NL2009256C2

NL2009256C2 - Age determination of avian embryos in ovo.

Info

Publication number: NL2009256C2
Application number: NL2009256A
Authority: NL
Inventors: Wouter Sebastiaan Bruins; Wil Marijn Stutterheim
Original assignee: Wouter Sebastiaan Bruins
Priority date: 2012-07-30
Filing date: 2012-07-30
Publication date: 2014-02-03

Description

AGE DETERMINATION OF AVIAN EMBRYOS IN OVO Field of the Invention

The present invention relates to a process for the determination of the 5 development stage, or age of an avian embryo in ovo, by determining the presence of developmental markers in the egg, more specifically in the allantoic fluid. The present process further refers to a process for the determination of viability of an avian embryo, and the determination of the developmental stage during incubation, and to the production of vaccines 10 and/or chicks using eggs selected according to the process.

Background of the Invention

Incubation of fertilized eggs of most avian species, in particular those reared commercially, such as domesticated chicken (Gallus gallus 15 domesticus), ducks, geese and turkeys tends to take a certain period of time, typically 21 days for chicken, over 28 days for turkeys and ducks, up to about 42 days for ostrich eggs.

In industrial hatcheries, eggs that are laid throughout a period of one to three days are typically collected, and subjected to an incubation process 20 whereby during the final phase of the incubation, typically the last three days, eggs are subjected to heat at a constant temperature and humidity in artificial incubators, to mimic the heat of the breeding hen. However, since eggs from different laying periods are collected to form larger groups of eggs, this results in earlier and later hatching of birds, resulting in the exposure of freshly 25 hatched birds to up to three days under the conditions of the artificial incubators, without access to food or water. Yet further, eggs that are later in their development will likely not hatch during the incubation period, while after the hatching process is completed, unhatched eggs are typically disposed off.

The fact that early hatched birds are subjected to hostile conditions, as 30 well as the fact that late hatchers are typically discarded, results in a strongly reduced efficiency of the hatching and incubation process, as well as to lower quality of the hatched chicks that have survived the incubation.

As a result, an incubation capacity of present chick farms is required that is larger than necessary. If a process was available that would allow to 2 select eggs based on the development of the embryo, the existing capacity could be exploited more effectively. Yet further, the obtained chicks should have a higher quality, leading to a multitude of healthier chicks, and hence a more effective rearing process.

5 Accordingly, it would be of great value for the environment, by reduction of the amount of energy and other resources required if an early stage method was available that allowed to determine the developmental stage of avian embryos prior to the incubation phase, also permitting to strongly increase the capacity of hatcheries.

10 A further benefit would be if the method also permitted to select viable embryos over unfertilized and/or otherwise nonviable eggs, increasing the efficiency of the hatching process further.

Summary of the Invention 15 Accordingly, the present invention relates to a process for the determination of the developmental stage of an avian embryo in ovo, comprising (a) detecting at least a first developmental marker compound selected from amino acids, precursors and metabolites thereof in an egg; (b) measuring the amount of the at least first detected developmental marker 20 compound, and (c) comparing the amount to a base line established for the developmental stage from laying until hatching, to determine the time until likely hatching.

Detailed Description of the Invention 25 The present invention now is described more fully hereinafter with reference to the accompanying drawing, in which a preferred embodiment of the invention is shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure 30 will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology 3 used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "avian" and "bird" as used herein, include males or females of any avian species, but are primarily intended to encompass poultry which 5 are commercially raised for eggs or meat. Accordingly, the terms "bird" and "avian" are particularly intended to encompass chicken, turkeys, ducks, geese, quail, pheasant, guinea, ostrich, pigeon, and grouse.

The term “incubation” refers to the period of the development of an embryo in ovo. The incubation period herein refers to the uninterrupted time 10 during which a particular egg is subjected to conditions emulating the brooding until the hatching, i.e. emergence of the birds, including any handling or transfers from e.g. an incubator to a hatchery unit, provided the development of a bird is not stalled

The overall incubation period may vary from species to species, 15 whereby typically larger species also have a longer incubation period. Typical incubation times range from canary at 13 days, pigeons at 18 days, parakeet at 19 days; different quail species of from 16 days to 23 days; Chicken at 21 days, pheasant, prairie chicken and grouse at 23 days, guinea at 27 days, ducks and turkeys at 28 days; Peafowl at 29 days, muscovy duck at 36 days, 20 geese at 32 to 35 days, to ostrich at 42 days.

The term "in ovo" as used herein, refers to bird embryos contained within an egg prior to hatch. The present invention may be practiced with any type of bird egg, including, but not limited to, (domesticated) chicken, turkey, duck, goose, quail, and pheasant eggs.

25 The terms "injection" and "injecting" herein encompass methods of inserting a device (typically an elongate device) into an egg or embryo, including methods of delivering or discharging a substance into an egg or embryo, methods of removing a substance (i.e. a sample) from an egg or embryo, and/or methods of inserting a detector device into an egg or 30 embryo.

The term "allantoic fluid" herein encompasses allantoic fluid with or without the presence of other egg materials. For example, the term allantoic fluid may include a mixture of blood and allantoic fluid. Embodiments of the present invention are not limited to extracting material from the allantoic fluid or from 4 areas near the upper surface of an egg. Removal of material from the allantoic fluid as described herein is provided as merely one example of possible embodiments of the present invention. Various materials including but not limited to amnion, yolk, shell, albumen, tissue, membrane and/or blood, may 5 be extracted from an egg and assayed to identify one or more developmental markers, as described below. Material may be extracted from eggs having virtually any orientation.

The term "predetermined location" herein indicates a fixed position or depth within an egg. For example, a device may be injected into an egg to a fixed 10 depth and/or fixed position in the egg. In alternative embodiments, the injection may be carried out based on information obtained from the egg, e.g., regarding the position of the embryo or the subgerminal cavity within the egg. Processes and apparatus according to embodiments of the present invention may be utilized for identifying one or more characteristics of an egg at any 15 time during the embryonic development period, also referred to as the incubation period thereof. Embodiments of the present invention are not limited to a particular day during the embryonic development period.

In the present process the at least first developmental marker compound is detected in an egg material, preferably an embryonic fluid of an 20 egg, more preferably the allantoic fluid.

Preferably, at least a second marker is also detected in step (a), and the at least first and second markers are analysed and compared to the base line, and to each other to establish a developmental marker ratio. More preferably, at least a first and a second developmental marker are detected 25 and analysed, and wherein the absolute amounts and the ratio of the at least first to second markers is employed to determine the developmental stage.

Preferably, the avian species is Gallus gallus domesticus, and the developmental markers are selected from Trimethylglycine; Aspartate and/or Asparagine; Glutamate and/or Glutamine, and/or Proline.

30 In the present process, the developmental markers may preferably be analysed invasively or non-invasively.

In the present process, the developmental markers may preferably be analysed invasively or non-invasively.

5

If the analysis is performed invasively, this typically includes the extraction of a sample of egg material. The sample is preferably taken from an embryonic fluid, preferably from the allantoic fluid, since this will least likely harm the embryo. The allantoic fluid typically is an excretory medium for the 5 nitrogenous metabolites of an avian embryo. ” The allantoic fluid begins to form around Day 3 of incubation, as disclosed by Hamburger, V and Hamilton, HL (1951). "A series of normal stages in the development of the chick embryo". Journal of Morphology 88 (1): 49-92.

Herein is indicated that the allantois was distinguishable at 65 hours 10 after incubation, as a short, thick-walled pocket; not yet vesicular. After 72 hours, the allantois was vesicular, variable in size; on the average of the size of the midbrain, indicating that the allantois and the allantoic fluid are present as of day 3.

It attains a maximum volume on day 13 of incubation and then wanes 15 in volume as incubation continues dues to moisture loss and fluid resorption, but is still present in significant volumes on day 18 of incubation.

The allantoic fluid is separated from the eggshell by the inner and outer shell membranes and the chorioallantoic membranes. Although the allantoic fluid encompasses the entire periphery of an embryonated egg, it typically 20 accumulates at the top of an egg directly underneath the membranes overlying the air cell.

The accumulation of the allantoic fluid at the top of the egg is due to gravity and displacement by the dense embryo and yolk sac. Attempting to accurately sample the allantoic fluid through the top of an egg while the egg is 25 upright may be difficult due to the variability of the air space from egg to egg. Gravity can be used to pool the allantoic fluid in a localized site. When an egg is turned on its longitudinal axis, the allantoic fluid will pool at the top side of the egg, directly underneath the shell. Laying the egg on its longitudinal axis renders the allantoic fluid useful for extraction of a sample.

30 The extraction of material, such as allantoic fluid, from eggs may be performed in various ways, including penetrating the egg shell, and inserting a sampling cannula trough the membranes. A sample of the fluid to be sampled may then be retrieved, while the membrane is actively sealed, or allowed to seal itself.

6

Suitable methods and apparatus for the penetration of eggs and invasively sampling of egg material are disclosed for instance in US-A-20070137577, WO-A-OO/22921 or WO-A-99/34667. The sample is then preferably subjected to a suitable protocol to permit the detection of the 5 developmental markers, and an analysis of the relative and/or absolute amounts of developmental markers present.

The sample may be analysed by any method suitable to detect and to quantify the developmental marker or markers. Preferably, the analysis is performed by a magnetic resonance imaging method including nuclear 10 resonance methods; spectral resonance methods including infrared or Raman spectroscopy, and/or analytical methods such as GC or HPLC coupled with suitable detectors, fluorescence spectroscopy, and/or enzyme-linked immunosorbent assay, including wet and dry methods, such as using a dipstick method. While the invasive methods permit to take a sample directly, 15 and to subject the sampled fluid to an analysis, preferably the analysis is performed non-invasively due to the efficiency of such analysis method, and to the fact that the eggshell and membranes therein remain imperforated.

Any suitable method may be employed to perform such non-invasive analysis. Typically, quantitative spectral resonance methods including infrared 20 or Raman spectroscopy may be employed, preferably using secondary spectra for the determination of the presence and absolute and/or relative amounts of developmental markers present in an egg. While several publications have disclosed the use of non-invasive methods, e.g. US-A-2011/144473 and US-A-7950349, these publications only vaguely describe 25 overall emission spectra; which in practice do not permit to select the development stage the viability and/or the gender of an embryo. The present process differs in particular from the disclosed methods in that the presence of specific components in the egg is determined, which may advantageously be done by using secondary derivative spectra that allow to selectively seek for 30 the absolute and relative amounts of one or more developmental marker compound(s).

In particular differential second-derivative Fourier transform infrared (FTIR) and FT-Raman spectroscopy, or combination thereof may advantageously be employed to achieve the necessary accuracy and 7 repeatability, while nuclear magnetic resonance methods may suitably be employed to determine the nature of the developmental makers, and to establish a base line to calibrate the system.

The developmental markers according to subject invention preferably 5 are selected from amino acids, and their respective metabolites and/or precursors.

Applicants found that in particular the absolute and relative amounts of amino acids, more preferably Trimethylglycine, also known as Betaine, Aspartate and Asparagine, Glutamate and Glutamine and Proline could be 10 directly related to the developmental stage of an avian embryo in ovo. This is highly relevant, since there are few features that allow determining the developmental stage, or age, of an avian embryo, in particular at early stages of the development.

The present process permits to select eggs of essentially the same 15 developmental stage, or actual age, and to combine a multitude of eggs of essentially the same age, to subject the eggs to a controlled incubation. The resulting hatching period should be more uniform, resulting in a more uniform chick population, and reducing the number of early hatched bird that are subject to prolonged exposure to the artificial incubation conditions.

20 The resulting hatched bird population will show a higher quality, resulting in a higher yield, and reduced follow-up requirements for the chick population, e.g. through reduced amounts of treatments.

Similarly, the use of age selected populations of eggs will also permit to prepare vaccines more efficiently, since herein the developmental stage of an 25 avian embryo is relevant for the point in time when most efficiently a virus is injected, and a vaccine harvested.

Preferably the determination is performed at a period of from 1 to 15 days, more preferably of from 2 to 14, yet more preferably of from 3 to 13, and even more preferably of from 4 to 12 days after the incubation is started. This 30 permits to avoid the costs involved in incubating eggs that are either no viable and/or not the desired gender. Furthermore, the actual developmental stage of an egg can be determined. For species with shorter or longer incubation times than those of domesticated chicken, other periods may apply, as suitable.

8

The present process advantageously also allows to determine whether an embryo is viable. Yet further, the present process advantageously also allows to determine whether an embryo is male or female. Either of these determinations is performed by selecting appropriate development markers, 5 e.g. sugars, amino acids and precursors, and/or derivatives thereof, but also sex related hormones such as for instance those disclosed in US-A-2003/009631. Preferably, though, the appropriate development markers are selected from sugars, amino acids, and precursors, and/or derivatives thereof.

Furthermore, preferably the subject process also preferably permits to 10 detect the viability and/or gender of an embryo in ovo.

For gender analysis, applicants found that developmental makers of particularly importance included Glucose, Choline and Valine, each of which had a statistically significant influence on the determination of the gender of the avian embryo.

15 Without wishing to be bound to any particular theory, Choline and ,

Trimethylglycine, its amino acid derivative, are considered to be particularly used to support the foetus’s developing nervous system. It was found that the Choline and Trimethylglycine (Betain) ratio differs strongly between male and female embryos, while also the absolute amounts of Choline were higher in 20 the allantaic fluid of female embryos. Generally, Choline and its metabolites are needed for three main physiological purposes: structural integrity and signaling roles for cell membranes, cholinergic neurotransmission (acetylcholine synthesis), and a major source for methyl groups via its metabolite, Trimethylglycine (Betaine) which participates in the S-25 adenosylmethionine (SAMe) synthesis pathways. Valine and Glucose on the other hand were also found to vary significantly between male and female embryos.

Where the avian species is Gallus gallus domesticus, preferably a first or further developmental marker is glucose in absolute amount for a female 30 embryo in the range of from 30 pm/ml to 70 pm/ml, and for a male embryo of from 1 pm/ml to 30 pm/ml in the allantoic fluid.

A further first or further preferred developmental marker for Gallus gallus domesticus embryos is Choline, in an absolute amount for a female 9 embryo tin the range of from 110 μηη/ml to 130 μηη/ml, and for a male embryo of from 90 μηη/ml up to, but not including 110 μηη/ml, in the allantoic fluid.

Yet a further first or further developmental marker for Gallus gallus domesticus embryos is preferably is Valine, in an absolute amount for a 5 female embryo in the range of from 110 pm/ml to 130 pm/ml, and for a male embryo of from 90 pm/ml up to, but not including 110 pm/ml, in the allantoic fluid.

Preferably, in the subject process at least a second marker is detected in step (a), and wherein the at least first and second markers are analysed 10 and compared to the base line, and to each other to establish a developmental marker ratio.

By correlating the analysis of two or three markers, the selectivity of the determination of viability and gender may advantageously be improved further. Accordingly, preferably at least a first and a second and/or further 15 developmental marker are detected and analysed, wherein the absolute amounts and the ratio of the at least first to second and/or further markers is employed to determine the gender and/or viability.

The present process further advantageously comprises determining whether an embryo in an egg is viable and male, or viable and female, and 20 separating a multitude of viable male eggs from a multitude of viable female eggs, to form a predominantly male or predominantly female egg selection.

The thus formed viable female or male egg selections may advantageously be subjected to an artificial incubation and hatching process to form a predominantly female or male chick population of essentially the 25 same age.

The present process further preferably comprises injecting a virus or virus-like material into each egg identified as containing a live embryo and male or female of essentially the same age, and preferably, after incubation comprises isolating the obtained vaccine from the incubated eggs.

30 After injection with a seed virus, the eggs containing live embryos are preferably transferred to an incubator for a predetermined period of time. At the end of this period of time, the eggs are transferred to a vaccine harvesting station where material from each egg, e.g. amniotic fluid, is extracted.

10

Accordingly, the present process preferably also comprises the steps of euthanizing an embryo in the infected egg, and harvesting amniotic fluid from each euthanized egg, wherein the amniotic fluid comprises vaccine; and preferably isolating a vaccine from the amniotic fluid. Preferably the virus 5 comprises human influenza virus, and hence the harvested amniotic fluid comprises human influenza vaccine.

The present invention also relates to egg selections, and after hatching, to a chick or a chick population obtainable by the process, having essentially the same age.

10 The following, non-limiting examples are provided to illustrate the invention.

Example 1

Protocol metabolic profiling in ovo

Gallus gallus domesticus eggs were incubated at 37.8°C, turning every 15 hour, in an incubator from MS hatching machines, model 50.

One group of 12 eggs was incubated for 9 days, a second group of 12 eggs for 10 days, and a third group of 12 eggs for 11 days.

Eggs were taken out of the incubator, placed in a paper holder, under a microscope, with the air sack up. The shell and membranes were punctured 20 and broken open around the air sack, leaving the inner membranes intact. Using light, the blood vessels running over the inner shell membrane were located, and a small puncture avoiding the blood vessels was made through the inner and outer membranes into the allantoic cavity.

The egg was skewed and a 1ml pipette was then used to blow air into 25 the cavity, after which 1.5 to 2 ml of allantoic fluid was extracted using the pipette. This was transferred into a cryovial, which was immediately plunged into liquid nitrogen. The samples were then taken out and stored in -80°C.

The embryo was taken out of the egg, by cutting away the membranes and by using a small spoon. It was put in a falcon tube filled with 96% ethanol 30 and on ice and stored in a dark place at room temperature.

The allantoic fluid was taken out of -80°C, defrosted and a sample of 1 ml was taken out and put in a glass vial.

1 ml of chloroform and 1 ml of a mixture of methanol and water (1:1) were added to the sample, using glass Pasteur pipettes. The vials were 11 closed using a cap and then shaken for 20 seconds, then placed at 4°C for 10 minutes. Using a glass Pasteur pipette, 1ml of the upper part of the mixture was taken out and transferred to a cryovial. The cap of this cryovial was punctured and it was freeze dried overnight. This freeze dried product was 5 employed as NMR sample.

Gender determination - Verification:

The chicken embryo was taken out of the ethanol and left out to dry for 10 minutes at room temperature. A small portion of the left leg was cut off and this was used to extract DNA, using a DNA extraction kit (commercially 10 obtainable as Qiagen DNeasy kit), after which the amount was measured using a nanodrop device.

PCR using primer pair 1272H and 1237L, commercially obtainable acquired from Sigma, was used to determine the gender of the embryo. The PCR program used was, 95°C for 5 minutes, then 36 times 95°C for 45 15 seconds, 56°C for 45 seconds, 72°C for 1 minute, after which one run was done at 72°C for 5 minutes.

The gender of the embryo was determined based on the resulting PCR product, identified by using a 2% agar gel.

NMR Sample preparation 20 50-100 mg of sample material obtained as described above were subjected to two-dimensional (2D)- 1H-1H -J-resolved NMR measurements, using 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP) as an internal standard, as disclosed in Nature Protocols, Vol.5, No.3, 2010, pages 536-549, and Phytochemistry 71,2010, 773-784.

25 The obtained data is depicted in Table 1: 12

Table 1: Measured Data for Days 9 to 11 9 days (μΜ/mL,

Standard

Development deviation in 10 days

Marker brackets)) (μΜ/mL) 11 days (μΜ/mL)

Trimethylglycine (Betaine) 0.75(0.27) 0.65(0.2) 0.43(0.13)

Aspartate and

Asparagine 9.35(3.2) 7.72 (2.75) 5.34(3.91)

Glutamate and

Glutamine 41.5(7.71) 37.1 (3.94) 31.7 (5.33)

Proline 12.7(2.69) 11(1.58) 9.02 (1.59)

Multivariate data analysis A the partial least square modelling, an unsupervised multivariate data 5 analysis, was employed for the 1H NMR data to group samples based on all the metabolites detected in 1H NMR. The most important information obtained was the correlation between two data sets, i.e. the measured 1H NMR signals (metabolites) and the sample classification (group information). The outcome was that the above developmental markers were all statistically significant.

10 The analysis not only revealed the absolute amounts of developmental markers for the developmental stage, but also the relative amounts. When a second, third and fourth marker were added, respectively, the selectivity of the test increased further.

The examples above clearly show the advantages of the process and 15 materials of the present invention.

Although several specific embodiments of the present invention have been described in the detailed description above, this description is not intended to limit the invention to the particular form or embodiments disclosed herein since they are to be recognised as illustrative rather than restrictive, 20 and it will be obvious to those skilled in the art that the invention is not limited to the examples.

Claims

A method for determining the developmental phase of a bird embryo in an egg, comprising: a) detecting at least a first developmental marker compound selected from amino acids, precursors, and their metabolites in an egg; b) measuring the amount of the at least one first detected development marker compound; and c) comparing the amount with a reference line established for the development phase, from egg laying to hatching, to determine the age of the embryo, as well as the time until the egg is likely to hatch . 15

The method of claim 1, wherein the at least first development marker compound is detected in an embryonic fluid of an egg, preferably the allantoic fluid.

The method of claim 1 or claim 2, wherein at least one second marker is detected in step a), and wherein the at least first and second markers are analyzed and compared with the reference line, as well as mutually compared to determine a development marker ratio. set.

The method of any one of claims 1-3, wherein the bird species is Gallus gallus domesticus and wherein the developmental markers are selected from trimethylglycine, aspartate and / or asparagine, glutamate and / or glutamine, and / or proline.

5. Method as claimed in any of the foregoing claims, wherein at least a first and a second development marker are detected and analyzed, and wherein the absolute amounts and the ratio of the at least first to the second marker is used to determine the development phase.

6. Method as claimed in any of the foregoing claims, comprising the step of performing an invasive or non-invasive analysis of the embryonic fluid.

The method of claim 6, wherein the analysis is performed by a magnetoresonance method, including nuclear resonance methods; spectral resonance methods including infrared or Raman spectroscopy, and / or analytical methods such as GC or HPLC in combination with suitable detectors, fluorescence spectroscopy and / or enzyme-linked immunosorbent assays.

8. Method as claimed in any of the foregoing claims, furthermore comprising selecting the eggs from substantially the same development phase, as well as subjecting the selected eggs to incubation and hatching in accordance with the development phase, in order to obtain a chick population with essentially the same age.

9. Method as claimed in any of the foregoing claims, further comprising determining whether an embryo in an egg is viable or not, as well as separating the non-viable eggs.

10. Method as claimed in any of the foregoing claims, further comprising determining whether an embryo is male or female, and separating a plurality of male eggs from a plurality of female eggs, in order to produce a substantially male or substantially female to form egg selection.

The method of claim 9 or claim 10, further comprising injecting a virus or virus-like material into each egg that has been determined to contain a male or female living embryo, in accordance with the developmental stage of the embryo.

12. A method according to claim 11, further comprising isolating the obtained vaccine from the incubated eggs.

A chick or chick population, obtainable by a method according to any one of the preceding claims.