# Fecal and Salivary Cortisol Concentrations in  Woolly(Lagothrix ssp.) and Spider Monkeys (Ateles spp.)

Authors: Kimberly D. Ange-van Heugten; Eric van Heugten; Saskia Timmer; Guido Bosch; Abahor Elias; Scott Whisnant; Hans J. M. Swarts; Peter Ferket; Martin W. A. Verstegen
Journal: International Journal of Zoology
 (2009)

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## Abstract

Detrimental physiological effects due to stressors can contribute to the low captive success of primates. The objective of this research was to investigate the potential impact of diet composition on cortisol concentrations in feces and saliva in woolly (n=27) and spider monkeys (n=61). The research was conducted in three studies: the first investigated spider monkeys in the United States, the second investigated spider monkeys within Europe, and the third investigated woolly monkeys within Europe. Fecal cortisol in spider monkeys in US zoos varied (P=.07) from 30 to 66 ng/g. The zoo with the highest fecal cortisol also had the highest salivary cortisol (P≤.05). For European zoos, fecal cortisol differed between zoos for both spider and woolly monkeys (P≤.05). Spider monkeys had higher fecal cortisol than woolly monkeys (P≤.05). Zoos with the highest dietary carbohydrates, sugars, glucose, and fruit had the highest cortisol. Cortisol was highest for zoos that did not meet crude protein requirements and fed the lowest percentage of complete feeds and crude fiber. Differences among zoos in housing and diets may increase animal stress. The lifespan and reproductive success of captive primates could improve if stressors are reduced and dietary nutrients optimized.


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## Body

## 1. Introduction

Spider(Ateles spp.) and woolly (Lagothrix ssp.) monkeys are two of the
largest New World primates with a weight range
of 5.5 to 11 kg in the wild [1]. They live in South American rainforests in Brazil, Colombia,
Peru, and Ecuador. Approximately 1000 spider
monkeys are reportedly housed in captivity worldwide and only 85 captive woolly
monkeys are reported worldwide [2].
Both spider and woolly monkeys are considered threatened species in the wild [3, 4]. Woolly monkeys are considered extremely difficult to breed
and successfully maintain in captivity [5–8]. The natural diets of both
spider and woolly monkeys are primarily frugivorous. They rely on more than 80%
ripe fruits in their diet [9–12].
Zoological institutions typically feed the majority of woolly and spider monkeys
diets as fruit. It is suspected, however, that human cultivated fruits differ
significantly when compared to wild fruits [13, 14]. Wild fruits
have higher contents of fibers, minerals, proteins, and vitamins as well as a
lower content of total sugar [14, 15]. In addition, there is
reportedly less sucrose and more fructose and glucose in wild fruits than
cultivated fruits [14, 15]. Although zoological institutions attempt to replicate dietary items consumed
in the wild, the actual dietary nutrients fed to the monkeys in captivity may
be very different from that. This is especially true with spider and woolly
monkeys which do not have their free-ranging dietary items analyzed for
nutrient content within available published literature. The seasonality of wild
fruits cause the nutrient content of diets for free-ranging monkeys to differ
substantiality over time due to both item availability and composition and this
seasonal variation is not typically reflected in human-cultivated fruits [14].Elevated levels of dietary sugars
and fats may increase sympathetic nervous system release of cortisol in mammals
[16]. Increased concentrations of cortisol for chronic or
long-term periods of time have been associated with negative health conditions
such as hypertension, immune system suppression, insulin-resistant diabetes,
and poor reproductive success [17–21]. These negative
conditions are also associated with the poor life expectancy of woolly monkeys
in captivity [5]. Various other management factors
within captive primate populations such as housing space, competition for
resources, age, and gender can also cause elevated levels [17, 22–24]. Studies
have successfully evaluated cortisol concentrations in both feces [24–26] and saliva as a measure of
stress in primates [27–30].The objectives of the current research were to
(1) investigate the fecal and salivary cortisol concentrations at multiple
zoological institutions in woolly and spider monkeys, (2) compare cortisol
measurements between spider and woolly monkeys and how they relate to diet
composition and zoo management. It was hypothesized that diets high in
sugar are associated with high fecal and salivary cortisol levels and that due
to their noted poor success in captivity, woolly monkeys are more responsive to
diet as measured by cortisol than spider monkeys.


## 2. Materials and Methods

### 2.1. Animal Housing and Management

Due to the scarcity of captive woolly monkeys and the challenges of transporting
biological samples from endangered primates, the current research was completed
in three separate studies. In all three of the studies, the age, gender, animal exhibit dimensions, species
and subspecies of monkey, and birth location (whether born in captivity or in the
wild) were noted for every monkey. Age was organized into three groups (Group 1
was 0 to 6 years; Group 2 was 7 to 20 years; Group 3 was 21+ years). In ascending
order, these groups are broadly considered youth, adult, and geriatric [1, 10].


### 2.2. Diet Collection and Analyses

For all
studies, zoos maintained the same daily monkey diets for at least three days
prior to data collection and animal keepers did not change the diets during the
research period. At all institutions, except number 4 and number 5, diet
consumption data were collected consecutively for three days (during which
samples for cortisol analyses were also collected). For institutions number 4
and number 5, diet consumption data were collected for only one day and samples
for cortisol analyses were also collected for one day. The diet
disappearance study consisted of a measure of the exact amounts of dietary items provided for 24 or 72 hours minus the dietary
items they did not consume. Evaporative losses were calculated for all
remaining feed items. The daily consumption data were then entered into diet analysis
software to determine percentages of nutrients in the daily diet. Two separate
diet analyses programs were used: Zootrition  (St. Louis Zoo, St. Louis, Mo, USA) and Zoo
Diet Analysis (ZDA; Allen and Baer Associates, Michigan State University (East Lansing, Mich, USA),
and Zoological Society of San Diego (San Diego, Calif, USA)). Depending on the foods used in the monkey diets,
these software programs use both nutrient percentages listed in table values as
well as laboratory measured values to calculate complete diet nutrient
composition. The Zootrition software program was used to calculate crude fat,
crude fiber, crude protein, fructose, glucose, sucrose, and total carbohydrates;
ZDA was used to calculate total sugars (the sum of disaccharides and
monosaccharides). Diet items were grouped into food categories, consisting of
breads and grains, fruits, nutritionally complete primate diet, vegetables, and
miscellaneous items.


### 2.3. Animals and Zoological Facilities

Study 1
Five
zoological institutions in the United States that housed spider monkeys
contributed data to this research study: Gladys Porter Zoo, Brownsville, Tex, USA(n=16); Omaha’s Henry Doorly Zoo,
Omaha, Neb, USA (n=6); Highwater Farms, Kipling, NC, USA (n=5); Little Mans Zoo, Chadbourne, NC, USA (n=8); Little Rock
Zoo, Little Rock, Ark, USA
(n=10). Zoo identity was blinded by giving them a random number. The zoos are hereafter
referred to as Zoos 1 to 5 in random order. Four zoos contributed data for the
fecal collection and four zoos also contributed to the saliva collection. Three
zoos contributed to both saliva and fecal collections. Three species of spider
monkey were used in this research project (Ateles
chamek, Ateles fusciceps, and Ateles
geoffroyi). All animals had access to both indoor and outdoor exhibits
during the study period. Samples were collected in the fall of 2005.Study 2
Two European zoos housing spider monkeys contributed data to this
research study: Apenheul Primate Park, Apeldoorn, The
Netherlands(n=9) and Twycross Zoo, Birmingham,
England, UK
(n=10). To preserve zoo
identity, the zoos are hereafter referred to as Zoos 6 and 7. Three species of
spider monkey were used in this research project (Ateles belzebuth, Ateles fusciceps, and Ateles geoffroyi). All animals had access to both indoor and
outdoor exhibits during the study period. Samples were collected during the
summer of 2006.Study 3
Three zoological
facilities housing woolly monkeys (Lagothrixssp.) contributed data to this
research study: Apenheul Primate Park, Apeldoorn, The
Netherlands (n=7), The Monkey Sanctuary, Looe,
UK (n=10), and Twycross
Zoo, Birmingham, England,
UK
(n=10). Zoos are hereafter referred to as 6, 7, and 8 in random order. All
animals had access to both indoor and outdoor exhibits during the study period.
Samples were collected during the summer of 2006.
It should be
noted that the two zoos in study 2 housing spider monkeys were also two of the
three zoos within study 3 with woolly monkeys (Zoos 6 and 7). Samples from both
monkey types were collected at the same time.


### 2.4. Fecal Sampling and Analyses

In
all studies, fecal samples were only collected if they were fresh (as quickly
after voiding as possible) and not contaminated with urine. Researchers and
monkey keepers routinely watched the monkeys and collected feces from
defecations they witnessed. The animal enclosures were cleaned at least twice
daily to ascertain freshness of the samples. The monkeys were housed in either
pairs or groups and, therefore, it was not possible to isolate fecal samples
from all monkeys or confirm that each monkey contributed a sample. Due to the
complications of transporting fecal samples from three European zoos to the USA, two separate laboratories were
used to analyze fecal samples for cortisol. Sample analyses procedure was
duplicated between laboratories. After the samples were collected, they were
immediately frozen and shipped overnight using dry ice to either North Carolina State University (Raleigh, NC, USA) or Wageningen University (Wageningen, The Netherlands). Samples were immediately stored at−20C° until ready for assay. For analysis, 0.5 g of dried
feces was mixed with 4.5 mL of 90% methanol in deionized water by shaking for
40 minutes. The mixture was then centrifuged at 2500 × g for 15 minutes at 4C°.
The supernatant was transferred to another tube and then evaporated to dryness
under nitrogen gas (99.9% purity) and then reconstituted in 0.15 mL of cortisol
zero calibrator (25COZ, Siemens Medical Diagnostics, Los Angeles, Calif, USA). Spiked
samples with 2.5, 5.0, 10.0, or 20.0 ng of cortisol added to them were tested
for recovery, which averaged 87%. Serial dilutions of pooled fecal extracts
were done and exhibited parallelism with the standard curve. Cortisol
concentrations were determined using the Coat-A-Count cortisol kit (Siemens
Medical Diagnostics, Los Angeles, Calif, USA) according to the
instructions provided by the manufacturer. Fifty μL of the reconstituted sample
was used and samples were assayed in duplicate. Inter- and intra-assay
coefficients of variation were 10.4% and 5.6%, respectively. Sensitivity of the
assay was 0.2 μg/dL.


### 2.5. Saliva Sampling and Analysis

Study 1
Spider
monkeys from the four zoos willing to collaborate with the salivary portion of
this research study collected samples for three consecutive days. Salivary
collections were attempted in the morning before monkey feeding time and in the
afternoon before the last daily feeding time. If possible, additional samples
were occasionally taken during the day. During the collection period, the
monkeys remained in their cages and saliva collection was completely voluntarily.
Not every monkey contributed salivary samples. As described previously [30], saliva was collected by letting monkeys chew on one inch
sections of cotton dental rope (Richmond Dental, Charlotte, NC, USA) held by
metal clamps. The monkey had to chew on the rope for a minimum of one minute
for the sample to be considered suitable for analyses. If more than one monkey
contributed to a sample, it was discarded.
Immediately
after collection, the saturated dental ropes were placed in Salivette tubes
(Sarstedt, Nuernbrecht, Germany) and centrifuged for 15
minutes at 2500× g at 4C° to remove the saliva. The extracted saliva was
then frozen at −20C° and shipped overnight to North Carolina State University, Raleigh, NC, USA,
until it
was analyzed for cortisol. Samples were thawed and again centrifuged at
2500 × g for 15 minutes at 4C°. Fifty μL samples were assayed using the Coat-A-Count
kit according to the manufacturer’s instructions with the following exception.
Another standard point (0.5 ng/mL) was created by diluting a portion of the
provided 10 ng/mL standard. The inter- and intra-assay coefficients of
variation were 8.7 and 6.1%, respectively.
From
preliminary studies and previous published literature, it was noted that
monkeys were more willing to offer salivary samples if a small food incentive
was added to the dental rope [27, 30].
Thus, the dental rope was lightly dipped into grape jelly or touched to a slice
of banana prior to sample collection. This was completed by the same researcher
to minimize variation. To account for dilution effects of jelly or
banana, a conversion factor was established using 24 human volunteers. Each
volunteer chewed on three pieces of dental rope in random order for one
minute. The three pieces included one without food additive, one with banana,
and one with grape jelly. Samples with grape jelly had salivary cortisol
concentrations that were 17% lower than samples without food additive and
samples with banana added had salivary cortisol concentrations that were 51%
lower than those without additive. Cross et al. [27] had previously
developed correction factors for banana added to dental rope using marmosets (Callithrix jacchus) in a similar manner. To
correct for food adhesion, cortisol concentrations obtained using dental rope
dipped in jelly or banana were multiplied by 1.17 and 1.51, respectively. Of
the 66 samples analyzed, 61 were collected using jelly and 5 were collected
using banana.


### 2.6. Statistical Analyses

Study 1
Multiple
samples were collected for some animals and data were averaged by animal
identification (ID) number such that each monkey contributed to the data only
once to avoid skewing of the data. Statistical analyses were conducted
using general linear models procedures of SAS (Cary, NC, USA). The model included zoo.
The least square means procedure was used to calculate fecal and salivary
cortisol means and SEM by zoo. Significances were noted atP≤.05
and tendencies were considered at .05<P≤.10.Studies 2 and 3
Animal ID could not be preserved for samples collected in these studies;
therefore, each sample was considered a unique observation in the data
analysis. Statistical analyses were conducted using general linear
models procedures of SAS (Cary, NC, USA).
The model included zoo. The least square means procedure was used to calculate
fecal cortisol means and SEM by zoo for the spider monkeys in study 2 and the
woolly monkeys in study 3. Significances were noted atP≤.05 and
tendencies were considered at .05<P≤.10.
In
addition, two zoological institutions in studies 2 and 3 both held spider and
woolly monkeys and sample collections were conducted at the same time for both
species. Data from these institutions were analyzed using the general linear
models procedures of SAS using zoo, monkey species, and the zoo by monkey
species interaction in the model. Least square means were calculated by zoo and
monkey species to compare fecal cortisol concentrations between species within
zoos.


## 2.1. Animal Housing and Management

Due to the scarcity of captive woolly monkeys and the challenges of transporting
biological samples from endangered primates, the current research was completed
in three separate studies. In all three of the studies, the age, gender, animal exhibit dimensions, species
and subspecies of monkey, and birth location (whether born in captivity or in the
wild) were noted for every monkey. Age was organized into three groups (Group 1
was 0 to 6 years; Group 2 was 7 to 20 years; Group 3 was 21+ years). In ascending
order, these groups are broadly considered youth, adult, and geriatric [1, 10].


## 2.2. Diet Collection and Analyses

For all
studies, zoos maintained the same daily monkey diets for at least three days
prior to data collection and animal keepers did not change the diets during the
research period. At all institutions, except number 4 and number 5, diet
consumption data were collected consecutively for three days (during which
samples for cortisol analyses were also collected). For institutions number 4
and number 5, diet consumption data were collected for only one day and samples
for cortisol analyses were also collected for one day. The diet
disappearance study consisted of a measure of the exact amounts of dietary items provided for 24 or 72 hours minus the dietary
items they did not consume. Evaporative losses were calculated for all
remaining feed items. The daily consumption data were then entered into diet analysis
software to determine percentages of nutrients in the daily diet. Two separate
diet analyses programs were used: Zootrition  (St. Louis Zoo, St. Louis, Mo, USA) and Zoo
Diet Analysis (ZDA; Allen and Baer Associates, Michigan State University (East Lansing, Mich, USA),
and Zoological Society of San Diego (San Diego, Calif, USA)). Depending on the foods used in the monkey diets,
these software programs use both nutrient percentages listed in table values as
well as laboratory measured values to calculate complete diet nutrient
composition. The Zootrition software program was used to calculate crude fat,
crude fiber, crude protein, fructose, glucose, sucrose, and total carbohydrates;
ZDA was used to calculate total sugars (the sum of disaccharides and
monosaccharides). Diet items were grouped into food categories, consisting of
breads and grains, fruits, nutritionally complete primate diet, vegetables, and
miscellaneous items.


## 2.3. Animals and Zoological Facilities

Study 1
Five
zoological institutions in the United States that housed spider monkeys
contributed data to this research study: Gladys Porter Zoo, Brownsville, Tex, USA(n=16); Omaha’s Henry Doorly Zoo,
Omaha, Neb, USA (n=6); Highwater Farms, Kipling, NC, USA (n=5); Little Mans Zoo, Chadbourne, NC, USA (n=8); Little Rock
Zoo, Little Rock, Ark, USA
(n=10). Zoo identity was blinded by giving them a random number. The zoos are hereafter
referred to as Zoos 1 to 5 in random order. Four zoos contributed data for the
fecal collection and four zoos also contributed to the saliva collection. Three
zoos contributed to both saliva and fecal collections. Three species of spider
monkey were used in this research project (Ateles
chamek, Ateles fusciceps, and Ateles
geoffroyi). All animals had access to both indoor and outdoor exhibits
during the study period. Samples were collected in the fall of 2005.Study 2
Two European zoos housing spider monkeys contributed data to this
research study: Apenheul Primate Park, Apeldoorn, The
Netherlands(n=9) and Twycross Zoo, Birmingham,
England, UK
(n=10). To preserve zoo
identity, the zoos are hereafter referred to as Zoos 6 and 7. Three species of
spider monkey were used in this research project (Ateles belzebuth, Ateles fusciceps, and Ateles geoffroyi). All animals had access to both indoor and
outdoor exhibits during the study period. Samples were collected during the
summer of 2006.Study 3
Three zoological
facilities housing woolly monkeys (Lagothrixssp.) contributed data to this
research study: Apenheul Primate Park, Apeldoorn, The
Netherlands (n=7), The Monkey Sanctuary, Looe,
UK (n=10), and Twycross
Zoo, Birmingham, England,
UK
(n=10). Zoos are hereafter referred to as 6, 7, and 8 in random order. All
animals had access to both indoor and outdoor exhibits during the study period.
Samples were collected during the summer of 2006.
It should be
noted that the two zoos in study 2 housing spider monkeys were also two of the
three zoos within study 3 with woolly monkeys (Zoos 6 and 7). Samples from both
monkey types were collected at the same time.


## 2.4. Fecal Sampling and Analyses

In
all studies, fecal samples were only collected if they were fresh (as quickly
after voiding as possible) and not contaminated with urine. Researchers and
monkey keepers routinely watched the monkeys and collected feces from
defecations they witnessed. The animal enclosures were cleaned at least twice
daily to ascertain freshness of the samples. The monkeys were housed in either
pairs or groups and, therefore, it was not possible to isolate fecal samples
from all monkeys or confirm that each monkey contributed a sample. Due to the
complications of transporting fecal samples from three European zoos to the USA, two separate laboratories were
used to analyze fecal samples for cortisol. Sample analyses procedure was
duplicated between laboratories. After the samples were collected, they were
immediately frozen and shipped overnight using dry ice to either North Carolina State University (Raleigh, NC, USA) or Wageningen University (Wageningen, The Netherlands). Samples were immediately stored at−20C° until ready for assay. For analysis, 0.5 g of dried
feces was mixed with 4.5 mL of 90% methanol in deionized water by shaking for
40 minutes. The mixture was then centrifuged at 2500 × g for 15 minutes at 4C°.
The supernatant was transferred to another tube and then evaporated to dryness
under nitrogen gas (99.9% purity) and then reconstituted in 0.15 mL of cortisol
zero calibrator (25COZ, Siemens Medical Diagnostics, Los Angeles, Calif, USA). Spiked
samples with 2.5, 5.0, 10.0, or 20.0 ng of cortisol added to them were tested
for recovery, which averaged 87%. Serial dilutions of pooled fecal extracts
were done and exhibited parallelism with the standard curve. Cortisol
concentrations were determined using the Coat-A-Count cortisol kit (Siemens
Medical Diagnostics, Los Angeles, Calif, USA) according to the
instructions provided by the manufacturer. Fifty μL of the reconstituted sample
was used and samples were assayed in duplicate. Inter- and intra-assay
coefficients of variation were 10.4% and 5.6%, respectively. Sensitivity of the
assay was 0.2 μg/dL.


## 2.5. Saliva Sampling and Analysis

Study 1
Spider
monkeys from the four zoos willing to collaborate with the salivary portion of
this research study collected samples for three consecutive days. Salivary
collections were attempted in the morning before monkey feeding time and in the
afternoon before the last daily feeding time. If possible, additional samples
were occasionally taken during the day. During the collection period, the
monkeys remained in their cages and saliva collection was completely voluntarily.
Not every monkey contributed salivary samples. As described previously [30], saliva was collected by letting monkeys chew on one inch
sections of cotton dental rope (Richmond Dental, Charlotte, NC, USA) held by
metal clamps. The monkey had to chew on the rope for a minimum of one minute
for the sample to be considered suitable for analyses. If more than one monkey
contributed to a sample, it was discarded.
Immediately
after collection, the saturated dental ropes were placed in Salivette tubes
(Sarstedt, Nuernbrecht, Germany) and centrifuged for 15
minutes at 2500× g at 4C° to remove the saliva. The extracted saliva was
then frozen at −20C° and shipped overnight to North Carolina State University, Raleigh, NC, USA,
until it
was analyzed for cortisol. Samples were thawed and again centrifuged at
2500 × g for 15 minutes at 4C°. Fifty μL samples were assayed using the Coat-A-Count
kit according to the manufacturer’s instructions with the following exception.
Another standard point (0.5 ng/mL) was created by diluting a portion of the
provided 10 ng/mL standard. The inter- and intra-assay coefficients of
variation were 8.7 and 6.1%, respectively.
From
preliminary studies and previous published literature, it was noted that
monkeys were more willing to offer salivary samples if a small food incentive
was added to the dental rope [27, 30].
Thus, the dental rope was lightly dipped into grape jelly or touched to a slice
of banana prior to sample collection. This was completed by the same researcher
to minimize variation. To account for dilution effects of jelly or
banana, a conversion factor was established using 24 human volunteers. Each
volunteer chewed on three pieces of dental rope in random order for one
minute. The three pieces included one without food additive, one with banana,
and one with grape jelly. Samples with grape jelly had salivary cortisol
concentrations that were 17% lower than samples without food additive and
samples with banana added had salivary cortisol concentrations that were 51%
lower than those without additive. Cross et al. [27] had previously
developed correction factors for banana added to dental rope using marmosets (Callithrix jacchus) in a similar manner. To
correct for food adhesion, cortisol concentrations obtained using dental rope
dipped in jelly or banana were multiplied by 1.17 and 1.51, respectively. Of
the 66 samples analyzed, 61 were collected using jelly and 5 were collected
using banana.


## 2.6. Statistical Analyses

Study 1
Multiple
samples were collected for some animals and data were averaged by animal
identification (ID) number such that each monkey contributed to the data only
once to avoid skewing of the data. Statistical analyses were conducted
using general linear models procedures of SAS (Cary, NC, USA). The model included zoo.
The least square means procedure was used to calculate fecal and salivary
cortisol means and SEM by zoo. Significances were noted atP≤.05
and tendencies were considered at .05<P≤.10.Studies 2 and 3
Animal ID could not be preserved for samples collected in these studies;
therefore, each sample was considered a unique observation in the data
analysis. Statistical analyses were conducted using general linear
models procedures of SAS (Cary, NC, USA).
The model included zoo. The least square means procedure was used to calculate
fecal cortisol means and SEM by zoo for the spider monkeys in study 2 and the
woolly monkeys in study 3. Significances were noted atP≤.05 and
tendencies were considered at .05<P≤.10.
In
addition, two zoological institutions in studies 2 and 3 both held spider and
woolly monkeys and sample collections were conducted at the same time for both
species. Data from these institutions were analyzed using the general linear
models procedures of SAS using zoo, monkey species, and the zoo by monkey
species interaction in the model. Least square means were calculated by zoo and
monkey species to compare fecal cortisol concentrations between species within
zoos.


## 3. Results

### 3.1. Animal Housing and Management

Details
on monkey
management and housing information from the zoological institutions are given in Table1 for
all three studies, including number of monkeys studied at each zoo, monkey species, number of
species per zoo, average space per monkey, percentage born in captivity, male
to female ratio, number of monkeys per age group, and laboratory used for
cortisol analyses. The five zoos that participated in study 1 held 45 spider
monkeys while the two zoos that participated in study 2 held 19 spider monkeys.
Study 3 had three contributing zoos holding a total of 27 woolly monkeys. The
same zoo in study 1 (zoo 1) was the only institution that housed more than one
spider monkey species as well as housing monkeys that had been born in the
wild. In study 2, the space allotted per monkey varied greatly between the two
zoos (35 m3 versus 150 m3) and only one of the
zoos held more than one species of spider monkeys. In study 3, the space
allotted per woolly monkey also varied greatly (60 m3–250 m3)
and only Zoo 6 had woolly monkeys that were born in the wild.Table 1
Monkey management and housing information from the
zoological institutions housing spider and woolly monkeys.
ZoonumberNMonkey typeSpecies housedAverage space(m3)*Percentage captive bornGender ratio male : femaleYoung age groupAdult age groupGeriatric age groupLab usedStudy 1116Spider245345:11286126Spider1341002:43211310Spider1561002:8064145Spider1261002:3131158Spider1391006:25301Study 269Spider135702:72252710Spider315070†6:41632Study 367WoollyNA60712:53402710WoollyNA2501008:23252810WoollyNA1251007:31902Average* space per individual monkey.
Zoo† 7 had 7 spider monkeys born in captivity, 1 born wild,
and 2 with unknown birth locations.


### 3.2. Diet Composition and Nutrient Content

Food
groups used in the monkey diets and nutrient percentages varied substantially
among zoological institutions for American zoos housing spider monkeys within
study 1 (Table2) and European zoos housing spider and woolly monkeys within
studies 2 and 3 (Table 3). Most notably, total sugars ranged from 17 to 42% of
the diet among American zoos housing spider monkeys and from 20 to 41% among
the zoos housing European spider and woolly monkeys. There was large variation
between zoos in the amounts of fruits, vegetables, nutritionally complete
primate feeds, breads and grains, and treats. The fruit category ranged from 34
to 83% within study 1, 30 to 49% in study 2, and 19 to 44% in study 3.
Similarly, the percentage of vegetables fed ranged from 0 to 68% when
considering all three studies and the monkey complete feed ranged from 0 to 22% for the three studies. The nutritionally complete primate
feeds utilized by each zoo also varied substantially. Zoo 1 fed Mazuri High
Protein Primate (PMI Nutrition International, St. Louis, Mo, USA); Zoos 2 to 5 fed Mazuri New World
Primate (PMI Nutrition International, St. Louis, Mo, USA);
Zoo 5 also fed Mazuri Old World Primate (PMI Nutrition International, St. Louis, Mo, USA). Zoos 6 and 7 fed Leaf Eater Primate (Mazuri Zoo Foods,
Witham, Essex, UK) and Zoo 8 did not feed a
nutritionally complete primate diet at all. All US zoos in study 1 fed the animals twice daily
while European Zoo 6 fed twice daily and Zoos 7 and 8 fed three meals per day.Table 2
Spider monkey diet nutrient analyses, food group percentages,
and fecal and salivary cortisol concentrations (±SEM) from five zoological
institutions in the US.
Study 1Zoo 1Zoo 2Zoo 3Zoo 4Zoo 5Dietary nutrients, % (dry matter basis)Protein1417211117Fat79445Fiber3.84.16.54.04.0Carbohydrates7164677868Total sugar4217182520Sucrose5.34.01.06.09.1Fructose13.46.02.46.25.0Glucose6.44.11.04.14.0Diet food group, % (as fed)Fruit8342443460Vegetable52732570Nutritionally completefeed‡101822920Breads and grains252011Miscellaneous08009Cortisol analysesFecal cortisol, ng/g*66±10.949±40.630±15.4NA€31±18.2Salivary cortisol,μg/dL£17±3.7aNA7±3.2b11±5.9a,b2±3.2bZoo‡ 1 fed Mazuri High Protein
Primate (PMI Nutrition International, St. Louis,
Mo, USA);
Zoos 2 to 5 fed Mazuri New World Primate (PMI Nutrition International, St. Louis, Mo, USA);
Zoo 5 also fed Mazuri Old World Primate (PMI Nutrition International, St.
Louis, Mo, USA).
Fecal* cortisol is measured on a DM basis.
Significant£ difference in salivary cortisol concentration among institutions (P<.05) is indicated by superscripts a, b.
NA€: information
not available.Table 3
Spider and woolly monkeys diet nutrient analyses, food group
percentages, and fecal cortisol concentrations (±SEM) from three zoological
institutions in Europe.
Zoo 6Zoo 7Zoo 6Zoo 7Zoo 8Study 2 spider monkeysStudy 3 woolly monkeysDiet nutrients, % (dry matter basis)Protein1812221512Fat19812127Fiber8.54.19.08.35.6Carbohydrates5475556275Total sugar2035222841Sucrose4.83.73.23.74.9Fructose10.79.96.78.013.8Glucose6.58.13.54.66.9Diet food group, % (as fed)Fruit3049193044Vegetable6042686048Nutritionally completefeed‡64540Breads and grains25026Miscellaneous20842Cortisol analysesFecal cortisol, ng/g*,£115±40.6a227±40.6b87±26.8x122±20.3x269±20.8yThe‡ nutritionally complete feed utilized by Zoos 6 and 7 was Leaf Eater Primate (Mazuri Zoo Foods,
Witham, Essex, UK).
Fecal* cortisol is measured on a DM basis.
Significant£ difference in fecal cortisol concentration
between spider monkey institutions (P<.05) are indicated by
superscripts a, b and among woolly monkey institutions by superscripts x, y.


### 3.3. Fecal and Salivary Cortisol Concentrations

For
all three studies, fecal cortisol concentrations were highest within each study
when total sugars and fruit percentages were highest and fiber was lowest
(Tables1 to 3). Fecal cortisol concentrations were also highest for all three
studies in the zoos with the highest levels of carbohydrates. Finally, for
fecal cortisol comparisons, the zoos with the lowest percentage of
nutritionally complete primate diet had the highest cortisol concentrations within
all three studies.Study 1
Fecal samples were collected from four zoological institutions. A total of
113 fecal samples were taken. There was no overall difference in fecal (main
zoo effect,P=.21) cortisol concentrations among the zoological
institutions. However, Zoo 1 cortisol concentration (66±10.9 ng/g) was twice the concentrations of Zoos 3 (30±15.1 ng/g; P=.07) and 5 (31±18.2 ng/g; P=.12).
Saliva
samples were taken in four institutions, holding together 37 spider monkeys. A
total of 66 samples were taken and analyzed. Salivary cortisol concentrations
differed among zoological institutions with Zoo 1 concentrations being higher than
Zoo 3 and Zoo 5 (P=.05).Studies 2 and 3
There were 39 fecal samples analyzed for the European spider monkeys
in study 2 and 120 fecal samples for the woolly monkeys in study 3. The spider
monkeys in study 2 differed with fecal cortisol concentrations for Zoo 7 being
higher than Zoo 6 (Table3). The woolly monkeys in study 3 also differed with
fecal cortisol concentrations for Zoo 8 being higher than both Zoos 6 and 7
(Table 3).
When
both of the zoos that housed spider and woolly monkeys were compared by zoo,
overall fecal cortisol concentrations were higher at Zoo 7 (142±12.1) than at Zoo 6 (91±15.8) (P≤.003). In addition, spider monkey fecal cortisol
concentration means were higher (171±15.4) than woolly monkey
means (62±12.6) (P≤.0001).


## 3.1. Animal Housing and Management

Details
on monkey
management and housing information from the zoological institutions are given in Table1 for
all three studies, including number of monkeys studied at each zoo, monkey species, number of
species per zoo, average space per monkey, percentage born in captivity, male
to female ratio, number of monkeys per age group, and laboratory used for
cortisol analyses. The five zoos that participated in study 1 held 45 spider
monkeys while the two zoos that participated in study 2 held 19 spider monkeys.
Study 3 had three contributing zoos holding a total of 27 woolly monkeys. The
same zoo in study 1 (zoo 1) was the only institution that housed more than one
spider monkey species as well as housing monkeys that had been born in the
wild. In study 2, the space allotted per monkey varied greatly between the two
zoos (35 m3 versus 150 m3) and only one of the
zoos held more than one species of spider monkeys. In study 3, the space
allotted per woolly monkey also varied greatly (60 m3–250 m3)
and only Zoo 6 had woolly monkeys that were born in the wild.Table 1
Monkey management and housing information from the
zoological institutions housing spider and woolly monkeys.
ZoonumberNMonkey typeSpecies housedAverage space(m3)*Percentage captive bornGender ratio male : femaleYoung age groupAdult age groupGeriatric age groupLab usedStudy 1116Spider245345:11286126Spider1341002:43211310Spider1561002:8064145Spider1261002:3131158Spider1391006:25301Study 269Spider135702:72252710Spider315070†6:41632Study 367WoollyNA60712:53402710WoollyNA2501008:23252810WoollyNA1251007:31902Average* space per individual monkey.
Zoo† 7 had 7 spider monkeys born in captivity, 1 born wild,
and 2 with unknown birth locations.


## 3.2. Diet Composition and Nutrient Content

Food
groups used in the monkey diets and nutrient percentages varied substantially
among zoological institutions for American zoos housing spider monkeys within
study 1 (Table2) and European zoos housing spider and woolly monkeys within
studies 2 and 3 (Table 3). Most notably, total sugars ranged from 17 to 42% of
the diet among American zoos housing spider monkeys and from 20 to 41% among
the zoos housing European spider and woolly monkeys. There was large variation
between zoos in the amounts of fruits, vegetables, nutritionally complete
primate feeds, breads and grains, and treats. The fruit category ranged from 34
to 83% within study 1, 30 to 49% in study 2, and 19 to 44% in study 3.
Similarly, the percentage of vegetables fed ranged from 0 to 68% when
considering all three studies and the monkey complete feed ranged from 0 to 22% for the three studies. The nutritionally complete primate
feeds utilized by each zoo also varied substantially. Zoo 1 fed Mazuri High
Protein Primate (PMI Nutrition International, St. Louis, Mo, USA); Zoos 2 to 5 fed Mazuri New World
Primate (PMI Nutrition International, St. Louis, Mo, USA);
Zoo 5 also fed Mazuri Old World Primate (PMI Nutrition International, St. Louis, Mo, USA). Zoos 6 and 7 fed Leaf Eater Primate (Mazuri Zoo Foods,
Witham, Essex, UK) and Zoo 8 did not feed a
nutritionally complete primate diet at all. All US zoos in study 1 fed the animals twice daily
while European Zoo 6 fed twice daily and Zoos 7 and 8 fed three meals per day.Table 2
Spider monkey diet nutrient analyses, food group percentages,
and fecal and salivary cortisol concentrations (±SEM) from five zoological
institutions in the US.
Study 1Zoo 1Zoo 2Zoo 3Zoo 4Zoo 5Dietary nutrients, % (dry matter basis)Protein1417211117Fat79445Fiber3.84.16.54.04.0Carbohydrates7164677868Total sugar4217182520Sucrose5.34.01.06.09.1Fructose13.46.02.46.25.0Glucose6.44.11.04.14.0Diet food group, % (as fed)Fruit8342443460Vegetable52732570Nutritionally completefeed‡101822920Breads and grains252011Miscellaneous08009Cortisol analysesFecal cortisol, ng/g*66±10.949±40.630±15.4NA€31±18.2Salivary cortisol,μg/dL£17±3.7aNA7±3.2b11±5.9a,b2±3.2bZoo‡ 1 fed Mazuri High Protein
Primate (PMI Nutrition International, St. Louis,
Mo, USA);
Zoos 2 to 5 fed Mazuri New World Primate (PMI Nutrition International, St. Louis, Mo, USA);
Zoo 5 also fed Mazuri Old World Primate (PMI Nutrition International, St.
Louis, Mo, USA).
Fecal* cortisol is measured on a DM basis.
Significant£ difference in salivary cortisol concentration among institutions (P<.05) is indicated by superscripts a, b.
NA€: information
not available.Table 3
Spider and woolly monkeys diet nutrient analyses, food group
percentages, and fecal cortisol concentrations (±SEM) from three zoological
institutions in Europe.
Zoo 6Zoo 7Zoo 6Zoo 7Zoo 8Study 2 spider monkeysStudy 3 woolly monkeysDiet nutrients, % (dry matter basis)Protein1812221512Fat19812127Fiber8.54.19.08.35.6Carbohydrates5475556275Total sugar2035222841Sucrose4.83.73.23.74.9Fructose10.79.96.78.013.8Glucose6.58.13.54.66.9Diet food group, % (as fed)Fruit3049193044Vegetable6042686048Nutritionally completefeed‡64540Breads and grains25026Miscellaneous20842Cortisol analysesFecal cortisol, ng/g*,£115±40.6a227±40.6b87±26.8x122±20.3x269±20.8yThe‡ nutritionally complete feed utilized by Zoos 6 and 7 was Leaf Eater Primate (Mazuri Zoo Foods,
Witham, Essex, UK).
Fecal* cortisol is measured on a DM basis.
Significant£ difference in fecal cortisol concentration
between spider monkey institutions (P<.05) are indicated by
superscripts a, b and among woolly monkey institutions by superscripts x, y.


## 3.3. Fecal and Salivary Cortisol Concentrations

For
all three studies, fecal cortisol concentrations were highest within each study
when total sugars and fruit percentages were highest and fiber was lowest
(Tables1 to 3). Fecal cortisol concentrations were also highest for all three
studies in the zoos with the highest levels of carbohydrates. Finally, for
fecal cortisol comparisons, the zoos with the lowest percentage of
nutritionally complete primate diet had the highest cortisol concentrations within
all three studies.Study 1
Fecal samples were collected from four zoological institutions. A total of
113 fecal samples were taken. There was no overall difference in fecal (main
zoo effect,P=.21) cortisol concentrations among the zoological
institutions. However, Zoo 1 cortisol concentration (66±10.9 ng/g) was twice the concentrations of Zoos 3 (30±15.1 ng/g; P=.07) and 5 (31±18.2 ng/g; P=.12).
Saliva
samples were taken in four institutions, holding together 37 spider monkeys. A
total of 66 samples were taken and analyzed. Salivary cortisol concentrations
differed among zoological institutions with Zoo 1 concentrations being higher than
Zoo 3 and Zoo 5 (P=.05).Studies 2 and 3
There were 39 fecal samples analyzed for the European spider monkeys
in study 2 and 120 fecal samples for the woolly monkeys in study 3. The spider
monkeys in study 2 differed with fecal cortisol concentrations for Zoo 7 being
higher than Zoo 6 (Table3). The woolly monkeys in study 3 also differed with
fecal cortisol concentrations for Zoo 8 being higher than both Zoos 6 and 7
(Table 3).
When
both of the zoos that housed spider and woolly monkeys were compared by zoo,
overall fecal cortisol concentrations were higher at Zoo 7 (142±12.1) than at Zoo 6 (91±15.8) (P≤.003). In addition, spider monkey fecal cortisol
concentration means were higher (171±15.4) than woolly monkey
means (62±12.6) (P≤.0001).


## 4. Discussion

Although termed frugivorous, spider
and woolly monkeys typically do not live on fruit alone. In the wild, they
procure various animal and plant sources to acquire additional nutrients
throughout different seasons [12, 31]. In
captivity, the fruit diets are supplemented by nutrients within breads and
grains, nutritionally complete primate diets, vegetables, and other
miscellaneous items. Most of the food items consumed by nonhuman primates in
the wild are not available for captive primates commercially. Products grown
for human consumption, which are fed routinely to captive primates, do not
typically have the same nutrient profile as similar items consumed in the wild.
This is because products such as fruits and vegetables available to captive
primates are traditionally higher in water and sugars, lower in fiber, and more
digestible than the natural diet of the animal [13, 14]. Analyses of the composition of wild primate foods for
comparison, however, are scarce. Often captive animal diets are formulated by
trying to equate wild food groups to what can be fed in captivity. Wild
monkey food selection criteria are not based on food groups. They choose
different plant parts based on resource availability and nutrient content [32].The large variation in  diet food
category percentages and nutrients between and within zoos from all three
studies is a potential for concern. The high levels of fruit and low levels of
nutritionally complete foods are not in agreement with the National Research
Council (NRC) nonhuman primate nutrient requirements [33]. Some zoos had
their monkeys consuming as little as 19% fruit while other zoo’s monkeys
consumed as high as 83%. Similarly, some zoos fed no vegetables while others
had their monkeys consuming up to 68% of their diet from vegetables. In
addition, some zoos did not feed any bread or grain items or any primate
nutritionally complete foods. Within study 1, the zoo that fed the highest
percentage of the daily diet as fruit had the highest fecal and salivary
cortisol concentrations in the studied animals. Monkeys in both study 2 and
study 3 also had the highest fecal cortisol concentrations within the zoo that
had the highest fruit content. The fecal cortisol concentrations were also the
highest for each of the three studies within the zoos with the greatest fruit
percentage, the highest carbohydrates, highest total sugar, highest glucose,
and lowest total fiber. There was not a clear relationship with high sucrose
levels having higher cortisol levels although previous research has indicated
that wild fruits consumed by primates have decreased sucrose content compared
to cultivated fruits [14]. Interestingly, the highest fecal cortisol concentrations were also found
at the zoos that fed the least amount of nutritionally complete primate feed.
There did not appear to be a relationship for cortisol concentrations for any
of the studies with respect to protein, fat, or breads and grains percentages
within the zoo diets.The current research shows that
within each of the three current studies, the zoos with the highest
concentrations of nutritionally complete feeds actually had the lowest cortisol
concentrations. In conjunction, low levels of protein were associated with
increased fecal and salivary cortisol concentrations. Being that the majority
of the fruits and vegetable consumed by the monkeys are low in protein, these
animals typically acquire a large percentage of their protein from the
nutritionally complete feeds. It has been estimated that New World primates
require 15% of their diet as crude protein [33]. The highest
concentrations of fecal and salivary cortisol were observed in zoos that did
not meet the crude protein requirement. In addition, in study 1, the highest
concentrations of salivary cortisol were measured in the two zoos that did not
meet the crude protein requirement.Seematter et al. [16] showed that
increased cortisol concentrations may lead to visceral fat deposition, with
adverse metabolic consequences such as
decreasing insulin sensitivity. It has long been recognized that chronic activation of the
hypothalamic pituitary adrenal axis resulting in increased cortisol levels can have deleterious
physiological effects. These effects include the induction or worsening of
hypertension, type 2 diabetes, ulceration in the gastrointestinal tract,
decreased reproduction, osteoporosis, and immunosuppression [17, 19–21]. It appears that the zoo
diets among all three studies may be a contributing factor to the elevation of
cortisol levels and potential health concerns. Future diet formulations for
these species should attempt to reflect nutrient needs instead of trying to
copy food group percentages.Several studies have previously
measured primate cortisol concentration in feces
[24–26, 34] and saliva to determine
stress levels [27–30]. New World primates and Old
World primates reportedly differ in their circulating cortisol
levels as well the metabolism of cortisol [35]. New World primates typically have a 10-fold
higher concentration than the Old World primates [36]. Spider and woolly monkeys are both considered New World primates. The spider monkey salivary cortisol
range reported within study 1 (2–17 μg/dL) was
lower than the New World monkey values previously reported for squirrel monkeys
(Saimiri sciureus) (28±2.3 μg/dL) but was
approximately 10-fold higher than the Old World monkey range (0.3–1.8 μg/dL)
reported for rhesus monkeys (Macaca mulatta) [29, 30]. Thus, circulating levels differ among monkey
species and it is important to establish normal concentrations for all species.Since spider and woolly monkeys
are both New World primates and extremely
closely related [7, 8], differences in fecal
cortisol concentrations between the two were not expected. In addition, the
higher fecal cortisol concentration for spider monkeys was also unexpected
since these species tend to be more successful in captivity with regard to
maintenance and reproduction when compared to woolly monkeys.Salivary cortisol concentrations
were only collected for study 1. However, it is noteworthy that the highest
fecal and salivary cortisol measures within spider monkeys in this study came
from the same zoo. Saliva samples were more difficult to obtain compared to the
fecal samples and the stress caused by the sampling could potentially skew
subsequent results. While some animals were excited to contribute saliva
samples, others were frightened by the procedure and some dominant animals
would not allow the subordinate ones near the collection ropes. Thus,
collection of salivary samples was discontinued in studies 2 and 3. In general,
fecal cortisol represents secretion and metabolism over a number of hours which
can be different than measures of stress from the single moment in time
estimate provided by salivary cortisol
[37]. Although fecal
cortisol is not as sensitive to the intensity of an acute event as serum and
salivary cortisol concentrations, they have the advantage of being easier to
collect and allow more samples to be collected without disturbing behavior
[24, 37].It has been reported that physical
stress, insufficient living space, and obesity are all factors that can cause
hypertension and increase cortisol concentrations [16, 38, 39]. Housing and management differed
between zoos and could have impacted cortisol concentrations measured within
the current research. It is interesting that for study 1, the only zoo to house
spider monkeys born in the wild as well as more than one species of spider
monkey had the highest fecal and salivary cortisol results. Decreased amount of
space per individual monkey did not appear to increase cortisol being that some
monkeys with the most space had the highest cortisol measures while conversely
some of the monkeys with the least space had the lowest cortisol measures.
There were not enough representatives from all enclosure size categories to
statistically analyze the effect of space on the cortisol data. Similarly, the
time of day that the cortisol samples were taken was not analyzed statistically due to the reasons
previously described. However, previous work has shown that spider monkey fecal
samples do not appear to change with respect to the time of day the sample is
taken [40]. Similarly, the possible effects of
gender and age were not able to be examined within this study. These factors
can possibly also influence cortisol concentrations and cortisol metabolism [35]. Due to these possible influencing variables and the fact that study
1 cortisol concentrations were analyzed in an American laboratory while studies 2
and 3 were analyzed in European laboratories, we did not compare the fecal analyses between and
among all zoos within both countries holding spider monkeys (study 1 versus study
2).In addition, the subspecies of
spider monkeys varied slightly among the two studies and this could have
provided cortisol variations. It is important to note, however, that all zoos and
all studies did hold monkeys from both genders and most age groups.Future studies further evaluating the effects of animal housing and
management are recommended before making concrete conclusion about the zoo diet
composition being the only cause of increased cortisol. However, as previously
suggested [41], diet alterations such as changing the monkey daily
feedings so that the monkeys do not have large quantities of sugar (or glucose)
available at any one point in the day or drastically reducing the total sugars
available to the woolly monkeys could potentially decrease the captive health
problems affecting this species.


## 5. Conclusion

This work demonstrates that large
differences exist between zoos with respect to housing facilities and diets of
spider and woolly monkeys. Measuring cortisol concentrations seems to be a
reliable method to compare the cortisol levels of both spider and woolly
monkeys. It can be hypothesized that high amounts of carbohydrates, total
sugars, glucose, and fruits and low amounts of nutritionally complete diets may
cause spider and woolly monkeys to be more susceptible to stress which can in
turn cause metabolic, reproduction, and cardiovascular problems. The lifespan
and breeding success of captive woolly and spider monkeys may improve if the
stressors and negative effects of nutrition on the health status can be reduced
and dietary nutrients can be optimized.



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*Source: 127852-2009-01-08.xml*