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5. COLLECTION AND INCUBATION OF EGCS

5.1 The Optimum Time for Egg Collection

Experience in the 1970s led some authorities to favor the collection of eggs early in the incubation period. Others preferred to wait until incubation was welladvanced. This difference of opinion, based on experience, can now be explained following detailed studies carried out on alligator eggs (Ferguson, 1981, 1982. Joanen & McNease, 1981) and, more recently, on other species (Webb and others 1985 b).

When the eggs are laid there is a tiny disc on the surface of the yolk. It is from this disc that the embryo will develop. During the 24 hours after an egg is laid the yolk can move inside the egg without damage to the disc but after the first day the disc becomes attached to the eggshell membrane. Movement of the egg after this stage can tear the developing embryo away and ruin the egg.

After about 3-4 weeks of incubation the embryo and its membranes have become strong enough to withstand gentle movement. From these findings one Must conclude that if eggs cannot be collected soon after laying they should be left in the nest for at least 3 weeks or else handled with extreme care.

In practice it will not often be possible to collect eggs from the wild shortly after laying. Thus, in Zimbabwe, it is customary to gather eggs after about 50-60 days of incubation (Blake, 1982). Where it is possible very early collection is preferable in order to avoid losses from predators or flooding.

5.2 Location of Nests

The same nesting sites, at least in several species, are used year after year and may be well known to local people. Nesting females may be located by air survey. In dense vegetation crocodile paths between water and nest are most easily seen from a boat. On known nesting grounds hole nests in sand can be located by probing with a length of wire. In earth the diggings of recent hole nests may be visible. The topmost eggs are commonly about 30 cm below the surface but the writer has seen Nile crocodile nests in very stony earth and on steep banks where eggs have been only a few centimeters below the surface.

Mound nests are conspicuous and can be seen from the air when they are not obscured by trees.

Parent crocodiles, on guard, usually leave the nest at the approach of humans but great care should be taken with dangerous species such as the estuarine and Nile crocodile; they may be close to a nest but well hidden.

Eggs must be shaded from hot sun when a nest is opened.

5.3 Estimating the stage of Incubation from the Eggshell

A freshly laid crocodile egg is a translucent white. Where and when the embryonic disc attaches to the inside of the eggshell a more opaque white spot appears on the outside. As the egg lies on its 'side' in the nest this spot will nearly always be on the upper surface. It has been shown in alligators (Ferguson, 1981, 1982) that in healthy eggs the opaque spot expands to form a band around the middle of the egg. The band gradually gets wider and by day 30 it is so broad that only the ends of the egg remain translucent. Banding is best seen by 'candling' - i.e. shining a light through the egg. (Fig.1). After 45 days the entire egg has become opaque. Hatching occurs in alligator eggs after about 65 days.

A similar pattern of egg banding has been described for the saltwater and Australian freshwater crocodiles by Webb and others (1983a and 1983b).

Infertile eggs do not become banded; they remain translucent. The banding is caused by a combination of factors and is influenced by the development of the embryo.

Expansion of the band towards the ends of the eggs corresponds exactly with the expansion of embryonic membranes which contain blood vessels. The eggshell membrane beneath the band also becomes chalky white and it is known that calcium passes from the shell, through the eggshell membrane and into the embryo.

.Banding can be a useful indicator of development during the early stages of incubation and its mere presence confirms that an egg is fertile. After about 50 days in alligator eggs the eggshell begins to flake away when handled so that the pliable eggshell membrane is exposed. In C. porosus eggs examined by the writer this stage was reached when incubation was about five sixths completed.

In mound nests eggs can become deeply stained by rotting vegetation so that it may be difficult to decide whether eggs are translucent and infertile or fully opaque but not yet flaking. In this case an egg must be broken open.

5.4 Estimating Stage of Incubation from the Embryo

Ferguson (1985) has developed a staging scheme for crocodile embryos in which 28 stages of development are distinguished on the basis of physical features. The stages are related to the age of the embryo in the three species studied (American alligator, saltwater and Australian freshwater crocodile) and the age at each stage is almost identical across the species up to stage 24 - at which point the embryos are fully formed. In the last four stages no new structures appear and the embryos continue their growth at different rates. It seems probable that age and stage will be similarly related in other species .

The staging scheme was, necessarily, worked out under standardized incubation conditions. A temperature of 30C and 90-100% humidity was maintained. The relationship between age and stage of development is affected by incubation conditions so that eggs in outdoor nests may take a few days more (or less) to reach a particular stage of development.

Another practical problem is that the early stages of development can only be seen with a microscope. The same problems exist if embryos are aged according to size although very close relationships have been shown between head length and age/stage of development.

It should be borne in mind that it is easier to estimate laying date from the stage of an embryo than it is to predict the hatching date. This is not only because the later stages of growth are more variable but because hatching is influenced by factors other than embryo completion.

The following notes and drawings describe a few stages as they appear in simple examination with a hand lens. The notes and drawings were made from C. porosus embryos incubated in an outdoor nest at about 29C. The number in brackets, after age in days indicates the developmental stage described in detail by Ferguson.

Less than one week (1-6)

To find an early embryo the eggshell must be very carefully broken away from the upper surface of the egg without tearing the shell membrane. Beneath the shell membrane a blood spot can be vaguely seen on the yolk after a few days of incubation. This marks the position of the embryo.

With care it is possible to cut through the shell membrane and collect the embryo on the tip of a blade but this is much easier to do if the egg has been injected with a fixative. The embryo is actually transparent and about 1cm long.

7 days (7)

Embryo still transparent and very difficult to see but after a week the blood area is obvious and blood islands appear as separate red dots. Possible to lift embryo on tip of blade and float in a spoonful of water. Embryo about 8 mm long in a curved position with prominent heart bulge and limb buds just beginning to swell.

Fig. 1. Opaque banding on American alligator eggs from day laid through day 52 of incubation, most easily seen with light held beneath eggs. From Ferguson (1981).

T - top view
B - bottom view
numerals - stage of incubation in days


Fig 2a C. porosus at 14 days (14 of 92). Length (in position shown) 13.5mm. digits obvious and limbs flexed at elbow and knee

Fig 2b. C. porosus at 28 days (28 of 92) Length (in position shown) 23mm.


Fig 2c. C. porosus at 38 days (38 of 92). Colour pink with faint scale pattern. Length (in position shown) 35mm.

Fig 2d. C. porosus at 49 days (49 of 92). Colour pink with patches of purplish pigment. Total length (extended) 118mm.

 

Fig 2e. C. porosus at 61 days (61 of 92) Colour purplish with darker markings. Large scutes still soft to touch. Total length 16cm.

 

14 days (11)

A streak of blood nearly 2cm long now has obvious blood vessels extending from it like dotted lines. Embryo eye is pigmented and is distinct as a blackish spot about 1.5mm in diameter. The embryo is still extremely delicate and mostly transparent but limb buds are easy to see with a lens. Fig 2a.

24 days (17)

Blood area very elongated with formed blood vessels extending as a network from both ends. Embryo 22 mm long in its natural posture with tail curled. Limbs have begun to bend at elbow and knee. Hands and feet appear as expanded discs with faint outline of fingers and toes. Embryo whitish not transparent. Fig 2b

38 days (21)

Embryo 30mm, long in natural posture. Webbing can now be seen between toes. Body pink in colour with pattern of scutes along back and tail. Sections of brain are distinct and white not transparent. Head flattened not rounded. Upper jaw long and shaped at tip. Jaw and ear regions outlined in white (cartilage). Eye very large with eyelids and nictitating membrane partly formed. Toe nails visible with lens and soft to touch. Shape of muscles on limbs. Fig. 2c

49 days (22)

Purplish patches of pigment on sides and tail which otherwise pink. Brain showing division along length. Heart partly or wholly within body cavity which still open. Scale patterns appearing on limbs. Nails still soft. Egg tooth prominent. Tip of tail kinked. Fig. 2d

61 days (23)

Embryo purplish overall with pattern of adult markings darker and distinct. Scutes of back still whitish and soft. Eyes appear fully formed. Brain can still be seen through thin "window" in roof of skull. Egg tooth hard and points of nails can be felt. Pattern of teeth obvious but teeth can not be felt. Yolk now a solid lump. Fig. 2e

77 days (25)

Colour as for normal hatching, including scutes (non white). Roof of skull firm but not hard - easily depressed at centre.

84 days (26-27)

Yolk mass being taken into belly so that belly wall appears to be pushing outwards to surround it. Teeth erupted and can be felt hard and sharp. Roof of skull firm to touch even at centre.

92 days (28)

Full term. All yolk within body.

5.5 Collecting the Eggs

In Louisiana it has been found convenient to use 19 litre plastic buckets when collecting on foot and 132 litre garbage bins when collecting from boats. In Zimbabwe eggs are usually carried in small styrofoam boxes. Clutches of gharial eggs were successfully collected and transported overland from Nepal to India in stout wooden boxes with screwdown lids. The boxes were packed to capacity with damp sand from the nesting site and were heavy. Obviously, the choice of container is a matter of convenience but it must be rigid and where weight is important a light packing material such as damp vermuculite, peat or rotted leaves should be carried to the site.

As each egg is uncovered it should be marked on the upper surface with a felt pen. It must be lifted very gently and placed, with the mark still uppermost in the box. The eggs should be separated from each other by packing. If an egg is found to be standing on end in the nest it should be laid on its side for transport and subsequent incubation but rotating or jolting must be avoided.

Clutches should either be packed separately or marked differently so that they can be incubated together for uniform hatching. Useful records can also be kept to reveal relationships between clutch size and size of parent, for example, or hatching success and details of nesting/egg collection.

The eggs should be kept damp and temperature changes avoided as far as possible. Plenty of packing material will act as insulation as well as serving to cushion the eggs. Transport to the incubator should be as swift and smooth as can be managed.

5.6 Incubation

Damaged eggs should be discarded at the outset. The requirements for successful incubation are:

- a correct and constant temperature

- high humidity

- eggs correctly oriented and undisturbed.

These factors are most important during the first half of the incubation period. Under these conditions chemical breakdown of the eggshell should proceed naturally as the embryo comes to term. Various constructions and techniques can be used for incubation of collected eggs but they may be categorised according to whether or not a powered incubator is used.

5.6.1 Incubation Using Artificial Nests

Successful incubation can be achieved by copying the natural situation. Eggs of the Nile crocodile have been successfully hatched by reburying clutches in suitable ground. Pooley (1971) has described such a system in which the incubation site was enclosed by wire netting and the clutches were buried in rows. The earth was made damp enough to keep its shape when squeezed. Temperature recordings were taken from the nests shortly after burying the eggs and every 2 hours thereafter, each time adding more soil above the nest, or taking some away as indicated, until the temperature stayed within the required range. Pooley found that the necessary depth from the soil surface to the top layer of eggs depended on soil type. In South Africa it needed to be about 15-20 cm in loam or clay but 30-45cm in fine sand. Soil moisture was checked regularly and water was sprinkled over the nests as necessary to keep the earth damp. Detailed records were kept of each clutch so that every egg could later be accounted for.

In Hyderabad, India, a slightly different technique was used for mugger eggs. Within an enclosure rows of cells were built with walls of loose bricks (i.e. no mortar). Each cell or compartment measured 1 x 1 x 1 m and was nearly filled with sand. A single nest was then dug in each cell (De Vos, 1982).

The nests of mound-building species are easily copied and artificial ones have been used successfully, at least on a small scale, in many parts of the world. The nests of C. porosus are commonly 2 metres or more in diameter and may be about 70-80 cm in height at first but they sink and are compressed to lower mounds during the incubation period. Straw, leaves, leaf litter and mud should be mixed together in building a mound nest.

The temperature in the centre of both mound and ground nests remains remarkably constant. Sand nests receive heat only from the sun but mound nests also generate their own heat (like compost heaps) from the decomposition of vegetation. In both cases material can be added or removed from the nest according to indications from probe thermometers. In both cases the nests must be kept damp, enclosed against predators, safe from flooding and preferably sheltered from the wind.

From artificial nests it is a small advance to placing clutches of eggs in boxes of sand or rotting plant material. The boxes can then be kept on racks inside an incubation room. This occupies less floor space, makes eggs easier to inspect and control at hatching time and offers independence from the vagaries of the weather. A danger is that, because the eggs are less well insulated, their temperatures will. fluctuate unless the incubation room is properly managed with regard to temperature.

An incubation room can be built from wood, brick or concrete block provided that there is ample control over ventilation. Windows and doors (with wire screens) can then be open by day and closed by night as the thermometers direct. The most effective routine should be found by trial and error (with thermometers in boxes of sand and nest material) before eggs are collected. A metal roof, for example, may need to be partially shaded or insulated. A small heater might be needed during cold nights. The aim should be to maintain a temperature of  31 - 32 C as constantly as possible inside the nest boxes - the contents of which must always be damp.

The boxes themselves may be of wood or styrofoam but must be well-drained. Small holes should be drilled in the bottom and sides. Wire mesh containers are also suitable if rotting plant material is used. Indeed, care must be taken to avoid overheating by decomposition in a closed box if the incubation room is warm. Temperatures should always be taken by probe thermometers close to the eggs.

In India, wooden boxes with internal dimensions of 54 x 34 x 34 cm have been found convenient for mugger eggs. Such boxes hold 40 eggs in two layers with 6-8 cm of sand below, between and above the layers. Each layer is made up of four rows of five eggs placed a finger-width apart with a margin of 6 cm between the eggs and the sides of the box (Whitaker & Whitaker 1977). Smaller styrofoam boxes are used in Zimbabwe but they are kept in rooms with a controlled temperature. Where temperature control is difficult or in doubt it is safer to use bigger boxes and more nest material as insulation. On the other hand, if temperature and humidity can be accurately controlled with sophisticated equipment it is possible to dispense with boxes and nest material altogether by using incubators.

5.6.2 Powered Incubators

The advantages of these are that eggs can be observed throughout the incubation period and all conditions are known and precisely controlled. Such sophistication is ideal for research and it has been extensively used in America by the Louisiana Dept. of Wildlife and Fisheries (Joanen & McNease 1976). For the purposes of commercial crocodile farming the requirements have been summarised by Ferguson (1982a):

A reliable supply of electricity is essential and assuming that this is available a laboratory tissue culture incubator is suitable. It may be worth buying 2 or 3 smaller ones rather than a single large model to safeguard against breakdowns and so that eggs can be incubated at slightly different temperatures (see 5.7). Tissue culture incubators are available commercially but there are various designs. Some are more suitable than others for crocodile egg incubation and there is one constraint common to all: they can only be used where the room temperature does not rise above 28C. In other words they can be used as heaters but not coolers. Incubators with a cooling coil work by drawing moisture out of the air inside the incubator and will dehydrate the eggs.

The same problem exists with cabinet incubators for hen eggs. These can be used for crocodile eggs only if they are placed in a cool (eg air conditioned) room where the temperature does not exceed 28C. Crocodile eggs must not be moved so if the turning mechanism (for hen eggs) is automatic it will have to be disconnected. Modern incubators have good air circulation but an extra tray of water may have to be fitted to maintain high humidity. Tissue culture incubators may have no fan - in which case a small fan will have to be fitted to the incubator ceiling and wired through a hole in the top. Good air circulation is essential. In general, water jacketed incubators maintain a more constant temperature than insulated ones.

One large, progressive crocodile farm in Papua New Guinea uses a specially designed walk-in incubator in which the correct temperature and humidity are automatically controlled by a system which blows air through heated/cooled water.

Whatever method is used the basic requirements are the same. Relative humidity should be 90-100% to prevent eggs from losing water and to help in chemical breakdown of the eggshell. Ventilation and air circulation must be adequate so that oxygen can enter the egg and carbon dioxide can leave it. Temperature should be optimal (31-32C) or at least within safe limits (28-34C).

Eggs (felt pen marks up) should be placed in rows on the incubator shelves, touching each other. In this way more than 250 eggs will fit on a square metre of shelf space. There need be no nest material of any kind but eggs which are stained or dirty from the nest must be left as they are, not washed.

Of course, if space permits, nest material can be used inside an incubator and, for alligators at least, the acids from rotting vegetation may help in the chemical breakdown of the shell (Ferguson 1981a). For other species, however, the value of nest material in incubators has been questioned and it can even be detrimental if it becomes sodden and restricts the flow of gases through the egg. The eggs, in effect, can be drowned.

5.7 Determining Sex by Incubation Temperature

It has been demonstrated (Ferguson and Joanen 1982, 1983, Ferguson 1985, Webb and others 1985a) that the sex 0 of crocodiles depends on the temperature of egg incubation. Alligator eggs incubated at 30C or below produce all females whereas eggs incubated at 34C or above produce all males. There is a temperature sensitive period which depends on the "run-in" temperature. For example, in eggs incubated at 30C and then switched up to 34C the temperature sensitive period is around 14-21 days after egg laying. Only during this period does the switch determine sex. At 32C throughout incubation, the sex ratio is approximately 80% female and 20% male.

In C. niloticus and C. porosus high and low temperatures also produce males and females respectively. The Australian freshwater crocodile shows a different response in that females are produced by both high and low temperature and males are produced at intermediate temperatures.

It is a common observation in crocodile farms that males of a species grow faster than females. Research has shown that during the first few months the sexes grow at a similar rate but males then begin to outgrow the females. Given the choice, crocodile farmers would obviously prefer to rear males but there are other factors which complicate the issue.

In Louisiana Joaned and McNease have obtained best results, in general, from alligator eggs incubated at 31-31.7C(Joanen & McNease 1976, 1979,1981).

In more recent studies (Joanen, McNease and Ferguson, 1985) it was found that at 31.7C (89F) the hatchlings were 74% males. Eggs incubated at 32.8C (91F) produced 99% males but these hatchlings grew less rapidly and had a higher proportion of runts 0 than those produced at 31.7C. Moreover, after 18 months, the females produced at 31.7 C were slightly heavier and longer than the males produced at 32.8C. Obviously if these results had been obtained in a commercial situation, the policy of producing all males by incubating at high temperatures would have been wrong. Incubation at 29.4 C (85F) not surprisingly, would have been even worse, resulting in 100% females, slowest hatchling growth and an even higher proportion of runts.

The exact relationship between incubation temperature, sex and subsequent growth is still not clear. Perhaps the best results could be obtained by switching the incubation temperature to the production of males for the critical period but incubating at optimal temperatures for the rest of the time. But without detailed knowledge of the species involved, and without complete confidence in the incubation equipment, the best policy must be to incubate at the optimum temperature which for the species most studied is 31-32 C.

5.8 Hatching

Depending upon temperature, and other factors, incubation time can vary by as much as three weeks. As the time approaches (it will not often be less than 8 weeks) inspections should be made every other day. The first sign that hatching is close will often be the sound of the young calling from inside the egg. If there has been no sign of hatching after the average incubation time I/ one may try patting or scraping at the nest. This may stimulate the young to call. It is a distinctive sound, easy to imitate but difficult to describe - a sort of high-pitched croak. Referring to Nile crocodile eggs buried in the ground Pooley (1971) suggested that after ninety days, if hatching had still not begun, the nests could be opened for an hour in the early morning as a further stimulus to hatching.

It is not advisable to try to induce hatching before the average incubation time has passed. Premature hatchlings are much less robust than full term ones. Hatching is, perhaps, most easily managed with eggs in incubation boxes and soft hay as the incubation medium. In this case a space of about 8-10cm can be left at the top of the box and a cover of fine mesh fitted. The hatchlings can easily emerge and should be left to walk about the box for 24-48 hours to allow the membranes to dry and shrivel. This was the practice adopted by Joanen and McNease with eggs in mesh-covered trays inside environmental chambers. The hatchlings were left in the trays for at least 24 hours. With eggs simply standing on shelves in an incubator, Ferguson (1982a) recommends removing hatchlings as they emerge.

Eggs which do not hatch after others in the clutch have produced normal hatchlings can be left for a week to ten days more. Alternatively, after 70% have hatched (if the young are not premature and all eggs are supposed to be at a similar stage of development) the remaining eggs can be carefully broken open. Ferguson (1982a) recommends the latter practice for eggs in incubators without nest material.


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