Tajda GREDAR & Lilijana BIZJAK MALI: Cultivation and morphology of blood cells... / »SOS Proteus« – FIELD NOTE 
Biotehniška fakulteta Univerze v Ljubljani in Nacionalni inštitut za biologijo, Ljubljana, 2017 
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Cultivation and morphology 
of blood cells of the olm 
Proteus anguinus 
 
Gojenje in morfologija krvnih celic 
močerila Proteus anguinus 
 
Tajda GREDAR & Lilijana BIZJAK MALI, 
Department of Biology, Biotechnical Faculty, 
University of Ljubljana, Jamnikarjeva 101,  
SI-1000 Ljubljana, Slovenia; E-mails: 
tajda.gredar@bf.uni-lj.si,  
lilijana.bizjak-mali@bf.uni-lj.si 
 
Sex identification in Proteus anguinus using 
external morphological characteristics is not 
possible because of the lack of sexual dimorphism. 
We can identify their sex only during the 
reproductive season when mature eggs are visible 
through the non-pigmented skin of females and 
the cloaca is swollen in males. Accurate sex 
identification is crucial for demographic studies in 
natural populations, and would also be important 
for the establishment of a successful captive 
breeding program that could be used for 
evolutionary developmental studies as well as 
conservation measures. 
 
We have optimized a non-destructive method, 
derived from human cytogenetics, to visualize 
chromosomes in order to search for potential 
dimorphism in sex chromosomes (Gredar 2016). 
We initially assumed that P. anguinus has similarly 
heteromorphic X and Y sex chromosomes as its 
closest living relative the North American 
mudpuppy, Necturus maculosus (Sessions 1980). 
Our cell culture approach was based on a 
previously described method used in N. maculosus 
(Seto et al. 1964) involving in vitro cultivation of 
blood cells in primary cell culture, but extensive 
modifications of in vitro cell culturing conditions 
are required. The first step is optimization of 
growth conditions, including the correct choice of 
culture medium and its osmolality, and the optimal 
incubation temperature. It was also necessary to 
determine the optimal concentration and time 
course of treatment with mitogen 
(phytohemagglutinin or. PHA-M) and antimitotic 
(colchicine) for the adequate stimulation of cell 
divisions of lymphocytes in cell culture and 
subsequent arrest in metaphase. We determined 
that the concentrations of mitogen and antimitotic 
in P. anguinus must be at least 3 or 4 times 
higher, with much longer treatment times, than 
are routinely used in human cytogenetics. One of 
our most surprising results is that the cultivation of 
cells was successful at an incubation temperature 
of 25oC indicating that P. anguinus cells can 
tolerate in vitro higher temperatures than 
previously believed. We successfully established a 
viable blood cell culture and were able to visualize 
the chromosomes. However, a recent study in our 
laboratory showed that P. anguinus is 
characterized by a translocation between the X and 
Y sex chromosomes so that both males and 
females have identical-looking chimeric sex 
chromosomes (Sessions et al. 2016). Consequently 
it is not possible to identify their sex 
chromosomally. Nevertheless, the establishment of 
successful cell culture methodologies potentially 
enables other kinds of studies including cell 
biology, cell physiology, genetics, biochemistry, 
toxicology and others in a way that does not 
require sacrificing the animal. This is especially 
important for protected and vulnerable species like 
P. anguinus.  
 
Very little is known about the morphology of blood 
cells and hematological parameters in P. anguinus. 
Therefore we used blood smears for a detailed 
morphological description of proteus blood cells as 
well as for differential counts. The relative 
abundance of different types of white blood cells 
(WBC) can provide important information about 
the physiological condition of animals and, by 
extension, problems with the surrounding 
environment (Allender & Fry 2008). 
 
In almost all Urodeles the erythrocytes (red blood 
cells or RBC) mature in the peripheral blood 
because of lack of bone marrow (Turner 1988). 
The same is true for P. anguinus, and the number 
of erythroblasts, that are immature RBC, is quite 
high (3.9–10.5% of all cells; N = 3) in comparison 
with axolotl (0.1–0.35%; N = 3) (Gredar 2016). 
The RBCs of P. anguinus are relatively large  
(52.7 ± 0.48 μm × 29 ± 0.24 μm; N = 300) and 
are among the largest in amphibians due to the 
large genome size, which is about 15 times that of 
the human genome. RBC sizes in vertebrates show 
a positive correlation with genome size  
(Gregory 2001) and a negative correlation with 
metabolism in animals, thus it is not surprising that 
metabolic rate in P. anguinus is considerably lower 
than that of the most surface dwelling amphibians 
(Hervant et al. 2001). The differential count of 
Tajda GREDAR & Lilijana BIZJAK MALI: Cultivation and morphology of blood cells... / »SOS Proteus« – FIELD NOTE 
NATURA SLOVENIAE 19(1): 29-30 
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leukocytes (WBC) in P. anguinus shows that 
lymphocytes and neutrophils are the predominant 
types (53.5–90.1% and 6.9–37.2%, respectively 
(N = 3)), while the eosinophils and monocytes are 
less numerous (0.0–7.0% and 1.7–3.0%, 
respectively (N = 3)) and basophils are almost 
absent. The low ratio between neutrophils and 
lymphocytes (0.20) indicates that animals were not 
under stress during captivity (Davis & Maerz 
2008). This research was done on a small sample 
size and further studies are needed to obtain 
baseline values of WBC in P. anguinus. Also, more 
analyses are needed for accurately interpreting 
hematological data and for identifying 
abnormalities. Future research will focus on 
deviations from baseline values of the WBC profile, 
which can signal various physiological problems. 
 
References 
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Davis A.K., Maerz J.C. (2008): Comparison of 
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