Artificial incubation techniques in relation to Testudo graeca & T. hermanni with notes on embryonic anoxia as a possible factor in
hatchling mortality in captive breeding programs.
A C Highfield
Incubator Design
The incubators used by the Tortoise Trust and in the Tortoise Survival
Project captive breeding program are based upon the design shown in
plate 1. This comprises a faced chipboard box measuring approximately
18" X 24" X 12". Heat is provided by a 60W industrial quality ceramic
heating element located at one end, and temperature control is provided
by a proportional electronic module which avoids the major fluctuations
experienced with simple on-off type thermostats (Highfield, 1987). The
design is capable of maintaining temperatures to +/- 0.25 degrees
centigrade if required (this facility can prove useful in Environmental
Sex Determination experiments). The probe from the temperature module
is placed at the same height as the egg trays, and a second probe leads
to a digital thermometer located on the front panel. This can include a
high-low warning if required.
Comparative egg morphology - Testudo species
There are considerable morphological differences in the construction of
eggs between various species. These differences are consistent and can
be of use in taxonomic diagnosis (Highfield, in press). For example,
the eggs of the east European Testudo hermanni boettgeri MOJSISOVICS
1889 (Bour, 1987) typically measure some 38mm X 30mm and are markedly
elongate. By comparison the eggs of Testudo ibera PALLAS 1814
typically measure 36mm X 29mm and those of the true Testudo graeca
LINNAEUS 1758 from Oran, Algeria are a diminutive 29mm X 28mm and are
roundish rather than obviously elongate. The more easterly Algerian
Testudo whitei BENNETT 1836 (Highfield & Martin, 1989) produces eggs
which are very much larger and a different shape than those of T.
graeca at 36mm X 28mm. There are similar differences to be seen in the
hatchlings, with true Testudo graeca L. 1758 typically emerging at
27mm/8g, Testudo ibera PALLAS 1814 at 33m/13g, T. hermanni boettgeri
MOJSISOVICS 1889 at 33mm/12g and Testudo whitei at 34mm/14g. Early
phase growth is also variable between species, but very consistent
within species (Highfield, in press). A recently discovered species of
land tortoise endemic to Tunisia lays eggs which are the smallest of
any known terrestrial chelonian at only 15mm long by 13mm broad.
Oxygenation & incubation conditions
One potential problem area often overlooked by those involved in
captive breeding chelonians is the requirement of developing eggs for
oxygen; admittedly this demand is low in the early phase of development
but it does increase (Bellairs, 1969). Where anoxia of eggs is allowed
to occur an increased number of 'dead-in-shell' incidents will be
noted. Embryonic anoxia can also result in hatchlings leaving the egg
early (it seems very probable that a rise in blood Co2 levels may act
as the 'trigger' for emergence). Where eggs are incubated in
conditions where oxygenation and gas-exchange potentials are limited
hatchlings will be seen to leave the eggs in a weak, feeble condition
often bearing unusually large egg-sacs. Incubation in sealed
incubators or in inadequately ventilated containers can drastically
increase both pre-hatching and post-hatching mortalities. It may also
contribute to neo-natal deformities. The practice of incubating eggs
in sealed or almost-sealed containers is therefore to be strongly
discouraged. Conversely, the airflow of most bird egg incubators is far
too high for the successful incubation of chelonian eggs and can easily
lead to excessive drying and even to embryonic dehydration. Infertile
eggs dehydrate most rapidly due too the lack of formation of protective
internal membranes, but even fertile eggs can suffer fatal dehydration
if subjected to extended periods of high airflow. We have found that
if the incubator is opened once per day for a 30 second ventilation
period this is quite adequate to flush any Co2 build-up and prevents
any problems of this nature manifesting.
Hatchling diet
To avoid secondary nutritional osteodystrophy and osteoporosis it is
extremely important that the chemical profile of the diet conforms as
closely as possible to that experienced in the wild. In respect of
Testudo species (and virtually all other herbivorous terrestrial
chelonians) this essentially means that it should be low in protein,
high in fibre, low in fat, rich in minerals and adequate in vitamins.
The Ca:P ratio should be 5:1 or higher. Artificially high protein diets
result in enhanced blood urea levels and subsequent renal problems,
fatty infiltration of the liver (steatitis) and excessive growth of
keratin resulting in severely 'lumpy' and deformed carapaces
(Highfield, 1989). All of these problems are 100% preventable if a
sensible dietary regime is adhered to. Our own hatchlings receive a
diet based largely upon wild plant foods supplemented with additional
green leaf vegetables and fruits. 'Vionate' boosted with additional
calcium is provided at every meal. No meat products (e.g dog food or
cat food) are given either to the hatchlings or the adults - it is
detrimental to their health and development and is not required. The
average protein content of most Testudo species diets in the wild is
circa 4%. Anything consistently in excess of this can and does cause
serious developmental problems.
References:
The incubators are fitted with two lids, one made of heat resistant
transparent plastic and the second of cork-lined plywood. To inspect
the eggs only the outer lid need be lifted, thus preserving temperature
stability during extended periods of observation (but see notes re;
ventilation). The eggs are not buried but rest in plastic ice-cream or
margarine containers on a substrate of sterile peat, light soil and
gravel. A third transparent plastic lid rests gently on each (to
prevent unexpected hatchlings climbing out and injuring themselves).
This is perforated to permit air to reach the eggs.
Humidity is provided by placing a tray of water containing sponges in
the incubator alongside the egg trays. The level of humidity does not
appear to be especially critical with the hard-shelled eggs of Testudo
species, but we usually maintain it at around 70-80%. Each incubator
holds approximately 30 eggs - we prefer to use multiples of these
smaller incubators rather than use fewer larger incubators and
(literally) place all our eggs in one basket!. So far, an incubator
has never failed but it is obviously far safer to distribute any risk
in this way. It also facilitates incubating batches at different
temperatures. As a general starting point, 31 degrees C. is suggested
for most Testudo species.
Whilst it is easier to maintain high levels of humidity in a sealed
environment tests have shown that Co2 levels accumulate rapidly in such
situations. Embryonic anoxia is quite possibly the hidden factor
responsible for many "dead-in-shell" and premature hatchlings
encountered by captive breeders where other explanations (e.g incubator
failure or genetic mismatches) can be discounted.
Where a sealed incubation environment has been used and high levels of
mortality encountered, a change to a more natural and aerated
incubation method will often produce an immediate and dramatic
improvement in survivorship. It is a myth that because eggs are buried
underground they require no oxygen or do not need to ventilate waste
gasses - in fact, soil oxygen levels of nesting sites are usually good
and permeability comparatively high.
Although primarily concerning the incubation of eggs from Testudo
species, most (if not all) of the above observations apply equally to
other species. Obviously soft-shelled eggs frequently require higher
levels of ambient humidity, but the same design of incubator and
incubation environment has proved successful with many Geochelone
species. At present, eggs from Malacochersus tornieri, Chelonoidis
carbonaria and T. horsfieldi are incubating under similar conditions.