Stress in farmed saltwater crocodiles (Crocodylus porosus): No difference between individually- and communally-housed animals

Abstract

Minimising stress in farmed crocodiles is not only important for improving animal welfare, but may also improve skin blemish healing and infection resistance, which influence the quality of the final skin product. Forty near-harvest size saltwater crocodiles (1.6-1.8 m TL) from two Australian farms were sampled to evaluate the effect of different pen types (communal pens n=20; individual pens n=20) on stress as indicated by plasma corticosterone. Blood samples were taken within three minutes of immobilisation and analysed using a commercial enzyme immunoassay kit. There was no relationship with animal size (P=0.16), between farms (P=0.86), pen types (P=0.69), communal pens between farms (P=0.28) or individual pens between farms (P=0.24). Based on corticosterone levels, it appears that individual pens do not cause significantly more stress on harvest-size animals than communal pens. Individual pens meet their design specifications by achieving comparable healing rates of belly skin blemishes as communal pens without compromising animal welfare and minimising the possibility of new blemishes.

Full text

RES E A R C H Open Access
Stress in farmed saltwater crocodiles (Crocodylus
porosus): no difference between individually- and
communally-housed animals
Sally R Isberg
1,2*
and Cathy M Shilton
3
Abstract
Minimising stress in farmed crocodiles is not only important for improving animal welfare, but may also improve
skin blemish healing and infection resistance, which influence the quality of the final skin product. Forty near-
harvest size saltwater crocodiles (1.6- 1.8 m TL) from two Australian farms were sampled to evaluate the effect of
different pen types (communal pens n=20; individual pens n=20) on stress as indicated by plasma corticosterone.
Blood samples were taken within three minutes of immobilisation and analysed using a commercial enzyme
immunoassay kit. There was no relationship with animal size (P=0.16), between farms (P=0.86), pen types (P=0.69),
communal pens between farms (P=0.28) or individual pens between farms ( P=0.24). Based on corticosterone levels,
it appears that individual pens do not cause significantly more stress on harvest-size animals than communal pens.
Individual pens meet their design specifications by achieving comparable healing rates of belly skin blemishes as
communal pens without compromising animal welfare and minimising the possibility of new blemishes.
Keywords: Communal pens; Corticosterone; Individual pens; Saltwater crocodile; Stress
Introduction
Quality specifications of raw crocodile skins imposed
by skin buyers have become increasingly important
over recent years a s the worlds ’ production of crocodil-
ian skins increases. Although saltwater crocodile
(Crocodylus porosus) belly skin remains in high demand
duetoitsdesirabletraitsofevenlydistributedsmall
scales, the definition of a “blemish-free” skin (Isberg
et al. 2004) is becoming increasingly rigorous. Howe ver,
publications detailing the pathological and epidemio-
logical aspe ct s of pre-har vest crocodilian skin blem-
ishes are scarce. Anecdotal evidence suggest s that
many belly skin blemishes are superficial scratches and
punctures that have been caused by non-aggressive in-
teractions between conspecifics within communal pens
(Huchzermeyer 2003). The majority of these scratches
and punctures only penetrate the upper keratin (scale)
layer, whilst some go slightly deeper into the underlying
epidermal and dermal layers. Few penetrate through the
skin into the underlying musculature, although aggressive
interactions can cause these more severe wounds. Other
factors such as rough concrete can also cause skin damage
(Huchzermeyer 2003).
Experience with C. porosus has show n that the major-
ity of superficial blemishes on the belly skin heal given
adequate time. As a result, many saltwater crocodile skin
producers are now using individual pens as a finishing
production stage. These individual pens allow blemishes
to heal without the risk of more being added by conspe-
cifics. Although juvenile and adult salt water crocodiles
are largely solitary and intolera nt of conspecifics in the
wild (Webb and Messel 1977, 1978), on farms they are
reared in groups from hatching until finishing. It is
therefore possible that being placed in a solitary situ-
ation at the finishing stage may increase stress.
Prolonged stress and resultant chronic exposure to
glucocorticoid stress hormones in mammals is associated
with a myriad of negative health effects, including de-
creased infection resistance and altered wound healing
(Schobitz et al. 1994; Christian et al. 2006; Capen 2007;
Marketon and Glaser 2008; Poetker and Reh 2010).
* Correspondence: sally@crocfarmsnt.com
1
Centre for Crocodile Research, PO Box 329, Noonamah, Northern Territory
0837, Australia
2
Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006,
Australia
Full list of author information is available at the end of the article
a SpringerOpen Journal
© 2013 Isberg and Shilton; licensee Springer. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Isberg and Shilton SpringerPlus 2013, 2:381
http://www.springerplus.com/content/2/1/381

Crocodilians have a similar hypothalamic-pituitary-adrenal
system and secrete the glucocorticoid hormone cortico-
sterone in response t o stress (Lance et al. 2000). There
is some evidence that stress causes immunosuppression
and altered wound healing in crocodilians and lizards
(Morici et al. 1997; L ance et al. 2000; French et al. 2006).
Wound infection and slow or altered healing dynamics in
farmed crocodiles near the finishing stages can negatively
influence the quality of the final skin product. The purpose
of this study was to investigate if housing harvest-size
saltwater crocodiles individually increases stress, as indicated
by corticosterone, compared to communal housing.
Methods and materials
Experimental housing
This study was conducted on two crocodile farms in the
Northern Territory, Australia. On both farms, saltwater
crocodiles were raised in communal pens from hatching
until slaughter size (1.6 – 1.8 m), and thereafter in individ-
ual pens for finishing. The communal pens on both farms
were similar in design (concrete floors (60:40 land:water ra-
tio), corrugated iron walls and canvas roof covering), with
anorth–south orientation and the same stocking density
(0.57 m
2
/animal). However, the communal pens on Farm 1
(CP1) have only one body of water whereas the grow-out
pens on Farm 2 (CP2) have two bodies of water. The indi-
vidual pens on both farms (Farm 1: IP1; Farm 2: IP2) were
also similar in design constructed of concrete blocks with a
70:30 land:water ratio and allow 1.22 m
2
/animal. All animals
were fed chicken heads in excess in the late afternoon/early
evening with uneaten food removed the following morning,
and the pens cleaned with a sodium hypochloride solution
(Chlorfoam, Reward Distribution, Darwin) and the water
changed. Crocodiles were fed either 2 (individual pens) or 3
(communal pens) times a week during the wet (hot, humid)
season and this was reduced to 1 (individual pens) or 2
(communal pens) times a week during the dry ( cool, dry)
seas o n as foo d co n s u m pt i o n dec l i n e d .
Experimental animals
From each farm, ten crocodiles from individual pens
(IP1: 10; IP2: 10) and ten from communal pens (CP1:
10; CP2: 10) were sampled (total of 4 0). Blood (2-3 ml)
was taken from the occipital sinus as described by
Lloyd and Morris (1999) using 18 gauge 1” needles into
serum vac utainers within three minutes of ele ctrical
immobilisation a s described by Franklin et al. (2003) to
ensure ba sal corticosterone levels were obtained. From
the communal pens, five animals were sampled within
ten minutes from two differe nt pens to minimise the
possible effect of prolonged human presence on basal
corticosterone levels. This also provided a more repre-
sentative sample of animals in communal pens. Total
length (TL) wa s measured on e ach crocodile from the
tip of the snout to the tip of the tail to investigate any
effect of size on serum corticosterone (Table 1).
Corticosterone assay
Serum corticosterone was measured using the OCTEIA
Corticosterone HS enzyme immunoassay kit (IDS Ltd.,
Tyne & Wear, UK) as per kit instructions. The limits of ac-
curate detection for the kit, according to the kit specifica-
tions, are 0.17-15 ng/ml. As such, sample results less than
0.17 ng/ml were set to 0, and to accurately interpret sam-
ples that may have exceeded the 15 ng/ml upper limit, test -
ing was performed on neat and diluted (either 1:2 or 1:10)
aliquots. An appropriate model for conversion of percent-
age binding values to corticosterone in ng/ml was deter -
mined using the kit calibrator values. CurveExpert (2009,
version 1.4) curve fitting software was used to model the
calibration curve with best-fit curves determined by their
standard error and correlation coefficients. The correlation
of the undiluted and the diluted serum corticosterone was
0.94 showing the validity of using the kit in this manner.
Statistical analysis
The data were log-transformed and analy sed using
Generalised Linear Models (GLM) and analysis of variance
in Genstat (2011, version 14) using variations of the
following model.
LnCort
ijkl
¼ μ þ β
TL
TL
i
þ Pen type
j
þ Farm
k
þ SamplingOrder
il
þ ε
ijkl
ð1Þ
where LnCort
ijkl
is the natural logarithmic transform-
ation of corticosterone (ng/ml); μ is the overall mean;
TL
i
=totallength(TL)oftheith individual; β
TL
=re-
gression coefficient for TL; Pen type
j
is t he fixed effe ct
of either communal or individual pen on the respe ctive
farm (j = CP1, CP2, IP1, IP2); Farm
k
is the fixed effect
of the kth farm (k = 1,2); SamplingOrder
il
is t he effect
of the sampling order (l = 1,..,5) on the ith individual;
and ε
ijkl
is th e random error [assumed N(0,)].
Table 1 Average (standard error of the mean; SEM) total
length and corticosterone (ng/ml) in the communal (CP)
and individual pens (IP) at Farms 1 and 2
Pen type N Total length (cm) Corticosterone (ng/ml)
Farm 1 CP1 10 158.0 (2.3) 8.69 (5.53)
IP1 10 159.4 (3.3) 3.17 (1.44)
Farm 2 CP2 10 165.3 (3.2) 3.04 (0.96)
IP2 10 174.8 (2.4)** 6.34 (2.05)
Overall average 40 164.4 (1.7) 5.31 (1.52)
N is the number of animals in each group.
**indicates significantly difference (P<0.01) in animal size.
Isberg and Shilton SpringerPlus 2013, 2:381 Page 2 of 6
http://www.springerplus.com/content/2/1/381

Results
Animal size versus corticosterone value
The overall average corticosterone value for all crocodiles
on both farms was 5.31 ± (SEM) 1.52 ng/ml. The individ-
ual pen animals from Farm 2 (IP2) were significant larger
than the other crocodiles (P<0.01; Table 1). Irrespective, as
Figure 1 shows, there is no relationship between animal
size (total length; TL) and corticosterone value (P=0.16).
Farm 1 versus farm 2
To evaluate any significant farm effects on the animals, a
basic analysis of Farm 1 versus Farm 2 including all ani-
mals irrespective of pen type was conducted. There were
no significant differences between farms (P=0.86).
Communal versus individual pens
After establishing there was no difference between farms,
an overall analysis was run to evaluate if there were any
differences between communal and individual pe ns. No
relationship was found (P=0.69).
Pen type between farms
By sub-setting the data into communal and individual pen
types, the overall average corticosterone value of communal
pen animals was 5.86 ± 2.81 ng/ml (range 0–56.67 ng/ml)
whilst the average corticosterone value of individual pen
animals was 4.75 ± 1.27 ng/ml (range 0.66-16.16 ng/ml).
An analysis comparing communal and individual pens be-
tween Farms 1 and 2 showed there were no significant dif-
ferences between farms (P=0.28 and P=0.24, respectively).
Records on duration in individual pens were available for
Farm 1. There was no relationship between how long an
animal had been in an individual pen and their cortico-
sterone value (P=0.83, Figure 2).
Sampling order
The order each animal was sampled within communal
pens did not significantly effect corticosterone levels
(P=0.08). However, there was a trend towards increased
corticosterone with prolonged human presence in the
pen (Figure 3).
Discussion
Previous studies of stress in crocodilians have shown a
negative relationship between corticosterone and im-
mune function (Morici e t al. 1997), juvenile growth
rates ( Elsey et al. 1990; Moric i et al. 1997; Turton et al.
1997), juvenile mortality (Morici et al. 1997), the major
reproductive hormones (testosterone – Lance and Elsey
1986; estradiol – Elsey et al. 1991) and reproductive
success (Lance 1994). Plasma corticosterone ha s also
been used to quantify crocodilian stress imposed by
handling (Gist and Kaplan 1976; Lance and Elsey 1999),
different restraint methods (manual versus immobilisa-
tion; Franklin et a l. 2003), different stocking densities
(Elsey et al. 1990), environmental salinity (Lauren 1985;
Lance et al. 2010) and betwe en healthy and runt croco-
diles of similar age (Isberg et al. 2009). In this study, no
significant differences were found in c orticosterone
levels , and thus stress , between harvest-size saltwater
crocodiles (1.6-1.8 m TL) housed in communal or indi-
vidual pens.
The only other study that has examined corticosterone
levels in saltwater crocodiles of a similar size to this study
and in a farm situation was Franklin et al. (2003), whilst in-
vestigating the the effect of immobilisation compared to
manual restraint. Franklin et al. (2003) reported baseline
corticosterone values of 1.09 ± 0.28 and 1.08 ± 0.14 ng/ml
for the immobilised and manual restraint groups respect-
ively, with a maximum average corticosterone value of 2.25
Figure 1 Relationship between animal size (total length; TL) and corticosterone value (ng/ml) according to pen type: communal (Farm
1 – solid triangles; Farm2 – open triangles) and individual pens (Farm 1 – solid circles; Farm 2 – open circles) (P=0.16).
Isberg and Shilton SpringerPlus 2013, 2:381 Page 3 of 6
http://www.springerplus.com/content/2/1/381

ng/ml half an hour following manual restraint. All of
the animals sample d in Franklin et al. (2003) we re
housed in individual pens for three months prior to
sampling. If only the individual pen animals from this
study are considered, the results pre sented herein show
higher overall corticosterone levels (average 5.31 ± 1.52
ng/ml) compared to Franklin et al. (2003). The reason
for this disc repancy is unclear. Howe ver, the ambient
air temperatures (average 22.9°C; range 14-32°C ) in
Franklin et al. (2003) were much lower than in the
current study (33.2°C and 32.9°C for Farms 1 and 2, re-
spectively; www.bom.gov.au). Further work is underway
to establish a relationshipbetween temperature/seasonal
effectsoncrocodilecorticosteronele vels.
There was no statistically significant relationship be-
tween length of time in individual pens and cortico-
sterone level on Farm 1 (Figure 2). However, if there
were more da ta available, it is conceivable that Figure 2
could represent the return to baseline corticosterone
values after moving from a communal to individual
pen. That is, one animal sampled 11 days after being
placed in an individual pen had a cortic osterone value
of 6.11 ng/ml, another after 20 days in an individual
pen had a value of 2.04 ng/ml and then values stabilise
Figure 2 Corticosterone levels (ng/ml) of crocodiles in individual pens from Farm 1 against the number of days in the pen (P=0.83).
Figure 3 The order crocodiles were blood sampled (Sampling Order) from communal pens for corticosterone assay (ng/ml). Communal
animals from Farm 1 (CP1; solid triangles) and Farm 2 (CP2; open triangles) (P=0.08).
Isberg and Shilton SpringerPlus 2013, 2:381 Page 4 of 6
http://www.springerplus.com/content/2/1/381

around 1.17 ± 0.15 ng/ml (n = 7; 28–158 days in indi-
vidual pens). The one exception from Farm 1 was an
animal that had been in its individual pe n for 118 days
(corticosterone value of 15.35 ng/ml).
The remainder of the literature on corticosterone
levels in crocodilians concerns either alligators ( Alliga-
tor mississippiensis ) and/or animals of a different age
than those used in this study. In a study involving cor-
ticosterone implants in alligators less than 12 months
old, the placebo (control) group showed high variation
in serum corticosterone, ranging between 3.8-42.8 ng/ml
(Morici et al. 1997). A similar range of corticosterone values
have been reported in adult alligators at different stocking
densities (Elsey et al. 1990) and alligators (<12 months old)
subject to restraint (Lance and Elsey 1999). In sexually ma-
ture adult all igators, published baseline corticosterone
values range from 0.07–1.86 ng/ml in females (Elsey et al.
1991) and from 0.27-1.99 ng/ml in males (Lance and Elsey
1986). In saltwater crocodiles, Turton et al. (1997) reported
average corticosterone levels of 6.82 ± 0.3 ng/ml, with a
range between 0.24 and 15.29 ng/ml, for less than 14 week
old saltwater crocodile hatchlings. In 5–7 month old salt-
water crocodiles, corticosterone averaged 10.13 ng/ml in
normal crocodiles and 16.18 ng/ml in animals that were
small for their age (Isberg et al. 2009). This large vari-
ation in serum corticosterone prese nted in the pub-
lished literature and refle cted in the present study
indicates there is still considerable work to be done to
understand the underlying dynamics of crocodilian
corticosterone se cretion.
Corticosterone levels of saltwater crocodiles in this
study are generally comparable to published values for
this species and alligators in captivity. Our results suggest
there is no difference in corticosterone levels between salt-
water crocodiles housed communally or individually. As
maintaining low stress levels may be important for general
well-being, prevention of infections and healing of existing
skin blemishes in crocodiles, housing saltwater crocodiles
of this size in individual pens is not considered detri-
mental. Individual housing also has the advantage of
preventing new skin blemishes caused by conspecifics
from occurring.
Competing interests
S.R.I was funded by Porosus Pty Ltd and Lagoon Crocodile Farm to conduct
this study. However, the corticosterone immunoassays were conducted
independently by Berrimah Veterinary Laboratory. SRI designed the
experimental protocol, collected the samples and performed the data
analysis. CMS arranged the corticosterone kits and assays. All authors
participated in drafting the article and have read and approved the final
manuscript.
Acknowledgements
The authors thank the management and staff at Lagoon Crocodile Farm,
Porosus Pty Ltd and Berrimah Veterinary staff (Ms Sue Aumann) and
volunteers for assistance with blood collection and corticosterone assays. We
also wish to thank Dr Greg Brown for help generating the standard
corticosterone curves and Prof Grahame Webb for critical review of analytical
techniques.
Author details
1
Centre for Crocodile Research, PO Box 329, Noonamah, Northern Territory
0837, Australia.
2
Faculty of Veterinary Science, University of Sydney, Sydney,
NSW 2006, Australia.
3
Berrimah Veterinary Laboratories, Department of
Primary Industry and Fisheries, Northern Territo ry Government, Berrimah,
Northern Territory 0800, Australia.
Received: 27 June 2013 Accepted: 8 August 2013
Published: 13 August 2013
References
Capen CC (2007) Endocrine Glands. In: Maxie MG (ed) Jubb, Kennedy, and
Palmer’s Pathology of Domestic Animals, 5th edn. Elsevier Limited,
Philadelphia
Christian LM, Grahamm JE, Padgett DA, Glaser R, Kiecolt-Glaser J (2006) Stress
and wound healing. Neuroimmunomodulation 13:337–346
Elsey RM, Joanen T, McNease L, Lance V (1990) Growth rate and plasma
corticosterone levels in juvenile alligators maintained at different stocking
densities. J Exp Zool 255:30–36
Elsey RM, Lance VA, Joanen T, McNease L (1991) Acute stress suppresses plasma
estradiol levels in female alligators (Alligator mississippiensis). Comp Biochem
Physiol 100A:649–651
Franklin CE, Davis BM, Peucker SKJ, Stephenson H, Mayer R, Whittier J, Lever J,
Grigg GC (2003) Comparison of stress induced by manual restraint and
immobilisation in the estuarine crocodile, Crocodylus porosus. J Exp Zool
298:86–92
French SS, Matt KS, Moore MC (2006) The effects of stress on wound healing in
male tree lizards (Urosaurus ornatus). Gen Comp Endo 145:128–132
Gist DH, Kaplan ML (1976) Effects of stress and ACTH on plasma corticosterone
levels in the Caiman Caiman crocodilus. Gen Comp Endo 28:413–419
Huchzermeyer F (2003) Crocodiles: Biology, husbandry and diseases. CABI
Publishing, UK
Isberg SR, Thomson PC, Nicholas FW, Webb GJW, Manolis SC, Barker SG, Moran C
(2004) Quantitative analysis of production traits in saltwater crocodiles (Crocodylus
porosus): IV. number of scale rows. J Anim Breed Genet 123:48–55
Isberg S, Shilton C, Thomson P (2009) Improving Australia’s crocodile industry
productivity: understanding runtism and survival. Rural Industries Research
and Development Corporation, Canberra, Australia, Available via https://rirdc.
infoservices.com.au/items/09-135. Accessed 25 July 2013
Lance VA, Morici LA, Elsey RM (2000) Physiology and endocrinology of stress in
crocodilians. In: Grigg GC, Seebacher F, Franklin CE (eds) Crocodilian Biology
and Evolution. Surrey Beatty & Sons, Chipping Norton, NSW, Australia
Lance VA (1994) Life in the slow lane: hormones, stress and the immune system
in reptiles. In: Davey KG, Peter RE, Tobe SS (eds) Perspectives in comparative
endocrinology. Ottawa, National Research Council of Canada
Lance VA, Elsey RM (1986) Stress-induced suppression of testosterone secretion
in male alligators. J Exp Zool 239:241–246
Lance VA, Elsey RM (1999) Plasma catecholamines and plasma corticosterone
following restraint stress in juvenile alligators. J Exp Zool 283:559–565
Lance VA, Elsey RM, Butterstein G, Trosclair PL III, Merchant M (2010) The effects
of hurricane Rita and subsequent drought on alligators in southwest
Louisiana. J Exp Zool 313A:106–113
Lauren DJ (1985) The effect of chronic saline exposure on the electrolyte
balance, nitrogen metabolism, and corticosterone titer in the American
alligator, Alligator mississippiensis. Comp Biochem Physiol 81A:217–223
Lloyd M, Morris PJ (1999) Phlebotomy techniques in crocodilians. Bull Assoc Rept
Amphib Vet 9:12–14
Marketon JI, Glaser R (2008) Stress hormones and immune function. Cellular
Immunology 252:16–26
Morici LA, Elsey RM, Lance VA (1997) Effects of long-term corticosterone implants
on growth and immune function in juvenile alligators, Alligator
mississippiensis. J Exp Zool 279:156–162
Poetker DM, Reh DD (2010) A comprehensive review of the adverse effects of
systemic corticosteroids. Otolaryngol Clin N Am 43:753–768
Schobitz B, Reul JMHM, Holsboer F (1994) The role of the hypothalamic-pituitary-
adrenocortical system during inflammatory conditions. Crit Rev Neurobiol
8:263–
291
Isberg and Shilton SpringerPlus 2013, 2:381 Page 5 of 6
http://www.springerplus.com/content/2/1/381

Turton JA, Ladds PW, Manolis SC, Webb GJW (1997) Relationship of blood
corticosterone, immunoglobulin and haematological values in young
crocodiles (Crocodylus porosus) to water temperature, clutch of origin and
body weight. Aust Vet J 75:114–119
Webb GJW, Messel H (1977) Abnormalities and injuries in the estuarine crocodile,
Crocodylus porosus. Aust Wildl Res 4:311–319
Webb GJW, Messel H (1978) Movement and dispersal patterns of Crocodylus
porosus in some rivers of Arnhem Land, Northern Australia. Aust Wildl Res
5:263–283
doi:10.1186/2193-1801-2-381
Cite this article as: Isberg and Shilton: Stress in farmed saltwater
crocodiles (Crocodylus porosus): no difference between individually- and
communally-housed animals. SpringerPlus 2013 2:381.
Submit your manuscript to a
journal and benefi t from:
7 Convenient online submission
7 Rigorous peer review
7 Immediate publication on acceptance
7 Open access: articles freely available online
7 High visibility within the fi eld
7 Retaining the copyright to your article
Submit your next manuscript at 7 springeropen.com
Isberg and Shilton SpringerPlus 2013, 2:381 Page 6 of 6
http://www.springerplus.com/content/2/1/381