Issue No.37 / July 2011

Physiological control mechanisms during late

embryogenesis and during pipping and

hatching

Pr. E. Decuypere & H. Willemsen

Laboratory of Physiology and Immunology of Domestic Animals,

Faculty of Applied Biosciences and Engineering,

Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, B-3001 Heverlee, Belgium.

THE HATCHING PROCESS

Hatching marks the termination of prenatal life in the bird and represents a drastic change from a well-protected

aqueous environment to a more hazardous life outside the egg.

A chick’s emergence from the shell does not occur until the change to pulmonary respiration is complete, the blood

has drained from the chorioallantois, and the yolk sac is normally fully withdrawn into the body cavity.

In birds, as in reptiles, there is a period during development when the chorioallantois and lungs are both functional.

During this period, the parafetal or perinatal period, the onset of breathing probably starts from the time the beak

penetrates the inner shell membrane (internal pipping, IP) which delineates the air cell. The shell over the air cell is

then cracked (external pipping, EP) by the egg tooth on the beak (± 8-9 h after breathing is established in Gallus

domesticus).

Indeed, in most bird species the upper mandible bears near its

tip the egg tooth, a pointed-like structure of keratinous

material, during the later stages of incubation. Occasional

coordinated head and body movements bring the tip of the egg

tooth into contact with the shell. Eventually cracks or “pips” are

produced in the shell.

The first pip is visible on the outer surface of the shell, generally as a raised comical area of small cracks. It is

noteworthy to make a distinction between a cracked shell (or external pipping) and/or outer membrane perforation

since the former can occur without and prior to outer shell membrane perforation, because the mechanical

pressure from the beak is apparently able to pip the shell without rapturing the membrane. The term “external

pipping” is used for a cracked shell and it is often assumed that hypoxia is relieved at the same time. However the

marked hypoxia and hypercapnia which occur at the end of incubation are relieved only when the embryo gains

access to atmospheric air, after penetration of the shell as well as the outer shell membranes, and there may be a

lot of variability in strength of both between species, breeds and individuals.

A temporal relationship, and in that order, between inner membrane perforation, lung inflation, pipping, fully

functional lungs and hatching is often put forward, suggesting at the same time a functional relationship between

internal pipping and lung ventilation. However a careful study of the time relationship between all these events by

EL-Ibiary and colleagues in 1966 showed that in about 35 % of the cases in hatching chickens lung inflation

occurred before outer membrane perforation while in the other 2/3 the lungs were inflated after access to

atmospheric air. If these observations are representative of the normal processes, then the air cell has no causal

relation to lung inflation and that shell cracking does not necessarily occur a certain time after breathing is

established as mostly represented.

The hatching climax is a chick’s escape from the shell at an appropriate stage of development and is characterised

by great activity. The chick makes many coordinated rhythmic movements consisting of vigorous oblique thrusts of

the beak against the shell together with simultaneous strong pressure of shoulder and thorax as well as tarsal joints

against the shell followed by a leftward rotatory movement of the whole body within the shell.

As a consequence, the beak thrusts chip the shell around or circumference thereby separating a cap from the rest

of the shell. When this is realized over 2/3 of the shells’ circumference, the cap is removed by the pushing

movements of thorax and joints, and the chick is able to leave the egg.

These events in the hatching process are dependent on the proper development of the supporting musculature

such as the hatching muscle (M. complexus) and the breast muscle (M. pectoralis).

As the process of pipping and hatching is a drastic changeover from a well-protected aqueous environment to a more

hazardous life outside the egg, which is largely comparable with the process of birth in mammals, it has however

received much less attention. This is to some extent linked with the view that hatching is the chicks’ escape from a rigid

container in which embryogenesis occurs as well as with the fact that hatcheryman, until rather recently, considered the

hatch percentage as the ultimate criterium of success – meaning that the % of dead embryos was the ultimate form of

embryo-hatcheryman communication. Now we realize that we could pay attention to the embryogenesis a little bit earlier

and we are looking for embryo-chick signals in the egg about their stage of development, their performance, their

reaction upon environmental conditions or stimuli, their readiness for postnatal life. Moreover, we realized since long that

the eggshell and membranes are more than a rigid container but a mediating boundary between embryo and

environment through which there is exchange of respiratory gases, conservation of water, prevention of infection, and to

a lesser extent also a heat transfer barrier. For the latter, the boundary layer of still air (depending on wind speed and

ventilation rate) is the major barrier to the exchange of heat. The mechanical properties of eggshells were also probably

subjected to selection such that the functional integrity of the shell was not damaged by cracks resulting from shocks or

pressure generated by the brooding parent during incubation, or were deliberately selected for increased strength in

view of mechanical handling of eggs in poultry production systems.

Among different bird species, different hatching strategies may

be interpreted in terms of the particular mechanical properties of

the shell and membranes from which the chick has to escape

(Bakhuis, 1977).

The most common hatching method, including all domesticated

bird species, is the symmetrical method characterized by rotation

of the chick in the egg during hatching climax, starting from the

initial pip hole. The asymmetrical method is used by a few long-

billed species and involves little or no rotation of the chick in the

egg and produces asymmetrical shell remains.

Parental assistance has been observed in some species, but only as an auxiliary to either the symmetrical or

asymmetrical method (examples are the blackbird, buzzard, flamingo, some owls, tits, a.o.). Megapodes have

developed a unique hatching method in response to their unusual incubation conditions linked with a much reduced

eggshell thickness (1/3 and enlarged pores) compared to eggs of other birds with comparable mass. Here a series

of stretching and tearing movements by the claws, together with chick rotation, is sufficient to break open shell and

membranes and to hatch. Among the species adopting the symmetrical method, there is considerable variability as

to how far the chick turns in the egg during hatching process. Chicks usually start pushing only after the amount of

chipping characteristics of that species has been completed. This is about 180-200°for swan, 200-240°for goose,

240-270° for domestic hen, ducks, pheasants, 300-360° for quail and pigeon. These differences seem to be an

adaptative feature forced on a chick by the mechanical properties of the integument, partly from the shell and partly

from the membranes. This combined integument has been shown tougher indeed for quail and pigeon than that of

eggs from domestic hen and duck (Bond et al., 1988).

FACTORS AFFECTING THE HATCHING PROCESS

Many factors, both endogenous as well as environmental, affect total incubation time as measured by hatching time,

but these factors not merely retard or advance the process but may act differentially as well. In other words, not all

components of these processes are affected in a similar way. Some examples will be given and these studies may

help us to clarify what differential causal mechanisms are stimulatory for the different processes of internal and

external pipping, hatching and of other physiological changes occurring during this crucial transition period.

INCUBATION DURATION (h)

460 470 480 490 500 510

N° OF HATCHED CHICKS

0

2

4

6

8

10

12

14

16

18

20

NON-VENTILATED INCUBATION

VENTILATED INCUBATION

µ = 480

s = 5.36

µ = 496

s = 6.97

A natural build-up of CO2 level due to air tight closure of the

incubator during the first ten days of incubation will result in an

earlier start of the hatching process compared to standard

incubation (De Smit et al., 2006; Witters, 2009). In addition,

Witters (2009) also found a significant interaction between CO2

incubation and age of the broiler breeder. It appears that the

eggs of broiler breeders with an age situated in the optimal

zone profit more from the positive effects of CO2 incubation.

It is known for a long time that storage of eggs before incubation

results in a delayed pipping and hatching time with a linear

relationship between both and a delay of a little less than one hour

per day of storage.

Also, the effect of the age of the broiler breeder has a clear effect on

the hatching process. It has been shown that eggs from a young and

old breeder hatch later than those of a breeder with an age in the

‘optimal zone’ which is situated around 42 weeks of age.

However, there also exist a significant interaction effect between the age of the broiler breeder and storage duration

(Witters, 2009) whereby eggs of young broiler breeders experience the most problems during the hatching process

when stored for up to 17 days before start of incubation. In addition, if we look at the duration of the different

processes of IP, EP and H as a function of storage (3 or 18 days) or age of the breeders (38 or 58 weeks of age) then

it is clear that duration of IP is affected by storage duration while duration of EP is only affected by the age of the

breeders. The first is related to the different levels of pCO2 into the egg, which is lower both at a similar physiological

stage (IP) in the longer stored eggs. The latter is related with different thyroid hormone levels in embryos from young

vs. old breeders. Storage time also influenced chick quality as well as postnatal growth but mechanisms behind are

still unclear.

In general, temperature manipulations during the first week of incubation will shorten the total incubation duration

according to the effect of Van’t Hoff on biochemical reactions. However, in the last week of incubation, this principle

of Van’t Hoff will be overruled by the physiological processes of the embryo making the effect of an increased

incubation temperature on hatching time, dependent on duration, amplitude and period of incubation temperature

manipulation. Experiments in our laboratory have confirmed this hypothesis. When manipulating temperature

continuously (24h/day) with 1°C higher and lower than standard incubation temperature from ED 3 – ED 14 and/or

ED 14 – ED 18, following observations about importantce of period and amplitude of manipulation can be made:

• A higher than standard incubation temperature from ED 3 – ED 14 results in an earlier start of the

hatching process, while from ED 14 – ED 18 no effect was observed. When manipulating temperature from ED 3 –

ED 18, the same shift forward in hatching process occurs as when manipulating during the early part of incubation

• A lower than standard incubation temperature results in an big delay in hatching process (20

hours) when performed from ED 3 – ED 14 and only a slight delay when performed from ED 14 – ED 18. When the

incubation temperature is manipulated throughout incubation (ED 3 – ED 18) the delay is slightly higher than the

manipulation early in incubation.

• Also a higher than standard incubation temperature during the second part of incubation can not

undo the delay in hatching process created by the low temperature treatment during the first half of incubation. In

addition, lowering the standard incubation temperature from ED 14 – ED 18 will reduce the enormous shift forward

of high incubation temperature treatment during the first half of incubation slightly.

Other experiments performed in our laboratory showed that manipulating incubation temperature continuously for

only a few days late in incubation (ED 16 – ED 18.5), the delaying effect is aggravated by the amplitude of the

manipulation. A 2°C lower or higher than standard temperature manipulation resulted in a 3-4 hour delay. A

manipulation of 3°C lower or higher than standard almost tripled this delay to 9-12 hours. In contrast, when

manipulating during the same period and with the same amplitude, but only for 4 hours on each day, the delay in

hatching process was only 1-3 hours.

When comparing different genetic broiler lines (S, E and L) again the line with the shortest duration of

incubation (S) had the highest pCO2 in the air chamber at IP and this was going together with higher corticosterone

as well as T3 levels. This brings us to the chronology of endocrine changes before hatching and their role in the

different physiological processes that take place in the perinatal period as will be developed in the next section.

However, the relationship between age of breeders and incubation duration is confounded with the effects of egg

mass and shell conductivity on this parameter, as both (egg mass and shell conductivity) are a function of the age

of the hens. This was studied in detail by Bamelis (2005).

CHRONOLOGY OF ENDOCRINE CHANGES BEFORE HATCHING

The chick embryo provides a unique system for examining the ontogeny of the endocrine system. Considerable

attention has been focussed on:

• the genesis of endocrine glands, including differentiation and maturation of individual cell types

• development of hormone synthesis and release

• acquisition of endocrine cells or glands of the capability to respond to secretagoques.

• evolution of control systems

• specific endocrine phenomena in the embryo

Since it is impossible to deal with all these aspects in detail within the framework of this talk and topic, I like to refer

to some reviews on the topic by Scanes et al. (1987), Decuypere et al. (1986, 1990), Thommes & Woods (1993).

Immunocytochemical methods have been utilized to study the time of initial appearance of hypothalamic and

hypophyseal hormones during incubation and this is extensively dealed with in the reviews of Scanes et al. (1987)

and Thommes & Woods (1993).

It has to be mentioned that large differences in this respect have been found between precocial and altricial

species. Precocial species (as the chicken) are born at a relatively advanced stage of development and synthesize

their pituitary and other hormones early during development. Altricial species (as the pigeon) on the other hand, are

born at a less developed stage and their hormones are synthesized at a much later time.

Whatever the time of appearance of pituitary hormones, this is occurring prior to the time of establishment of the

hypothalamo-adenohypophysical portal vascular plexus which is established in the chick embryo at day 12 of

incubation. This neuroendocrine unit and pituitary responsiveness matures differentially over developmental time

for the different endocrine axes but is about completely functional 5-6 days before hatching in the chicken.

We will focus in the next section especially on the hypothalamo-hypophyseal-thyroid axis and –adrenal axis

because of their strong and mutual interactive involvement in several perinatal processes such as yolk sac

retraction, lung maturation, pipping and hatching, thermoregulation …

Of course, several other hormonal levels increase rapidly at the end of incubation such as prolactin, mesotoxin

(MT) and agrinine vasotocin (AVT): a possible role in amnion resorption and a causal relation with the observed

decreasing hematocrit values at the end of incubation is suggested (Nouwen et al., 1983). Hormones or factors

related to calcium homeostasis are also changing during the last incubation week such as calcitonin, plasma levels

and CAM-binding of 1,25 (OH)2-cholecalciferol (VitD3), and this when large amounts of calcium enter the embryo

from the shell. These aspects will not be further discussed in the framework of this overview.

In late embryonic development, the plasma concentration of T3

rises dramatically. This increase in plasma T3 occurs considerably

later than that of thyroxine (T4), hence the ratio T3/T4 increases.

This is due partly to a shift from 5 to 5’-monodeiodinase activity in

ovo, but also and even mainly to an inhibition of the T3 degrading

activity.

Indeed, in the chick embryo glucocorticoids, which are also

increasing at the end of incubation, effectively increase plasma T3

concentration by reducing the hepatic T3 degrading activity. The

interaction of the thyroid and adrenal axis also extends to the

hypothalamo-pituitary axis as corticotropin releasing hormone

(CRH) induces an elevation of glucocorticoids but also of

thyrotropin (TSH) and hence raises T4 concentrations which are a

substrate for T3 production. This is illustrated by in ovo injections

of CRH, ACTH and dexamethasone. This mutual interaction

initiates and enhances a number of important physiological

processes in the perinatal period.

INVOLVEMENT OF THYROID HORMONES AND ADRENAL HORMONES IN

PERINATAL PROCESSES

In late embryonic development, the plasma concentration of T3 rises dramatically. This increase in plasma T3

occurs considerably later than that of thyroxine (T4), hence the ratio T3/T4 increases. This is due partly to a shift

from 5 to 5’-monodeiodinase activity in ovo, but also and even mainly to an inhibition of the T3 degrading activity.

Indeed, in the chick embryo glucocorticoids, which are also increasing at the end of incubation, effectively

increase plasma T3 concentration by reducing the hepatic T3 degrading activity. The interaction of the thyroid

and adrenal axis also extends to the hypothalamo-pituitary axis as corticotropin releasing hormone (CRH)

induces an elevation of glucocorticoids but also of thyrotropin (TSH) and hence raises T4 concentrations which

are a substrate for T3 production. This is illustrated by in ovo injections of CRH, ACTH and dexamethasone. This

mutual interaction initiates and enhances a number of important physiological processes in the perinatal period.

These phospholipids will act to reduce the surface tension of the lungs once air breathing has begun and hence

facilitate lung function (Hylka & Doneen, 1982). This surfactant system and its maturation has a certain degree of

plasticity as hypoxia induces a change in the onset and rate of development of surfactant, probably via endogenous

glucocorticoids.

Indeed, both hypoxia as well as a long acting exogenous corticosteroid, dexamethasone are able to accelerate

maturation of the surfactant lipid composition, while exposure to hypoxia during critical developmental windows

(day 16 of incubation) is probably accelerating corticosterone production (Blacker et al., 2004) as conditions with

increased pCO2 in the air chamber also increase corticosterone levels (Tona et al., 2003).

During transition from prenatal to postnatal life also pulmonary vascular resistance is reduced so that blood starts

to flow preferentially to the lungs. This is dependant on the concentration of ornithokinin (the avian counterpart of

mammalian bradykinin) and the balance of factors stimulating its production or its degradation. Thyroid hormones,

hypoxia and probably also corticosteroids play a role in this processes leading to an increased ornithokinin and

pulmonary vasodilation (Decuypere et al., 1990).

We referred already in a previous section to the amion fluid resorption; indeed, at the end of incubation this removal

of the fluid invading the respiratory tract is a prerequisite for breathing and there is some evidence for a role of Prl,

AVT and MT in these embryonic osmoregulatory processes.

Complete retraction of the yolk sac before hatching is also important for the livability as well as for the quality of the

hatched chick. Retraction of yolk sac has been linked with thyroid hormones (Wishart et al., 1977; Decuypere et al.,

1982, 1990).

Glucocorticoids and thyroid hormones are also involved in another way in the preparation for hatching, namely in

the increased blood supply of lungs as well as in the development of the surfactant system in the embryonic lung

in the chicken.

Glucocorticoids trigger the synthesis of surfactant phospholipids, including both phosphatidylcholine and

disaturated phosphatidylcholine by the embryonic lung.

SOME CONSEQUENCES OF DIFFERENTIAL HATCHING

Depending on the spread of the hatching curve together with the place in the sequence of hatching (early or late),

and in interaction with quality of setted eggs, storage, age of breeders ….. there will be a period between hatch and

first feeding of variable length. This may have repercussion on yolk uptake and utilization, development of the

gastrointestinal tract, metabolic level, immune system development and IgG-uptake and overall growth as all these

parameters may be influenced by the time between hatch and first food uptake.

This is related to some crucial hormone levels and enzyme activities for growth that are strongly influenced by post-

hatch food intake, such as insulin and p70S6 kinase activity, a rate-limiting step for protein synthesis.

Several reports have demonstrated that delay in feed intake after hatch adversely affects post-hatch performance

of chicks, especially growth (Pinchasov & Noy, 1993; Gonzales et al., 2003; Bigot et al., 2003).

However, additional factors may aggravate this effect of delay in feed access. Recent data from our research group

conclusively showed that delay in feed access after hatch depresses relative growth rate of chicks, but synergic

effects can be seen when one compares chicks from eggs stored for short vs. long duration before incubation, or

early hatched chicks vs. late hatchers. The magnitude of the effect of delayed feeding is therefore dependent on the

spread of hatching as well as on the hatching period within the hatching window. This may be related to the

different intrinsic quality or characteristics of chicks hatching from short or longer stored eggs, or early vs. late

hatchers as was shown also by their respective hormonal levels. The latter may be a causative factor for the actual

hatching time within the hatching window as well as for the later intrinsic quality of the hatched chick.

This is a factor that has so far been ignored in previous studies and in hatchery practice.

Time

median

early late hatchers

Hatching

Time

median

early late hatchers

Hatching

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