CLINICAL & BASIC RESEARCH

SQU Med J, May 2011, Vol. 11, Iss. 2, pp. 236-244, Epub. 15th May 11
Submitted 5th Dec 10
Revision ReQ. 12th Jan & 2nd Mar 11, Revision recd. 23rd Jan & 8th Mar 11
Accepted 16th Mar 11

1Biomolecular Sciences, Faculty of Health & Life Sciences, Coventry University, Coventry, UK; 2Department of Physiology, Sultan 
Qaboos University Hospital, Muscat, Oman; 3Dept. of Medical & Social Care Education, Leicester Medical School, University of 
Leicester, Leicester, UK.
*Corresponding Author email: apx301@coventry.ac.uk

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


P < 0.0510– 3
P < 0.001P < 0.05 KATP
P < 0.00189HP < 0.001
P < 0.001
P < 0.00189H
P < 0.01P < 0.05LNAME



:مفتاح الكلمات

aBstract: Objectives: Hydrogen sulphide has been identified as a gas signalling molecule in the body, and has 
previously been shown to have vasorelaxant properties. The aim of the study was to investigate the effects of sodium 
hydrosulphide (NaHS), a hydrogen sulphide donor, on heart rate (HR), left ventricular developed pressure (LVDP) 
and coronary flow (CF) in the isolated perfused rat heart. Methods: A Langendorff isolated heart preparation was 
used to investigate the effect of a dose range of sodium hydrosulphide, in the presence and absence of inhibitors, on 
heart rate, left ventricular developed pressure and coronary flow. Results: Sodium hydrosulphide caused a significant 
decrease in heart rate at a concentration of 10-3 M (P <0.001). This decrease was partially inhibited by glibenclamide, 
a KATP channel blocker (P <0.05); L-NAME, a nitric oxide synthase inhibitor (P <0.001), and methylene blue (P 
<0.001), but not by H-89, a protein kinase A inhibitor. Sodium hydrosulphide significantly increased coronary flow 
at concentrations of 10-4 – 10-3M (P <0.05). This response was significantly increased in the presence of L-NAME 
(P <0.001) and methylene blue (P <0.001), whereas H-89 inhibited the increase in coronary flow due to sodium 
hydrosulphide (P <0.001). Sodium hydrosulphide significantly decreased LVDP at all concentrations (P <0.001). 
In the presence of glibenclamide and H-89, the time period of the decrease in LVDP due to sodium hydrosulphide 
was extended (P <0.001), whereas methylene blue and L-NAME caused a significant reduction in the response to 
sodium hydrosulphide (P <0.05, P <0.01 respectively). Conclusion: Sodium hydrosulphide reduced heart rate and 
LVDP, and increased coronary flow in the isolated perfused rat heart; however, the mechanisms of action could not 
be fully elucidated. 

Keywords: Hydrogen sulphide; Heart; Langendorff; H2S gasotransmitter; Vasorelaxation

Effects of Hydrogen Sulphide on the Isolated 
Perfused Rat Heart 

*Afthab Hussain,1 Helen Maddock,1 Hajar Al-Rajaibi,2 Ray J Carson3

Advances in Knowledge
1. This study identifies a sodium hydrosulfide dependant decrease in heart rate and left ventricular pressure and an increase in coronary 

flow. 
2. This study also identifies the potential cell signalling pathways via which these physiological changes take place.



Afthab Hussain, Helen Maddock, Hajar Al-Rajaibi and Ray J Carson

Clinical and Basic Research | 237

It is now well established that  hydrogen sulphide (H2S) gas has effects on the heart; however, to date, the cardiovascular 
effects of both endogenous and exogenous H2S have 
not been fully elucidated. Zhao et al. reported that 
an intravenous bolus injection of H2S at 2.8 and 14 
µmol/Kg body weight caused a significant transient 
decrease in blood pressure in anaesthetised rats, 
which was partially antagonised by glibenclamide 
(a potassium adenosine triphosphate [KATP] 
ion channel blocker).1 The heart rate was not 
significantly affected by H2S injection. An early study 
on sulphydryl reagents on the isolated perfused  
guinea-pig heart reported an increase in coronary 
flow and heart rate.2 Geng et al. reported that sodium 
hydrosulphide (NaHS), a H2S donor, inhibited 
left ventricular developed pressure in the isolated 
perfused rat heart in a concentration-dependent 
manner over a range of 10-6–10-3 M.3 However, 
they found a decrease in heart rate and coronary 
flow only at a concentration of 10-3 M NaHS 
which was partially inhibited by glibenclamide.3 
Sun et al. found that NaHS impeded contraction 
of isolated rat cardiomyocytes by inhibiting L-type 
calcium channels.4 No effects on KATP channel 
currents or on levels of cAMP (cyclic adenosine 
monophosphate) or cGMP  (cyclic guanosine 
monophosphate)  were found. H2S was found to 
have a negative chronotropic action on pacemaker 
cells in the sinoatrial node of rabbit heart, possibly 
via the opening of KATP channels.

5

A number of recent studies have shown that 
H2S is cardioprotective in myocardial ischaemia 
and ischaemia-reperfusion injury.6-12 Some of these  
studies found evidence that the cardioprotective 
mechanism is mediated via the opening of KATP 
channels;7,10,11,13,14 however, it is controversial whether 
mitochondrial KATP channels are involved.

11,13,15 
Inhibition of nitric oxide production attenuated 
the cardioprotective effects of NaHS, suggesting 
some synergy between nitric oxide (NO) and H2S.

13 
There is some evidence that the protein kinase C 
pathway,15,16 upregulation of cyclooxygenase-217217 
or upregulation of heat shock protein 7212 could be 

involved in cardioprotection.  
H2S is produced endogenously from the amino 

acid L-cysteine by two enzymes, cystathionine 
β-synthase (CBS) and cystathionine γ-lyase (CSE). 
CBS has been shown to have no activity or expression 
in human cardiovascular related tissues.18,19 
Conversely, CSE expression and endogenous 
production of H2S have been shown in the rat portal 
vein, thoracic aorta20 and heart.3,6 The expression of 
CSE has been identified in vascular smooth muscle 
cells, but not in the endothelium, whereas CBS 
expression was not detected in vascular tissue.1 

There appears to be some similarities between 
H2S, NO and carbon monoxide (CO) in terms of their 
effects, and mechanisms of action and interactions 
between them have been reported.21Hosoki et al. 
were the first to suggest synergy between H2S and 
NO in relaxing vascular smooth muscle.19 They 
demonstrated a left-ward shift in the dose-response 
curve for relaxation of rat thoracic aorta by NaHS 
in the presence of two different NO donors, 
sodium nitroprusside and morpholinosydnonimine.  
They reported that a low concentration of H2S 
enhanced the smooth muscle relaxant effect of NO 
by up to 13-fold.  However, Zhao and Wang found 
that low doses of NaHS shifted the dose-response 
relaxation curve for sodium nitroprusside to the right 
in rat aortic rings, suggesting that H2S inhibited the 
vasorelaxant effect of NO.22

The primary aim of this study was to investigate 
the effects of a concentration range of NaHS, a 
H2S donor, on heart rate, left ventricular developed 
pressure and coronary flow in the isolated perfused 
rat heart using the Langendorff model. 

Methods
Male Sprague-Dawley male rats (250–300g body 
weight) were used in all the experiments. All 
animals were from the same source, fed a standard 
diet and housed in the same conditions. All animals 
received humane care in accordance with the 
UK Guidance on the Operation of the Animals 
(Scientific Procedures) Act 1986. Animal protocols 

Application to Patient Care
1. Hydrogen sulphide has been identified as a gaseous signalling molecule alongside nitric oxide and carbon monoxide mediating a range 

of physiological processes. 
2. Hydrogen sulphide has been shown to abolish the effects seen during myocardial ischaemia reperfusion injury and has the potential of 

being translated into a clinical setting.



Effects of Hydrogen Sulphide on the Isolated Perfused Rat Heart 

238 | SQU Medical Journal, May 2011, Volume 11, Issue 2

were approved by the Committee of Animal Care 
and Supply of Coventry University, UK. 

All chemicals were obtained from Sigma (Poole, 
UK). The Krebs Heinseleit buffer contained (mM) 
NaCl 118.5, NaHCO3 25, KCl 4.8, MgSO4 1.2, 
KH2PO4 1.2, CaCl2, 1.7, and glucose 12, methylene 
blue (a guanylate cyclase inhibitor), glibenclamide 
and H-89 (a protein kinase A inhibitor) were 
dissolved in dimethyl sulphoxide (DMSO) initially 
and then diluted with a KH buffer. L-nitro-arginine-
methyl ester (L-NAME) was dissolved in distilled 
water. NaHS (a H2S donor) was dissolved in a KH 
buffer. The rate of infusion of agents was 1% of 
coronary flow to achieve the final concentrations 
indicated.

The animals were killed by cervical dislocation 
followed by exsanguination. Hearts were rapidly 
excised and placed in ice-cold Krebs-Heinseleit 
(KH) buffer solution. The aortic stump was rapidly 
mounted onto a cannula attached to a standard 
Langendorff set up. Hearts were perfused with 
KH buffer, gassed with 95% O2 5% CO2 (pH 7.4) 
and maintained at 37.5°C. The temperature was 
constantly monitored by a thermocouple inserted 
into the right ventricle. 

A latex balloon was positioned in the left ventricle 

via an insertion performed in the left atrial appendage 
and inflated to 5–10 mmHg. The balloon was attached 
to a pressure transducer connected to a Harvard 
amplifier to determine the left ventricular developed 
pressure (LVDP). Heart rate was monitored via the 
electrocardiogram (ECG) recorded using electrodes 
connected to a Harvard isolated preamplifier. Hearts 
exhibiting arrhythmia were discarded. Readings 
were taken at 5 min intervals using a Thermo 
array recorder WR7700 (Western Graphtec, USA). 
Coronary flow was measured at 5 mins intervals by 
collecting the perfusate draining from the perfused 
heart over a fixed time period.  

Hearts were allowed to stabilise for 20 mins 
following mounting on the Langendorff set up. 
Hearts were randomly assigned to the following 
protocols: 

a) Control group (n = 5): hearts were infused 
using a Harvard infusion/withdrawal pump with KH 
buffer at room temperature without the addition of 
any agents for 15 minutes at an infusion rate of 0.13 
ml/min and allowed to recover for a further 20 mins; 
b) Concentration-effect group: hearts were infused 
with a range of concentrations of NaHS (M) of 10-5  
(n = 6), 10-4 (n = 5) and 3x10-3 (n = 5) for a period 
of 15 minutes, followed by a further 20 minutes of 
recovery; c) Hearts were allowed to stabilise for 
20 mins with KH buffer perfusion and were then 

Figure 1a: Percent change in heart rate over time due 
to infusion of Sodium hydrosulphide (NaHS) (10-3 M) 
in the presence of glibenclamide (10 µM), H89 (1 µM), 
methylene blue (10 µM) and L-nitro-arginine-methyl 
ester (L-NAME) (10 µM) (mean ± Standard error of 
the mean (SEM), n = 5). The initial heart rate in each 
case was taken as 100%. Hearts were infused with the 
inhibitors for 15 minutes, followed by NaHS for 15 
minutes and then recovery was monitored for a further 
20 minutes. 
Note: ***P <0.001 vs. Control; * P <0.05 vs. NaHS; ### P <0.001 
vs. NaHS.

Figure 1b: Percent change in heart rate over time due 
to infusion of glibenclamide (10 µM), H89 (1 µM), 
methylene blue (10 µM) and L-nitro-arginine-methyl 
ester (L-NAME) (10 µM) (mean ± Standard error of 
the mean (SEM), n = 5). The initial heart rate in each 
case was taken as 100%. Hearts were infused with the 
inhibitors for 35 minutes. H89 alone caused a significant 
increase in heart rate compared to control (P <0.02).
Note: * P <0.05 vs. Control. 



Afthab Hussain, Helen Maddock, Hajar Al-Rajaibi and Ray J Carson

Clinical and Basic Research | 239

exposed with either 10 µM glibenclamide (n = 5), 
or 10 µM MB (a guanylate cyclase inhibitor) (n = 
5), or 10 µM L-NAME  (n = 5), or 1 µM H-89 (n = 
5) for 35 mins; d) Hearts were allowed to stabilise 
for 20 mins with KH buffer and were then exposed 
to either 10 µM glibenclamide (n = 5), 10 µM MB 
(n = 5), 10 µM L-NAME (n = 5) or 1 µM H-89 (n = 
5) for 15 minutes. Hearts were then exposed to 10-3 

M NaHS for 15 mins followed by a further 20 mins 
for recovery.  

Statistical analysis was conducted using the 
Statistical Package for the Social Sciences (SPSS, 
Version 10.5). Data were compared using analysis 
of covariance (ANCOVA) or one-way analysis of 
variance (ANOVA) with Fisher’s protected least 
mean squares difference post hoc test. Differences 
were considered significant where P <0.05. All data 
are expressed as mean ± standard error of the mean 
(SEM). 

Results
Results are presented as the percentage mean of 
the stabilisation period. NaHS caused a significant 
decrease in heart rate compared with control at 
a concentration of 10-3 M (P <0.001) [Figure 1a]. 

This decrease was partially, but significantly, 
inhibited by glibenclamide (P <0.05), by L-NAME 
(P <0.001), and by methylene blue (P <0.001), but 
not by H-89 [Figure 1a]. Glibenclamide, L-NAME 
and methylene blue alone had no significant effect 
on heart rate, but H-89 alone caused a significant 
increase in heart rate compared to control subjects 
(P = 0.05) [Figure 1b]. 

NaHS caused a significant increase in coronary 
flow compared to control at a concentration of 10-3 M 
(P <0.05) [Figure 2a]. Glibenclamide did not block 
the response to NaHS [Figure 2a]. H-89 alone caused 
a significant increase in coronary flow (P <0.001) 
[Figure 2b], but it significantly inhibited the increase 
in coronary flow due to NaHS (P <0.001) [Figure 
2a]. Methylene blue alone had no significant effect 
on coronary flow, but it significantly augmented 
the increase due to NaHS (P <0.001) [Figure 2a].  
L-NAME alone had no significant effect on coronary 
flow [Figure 2b], however L-NAME with NaHS 
caused a profound, highly significant increase in 
coronary flow compared to control (P <0.001) and 
compared to NaHS alone (P <0.001) [Figure 2b]. 

NaHS significantly decreased LVDP at all 
concentrations (P <0.001) compared with control 
(data not shown). Glibenclamide did not affect the 
initial response to NaHS, but significantly inhibited 
the recovery in LVDP following NaHS treatment (P 

Figure 2b: Percent change in coronary flow above over 
time due to infusion of glibenclamide, H89, methylene 
blue and L-nitro-arginine-methyl ester (L-NAME) 
(mean ± Standard error of the mean (SEM), n = 5). The 
initial coronary flow in each case was taken as 100%. 
Hearts were infused with the inhibitors for 35 minutes. 
H-89 alone caused a significant increase in coronary 
artery flow (P <0.001).
Note: *** P <0.001 vs. Control.

Figure 2a: Percent change in coronary flow over time 
due to infusion of Sodium hydrosulphide (NaHS) (10-
3 M) in the presence of glibenclamide (10 µM), H89 
(1 µM), methylene blue (10 µM) and L-nitro-arginine-
methyl ester (L-NAME) (10 µM) (mean ± Standard 
error of the mean (SEM), n = 5). The initial coronary 
flow was taken as 100% in each case. Hearts were 
infused with the inhibitors for 15 minutes, followed by 
NaHS for 15 minutes and then recovery was monitored 
for a further 20 minutes.  
Note: *** P <0.001 vs. NaHS; ### P <0.001 vs. Control.



Effects of Hydrogen Sulphide on the Isolated Perfused Rat Heart 

240 | SQU Medical Journal, May 2011, Volume 11, Issue 2

<0.001) (Figure 3a). H-89 alone caused a significant 
decrease in LVDP over time (P = 0.03) [Figure 3b], 
and H-89 significantly decreased the recovery in 
LVDP after NaHS treatment (P <0.001) [Figure 3a]. 
Methylene blue caused a significant reduction in the 
response to NaHS (P <0.05) [Figure 3a]. L-NAME 
alone caused a significant reduction in LVDP over 
time (P = 0.03) [Figure 3b], but it significantly 
reduced the effect of NaHS on LVDP (P = 0.01) 
[Figure 3a].  

Discussion
The H2S donor NaHS caused significant reductions 
in heart rate and LVDP, but increased coronary 
flow. The effect on LVDP concurs with the known 
role of H2S as a muscle relaxant,

1,20,22,23 and suggests 
that it has a similar effect on cardiac muscle. The 
effects of NaHS on LVDP reported here support 
the findings of Geng et al.3 In a study on isolated 
rat cardiomyocytes, Sun et al. reported that 
NaHS reduced contraction and inhibited L-type 
calcium channels.4 The vasodilatory effect of H2S 
in increasing coronary flow agrees with reported 
effects on peripheral blood vessels in vitro.1,20,22 
However, Geng et al. reported a decrease in 

coronary flow in a similar model.3

The negative chronotropic effect on heart rate, 
which agrees with the findings of Geng et al.3 must 
presumably be due to H2S influencing ion channels 
and ion currents in the pacemaker cells. Xu et al.5 
have reported that NaHS decreases the rate of 
pacemaker firing in the sinoatrial node in rabbit. In 
previous reports of the effects of H2S on the heart in 
vivo, Zhao et al.1 and Geng et al.3 reported that an 
intravenous bolus injection of H2S or NaHS caused 
a significant transient decrease in blood pressure, 
but the heart rate was not significantly affected. The 
heart may well respond differently in vivo compared 
to in vitro. There are probably differences in dose 
between these experiments and the present study. A 
crude estimate of the maximum dose range the heart 
was exposed to in the in vivo experiments of Zhao et 
al. is 4.5 x 10-5 – 2.4 x 10-4 M, assuming a rat body 
weight of 375 g and a blood volume of 22 ml.1 Geng 
et al. used a dose of 2.8 µmol/Kg body weight.3   

In the present study, the effect of H2S on heart 
rate seems to partially involve KATP channels, as 
it was inhibited by glibenclamide. This agrees 
with the findings of Xu et al. who suggested that 
H2S influences pace maker cell depolarisation by 
opening KATP channels.

5 H2S, acting by opening 
KATP channels, would hyperpolarise the membranes 
of pacemaker cells, thus reducing their rate of 
depolarisation and decreasing heart rate. The lack 

Figure 3a: Percent change in Left ventricular 
Developed Pressure (LVDP) over time due to different 
concentrations of Sodium hydrosulphide (NaHS) (10-
3 M) in the presence of glibenclamide, H89, methylene 
blue and L-nitro-arginine-methyl ester (L-NAME) 
(mean ± Standard error of the mean (SEM), n = 5). The 
initial LVDP was taken as 100% in each case. Hearts were 
infused with the inhibitors for 15 minutes, followed by 
NaHS for 15 minutes and then recovery was monitored 
for a further 20 minutes.  
Note: *** P <0.001 vs. Control; ### P <0.001 vs. NaHS; * P <0.05 
vs. NaHS; ** P <0.01 vs. NaHS.

Figure 3b: Percent change in Left ventricular 
Developed Pressure (LVDP) over time due to infusion 
of glibenclamide, H89, methylene blue and L-nitro-
arginine-methyl ester (L-NAME) (mean ± Standard 
error of the mean (SEM), n = 5). The initial LVDP in 
each case was taken as 100%. Hearts were infused with 
the inhibitors for 35 minutes. 
Note: * P <0.03 vs. Control.



Afthab Hussain, Helen Maddock, Hajar Al-Rajaibi and Ray J Carson

Clinical and Basic Research | 241

of effect of H-89 suggests that H2S is not exerting 
its negative chronotropic effect via the Cyclic 
adenosine monophosphate/protein kinase A (cAMP/
PKA) pathway. The significant increase in heart 
rate due to H-89 alone might be due to the fact that 
H-89 is not very specific in its action of inhibiting 
PKA. Alternatively, the normal control of heart 
rate could involve the (cAMP/PKA) pathway. The 
effect of L-NAME and methylene blue in inhibiting 
the action of H2S in decreasing heart rate suggests 
that the mechanism involves the stimulation of 
production of NO and the guanylate cyclic guanosine 
monophosphate (cGMP) pathway. In comparison, 
Kojda et al. found that L-arginine had a positive 
chronotropic effect in rat heart, whereas methylene 
blue reduced heart rate.29 

H2S has been previously shown to have 
vasodilatory effects in the rat heart1,20,22 and, in 
the present study, H2S increased coronary flow 
in isolated perfused hearts indicating dilation of 
coronary blood vessels. Conversely, Geng et al. 
reported a decrease in coronary flow by NaHS in 
a similar model.3  Blockade of KATP channels with 
glibenclamide, in the present study, did not inhibit 
the vasodilatory effect of H2S, suggesting that the 
mechanism of action of H2S was not via opening KATP 
channels. This is in contrast to the findings of Zhao 
et al. using peripheral blood vessels.1 H-89 alone 
significantly increased coronary flow which could be 
due to its other actions apart from inhibiting PKA. 
However, H-89 inhibited the vasodilatory effect of 
NaHS, suggesting that the mechanism of action of 
H2S in this case is via the cAMP/PKA pathway. This 
finding agrees with that of Kimura who showed that 
physiological concentrations of H2S increased the 
production of cyclic AMP in neurones and oocytes 
in culture.27 

Methylene blue and L-NAME both caused a 
highly significant increase in the vasodilatory effect 
of NaHS, which shows that inhibition of nitric 
oxide synthesis and signalling via guanylate cyclase 
augmented the effect of H2S. These findings were 
reproducible and were highly statistically significant. 
This suggests that endogenous production of NO 
inhibits the vasodilatory effect of H2S in coronary 
blood vessels; however, it is well known that NO is 
a vasodilator of coronary blood vessels. Zhao et al. 

showed that L-NAME, an inhibitor of endogenous 
NO production, significantly shifted the H2S dose-
response relaxation curve to the right, decreasing 

the potency of H2S, in rat aortic rings, and similar 
effects were obtained by removing the endothelium 
from the aortic rings.1 They also showed that the 
addition of a specific inhibitor of soluble guanylate 
cyclase, 1 H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1- 
one,  significantly increased the relaxant effect of 
H2S; however, synergy between NO and H2S has also 
been reported. Hosoki et al., demonstrated a left-
ward shift in the dose-response curve for relaxation 
of rat thoracic aorta by NaHS in the presence of 
two different NO donors, sodium nitroprusside 
and morpholinosydnonimine.15 Cheung and Schulz 
reported that glutathione and glutathione disulphide 
caused coronary vasodilation which was mediated by 
a NO and guanylate cyclase-dependent mechanism.26 
It is possible, in the current study, that endogenously 
produced NO could stimulate the production of a 
factor which opposes the actions of H2S. 

Both H-89 and L-NAME caused a significant 
reduction in LVDP. The reduction in LVDP by H-89 
could be due to effects other than on PKA. The 
depressor effect of L-NAME would suggest that the 
maintenance of LVDP requires the production of 
NO, although NO is generally known to be a muscle 
relaxant. It has been previously reported that a NO 
donor SPM3672 increased maximal left ventricular 
pressure in the isolated rat heart and increased levels 
of cGMP in rat cardiomyocytes.28 The positive 
inotropic effects of NO donors in rat heart have 
been confirmed by the same group and others.25,26 
H-89 extended the time period of the decrease 
in LVDP following NaHS treatment, suggesting 
that normal recovery involved the cAMP/PKA 
pathway. L-NAME and methylene blue significantly 
inhibited the response to NaHS, suggesting that 
the mechanism of relaxation of cardiac muscle 
by H2S involves NO production and guanylate 
cyclase. As H2S binds to heme groups, similarly to 
NO, it is possible that guanylate cyclase is a target. 
Glibenclamide did not inhibit the initial depression 
of LVDP by NaHS, suggesting that the mechanism of 
action did not involve the opening of KATP channels. 
Indeed, glibenclamide extended the time period 
of the depression of LVDP due to NaHS and thus 
inhibited recovery of the heart from NaHS treatment. 
This could be interpreted as an involvement of the 
opening of KATP channels in the recovery from H2S 
treatment. However, it is unclear how the opening 
of KATP channels and the resultant hyperpolarisation 
could increase LVDP. 



Effects of Hydrogen Sulphide on the Isolated Perfused Rat Heart 

242 | SQU Medical Journal, May 2011, Volume 11, Issue 2

Conclusion
NaHS, an H2S donor, significantly reduced heart 
rate and LVDP, and increased coronary flow in 
the isolated perfused rat heart; however, the 
mechanisms of action could not be fully elucidated.  

c o n f l i c t o f i n t e r e s t
The authors reported no conflict of interest.

a c k n o w l e d g e m e n t s

The authors are grateful to Mark Bodycote and 
Adrian Wallen of Coventry University, UK, for their 
expert care of the animals.

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