Concentrations of snake venom used for each venom.
Venomous snake bites in pregnant women can lead to poor survival rates in both the foetus and mother; early bites can precipitate teratogenesis, miscarriages, preterm delivery, foetal death and antepartum haemorrhage. The chicken embryo poses as a valuable research model for venom research due to its advantages such as ease of availability, economic feasibility and its non-invasiveness. This study evaluates the embryotoxic effects of Puff adder venom (Bitis arietans) from Namibia, Kenya, South Africa and non-specified region of Africa at varying concentrations. The venoms were applied to chicken embryos on the fourth day of incubation and assessed on a ninth day, focusing on body weight, heart weight, liver weight and mortality rate. Nile blue staining was also performed to observe the occurrence of apoptosis amongst the venoms at the strongest concentrations. The information provided from our results suggested that there was a regional variation in venom toxicity, with the Kenyan venom producing the largest weight changes, whereas the non-specified African venom proved the most lethal across the concentrations. Further studies to assess venom protein concentrations in comparison with regional diet disparities are required.
- Bitis arietans
- chicken embryo
- snake venom
1. Chicken embryo as an animal model for biomedicine
The chick embryo has established itself within biomedical science as a crucial research model due to its similarity on a cellular and anatomical level to human embryos along with its rapid development, easy visibility and manipulation . The cardiovascular system in the avian develops rapidly with the added benefit of the embryo remaining transparent during morphological development of primary organs, allowing visibility of vasculature formation using simple microscopy techniques .
1.1 Transgenic chicken egg as a bioreactor
The use of a chick egg as a bioreactor has been promoted over the use of standard large-scale fermenters due to its ability to produce large quantities of complex proteins and cheaper running costs . A 2002 study placed a retroviral vector based on avian leucosis virus into the chicken genome and noted that biologically active enzymes were secreted into the serum and egg white of four generations of chickens. The levels of enzyme expression remained steady through each successive generation and remained constant for over 16 months in the magnum of hens indicating the expression of this enzyme was stable. This supports the possibility of chicken eggs in the role of a bioreactor for the production of biologically active proteins for therapeutic interest . Human interferon a2b and β1a along with human granulocyte-colony stimulating factor have been successfully harvested from this method . The egg is an ideal model for the recovery of these proteins as the content is sterile and provides a long half-life . Production of human proteins in hens could become the method of choice for proteins that are toxic to mammals, an example of this is human erythropoietin which is damaging to the mammary gland of rabbits but remains inactive in chickens .
1.2 Chicken egg used for drug screening and production
The avian chorioallantoic membrane (CAM) refers to the outermost membrane that is highly vascularised for gas exchange and calcium transportation between the embryo and its environment . It has been widely used to discover targets and measure the in vivo angiogenic effects of factors such as vascular endothelial growth  and P13 kinase . The chorioallantoic membrane provides advantageous exposure to the embryo with minimal invasiveness . The chicken egg is a reliable candidate in assessing angiogenic responses to drugs as well as being cost and time-effective . In 1984, inactivated rabies vaccine for human use was developed which had been adapted to proliferate in primary chick egg cultures . This vaccine provided similar antigenicity to the human diploid cell strain with the advantages of cheaper and faster production. This purified chick embryo cell culture vaccine has now been licenced in over 60 countries worldwide for 30 years . The influenza vaccine has been developed in the embryonated chicken eggs since the 1940s, the egg provides high titre capabilities and large-scale production opportunity which has led to the streamlined process of today which provides vaccination for millions worldwide .
1.3 Chicken egg used in retinal development
The chicken embryo is advantageous in studying retinal development due to its significant similarity to the human embryo at a molecular, cellular and anatomical level and its rapid development . It is an important model in regenerative research as the neural retina can regenerate from the pigmented epithelium as late as stage 24 . The regeneration can occur from the cup margin or patches of pigmented epithelium far from the margin. The lens does not have to be present for the regeneration, and the regenerating tissue develops faster than normal . The chicken model is used in the vision research community to study human retinogenesis and develop new ways to battle human blindness and eye disease by allowing scientists an insight into the complex regulatory network of neurons and glial cells . In America, diabetic retinopathy is the leading cause of blindness amongst over 40-year-olds  with all type 1 diabetes developing DR and more than 60% with type 2 . Chickens, like humans, are diurnal with complex colour vision and as their retina is cone-dominant it makes them an ideal model for photoreceptor-degenerative blindness . In a 2014 study, chicken embryos were injected with either streptozotocin or high concentration of glucose at day 11; in both groups, cataracts occurred in varying degrees making them a promising animal model for diabetes research .
1.4 Chicken embryo as an animal model for research in Alzheimer’s disease
Studies by Murphy and LeVine  and Carrodeguas et al.  revealed that the chick embryo has an active process for the formulation and degeneration of the amyloid-beta peptide deposits associated with Alzheimer’s disease. The distribution of the peptide sequence in chick embryos is similar to humans, predominating in the nervous system. This study also discovered that the chick embryo produces neprilysin (ADAM-17), a protease that degrades the peptide . Due to the easy access of chick embryos, it’s an ideal candidate for developing a potential system for drug regulation and regression for this neurodegenerative disease . In a 2003 study, the chick embryo was a model for supporting the use of
1.5 The role of the chicken embryo in oncology research
The chick embryo allows the unique research of oncology in vivo. Along with being highly accessible and easy to manipulate, the CAM is naturally immune-deficient which can support engraftment of both normal and cancerous tissue up until developmental day 18 . The CAM has also been used for research into carcinogenesis  and tumour angiogenesis . In 1911, by inoculating the Rouse Sarcoma virus into chick embryos, it was suggested for the first time that viruses could cause cancer . It is now estimated that 15–20% of human cancers worldwide involve tumour inducing viruses, such as T-cell leukaemia virus type 1 and hepatitis C . In 2002, Zijlstra et al.  developed a highly sensitive assay to monitor metastatic dissemination of human cancer cells in the chick embryo by using the CAM as an established biological platform. In 2014, Mu et al. concluded that chicken embryo extract may promote the reversion of metastatic phenotypes of osteosarcoma cells, which could lead to tumour reversion through epigenetics .
1.6 Role of the chicken embryo in stem cell research
In 1996, Pain et al. displayed that early chicken blastoderm cells or avian embryonic stem cells can be maintained in vitro for long term culture. These cells model traits of murine stem cells in morphology, reactivity to antibodies and high telomerase activity along with the high capability to differentiate into various cell types . When blastoderm embryonic stem cells are grafted onto the CAM, they organise into complex structures such as embryoid bodies and derivatives of the three primary germ layers. These cells can provide an in vitro model of cell differentiation and maturation along with means of targeted genome control . Germline stem cell research is associated with the manufacture of transgenic animals using male spermatogonial stem cells  for the production of pharmaceutical proteins . Recently direct reprogramming has been created to convert differentiated somatic cells into pluripotent embryo stem-like cells; this is an advantageous medical method avoiding ethical issues surrounding human egg use . Amniotic stem cells isolated from the amniotic cavity can create clonal cell lines and due to their ease of isolation, are considered a potential source for regenerative medicine application .
1.7 Chick embryotoxicity screening test
Developed by Jelinek in 1977 the chick embryotoxicity screening test (CHEST) is a standardised technique to allow administration of small amounts of test compounds and the quantitative measurement of results. This method is advantageous as it requires limited materials. Fertilised eggs, micropipettes for administration, incubator and a dissection kit are all that is needed. The CHEST test allows the use of a chick embryo to study experimental teratogenicity. The number of dead, malformed, and growth-retarded foetuses (weight <650 mg and with no malformation) are totalled for each concentration and stage of the embryo at the time of exposure. The proportion of affected embryos is plotted against the concentration of test compound, and the embryo-stage of administration. The proportion of effects on particular organs in surviving embryos is considered separately to establish a profile of effects for each compound . The screening test has been used to determine the toxicity of many substances, establishing parameters of dose–response and stage-response .
2. Snake venom
2.1 The role of snake venom
Venom is defined as ‘a secretion, produced in a specialised gland in one animal and delivered to a target animal through the infliction of a wound, which contains molecules that disrupt normal physiological or biochemical processes to facilitate feeding or defence’ .
Venom is composed of proteins and polypeptides, it has two primary functions—to paralyse their prey and start the digestive process with hydrolysing proteins leading to tissue necrosis and blood clotting . These venom components can be grouped according to the mode of action:
Binding to cholinergic receptors, often leading to respiratory muscle paralysis.
Inhibition or increase release of acetylcholine, causing muscle cells to not react to nerve stimulus, leading to spasms, damage of the skin and disruption connective tissues.
Cytotoxic and cardiotoxic, which harm cell membranes and disrupt the transport of substances across the membranes .
In broad terms, snake venoms are classified as inflammatory, cytotoxic, neurotoxic or haemotoxic .
2.2 The venom delivery system
Many snakes use fangs, specialised dentition associated with the venom gland (Duvernoy gland) to introduce venom into prey. Fangs can either be posteriorly (grass snake) or anteriorly (vipers) positioned within the upper jaw .
Many poisonous snakes possess different types of dentition; vipers and atractaspidids have a shortened maxilla which rotates allowing the fangs to move; whereas elapids fangs are fixed at the front of the maxilla. Colubrids can possess an enlarged rear positioned fang or no fang at all. The venom gland, also known as the Duvernoy gland in posteriorly positioned fanged species, is innervated by the maxillary branch of the trigeminal nerve, facial nerve and supplied by the internal carotid artery . The Duvernoy gland is positioned posteriorly to the eye, encased in a thin layer of connective tissue and consists mainly of serous cells; a single duct extends from the gland to the base of the fang; whereas the venom gland is encased in a fibrous sheath varying in position depending on species. The viperid gland is large, isosceles with the longest side along the upper lip directed dorsally; the gland is divided into lobules by the outer sheath with the lumen becoming a primary duct which passes through a mucous accessory duct into a secondary duct extending the length of the fang . In contrast, the elapid venom gland is oval made from many branching tubules, its lumen is narrow and therefore most of the venom is stored in the surrounding cells . The atractaspidid species possess a cylindrical gland extending posteriorly beyond the head, its lumen has a characteristic pattern of unbranched tubules radiating outwards .
2.3 Snakebite classification
Bites can be classified as cytotoxic bites characterised by a painful swelling with watery blood leaking from the wound followed by shock, blistering and discolouration. Venomous snake bites in pregnant women can lead to poor survival rates in both the foetus and mother; early bites can precipitate teratogenesis, miscarriages, preterm delivery, foetal death and antepartum haemorrhage . The bite will cause severe pain to the limb affected. Species associated with this bite include the puff adder, Gaboon adder and spitting cobras. Black and green mambas along with non-spitting cobras produce a neurotoxic bite in victims which leads to moderate swelling, cold and clammy extremities, dilated pupils and drooping of the eyelids. Patients suffering these bites will develop swollen lymph glands, vomiting, ptyalism and breathing difficulties. Haemotoxic bites associated with boomslang and vine snakes cause bleeding from the gums, nose, corner of eyes and old wounds and scratches. Certain snake species can produce more than one type of bites such as the
2.4 Composition of snake venom
Medically significant venomous snakes are all front-fanged and are classified into three families: Atractaspididae, Elapidae and Viperidae; the glands of these snakes are homologous , with current evidence suggesting these evolved from non-front fanged venomous snakes . These glands contain the snake venom, made from a combination of different protein families, each containing a variety of toxin isoforms along with carbohydrates, lipids, nucleosides and metals. Although homologous, the venom proteome is not due to the influence of genetic mutations and natural selection . In a 2017 review, 59 protein families were identified in these three species, with four dominant proteins: phospholipase A2s, metalloproteases, serine proteases and three-finger toxins; six secondary families: cysteine-rich secretory proteins, L-amino acid oxidases, Kunitz peptides, c-type lectins, disintegrins and natriuretic peptides; nine minor proteins and 36 rare. Results revealed that elapid venom contained a less diverse range of protein families than the others, mostly consisting of phospholipases and three-finger toxins. Viper venom showed to scarcely contain three-finger toxin .
2.4.1 Phospholipase A₂
These enzymes play an important role in the regulation of phospholipid turnover, membrane permeability, cell maintenance and growth, apoptosis and the production of leukotrienes and prostaglandins . Of the four types of PLAs, only type one and two are found in snake venom with several isoforms. These PLAs target the motor nerve terminal and the terminal part of the motor axon, by initiating hydrolysis of the lipids of the outer leaf of the plasma membrane of the nerve terminal resulting in its depolarization. Synaptic vesicles are also destroyed by the PLA and the products of lipid hydrolysis . Exposure of skeletal muscle to venom PLAs causes a severe inflammatory degenerative response, with the first clinical signs apparent less than 1 hour after inoculation, with affected fibres rapidly depolarising .
Haemorrhaging is a common clinical sign associated with viper and crotaline snake bites and has been associated with the proteolytic activity of metalloprotease . The proteases induce direct damage of the microvessels . Snake venom metalloproteases are classified into four main groups based on their domain structure: PI—possesses a metalloprotease domain only, PII—consists of both metalloprotease and disintegrin-like domains, PIII—compromised of metalloprotease, disintegrin-like and high-cysteine domains, PIV—in addition to metalloprotease, disintegrin and cysteine also possesses a lectin-like polypeptide . The disintegrin-like domain inhibits platelet aggregation by binding to the fibrinogen receptor in platelet plasma membranes and although the role of the other domains is not clear, venoms with cysteine and disintegrin domains are more active at inducing haemorrhage than enzymes with only metalloprotease domains . Investigations suggest pathogenesis is associated with a per rhexis mechanism whereby endothelial cells of capillary blood vessels rapidly thin and detach from surrounding basal lamina, progressive degeneration of these vessels leads to breaks in the endothelial lining, allowing blood to enter interstitial space .
2.4.3 Serine protease
Serine proteases are major components of snake venom, mostly identified in snakes of the Viperidae family and certain Elapidae, Colubridae and Hydrophiidae families . These venom enzymes affect the haemostatic system by acting on the coagulation cascade and possess strong pro-coagulant effects through the activation of platelets, production of thrombin-like enzymes which clot fibrinogen and the creation anti-coagulant enzymes such as protein C , these serine protease produced thrombins are not susceptible to hirudin or heparin, other proteases possess kininogenase activity which releases hypotensive bradykinin .
2.4.4 Three-finger toxin (3FTs)
This family of non-enzymatic polypeptides exhibit potent toxic effects. Based on their biological properties, they can be classified as postsynaptic neurotoxins targeting the nicotinic and muscarinic acetylcholinesterase receptors; fasciculins targeting acetylcholinesterase; calciseptins and FS2 toxins targeting L-type calcium channels; anticoagulants; β-blockers targeting β1 and β2 adrenergic receptors; dendroaspin targeting specific glycoproteins; cardiotoxin A5 targeting integrins and antagonists of α1A and α2A adrenergic receptors . A large number of 3FTs are neurotoxic, interfering with cholinergic transmission at post-synaptic sites in the peripheral and central nervous system; mipartoxin-I, a 3FT, is the most abundant protein found in coral snake venom from northern South America and possesses a lethal effect in mice and a clear neuromuscular blockade in avian and mice subjects with an affinity for the cholinergic nicotinic receptor . A group of cardiotoxic 3FTs found only in cobra venom is the second-largest group, at low concentrations they elevate heart rates and at high concentrations, cause death by cardiac arrest .
2.5 The use of
Bitis arietans snake venom in biopharmaceutical research
Biscetin, a platelet adhesion inducer, isolated from the venom of
2.6 The Puff adder (
The Puff adder (
2.7 The occurrence of snake bites
A global review by White in 2000 estimated that between 1.25 and 5.5 million snakebites occur annually . These primarily occur in developing countries where there is a dense population of humans, an abundance of snakes and a lack of medical treatment facilities. In 1987 in South Africa, pregnant women accounted for 0–4% of cases admitted to hospital, in India, they accounted for 1% of admissions . Venomous snakebites may lead to poor foetal development and adversely affect the mother’s health; previous studies determined that foetal death occurred in 38–43% of snakebite cases, whereas maternal death accounted for 10% . The African puff adder, along with the carpet viper, is the two most common species responsible for fatalities following bites in Africa. These adder venoms possess cardiotoxic and haemorrhagic effects which can lead to hypovolemic shock, necrosis, systemic haemorrhage and arrhythmias .
2.8 The geographical variations in snake venom
Variation in venom composition occurs between species of snake as well as within a species. These variations can have a significant impact on venom toxicity and medical management. Different toxin-encoding genes in the genome attribute to these variations , however the mechanisms by which these gene expressions are controlled are poorly understood . Results from Barlow et al.  provides evidence for the theory of diet affecting venom composition, by observing
3. Materials and methods
3.1 Ethics and morality of research
In accordance with the European Union Directive 2010/63/EU section nine, embryos were not developed beyond the first two-thirds of development. This also corresponds with mandates of The Institutional Animal Care and Use Committee, Association of New England Medical Centre (Tufts) and the National Institute of Health, USA which dictate that a chick embryo that has not reached the 14th day of its gestation period will not experience pain and therefore can be used for experimentation without ethical restrictions.
3.2 CHEST II
For this research, the chick embryotoxicity screening test was used. This standardised technique allows for the administration of small amounts of test compounds into the fertilised egg on 4th embryonic day (ED4) and the measurement of a quantitative endpoint on ED9.
Fertilised eggs: alive and morphologically normal (breed Lohmann Brown, Hatchery Farm Párovské Háje, Nitra, Slovakia).
Incubator (ART 549/A).
Dissection kit: including tissue forceps, dissecting scissors and blunt scissors.
Micropipette with disposable tips.
Stereomicroscope (Olympus SZ 61 with digital camera ARTCAM-300MI).
Observing dish with fixing pins.
Distilled water: to aid observation.
3.3 Application of snake venom: CHEST II
Eggs were placed in an incubator on day 0 at 37–38°C with a 50–60% relative humidity and rotated periodically until (ED4). Then eggs were removed and blunt end of eggs was cleaned with 70% alcohol and covered by a transparent adhesive tape. Subsequently, using serrated scissors (FST 14071-12), an opening was cut for application of the respective doses of snake venom (100 μl). The tested concentration was applied directly over the embryo on the top of inner shell membrane (
|Snake venom||Concentrations (mg/ml)|
|Control (sterile distilled water)|
|B.A. nonspecific region||100.00||10.00||1.00||0.10|
|B.A. South Africa||100.00||10.00||1.00||0.10|
|Snake venom||Dose (mg/ml)||Dead embryos||Mortality (%)||Mean body weight (g)||Mean heart weight (mg)||Mean liver weight (mg)|
|Bitis arietans—South Africa||0||10||1||10||1.62||18.0||28.8|
3.4 Preparation of snake venom concentrations
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. Before use, it was ensured the venoms were continuously kept in cold storage to ensure that they retained their full toxicological potential. The venoms were diluted with sterile distilled water to give equal concentrations (based on molecular weights) of E-1, E-2, E-3 and E-4 when required (Table 1). The composition of tested snake venoms used in our study has been already described and determined in previous studies [75, 76].
3.5 Nile blue staining
For this staining method, embryos with the highest concentration of snake venom (10-1) were used. The blue stain was applied on ED4. The embryos were then dissected on day ED6 and incubated in a 1/8000 solution of Nile Blue A (Sigma) and PBS for 15 minutes at 37°C in an incubator. Following this, embryos were transferred to cold PBS (4 °C) and washed for 4 hours. Embryos were then photographed with a stereomicroscope Olympus SZ 61 with digital camera ARTCAM-300MI and Quick Photo 2.3 software. These embryos were lastly compared with control embryos (sterile distilled water applied only) dissected on ED6.
The number of dead or growth-retarded embryos (those <650 mg) were totalled for each concentration. The proportion of effects on heart and liver in surviving embryos were considered separately to establish a profile of each concentration. The result of the CHEST was rank venom in order of their teratogenic potency in chickens; these ranges can be compared for an assessment of human risk. For statistical analysis, the programme GraphPad Prism 6.0 was utilised (one-way ANOVA, with a
The following results were analysed—body weight, heart weight and liver weight. These results were compared between control samples and the venom of
4.1 Body weight
All living embryos were weighed on ED 9 in grams. Figure 2 presents the following concentrations E1 (10-1), E2 (10-2), E3 (10-3) and E4 (10-4).
The average body weight of the control embryos was 1.73 g, whereas embryos infused with venom concentration 10-1 (E1), 10-2 (E2) and 10-3 (E3) were significantly reduced (
The embryos injected with venoms of the Namibian snake showed no statistically significant changes in body weight in comparison with the control embryos. E1 body weight averaged at 1.4 g in comparison with the 1.72 g of control embryos. E2 average body weight was 1.7 g whereas E3 averaged at 1.6 g. E4 embryos body weight was less than E2 and E3 at 1.54 g.
4.1.3 South Africa
None of the embryos impregnated with South African Puff adder venom showed a statistically significant change in body weight compared to the control group. E1, E2 and E3 body weights were all similar, averaging at 1.67, 1.62 and 1.66 g respectively. E4 averaged the smallest body weight at 1.43 g.
4.1.4 Non-specific region
None of the embryos infused with the non-specific venom showed significant differences in body weight compared to the control group. E1 averaged the smallest body weight at 1.42 g, E2 averaged at 1.66 g, E3 at 1.58 g and E4 at 1.66 g.
4.2 Heart weight
Hearts were cut from surviving embryos at the level of the aorta and weighed in milligrams (Figure 3).
The control heart mean weighed 23 mg; the embryo hearts infused with venom were significantly smaller in E1, E2 and E3. E1 and E2 weighed in at 13 mg, E3 had a slight increase in weight at 15 mg. There was a large increase between E3 and E4 with the latter averaging 18 mg.
None of the concentrations had a significant effect on heart weight in comparison to the control group. E1 mean heart weight came in at 17 mg. E2 was the largest weights at 21 mg, E3 and E4 averaged 20 mg.
4.2.3 South Africa
The largest average heart weight was seen in E3 and E2 at 19.8 mg. E1 averaged at 18.5 mg. E4 had the lowest body weight of 15 mg; this result was statistically significant.
4.2.4 Non-specified region
E1 and E3 results were both deemed statistically significant. E1 averaged the lowest heart weight of 16 mg. E2 averaged 18 mg whereas E3 mean was lower than E2 at 17 mg. E4 weighed in with the largest heart weight of 18 mg.
4.3 Liver weight
Livers were cut at the level of the portal vein, cut from surviving embryos and weighed in milligrams (Figure 4).
All four concentrations of snake venom s were proven statistically significant. The mean control group liver weighed 31.6 mg, larger than all four test groups. E1 liver weight mean was recorded as 15.5 mg, E2 weighed in at 14.8 mg; E3 was the lowest average liver weight of 14 mg. E4 average liver weight was the largest at 20 mg.
Venom concentration E1 was the only statistically significant result in this test group, with its average weight calculated at 20 mg. E2 liver weight averaged heavier than the control group (31.6 mg) with 35 mg. E3 mean weight was similar to the control at 31.4 mg and E4 averaged 30 mg.
4.3.3 South Africa
E1, E2 and E4 concentrations were proven statistically significant; whereas E3, with the largest liver weight of 26 mg, was not. E1 had the lowest average liver weight of 19.2 mg; E2 was marginally heavier at 19.8 mg. E4 liver weight averaged at 20.7 mg.
4.3.4 Non-specified region
Three of the four concentrations of non-specified venom had a statistically significant effect on the liver weight (E2, E3 and E4). E1 mean result weighed in at 20 mg; E2 was faintly heavier at 20.9 mg. E3 weighed the least with 19.9 mg. E4 had the heaviest weight within the test group at 22.2 mg.
4.4 Mortality rates
The highest mortality rate was seen in the highest concentration, in this maximum venom concentration (E1) the non-specific venom samples had a 70% death occurrence (embryos not surviving to day 9); the Kenyan, Namibian and South African E1 all had a 20% mortality rate. All groups at each concentration had a fatality, except the South African E4 group.
The non-specified venom produced the highest overall amount of fatalities, with the Kenyan being second, Namibian third and South African venom being the least lethal. The 10 mg/ml (E2) concentration saw the highest mortality rates overall, with the Kenyan test group showing a 40% mortality, the Namibian and non-specific test group perceiving 30% mortality and the South African group a 20% mortality. E3 showed 20% fatality in the South African and non-specific test groups, in Namibia and Kenyan groups only 10% fatality rate was recorded. In the lowest concentration (E4) the highest mortality rate was recorded in the Kenyan group with 30%. No fatalities were recorded in the South African group. The Namibian test group showing a 20% mortality and the non-specific test group a 10% mortality (Figure 5, Table 2).
4.5 Nile blue staining
The embryos were stained on ED4 and removed from their eggs on ED6; all were envenomed with the strongest concentrations of venom (10-1; Figure 6).
The arrows on the above images highlight the darkened areas of apoptotic cells. These images show that initial cell death is initiated on the wing buds. The control embryo (a), infused with distilled sterile water only, demonstrates no apoptosis. The nonspecific venom (b) indicates a small amount of apoptosis located on the wing buds. Embryos envenomed with South African (c) and Kenyan (d) B.A. showed significant apoptosis along with the entire wing buds, with the Kenyan spreading to the extremities. The Namibian embryo showed less severe cell apoptosis than (c) and (d) along with the wing buds.
Although the body weights varied amongst regional snakes and concentrations, they all presented similarly which was anticipated as they are the same species of snake. Generalised haemorrhaging was present across all four venom groups, which was expected due to the haemotoxic nature of
5.2 Heart weight
All heart weights of embryos envenomed with
5.3 Liver weight
All groups had a minimum of one statistically significant concentration, suggesting the liver was the most affected parameter (in comparison with heart and body weight). In furtherance with the proposal that the Kenyan venom has the highest potency, E1, E2 and E3 all proved statistically significant as well as being of the lowest median body weights of the groups surveyed. All livers exhibited a decrease in mean weight, varying yellow colour change along with increased susceptibility to disintegration on removal; this substantiates the results from a previous study on the effects of
The highest incidence of mortality was observed in E1 by the non-specified test group, with a 70% mortality; whereas Kenya, Namibia and South Africa observed a 40–10% death rate. This evidence proposes that although the Kenyan venom has the most significant impact on development, the non-specific venom has the strongest potency. Furthers study with a larger specimen pool is needed to confirm these findings.
5.5 Apoptotic cell appearance (Nile blue staining)
A 1993 study observed that haemorrhagic snake venom (
In conclusion, it was seen that the test group envenomed by
These results suggest a geographical variation in potency of
This work was realized within the framework of the project of the Ministry of Education VEGA No. 1/0050/19 and APVV-17-0017.
Vergara MN, Canto-Soler MV. Rediscovering the chick embryo as a model to study retinal development. Neural Development. 2012; 7:22. DOI: 10.1186/1749-8104-7-22
Bressan M, Mikawa T. Avian as a model system of vascular development. Methods in Molecular Biology. 2015; 1214:225-242. DOI: 10.1007/978-1-4939-1462-3_14
Lillico SG, McGrew MJ, Sherman A, Sang HM. Transgenic chickens as bioreactors for protein-based drugs. Drug Discovery Today. 2005; 10:191-196. DOI: 10.1016/S1359-6446(04)03317-3
Harvey AJ, Speksnijder G, Baugh LR, Morris JA, Ivarie R. Expression of exogenous protein in the egg white of transgenic chickens. Nature Biotechnology. 2002; 20:396-399. DOI: 10.1038/nbt0402-396
Rashidi H, Sottile V. The chick embryo: Hatching a model for contemporary biomedical research. BioEssays. 2009; 31:459-465. DOI: 10.1002/bies.200800168
Harvey AJ, Ivarie R. Validating the hen as a bioreactor for the production of exogenous proteins in egg white. Poultry Science. 2003; 82:927-930. DOI: 10.1093/ps/82.6.927
Steinlein P, Deiner E, Leutz A, Beug H. Recombinant murine erythropoietin receptor expressed in avian erythroid progenitors mediates terminal erythroid differentiation in vitro. Growth Factors. 1994; 10:1-16
Kue CS, Tan KY, Lam ML, Lee HB. Chick embryo chorioallantoic membrane (CAM): An alternative predictive model in acute toxicological studies for anti-cancer drugs. Experimental Animals. 2015; 64:129-138. DOI: 10.1538/expanim.14-0059
Wilting J, Christ B, Bokeloh M, Weich HA. In vivoeffects of vascular endothelial growth factor on the chicken chorioallantoic membrane. Cell and Tissue Research. 1993; 274:163-172. DOI: 10.1007/bf00327997
Nowak-Sliwinska P, Segura T, Iruela-Arispe ML. The chicken chorioallantoic membrane model in biology, medicine and bioengineering. Angiogenesis. 2014; 17:779-804. DOI: 10.1007/s10456-014-9440-7
Barth R, Gruschkau H, Jaeger O, Milcke L, Weinmann E. Purified chick embryo cell (PCEC) rabies vaccine for human use. Laboratory data. Behring Institute Mitteilungen. 1984; 76:142-154
Giesen A, Gniel D, Malerczyk C. 30 years of rabies vaccination with Rabipur: A summary of clinical data and global experience. Expert Review of Vaccines. 2015; 14:351-367. DOI: 10.1586/14760584.2015.1011134
Hegde NR. Cell culture-based influenza vaccines: A necessary and indispensable investment for the future. Human Vaccines and Immunotherapeutics. 2015; 11:1223-1234. DOI: 10.1080/21645515.2015.1016666
Coulombre JL, Coulombre AJ. Regeneration of neural retina from the pigmented epithelium in the chick embryo. Developmental Biology. 1965; 12:79-92. DOI: 10.1016/0012-1606(65)90022-9
Langouet-Astrie CJ, Meinsen AL, Grunwald ER, Turner SD, Enke RA. RNA sequencing analysis of the developing chicken retina. Scientific Data. 2016; 3:160117. DOI: 10.1038/sdata.2016.117
Ola M, Mawaz M, Siddiquei M, Al-Amro S, Abu El-Asrar A. Recent advances in understanding the biochemical and molecular mechanism of diabetic retinopathy. Journal of Diabetes and Its Complications. 2012; 26:56-64. DOI: 10.1016/j.jdiacomp.2011.11.004
Fong DS, Aiello L, Gardner TW, King GL, Blankenship G, Cavallerano JD, et al. Retinopathy in diabetes. Diabetes Care. 2004; 27(Suppl 1):84-87. DOI: 10.2337/diacare.27.2007.s84
Valdez DJ, Nieto PS, Díaz NM, Garbarino-Pico E, Guido ME. Differential regulation of feeding rhythms through a multiple-photoreceptor system in an avian model of blindness. FASEB Journal. 2013; 27:2702-2712. DOI: 10.1096/fj.12-222885
Shi L, Ko ML, Huang CC, Park S, Hong M, Wu C, et al. Chicken embryos as a potential new model for early onset type I diabetes. Journal of Diabetes Research. 2014; 2014:1-10. DOI: 10.1155/2014/354094
Murphy MP, LeVine H 3rd. Alzheimer’s disease and the amyloid-beta peptide. Journal of Alzheimer’s Disease. 2010; 19:311-323. DOI: 10.3233/JAD-2010-1221
Carrodeguas JA, Rodolosse A, Garza MV, Sanz-Clemente A, Pérez-Pé R, Lacosta AM, et al. The chick embryo appears as a natural model for research in beta-amyloid precursor protein processing. Neuroscience. 2005; 134:1285-1300. DOI: 10.1016/j.neuroscience.2005.05.020
Sarasa M, Pesini P. Natural non-trasgenic animal models for research in Alzheimer’s disease. Current Alzheimer Research. 2009; 6:171-178
Ahlemeyer B, Krieglstein J. Pharmacological studies supporting the therapeutic use of Ginkgo biloba extract for Alzheimer’s disease. Pharmacopsychiatry. 2003; 36:8-14. DOI: 10.1055/s-2003-40454
Kain KH, Miller JWI, Jones-Paris CR, Thomason RT, Lewis JD, Bader DM, et al. The chick embryo as an expanding experimental model for cancer and cardiovascular research. Developmental Dynamics. 2014; 24:216-228. DOI: 10.1002/dvdy.24093
Bader AG, Kang S, Vogt PK. Cancer-specific mutations in PIK3CA are oncogenic in vivo. Proceedings of the National Academy of Sciences. 2006; 103:1475-1479. DOI: 10.1073/pnas.0510857103
Ribatti D. The chick embryo chorioallantoic membrane in the study of tumor angiogenesis. Romanian Journal of Morphology and Embryology. 2008; 49:131-135
Rous P. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. The Journal of Experimental Medicine. 1911; 13:397-411. DOI: 10.1084/jem.13.4.397
Javier RT, Butel J. The history of tumour virology. Cancer Research. 2008; 68:7693-7706. DOI: 10.1158/0008-5472.CAN-08-3301
Zijlstra A, Mellor R, Panzarella G, Aimes RT, Hooper JD, Marchenko ND, et al. A quantitative analysis of rate-limiting steps in the metastatic cascade using human-specific real-time polymerase chain reaction. Cancer Research. 2002; 62:7083-7092
Mu X, Sultankulov B, Agarwal R, Mahjoub A, Schott T, Greco N, et al. Chick embryo extract demethylates tumor suppressor genes in osteosarcoma cells. Clinical Orthopaedics and Related Research. 2014; 472:865-873. DOI: 10.1007/s11999-013-3104-6
Pain B, Clark ME, Shen M, Nakazawa H, Sakurai M, Samarut J, et al. Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities. Development (Cambridge, England). 1996; 122:2339-2348
Petitte JN, Liu G, Yang Z. Avian pluripotent stem cells. Mechanisms of Development. 2004; 121:1159-1168
Intarapat S, Stern CD. Chick stem cells: Current progress and future prospects. Stem Cell Research. 2013; 11:1378-1392. DOI: 10.1016/j.scr.2013.09.005
Naito M. Development of avian embryo manipulation techniques and their application to germ cell manipulation. Animal Science Journal. 2003; 74:157-168. DOI: 10.1046/j.1344-3941.2003.00101.x
Bajada S, Mazakova I, Richardson JB, Ashammakhi N. Updates on stem cells and their applications in regenerative medicine. Journal of Tissue Engineering and Regenerative Medicine. 2008; 2:169-183. DOI: 10.1002/term.83
Davies WJ, Freeman SJ. Chick embryotoxicity screening test (CHEST I and II). Methods in Molecular Biology. 1995; 43:307-310. DOI: 10.1385/0-89603-282-5:307
Jelinek R, Peterka M, Rychter Z. Chick embryotoxicity screening test—130 substances tested. Indian Journal of Experimental Biology. 1985; 23:588-595
Casewell NR, Wüster W, Vonk FJ, Harrison RA, Fry BG. Complex cocktails: The evolutionary novelty of venoms. Trends in Ecology and Evolution. 2013; 28:219-229. DOI: 10.1016/j.tree.2012.10.020
Vyas VK, Brahmbhatt K, Bhatt H, Parmar U. Therapeutic potential of snake venom in cancer therapy: Current perspectives. Asian Pacific Journal of Tropical Biomedicine. 2013; 3:156-162. DOI: 10.1016/S2221-1691(13)60042-8
Langley RL. Snakebite during pregnancy: A literature review. Wilderness and Environmental Medicine. 2010; 21:54-60. DOI: 10.1016/j.wem.2009.12.025
Jackson K. The evolution of venom-conducting fangs: Insights from developmental biology. Toxicon. 2007; 49:975-981. DOI: 10.1016/j.toxicon.2007.01.007
Kochva E. The development of the venom gland in the Opisthoglyph snake Telescopus fallaxwith remarks on Thamnophis sirtalis(Colubridae, Reptilia). Copeia. 1965; 2:147-154. DOI: 10.2307/1440716
Kochva E, Gans C. The venom gland of Vipera palaestinae with comments on the glands of some other viperines. Cells, Tissues, Organs. 1965; 62:365-401. DOI: 10.1159/000142760
Rosenberg HI. Histology, histochemistry, and emptying mechanism of the venom glands of some elapid snakes. Journal of Morphology. 1967; 123:133-155. DOI: 10.1002/jmor.1051230204
Underwood G, Kochva E. On the affinities of the burrowing asps Atractaspis (Serpentes: Atractaspididae). Zoological Journal of the Linnean Society. 1993; 107:3-64. DOI: 10.1006/zjls.1993.1002
Adewole AA, Ugiagbe OA, Onile TG, Joseph GA, Kassim OD, Medupin PF, et al. Case reports in Women’s Health Snake bite in third trimester of pregnancy with systemic envenomation and delivery of a live baby in a low resource setting : A case report. Case Reports in Women’s Health. 2017; 16:14-17. DOI: 10.1016/j.crwh.2017.10.001
Chisale M. Snake and snakebites–Part 2. WHO Pharmaceuticals Newsletter. 2008; 5:1-4
Jackson TNW, Young B, Underwood G, McCarthy CJ, Kochva E, Vidal N, et al. Endless forms most beautiful: The evolution of ophidian oral glands, including the venom system, and the use of appropriate terminology for homologous structures. Zoomorphology. 2017; 136:107-130. DOI: 10.1007/s00435-016-0332-9
Vonk FJ, Admiraal JF, Jackson K, Reshef R, de Bakker MAG, Vanderschoot K, et al. Evolutionary origin and development of snake fangs. Nature. 2008; 454:630-633. DOI: 10.1038/nature07178
Harris JB, Scott-Davey T. Secreted phospholipases A2 of snake venoms: Effects on the peripheral neuromuscular system with comments on the role of phospholipases A2 in disorders of the CNS and their uses in industry. Toxins. 2013; 5:2533-2571. DOI: 10.3390/toxins5122533
Tasoulis T, Isbister G. A review and database of snake venom proteomes. Toxins. 2017; 9:E290. DOI: 10.3390/toxins9090290
Rigoni M, Caccin P, Gschmeissner S, Koster G, Postle AD, Rossetto O, et al. Equivalent effects of snake PLA2 neurotoxins and lysophospholipid—fatty acid mixtures. Science. 2005; 310:1678-1680. DOI: 10.1126/science.1120640
Gutiérrez JM, Rucavado A. Snake venom metalloproteinases: Their role in the pathogenesis of local tissue damage. Biochimie. 2000; 82:841-850
Baldo C, Jamora C, Yamanouye N, Zorn TM, Moura-da-Silva AM. Mechanisms of vascular damage by hemorrhagic snake venom metalloproteinases: Tissue distribution and in Situhydrolysis. PLoS Neglected Tropical Diseases. 2010; 4:E727. DOI: 10.1371/journal.pntd.0000727
Bjarnason JB, Fox JW. Hemorrhagic metalloproteinases from snake venoms. Pharmacology & Therapeutics. 1994; 62:325-372
Moreira L, Borkow G, Ovadia M, Gutiérrez JM. Pathological changes induced by BaH1, a hemorrhagic proteinase isolated from Bothrops asper(Terciopelo) snake venom, on mouse capillary blood vessels. Toxicon. 1994; 32:976-987
Serrano SMT, Maroun RC. Snake venom serine proteinases: Sequence homology vs. substrate specificity, a paradox to be solved. Toxicon. 2005; 45:1115-1132. DOI: 10.1016/j.toxicon.2005.02.020
Markland FS. Review paper snake venoms and the hemostatic system. Toxicon. 1998; 36:1749-1800
Matsui T, Fujimura Y, Titani K. Snake venom proteases affecting hemostasis and thrombosis. Biochimica et Biophysica Acta. 2000; 1477:146-156. DOI: 10.1016/s0167-4838(99)00268-x
Girish VM, Kumar S, Joseph L, Jobichen C, Kini RM, Sivaraman J. Identification and structural characterization of a new three-finger toxin hemachatoxin from Hemachatus haemachatus venom. PLoS One. 2012; 7:E48112. DOI: 10.1371/journal.pone.0048112
Rey-Suárez P, Floriano RS,Rostelato-Ferreira S, Saldarriaga-Córdoba M, Núñez V, Rodrigues-Simioni L, et al. Mipartoxin-I, a novel three-finger toxin, is the major neurotoxic component in the venom of the redtail coral snake Micrurus mipartitus (Elapidae). Toxicon. 2012; 60:851-863. DOI: 10.1016/j.toxicon.2012.05.023
Dufton MJ, Hider RC. Structure and pharmacology of elapid cytotoxins. Pharmacology and Therapeutics. 1988; 36:1-40
Matsui T, Hamako J, Matsushita T, Nakayama T, Fujimura Y, Titani K. Binding site on human von Willebrand factor of bitiscetin, a snake venom-derived platelet aggregation inducer. Biochemistry. 2002; 41:7939-7946. DOI: 10.1021/bi020004b
Knight LC, Baidoo KE, Romano JE, Gabriel JL, Maurer AH. Imaging pulmonary emboli and deep venous thrombi with 99mTc-bitistatin, a platelet-binding polypeptide from viper venom. Journal of Nuclear Medicine. 2000; 41:1056-1064
Calvete JJ, Harrison RA, Casewell NR, Wagstaff SC, Bolton FMS, Sanz L, et al. Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms. Proceedings of the National Academy of Sciences. 2014; 111:9205-9210. DOI: 10.1073/pnas.1405484111
Warrell DA. Venomous and poisonous animals. In: Farrar J, Hotez PJ, Junghanss T, Kang G, Lalloo D, White NJ, editors. Manson’s Tropical Diseases. 23rd ed. China: Elsevier; 2014. pp. 1096-1127. DOI: 10.1016/B978-0-7020-5101-2.00076-5
Glaudas X, Graham A. Natural history notes: Bitis arietans(puff adder). Foraging behavior. Herpetelogical Review. 2014; 45:139-140
White J. Bites and stings from venomous animal: A global overview. Therapeutic Drug Monitoring. 2000; 22:65-68
Dunnihoo DR, Rush BM, Wise RB, Brooks GG, Otterson WN. Snake bite poisoning in pregnancy. A review of the literature. The Journal of Reproductive Medicine. 1992; 37:653-658
Pollach G. A 14-year-old boy from Malawi who has been bitten by a snake. In: Rothe C, editor. Clinical Cases in Tropical Medicine. 1st ed. China: Elsevier; 2014. pp. 126-129. DOI: 10.1016/C2012-0-03630-7
Currier R. Investigating venom synthesis: Exploring the composition, variation and gene expression dynamics of Bitis arietansvenom. [PhD thesis]. University of Liverpool. 320 p
Barlow A, Pook CE, Harrison RA, Wüster W. Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution. Proceedings of the Royal Society B: Biological Sciences: The Royal Society. 2009; 276:2443-2449. DOI: 10.1098/rspb.2009.0048
Sánchez LV, Pla D, Herrera M, Chippaux JP, Calvete JJ, Gutiérrez JM. Evaluation of the preclinical efficacy of four antivenoms, distributed in sub-Saharan Africa, to neutralize the venom of the carpet viper, Echis ocellatus, from Mali, Cameroon, and Nigeria. Toxicon. 2015; 106:97-107. DOI: 10.1016/j.toxicon.2015.09.027
Dissanayake DSB, Thewarage LD, Waduge RN, Ranasinghe JGS, Kularatne SAM, Rajapakse RPVJ. The venom of spectacled cobra (Elapidae: Naja naja): In Vitrostudy from distinct geographical origins in Sri Lanka. Journal of Toxicology. 2018; 2018:1-14. DOI: 10.1155/2018/7358472
Fasoli E, Sanz L, Wagstaff S, Harrison RA, Righetti PG, Calvete JJ. Exploring the venom proteome of the African puff adder, Bitis arietans, using a combinatorial peptide ligand library approach at different pHs. Journal of Proteomics. 2010; 73:932-942. DOI: 10.1016/j.prot.2009.12.006
Mackessy SP. Handbook of Venoms and Toxins of Reptiles. Florida: CBC Press; 2010. 528 p. ISBN: 978-0-8493-9165-1
Aznaurian AV, Amiryan SV. Histopathological changes induced by the venom of the snake Vipera raddei(Armenian adder). Toxicon. 2006; 47:141-143. DOI: 10.1016/j.toxicon.2004.11.012
Mohamed AH, Nawar NN, Hanna MM. Some effects of Naja nigricollisenvenomation on developing fetal tissue. Toxicon. 1974; 12:477-480
Jarrar BM. Histological alterations and biochemical changes in the liver of sheep following Echis coloratusenvenomation. Saudi Journal of Biological Sciences. 2011; 18:169-174. DOI: 10.1016/j.sjbs.2010.12.002
Araki S, Ishida T, Yamamoto T, Kaji K, Hayashi H. Induction of apoptosis by hemorrhagic snake venom in vascular endothelial cells. Biochemical and Biophysical Research Communications. 1993; 190:148-153. DOI: 10.1006/bbrc.1993.1023
Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. Journal of Morphology. 1951; 88:49-92
Lewis JH, Wolpert L. The principle of non-equivalence in development. Journal of Theoretical Biology. 1976; 62:479-490. DOI: 10.1016/0022-5193(76)90132-6
Krabbenhoft KM, Fallon JF. The formation of leg or wing specific structures by leg bud cells grafted to the wing bud is influenced by proximity to the apical ridge. Developmental Biology. 1989; 131:373-382. DOI: 10.1016/s0012-1606(89)80011-9