Microtus pennsylvanicus), red-legged grasshoppers (Melanoplus femurrubrum), quails (Coturnix coturnix japonica) and guinea pigs (Cavia porcellus).

The gross energy (Table 1) contained in the different prey items are not significantly different from one another. Day old chicks appear to contain the highest percentage of energy but it is closely followed by chickens, mice and house sparrows.

Table 1. The gross energy (Kcal/g DM) content in different domestic prey items (modified from data collected by Bird and Ho (1976) and Bird et al. (1982)) Rats Mouse Chickens Day Old House Meadow Grasshopper Chicks Sparrow Vole Gross energy 5.78 5.84 5.93 6.02 5.39 4.15 5.02 (Kcal/g DM) DM: Dry matter

Crude protein (% dry matter (DM)) (Table 2) content in domestic species was analyzed and varied between the studies. The study done by Clum et al. (1997) found that quails had the highest concentration of crude protein, followed by mice, chickens and rats. This study also recovered overall higher concentrations of crude protein in the animals compared to the study done by Bird and Ho (1976). The study done by Bird and Ho (1976) also found that rats contained the highest concentration of crude protein, followed by day old chicks, chickens and mice.

Even although rats contained the highest concentration of crude protein in the study done by Bird and Ho (1976) compared to the second lowest concentration of crude protein, in the study done by Clum et al. (1997), the variation between the two studies was 0.6 % DM, which is very low.

Table 2. Crude protein (% DM) content measured in different domestic prey items (modified from data collected by Bird and Ho (1976), Bird et al. (1982) and Clum et al. (1997)) Rat Mouse Chicken Quail Guinea Day Old House Meadow Grass- Pig Chick Sparrow Vole hopper Clum et al. 63.4 64.4 64 67.6 58.9 (1997) Bird and Ho 62.8 56.1 56.7 62.4 (1976) Bird et al. 64.58 57.29 75.70 (1982) DM: Dry matter

Due to the high protein concentrations found in the red-legged grasshopper (75.70 % DM), they could be excellent food sources for merlins (Falco columbarius), but for raptors, birds that need high energy food sources, the low gross energy counts could be insufficient to live on. The low gross energy can be due to the low crude fat concentrations (Table 3), which means that for raptors who require higher energy dense food sources that the red-legged grasshopper would not be the ideal food source.

The meadow vole measured the lowest concentration of gross energy (4.15 Kcal/g DM) with crude fat (6.01 % DM), and crude protein (57.29 % DM). Compared to Bird and Ho (1976), this is relatively high since mice (56.1 % DM) and chickens (56.7 % DM) contained lower concentrations of crude protein. But compared to the study done by Clum et al. (1997), the meadow vole contained the lowest crude protein. Since the meadow vole also contained the lowest crude fat and gross energy, it might suggest that meadow voles are not the best supply of energy for raptors.

When considering house sparrows as a possible food item, gross energy intake of 5.39 Kcal/g DM with high protein intake (64.58 % DM) could contribute to being a good food source. Crude fat content (15.93 % DM) is below average when compared to the mammals tested. The low crude fat can contribute to the relatively low gross energy concentration.

The varying results in the different studies indicated variation of prey composition within the species. This variation could be due to different reasons, for instance age of the animal, gender, type of diet it was given before being fed to raptors and environment it was housed in.

The concentration of crude fat (% DM) (Table 3) was found to have a broader variation between the different species. Clum et al. (1997) found the highest concentration to be in chickens and guinea pigs, followed by rats, quails and mice. Bird and Ho (1976) found the highest concentration crude fat in chickens and mice, followed by day old chicks and rats. Clum et al. (1997) found an overall higher concentration of crude fat in the animals.

The concentration of crude fat found in mice was higher in the study done by Bird and Ho (1976) than in the study done by Clum et al. (1997). As in the case of rats and crude protein, the variation between the two studies was 1.2 % DM, which is relatively small. Both studies indicated that chickens contained the highest concentration of crude fat, which probably contributed to the high concentration of gross energy (Table 1).

Table 3. The crude fat (% DM) content in different domestic prey items (modified from data collected by Bird and Ho (1976), Bird et al. (1982) and Clum et al. (1997)) Rat Mouse Chicken Quail Guniea Pig Day old House Meadow Grass- chick Sparrow Vole hopper Clum et al. 34.9 23.7 47.2 29.7 45.4 (1997) Bird and Ho 22.1 (1976) 22.1 24.9 26.9 23.8 Bird et al. 15.93 6.01 6.03 (1982) DM: Dry matter

The findings illustrate that when a prey item contains a high fat proportion and is given incorrectly, for instance too much of one item, it will result in a high energy diet. This type of diet can be appropriate in raptors with a short digestive tract, needing high energy food sources, such as the European kestrel, but can be devastating in raptors that need low energy food sources, such as the merlin (Forbes,

20143). Merlins normally live on an insect-based diet, and when they are kept on a high fat diet, they are prone to develop “Fatty Liver Kidney Syndrome” (Forbes, 20143). Another disorder that can be related to a high fat diet is atherosclerosis (Forbes, 20143). In comparison, when raptors with a shorter digestive tract are kept on a diet containing low fat proportions, these raptors will be unable to maintain their body mass even though they are consuming enough food (Taylor et al., 1991).

Raptors feed on diverse animals that vary in size. The size of the prey and frequency of hunting or feeding on animals seems to be correlated to the size of the raptor. Smaller raptors, like American kestrel and eastern screech-owl (Megascops asio), are known to hunt on smaller animals and will hunt more frequently to maintain a normal body weight. They have been observed hunting small rodents, birds and insects (Duke and Houston, 2007; Murray, 2014), while the barn owl and the red-tailed hawk, which are larger than the above-mentioned raptors, have been observed hunting larger mammals, small rodents and birds (Duke and Houston, 2007). The barred owl (Strix varia), which is an even larger raptor, has been observed hunting small rodents, larger mammals, insects and amphibians (Duke and Houston, 2007; Murray, 2014).

Raptors kept in captivity such as rehabilitation centers, zoos or with private breeders, are provided a diet that consists mainly of domestic animals, mostly due to necessity. This diet is however, less diverse than the diet of wild raptors (Clum et al., 1997). To ensure that the raptors maintain their body mass they must be fed prey items that contain the necessary nutrients and are of appropriate size. Owls prefer to swallow their prey whole, if they are given a prey item that is to big they will not be able to obtain the necessary nutrients, which will result in undernutrition. The diet must also be appropriate since nutritional status is correlated to the health (Gershwin et al., 1985; Sklan et al., 1995), growth (Lavigne et al., 1994), reproduction (Naber and Squires, 1993) and longevity (Good and Gajjar, 1986) of the raptors. The nutritional status can also influence the success of reintroduction programs (Clum et al., 1997).

Due to limited variety and type of prey, in the diets of captive raptors, this diet is considered unnatural. This is even more so in vultures, which usually feed on carrion, consuming large quantities of freshly killed animals or rotting meat in large groups, while captive vultures are usually fed small domestic rodents and different types of domestic bird species, which is a completely different from their diet in the wild (Murray, 2014).

The frequency of hunting can be correlated to the activity of the raptor and surrounding temperature (Duke and Houston, 2007). The activity can be affected by different factors such as photoperiod, availability of prey, breeding and disturbances (Duke and Houston, 2007).

2.6 Protein catabolism

Catabolism of proteins is the process whereby proteins (macromolecules) are cleaved into amino acids for the purpose of producing energy (ATP), red and white blood cells, new proteins for the epithelium and micro-organisms and repairing tissues (Mortimore and Pösö, 1987; Wu, 2010). The process takes place in an environment that is favorable for proteolysis, for instance an environment containing low pH and high temperatures like the stomach and small intestine. Catabolism of proteins normally occurs

3 Forbes: https://www.scribd.com/document/374794681/Practical-Raptor-Nutrition-Neil-Forbes-pdf (20/05/2018)

in an aerobic environment by the local aerobe microbiota and epithelium cells by utilizing oxygen, but can also take place in an anaerobic environment.

Animal proteins and polypeptides are comprised of 22 different amino acids, which are bound by peptide bonds (Wu, 2009). Ten of the 22 are essential and must be supplemented in the diet. These are: arginine, histidine, isoleucine, leucine, lysine, methionine, threonine, tryptophan, phenylalanine and valine. Amino acids are precursors of different functions in the body, the precursor of nucleic acids being the most important one (Gutiérrez-Preciado et al., 2010). They also play an important role in cell signaling, regulating gene expression, regulating protein phosphorylation cascade, production of hormones and production of low-molecular weight nitrogenous substances (Wu, 2009).

Functional amino acids, such as arginine, cysteine, glutamine, leucine, proline and tryptophan are necessary for growth, immunity, development, reproduction, maintenance and replacements in the body (Duke, 1997; Wu, 2009). When peptide bounds are cleaved by enzymatic digestion in the lumen of the digestive tract, short peptides and mono-amino acids are formed which are absorbed by the epithelium cells where it can either be escorted into the blood stream (portal vein) or oxidized into energy (Wu, 1998; Rietdijk et al., 2007).

Raptors feed on animal proteins, most importantly the muscles, containing large quantities of free and peptide bound amino acids. Since catabolism of proteins mainly occurs intracellularly, peptide bound amino acids (proteins) are transported intracellularly, where they are cleaved into mono-amino acids by enzymes like proteases, this process is called proteolysis. Proteolysis is known as the first step in protein catabolism. Before the cleaved amino acids can be re-used for different processes, such as production of new proteins, conversion to different amino acids or catabolism of amino acids, they must pass through different cellular membranes.

The catabolization of amino acids results in different metabolites, namely short-chain-fatty-acids

(SCFA), long-chain-fatty-acids (LCFA), ammonia, carbon dioxide (CO2), uric acid, glucose, ketone bodies, nitric oxide (NO) poly-amines and nitrogenous substances (Blachier et al., 2007; Morris, 2007; Rider et al., 2007; Sugita et al., 2007; Montanez et al., 2008). The most important metabolic pathways of catabolization of amino acids are through the Krebs or Citric Acid or tricarboxylic acid (TCA) cycle or through glycolysis. Both pathways result in the production of energy, either used by the epithelium or the microbiota. The TCA cycle receives amino acids from different processes, for instance deamination, decarboxylation, transamination and dehydrogenation, while in glycolysis amino acids are derived from oxidative deamination to form sugars that internally can also end up in the TCA cycle.

The TCA cycle (Fig. 3) is a metabolic pathway that is able to metabolize proteins, carbohydrates and fats into acetyl coenzyme A (acetyl-CoA) that can be converted into SCFA and will lead to ATP production. During the TCA cycle, a large amount of water and acetate is used. Acetate is oxidized to produce acetyl-CoA which can be oxidized to produce ATP.

Glycolysis (Fig. 3) starts by removing the amino group from the amino acid and convert it into ammonia, which will leave the body through the urea cycle. The remaining part of the amino acids is oxidized and produces alpha-keto acid, which is used in the TCA cycle or glycolysis where it can be converted into pyruvate. The pyruvate can then be transformed into acetyl-CoA used in the TCA cycle for ATP production (Herzig et al., 2012).

Phosphoenol- pyruvate (PEP)

Pyruvate

A B

Figure 3. Protein catabolism. A represent the Krebs (TCA) cycle and B represent glycolysis (Taken out of PLOS ONE written by Phong et al., 2013)

When acquiring amino acids through transamination, the amino group is transferred to alpha- ketoglutarate and is not lost as ammonia through the urea cycle. Alpha-ketoglutarate can be transformed into glutamate, which transfers the amino group to oxaloacetate, and can eventually be transformed into other amino acids or aspartate (Wu, 2010). This process eventually undergoes oxidative deamination with the end products being alpha-keto acid and ammonia. The unused amino acids are either used in glycolysis or in the TCA cycle.

Proteins are constantly digested and reconstructed depending on the nutritional need of the cells or local microbiota. Since proteins have different half-live lengths, namely short and long half-lives, they can be utilized in different ways. For instance, short half-live proteins can accommodate changes in the epithelium which are caused by the different metabolic processes. Due to the short half-lives, these proteins need to be reconstructed regularly and build into the epithelium, resulting in accommodating changes that occur due to the reactions of the different processes (Bojkowska et al., 2011).

Bacteria can also utilize mono-amino acids or peptide bound amino acids in the production of microbial proteins and for the oxidation of amino acids in the production of ATP. But before peptide bound amino acids can be used by bacteria, they must undergo proteolysis since proteins are too large to enter the bacterial plasma membrane. After proteolysis, amino acids will converge in the cytoplasm of bacteria to form functional proteins or are used as energy which bacteria can use to function. This

process can take place in both aerobic and anaerobic bacteria. The difference though is that in anaerobe bacteria, pyruvate is converted into lactic acid, while in aerobic bacteria pyruvate is converted into SCFA.

Proteolysis can also occur spontaneously in low a pH and in high temperatures without the use of enzymes. This environment helps to speed up the digestion of animal proteins.

2.7 Microbial fermentation

Microbial fermentation primarily occurs in the caeca of raptors. In mammals, fermentation mainly occurs in the colon, except in ruminant mammals that primarily use their forestomach for fermentation. Fermentation is the process whereby anaerobic bacteria or yeast converts carbohydrates and dietary proteins into SCFA, branched-chain-fatty-acids (BCFA), ammonia (NH3), phenol, indole, CO2 and H2, which are used in ATP production (Annison et al., 1968; Stevens and Hume, 1998; Price et al., 2014; Koh et al., 2016).

Fermentation works as follows: digested and intact dietary proteins (digested muscle and/or bones) enter the caeca where the local microbiota breaks the dietary proteins down into smaller molecules, mono-amino acids and short peptides. After removing the amino group, the remaining amino acids are oxidized and produce alpha-keto acids. During glycolysis, alpha-keto acids are converted into pyruvate. Pyruvate can then be transformed into acetyl-CoA and is fermented into butyrate, acetate, lactate and formate. Even though propionate is most abundantly found in the digestive tract, it is produced slightly different compared to the rest of SCFAs. Propionate is produced during the TCA cycle where it is part of the cycle, and not an end-product and it does not need acetyl-CoA for its production. Proportionate can be converted into pyruvate where it can internally be converted into acetyl-CoA. SCFAs are then - + absorbed by the enterocytes in exchange for bicarbonate (HCO3 ) and hydrogen (H ). This process can occur in one of two ways: passive or active absorption. During passive absorption, sodium (Na+) is + - exchanged for H and during active absorption carrier-mediated transport exchanges SCFA for HCO3 . The carrier-mediated transporters are found in the basolateral and apical membranes. The basolateral - - - transporters exchanges HCO3 and chlorine (Cl ) for SCFAs whereas in the apical membrane only HCO3 is exchanged for SCFAs (Stevens and Hume, 1998; Józefiak et al., 2004).

Birds are known to have a shorter digestive tract compared to other vertebrates. This smaller tract is compensated with proportionately more villi per enterocyte that efficiently increases the surface area. Due to the deficient carrier-mediated transportation, birds rely on paracellular transportation. Paracellular transport occurs when molecules (nutrients and water-soluble nitrogenous components) are transported between cells, through the tight-junctions and not intracellular (transcellular transport). This occurs through diffusion or solvent drag (Fig. 4). Paracellular transport has several beneficial functions namely absorption is a continuous process, there is no carriers saturation, and transport occurs passively without using energy. Transportation of molecules through the tight- junctions is dependent on size and load of the molecules. Birds primarily use paracellular transport for nutrient absorbtion. This also means that the digestive tract is more exposed to water-soluble dietary toxins (Price et al., 2014). Price at al. (2014) also indicated that when the body mass in birds increases, the level of paracellular transportation decreases, meaning that bigger raptor species are less likely to rely on paracellular transport and more likely to rely on transcellular transport.

Figure 4. Transportation of nutrients in vertebrates. Small, water-soluble molecules and water diffuse between enterocytes by means of paracellular transport. Glucose and fructose are absorbed by carrier-mediated transport into the enterocytes, where glucose is then transported to the bloodstream. SCFA are absorbed by the epithelium through passive diffusion and serve as energy for the epithelium (reproduced from Physiology and written by Price et al., 2014) Pancreatic enzymes like hydrolytic enzymes, are either located in the lumen or bound to the apical membrane of the small intestines and are necessary to digest dietary proteins into smaller molecules. These smaller molecules are then either transported transcellularly, paracellular or further fermented by the local microbiota. Although transcellular transport is inferior to paracellular transport in birds, it still occurs but less efficiently than in other vertebrates (Price et al., 2014).

As said earlier, microbiota are capable of fermenting the short peptides and mono-amino acids into SCFA including 1) butyrate (butyric acid), 2) acetate (acetic acid), 3) propionate (propionic acid), 4) formate (formic acid), and 5) valerate (valeric acid). The production of the different SCFAs transpires through different bacteria and synthesis cycles and is controlled by different factors such as diet, host, time of fermentation, mean retention time and local microbiota (Rehman et al., 2007).

2.7.1 Short chain fatty acids (SCFA)

Butyrate is the most important SFCA, and it functions as the energy source for local cells, helps in preventing cancer and in anti-inflammatory processes (Stevens and Hume, 1998; Guilloteau et al., 2010; Canani et al., 2011). Acetate is most commonly produced from pyruvate followed by propionate and butyrate (Annison, et al., 1968; Stevens and Hume, 1998; Price et al., 2014).

Acetate can also internally be used to produce butyrate. When acetate is found in low concentrations it has a positive effect on the host but when it is found in larger quantities it can be carcinogenic (Koh et al., 2016).

Propionate is produced through the Krebs (TCA) cycle in the digestive tract in the epithelium cells or in the local microbiota. Phosphoenolpyruvate (PEP) can be utilized by different bacteria to finally produce propionate. Propionate can then either passively diffuse through the enterocytes or can be further

utilized by the same or different bacteria to produce pyruvate. Pyruvate is utilized to produce butyrate, acetate, formate, lactate and ethanol. These molecules are produced by means of acetyl-CoA.

When bacteria ferment amino acids, not only will SCFAs be produced but also ammonia and biogenic amines (phenol and indole). SCFAs use passive diffusion to enter the enterocytes where it is used as an important source of energy. SCFAs not only provide the necessary energy but also help with water and sodium (Na+) absorption (Stevens and Hume, 1998). A study by Kim et al. (2016) suggests that SCFAs plays an important supportive role in maintaining the integrity of the epithelium, immunomodulation and regulatory and effector T-cells. The same study showed that dietary fiber and SCFAs have a positive effect on intestinal IgA and systematic IgG response; that SCFAs are directly responsible for the regulation of B-cell and skew gene expressions for the production of antibodies. SCFAs also positively affect cellular acetyl-CoA levels, mitochondrial respiration and lipid droplets in B- cells. SCFAs increase the production of antibodies by the human B-cells and SCFAs assist in pathogen- specific antigen response. SCFAs are also known to help out with the secretion and modulation of bile acid and pancreatic enzymes, production of mucus and the expression of genes (Rehman et al., 2007).

BCFA are fatty acids which are typically saturated with a methyl group on the carbon chain, such as isobutyrate (isobutyric acid) and isovalerate (isovaleric acid). Microbiota are able to saturate fatty acids with groups other than methyl. Mono-methyl-branched fatty acids are most frequently formed, but di- and poly-methyl-branched fatty acids can also be found (Ran-Ressler et al., 2008; Christie, 2012). BCFAs functions in the membranes of cells and bacteria, and may increase the flexibility of lipids in the membranes (Christie, 2012).

Saccrolytic bacteria are primarily responsible for fermentation of dietary starches and sugars into usable energy for the enterocytes and are found in low quantities (Mead, 1989; Vispo and Karasov,

1997). Ethanol, lactose, succinate, acetate, CO2 and H2 are produced by Escherichia and Salmonella. Bacteroides found in the caeca are commonly associated with the production of proteins, amino acid and lactose, while Bacillus coli is only capable of fermenting lactose. Clostridium sp., Enterococcus sp. and Bacteroides sp. (proteolytic bacteria) are responsible for the production of ammonia and possibly toxic metabolites such as phenol and indole (Rehman et al., 2007).

2.7.2 Microbial fermentation

SCFAs are produced by the local microbiota and can be used as an index for microbial fermentation. This is due to the blood and prey of raptors naturally containing very little SCFAs. Annison et al. (1968) found that SCFAs were detected along the digestive tract with the highest concentrations found in the caeca, which correlates with the finding that the caeca contains the highest concentration of bacteria.

Lactic acid (lactate) is the second most important marker that can be used as an index for microbial fermentation; it is produced by Streptococcus, Lactobacillus and Bacillus (Stevens and Hume, 1998), and by Megamonas sp. and Clostridium sp. in chickens (Mead, 1997). It is not only produced after fermentation of lactose but also after the fermentation of proteins. Lactate has been found to be important in the growth of enterocytes (Fu and Mathews, 1999), and is an important glucose precursor in birds (Brady et al., 1979; Ogata et al., 1982; Franson et al., 1985).

A study by Mead (1997) on chickens identified some of the important producers of glucose, namely Coprococcus, Streptococcus, Peptostreptococcus, Bacteroides sp., Eubacterium sp., Fusobacterium sp.,

Megamonas sp., Bifidobacterium sp., Clostridium sp. and Gemmiger. Glucose is an important source of energy for most vertebrates, in contrast to birds which rely more on proteins for their energy.

Absorption of SCFA by enterocytes occurs faster than absorption by bacteria. This ensures that the host is supplied with the necessary nutrients before bacteria are supplied.

Dietary endogenous carbohydrates, dietary nitrogenous and endogenous nitrogenous compounds can be utilized to produce SCFAs, while nitrogenous compounds can also produce ammonia (NH4+) and microbial proteins. Ammonia can either be absorbed or excreted with the rest of the digesta. When ammonia is absorbed by the enterocytes, water and nitrogen can be preserved. Endogenous nitrogen compounds can be uric acid, creatinine, mucus, enzymes involved in digestion and dietary starch fragments (Stevens and Hume, 1998).

2.8 Fermentation of uric acid

Uric acid is produced as the final oxidation product of purine metabolism, but in birds it is also a byproduct of protein synthesis. It is an important waste product of nitrogen (Laverty and Skadhauge, 2008) and is produced by vertebrate mammals and birds (Stevens and Hume, 1998). In mammals, ammonia is transported to the liver, where the liver transforms ammonia into urea that is less toxic, which is then transported to the kidneys and excreted in urine. In birds the uric acid is also excreted through the kidneys, but since birds lack a bladder, urine is transported into the urodeum in the cloaca. When in the cloaca, urine can directly be excreted, as a dry mass, or it can be refluxed with the rest of the digesta. By refluxing urine, percentage of available water increases in the colon and the caeca and this water can then be absorbed through paracellular transport back into the body.

Local microbiota, such as ureolytic bacteria, in the colon and caeca are able to ferment uric acid into ammonia (NH3) and microbial proteins and aid in the production of SCFA (Thomas and Skadhauge, 1988). Microbiota involved in fermentation, primarily consist of anaerobic bacteria, which include species of Coprococcus, Streptococcus, Peptostreptococcus, Bacteroides, Fusobacterium, Eubacterium and Clostridium and yeast, but an earlier study done by Rouf and Lomprey (1968) also observed aerobic bacteria fermenting uric acid. Bacteria involved were Aerobacter aerogenes, Klebsiella pneumoniae, Serratia kiliensis, Serratia marcescens, Pseudomonas aeruginosa, Bacillus sp., Bacillus subtilis and in low quantities Mycobacterium phlei. They were found to utilize uric acid to produce nitrogen, carbon and energy.

By utilizing ammonia and microbial proteins, bacteria can produce amino acids. Ammonia concentrations are higher in the caeca compared to the other segments of the digestive tract (Rehman et al., 2007). By producing amino acids, endogenous nitrogen can be preserved (Mead 1989; Vispo and Karasov, 1997; Stevens and Hume 1998; Józefiak, 2004; Kohl, 2012). It was thought that amino acids were primarily utilized by local microbiota and were an internal aid in the production of SCFAs (Thomas and Skadhauge, 1988), but Skadhauge (1993) found that chickens placed on a high sodium diet displayed carrier-mediated transport of amino acids in the caeca and colon.

Uric acids can be excreted as a fluid or as urate salts, which can be observed as white crystals next to fecal matter (Klasing, 1999). According to Klasing (1998) water absorption only occurs in the colon and kidneys in raptors other than owls, this is due to the caeca being inactive in other raptors. But this statement is theoretical and has not yet been proven, which means the possibility exists that the caeca of other raptors might also be functional.

2.9 Digesta passage rate

The digesta passage rate is measured from the time a bolus (inert marker) is swallowed until it first appears in the fecal matter (Stevens and Hume, 1998). The passage rate of digesta can help determine concentrations and type of indigenous bacteria found in the digestive tract as well as level of microbial fermentation. This is done by measuring how long a marker is retained in a particular segment of the digestive tract.

2.10 Mean retention time (MRT)

The mean retention time (MRT) is the average time that digesta spends in the digestive tract and can be measured by using different markers like titanium dioxide (TiO2). Retention time is naturally prolonged in birds due to the presence of four different pacemaker regions along the digestive tract (Stevens and Hume, 1998; Klasing, 1999). This internally prolongs absorption time of water and electrolytes. Depending on the main site of fermentation, fluid and particles will vary in their MRT’s. This was proven by Steven and Hume (1998) in studies done on omnivores, herbivores and carnivores, where fluid markers and particulate markers were used.

In birds, it was proven that fluids have a shorter MRT compared to solids, which remained longer in the ventriculus, resulting in a longer MRT (Stevens and Hume, 1998). Fluid markers resulted in the observation that the caeca is able to selectively conserve fluid in a couple of species, resulting in a prolonged MRT. By increasing MRT in the caeca, food is retained longer in the raptor, resulting in a longer period of increased body mass and ultimately ensuring that less food is ingested to counteract increased body mass, since an increase in body mass makes it difficult for birds to fly. The increased retention time in the colon and caeca assists with the increased microbial population (Stevens and Hume, 1998).

2.10.1 Influence of quantity and type of diet

Different raptors on two different diets resulted in different MRT. Barton and Houston (1993) illustrated that when Eurasian buzzards and peregrine falcons were fed pigeons and rabbits at different points in time, MRT for pigeons was longer than for rabbits in both species. They also demonstrated that quantity of the food type had an effect on the MRT. When four different raptor species were given five chicks (large meal) compared to two chicks (small meal), peregrine falcon, honey buzzard, red kite and Eurasian buzzard all showed an increase in MRT for the larger meal. Other studies also indicated that the amount of food eaten per day by the birds was found to be influenced by MRT, enzymatic hydrolysis and absorption by the epithelium (Penry and Jumars, 1987; Karasov, 1990; Karasov and Levey, 1990).

2.10.2 Influence by body temperature

Not only diet but also the body temperature influence MRT. According to Steven and Hume (1998), when reptiles were recorded having lower body temperatures MRT increased, which was thought to accommodate for a decreased intake of nutrients and fermentation. Stevens and Hume (1998) compared MRT found in the study done by Guard (1980) with a study of their own, on rock ptarmigan (Lagopus muta), sooty albatross (Phoebetria fusca) and rockhopper penguin (Eudyptes chrysocome)

and found that reptiles have a MRT of 35-48 hours, while birds have a MRT of 3.8-9.9 hours, which is considerably shorter.

2.10.3 Influence by length of digestive tract

The length of the digestive tract plays an important role, meaning a longer tract has a longer MRT. Barton and Houston (1993) found that Eurasian sparrow hawk, peregrine falcon and Eurasian hobby (Falco subbuteo) have relatively short digestive tracts resulting in short MRT, while Eurasian buzzard, European kestrel and red kite have longer digestive tracts resulting in longer MRT. The short digestive tract together with a short MTR can result in insufficient enzymatic hydrolysis and can therefore results in the insufficient absorption of nutritional end-products (Barton and Houston, 1993). A longer digestive tract will aid in longer presentation of digesta to the digestive tract and caeca, resulting in an increased digestibility of digesta and microbial fermentation, which will result in an increased absorption of nutrients and the production of SCFA (Swart et al., 1993). The increased retention time in the colon and caeca assists with the increased microbial population (Stevens and Hume, 1998). Birds with shorter tracts have a decreased microbial fermentation and digest food less efficiently, resulting in the loss of energy in the excreta. To compensate for this disadvantage, these birds will increase the frequency of meals per day. By eating more meals per day, the required nutrients and energy per day is still achieved.

2.11 Inert markers

In the past, chromic oxide (Cr2O3) was used as an inert marker but since TiO2 is easier to analyze, Cr2O3 has been replaced with TiO2 (Barton and Houston, 1991).

Titanium dioxide (TiO2) is a white, unflavored metal oxide that can easily be administered to raptors and mammals and used as an inert marker to determine efficiency of digestion, food passage rates and MRT. Since it is assumed that TiO2 is not absorbed when passing through the digestive tract, TiO2 can be used as a marker for food intake and efficiency of digestion by determining the ratio marker in food and in fecal samples. But since the study conducted by Barton and Houston (1991) suggested that

TiO2 is not a worthy marker to determine digestibility due to the incomplete recovery of TiO2, different markers might be better suited, such as stable isotopes.

Trophic isotopes, also known as stable isotopes, carbon (C), nitrogen (N), sulfur (S) and hydrogen (H) are found in everything, everywhere and are known to have a light and heavy mass (12C and 13C, 14N and 15N, 33S and 34S, and 1H and 2H). To determine the isotope ratio, heavy isotopes are more often used than light isotopes. Every living organism has its own isotope ratio (δ13C, δ15N, δ34S and δ2H) which is determined by percentage 13C, 15N, 34S and 2H found. Percentage stable isotopes and isotope ratio increases with organisms higher up in the food chain. Plants have the lowest ratio δ 13C and δ 15N while carnivores have the highest ratio δ 13C and δ 15N (McCutchan et al., 2003).

Studies have shown that the diet influences stable isotope ratio, thus when switching diets, isotope ratio will change in the predator, meaning that there is a trophic shift between prey and predator (Klaassen et al., 2004). Trophic shifts occur due to differential digestion or fractionation and indicate that there is an interaction of isotopes between predator and prey.

Animals with higher metabolic rates are believed to absorb C and N with higher frequencies (Trieszen et al., 1983; Carleton and del Rio, 2005). Trieszen et al. (1983) found that every tissue has its own incorporation rate, which would suggest that tissues with high metabolic rates could incorporate C and

N with a higher frequency leading to tissues with different isotope ratios (DeNiro and Epstein, 1981; Hobson and Clark, 1992).

Feathers have been found to be good indicators for protein intake in a diet, due to largely being comprised of keratin, and since raptors almost exclusively feed on animal proteins, feathers are excellent indicators to use when measuring different isotropic markers (Mizutani et al., 1992). This is helpful since feathers are easily obtained and are large enough to homogenize and measure the isotropic markers (Mizutani et al., 1992). Even although feathers are mostly comprised of keratin, containing high concentrations of sulfur and low concentrations of lipid and water, it is believed that small concentrations of lipid (and not high concentrations of sulfur) can influence the results. The low concentration of lipids is thought to lower the δ13C of the feathers due to their own low δ13C (DeNiro and Epstein, 1977), but Mizutani et al. (1992) found no significant differences between feathers with and without oil.

2.11.1 C isotopes

The atmosphere contains a natural concentration of 12C and 13C, which is respectively 98.9% and 1.1% 13 and results in bicarbonate (CO2) having a δ C of 8‰ (Bocherens and Drucker, 2003; Marshall et al., 2007). Due to mass-dependent effects in plants during photosynthesis, plants contains less 13C than the atmosphere, and since plants can be categorized in three groups according to their photosynthesis 13 process, δ C will vary according to the plant. Plants needing only three C-ions (C3-plants) to make CO2 13 13 have a δ C of -26‰, while plants needing four C-ions (C4-plants) to make CO2 have a δ C of -12‰.

C isotope ratios in the predator (raptors) are related to the isotope ratio of their prey (rats, mice, chickens, quails and rabbits) which in turn is related to the preys’ diet (McCutchan et al., 2003). When prey change diets, this will have an indirect effect on the predator as well. Herbivorous animals can change food sources which will result in trophic shift within the animal. Due to plants having low δ 13C, the trophic shift in the animal will be limited. Trophic shifts are larger in omnivore animals due to the significant difference between the isotope ratio in animals and plants. In carnivorous animals, trophic shifts will be less extreme, due to feeding on animals, which all have a higher isotope ratio.

2.11.2 N isotopes

The natural concentration of 15N and 14N in the atmosphere is, respectively 0.37% and 99.63%. δ 15N has been found to systematically increase per trophic level with approximately 3‰ (DeNiro and Epstein, 1981; Kelly, 2000; Kuitems, 2007). This means when a herbivore feeds on a plant, δ 15N will increase in the herbivore when compared to the original δ 15N in the plant, thus when a carnivore eat the herbivore, δ 15N will increase in the carnivore compared to that of the herbivore. This indicates that diet can have an effect on δ 15N levels in an organism. Not only does diet have an important effect so does sea- or saltwater. Marine organisms have higher δ 15N compared to terrestrial plants and animals living near the sea or salt-water bodies have higher δ 15N then animals that do not, which means that raptors which frequently hunt in the sea or salt waters have higher δ 15N then raptors hunting terrestrial animals. Fish-based diet leads to higher δ 15N than meat-based diets (Schoeninger and DeNiro, 1984). N isotope ratio is also determined by the diet of the predator. The trophic shift for N was found to be highest in animal diets containing high amounts of N, and was found to be influenced by the production of excreta containing nitrogenous end-products like ammonia and uric acid (McCutchan et al., 2003).

According to different studies (Ambrose, 1993; Hobson et al., 1993; Cormie and Schwarcz, 1996; Klaassen et al., 2004), a period of high protein absorption can likely result in higher N intake and can result in higher δ 15N. This means that not only the isotope ratio in the diet itself, but also the protein percentage in diet has an effect (Klaassen et al., 2004).

2.11.3 S isotopes

S isotope is contained in amino acids. Even though there is no fractionation involved when amino acids are absorbed by the epithelium, fractionation of organic S or other processes might be important (Mekhtiyeva et al., 1976; McCutchan et al., 2003).

Sulfur ratio in animal tissues can be acquired by organic and inorganic S. Organic S is obtained from the diet while inorganic S can be obtained from the environment. Both types of S contributes to the S- ratio (Mekhtiyeva et al., 1976).

2.11.4 H isotopes

H isotopes are not often used but have been used to study migratory patterns of birds (Hobson and Wassenaar, 1997) and are determined not only by the diet. H isotopes indicate a small shift between diet and prey but it is insignificant when compared to the variation in the environment (DeNiro and Epstein, 1981).

3. Hypothesis

Raptors are known to have different digestive tracts depending on the species, for instance a well- developed crop or no crop, appendicular or vestigial caeca and short or long digestive tract. It is still unclear why this difference exists since the diet of raptors is mostly the same, all typically including high concentrations of animal tissues.

3.1 Function of the vestigial caeca

The most important question to address is why did the caeca become vestigial in all raptors except the owl? By providing an answer to this question, it may provide insight as to why there is a difference, how the digestive tract compensates for the vestigial caeca and whether the vestigial caeca are involved in fermentation.

3.2 Efficiency of diet utilization

It is well documented that the length of the digestive tract can be correlated to the efficiency of digestion and on the frequency of hunting. Previous studies are based on either eagles, falcons, buzzards or owls, but very few studies have included vultures, thus the efficiency of their digestion remains unknown.

3.3 Speed of excretion

Each food source has its own nutritional value and depending on this value, food will be excreted with a certain speed. Food sources with high nutritional value tend to ensue longer time in the host, resulting in the production of SCFA and energy.

Each food source is consists of nutritional valuable and invaluable particles. The digestive tract must determine which particles are nutritionally valuable and which are not since it needs to obtain the highest concentration of energy, while using the least amount of energy, to digest the food.

The reason why some nutritional particles remain longer in the host is however, still unknown. Determining whether a separation mechanism, that is capable of separating nutritionally valuable from invaluable particles, by selectively pushing these valuable particles into the caeca, is involved, can help in deciding what food sources are best suited for raptors. This mechanism may also provide insight in the functionality of the vestigial caeca including which part it plays in fermentation.

4. Aim

The goal of this study was to investigate a range of digestive function parameters in raptors, specifically vultures, and to determine whether the vestigial caeca is still active in fermentation.

4.1 Objective 1: Food intake and excretion pattern

Food intake and excretion patterns will shed insight on how much and how frequently vultures need to eat and whether different fecal samples can be found.

To determine nutrient intake profile, mainly focusing on dietary fiber by weighing food before and after offering.

To determine differences in fecal samples by photographing each fecal sample after which color and consistency will be methodically recorded.

4.2 Objective 2: Efficiency of diet utilization

By measuring stable isotopes (ratio C and N) in predator (feathers of vultures) and prey (ear of a rat) and comparing these measurements with measurements of the fecal samples, it will provide insight in how efficiently the prey was digested. When the stable isotopic ratio lies closer to that of the prey, less prey was absorbed by the predator, whereas when the ratio lies closer to that of the predator more prey was absorbed by the host and fecal samples mostly contain cells from the predator.

To determine efficiency of digestion in vultures, by measuring stable isotopic markers in the fecal samples of vultures as well as in the food source (rats), in the feathers of the vultures and in the fecal sample.

4.3 Objective 3: Passage rate and mean retention time (MRT)

To determine whether the vestigial caeca is still active, passage rate and MRT will be determined. By providing an answer to this question, the function of the vestigial caeca may be better understood.

To determine passage rate in vultures, a one-time titanium oxide (TiO2) bolus will be given as a model to determine the excretion pattern. This model will help to determine movement patterns of food in the digestive tract, which include time of food spends in the digestive tract and whether food enters the caeca. The maximum TiO2 in the fecal samples correlates with the MRT. MRT gives insight in the average time it takes food to be digested and excreted which is linked to efficiency of digestion.

5. Material and Methods

The study was conducted in ‘Kalba Bird of Prey Centre’ in the United Arab Emirates. Four griffon vultures (Gyps fulvus), all female, and three lappet-faced vultures (Torgos tracheliotos), one female and two males, were used in this study (Table 4). The vultures were first weighed before being temporarily placed inside individual enclosures. The four griffon vultures are normally housed outside in a group aviary setting, while the lappet-faced vultures are normally tethered in mews, outside under shelters next to each other. Their ages ranged from around 1 year to 4 years (some were of unknown age) (Table 4).

The vultures were used during a four-day trial. During the first 3 days, they were kept on a diet consisting of rats, while on the fourth day, the diet was changed to a chicken diet. Both these prey items are a normal dietary component of these birds. The change from rat to chicken was done to ensure the ingestion of the TiO2.

Vultures were stimulated to eat all the food that was provided by hand feeding the vultures.

Table 4. Animal details of all the vultures used in the 4-day study Names Species Gender Age Weight (kg) Housing ZE Griffon Female 3y (2015) 6 Group ZJ Griffon Female 4y (2014) 7 Group ZF Griffon Female Unknown 6.5 Group AE Griffon Female 1y (2017) 7 Group AD Lappet-faced Female Unknown 7 Individual AB Lappet-faced Male 3y (2015) 7 Individual ED Lappet-faced Male 2y (2016) 7 Individual

5.1 Food intake and excretion pattern

The normal diet of the vultures prior to the trial, consisted of a variation of food rotating between rats, quails, chickens, one-day old chicks and red meat (beef). However, for the trial, the diet was changed to rats only.

The rat diet (3 days) consisted of two rats per day, weighing around 183g each, although the second rat was only given after the first rat was ingested. Rats were weighed after the tail was removed, after which they were placed in a bowl and offered to the vultures. At the end of each day, leftover food was collected, origin was recorded and weighed.

Water was freely available from the second day onwards.

During the 3-day trial, vulture enclosures were checked on the hour, any fecal sample detected was photographed, collected, marked, sun-dried and placed in a freezer at -19°C. Sampling stopped at 5- 6pm and restarted the next day at 7am, at which time all the samples that were produced during the night were collected as one sample. After the first sample of the day, normal sampling continued as described above.

5.1.1 Color variation

To determine color variation in fecal samples of the vultures, photographs were taken of each sample. A color scheme (Fig. 5) was created from the photos taken during the experiment as reference. Three different colors were identified: green, brown and dark.

The color ranged from light brown to dark green. Brown was divided into three different variations ranging from 1-1.8, dark fecal samples were given 2, green fecal samples were divided into four variations ranging 3-3.9 and when urates covered the sample, it was given 4.

A B C D

E F G H I . Figure 5. Color variation identified of fecal samples collected from vultures A. light brown (1), B. brown (1.4), C. darker brown (1.8), D. dark brown (2), E. light green (3), F. green (3.3), G. darker green (3.6), H. dark green (3.9), I. white (4) due to urates covering the sample

5.1.2 Consistency variation

To determine the consistency of fecal samples (Fig. 6), the same photographs were used to create a consistency chart to be used as a reference. The chart ranged from fluid (1-4), soft (5-7) to firm (8-10) samples.

A B C D . . .

E F G . . .

H I J . Figure 6. Fecal samples collected from vultures indicating the variation in consistency, ranging from fluid to firm samples. A-D. Fluid samples, E-G. Soft samples and H-J. Firm samples

5.1.3 Statistical analyses

In the case of the excreta consistency scores, a repeated-measures analysis of variance was used, with score as within-subject variables and vulture species as between-subject variables.

To estimate the relationship between percentage of a certain fecal sample color and food intake, a linear regression analysis was performed. All statistical analyses were performed in SPSS 25. Significance is accepted at P < 0.05.

5.2 Efficiency of diet utilization

To estimate difference in efficiency of digestion between the vulture species, the excreta samples collected as explained above (5.1) were tested for stable isotope ratios, 15N to 14N and 13C to 12C, standardized using international standards. Individual feathers of each vulture and a piece of a rat ear were obtained, sealed in plastic bags, marked, stored and sent to the ISOFYS laboratory of the Faculty of Bioscience Engineering at Ghent University (Belgium), where the stable isotope ratios were measured according to the technique of Peterson and Fry (1987). The feathers were taken to determine the stable isotope profile of the vultures, whereas the rat ear was taken to represent the stable isotope profile of the diet (which consisted of rats solely).

5.2.1 Determining stable isotopes

Samples were first freeze-dried, then weighed and homogenized using a ball mill for 1 min. (PM 400 Retsch, Haan, Germany). To analyze 13C to 12C ratio and 15N to 14N ratio, an element analyzer (EA) was coupled to an isotope ratio mass spectrophotometer (IRMS).

Nitrogen was standardized against atmospheric nitrogen gas whereas carbon was standardized against Pee Dee limestone deposit.

When measuring stable isotopes, the results automatically include the total concentration of nitrogen (N) (%N) and carbon (C) (%C), as the results of the element analysis.

All carbon and nitrogen measurements were reported in permil (‰, parts per thousand) units relative to international standard for the isotopes, δ15N or δ13C (‰) = (Rsample-Rstandard/Rstandard) x 1,000, where R is the ratio of the heavier to lighter isotope.

Because the type of diet and the setting did allow neither total quantitative excreta collection, nor the homogenous diet mixing of a marker, a novel approach was used to estimate differences in digestive efficiency between the vulture species. The principle applied with the values of δN and δC is that the closer the value of the excreta is to the value of the diet (hence farther from the vultures’ value), the less efficient diet utilization was. Therefore, this estimator of diet utilization was calculated:

Protein utilization efficiency (DUE) (%) = 100 x (δNexcreta – δNdiet)/( δNvulture – δNdiet);

Organic matter utilization efficiency (DUE) (%) = 100 x (δCexcreta – δCdiet)/( δCvulture – δCdiet).

The total concentrations of N and C in the dry matter of the excreta may indicate how efficiently crude protein and organic matter are digested in the digestive tract.

When N is higher compared to C, less N, which comes from proteins, is retained in the host, indicating that the digestion of crude protein is inefficient. Determining the N:C ratio, therefore provides insight in the use of protein compared to other nutrients. When the ratio is higher, less proteins will be used compared to other nutrients.

The utilization efficiencies using δ15N and δ13C as mentioned above should theoretically provide results between 0-100percent, although already one factor may cause deviation, which is the “discrimination factor”. This factor indicates to which extent the dietary stable isotope profile is accumulating in the host, and this factor is not known for vultures. Yet, in this study, we assumed that the factor would not be substantially different between the two vulture species, hence not causing a bias in the interpretation of relative differences between the species.

5.3 Passage rate

To determine the digestive passage rate in vultures, titanium dioxide (TiO2) was used as a non- digestible marker. Four gram of TiO2 was placed insight a chicken skin (Table 9), weighing between 33- 69g which were subsequently offered to all the vultures around 8am as a one-time bolus on day 4.

After the skin were ingested, the vultures were given a chicken breast including hind legs, which weighed between 202-205g.

For the following 24 hours, all fecal samples were individually collected on the hour, placed in plastic bags, marked, sundried and placed in the freezer at -19°C. At the end of the day, leftover food was collected and weighed. After 5-6pm, the sample collection stopped until the following day at 7am when all the samples were collected as one sample, placed in plastic bags, marked, weighed, sun-dried and placed in freezer at -19°C.

The collected samples were brought to the laboratory of the Department of Nutrition, Genetics and Ethology at Ghent University (Belgium) with permission of Belgian authorities (FAVV). There, the samples were defrosted and further dried. Samples collected at a specific hour were used to make a fecal mixture, which were evaluated for TiO2 according to the technique of Myers et al. (2004).

5.3.1 Determining TiO2 concentration

The samples were first weighed, then 0.5g of each sample was placed inside 250ml macro-Kjeldahl digestion tubes. A control sample was included for background correction. A reaction catalyst was added to each tube, including the control tube, which contained 3.5g K2SO4 and 0.4g CuSO4. Thirteen ml of concentrated H2SO4 was added to each tube and placed in an oven at 420°C for 2 hours to digest. After 2 hours, tubes were removed from the oven and allowed to cool for a minimum of 30min.

Subsequently, 10ml of 30% H2O2 was added to each tube and allowed to cool for another 30min. Thereafter, distilled water was added to each tube until 100g was reached. Then the content of the tubes was individually filtered through Whatman No 541 filter paper to remove all the excess precipitate. After filtration, samples were measured with a spectrophotometer at 410nm to determine absorbance. The spectrophotometer was first calibrated, by adding 0, 2, 4, 6, 8 and 10mg of TiO2 to blank tubes, which were prepared as explained above. The 0 mg standard was used to zero the spectrophotometer.

5.3.2 Calculation and statistical analysis

To determine the mean retention time (MRT) of TiO2 a calculation was done according to Thielemans et al. (1978).

MRT (h) = ∑ ti Ci ∆ti /∑ Ci ∆ti

MRT, in hours, is based upon 3 factors, namely C푖 which is the marker concentration (TiO2), t푖 is the interval indicated by time (hours after marker was ingested by the vultures) and ∆t푖, is the interval of the concentration sample and was measured as followed:

∆ti = ((ti + 1 - ti) + (ti - ti -1))/2

To have an indication of potential differences in the TiO2 excretion profile independent of MRT, per bird the average TiO2 excretion was calculated before and after 10 hours after the feeding of the TiO2 bolus. The values were used in a repeated-measured analysis of variance, with hour as within-subject variable and vulture species as between-subject variable. Statistical analysis was performed in SPSS 25. Only two analyses were done, one at the beginning of the day and one at the end of the day, since data in the middle of the day was inconclusive, resulting in a disfigured table.

6. Results

6.1 Food intake and excretion pattern

6.1.1 Total amount eaten per day

Even though less food was offered to the griffon vultures as a response to their lower intake (Table 5), they had higher amounts of leftover food, which contributed, together with the reduced voluntary intake of food, to the total amount ingested being significantly lower (P=0.016) than that of the lappet- faced vultures.

Table 5. The daily mount of given food, leftovers and eaten food (rats) in the study with griffon vultures (n=4) and lappet-faced vultures (n=3) Given (g/kgBM/d) Leftovers (g/kgBM/d) Eaten (g/kgBM/d) Griffon vulture 44.5 21.42 23.23 Lappet-faced vulture 58 5.57 52.57 P 0.024 0.035 0.016 SEM 27 26 51 g/kgBW/d = gram/kilogram bodyweight/day SEM: standard error of the mean

6.1.2 Color and consistency variation

During the trial, three different colors of fecal samples were identified: brown, green and dark. It was observed that the intensity of the colors varied from a light color to a very dark color. It was also noted that all fecal samples observed were relatively wet, with some containing very little urine and other samples containing a lot of urine.

6.1.2.1 Color variation

Results (Fig. 6) indicate that the color profile of fecal samples was significantly different between the two species. Griffon vultures had a significantly lower percentage of brown samples (P=0.006) and a significantly higher percentage of green samples (P=0.040) compared to the lappet-faced vultures. Percentage of dark and white samples was not significantly different (P=0.298 and P=0.930 respectively).

In lappet-faced vultures, color of the fecal samples tended to follow a patter namely in the morning (7-9 am) fecal samples were light brown and during the day the samples turned progressively darker. This pattern was repeatedly seen during the three days and was observed for all three lappet-faced vultures (Fig. 7, Appendix 1). Griffon vultures did not seem to follow any distinct pattern when observing fecal samples.

Figure 6. Color profile of fecal samples collected from griffon (n=4) and lappet-faced (n=3)

Lappet-faced vulture 3 day trial

1.5

Color Color variation 0.5

0 8u 9u 10u11u12u14u15u16u17u 7u 8u 9u 10u11u12u14u15u16u17u 7u 8u 9u 10u11u14u15u16u17u

Table 7. Color variation observed in fecal sample of on lappet-faced vultures. In the morning the fecal samples were light brown (1), while during the day the fecal sample became progressively darker (2)

6.1.2.2 Consistency variation

Consistency of fecal samples was similar for both vulture species, indicating no difference between the two species. Soft fecal samples, ranging between 5-7, were observed most abundantly in both species (Fig. 8).

Light brown samples were mostly observed containing a soft to firm consistency, dark brown samples were mostly fluid to soft samples and green samples ranged from fluid to firm with no clear connection to the variation in green.

40 Lappet-faced vulture 35 Griffon vulture 30 25 20 15 10

Proportionofscore (%) 5 0 0 2 4 6 8 10 12 -5 Excreta consistency score

Figure 8. Distribution of excreta consistency scores in griffon vultures (n=4) and lappet-faced vultures (n=3) feeding on rats (error bars represent standard deviations)

6.1.3 Correlation between food intake and color of fecal samples

The difference in amount of food ingested (Fig. 9) was significantly correlated with color of the fecal samples. As the food intake increased, production of brown fecal samples increased. However, because the lappet-faced vulture had a significantly higher food intake and also produced much more brown fecal samples, this may just be an accidental feature and needs to be further investigated.

. 80 70 y = 0.2158x - 24.972 R² = 0.7854 60 50 40 30 20 10 0 -10 0 100 200 300 400 500 Proportionexcretaofbrown (%) -20 Food intake (g/d)

Figure 9. Amount of food ingested by vultures correlated to the proportion of brown fecal samples

6.1.4 Leftover food

The majority of the leftover pieces (Table 6) collected included heads, legs, skin, spinal cords, thorax and pelvis of rats. The majority of rat heads were untouched and when legs were found, they still contained skin and muscles. Amount of muscle attached to the spinal cord and the thorax varied. Some vultures cleared all the muscle from the bones, while others ate very little of the muscles between the

ribs. According to leftover pieces found, skin and spinal cord were the least favorable pieces, while the most commonly eaten organs were the intestines.

Griffon vultures abandoned the food quicker, leaving more leftover pieces. The findings are correlated to the amount of food ingested by the vultures. Griffon vultures ingested the least amount of food and therefore obtained the highest amount of leftover food (Table 6).

Lappet-faced vultures more often ate all the food offered although one of the three vultures did leave some leftover food on each day as can be seen in Table 6.

Table 6. Organ pieces leftover by the vultures Name Species Day 1 Day 2 Day 3 ZE Griffon vulture Heads, legs, skin, spinal Heads, legs, thorax, Heads, legs, skin, spinal cord, pelvis, intestines, skin, spinal cord with cord, ribs with pieces of small piece of the liver some muscle attached, the muscles. intestines ZJ Griffon vulture Two rats with an open Heads, legs, thorax, Heads, legs, skin, spinal abdomen skin, spinal cord, cord, ribs with pieces of intestines the muscles. ZF Griffon vulture Heads, legs, skin, thorax, Heads, legs, skin, spinal No leftovers spinal cord with pieces of cord muscle attached, second rat almost untouched, open abdomen with uneaten intestine AE Griffon vulture Heads, legs, skin, thorax, Heads, legs, skin, spinal Very little skin, one leg, spinal cord with pieces of cord spinal cord muscle attached AD Lappet-faced No leftovers No leftovers No leftovers vulture AB Lappet-faced No leftovers No leftovers No leftovers vulture ED Lappet-faced Spinal cord Skin, spinal cord Head, 2 legs, skin, spinal vulture cord

6.2 Efficiency of diet utilization

Table 7 indicates δ 15N and δ 13C isotopes recorded in the ear of the rat, in feathers and fecal samples of the griffon and the lappet-faced vultures. The δ 15N isotope for the ear of the rat was 5.11‰, while the average δ 15N for the feather of the griffon vultures (n = 4) was 8.29‰ and 6.89‰ for the feathers of lappet-faced vultures (n=3).

The average δ 15N isotope for fecal samples collected from griffon vultures was 4.66‰ while it was 4.91‰ for the lappet-faced vultures. These findings indicated that the δ 15N measured in the fecal samples was more close to δ 15N of the rat than that of the feathers of both species. This would indicate an inefficient ingestion of nitrogen and thus proteins by both vulture species.

δ 13C isotopes measured were 21.30‰ in the rat’s ear, 20.28‰ and 20.81‰ in feathers and 21.77‰ and 21.49‰ in fecal samples for the griffon and lappet-faced vultures respectively. The same tendency as for the N isotopes was observed, with the value of the food source (rat’s ear) being more similar to the value in fecal sample.

The total concentration N and C in the rat was 13.04%N and 46.99%C, in griffon and lappet-faced vultures 15.53%N and 11.98%N and 54.15%C and 43.15%C respectively. The total concentration N in the fecal samples of both vultures were 17.80%N and 12.33%N respectively and the total concentration C were 21.66%C and 21.54%C respectively.

In both species %N was lower than %C which indicates that crude proteins were for some part absorbed in both species.

Table 7. Stable isotopes δ 15N and δ 13C, and C% and % N Mean recorded in the feathers and fecal samples of griffon (n=4) and lappet-faced (n=3) vultures and a rats ear (n=1) %N Mean + SD δ 15N in ‰ versus AIR % C Mean + SD δ 13C in ‰ versus PDB Mean + SD Mean + SD Rat Ear Rat ear 13.04 + 0.23 5.11 + 0.07 46.99 + 1.16 -21.30 + 0.09 Average Feathers Griffon 15.53 + 0.89 8.29 + 0.17 45. 15 + 1.49 -20.28 + 1.63 Lappet 11.98 + 4.26 6.89 + 1.92 43.15 + 4.45 -20.81 + 5.14 P-value 0.224 0.273 0.496 0.874 Average Fecal samples Griffon 17.80 + 1.42 4.66 + 0.51 28.87 + 2.98 -21.77 + 0.26 Lappet 12.79 + 1.30 4.91 + 0.47 21.84 + 2.47 -21.49 + 0.36 P-value 0.005 0.531 0.019 0.304 SD= standard deviation

25.000 35.000

30.000 20.000 25.000 15.000 20.000

10.000 15.000

% N % in excreta %C %C inexcreta 5.000 10.000 5.000 0.000 Lappet-faced Griffon vulture 0.000 vulture Lappet-faced vulture Griffon vulture

Figure 10. Percentage nitrogen (N) and carbon (C) measured in fecal samples of griffon (n=4) and lappet-faced vultures (n=3)

There was a significant difference between %N and %C found in the fecal samples among both species (Fig. 10). Fecal samples of griffon vultures contained 17.80%N and 28.87%C, while fecal samples of lappet-faced vultures contained 12.79%N and 21.84%C, which was significantly less than the griffon vultures.

Table 8. Nitrogen to Carbon (N:C) ratio, measured in fecal samples of griffon (n=4) and lappet-faced vultures (n=3) Species N:C in fecal samples N dig fecal samples C dig fecal samples Griffon vultures 0.62 + 0.05 -14.50 + 17.21 -4.55 + 41.38 Lappet-faced vultures 0.59 + 0.02 15.75 + 37.12 -8.30 + 10.32 P-value 0.30 0.25 0.86

The N:C ratio between the two species did not differ significantly as can be seen in Table 8 and Fig.11 with a value lower than 1 indicating that N was lower than C thus crude protein is efficiently digested. The higher the N:C ratio becomes the less proteins will be used compared to other nutrients.

δ 15N dig and δ 13C dig was not significantly different between the two species. Normally, values are between 0 and 100 percent but since the variability was too high, some results were negative.

0.7000

0.6500

0.6000

0.5500 N C to in excreta ratio 0.5000 Lappet-faced vulture Griffon vulture

Figure 11. Nitrogen to Carbon ratio measured in fecal samples of griffon (n=4) and lappet-faced (n=3) vultures

6.3 Passage rate

During the passage rate experiment (Day 4) (Appendix 4), all vultures received more or less the same amount of food (237g-271g), but they produced different amounts of leftover food and therefore different amounts of ingested food per day.

The average ingested food by griffon vultures was 6.50g/kgBM and by lappet-faced vultures it was 7.80g/kgBM. Lappet-faced vultures had only 0.30g/kgBM of leftovers (an average of 0.1g/kgBM per bird) while the four griffon vultures had a total of 3.51g/kgBM of leftovers (an average of 0.90g/kgBM per bird).

6.3.1 Mean retention time (MRT)

Visual analyses of the key data of MRT showed no apparent skewness or kurtosis of the data distribution, from which we assumed a normal distribution of the population. Statistical analysis found no significant difference (P=0.117) for the MRT between the species. MRT in griffon vultures seemed to be longer than that of lappet-faced vultures even though statistical analysis of MRT data showed no significant differences (Fig.12).

Taken into account that griffon vultures ate less food than lappet-faced vultures, it would seem as if griffon vultures digest food longer and maybe more effective than the lappet-faced vulture.

15 (h) 10

5 MeanRetention Time(MRT) 0 Griffon vulture Lappet-faced vulture

Figure 12. Mean retention time (MRT) of griffon vultures (n=4) and lappet-faced vultures (n=3)

6.3.2 TiO2 concentration

The TiO2 concentrations were determined for all the samples collected on the hour after ingestion of the bolus.

No significant differences could be detected in the concentrations of TiO2 collected at the beginning (P=0.272) of the experiment (Fig. 13 A) or at a late point in time (P=0.550) (Fig. 13 B) between the two species.

However, TiO2 concentration in griffon vultures started at less than 50g/kg compared to 115g/kg in the lappet-faced vultures. At the end, TiO2 concentration was 1100g/kg in the griffon vultures compared to just over 500g/kg in the lappet-faced vultures. Although not significant, TiO2 concentration in the griffon vultures was almost double that of the lappet-faced vulture. This after starting with a lower concentration at the beginning of the experiment.

Although no significant differences were observed between the TiO2 concentrations for both species, there seemed to be a tendency that the passage rate of digesta for griffon vultures was higher than for lappet-faced vultures.

200 2000

150 1500

100 1000

concentration

late phase late

early phase early

- -

50 500

(g/kg) (g/kg) (g/kg) (g/kg)

0 0

Excreta TiO Excreta TiO Griffon vulture Lappet-faced Griffon vulture Lappet-faced vulture vulture

Figure 13. Titanium dioxide (TiO2) concentration measured in fecal samples of griffon vultures (n=4) and lappet- faced vultures (n=3), Left: collected at the beginning of the trial and Right: collected late in the trial

7. Discussion

7.1 Food intake and excretion pattern

7.1.1 Total amount eaten per day

The distinctly higher voluntary food intake in lappet-faced vultures compared with griffon vultures, despite their comparable body weight, suggests that the latter either more efficiently digest rats or have a lower metabolic rate.

In nature, lappet-faced vultures are one of the larger Old World vultures (Bouglouan, 20104), and are capable of tearing carrions apart, suggesting that these vultures can be less selective when ingesting harder to digest pieces. The higher food intake might be associated with ingestion of proportionately tougher tissues, “fibrous” parts in the selected diet, such as skin, bones and tendons. This was observed when comparing the leftover pieces between the two species. Griffon vultures favored softer tissues and almost excluded the ingestion of skins, while lappet-faced vultures had no particular favoritism and ingested almost everything. Only one lappet-faced vulture produced leftovers and these were mainly of the spinal cord.

In the present study setting, it was however not possible to compare the animal fiber content of the leftovers with the originally offered diet. Future work could investigate this partial hypothesis.

7.1.2 Color and consistency variation

The color of fecal samples is known to vary according to the diet of the raptors. The same can be said for consistency of the fecal sample and the amount of fecal samples produced. Brown samples are most frequently observed although dark and green samples are also commonly observed according to literature (Ash and Needham, 20065). Fecal samples of digested rat have a very unique color and consistency, and the most frequently observed color is light brown (Fig. 5A) while its consistency is between soft and firm (Fig. 6I) (Ash and Needham, 20064). This type of fecal samples was commonly observed in lappet-faced vultures during the three day trial period, however it was only observed once in the griffon vultures namely at the beginning of the trial. Also, light brown samples were not observed immediately after ingestion of rat but only the next morning, which could indicate that digestion of rat occurred at night therefore only appearing in the fecal samples the next morning. During the day, the majority of samples were dark brown. This was observed even after ingestion of the rat. These samples would seem to consist of something other than rat or could be caecal droppings especially since dark brown samples were accompanied with a consistency ranging between fluid to soft. This could however not be confirmed since it is unclear what caecal droppings look like in vultures. Although light brown fecal samples are described as a normal feature in vultures, results from this trial would indicate that dark brown fecal samples are just as common since they were observed very frequently.

4 Bouglouan: http://www.oiseaux-birds.com/article-old-world-vultures.html (20/05/2018) 5 Ash and Needham: http://www.themodernapprentice.com/mutes.htm (20/05/2018)

It is unclear what is happening in the digestive tract of lappet-faced vultures causing the fecal samples to change in color from light brown to much darker and further research is needed. It is anyhow remarkable that such a color shift was not appearing in the griffon vulture excreta, and may be related to the higher intake of fibrous animal matter in the former. De Cuyper et al. (2018) recently demonstrated the presence of a digestive separation mechanism in a mammalian carnivore, the dog, when fed complete prey. The appearance of a color shift throughout the day in the lappet-faced vultures may therefore reflect such a separation mechanism as well. Although light brown is the normal color of digested rat, which is the only diet the vultures received why was the fecal sample produced by griffon vultures green? Green fecal samples are also considered to be normal (Ash and Needham, 20064). And it is most likely caused by biliverdin which is a bile pigment (Mateo et al., 2004). Green fecal samples can also be observed after fasting or have a pathological origin, such as when a raptor is anorexic or during overproduction of bile (Ash and Needham, 20064). But during our experiment, all vultures used were in a healthy condition which was confirmed by regular blood works and a stable body weight.

Two of the griffon vultures used in our experiment had fasted for 1 day prior to the experiment, which could account for the production of green fecal samples although this should have changed later in the experiment. Also, the other griffon vultures produced the same color fecal samples, and had not been fasting. It is therefore unlikely that fasting had a significant contribution in the green color production.

Since there was a significant difference between green and brown fecal samples in griffon vultures, with green being produced most abundantly, it could be considered that green is a normal feature in griffon vultures.

As said earlier, light brown samples are typical for digested rat. If so, it is significant that griffon vultures rarely produced light brown samples, which could indicate that rat was inefficiently digested in griffon vultures.

Consistency of fecal samples between the two species did not differ. There was no clear pattern in the production of fluid to soft and soft to firm fecal samples. The majority of fecal samples in both species were soft samples. When observing normal fecal samples, they should present as soft to firm samples without excess of fluids and never be hard. Caecal droppings on the other hand should present as soft to fluid-like samples, which is caused by the excess of water, SCFA and other molecules. Dark brown samples with a consistency of fluid to soft were collected and it is possible that these were caecal droppings. This could however not be confirmed at this time, and more research is needed to substantiate this observation.

7.1.3 Correlation between food intake and color of fecal samples

When considering a correlation between the color of fecal samples and amount of food ingested, it would seem that with more food ingested, a higher incidence of brown fecal samples was observed. This was confirmed in all the lappet-faced vultures and in one griffon vulture.

As mentioned earlier, light brown samples are indicative of digested rat while dark brown samples could be indicative of caecal content. This would match the idea that lappet-faced vultures can eat higher amounts of fibrous tissue and at least partly ferment it.

However, since lappet-faced vultures ingested the highest amount of food, produced more fecal samples with a majority of brown fecal samples, it could just be a common feature that is only expressed when food intake is high. This should be further investigated.

7.1.4 Leftover food

Vultures and other raptors in captivity are normally kept on a diet that is composed based on literature studies, since no protocol exists to feed captive raptors. The data whereby nutritional intake is determined is mostly based on the measurements of the entire prey.

However, when considering our results, it is clear that griffon vultures rarely ingested the entire rat, even though they have large beaks and are fully capable of swallowing an entire rat. This indicates that when whole prey items are used to evaluate or calculate nutrition value, it will provide inaccurate results since the actual nutritional intake is probably often lower.

Griffon vultures tended to avoid ingesting skin and spinal cord, mostly ingesting softer tissues, while lappet-faced vultures ate everything without any clear favoritism for a particular organ. The finding could agree with the hypothesis that lappet-faced vultures are able to digest tougher tissues while griffon vultures prefer tissue that is easily digestible.

Literature states that stress could also be a cause for not ingesting all the food. Stress could be caused by the new environment, human interaction or being housed alone. However, it is believed that even although some stress might be experienced by the vultures, our observation that griffon vultures allowed more leftover food than the lappet-faced vultures was not mainly caused by stress.

7.2 Efficiency of diet utilization

Since every being has its own stable isotope ratio, stable isotopes can be used to measure how efficiently food is digested in an organism. Since it is known that the predator has a higher δ 15N than its prey, the ratio measured in fecal samples of the predator could explain how efficiently prey was digested.

When comparing fecal samples with stable isotopes of the feathers and the rat’s ear, data measured suggested that digestion of the rat was inefficient since the ratio of the stable isotopes in the fecal samples were closer to the ratio of the rat than that of the vultures. There was also no difference measured between ratios for both species, which seemed to indicate the efficiency of digestion was similar for both species.

When considering the observed fecal samples, the results are peculiar, since fecal samples collected from both vulture species seemed to indicate that digestion was efficient since no bones, fur or other tissues were visible in the fecal samples. Fecal samples in lappet-faced vultures contained light brown fecal samples representing the digestion of rat, and therefore should be efficiently digested.

When stable isotopes are measured in fecal samples, a discrimination factor can emerge, meaning that stable isotopes can accumulate in the host at different rates depending on the species and can present as if digestion was inefficient. When the two vulture species were compared, δ 15N in the fecal samples and the feathers was similar. When ignoring the presence of a discrimination factor, this would seem as if digestion had been completely inefficient. This is highly unlikely, suggesting that the discrimination

factor is likely high in these birds. Anyhow, based on the absence of difference between the vulture species, the isotope analysis could not indicate differences in digestive efficiency.

The higher N concentration in the griffon vulture excreta points to a more efficiently digested protein in lappet-faced vultures than in griffon vultures.

The same was found for %C, which indicated that the digestion of organic matter was more efficient in lappet-faced vultures compared to griffon vultures.

The findings contribute to the hypothesis that lappet-faced vultures are thought to consume tougher tissues, therefore the digestive tract has evolved to digest and metabolize the tough tissues (fibrous tissues). This could be due to a higher fermentative capacity of fibrous prey matter, such as skin, tendons and bones. In this case, “animal fiber” is actually protein.

The lack of difference in fecal samples N:C ratio between the species indicates that eventual differences in digestive efficiency are not different for protein than for other organic matter.

7.3 Passage rate

7.3.1 Mean retention time

MRT is the average time digesta spends inside the digestive tract and can also provide information on efficiency of the digestive tract. In lappet-faced vultures, a greater variability of MRT was seen compared to griffon vultures. This could mean that digestion or production of fecal samples occur at different rates within a species and could indicate that the efficiency of digestion varies and is not stable within the specie.

7.3.2 TiO2 concentration

The tendency to a higher passage rate in griffon vultures suggest differences in the processing of whole carcass diet between the two species. It again supports the idea of the lappet-faced vultures spending more effort in trying to digest more fibrous material, whereas the griffon vulture may select for more digestible parts, hence developing a strategy for higher throughput of highly digestible material.

8. Conclusion

The trial compared two vulture species with similar weight but with different presence of vestigial caeca. Even though the function is still unclear, differences were found between the species. Griffon vultures ingested significantly less food and produced the highest amount of leftovers compared to lappet-faced vultures. The color of the fecal samples were significantly different, with lappet-faced vultures producing mostly brown samples and griffon vultures producing mostly green samples. Soft fecal samples were observed in a higher frequency than fluid or firm samples but was not significantly different between the species. Lappet-faced vultures had a significantly lower %N and %C than griffon vultures indicating they are capable of digesting crude protein and organic matter more efficiently. Digestive passage rate was similar despite different dietary intake, supporting the idea that lappet- faced vultures have more evolved to a high capacity to digest fibrous matter from animal diets. The different color profiles are again in support of such a strategy. These first explorations in vulture

digestion warrant further investigation as they seem to point to different nutritional needs among raptors, even among vulture species, and may depend on their digestive anatomy.

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10. Attachments

Appendix 1.

The variation in the color of fecal samples of lappet-faced vultures (n=3). The tables indicate in the mornings the production of light brown samples (1) while during the day the samples became progressively darker. At night all three lappet-faced vultures produced dark samples. The color variation were divided into four numbers: 1=light brown, 2=dark brown/dark, 3=green, 4=white, covered with urates

Lappet-faced vulture (AD) - Day 1-3

4 3.5 3 2.5 2 1.5

Color Color variation 1 0.5 0 9u 11u12u14u15u16u17u18u 7u 8u 9u 10u11u12u14u15u16u 7u 8u 9u 10u11u12u14u15u16u17u

Lappet-faced vulture (AB) - Day 1-3

1.5

Color Color variation 0.5

0 7u 8u 9u 10u11u12u14u15u17u 7u 8u 9u 10u11u12u14u15u17u 7u 8u 9u 10u11u14u15u16u17u

Lappet-faced vulture (ED) - Day 1-3

1.5

Color Color variation 0.5

0 8u 9u 10u11u12u14u15u16u17u 7u 8u 9u 10u11u12u14u15u16u17u 7u 8u 9u 10u11u14u15u16u17u

Griffon vulture (ZE) - Day 1-3

1 Color Color variation 0 7u 8u 11u 14u 15u 16u 17u 7u 8u 9u 10u 11u 12u 14u 16u 17u 19u 7u 8u 9u 12u 14u 15u 16u 17u

Griffon vulture (ZJ) - Day 1-3

variation 2

1 Color Color 0 7u 8u 9u 14u 16u 7u 8u 10u 11u 14u 15u 17u 19u 7u 8u 9u 10u 11u 12u 14u 15u 16u 17u

Griffon vulture (ZF) - Day 1-3

1 Color Color variation

0 10u 15u 7u 8u 10u 11u 12u 14u 16u 17u 7u 8u 9u 10u 11u 12u 14u 15u 16u 17u

Griffon vulture (AE) - Day 1-3 4 3.5 3 2.5 2 1.5

1 Color Color variation 0.5 0 10u11u12u14u15u16u17u 7u 8u 9u 10u11u12u14u15u16u17u 7u 8u 9u 10u11u12u14u15u16u17u18u

Appendix 2.

Table. Stable isotopes recorded in the feathers and fecal samples of griffon (n=4) and lappet-faced (n=3) vultures and a rats ear (n=1) %N Mean + SD δ 15N in ‰ versus % C Mean + SD δ 13C in ‰ versus PDB AIR Mean + SD Mean + SD Rat Ear Rat ear 13.04 + 0.23 5.11 + 0.07 46.99 + 1.16 -21.30 + 0.09 Feathers ZE 14.57 + 0.29 8.44 + 0.07 45.88 + 1.19 -18.78 + 0.05 ZJ 16.34 + 0.09 8.34 + 0.14 43.44 + 0.77 -20.05 + 0.18 ZF 15.66 + 0.38 8.10 + 0.30 46.14 + 0.71 -22.02 + 0.05 AE 14.03 + 0.07 7.45 + 0.07 44.81 + 0.24 -18.94 + 0.05 AD 14.19 (5.63+ 0.73) 6.88 (4.05 + 0.25) (36.52 + 1.74) (-28.45 + 0.46) AB 14.72 + 0.05 7.95 + 0.30 46.03 + 0.37 -18.48 + 0.23 ED 13.54 + 0.54 8.13 + 0.70 45.22 + 0.45 -17.37 + 0.27 Average Feathers Griffon 15.53 + 0.89 8.29 + 0.17 45. 15 + 1.49 -20.28 + 1.63 Lappet 11.98 + 4.26 6.89 + 1.92 43.15 + 4.45 -20.81 + 5.14 P-value 0.224 0.273 0.496 0.874 Fecal samples ZE 17.28 + 1.19 4.76 + 0.05 30.89 + 0.25 -22.06 + 0.15 ZJ 19.41 + 0.55 5.11 + 0.06 30.27 + 0.00 -21.63 + 0.01 ZF 16.71 + 0.29 4.10 + 0.03 25.45 + 0.14 -21.61 + 0.04 AE 14.18 + 0.39 4.72 + 0.06 25.11 + 0.00 -21.34 + 0.04 AD 11.20 + 0.48 4.38 + 0.06 19.13 + 0.07 -21.13 + 0.00 AB 12.37 + 0.55 5.07 + 0.05 21.34 + 0.06 -21.96 + 0.09 ED 13.42 + 0.11 5.48 + 0.24 21.77 + 0.17 -21.52 + 0.09 Average Fecal samples Griffon 17.80 + 1.42 4.66 + 0.51 28.87 + 2.98 -21.77 + 0.26 Lappet 12.79 + 1.30 4.91 + 0.47 21.84 + 2.47 -21.49 + 0.36 P-value 0.005* 0.531 0.019* 0.304 SD= standard deviation

The feathers of AD, lappet-faced vulture, obtained 2 different results when N was calculated. The one feather measured 14.19 %N and 6.88 δ 15N without a standard deviation, while the other feather measured 5.63 %N and 4.05 δ 15N with a standard deviation. Even though there is no standard deviation for the first feather, it is more likely that this feather was of the vulture and the second feather was probably incorrectly collected and therefore placed in brackets. The first feather was inconclusive when % C and δ 13C were measured, therefore the results were placed in brackets.

Appendix 3.

Table. Titanium dioxide (TiO2) excretion patterns in the individual vultures

ZE-Griffon vulture 600 500 400 300 255.03 200 138.2 124.91 118.75 100 0 5.52 3.61 23.04 0 TIO2 CONCENTRATION (MG) CONCENTRATION TIO2 0 1 2 6 7 8 9 23 24 HOURS AFTER START (HOURS)

ZJ-Griffon vulture 500 400 300

200 126.22 139.09 100 33.95 54.55 0 1.51 2.16 0.88 3.63 0

TIO2 CONCENTRATION (MG) CONCENTRATION TIO2 0 1 6 7 8 22 23 24 25 26 HURS AFTER START (HOURS)

ZF-Griffon vulture 500

400

300

200

100 45.91 32.7 43.14 0 0.63 9.05 TIO2 CONCENRTATION (MG) CONCENRTATION TIO2 0 0 2 4 6 8 23 24 HOURS AFTER START (HOURS)

AE-Griffon vulture

3000 2538.18 2500 2000 1500 1000 500 0 0 0 23 TIO2 CONCENTRATION (MG) CONCENTRATION TIO2 HOURS AFTER START (HOURS)

AD-Lappet-faced vulture 800 700 600 500 593.3 400 300 346.21 200 251.15 100 0 3.57 57.63 33.31 0 0 2 3 5 7 8 22 25 TIO2 CONCENTRATION (MG) CONCENTRATION TIO2 HOURS AFTER START (HOURS)

AB-Lappet-faced vulture 500 431.98 450 428.49 400 337.2 350 300 250 200 150 100 TIO2 CONCENTRATION (MG) CONCENTRATION TIO2 50 0 3.8 15.79 11.71 5.3 2.11 3.93 0 0 0.33 1 2 4 6 7 8 9 23 HOURS AFTER START (HOURS)

ED-Lappet-faced vulture 600 500 400 300 231.85 176.01 200 100 36.37 0 9.48 5.09 9.03 4.27 14.43 20.45

TIO2 CONCENTRATION (MG) CONCENTRATION TIO2 0 0 1 2 3 4 6 7 8 9 23 24 HOURS AFTER START (HOURS)

Appendix 4.

Table 9. Amount of food provided to each vulture during the one-day experiment including a one-time 4g titanium dioxide (TiO2) rolled up in chicken skin, chicken breast and hind legs, leftover chicken and total amount consumed by each bird

Species TiO2 Chicken breast and Leftover Total (4g) + chicken skins hind legs (g/kgBM/d) (g/kgBM/d) (g/kgBM/d) (g/kgBM/d) Griffon vulture 6.45 30.65 3.51 33.60 Lappet-faced vulture 7.76 28.95 0.29 36.29 g/kgBM/d = gram/kilogram body mass/day