Toxicon 158 (2019) 69–76

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Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Evaluation of vasoactivity after haemotoxic snake venom administration T

a c c b b b,∗
R.B. Knight , S. Dvorcakova , L. Luptakova , K. Vdoviakova , V. Petrilla , E. Petrovova
a
Head and Head Veterinary Practice Ltd, Cornwall, TR13 0LW, United Kingdom
b
Department of Anatomy, Histology and Physiology, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81, Košice, Slovakia
c
Department of Biology and Genetics, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81, Košice, Slovakia

A R T I C LE I N FO A B S T R A C T

Keywords: The chick chorioallantoic membrane (CAM) is a widely used model in medical research and fulfils the re-
CAM assay quirements laid out in the 3 R's. The CAM, contains a dense network of blood vessels, essential for embryo
Chick embryo development but also makes the chick embryo an invaluable resource for the study of angiogenesis and the
Snake venom haemotoxicity of substances. Non-neurotoxic snake venom is responsible worldwide for numerous deaths but
Vasoactivity
also has been found to have a vast range of medicinal benefit. This study combines evaluating the time of onset
and intensity of effects of three whole viper venoms (Bitis aritans, Crotalus viridis, Agkistrodon contortrix) at
varying concentrations. They were applied onto the CAM, using the Luepke grading system as one method of
determining their rapid irritation potential. Regarding the principles of 3 R's, this method helps to evaluate the
haemotoxic effect of venom as an alternative method to the rodent tests. The information provided from these
results can be used as a rapid tool for both medical management of snakebite wounds and the potential use of
snake venom in medical treatments. Then, Luepke grading system can help to evaluate the haemotoxic effect of
venom in combination with other appropriate methods.

1. Introduction species and it's response in toxicological studies resembling that of

Animal models play key roles in basic medical research. However,
their application as animal models in experimental studies encounters
ethical, practical and technical problems, resulting in their use is lim-
ited in regards to research (Rashidi and Sottile, 2009). The avian em-
bryo represents an easily available and cost-effective alternative animal
model, which may be used in the area of testing various substances and
materials (Psychoyos et al., 2008). Chick embryo development takes
place outside the mother's body, and therefore the effect of a substance
or biomaterial is observed directly on the embryo without the influence
or detrimental effect on the mother's metabolism (Petrovova et al.,
2009).
Utilising chick embryos as an animal model has further major ad-
vantage in that they do not require approval for their use. This makes
their use both more appealing and feasible in comparison to other an-
imal models, which are protected by the horizontal legislation on the
protection of animals used for scientific purposes (2010/63/EU).
Furthermore, the chick embryo is a widely used and effective model
for the assessment of vasoactivity and angiogenesis. In particular, the
chorioallantoic membrane (CAM) has been confirmed as a suitable and
effective preliminary stage in experimental studies with research
showing the CAM's tissue responses resemble that of mammalian
other animal models (Kue et al., 2015; Valdes et al., 2002). Due to these
similarities, the CAM is used for a wider extent of research including
assessment of molecules regulating the formation of blood vessels for
use in the treatment of chronic inflammation, tumours, healing of
wounds and fractures, drugs delivery, toxicological analysis, angiogenic
and anti-angiogenic molecules (Kain et al., 2014; Ribatti, 2016).
The CAM is the outermost extra-embryonic membrane formed on
the 4th embryonic day by the fusion of the chorion and the allantois.
Histologically, the CAM is comprised of three levels: the ectoderm,
mesoderm, and endoderm. It is a highly vascularised membrane that
serves as both a respiratory and excretory organ (Ribatti, 2010; Bellairs
and Osmond, 2014). Due to the CAM being very thin with a transparent
matrix its very well-formed blood network is easily visible making it
ideal for angiogenesis studies (Richardson and Singh, 2003). By the 8th
embryonic day, the proliferation and differentiation of blood vessels are
already to a great extent underway, forming an extensive and easily
accessible arteriovenous system which is composed of the umbilical
arteries and veins (Ausprunk et al., 1974; Djonov et al., 2000).
Despite the early proliferation of the circulatory system within the
embryo, it has been repeatably shown that although pain receptivity
begins from embryonic day 7, the process is not complete with the brain
not fully functional until embryonic day 13 and the chick remaining

∗
Corresponding author.
E-mail addresses: eva.petrovova@uvlf.sk, lenka.luptakova@uvlf.sk (E. Petrovova).

https://doi.org/10.1016/j.toxicon.2018.11.430
Received 19 July 2018; Received in revised form 20 November 2018; Accepted 22 November 2018
Available online 04 December 2018
0041-0101/ © 2018 Elsevier Ltd. All rights reserved.
R.B. Knight et al.
only semi-conscious until a few days prior to hatching (IACUC, 2012;
Rosenbruch, 1997). This further supports the idea that this model re-
presents an interesting intermediate step between in vitro and in vivo
testing (rodents, mammals) of toxicity, which enables a reduction in the
number of animals in the in vivo experiment. With the chick embryo, we
can evaluate final concentrations of substances. Then, we can use them
for the testing in rodents and higher species (3R rules - replacement,
reduction and refinement; Flexnell, 2002).
Many reptiles produce venoms to enable them to immobilise and
kill their prey. This substance is typically produced in a specialised
gland and then inoculated into the target animal via secretory glands
and teeth (Vonk et al., 2008). The hemotoxic venoms of Viperinae and
Crotalinae are responsible for most of the evenomations in the world
(Calzia et al., 2011). Snakes from the family Viperidae have their fangs
directed forward, large glands and well developed striated muscle en-
abling them to instantaneously inoculate their venom after latching
onto their target (Mackessy and Baxter, 2006). Viperid venoms are
typically noted for the local and systemic haemorrhages in which they
cause due to their effects of zinc-containing metalloproteinases. These
metalloproteinases cause degradation and lysis of the proteins within
the basement membrane of the capillary endothelium, which presents
locally as pain, swelling and necrosis, systemically as widespread blood
loss, and ultimately death due to cardiovascular shock (Gutierrez et al.,
2016). Another important factor in Viper venom is phospholipase A2,
this has distinct myotoxic, neurotoxic and haemotoxic capabilities re-
sulting in skeletal muscle degeneration, the inhibition of neuromuscular
transmission via the blocking of acetylcholine receptors, and inhibition
of the blood coagulation factor cascade (Mackessy, 2010a; Schedel
et al., 2016).
This work aims to compare the three viper venoms and their effects
on the chick embryo CAM vessels. This then can allow the venoms to be
compared to previous findings on their vasogenic potential and allow a
critical analysis of the CAM assay as an appropriate alternative model.
2. Material and methods
2.1. Animal model
Broiler breed Ross 308, fertilised chicken eggs (120 pcs) were ob-
tained from a hatching farm (Párovské háje, Nitra, Slovakia). They were
put into an incubation store and held at 14 °C until day 1 of incubation.
Eggs were cleaned with 70% ethanol before being placed into an in-
cubation rack, with the tip pointing downwards. The incubator was set
to 37.5 °C and 55-60% relative humidity with the rotation of the egg
occurring every 3 h. On embryonic day 3 of incubation, a small hole
was made on the tip of each egg and 2 ml of egg white was extracted
from each egg with syringe and needle (20G), the hole was then sealed
with paraffin and the eggs were placed back into the incubator under
the standard conditions as stated above.
According to Luepke grading system, in every concentration of
venom (control as well) a series of four eggs was used (totally 12 eggs
per venom). The mean value of three sets makes possible an assessment
by a classification scheme (Table 2). However, eggs were incubated in
order to accommodate for the potential occurrence of un-embryonated
egg losses during incubation or damage to the CAM during preparation.
There is no need to request animal protocol approval for the chick/
quail embryo in ovo as an experimental model as they are exempt from
the horizontal legislation on the protection of animals used for scientific
purposes (2010/63/EU), as well as applicable laws in the United States.
2.2. Preparation of snake venom concentrations
Venoms from Bitis aritans, Crotalus viridis and Agkistrodon contortrix
were extracted in the breeding garden Pata near Hlohovec (Slovakia),
which had been designed for reptiles conservation of the gene pool
under the veterinary certificate No. CHEZ-TT-01. The breeding garden
70
Toxicon 158 (2019) 69–76
Table 1
Concentrations of snake venoms.
Snake venom Concentration (mg/ml)
E-2 E-3 E-4
Bitis arietans 10.310 1.031 0.103
Crotalus viridis 10.180 1.018 0.102
Agkistrodon contortrix 10.240 1.024 0.102
also serves as a quarantine station for imported animals and is an of-
ficial importer of exotic animals from around the world, having the
permission of the State Nature Protection of the Slovak Republic under
the No. 03418/06, the trade with endangered species of wild fauna and
flora and on amendments to certain laws under Law No. 237/2002.
A sterile plastic cup was used for venom extraction with plastic food
wrap, and rubber bands fixed the plastic wrap. For the application, we
used snake venoms immediately after their extracting. Prior to use, it
was ensured the venoms were continuously kept in cold storage to
ensure that they retained their full toxicological potential. The venoms
from Bitis aritans, Crotalus viridis and Agkistrodon contortrix were each
diluted with sterile distilled water to give equal concentrations (based
on molecular weights) of E-2, E-3 and E-4 when required (Table 1).
2.3. Application
On embryonic day 9, the eggs were removed from the incubator for
the application and evaluation of venom concentrations. Both un-em-
bryonated and dead embryos were discarded at this stage. The shell
membrane was then carefully dissected from the shell using forceps,
this then exposed the inner contents of the egg and the vessels of the
CAM. The first 4 randomly selected eggs where used as a control and
50 μl of sterile distilled water was applied gently onto the CAM vessels
(Fig. 1) with each egg being evaluated individually immediately after
application. After having conducted the control test, the same method
was utilised for the snake venom taking each venom, in turn, starting
from the strongest concentration and pipetting 50 μl of the corre-
sponding venom to the surface of the vessels. Photographs were then
taken at 0, 30, 120, 240 s to allow visualisation of the venoms effect
using stereomicroscope Olympus SZ61, digital camera ARTCAM-300MI
and software Quick Photo 2.3. It should be noted that after having used
each chick embryo for the examination, it was humanely killed by
decapitation with scissors and disposed of safely.
2.4. Evaluation
After acquiring all required images, the vasogenic effects and irrit-
ability of each venom at the various concentrations were evaluated
based upon the Luepke grading system for irritation potential (Luepke,
1985). This system (Table 2) provides a score based upon if and how
long it takes for hyperaemia, haemorrhage and clotting to develop.
Each of the four tests for each sample were to be graded according
to this table and from these scores an average cumulative score was
determined for each venom concentration. This average score then
correlates to an irritation potential as laid out in Table 3.
Table 2
Scoring scheme for irritation potential testing using CAM.
Time taken for manifestation of Score
irritation effect
Hyperaemia Haemorrhage Clotting
< 0.5 min 5 7 9
0.5-2 min 3 5 7
2-5 min 1 3 5
Luepke (1985).
R.B. Knight et al. Toxicon 158 (2019) 69–76

and Bitis arietans (E-2, E-3). Although each venom differed marginally
in its effect as was expected, there was a clear correlation between the
concentration of the venom and the irritation potential, with each of the
three venoms showing a decrease in effect as the venom become more
dilute (Table 4, Fig. 2).
In all evaluations, the control test showed no effects on the CAM
over the entire 240 s test time. This is reflected in its scoring as it was
graded 0's across the board and rated as having a negligible irritation
potential in the grading system. This allows it to be confirmed that any
changes to the CAM were induced from the venom application and not
linked to the method and external environment.

3.1. Bitis arietans

As seen in Table 5, Bitis arietans venom at concentrations of E-2 and

Fig. 1. Chorioallantoic membrane of chick embryo on embryonic day 6
E-3 respectively typically initiated haemorrhage within the first 30 s of
(arrow), scale bar: 2 mm. application. In both of these concentrations, haemorrhage and clotting
were visualised within this time span with a resultant grading of having
a strong irritation potential (Table 4; Figs. 3 and 4).
Table 3
The third and weakest dilution of venom, E-4, as expected showed
Classification of irritation potential based upon cumulative
significantly milder effects on the CAM. In all four tests, there was no
score.
induced clotting visualised and the haemorrhage typically became ap-
Cumulative Score Irritation Potential parent at 120 s post application resulting in this concentration of the
venom being rated as having only a slight irritation potential according
< 1.0 Negligible
1.0-4.9 Slight to the Luepke grading system (Table 4).
5.0-8.9 Moderate Although simultaneous with their onset and both being rated as
9.0-21.0 Strong having a strong irritation potential according to the Luepke grading
system, it was evident in the venom concentrations E-2 and E-3 (Figs. 3
Luepke (1985).
and 4). The venom concentration of E-2 caused a wider area and more
drastic degree of haemorrhage over the 240 s.
Statistical analysis of the correlation between the concentration of
This distribution of haemorrhage was seen to become even milder at
snake venoms and the irritation potential was performed using the Chi-
the third concentration (E-4) with only a mild localised area of affection
square test with significance level set at 0.5 (GraphPad Prism 6 for
(Fig. 5).
Windows).

3.2. Crotalus viridis

3. Results
Four sets of results for each concentration of the venoms were
evaluated to determine if and when the actions assessed in the Luepke's
grading system occurred. Based upon table no. 1 a score was allotted
and a mean score determined for each sample allowing a cumulative
score to be made and the irritation potential stated (Table 4).
Throughout the examination, none of the venoms caused hyper-
aemic changes on the CAM vasculature resulting in each of the three
venoms at each of the concentrations to be graded with 0 for this action.
However, some haemostatic changes could be seen. In his assessment,
Luepke does not report about the evaluation of haemostasis on the
vessels so this feature was not included in the grading. Haemostasis
could be visualised by a decreased diameter or visibility of the vessels
as blood flow has been impeded. This haemostatic action was seen
within 30 s of application of Agkistrodon contortrix venom (E-2, E-3, E-4)
In general, after the application of Crotalus Viridis venom at a con-
centration of E-2 the onset of both haemorrhage and clotting took only
30 s with only one test showing a slightly delayed response (Table 6).
The effects, as seen in figure no 6, became rapidly widespread following
this time. This rapid effect resulted in this venom concentration being
graded as having a strong irritation potential (Table 4).
Meanwhile, the more dilute concentration of E-3, took up to 120 s
for effects to occur (Table 6). Although it caused significant haemor-
rhaging in the localised area, as seen when comparing Figs. 6 and 7. The
effects are markedly milder than at the stronger concentration E-2.
Regardless of this, the concentration E-3 was also ranked as having a
strong irritation potential according to the Luepke grading system.
Alike with Bitis arietans, when diluted down to E-4, the Crotalus
viridis venom continued to cause haemorrhaging but failed to cause any
clotting effect. The haemorrhaging was particularly mild and localised,

Table 4
Average irritation potential of snake venoms at varying concentrations.
Venom Conc. Hyperaemia Haemorrhage Clotting Total Average Irritation Potential

Control N/a 0 0 0 0 Negligible

Bitis arietans E-2 0 6.5 8.5 15 Strong
E-3 0 6.5 6.75 13.25 Strong
E-4 0 4.5 0 4.5 Slight
Crotalus viridis E-2 0 6.5 8.5 15 Strong
E-3 0 5 7 12 Strong
E-4 0 5 0 5 Moderate
Agkistrodon contortrix E-2 0 5 7 12 Strong
E-3 0 5 7 12 Strong
E-4 0 4 0 4 Slight

71
R.B. Knight et al. Toxicon 158 (2019) 69–76

Fig. 2. Correlation between the concentration of snake venoms and the irritation potential.

Table 5
Evaluation of action of Bitis arietans venom at varying concentrations.
Conc. Sample No. Hyperaemia Haemorrhage Clotting Total Score

A B C A B C A B C

E-2 1 16

2 14

3 16

4 14

E-3 1 16

2 16

3 16

4 5

E-4 1 16

2 14

3 16

4 14

especially in comparison to the other concentrations from the same
species (Fig. 8). The first appearance of haemorrhage was typically
visualised at around 120 s.
Despite this concentration, ranking a score of 0 for two out of three
of the actions (hyperaemia and clotting) in the Luepke grading system,
it was still rated as having a moderate irritation potential.
3.3. Agkistrodon contortrix
comparison to the weaker (Figs. 9-11). Figs. 10 and 11 further reflect
the similarity between the venom at these concentrations with the de-
gree and distribution of both haemorrhaging and clotting over the 240-s
time span being visualised as a similar effect.
The effects of Agkistrodon contortrix venom at a concentration of E-4
produced extremely mild, localised areas of haemorrhage on the CAM
with the majority of blood vessels still being both visible and intact
(Fig. 11).

The third venom, Agkistrodon contortrix, in terms of time produced 4. Discussion

identical results at both E-2 and E-3 with both haemorrhage and clot-
ting appearing at 120 s in all test samples of each venom (Table 7,
Fig. 9). This resulting in both concentrations being graded as having a
strong irritation potential (Table 4).
The prolonged time taken to observe the effect of the venom further
to a concentration of E-4. Like the previous two venoms this further
dilution resulted in only haemorrhaging on the CAM. In the samples,
the effects varied with the onset of haemorrhaging being recorded be-
tween 120 and 240 s, with a corresponding grading of having a slight
irritation potential (Table 4).
Haemostasis was reported in all three concentration of this venom
but the effects were more pronounced in the stronger concentration in
Although the degree and severity of the effects varied between the
venoms all three venoms presented similarly, but this was to be ex-
pected due to the venoms coming from the same family of snakes. All
three venoms showed signs of haemorrhage and clotting at concentra-
tions of E-2 and E-3 but only haemorrhage when at E-4. A hypothesis
for why clotting failed to happen at the lower concentration can be
made from Eagle (1937), who showed how the concentration of pro-
teolytic enzymes within the venom affects the ability of the venoms to
cause coagulation and destruction of fibrinogen. Based on this prin-
ciple, it would be expected that as you dilute the venoms you subse-
quently dilute the level of proteolytic enzymes within the venom

72
R.B. Knight et al.
Fig. 3. Effect of Bitis arietans (E-2) venom on the CAM over 240 s: a; 0 s, b; 30 s,
haemorrhage (arrow); clotting (head arrow), c; 120 s, d; 240 s.
Fig. 4. Effect of Bitis arietans (E-3) venom on the CAM over 240 s: a; 0 s, b; 30 s,
haemorrhage (arrow); clotting (head arrow), c; 120 s, d; 240 s.
resulting in insufficient levels to induce clotting. The dilution of the
venom showed no effects on the presence of haemorrhaging only the
degree in which it occurred. Boviatsis et al. (2003), suggested that the
effects of the toxins within venom on the vessel walls and subsequent
damage it causes to the capillary endothelium results in this visible
leakage of blood into the surrounding tissues. Naturally, dilution of the
venom and hence subsequent dilution of the toxins results in less en-
dothelium damage and hence milder haemorrhage.
Both Bitis arietans and Agkistrodon contortrix venoms were seen to
induce haemostasis resulting in the blood flow being impeded to areas
on the CAM. Tay et al. (2012), used the Luepke grading system com-
bined with observing for signs of haemostasis by measuring vessel
diameter. They showed that although there were natural mild
73
Toxicon 158 (2019) 69–76
Fig. 5. Effect of Bitis arietans (E-4) venom on the CAM over 240 s: a; 0 s, b; 30 s,
c; 120 s, d; 240 s.
fluctuations in the blood vessels over time, these changes were negli-
gible, concluding that a visible haemostatic change must be induced by
the test substance. This supports that the haemostatic changes seen in
this study were induced by the application of the venom and potentially
could be an invaluable aid in medical research.
Campbell and Lamar (2004), noted how Agkistrodon contortrix in-
duced milder and most localised effects following envenomation which
can be clearly visualised on the CAM in this study. This suggests it is the
least haemotoxic of the three venoms and potentially the safest to use in
medically.
The grading system designed by Luepke can be evaluated as being
both invaluably effective but also may be regarded as less informative.
As seen above in Tables 2 and 3, the table clearly lays out the scoring
system for the initiation of hyperaemia, haemorrhage and clotting, this
then allows irritation potential to be determined based upon an accu-
mulation of scores determined by the time of onset. However, this
system fails to evaluate the intensity of the effect of an agent on the
CAM vasculature. This results in a substance which causes rapid but
mild effect potentially being graded as a higher potential irritant than
that of one whose effects are longer in onset but more intense. A sce-
nario reflecting this flaw was seen when examining the viper venoms,
as all three venoms at a concentration of E-2 were graded as having a
strong irritation potential. However when is compared the picture of
Bitis arietans to Agkistrodon contortrix at this concentration it is un-
doubtedly evident that the effect which Bitis arietans venom has on the
CAM blood vessels over the duration of the entire time (240 s) is
markedly more extreme than that of Agkistrodon contortrix regardless of
the fact that the onset of initial signs were both at the same time.
Protein components of the snake venoms, mainly serine proteases
and metalloproteinases could play an important role during our eva-
luation of vasoactivity after snake venoms administration on CAM
vessels. The composition of tested snake venoms used in our study has
been already described and determined in previous studies (Fasoli
et al., 2010; Mackessy, 2010b; Bocian et al., 2016).
Another imperfection in the Luepke grading system is that it only
accounts for three potential effects in which a substance can have on
the CAM blood vessels. This means that if an agent has a different effect
on the vasculature it fails to be evaluated and incorporated in the as-
sessment. Alike, in the Tay et al. (2012) studies where haemostasis had
R.B. Knight et al. Toxicon 158 (2019) 69–76

Table 6
Evaluation of action of Crotalus viridis venom at varying concentrations.
Conc. Sample No. Hyperaemia Haemorrhage Clotting Total Score

A B C A B C A B C

E-2 1 16

2 16

3 12

4 16

E-3 1 12

2 12

3 12

4 12

E-4 1 5

2 5

3 5

4 5

Fig. 6. Effect of Crotalus viridis (E-2) venom on the CAM over 240 s: a; 0 s, b; Fig. 7. Effect of Crotalus viridis (E-3) venom on the CAM over 240 s: a; 0 s, b;
30 s, haemorrhage (arrow); clotting (head arrow), c; 120 s, d; 240 s. 30 s, c; 120 s, d; 240 s.

to be measured using other methods, the Luepke grading system did not
account for or consider the haemostasis induced by Bitis arietans and
Agkistrodon contortrix venoms.
Palmeira-de-Oliveira et al. (2018), summed up that a Luepke
grading system is a brilliant tool for irritation testing with the potential
to provide information on toxicity, sensitivity, metabolic and im-
munopathological effects. CAM model provides rapid irritation testing
of snake venoms. Sells et al. (1998, 2001) tested non-neurotoxic ve-
noms using hens' eggs ED50 method, and it should be considered as an
alternative to the rodent ED50 test. Combination of these methods re-
duces suffering and number of required experimental animals with re-
spect to the principles of the 3R's (Doke and Dhawale, 2015). However,
they also stated that it is unable to completely replace other methods
due to some of the points mentioned above.
5. Conclusion
The CAM provides an ideal platform to allow the visualisation and
evaluation of the snake venoms effects on the blood vessels, whilst
conforming to the ethical 3R's approach to medical research. The results
of this study show that all three of the viper venoms have varied hae-
motoxic effects, which rapidly decrease in severity, as there is the di-
lution of the venom linked to the corresponding decreased dilution of
the proteolytic enzymes within the venom. It shows that in higher
concentrations, Bitis arietans and Crotalus viridis venom have a greater
irritation potential than Agkistrodon contortrix. However, once the

74
R.B. Knight et al. Toxicon 158 (2019) 69–76

Fig. 8. Effect of Crotalus viridis (E-4) venom on the CAM over 240 s: a; 0 s, b; Fig. 9. Effect of Agkistrodon contortrix (E-2) venom on the CAM over 240 s: a;
30 s, c; 120 s, d; 240 s. 0 s, b; 30 s, haemostasis (arrow) c; 120 s, haemorrhage (arrow); clotting (head
arrow), d; 240 s.

Table 7
Evaluation of action of Agkistrodon contortrix venom at varying concentrations.
Conc. Sample No. Hyperaemia Haemorrhage Clotting Total
Score
A B C A B C A B C

E-2 1 12

2 12

3 12

4 12

E-3 1 12

2 12

3 12

4 12

E-4 1 5

2 5

3 7

4 7

venoms are diluted down to E-4, although A. contortrix is still had the
lowest potential, the difference in irritation potential between the three
venoms is marginal. Although, a definitive advancement may not have
been made, it can be used as a foundation for further studies into other
families of snake or assessment of other effects of these venoms.
Fig. 10. Effect of Agkistrodon contortrix (E-3) venom on the CAM over 240 s: a;
0 s, b; 30 s, haemostasis (arrow) c; 120 s, d; 240 s.
No. 26220120066 “Centre of excellence for biomedical technologies”,
which is supported by the Operational Program “Research and
Development” financed through the European Regional Development
Fund and the project of Ministry of Education VEGA No. 1/0046/16.

Conflicts of interest
Transparency document
There is no conflict of interest.
Transparency document related to this article can be found online at
Acknowledgement https://doi.org/10.1016/j.toxicon.2018.11.430.

This work was realized within the framework of the project ITMS

75
R.B. Knight et al.
Fig. 11. Effect of Agkistrodon contortrix (E-4) venom on the CAM over 240 s: a;
0 s, b; 30 s, haemostasis (arrow) c; 120 s, d; 240 s.
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