Page 1 of 10 Original Research

The post-occipital spinal venous sinus of the Nile

crocodile (Crocodylus niloticus): Its anatomy and use for
blood sample collection and intravenous infusions
Authors: The post-occipital sinus of the spinal vein is often used for the collection of blood samples
Jan G. Myburgh1 from crocodilians. Although this sampling method has been reported for several crocodilian
Robert M. Kirberger2
Johan C.A. Steyl1
species, the technique and associated anatomy has not been described in detail in any
John T. Soley3 crocodilian, including the Nile crocodile (Crocodylus niloticus). The anatomy of the cranial neck
Dirk G. Booyse3 region was investigated macroscopically, microscopically, radiographically and by means of
Fritz W. Huchzermeyer1 computed tomography. Latex was injected into the spinal vein and spinal venous sinus of
Russell H. Lowers4
crocodiles to visualise the regional vasculature. The spinal vein ran within the vertebral canal,
Louis J. Guillette Jr1,5,6
dorsal to and closely associated with the spinal cord and changed into a venous sinus cranially
Affiliations: in the post-occipital region. For blood collection, the spinal venous sinus was accessed
1
Department of Paraclinical through the interarcuate space between the atlas and axis (C1 and C2) by inserting a needle
Sciences, University of angled just off the perpendicular in the midline through the craniodorsal cervical skin, just
Pretoria, South Africa
cranial to the cranial borders of the first cervical osteoderms. The most convenient method
2
Department of Companion of blood collection was with a syringe and hypodermic needle. In addition, the suitability
Animal Clinical Studies, of the spinal venous sinus for intravenous injections and infusions in live crocodiles was
University of Pretoria, evaluated. The internal diameter of the commercial human epidural catheters used during
South Africa
these investigations was relatively small, resulting in very slow infusion rates. Care should be
3
Department of Anatomy taken not to puncture the spinal cord or to lacerate the blood vessel wall using this route for
and Physiology, University of blood collection or intravenous infusions.
Pretoria, South Africa

InoMedic Health
Introduction
4

Applications, Kennedy Space

Center, United States Blood samples are routinely collected from crocodilians for general health examination purposes
(Huchzermeyer 2003), pharmacokinetic studies (Helmick et al. 2004a, 2004b; Martelli et al.
5
Department of Obstetrics
and Gynecology, Medical 2009), ecotoxicological monitoring (Bermudez et al. 2005; Milnes et al. 2002), endocrinological
University of South Carolina, investigations (Boggs et al. 2011; Edwards et al. 2004; Hamlin, Lowers & Guillette 2011) or other
United States research projects (Franklin et al. 2003). Laboratory investigations very often depend on the
ability of the clinician or scientist to collect appropriate blood samples as atraumatically as
6
Hollings Marine Laboratory,
Charleston, United States
possible from live crocodilians (Lovely, Pittman & Leslie 2007; Millan et al. 1997; Rossini et al.
2011; Zayas et al. 2011).
Correspondence to:
Jan Myburgh The most commonly used anatomical access points for venipuncture and blood collection from
crocodilians are: the dorsal neck (Bermudez et al. 2005; Jacobson 1984; Lloyd & Morris 1999;
Email:
[email protected] Martelli et al. 2009; Milnes et al. 2002; Olson, Hessler & Faith 1975; Pothiwong, Prachammuang
& Koykol 2000; Sykes & Klaphake 2008), ventral tail (Gorzula, Arocha-Piñango & Salazar 1976;
Postal address: Rossini et al. 2011), dorsal tail (Huchzermeyer 2003) and temporal (Samour et al. 1984) regions.
Private Bag X04, Blood can also be collected directly from the heart (Carmena-Suero et al. 1979; Jacobson 1984;
Onderstepoort 0110,
South Africa Lloyd & Morris 1999). MacLean, Lee and Wilson (1973) reported a simple method of obtaining
small blood samples from the orbital sinuses in reptiles. This method has not been described for
Dates: crocodilians.
Received: 28 Nov. 2012
Accepted: 27 Jan. 2014 Although the blood vessel in the post-occipital neck region (dorsal neck approach) is most
Published: 05 May 2014
often used (Bermudez et al. 2005; Boggs et al. 2011; Hamlin et al. 2011; Lovely et al. 2007; Martelli
et al. 2009; Millan et al. 1997; Milnes et al. 2002; Stacy & Whitaker 2000; Sykes & Klaphake 2008;
Zayas et al. 2011), the nomenclature associated with this vessel is unfortunately very confusing.
Zippel, Lillywhite and Mlandinich (2003) conducted an in-depth study of the anatomy of this
blood vessel, which is closely associated with the spinal cord, in several crocodilian species
Read online: (Alligator mississippiensis, Caiman latirostris, Crocodylus siamensis and Osteolaemus tetraspis) and
Scan this QR
code with your
smart phone or How to cite this article: Myburgh, J.G., Kirberger, R.M., Steyl, J.C.A., Soley, J.T., Boyse, D.G., Huchzermeyer, F.W. et al., 2014, ‘The post-
mobile device occipital spinal venous sinus of the Nile crocodile (Crocodylus niloticus): Its anatomy and use for blood sample collection and intravenous
to read online. infusions’, Journal of the South African Veterinary Association 85(1), Art. #965, 10 pages. http://dx.doi.org/10.4102/jsava.v85i1.965
Copyright: © 2014. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License.

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 Page 2 of 10 Original Research

named it the spinal vein. However, the post-occipital part of enrofloxacin (Baytril, 50 mg/mL) to healthy captive-reared
the spinal vein (area of venipuncture) has also been called estuarine crocodiles (Crocodylus porosus). Edwards et al. (2004)
the internal jugular (Huchzermeyer 2003; Jacobson 1984; used the same blood vessel for IV injections of ovine follicle
Olson et al. 1975), occipital sinus (Martelli et al. 2009; Stacey stimulating hormone (FSH) in 4-year-old male American
& Whitaker 2000), occipital venous sinus (Lloyd & Morris alligators.
1999), post-occipital sinus (Lovely et al. 2007), post-occipital
venous sinus (Millan et al. 1997), subdural sinus (Pothiwong The purpose of this study was to investigate and describe
et al. 2000), post-cranial supravertebral sinus (Boggs et al. the clinically relevant anatomy of the post-occipital spinal
2011; Sykes & Klaphake 2008), post-cranial supravertebral venous sinus, including external landmarks, together with
blood vessel (Bermudez et al. 2005; Edwards et al. 2004), the associated clinical procedures of blood sampling and IV
supravertebral occipital venous sinus (Zayas et al. 2011) and infusion in the Nile crocodile.
post-cranial vertebral vein (Milnes et al. 2002). In humans,
the equivalent blood vessel is referred to as the posterior Research method and design
internal vertebral venous plexus (Groen et al. 1997). Based on
the findings of this study, the term spinal venous sinus will Cervical and spinal venous sinus anatomy
be used whenever we refer to the post-occipital part (area of Three mature crocodiles, > 2.2 m in total length (TL), and
venipuncture) of the spinal vein. six young crocodiles (1.0 m – 1.5 m TL) were obtained from
a commercial crocodile farm (Izintaba Crocodile Farm, De
A basic knowledge of the anatomy of this blood vessel, Wildt, North West Province, South Africa). The three mature
especially in the post-occipital region, is essential if it is to crocodiles were received as fresh carcasses for necropsies by
be used for clinical procedures in crocodilians. However, the the Pathology Section, Department of Paraclinical Sciences,
anatomy of this blood vessel, especially in the Nile crocodile Faculty of Veterinary Science, University of Pretoria. The
(Crocodylus niloticus), has not been thoroughly investigated. three heads and necks (HaNs) were removed for further
Zippel, Lillywhite and Mlandinich (2001) described this processing during the necropsy investigations (Table 1). The
vascular system in eight snake species, representing three individual bones of one HaN were cleaned, reassembled
families, as an elaborate vertebral plexus extending from and glued in place to study the anatomy. Rubber latex was
the base of the skull to the tip of the tail. In a follow-up injected directly into the cervical spinal vein, from the caudal
investigation, the same authors (Zippel et al. 2003) reported end (cut surface), of the two remaining HaNs to investigate
that crocodilians possessed a well-developed spinal vein, the morphology of the blood vessel. This was achieved by
with caval and hepatic connections, that contributed cutting one of the heads along the midline from the snout
significantly to the return of blood to the heart from the rest to the end of the neck and by cutting the other head into
of the body. They also stated that the occipital sinus covered ± 2 cm-thick transverse sections using a commercial bandsaw
the dorsal and lateral surfaces of the crocodilian brain and (Table 1).
drained caudally through the foramen magnum into a
voluminous post-occipital sinus, after which it continued Six young crocodiles were humanely euthanised (project
caudally as a non-plexiform (single) extradural spinal vein, number: V041/05) with sodium pentobarbitone (Eutha-
remaining dorsal to the spinal cord for the entire length of naze; Bayer Animal Health, Isando, South Africa) injected IV
the vertebral column. and the HaNs removed to investigate cervical anatomy and
spinal venous sinus morphology (Table 1). In the case of one
Clinicians required to access the spinal venous sinus to of the HaNs, necrophagous insects were used to remove the
collect blood samples or give intravenous (IV) injections excess soft tissue to preserve the individual cervical bones
are faced with a dilemma as the spinal venous sinus is well- in their original positions. The preparation and processing
covered with soft tissue and venipuncture is limited by the procedures, and final examinations performed on the HaNs
surrounding osseous structures (Zippel et al. 2003). It is the are summarised in Table 1.
authors’ experience that only the tip of a perpendicularly
angled needle can access the blood vessel. This makes it
extremely difficult, specifically, to inject even small volumes Diagnostic imaging
into the venous sinus, or to insert an IV catheter. Some or Before being used to visualise the regional vasculature, the
even all of the liquid could easily be injected perivascularly if HaN of one of the latex-infused mature crocodiles underwent
the tip of the needle is moved slightly or if the animal is not radiographic and computed tomography (CT) examinations
kept completely still during the procedure. As the systemic (Emotion dual slice CT; Siemens, Erlangen, Germany) to
circulation can be accessed directly through the spinal venous assess the morphology of the cranial cervical vertebrae
sinus, IV injections or continuous infusions in crocodiles (Table 1). Additionally, a live young crocodile (1.42 m TL)
may be helpful in the treatment of individual cases or when from another research project (project number: V006/09) was
needed for specific research projects. Enrofloxacin (Baytril, used for a contrast enhanced CT scan. Information about this
22.7 mg/mL) (Helmick et al. 2004a) and oxytetracycline animal is not reflected in Table 1 because the CT scan was
(Liquamycin LA, 200 mg/mL) (Helmick et al. 2004b) were performed as part of a more complete clinical investigation of
injected IV into American alligators (A. mississippiensis). this healthy crocodile and it was not euthanised afterwards.
Martelli et al. (2009) also used this blood vessel to administer It was immobilised with 0.5 mL gallamine triethiodide

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TABLE 1: Summary of the preparation, processing and examination of the crocodilian heads and necks used in the study.
Size of crocodiles Number of heads Source of material Initial preparation of the heads and necks Final processing and examination of the heads
and necks processed and necks
Mature crocodiles 3 Carcasses of crocodiles • The HaNs were removed from the fresh • One HaN was boiled and the remaining soft
> 2.2 m TL received by pathologist carcasses by cutting transversely through the tissue removed. The individual vertebrae
for necropsies. caudal neck region with a commercial were dried, reassembled and glued in place
bandsaw. afterwards.
• The covering skin and some of the muscles • Rubber latex was injected into the cervical
were removed from the HaNs with a spinal vein from the caudal end (cut surface)
necropsy knife and scalpel blade. of the two remaining HaNs. These specimens
were subsequently fixed in 10% buffered
formalin for at least 2 weeks.
• One of the latex injected HaNs was used
for radiography and computed tomography
prior to being halved along the midline (from
the snout to the end of the neck) using a
bandsaw.
• The second latex injected HaN was cut
transversely into ± 2 cm sections.

Young crocodiles 6 Crocodiles humanely • Three crocodiles were euthanised by inserting • One HaN was boiled and the remaining soft
1.0 m – 1.5 m TL euthanised. an epidural catheter into the spinal venous tissue removed. The individual vertebrae
sinus of each crocodile and injecting sodium were dried, reassembled and glued in place
pentobarbitone through the catheter. afterwards.
• Three crocodiles were injected IV with • Necrophageous insects were used to remove
sodium pentobarbitone using the needles the remaining soft tissue from a HaN
(20G) routinely used for blood collection. leaving the vertebrae, still attached by their
• The HaNs were removed as described ligaments, in situ.
for the mature crocodiles. • The remaining four HaNs were fixed in 10%
buffered formalin for at least 2 weeks.
• Two of the formalin-fixed HaNs were halved
along the midline (from the snout to the
end of the neck) using a bandsaw. The other
two HaNs were cut transversely into ± 2 cm
slices.

Hatchlings 3 Carcasses of hatchlings • The HaNs were removed from the fresh • Three HaNs were fixed in 10% buffered
± 32 cm TL received by pathologist carcasses by cutting transversely through formalin and the bones decalcified with
for necropsies. the caudal neck region with a commercial formic acid.
bandsaw. • Transections of the post-occipital neck region
• The covering skin and some of the were routinely prepared for histological
muscles were removed from the HaNs examination.
using a scalpel.
TL, total length; HaN, head and neck; IV, intravenously.

(Flaxedil; Kyron Laboratories [Pty] Ltd, Benrose, South Africa) years (2007–2011) by the principal author (J.G.M.). A
intramuscularly before the procedure (Flamand, Rogers & hypodermic needle attached to a syringe was used and the
Blake 1992; Loveridge & Blake 1972). The CT scan of the live samples transferred to specific blood tubes immediately
animal was performed in order to record the post-occipital after collection. Different lengths (mm) and gauges (G) of
spinal venous sinus and the cervical spinal vein relative to hypodermic needles were evaluated to determine their
the rest of the circulation with the vascular system filled with suitability – 32 mm 22G (0.7 mm outer diameter [OD]),
blood. Iohexol 300 mg/mL (Omnipaque; GE Healthcare, 25 mm 20G (0.9 mm OD), 38 mm 20G (0.9 mm OD)
Weltevreden Park, South Africa) was injected into the spinal and 38 mm 18G (1.2 mm OD) – as well as an 88 mm 18G
(1.2 mm OD) spinal needle (Terumo Corporation, Tokyo,
venous sinus by means of an epidural catheter. Transverse
Japan). All the different hypodermic needles had regular
and multiplanar reformatted images were made in bone and
bevels and the spinal needle a short bevel. During the
soft tissue windows together with volume rendered images.
collection of blood samples relevant external landmarks
relative to the placement of the needle were identified. All
Microanatomy of the post-occipital cervical complications or side effects (e.g. paralysis) associated with
region blood sampling were recorded and further investigated.
Several hatchling carcasses were received from a commercial Complete necropsies were performed on affected animals to
crocodile farm for necropsies. The HaNs of three fresh examine possible damage to the wall of the spinal venous
carcasses were removed, fixed in situ in 10% buffered sinus and surrounding tissues.
formalin, decalcified with formic acid as described by
Bancroft and Gamble (2003) and routinely processed for Injections and infusions using the spinal
light microscopy (Table 1). Transections of the post-occipital venous sinus
region were stained with haematoxylin and eosin and Three of the six young crocodiles (Table 1) were euthanised
examined by bright field light microscopy. Images were with sodium pentobarbitone (Eutha-naze; Bayer Animal
digitally recorded, focusing on the spinal venous sinus in the Health, Isando, South Africa) injected directly into the
post-occipital region. spinal venous sinus using either 25 mm or 38 mm 20G
hypodermic needles (Table 1). Commercial human epidural
catheterisation sets were obtained and evaluated in four
Blood sampling young live crocodiles. Using this method, three animals
The post-occipital region was used for the collection of were euthanised with sodium pentobarbitone (Table 1) and
blood samples (± 350) from a wide range of Nile crocodiles one animal was infused with contrast medium for the CT
(0.5 m – 3.8 m TL) in southern Africa over a period of five scan (see subsection entitled ‘Diagnostic imaging’ above).

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10 Original
Original Research
Research

2 2

Note:
Note: The
The cranially
cranially situated,
situated, triangular
triangular bone
bone (arrow
(arrow 1)
1) and
and the
the overarching
overarching lateral
lateral bones
bones of
of the
the atlas
atlas (arrows
(arrows 2)
2) partially
partially cover
cover the
the intravertebral
intravertebral canal.
canal. The
The opening
opening between
between the
the atlas
atlas and
and axis,
axis,
through
through which
which entry
entry isis gained
gained to
to the
the post-occipital
post-occipital venous
venous sinus,
sinus, isis indicated
indicated with
with arrow
arrow 3.
3.
FIGURE
FIGURE 1:1: The
The base
base of
of the
the head
head and
and post-occipital
post-occipital cervical
cervical vertebrae
vertebrae of
of aa young
young Nile crocodile in
Nile crocodile in situ
situ after
after removal
removal of
of the
the surrounding
surrounding soft
soft tissue
tissue by
by necrophageous
necrophageous
insects.
insects.

Epidural needles (80 mm 18G) and the accompanying

920 mm 20G epidural catheters (B-D®® DurasafeTM
TM
18G 405096;
Becton Dickinson Benelux S.A., Erembodegem, Belgium)
were used.

Each epidural needle was inserted into the spinal venous

sinus with the tip opening pointing in a caudal direction. In
each crocodile, the correct positioning of the needle tip in the
blood vessel was confirmed by ready aspiration of venous
blood. The accompanying epidural catheter was immediately
inserted through the needle into the spinal venous sinus and
pushed caudally into the spinal vein. The exact position of
the three catheters was confirmed after death and following
removal of the HaNs (Table 1).

In respect of the young crocodile destined for the CT

examination (1.42 m TL), a similar epidural catheter was
Note:
Note: The
The gap,
gap, 3.5
3.5 mm
mm ×× 5.0
5.0 mm
mm inin this
this case,
case, just
just cranial
cranial to
to the
the spinous
spinous process
process of
of the
the axis
axis
inserted into the spinal venous sinus for the infusion of through
through which
which the
the hypodermic
hypodermic needle
needle isis passed
passed (arrow).
(arrow). The
The potential
potential interarcuate
interarcuate space
space isis
contrast medium. An administration set (Sabax paediatric partially
partially covered
covered by
by the
the cranial
cranial projection
projection ofof the
the spinous
spinous process
process (axis).
(axis).

solution administration set, AFC 0186; Adcock Ingram FIGURE

FIGURE 2:
2: Volume
Volume rendered
rendered computed
computed tomography
tomography image
image of
of the
the cranial
cranial cervical
cervical
region
region of
of aa young
young Nile
Nile crocodile.
crocodile.
Critical Care (Pty) Ltd, Johannesburg, South Africa),
60 drops/mL, was attached to the catheter and sterile saline
(Sabax sodium chloride 0.9%; Adcock Ingram Critical Care Results
(Pty) Ltd, Johannesburg, South Africa) administered, as fast
Anatomy of the post-occipital cervical region
as the catheter would allow, to confirm that the catheter
was correctly inserted in the blood vessel. After 250 mL of
and spinal venous sinus
saline was administered, the procedure was considered to Dorsally, a horizontally oriented, cranio-caudally inclined,
be a successful IV catherisation and the contrast medium flat triangular bone formed the cranial aspect of the roof
injected. of the atlas. This bone also covered the space between the

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 Page 5 of 10 Original Research

base of the skull and the remaining components of the connective tissue. Although unconfirmed, the cells forming
atlas. The base of the bone formed the cranial border of the this layer displayed what appeared to be myofibroblastic
dorsal opening or space present between the atlas and axis features. The outer wall was inseparable and histologically
(Figure 1 and Figure 2). Paired lateral bones made up the indistinct from the periosteum lining the vertebral canal.
sides and roof of the neural arch. These bones were complex Similarly, the inner wall of the sinus formed a continuum
in shape and articulated cranially with the occipital condyle with the dura mater ventrally. Considering the above
and caudally, at two points, with the cranial aspects of observations, the spinal venous sinus is either intradural or
the axis. The lateral bones flattened dorsally and arched epidural in location (Figure 4). The sinus was traversed by
medially, but only partially, towards each other to complete randomly positioned connective tissue trabeculae that were
the roof of the atlas. The paired lateral bones thus formed lined by a single layer of squamous epithelial cells continuous
the lateral borders of the dorsal opening described above. A with the rest of the endothelium. A typical arachnoid was
single ventral bone completed the structure of the atlas. This not observed beneath the dura mater, although a substantial
squat bone articulated cranially with the occipital condyle space was present between the latter and the pia mater
via a large articulation facet and also made contact dorso- surrounding the spinal cord. This space contained cerebro-
caudally with the odontoid process and with the cranio- AOSIS OpenJournals – Language Edited Version

ventral aspect of the axis. The axis was an elongated bone

with a well-developed spinous process. The cranio-dorsal
aspect of the axis formed the caudal border of the dorsal
opening described above. This opening (Figure 1 and Figure 2)
represented the point of access to the spinal venous sinus. In
older animals, the spinous process of the axis was observed
to project cranially, thus partially occluding the space.

The latex injected into the blood vessels of the HaNs of Note: The contrast medium in the spinal vein indicates its dorsal positioning in the vertebral
canal (arrows 1). Contrast medium is also obvious in the heart (arrow 2) and surrounding
both mature and young crocodiles (Table 1) confirmed that vasculature.
the spinal vein ran within the vertebral canal dorsal to, and FIGURE 3: Sagittally reconstructed computed tomography image following the
closely associated with, the spinal cord. The round spinal vein administration of contrast medium into the post-occipital venous sinus.
AOSIS OpenJournals – Language Edit
present in the caudal neck region was observed to change into
a sinus in the mid-to-cranial cervical region by progressively
enveloping the spinal cord in a nearly symmetrical way as
the foramen magnum was approached.

Diagnostic imaging findings

Radiographs of the occipito-atlanto-axial region were
unrewarding due to the superimposition of the clinically
relevant bones in the cranial post-occipital region. However,
CT gave good images of the region. The dorsal gap formed
by the lateral bones of the atlas and demarcated cranially by
the triangular-shaped dorsal bone of the atlas and caudally
by the axis was clearly visible (Figure 2).

The contrast medium injected via the IV catheter graphically

illustrated the spinal venous sinus, revealing that it almost
completely surrounded the spinal cord in the post-occipital
region. Communication between the spinal vein and the
general circulation was also confirmed by the presence of the *
contrast medium inside the heart approximately one minute
after infusion (Figure 3). The healthy crocodile used for
this procedure recovered uneventfully from the gallamine
triethiodide (Flaxedil) treatment, IV catheter placement and
iohexol (Omnipaque) injection.

Histology of the spinal venous sinus and

surrounding tissues
Microscopy confirmed that the spinal vein in the caudal
cervical region was a non-trabeculated cylindrical structure 200 μm
that cranially became progressively wider, forming a sinus
enveloping the spinal cord up to the level of the ventral Note: Haematoxylin and eosin stain = 20× magnification.
Dotted outline area, spinal venous sinus; arrow, dorsal interarcuate ligament; *, subarachnoid
spinal nerve roots (Figure 4). The sinus wall consisted of or subdural space; vertical dashed line, path of needle during venipuncture.
an endothelial lining supported by a 4–6 cell thick layer of FIGURE 4: Histological transection of the post-occipital region of a hatchling.

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 Page 6 of 10 Original Research

spinal fluid (CSF) and constituted the subarachnoid or AOSIS OpenJournals – Language E

subdural space (Figure 4).

Blood sampling technique

Crocodiles were not sedated or anaesthetised for the
blood collection procedures. They were either manually
restrained (wild crocodiles) or electrically immobilised
(farmed crocodiles) with a commercial crocodile stunner
(D7 Electronics, Pongola, South Africa). The mouths of all
crocodiles were properly tied and their eyes covered. Blood
sampling was usually completed in less than a minute per
crocodile.

With the crocodile in sternal recumbency the post-

Note: The landmarks are the dorsal midline and the cranial borders (white line) of the first
occipital spinal venous sinus was accessed by inserting dorsal osteoderms (arrows).
a needle, positioned nearly perpendicularly, through the FIGURE 5: Needle placement for accessing the post-occipital spinal venous sinus
dorsal cervical skin in the midline just cranial to the most demonstrated on a carcass received for necropsy.
cranial borders of the first two dorsal cervical osteoderms
(Figure 5). The needle was slowly pushed deeper whilst
regular attempts were made to aspirate blood (observed in
the needle hub). When traversing the interarcuate ligament
a gritty feeling could occasionally be felt, indicating that the
tip of the needle was in or near the blood vessel (Figure 4).
Blood was collected by creating a constant vacuum with the
syringe. It is critically important to prevent any movement
of the needle, both during the initial phase when the tip was
confirmed to be in the spinal venous sinus and during the
ongoing blood aspiration procedure. Maintaining the needle
tip in the blood vessel was achieved by grasping the needle
hub between the thumb and index finger (left hand in the case
of J.G.M.), whilst the same hand was rested on the neck of the
crocodile for stability. The other hand was used to create the
vacuum in the syringe. The depth of the spinal venous sinus
below the skin surface ranged from 2.5 cm – 3.5 cm in young
crocodiles (1.5 m – 2.0 m TL) to 7 cm in larger crocodiles
(> 3 m TL). Body condition of individual crocodiles affected Note: Needles from left to right are, 88 mm 18G spinal needle and 38 mm 18G, 38 mm 20G,
32 mm 22G hypodermic needles.
the depth of the blood vessel.
FIGURE 6: Examples of the bevels of the different needles used in the
investigation.
Most of the blood samples (± 350 samples) were collected
with 20 mm or 38 mm 20G needles. For crocodiles < 1.2 m TL,
fluid was occasionally aspirated whenever the needle was
a 20 mm 20G needle was found to be the most suitable, whilst
inadvertently inserted too deep. Figure 4 illustrates why
for slightly larger crocodiles (1.2 m – 2.2 m TL) a longer 20G
the aspiration of CSF is possible with this approach for
needle was required (38 mm). The 32 mm 22G needles were
venipuncture.
found to be unsuitable because the small internal diameter of
the needles slowed down aspiration and created unnecessary
turbulence and red blood cell damage. The potential damage Intravenous injections and infusions using the
to red blood cells by the 22G needles was not thoroughly spinal venous sinus
investigated, but is a perception and concern of the principal When using the post-occipital spinal venous sinus for IV
author (J.G.M.). It is also possible to use 38 mm 18G needles injections (Table 1), the sinus was accessed in the same way
in some crocodiles, but, in the authors’ opinion, blood as for blood sampling. Injections were performed slowly
collection was just as efficient with the 38 mm 20G needles with intermittent aspirations to confirm that the needle tip
and therefore the use of the latter was preferred. For large, was still in the blood vessel.
mature crocodiles (> 2.2 m TL) an 88 mm 18G spinal needle
was found to be most appropriate because of its length To introduce an epidural catheter into the blood vessel for
advantages. Needles with short bevels (e.g. spinal needles) IV infusions, the needle from the epidural set (Figure 7)
were more ideal (safer) for blood collection from the spinal was routinely inserted into the spinal venous sinus. When
venous sinus using the perpendicular approach (Figure 6). inserting the epidural needle, it was essential that the needle
tip opening pointed in a caudal direction. The catheter was
Clotting of blood in the needles or syringes was never a gently fed through the needle as soon as the tip was confirmed
problem and an anticoagulant was not used. Cerebrospinal to be in the blood vessel (blood aspiration). Commercial

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 Page 7 of 10 Original Research

epidural catheters were successfully inserted into three Complications

crocodiles to be euthanised (Table 1). A fourth crocodile was
successfully catheterised before the CT procedure described Complications were experienced during blood collection in
above and recovered without any side effects (see subsection three crocodiles (3/350; < 1%). In all three cases the crocodiles
entitled ‘Diagnostic imaging’ under ‘Research method and were between 1.5 m – 2.0 m TL and 38 mm 20G needles were
design’). used. The three crocodiles became paralysed within a few
minutes and were humanely euthanised by injecting sodium
Because the epidural needles were thicker than the needles pentobarbitone into the spinal venous sinus. Lacerations of
used for routine blood sampling from these size animals the ventral wall of the spinal venous sinus were confirmed
(usually 20G) and had a short bevel with a curved tip on necropsy in the three carcasses. The lacerations resulted in
(Figure 7), it was slightly more difficult to penetrate the blood being pumped into the subarachnoid spaces containing
tough skin and to access the spinal venous sinus. For the IV CSF through the damaged blood vessel walls, causing the
catheterisation procedures, the authors preferred to penetrate cranial cervical spinal cord and caudal brain regions to be
the skin slightly more cranially than normal with the needle covered with clotted blood (Figure 8). No obvious gross
tip, allowing the spinal venous sinus to be accessed more spinal cord damage was observed.
obliquely, which facilitated the feeding of the catheter
around the more obtuse angle created between the needle
and the blood vessel. An example of a commercial human
Ethical considerations
epidural catheterisation set (B-D® DurasafeTM 18G 405096; This study was approved by the Animal Ethics Committee
Becton Dickinson Benelux S.A., Erembodegem, Belgium) (AEC) of the University of Pretoria. The AEC approval
numbers for the anatomy and diagnostic imaging projects
that was used is illustrated in Figure 7.
are V041/05 and V006/09, respectively.

a b Discussion
Although the post-occipital neck region has been described
as a venipuncture site (Jacobson 1984; Lloyd & Morris 1999;
Sykes & Klaphake 2008) and is regularly used for blood
sample collection from crocodilians by scientists (Boggs
et al. 2011; Hamlin et al. 2011; Lovely et al. 2007; Millan et al.
1997; Rossini et al. 2011; Zayas et al. 2011), very little has
AOSISbeen
OpenJournals – Language
published on theEdited Version of the neck
anatomy 2013of crocodilians,
in general, and on the Nile crocodile in particular. As the
FIGURE 7: An (a) epidural needle and catheter (B-D® DurasafeTM 18G 405096; blood vessel is completely surrounded by the cervical
Becton Dickinson Benelux S.A., Erembodegem, Belgium), used for intravenous
infusions and (b) the short bevel of the epidural needle. vertebrae, the only way to access the spinal venous sinus is
to use the opening between the atlas and axis. To blindly find
this opening and the spinal venous sinus beneath, external
landmarks become important and essential. The external
landmarks that were identified during this investigation
(Figure 5) were found to be extremely good indicators of
the locality of the underlying interarcuate space between the
atlas and axis in the Nile crocodile.

By studying several HaNs (Table 1), the shape and size of

the opening between the atlas and axis was confirmed, as
well as the anatomy of the individual vertebrae. It was clear
(Figure 1 and Figure 2) that the opening between the atlas
and axis is slightly longitudinal in shape in a cranio-caudal
direction. If the external landmarks were used as the authors
have suggested (Figure 5), the tip of the needle should
penetrate the opening close to its caudal border. Therefore,
if the blood collector was unsuccessful in the first attempt
at this locality, the tip of the needle could be moved or re-
inserted slightly more cranially.

The most interesting observation concerning the atlas was that

it consisted of four individual bones. Interestingly, this has
also been observed in some dinosaurian fossils (Weishampel,
Dobson & Osmólska 2004). The triangular-shaped dorsal
Note: The arrows indicate the clotted blood ventrally in the caudal brain and cranial cervical bone of the atlas is thin and fragile in young crocodiles and
spinal cord regions. The neck was removed from the head – left side of picture.
FIGURE 8: Mid-sagittal section through the head and neck of a crocodile that
easily lost during the process of cleaning the neck vertebrae,
developed complications after blood sampling. as was experienced by two of the authors (J.G.M. and D.G.B.).

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 Page 8 of 10 Original Research

Chiasson (1962), in a study of the alligator, notes that this et al. (2000) reported such problems when collecting blood
bone could be a detached neural spine or a membranous from C. siamensis. It is important to establish that CSF is
bone that is not a true component of the atlas. not being aspirated by observing the hub of the needle
or the syringe for signs of the straw-coloured CSF. The
It was deduced from the HaN anatomy that a slight other important factor is to make sure that the tip of the
downward flexing of the neck would help to increase the needle is kept steady in the blood vessel during the entire
size of the opening between the atlas and axis. In addition, blood collection procedure. Any movement of the needle
it was experienced by the principal author (J.G.M.) that it to a deeper position would obviously increase the risk of
was more difficult to find the opening if the head was lifted penetrating the subarachnoid space. Accidental removal
upwards. This easily happens with wild crocodiles lying on of the needle tip from the spinal venous sinus most often
uneven surfaces. With the crocodile in sternal recumbency, it happens when exchanging syringes.
is important that the head of the crocodile (mandible) should
lie flat on the underlying surface (e.g. a table) and not be The principal author (J.G.M.) has aspirated clear CSF on
forced up by an uneven surface (e.g. uneven ground). Helpers several occasions when the needle tip was inserted too deep
must not put their hands under the mandible to control and penetrated the subarachnoid space. This technique could
the animal, as this would slightly lift the head. The ideal therefore be used, in theory, for the collection of CSF samples
situation is to keep the head down by pressing downwards from Nile crocodiles. However, the unnecessarily high risk
between the eyes and nostrils with the palm of an open hand. for serious complications negates the use of this technique
In the case of wild crocodiles the blood collector must move for routine CSF sample collection. Another disadvantage of
the crocodile to the most ideal position in which the neck is using this route for CSF collection from crocodilians is that it
flexed slightly downwards. Contrary to our observations, would be nearly impossible to collect a pure CSF sample as
Olson et al. (1975) suggested that a dorsal flexion of the neck the needle first has to pass through the spinal venous sinus
may help to enlarge the opening. A possible explanation for before entering the subarachnoid space.
this difference of opinion is that these authors suggested
penetrating the skin, not in the midline, but rather from a Excessive penetration with the tip of the needle may bring it
point lateral to the most cranial part of the spinous process of into contact with the spinal cord, which results in immediate
the axis. Both Figures 1 and 2 are helpful in visualising why involuntary muscle movement. Fortunately no permanent
a slight dorsal flexion of neck would support their approach. damage was observed in crocodiles where this behaviour
was noted during blood collection. A similar situation was
The spinal vein of the Nile crocodile clearly formed a sinus in experienced by one of the authors (R.M.K.) during canine
the post-occipital region. This was observed macroscopically cervical myelography, again with no deleterious effects
(dissections and latex casts), as well as in histology sections being noted afterwards.
(Figure 4) and following contrast medium administration.
It was also confirmed that the post-occipital spinal venous Using the post-occipital spinal venous sinus for venipuncture
sinus continues caudally as the spinal vein as described by has some advantages and disadvantages. Advantages are:
Zippel et al. (2003). the site of venipuncture is easy to determine using external
landmarks, the animal is restrained in a natural position
Pothiwong et al. (2000) suggested that the sinus is located (lying on its ventral surface) and the collection technique is
beneath the dura mater in C. siamensis and therefore referred relatively simple. In the case of large crocodiles, the collector
to it as a subdural sinus. The histological investigations in this may sit on the animal’s thoracic cavity and safely collect the
study confirmed that in the Nile crocodile it does not lie in sample from the post-occipital area. Potential disadvantages
the subdural space but is situated epidurally (extradural) or are: the blood vessel wall could be lacerated, the spinal cord
possibly even intradurally (Figure 4). The extradural location may be damaged, contamination of the blood sample by CSF
of the spinal vein in the vertebral canal was also reported by is a possibility and, in bigger crocodiles, the blood vessel
Zippel et al. (2003). will be comparatively deeper and the opening between the
atlas and axis, therefore, sometimes more difficult to find.
The physiological role of the spinal venous sinus in the The most likely explanation for the difficulty in finding
post-occipital region of the neck is unfortunately not clearly the interarcuate space in large mature crocodiles is that the
understood (Zippel et al. 2003). The authors observed that the manipulation of the tip of the needle becomes less effective
connective tissue component of the sinus wall displayed cells the deeper it is in the animal. Additionally, the observation
with apparent myofibroblastic features (Figure 4), suggesting that the spinous process of the axis projects more cranially
the possibility that these cells could provide the sinus wall in older crocodiles, and therefore occludes the interarcuate
with the ability to modify the diameter of the lumen and space more than in younger animals, warrants a more in-
therefore influence blood pressure. Zippel et al. (2003) also depth anatomical investigation.
speculated about the possible physiological functions of
the spinal vein in crocodilians without coming to definite Although CT has been used to study reptile anatomy (Banzato
conclusions. et al. 2013; Wyneken 2006), as far as could be ascertained it
has not previously been used in the Nile crocodile. Modern
Care should be taken during blood aspiration to prevent diagnostic technology holds great opportunities for future
collecting CSF with the blood as it could potentially veterinary procedures and specifically for crocodilian
influence specific blood chemistry parameters. Pothiwong research projects (Banzato et al. 2013; Ohlerth & Scharf

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 Page 9 of 10 Original Research

2007; Wyneken 2006). It was observed during the scanning it is important that the crocodile be kept completely under
procedure that the crocodile experienced discomfort at control. As indicated above, movement of the animal or of
the vibration of the CT apparatus. Although gallamine the needle whilst positioned within the blood vessel, caused
triethiodide was used to prevent movement, the crocodile in complications in three animals. Laceration of the ventral wall
the CT scanner reacted immediately when it was operated of the spinal venous sinus with the tip of the needle resulted in
(vibrations). General anaesthesia would therefore be more the accumulation of blood in the subarachnoid space, around
suitable when using the scanning procedure. the spinal cord and in the caudal parts of the brain (Figure 8).
Heard et al. (1988) reported that an African dwarf crocodile
Intravenous injections and infusions are not often performed was killed during an attempted blood transfusion using the
on crocodilians as intensive treatment of individual animals spinal venous sinus (named the internal jugular) as a result
is usually not attempted. As the post-occipital spinal venous of the compression of the spinal cord by blood entering the
sinus can only be penetrated with the needle positioned subarachnoid space through the damaged blood vessel wall.
at a nearly perpendicular angle, inadvertent paravenous Pothiwong et al. (2000) noted similar complications when
injections may result, particularly if the animal moves using this technique and described similar clinical signs to
during the procedure. It was unfortunately not possible those observed in the present study. Such complications may
in the present study to determine whether any fluid was be avoided by using other venipuncture sites, but because
inadvertently injected perivascularly in the crocodiles complications are rare and preventable, and this technique
euthanised with sodium pentobarbitone (Table 1). Edwards is relatively easy to perform, this venipuncture method is
et al. (2004) injected FSH directly into the spinal venous recommended.
sinus although the volume used was small. However,
larger volumes of commercial antimicrobials have also The length of the bevel of the needle could play an important
been injected successfully by the IV route into the American predisposing role in the development of complications.
alligator (Helmick et al. 2004a, 2004b) and estuarine crocodile A longer bevel would increase the risk of the blood vessel
(Martelli et al. 2009) for pharmacokinetic studies. The spinal wall being damaged during the blood collection procedure
venous sinus should therefore be considered an important IV (Figure 6). This is particularly relevant considering that the
route for crocodilians. length of the bevel, when inserted perpendicularly into the
blood vessel of a smaller crocodile, is nearly as long as the
The angle of penetration and depth of the blood vessel made total diameter of the venous sinus. This creates a situation
it extremely difficult to introduce an IV catheter into the where the sharp tip of the needle is already touching or
vessel around the nearly 90° angle. The only way to solve this penetrating the ventral wall of the blood vessel before the
problem was to use commercially available human epidural needle opening comes into contact with the blood. A possible
needles (Frölich & Caton 2001) and their accompanying solution is to find a commercial source of hypodermic
catheters. Zippel et al. (2003) also inserted catheters into needles with short bevels (similar to spinal needles) or
the spinal veins of several alligators for contrast medium to manufacture hypodermic needles with short bevels
administration using either modified Cournand style (18G) specifically for crocodilians.
or modified Seldinger (18G) needles. To further assist the
procedure, it was preferred to penetrate the skin slightly
more cranially than normal when inserting the specific
Conclusion
needle, allowing the spinal venous sinus to be accessed Although the spinal venous sinus is accessed blindly, the
more obliquely, which, in theory, should facilitate smoother use of the external landmarks identified in this study makes
feeding of the catheter through the curved needle tip (Frölich it possible to be successful during most attempts. It is
& Caton 2001) and into the blood vessel in a caudal direction. important, however, for the blood collector to appreciate the
Penetrating the tough skin could be overcome by making a basic anatomy of the post-occipital region before attempting
small incision in the skin. The use of a local anaesthetic is this procedure to increase the success rate and prevent
indicated. Although it was slightly more difficult to insert a complications.
commercial human epidural catheter into the blood vessel
than to access the spinal venous sinus with a hypodermic
needle, the authors’ opinion is that these epidural sets are
Acknowledgements
the most practical, and definitely suitable, for catherisation This study was supported by grants received from the
of crocodilian spinal veins. Norwegian Council for Higher Education’s Programme for
Development, Research and Education (NUFU 08/02) and
Although the epidural catheters were successfully inserted, the Royal Netherlands Embassy in South Africa (Prof. Nico
the internal diameter of the catheters was unfortunately Visser). The authors would like to acknowledge the support
relatively small, therefore preventing the administration of received from the Izintaba and Le Croc crocodile farms. Help
drip fluid at a high infusion rate. The injection of viscous and suggestions from Professors Christo Botha and Herman
liquids (e.g. oxytetracycline) through this catheter was also Groenewald, Faculty of Veterinary Science, University of
not achievable. Similarly, no aspiration of blood through the Pretoria are appreciated. Personnel from the Diagnostic
IV catheters was possible. Imaging Section, Onderstepoort Veterinary Academic
Hospital, Faculty of Veterinary Science helped with the
It is important that the blood collector be careful not to damage CT scanning. Dr Leon Venter assisted with the chemical
the blood vessel wall during the clinical procedure. Similarly, immobilisation of the crocodile that was used for the CT

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 Page 10 of 10 Original Research

scanning. The authors also would also like to acknowledge Hamlin, H.J., Lowers, R.H. & Guillette, L.J., 2011, ‘Seasonal androgen cycles in
adult male American alligators (Alligator mississippiensis) from a barrier island
the support received over the years from Jannie Coetzee, population’, Biology of Reproduction 85, 1108–1113. http://dx.doi.org/10.1095/
biolreprod.111.092692
Hannes Botha and Koos de Wet from the Mpumalanga
Heard, D.J., Jacobson, E.R., Clemmons, R.E. & Campbell, G.A., 1988, ‘Bacteremia
Tourism and Parks Agency. and septic arthritis in a West African dwarf crocodile’, Journal of the American
Veterinary Medical Association 192, 1453–1454.
Helmick, K.E., Papich, M.G., Vliet, K.A., Bennett, R.A. & Jacobson, E.R., 2004a,
Competing interests ‘Pharmacokinetics of enrofloxacin after single-dose oral and intravenous
administration in the American alligator (Alligator mississippiensis)’, Journal of
Zoo and Wildlife Medicine 35, 333–340. http://dx.doi.org/10.1638/03-002
The authors declare that they have no financial or personal
Helmick, K.E., Papich, M.G., Vliet, K.A., Bennett, R.A. & Jacobson, E.R., 2004b,
relationships which may have inappropriately influenced ‘Pharmacokinetic disposition of a long-acting oxytetracycline formulation after
them in writing this article. single-dose intravenous and intramuscular administrations in the American
alligator (Alligator mississippiensis)’, Journal of Zoo and Wildlife Medicine 35,
341–346. http://dx.doi.org/10.1638/03-001
Huchzermeyer, F.W., 2003, Crocodiles: Biology, husbandry and diseases, CABI
Authors’ contributions Publishing, Wallingford. http://dx.doi.org/10.1079/9780851996561.0000
Jacobson, E.R., 1984, ‘Immobilization, blood sampling, necropsy techniques and
J.G.M. (University of Pretoria), F.W.H. (University of Pretoria) diseases of crocodilians: A review’, Journal of Zoo Animal Medicine 15, 38–45.
and L.J.G. (University of Pretoria; Medical University of http://dx.doi.org/10.2307/20094678
South Carolina; Hollings Marine Laboratory) planned Lloyd, M. & Morris, P.J., 1999, ‘Phlebotomy techniques in crocodilians’, Association of
Reptilian and Amphibian Veterinarians 9, 12–13.
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(University of Pretoria) and D.G.B. (University of Pretoria) biochemistry of wild Nile crocodiles (Crocodylus niloticus) in the Okavango Delta,
Botswana’, Journal of the South African Veterinary Association 78, 137–144.
were responsible for the clinical work and anatomical http://dx.doi.org/10.4102/jsava.v78i3.305
investigations. Diagnostic imaging was performed by R.M.K. Loveridge, J.P. & Blake, D.K., 1972, ‘Techniques in the immobilization and handling of
the Nile crocodile, Crocodylus niloticus’, Arnoldia 5, 1–14.
(University of Pretoria). L.J.G. and R.H.L. (InoMedic Health
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Applications) taught J.G.M. the blood collection technique. from lizards’, Copeia 2, 338–339. http://dx.doi.org/10.2307/1442973
All authors contributed to the project and the preparation Martelli, P., Lai, O.R., Krishnasamy, K., Langelet, E., Marín, P., Laricchiuta, P. et al., 2009,
‘Pharmacokinetic behavior of enrofloxacin in estuarine crocodile (Crocodylus
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contributed substantially to the final editing of the article. and Wildlife Medicine 40, 696–704. http://dx.doi.org/10.1638/2009-0106.1
Millan, J.M., Janmaat, A., Richardson, K.C., Chambers, L.K. & Fomiatti, K.R., 1997,
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