Gender and fertilisation status determination of eggs

Abstract

Disclosed is a method of determining in-ovo gender and / or fertilisation status from an egg. The method comprises optically exciting the egg with light from a laser source; measuring autofluorescence of the egg over a period of time; calculating a fluorescence lifetime indicative of a decay of the autofluorescence over the period of time; and determining the in-ovo gender and / or fertilisation status based on the fluorescence lifetime. The fluorescence lifetime is an intrinsic property of the egg and indicative of the gender and / or fertilisation status of the egg. As such, determining the in-ovo gender and / or fertilisation status based on the fluorescence lifetime provides an accurate and non-invasive method for determining the in-ovo gender and / or fertilisation status.

Description

Gender and fertilisation status determination of eggsCross-Reference to Related Applications

[0001] The present application claims priority from Australian Provisional Patent Application No 2024904228 filed on 19 December 2024 and Australian Provisional Patent Application No 2025901983 filed on 22 May 2025, the contents of which are incorporated herein by reference in their entirety.Technical Field

[0002] This disclosure relates generally to determining in-ovo gender and / or fertilisation status from an egg.Background

[0003] Various industries rely on the gender of an animal for numerous aspects within the industry. One industry, in particular, is the poultry industry, where different segments of the industry sex chickens for various reasons. Prior to the development of modern broiler meat breeds, most male chickens were slaughtered for meat, whereas females would be kept for egg production. However, once the industry bred separate meat and egg-producing hybrids, there was no reason to keep males of the egg-producing hybrid. As a consequence, the males of egg-laying chickens are culled as soon as possible after hatching and sexing to reduce losses incurred by the breeder.

[0004] In the current poultry industry, all hatchery eggs are incubated, and chicks are sexed a few days / weeks after hatching when it is possible to determine gender. This occupies valuable hatchery resources and is very labour-intensive. From an industry perspective, incubating male eggs is a significant waste of resources. The subsequent culling of male chicks after hatching is a significant animal welfare concern for the industry and broader community.

[0005] Further, in the poultry industry, some eggs may be received which are unfertilised and it can be nearly impossible to determine whether an egg has been fertilised non-invasively. As such, both the fertilised and unfertilised eggs are incubated, and one must wait a few days before being able to determine whether an egg has been fertilised. Common practice is to wait until day 16 to check fertilisation status and any deformities. Similar to incubating male eggs, incubating unfertilised eggs is a waste of resources such as hatchery space, electricity and workforce.

[0006] There is a need for a non-invasive method for chicken egg sexing and determination of fertilisation status at an early stage of incubation before onset of embryo sensitivity. More specifically, there is a need for a non-invasive method that can determine gender and / or fertilisation status before incubation of the egg (i.e., day 0). Such a non-invasive method would not disturb or destroy the blastoderm (before incubation) / embryo (after incubation) from developing.

[0007] Moreover, such a non-invasive method would align with animal welfare priorities, such as ‘sustainable production’ of ‘chick welfare’, by greatly reducing the extent of culling of male chicks and ‘improvements in production systems’ through reduced waste in costs / time in hatching male chicks or incubating unfertilised eggs. Therefore, many benefits would result from the ability to sex eggs to determined fertilisation status, not only for hatcheries but for the industry in general. The procedure would reduce the cost of rearing chickens by 50% which in turn would reduce labour and feed expenses.

[0008] In particular, such a method would reduce the carbon footprint of the poultry industry by 50% by reducing the electricity and waste. For example, waste can be reduced by selling male eggs for vaccine production as the virus can be grown in the yolk of fertilised hens eggs. Also, the mental health of poultry industry workers (involved with gender separation, culling, shredded waste, for example) will be significantly improved, as well as public image of the industry as a whole.

[0009] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

[0010] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.Summary

[0011] According to the present disclosure, there is provided a method of determining in- ovo gender and / or fertilisation status from an egg, the method comprising: optically exciting the egg with light from a laser source; measuring autofluorescence of the egg over a period of time; calculating a fluorescence lifetime indicative of a decay of the autofluorescence over the period of time; and determining the in-ovo gender and / or fertilisation status based on the fluorescence lifetime.

[0012] It is an advantage to determine the in-ovo gender and / or fertilisation status based on the fluorescence lifetime as the fluorescence lifetime is an intrinsic property of the egg and indicative of the gender and / or fertilisation status of the egg. As such, determining the in-ovo gender and / or fertilisation status based on the fluorescence lifetime provides an accurate and non-invasive method for determining the in-ovo gender and / or fertilisation status.

[0013] In some embodiments, optically exciting the egg with light from the laser source and / or determining the in-ovo gender and / or fertilisation status occurs prior to incubation of the egg.

[0014] In some embodiments, the laser source is a low power laser source.

[0015] In some embodiments, calculating the fluorescence lifetime comprises calculating the fluorescence lifetime for multiple ranges corresponding to multiple respective components of the egg, and determining the in-ovo gender and / or fertilisation status is based on the fluorescence lifetime corresponding to a blastoderm component.

[0016] The method expresses the measurement in a parameter, but more importantly it clears the contribution of the other parts of the egg. The method can list the autofluorescence of all parts of the eggs in the order which is wavelength dependent and finding the best possible parameters to get the signature of the blastoderm. Otherwise, the signature may be hidden bellow the other parts.

[0017] In some embodiments, the fluorescence lifetime is a shorter component of the autofluorescence of the egg.

[0018] In some embodiments, determining the in-ovo gender is based on a difference in fluorescence lifetime between male and female blastoderm indicative of a difference in expression of proteins.

[0019] In some embodiments, measuring the autofluorescence comprises detecting photons emitted from the egg over the period of time.

[0020] In some embodiments, detecting photons emitted from the egg over the period of time comprises time-tagging the photons emitted from the egg by collecting the photons arriving on a detector at multiple time intervals over the period of time.

[0021] In some embodiments, calculating the fluorescence lifetime comprises fitting a double-exponential decay model to the photon collection at the multiple time intervals over the period of time, the fluorescence lifetime corresponding to a decay rate of the exponential decay model.

[0022] In some embodiments, the period of time is between 10 and 30 seconds.

[0023] In some embodiments, the laser source has a power below a photothermal damage threshold of the egg.

[0024] In some embodiments, the power of the laser source is between 50 pW to 0.5 mW.

[0025] In some embodiments, the light from the laser source is visible or near infrared (NIR).

[0026] In some embodiments, the light from the laser source has an optimal wavelength of between 580 to 610 nm.

[0027] In some embodiments, the measuring autofluorescence of the egg over a period of time comprises using one or more emission filters to filter desired wavelengths of the autofluorescence, the one or more emission filters being between 450 and 700 nm long-pass optical filters.

[0028] In some embodiments, the light from the laser source comprises light pulses with a pulse width of between 1 to 10 ps and a pulse frequency of between 10 and 30 MHz.

[0029] In some embodiments, the light from the laser source has a bandwidth of between 1 and 20 nm.

[0030] In some embodiments, the in-ovo gender determined is male upon the fluorescence lifetime being within a male range; or female upon the fluorescence lifetime being within a female range, wherein the male range and the female range are non-overlapping.

[0031] In some embodiments, the in-ovo fertilisation status determined is fertilised upon the fluorescence lifetime being within the male range or the female range; or unfertilised upon the fluorescence lifetime being outside of the male and the female range.

[0032] In some embodiments, the male range and the female range are dependent on the wavelength of the light from the laser source.

[0033] In some embodiments, the wavelength of the light corresponds to green light, wherein the male range is from 0.45 to 0.67 ns; and the female range is from 0.77 to 0.93 ns for brown eggs.

[0034] In some embodiments, the wavelength of the light corresponds to orange light, wherein the male range is from 0.53 to 0.65 ns; and the female range is from 0.77 to 0.93. ns for brown eggs.

[0035] In some embodiments, the method further comprises repeating the steps of optically exciting the egg with light from the laser source and measuring autofluorescence of the egg over a period of time for multiple locations on the egg, wherein the fluorescence lifetime is based on an average of the repeated measurements for egg components.

[0036] In some embodiments, the method further comprises excluding measurements originating from an egg shell of the egg, inner shell and white of the egg and / or a yolk of the egg from the repeated measurements before determining gender.

[0037] In some embodiments, excluding measurements from the repeated measurements comprises excluding measurements below 0.7 ns.

[0038] In some embodiments, the egg is a chicken egg.

[0039] In some embodiments, the method further comprises placing the egg in a horizontal position and rotating the egg to move the blastoderm of the egg towards the egg shell.

[0040] In some embodiments, the method further comprises, placing an objective relative to the egg where a long axis of the egg is tilted off the optical path of the objective and repeatedly performing the steps of exciting, measuring, calculating and rotating the egg or the objective about its long axis to acquire multiple measurements.

[0041] In some embodiments, the egg is placed in a blunt-end up position and the multiple measurements are acquired along a circle on the blunt end of the egg centred in the long axis of the egg.

[0042] In some embodiments, an objective is placed above the egg to illuminate from above the egg, and collect emission from above the egg targeting the blastoderm in the egg positioned blunt-end up.

[0043] According to the present disclosure, there is provided software for non-invasive determining in-ovo gender and / or fertilisation status from an egg that, when installed on a computer and executed by the computer, causes the computer to perform the method as hereinbefore described.

[0044] According to the present disclosure, there is provided a system of non-invasive determining in-ovo gender and / or fertilisation status from an egg, the system comprising: a laser source configured to emit light to optically excite the egg; a detector configured to measure autofluorescence of the egg over a period of time; and a processor configured to: calculate a fluorescence lifetime indicative of a decay of the autofluorescence over the period of time; and determine the in-ovo gender and / or fertilisation status based on the fluorescence lifetime.

[0045] Optional features provided in relation to the method, equally apply as optional features to the software and the system.Brief Description of Drawings

[0046] An example will be described with reference to the following drawings:

[0047] Fig. 1 illustrates a system for determining in-ovo gender and / or fertilisation status from an egg.

[0048] Fig. 2a illustrates a method for determining in-ovo gender and / or fertilisation status from an egg.

[0049] Fig. 2b illustrates the difference between egg components and whole egg.

[0050] Fig. 3a show an egg at day 1 of incubation.

[0051] Fig. 3b shows the egg at day 3 of incubation.

[0052] Fig. 3 c shows the egg at day 5 of incubation.

[0053] Fig. 4 illustrates an example system corresponding to the experimental setup of the confocal microscope.

[0054] Fig. 5a shows a representative 200 pm x 200 pm confocal image of washed embryo deposited on a glass slide.

[0055] Fig. 5b shows the fluorescence decay traces obtained and fitted to extract lifetime components for the white square marked regions of interest of Fig. 6a.

[0056] Fig. 6a shows shell and inner membrane of an egg on a glass slide prepared for imaging.

[0057] Fig. 6b shows a near-field confocal image of the outer shell.

[0058] Fig. 6c shows a near-field confocal image of the inner shell.

[0059] Fig. 7a shows the average fluorescence lifetime 1of a day 2 extracted and washed embryo - green laser excitation with 80 pW power used, where= 0.91±0.06 ns.

[0060] Fig. 7b shows the average fluorescence lifetime 2 T2of a day 2 extracted and washed embryo - green laser excitation with 80 pW power used, where T2=6.0±1.0 ns.

[0061] Fig. 8a shows lifetime 1 of brown shell eggs at day 0 of incubation-8 eggs.

[0062] Fig. 8b shows lifetime 1 of brown shell eggs after five days of incubation- 17 eggs.

[0063] Fig. 9 shows a comparison between optical measurements (lifetime) and PCR testing for eggs incubated for 5 days.

[0064] Fig. 10 shows a schematic of the final stage of the egg layer approach.

[0065] Fig. I la shows 200 pm x 200 pm confocal scans of the egg.

[0066] Fig. 1 lb shows the fluorescence decay traces were obtained for the cross marked regions of interest of Fig. 15a.

[0067] Fig. 11c shows lifetime values of the first component determined from the exponential decay, for 10 different fluorescent spots.

[0068] Fig. l id shows lifetime values of the second component determined from the exponential decay, for 10 different fluorescent spots.

[0069] Fig. 12 shows schematic of lifetime of individual egg components indicating their relative position for an excitation wavelength of 532 nm.

[0070] Fig. 13 shows transmission of the shell (brown egg) as a function of wavelength.

[0071] Fig. 14a shows the lifetime measurement of shell only.

[0072] Fig. 14b shows the lifetime measurement of all eggs layers.

[0073] Fig. 15 shows a schematic of lifetime of yolk, shell, male blastoderm and all eggs layers indicating their relative position for an excitation wavelength of 532 nm.

[0074] Fig. 16 shows a schematic of lifetime of individual egg components indicating their relative position for an excitation wavelength of 490 nm.

[0075] Fig. 17 shows a schematic of lifetime of individual egg components indicating their relative position for an excitation wavelength of 600 nm.

[0076] Fig. 18 shows a schematic indicating blastoderm position for the non-incubated eggs in the blunt-end up position.

[0077] Fig. 19 shows a schematic indicating blastoderm position for the non-incubated egg in the horizontal position.Description of Embodiments

[0078] This disclosure provides a non-invasive method of early-stage determination of the gender of fertilized eggs as well as the fertilisation status of an egg, using the egg’s intrinsic fluorescence signature. More specifically, using the intrinsic fluorescence lifetime of the egg, and more particularly, the intrinsic fluorescence lifetime of the blastoderm. The experiments disclosed herein demonstrate that the gender of an egg can be determined as the fluorescence lifetime falls within a specific and non-overlapping range for male and female. The blastoderm in eggs refers to the layer of cells formed during the early stages of embryonic development. It is a disc-shaped structure located on the yolk's surface and plays a role in the differentiation and development of the embryo. In fertilized eggs, the blastoderm contains cells that will develop into the embryo, while in unfertilized eggs, it remains inactive. In this disclosure, the term “blastoderm” refers to the component of the egg before incubation that will then form into the embryo. The term “blastoderm” is therefore used interchangeably with the term ’’germinal disk”. It is further noted that the term “gender” is used interchangeably with “sex” in this disclosure.

[0079] Importantly, it has been shown in the experiments disclosed herein that the fluorescence lifetime of the pre-incubated eggs (i.e., day 0) can be used to determine the in- ovo gender and hence, fertilisation status. As such, the gender and / or fertilisation status can be determined before incubation (i.e., day 0). This provides many benefits to the animal industry and would enable such industries to conform with animal welfare principles. For example, male chicken eggs can be sold for vaccine production rather than discarded, which minimises the waste of male chicken eggs.

[0080] This disclosure provides confocal imaging methods to measure egg autofluorescence, to produce a specific intrinsic fluorescence signature that can identify gender of the blastoderm and also decern contributions from other parts of the egg. In particular, the experimental results disclosed herein show that, at day 0, the blastoderm inside the egg has developed enough such that its intrinsic fluorescence lifetime is distinguishable between maleand female blastoderm / embryos. The difference in intrinsic fluorescence lifetime between male and female blastoderm is due to the differences in proteomes between each gender.

[0081] A proteome is the entire set of proteins that is dynamic and changes over time. The disclosed method is based on the difference in intrinsic fluorescence lifetime between male and female blastoderm that is due to the differences in at least 35 proteins between each sex. The disclosed method confirms maternal sex-allocation theory: “During the process of (from meiosis I to) fertilization, the sex was depended critically on Z or W chromosome in haploid oocytes (Chromosome- Assisted Sex Identification, CASI) and the molecular components (proteome and lipids) of matrix in vitellus were intelligently manipulated or identified by the hens (SA) (Uller et al., 2009). 2) During the formation of (from fertilization to) shell egg, zygote cells begin to grow, divide and partially slightly differentiate (SD); more importantly, it is worth mentioning that the contribution of sex-allocation, egg white, and shell were selfassembled and wrapped on the surface of vitellus according to its sex, gradually increasing until shell egg was formed (SA). 3) The effect of SA has disappeared completely during the period of incubation (from fertilized eggs to chicks) and the contribution of CASI and SD gradually increase over time.” (X. Xiang, et al, 2022 Poultry Science).

[0082] Moreover, while other parts of the egg can inhibit the detection of the fluorescence lifetime of the blastoderm, the experiments disclosed herein show that the fluorescence lifetime of the blastoderm can be measured accurately from outside the egg. This enables gender and / or fertilisation status determination without extracting the blastoderm from the egg-

[0083] The techniques described herein provide a fluorescence emission recording, without the need to use dyes and proteins for labelling, or any other chemical processing. Moreover, the disclosed method can be performed using low power laser excitation, well below the threshold for photothermal damage, making the disclosed method non -invasive. Further, all measurements can be performed at room temperature and the low laser power is consistent with low-cost laser diodes.

[0084] The disclosure provides a simple to implement method with a very fast turnaround time from sampling to result. In particular, using this methodology, it is possible to record fluorescence within minutes from sampling. Previous systems require complex technologythat requires a technician to use and analyse the results. Using a technician can be time consuming, expensive and logically difficult, particularly if the hatcheries are located remotely. The fast turnaround time also provides an optimised allocation of hatchery resources, which saves time and costs.

[0085] The disclosed system and method can also be employed with multiple parameter measurements, such as confocal images, intensity of emission, spectra, and lifetime. This provides a distinct advantage over other identification systems as, while bioimaging data can be derived from confocal images, identification based on images is insufficient and time- and skilled labour consuming. Moreover, the disclosed system and method can be integrated into a device, which simplifying chickening sexing in hatcheries, such as by providing a single digit display. One of the major advantages over other imaging techniques is that the disclosed system and method does not rely on image-based recognition, which would always include an expert. The disclosed system and method may provide a parameter (i.e., a number) to determine gender and / or fertilisation status, rather than relying on visual recognition.

[0086] From the experimental results described herein, gender determination of brown shell chicken egg layers has been demonstrated by optical fluorescence lifetime measurements. The shorter component of the lifetime (lifetime 1) of male blastoderm is significantly different from that of female blastoderm even prior to incubation (day 0) at all excitation wavelength considered, from blue to red laser excitation. Furthermore, lifetimes for all parts of egg (e.g., shell, yolk, blastoderm) were measured at different excitation wavelengths and their relative position to those of the male and female blastoderm were analysed. There is no overlap in the values of lifetime between different parts of the fertilized egg recorded. This feature of distinct values enabled a search for the optimal wavelengths at which the male or female optical signature was recorded and identified with 90 % certainty.

[0087] While disclosure is primarily directed towards to the application to chicken eggs, it is noted that the disclosed system and method is not limited to chicken eggs. The disclosed system and method are also applicable to other shell-based eggs, such as other poultry eggs. For example, the disclosed system and method can be used to determine the in-ovo gender and / or fertilisation status from turkey, geese and duck eggs. Moreover, the disclosed system and method are applicable to shell-based eggs other than poultry, such as reptile eggs.

[0088] The disclosure is also applicable to other reproductive material such as ovas, embryos, blastocyst or the like, that is derived from an animal. In particular, this includes in vitro reproductive materials, such as reproductive material that is prepared and / or fertilised in a petri dish. This is because, similar to shell-based eggs, such reproductive material also have an intrinsic fluorescence lifetime that can be used to determine gender and / or fertilisation status.System

[0089] Fig. 1 illustrates an example system 100 for determining in-ovo gender and / or fertilisation status from egg 101. Fig. 1 is one example of a configuration of system 100. However, system 100 is not strictly limited to this configuration and this may be one possible embodiment of system 100. In some embodiments, egg 101 may be a chicken egg, such as a white shell or a brown shell egg, for example. However, egg 101 is not limited to a chicken egg and may be a duck, fish or platypus egg, for example.

[0090] System 100 comprises a devices unit 102, consisting of a processor 103, which may be smartphone, computer or similar device. Processor 103 is connected to a time correlation unit 104 and a data acquisitioninterface 105. Processor is further connected to program memory 120 and data memory 121. Processor 103 receives data through all these interfaces, which includes memory access of volatile memory, such as cache or RAM, or non-volatile memory, such as an optical disk drive, hard disk drive, storage server or cloud storage. The program memory 120 is a non-transitory computer readable medium, such as a hard drive, a solid state disk or CD-ROM.

[0091] The system 102 includes a time-correlated single photon counting (TCSPC) module 104, implemented via a time correlation card and a data acquisition interface DAQ 105. The TCSPC module 104 may comprise a PicoQuant, TimeHarp 260, https: / / www.picoquant.com / products / category / tcspc-and-time-tagging-modules / timeharp- 260-tcspc-and-mcs-board-with-pcie-interface. The interface 105 processes a portion of the detected fluorescence signal, for example approximately 50%, to generate a spatial fluorescence intensity map corresponding to the surface of the sample. It is to be noted that the data interface 105 provides the data as a voltage signal on a physical bit line and processor 103 receives the fluorescence measurements via a memory interface.

[0092] The remaining percentage of the fluorescence signal is directed to the TCSPC module 104 for lifetime computation. The TCSPC module 104 records the temporal relationship between excitation laser pulses and detected fluorescence photons. Software, that is, an executable program stored on program memory 120 causes processor 103 to perform methods for determining in-ovo gender and / or fertilisation status from an egg or parts thereof. For example, once executed, the software causes processor 103 to receive measurements of autofluorescence of egg 101 over a period of time, calculate a fluorescence lifetime indicative of a decay of the autofluorescence over the period of time and determine the in-ovo gender and / or fertilisation status based on the fluorescence lifetime.

[0093] Upon each excitation event, the TCSPC module 104 assigns a precise time tag to individual photon arrivals relative to the corresponding laser trigger. These time-stamped photon events are compiled into a histogram representing photon counts as a function of delay time after excitation. The resulting fluorescence lifetime data are processed to extract optical characteristics of the egg or its components, and the processor 103 is further configured to determine the in-ovo gender and / or fertilisation status based on the fluorescence lifetime. The data memory 121 may store autofluorescence measurements or fluorescence lifetimes for later use. For example, processor 103 may receive autofluorescence measurements and store the autofluorescence measurements on data memory 121. Processor 103 may then calculate a fluorescence lifetime by retrieving the autofluorescence measurements from data memory 121, performing the calculation then may store the fluorescence lifetime on data memory 121. The autofluorescence measurements and fluorescence lifetimes may be stored on data memory 121 in the form of a XML file, JSON file or another equivalent data file. It is to be understood that any receiving step may be preceded by processor 103 determining or computing the data that is later received. For example, processor 103 may then store the fluorescence measurements on data memory 121, such as on RAM or a processor register. Processor 103 then requests the data from the data memory 121, such as by providing a read signal together with a memory address. The data memory 121 provides the data as a voltage signal on a physical bit line and processor 103 receives the fluorescence measurements via a memory interface.

[0094] System 100 further comprises a laser source 106 (e.g., fibre coupled laser source) which is configured to emit light 107 (represented graphically as grey bold lines withoutarrows). More specifically, laser source 106 is configured to emit light 107 to optically excite egg 101 and cause egg 101 to fluoresce (i.e., under spontaneous emission and emit photos). In some examples, laser source 106 may be, but not limited to, a Nd:YAG laser or a heliumneon laser.

[0095] In some embodiments, laser source 101 has a power below a photothermal damage threshold of egg 101. For example, the power of laser source 106 may be between 50 pW to 0.5 mW, which is well below the photothermal damage threshold of chicken eggs. However, the power of laser source 106 is not limited to this range and may be within a range that is well below the photothermal damage of other eggs, for example. Light 107 from laser source 106 may be visible or near infrared (NIR). More specifically, light 107 may have a wavelength of between 400nm and 1mm. Light 107 may also have a set bandwidth.

[0096] Processor 103 via DAQ 105 may establish a communication with laser source 106 using I / O port 108. In one example, processor 302 sends instructions to laser source 106 via I / O port 108 to emit light 107. Although I / O port 108 is shown as single entity, it is to be understood that any kind of data port may be used to receive data, such as a network connection, a memory interface, a pin of the chip package of processor 103, or logical ports, such as IP sockets or parameters of functions stored on program memory 101 and executed by processor 103. The parameters of functions may be stored on data memory 121 and may be handled by-value or by-reference, that is, as a pointer, in the source code.

[0097] Preferably, laser source 106 is a pulsed supercontinuum laser (for example, Fianium, WhiteLase SC400, NKT Photonics USA, https: / / www.nktphotonics.com / products / supercontinuum-white-light-lasers / superk-fianium / ). As such, light 107 from laser source 106 may be pulsed laser light, in the sense that light 107 is not in a continuous mode, but rather in the form of optical pulses (in other words, light flashes or light pulses). A set number of pulses with a set pulsed width may be emitted from laser source 106. For example, light 107 from laser source 106 may light pulses with a pulse width of between 1 to 20 ps and a pulse frequency of between 1 and 50 MHz.

[0098] System 100 further comprises detector 109 configured to measure autofluorescence 110 (represented graphically as black bold line without arrows) of the egg over a period of time. The detector 109 is an Avalanche Photodiode (APD) in the red to near infraredwavelength range, which can be used to measure the fluorescence intensity in units of photons detected per second. The Single Photon Avalanche Diodes (SPADs) may be a PicQuant PDM Series (https: / / www.picoquant.com / products / category / photon-counting-detectors / pdm-series- single-photon-avalanche-diodes) for example. However, detector 109 may be any other equivalent photodetector or a combination of photodetectors.

[0099] Device 102 further comprises another I / O port 111, enabling the device 102 to establish a communication with detector 109. I / O port 111 may have the same input and output functionally as I / O port 108. Thus, in some examples, the I / O port 108 and I / O port111 may be the same port, although they are drawn as distinct entities in Fig. 1. In some examples, I / O port 111 may be a wireless transmitter and / or receiver, that is configured to send and receive data.

[0100] Software may provide a user interface presented to the user on device 102. The user interface is configured to accept input (via buttons or text fields etc.) from the user, via a touch screen or a device attached to device 102 such as a keyboard or computer mouse. These devices may also include a touchpad, an externally connected touchscreen, a joystick, a button, and a dial. In an example, device 102 may display multiple instances of fluorescence measurements, and the user may choose one of the multiple instances of fluorescence measurements, by processor 103. The user may choose one of the multiple instances of fluorescence measurements by interacting with the touch screen or inputting the selection with a keyboard or computer mouse.

[0101] System 100 further comprises an optional neutral density filter (attenuator) to adjust the laser input power and an objective lens 112 to focus light 107 on egg 101. Objective lens112 may be set to a specific focus. System 100 further comprises dichroic 113 (such as a dichroic mirror) which filters autofluoresence 110 by blocking the excitation wavelength from interfering with the collection arm, before being detected. System 100 further comprises emission filters 114, 115. In some examples, emission filters 114, 115, may be selected for every excitation mode of autofluorescence 110 of egg 101. It is noted that each emission filter 114, 115 may correspond to a different excitation mode of autofluorescence 110. Further, while only two emission filters are depicted in Fig. 1, there may be one or more emission filters. System 100 further comprises pinhole 116, which spatially filters autofluoresence 110by acting as a low-pass filter for spatial frequencies in the image plane of autofluoresence 110.

[0102] It is noted that system 100 of Fig. 1 is only meant to illustrate an example and a preferred system which is capable of performing the disclosed method. Many other configurations of system 100 may equally perform the disclosed method.Method

[0103] Fig. 2a illustrates method 200 for determining in-ovo gender and / or fertilisation status from an egg. Fig. 2a, or part thereof, is to be understood as a blueprint for the software program and may be implemented step-by-step, such that each step in Fig. 2a is represented by a function in a programming language, such as Python, C++ or Java. The resulting source code is then compiled and stored as computer-executable instructions on program memory 104, which causes processor 103 to perform method 200.

[0104] In method 200, laser source 106 optically excites 201 egg 101 with light 107. More specifically, light 107 causes egg 101 to fluoresce by providing enough energy to the electrons of egg 101 to move from a ground state to an excited state. The electrons in the excited state then return to the ground state by undergoing spontaneous emission, thereby emitting autofluorescence 110 as photons. It is noted that the excited electrons may return to the ground state via intermediate energy states between the ground state and the excited state. As such, egg 101 may emit light 107 which corresponds to many photons of varying wavelengths. It is noted that autofluorescence 110 corresponds to the natural emission of light by biological structures as a result of absorbing light (e.g., light 107).

[0105] After egg 101 is optically excited 201, detector 109 measures 202 autofluorescence 110 of egg 101 over a period of time. In some examples, detector 109 measures autofluorescence of egg 101 by detecting photons emitted from egg 101 over the period of time. More specifically, detector 109 measures autofluorescence of egg 101 by time-tagging the photons emitted from egg 101 by counting the photons arriving on detector 109 at multiple time intervals over the period of time. The period of time may be any length of time but preferably is between 10 and 30 seconds. The multiple time intervals can be any length oftime shorter than the period of time, but preferably divides the period of time into equal intervals.

[0106] After detector 109 measures 202 autofluorescence 110 of egg 101 over a period of time, processor 103 receives the autofluorescence measurements. These measurements may correspond to time-resolved photoluminescence (PL) decay traces, for example. Processor 103 then calculates 203 a fluorescence lifetime indicative of a decay of the autofluorescence over the period of time. Processor 103 may calculate 203 the fluorescence lifetime using the autofluorescence measurements received from detector 109 and the period of time. For example, processor 103 may calculate fluorescence lifetime based on an average of the autofluorescence measurements.

[0107] In some embodiments, processor 103 calculates 203 the fluorescence lifetime by applying a mathematical operation, formula or algorithm to the autofluorescence measurement. For example, processor 103 may fit a model to the autofluorescence measurement and the fluorescence lifetime may be based on the result of the model. Such a model may be an exponential decay model. However, other methods can be equally applied.

[0108] An exponential decay model is an optimal model to use for calculating 203 the fluorescence lifetime, as the fluorescence lifetime is indicative of a decay of the autofluorescence over the period of time. In particular, the fluorescence decay can be modelled using the following bi-exponential equation:where a is a fitting constant, b is an amplitude, c is a short lifetime, d is an exponential fitting constant, e is an amplitude, f is a long lifetime and At is the time difference plotted on the horizontal axis (corresponding to time t). The fluorescence lifetime may be the short lifetime (as referred to as the shorter component of the fluorescence lifetime), the long lifetime (as referred to as the longer component of the fluorescence lifetime) or a combination thereof. Preferably, the fluorescence lifetime is the shorter component, as this provide greater accuracy of the in-ovo gender and / or fertilisation status, as will be shown experimentally later in the disclosure.

[0109] Finally, processor 103 determines 204 the in-ovo gender and / or fertilisation status based on the fluorescence lifetime. For example, there may be a one or more ranges that the fluorescence lifetime falls within for egg 101 to be classified as male, female, fertilised or unfertilised, noting that being classified as male or female means that egg 101 will be also classified as fertilised. Such one or more ranges may be determined experimentally. Preferably, the range for male and the range for female are non-overlapping, enabling a clear distinction between the in-ovo gender. This is indeed the case as will be discussed in the experimental sections. Having non-overlapping ranges for male and female means, intuitively that, if the fluorescence lifetime were to fall within either range, then egg 101 can be classified as fertilised. Similarly, if the fluorescence lifetime falls outside both ranges, then egg 101 can be classified as unfertilised. As such, both the in-ovo gender and fertilisation status can be determined from the same fluorescence lifetime.

[0110] Optically exciting 201 egg 101 and / or determining the in-ovo gender and / or fertilisation status may occur prior to incubation of egg 101. This enables one to incubate only the desired eggs, such as the female eggs. However, method 200 is applicable to an egg that has undergone incubation. It is shown experimentally later in the disclosure that both preincubated (i.e., day 0) eggs and incubated eggs, up to day 5 after incubation, show a distinctive fluorescence lifetime that can be used to determine the in-ovo gender and / or fertilisation status.

[0111] In one example, the ranges for lifetime values for male and female eggs is determined based on measurements of egg components, that is by breaking the egg, extracting / isolating the components and measuring the components separately. This may also be referred to as a calibration phase. This may involve multiple repeated measurements of each component to establish an average range. When the whole egg is tested (evaluation phase) in a non-invasive way, a single measurement can be used without averaging, which significantly improves throughput. Fig. 2b illustrates the difference between the calibration phase on the left and the evaluation phase on the right. In the calibrationphase, the steps of optically exciting 202 egg components 101 and measuring 202 autofluorescence of egg components are repeated over a period of time for multiple locations on egg components, such as by measuring 10-30 spots. Hence, the fluorescence lifetime may be based on an average of the repeated measurements. For example, processor 103 may calculate afluorescence lifetime for each of the repeated measurements and then calculate an average. In other examples, processor 103 applies a mathematical formula or model to calculate the fluorescence lifetime from the repeated measurements of the components.

[0112] During the evaluation phase, in which the whole egg is analysed, the steps of optically exciting 202 the egg 101 and measuring 202 autofluorescence of egg component 101 can be performed only once. With the ranges from the component measurement, the values for non-blastoderm components can be excluded and the fertilization status and sex determined from the value of the blastoderm. For example, processor 103 may order the lifetime values to extract the different components and then use the value for the blastoderm from male or female range.

[0113] As such, in some embodiments, processor 103 may exclude measurements originating from an egg shell of the egg, and / or a yolk of the egg from the repeated measurements in the determination of the fluorescence lifetime. In further embodiments, only the determination of a female may be desired and hence, processor 103 may exclude measurements originating from an egg shell of the egg, a male blastoderm of the egg, and / or a yolk of the egg from the repeated measurements in the determination of the fluorescence lifetime.

[0114] In some embodiments, processor 103 determines the in-ovo gender is male upon the fluorescence lifetime being within a male range; or female upon the fluorescence lifetime being within a female range. The male range and the female range are non-overlapping, which is shown experimentally. Similarly, in some embodiments, processor 103 determines the in- ovo fertilisation status is fertilised upon the fluorescence lifetime being within the male range or the female range; or unfertilised upon the fluorescence lifetime being outside of the male and the female range.

[0115] It is noted that the consideration of the individual spot lifetime (the lifetime from one of the multiple spots) is not an average measurement but an individual value for the whole egg measurement. This significantly reduces the computational complexity and hence, the evaluation time, which increases throughput when used in an industrial operation.

[0116] In some embodiments, the male range and the female range are based on the wavelength of the light from the laser source. For example, the male range and the female range are dependent on the wavelength of the light from the laser source. This is because different excitation wavelengths may excite different fluorophores. As such, the male and female range are dependent on the excitation wavelength. However, from the experimental results, there is not a significant change in the male and female range from different excitation wavelengths. Moreover, as shown experimentally, the male and female ranges are independent of the power of laser source 106.Experimental setup

[0117] Experiments were performed in two stages, where the methodology and results for each stage are reported later in the disclosure. Stage 1 focussed on the blastoderm only, while Stage 2 focussed on all layers of the egg. Stage 3 investigated determination of the gender and fertilization status of whole fertilized eggs in a non-invasive manner before incubation. Each stage used the following techniques and configurations to perform the fluorescence lifetime measurements. These measurements were performed using in-house built confocal microscope which used a pulsed supercontinuum laser (Fianium, WhiteLase SC400, NKT Photonics USA).

[0118] For Stage 1, an excitation wavelength of = 532 nm, emission filters (575 LP), bandwidth (BW) 10 nm were selected, and the laser beam was focused on the sample (i.e., the egg of part thereof) with a 100x0.9 NA objective lens. The fluorescence from the sample was filtered with a green (532) dichroic to block the excitation wavelength from interfering with the collection arm, before being detected. The power was in the range of 50 pW to 500 pW respectively.

[0119] For Stage 2, excitation wavelengths of = 490, 532, 580, 600, 625 nm were used with a bandwidth (BW) of 10 nm. Different emission filters were selected for every excitation mode including the 500 LP, 575 LP, 633 LP and 650 LP. The laser beam was focused on the sample with a 100x0.9 NA objective lens. For some measurements, laser beam was focused on the sample with a 40 x NA objective lens. The fluorescence from the sample was filtered with a dichroic to block the excitation wavelength from interfering with the collection arm,before being detected. The power was in the range of 50 pW to 200 pW respectively, well below the threshold for photothermal damage.

[0120] For Stage 3 excitation wavelengths of = 532, and 580 nm were used with a bandwidth (BW) of 10 nm. Different emission filters were selected for every excitation mode including the 575 LP, and 633 LP. The laser beam was focused on the sample with a 100x0.9 NA objective lens. For some measurements, the laser beam was focused on the sample with a 40x NA objective lens. The fluorescence from the sample was filtered with a dichroic to block the excitation wavelength from interfering with the collection arm, before being detected. The power was in the range of 100 pW to 200 pW respectively, well below the threshold for photothermal damage.

[0121] For all three stages, time-resolved photoluminescence (PL) decay traces were obtained by correlation card (Picoquant, TimeHarp 260). The repetition rate for the supercontinuum laser was set to 20 MHz with pulse width of 6 ps. Fluorescence decay traces were obtained by time-tagging the photon arrivals following the end of each pulse and averaging the signal over a total integration time of 30 s. The decay traces were then fitted to a stretched exponential decay model to extract the fluorescence lifetime. About 10 to 20 different points from various spots of each sample were collected. The confocal measurements take only few minutes and therefore this technique is highly applicable for real-time measurements. Note that samples were investigated at room temperature, without any chemical processing, pre-treatment, fixing, or staining of the samples.Stage 1 methodology and results (Blastoderm only)Aims

[0122] In this stage, an optical methodology to differentiate the gender of chicken eggs, non- invasively, based on their intrinsic fluorescence was investigated. Using confocal microscopy, the fluorescence properties of chicken eggs were investigated by measuring both the emission spectrum and fluorescence lifetime, rather than just intensity. This permitted the observation of clear differences between eggs that may enable fast identification with no post-processing required. Extremely small features can be differentiated by optical imaging, not only in confocal but in other microscopic methods. However, identification based on images isinsufficient and time- and labour consuming. Selectivity can be maximized by detecting specific optical signatures in spectra of single species. Further discrimination between different features is achieved using fluorescence lifetime measurements in addition to spectroscopic signatures.

[0123] In particular, this stage investigated and compared the optical properties of classes of fertilized eggs at different stages, from day 0 (prior to incubation) and day 1 to day 5 of incubation. For practical reasons, gender determination on day 0, prior to incubation, is the most preferred option because of the practical and ethical benefits. The first step in optical measurements involved investigating intrinsic properties of eggs using existing confocal microscopy. In the second step, the spectral difference between different samples was established. The third step involved lifetime analysis of fertilized eggs determining gender. In the last step, the samples were subjected to PCR testing measurements, to validate the results obtained using optical measurements.Materials and MethodologyEggs

[0124] Fertilized eggs supply

[0125] Fertilized eggs were supplied by Australian SPF Services Pty Ltd, Cadello, Victoria and by Specialised Breeders Australia (SB A), Kamarooka hatchery, Victoria. The first batch of 50 eggs were purchased from SPF were white leghorn with white shells. The second batch of 60 eggs supplied by SB A were Hy-line Brown with brown shell.

[0126] Egg handling

[0127] One fifth of eggs were immediately subjected to optical measurements at day 0 (as received). The remainder of the eggs were incubated over different durations, 1, 2, 3, 4 or 5 days. The incubation was performed in an automatic egg incubator (Rcom Maru 190 Deluxe) at 37°C and a humidity of 45 %, and eggs were turned every hour.

[0128] At day 0, 1, 2, 3, 4 or 5, the eggs were subjected to optical measurements followed by PCR measurements. The eggs were cracked by hand and the shells, egg white, egg yolkand the inner membranes were separated. The blastoderm for day 3 and onward was separated from the egg yolk using forceps under the portion and sharp pointed scissors to cut away the rest of the membrane. The smaller blastoderm for day 0 - 3 eggs were extracted via syringe. All blastoderms were transferred to glass slides and washed well with PBS prior to optical imaging.

[0129] Fig. 3 shows eggs at different days of incubation. Fig. 3a show an egg at day 1. Fig. 3b shows the egg at day 3. Fig. 3c shows the egg at day 5.

[0130] Confocal microscopy

[0131] It is noted that the term ‘ ‘ autofluorescence” used in this disclosure refers to the intrinsic fluorescence of biological compounds, as opposed to the fluorescence obtained by treating samples with exogenous fluorescent labels, dyes, and tags. Confocal microscopy systems can used to measure this intrinsic fluorescence. The confocal systems used in Stage 1 and Stage 2 are substantially different from the commercially available confocal microscopes. For example, the nanomaterials are not tagged, rather the intrinsic fluorescence from materials is relied upon. However, it has been shown that commercial microscopies can also be used in the disclosed method, provided the tagging step is avoided.

[0132] Near-field imaging

[0133] Fig. 4 illustrates example system 500 corresponding to the experimental setup of the confocal microscope. It is noted that system 500 is similar to system 100 of Fig. 1. However, detector 109 uses beam splitter 501, which splits autofluorescence to be received by DAQ 105 and time correlation card 104DAQ 105 is connected to device 503, which is configured to calculate and provide confocal scans of egg 101, while device 504is configured to calculate and provide the fluorescence lifetime of egg 101. It is noted that devices 503, 504may be the same device, despite being shown as separate entities in Fig. 4.

[0134] Confocal fluorescence scanning was performed using a customized confocal microscope. A 532 nm continuous wave frequency doubled Nd: YAG laser was used for sample illumination, through a 100, 0.95 NA objective lens. The samples on silicon (Si) and cover glass substrates were mounted on a computer-controlled stage facing the objectiveperpendicularly and equipped with an xyz closed loop positioner with 100 pm travel in each direction and step size resolution of 100 nm.

[0135] The in-plane optical resolution was approximately 300 nm. The fluorescence from the samples was collected with a SPAD in the red to near infrared wavelength range. SPADs were used to measure the fluorescence intensity in units of photons detected per second. Avalanche photodiodes are used for single photon counting with dark count rates well below 1 kcounts / s. The intensity traces have been optimized (at a range of power values from 500 pW to 0.5 mW).

[0136] It is noted that the axis of the egg is tilted from the optical axis of the objective. As shown at the bottom of Fig. 4, this can be achieved by tilting the egg or by tilting the objective, or both. Fig. 4 also shows how multiple spots are measured along a rink around the blunt end of the egg to locate the blastoderm. The application of the laser light to the multiple points can be achieved in various different ways, such as by multiple lasers, multiple optical guides, or mirrors, or a ring with integrated lasers or LEDs that can be placed on the blunt end of the egg.

[0137] Lifetime Measurements

[0138] About 10 to 30 different points from various spots of each sample were collected, as shown in Fig. 5. Fig. 5a shows a representative 200 pm x 200 pm confocal image of washed embryo deposited on a glass slide. Red regions represent higher intensity fluorescence embryo and blue is low intensity background. Fig. 5b shows the fluorescence decay traces obtained and fitted to extract lifetime components for the white square marked regions of interest of Fig.5a.

[0139] Reference testing

[0140] Reference sexing was obtained with genetic analysis on embryonic tissue based on polymerase chain reaction (PCR).

[0141] DNA extraction

[0142] Samples for DNA extraction and PCR were collected either directly from slides used to assess autofluorescence or using sterile utensils to collect material from eggs. This included using sterile metal forceps and sterile wide bore pipette tips to collect membranes and blastoderms. DNA extraction utilised the RNeasy Mini Kit (Qiagen). For extraction directly from slides, buffer RLT (containing guanidine thiocyanate with 2-mercaptoethanol) was added directly to the slides to resuspend the organic material, and then this resuspension was added to a 1.5 mL flip cap tube. For extraction from samples collected directly from eggs, the membranes or blastoderm was added to a 1.5 mL flip cap tube and buffer RLT was added to this tube, and extraction performed as per manufacturer’s instructions. For positive extraction controls, an extraction was undertaken of cultured Leghorn Male Hepatoma (LMH) cell line and a pool of harvested chicken embryo kidney (CEK) cells. These extractions would provide controls for male and female respectively for the 12S / SWIM PCR described below. For negative extraction control, an extraction of reagents only was included. For a subset of samples, DNA concentration and extract quality were assessed on a Nanodrop Spectrophotometer (Thermofisher).

[0143] Chicken Sexing PCR

[0144] Extracted DNA was subjected to a known PCR designed to differentiate male and female birds. PCRs were performed on samples with no prior knowledge of microscopy results (in the cases where samples that had been assessed). The PCR uses two sets of primers, one targeting 212 bp of the 12S region, which is a genomic region present in both male and female chickens, and one targeting a 131 bp region of the SWIM gene, which is a genomic region present only in female chickens (on the W chromosome). The PCR results in either one (male) or two (female) products, which can be visualised on an electrophoresis gel.

[0145] The PCR components for each reaction were as follows: 5 pL of DNA template, 200 nM of each forward and reverse primer for each target, 1.5 mM MgC12, 0.2 mM of each dNTP, 1.25 Units of GoTaq Polymerase (Promega), GoTaq Polymerase buffer, and nuclease free water to a final volume of 25 pL. A previously extracted and sequenced chicken cell line (LMH cells) was used as a positive PCR control, and water used as a no-template control. Reactions were run on a T100 thermocycler (Bio-Rad) with 94°C initial denaturing, followed by 35 cycles of 94°C for 30 sec, 55 °C for 30 sec, and 72°C for 30 sec. A final extension stepof 72°C for 5 minutes was conducted before samples were placed at 4°C. The resulting product was separated by electrophoresis on a SYBR Safe (Invitrogen) stained 1% w / v agarose gel, and viewed using a Chemidoc transilluminator (Bio-Rad).ResultsFluorescence

[0146] Here, all parts of the egg were disconnected and their autofluorescence properties were recorded separately. Near- field imaging was used to gather the result presented here.

[0147] Confocal imaging

[0148] There are four types of chicken eggshells (white, brown, light green, dark green). The colour pigment protoporphyrin IX (PPIX) is embedded in all four shell types are highly fluorescent monomers. Eggs examined here were white and brown shell eggs.

[0149] Fig. 6 shows near-field confocal images of the outer and inner shell of brown eggs, indicating bright red fluorescence as a result of being excited by green laser (532 nm). Fig. 6a shows shell and inner membrane of an egg on a glass slide prepared for imaging. Fig. 6b shows a near-field confocal image of the outer shell and Fig. 6c shows a near-field confocal image of the inner shell.

[0150] All parts of the chicken egg appear to be auto fluorescent in the visible part of spectrum. Although optical imaging is an important step in this study, image-based analysis is insufficient for detection and identification of blastoderm / embryo. Additionally, image-based analysis is time- and skilled labour consuming.

[0151] Lifetime measurement

[0152] In addition to spectral features, lifetime is an intrinsic property of a fluorescence emitter and a potential candidate for a single parameter-driven method of identification. For lifetime imaging, the decay in the fluorescence intensity is recorded for egg yolks and blastoderm / embryos separately after optical excitation with a short laser pulse (6 ps) at 532 nm as described earlier in the disclosure.

[0153] The representative results for lifetime measurements for egg yolk are plotted in Fig.7. There are two main components, - a short and a long lifetime emitter, denoted asor lifetime 1 in Fig. 7a and T2orlifetime 2 in Fig. 7b. Fig. 7a shows the average fluorescence lifetime 1 of a day 2 extracted and washed embryo - green laser excitation with 80 pW power used, where= 0.91±0.06 ns. Fig. 7b shows the average fluorescence lifetime 2 T2of a day 2 extracted and washed embryo - green laser excitation with 80 pW power used, where T2= 6.4±0.9 ns.

[0154] White eggs results

[0155] In total 50 white shell fertilized eggs, supplied by Australian SPF Services were tested. Day 0 (no incubation, as received) eggs were immediately prepared as described earlier and examined optically as described earlier. Subsequent PCR testing was performed as described earlier. The remainder of the eggs were incubated as described earlier. On day 1, 2, 3 and 5 eight eggs were removed for testing. Representative results for lifetime measurement are shown in Tables 1 to 3.

[0156] Table 1 contains representative results for the two components of the lifetime (lifetime 1 and lifetime 2) of the egg yolk for female and male embryos (or blastoderm if it measured at day 0) and non-fertile eggs. The first component of the lifetime is smallest for the egg yolk and largest for the female embryo / blastoderm This is a consistent trend in all measurements, regardless of the day of incubation. Second component lifetime shows the same trend for all day 0 eggs. There is a large gap between the lifetimes of different genders, in particular for lifetime 1.DayO Lifetime 1 (ns) Lifetime 2 (ns) SexTable 1 : White eggs lifetime. Representative results for fertilized eggs (blastoderm) prior to incubation - day 0. F stands for female, M for male and NF for non-fertile.

[0157] Table 2 contains representative results for day 1 and 2 incubated eggs, showing the same trend as for non-incubated eggs. The shortest lifetime 1 and 2 belong to egg yolk andlongest lifetimes belong to female eggs. The lifetime for yolk, female and male embryo are again well separated.Day 1-2 Lifetime 1 (ns) Lifetime 2 (ns) SexTable 2: White eggs lifetime. Representative results for fertilized eggs (embryo and yolk) after one and two days of incubation.

[0158] As an embryo is developing the lifetime for all components is increasing with time of incubation. This is illustrated in Table 3. Whereas the lifetime 1 increase is consistent in all samples measured, the increase in lifetime of the second component with incubation time is irregular. Therefore, the first (shorter) lifetime component should be used for embryo stage of development verification.Day Lifetime 1 (ns) Lifetime 2 (ns)Table 3: White eggs results. Comparison of lifetime for different days of incubation, here shown for female embryo. Lifetime 1 shows the increasing trend with time whereas lifetime 2 is not dependent on time.

[0159] Overall, the results from white shell fertilized eggs give strong indication that the lifetime of the shorter component can be used for gender determination. The values for male and femaleblastoderm / embryos are well separated, no overlap in their values have been measured even though it was expected. In addition to the aimed gender determination, there is strong indication that the stage of embryo development can be determined by this methodology too.

[0160] However, different type of eggs and those from a different supplier may lead to different conclusions. Therefore, it is necessary to investigate another set of eggs coming from a different type of hen and different supplier. As such, the results measured on brown shell fertilized eggs obtained from SBA are now presented.

[0161] Brown Eggs results

[0162] Brown shell fertilized eggs were supplied by Specialised Breeders Australia. In total 60 fertilized eggs were divided at delivery in group of eight for day 0 and day 1, 4 of incubation and group of 18 for day 3 and 5 of incubation. The choice to analyze the largest number of eggs on Day 3 and 5 of incubation was made due to the white shell eggs PCR testing results. Those results were more reliable after day 3 of incubation and consistent on day 5 of incubation as explained earlier.

[0163] Representative results for lifetime 1 and 2 for brown shell fertilized eggs on day 0 (no incubation), day 3 and 5 incubation are shown in Table 4.Day035Yolk (Day 1-3)Table 4: Brown eggs lifetimes. Representative results for male and female blastoderm / embryo prior to incubation (day 0) and after day 3 and 5 of incubation. F stands for female, M for male and NF for non -fertile.

[0164] The results for the lifetime 1 of brown shell eggs are in agreement with the results for white shell eggs. The shortest lifetime 1 is measured for egg yolks and the longest lifetime 1 is measured for female embryos. Note that non-fertilized egg (NF) have the same lifetime as for the egg yolk.Day 5 Lifetime 1 range (ns) Lifetime 2 range (ns)Male ] 0.49-0.573.2 -4.5Female | 0.71-1.16 [ 3.1-8.2Table 5: Brown eggs lifetimes. The range of lifetime for male and female eggs at day 5.

[0165] Fig. 8a shows lifetime 1 of brown shell eggs at day 0 of incubation-8 eggs. Fig. 8b shows lifetime 1 of brown shell eggs after five days of incubation- 17 eggs. Blue block indicates male and pink block indicates female blastoderms / embryos. Again, the lifetime 1 iscompletely consistent in terms of gender determination. There is no overlap in those two groups as indicated in Figs. 8a-b.

[0166] There is a larger number of female embryos in this set of day 5 eggs which is not intentional. The larger number of “male” embryos for day 1 eggs compensate for the gender unbalance of day 5 eggs. Overall, there is 50-50 percent “female” and “male” ratio of all fertilized eggs, excluding eggs classified as non-fertile.

[0167] There is one outcome for the brown shell egg lifetime different to the results obtained for white shell eggs. For the white shell eggs, the development stage could be established, with the lifetime increasing with incubation time. This was not established for the brown shell eggs. There is no apparent reason for this finding, and it should be addressed in future studies.PCR reference testing and comparison

[0168] SWIM PCR of DNA was extracted from the egg samples on the glass slides. M shows the base pair size markers (Hyperladder 100 bp). Samples from day 0 (lanes 1 to 4), day 1 (lanes 5 to 11), day 3 (lanes 12 to 15). Male (LMH) positive control (lane 17), female (CEK) positive control (lane 18) are extracted, along with the PCR negative and positive (female) controls (lanes 19 and 20) and a negative extraction process control (lane 16).

[0169] This PCR amplifies 2 products from female cells and one product from male cells. The smaller sized band is amplified in both male and female cells, and the larger band is detected only in female cells. It can be seen (lanes 12, 13, 16), the larger female band is not as strongly detected as the lower band. When samples were extracted from older blastoderms, (day 3, lanes 12 to 15) the PCR resulted in stronger bands and detection of male and female bands was seen, although there was significant sample to sample variation. Since less DNA is available for extraction in the younger eggs with fewer cell per blastoderm, PCR detection of both bands, particularly the female band, was more difficult from these samples. The lanes without any bands did not have sufficient DNA for testing.

[0170] While most day 0 eggs had enough DNA to show amplification of the lower band, it was not sufficient to confirm if they were male or female, given the relative weakness of thefemale band. Sex determination by PCR was much clearer by day 5. For day 5, the embryo should be starting to form and a rapid increase in blood vessels to form the chorioallantoic membrane, providing a larger number of cells and DNA for extraction and detection.Table 6: Comparison between optical measurement and PCR testing for male and female blastoderm / embryo prior to incubation (day 0) and after day 1, 3 and 5 of incubation. Note that optical testing is more reliable and consistent than PCR testing up to day 5, as a result of the PCR testing becoming more consistent than optical measurement due to appearance of blood vessels.

[0171] As pointed out above, optical measurement results are more reliable and consistent up to day 5 of incubation as a result of the PCR testing becoming more reliable and certain. Fig. 9 shows a comparison between optical measurements (lifetime) and PCR testing for eggs incubated for 5 days. Blue block indicates male and pink block indicates female embryos where the results agree for optical and PCR testing. Grey block indicates 29 % of differing results.

[0172] The results for day 5 optical and PCR test agreement are shown in Fig. 9 with the grey block indicating differing results. The fact that blood vessels are more apparent after day 4 of incubation is beneficial for PCR testing whereas for the confocal testing it is an obstacle. Some components in blood such as red blood cells, naturally fluoresce across multiple wavelengths which can mask or interfere with the fluorescence of natural gender “markers” in embryo. In addition, the effect of maternal sex allocation on which this method is based weakens during the period of incubation.Stage 2 methodology and results (all layers)

[0173] All egg components, outer and inner shell, albumen, yolk and blastoderm, are fluorescent and the main challenge is to address and read out the signal from the blastoderm while keeping other parts of the egg intact. This challenge is investigated in this stage.

[0174] Eggs were “reconstructed” from blastoderm to whole egg. Each of the parts of the egg were optically examined. “Reconstructing” fertilized egg from blastoderm to whole egg was done in stages. Each stage uses confocal microscopy measurements.Materials and MethodologyEggs supply

[0175] Fertilized eggs were supplied by Specialised Breeders Australia (SBA), Kamarooka hatchery, Victoria. The eggs supplied by SBA were Hy-line Brown with brown shell. All optical measurements were performed on fertilized eggs as received, at day 0 of incubation.Egg handling

[0176] At day 0 (as received) the fertilized eggs were subjected to optical measurements. The eggs were cracked by hand and the shells, egg white, egg yolk and the inner membranes were separated. The blastoderm for day 0 eggs were extracted via syringe. All blastoderms were transferred to glass slides and washed well with PBS prior to optical imaging.

[0177] Egg components were deposited on a glass slide including outer brown shell and inner thin membrane. Individual parts of the eggs were investigated separately for the inner membrane and outer shell.

[0178] Egg layers

[0179] The aim of this stage was to investigate the influence of the outer shell, inner membrane, yolk, and albumen i.e., the whole egg components. There are two main approaches: One is measuring lifetime of whole fertilized egg. However, because the egg is a complex optical object, it is hard to distinguish contributions from individual components. Therefore, the investigation was started reconstructing the egg from the blastoderm to shell instages, constructing egg’s component layers, one at the time. A schematic of the final stage of the egg layer approach is shown in Fig. 10.

[0180] Note that it is possible to follow a different approach, starting with the whole egg as received, measuring the fluorescence lifetime and proceeding marked eggs to incubation for 3-5 days, followed by PCR testing. As pointed out in Stage 1, PCR testing results were more reliable after day 3 of incubation and certain on day 5 of incubation when compared to day 0 to day 2 incubation period.

[0181] Confocal microscopy

[0182] The confocal systems and methods used herein are equivalent to the systems and methods describe in Stage 1.

[0183] Fig. I la shows 200 pm x 200 pm confocal scans of the egg. Fig. 1 lb shows the fluorescence decay traces were obtained for the cross marked regions of interest of Fig. I la. These decay traces were fitted to double exponential to extract lifetime components, as can been seen in Fig. 1 lb. Fig. 11c shows lifetime values of the first component determined from the exponential decay, for 10 different fluorescent spots. Fig. l id shows lifetime values of the second component determined from the exponential decay, for 10 different fluorescent spots. Note that in Stage 2 only the first and more fluorescent component Or) is used (even though both components are simultaneously measured). This corresponds to the shorter component of the fluorescence lifetime.Results

[0184] In contrast to Stage 1, Stage 2 focussed only on Day 0 (before incubation) measurements, which is the most practical and ethical strategy. Initially, lifetimes of all individual layers are measured with 532 nm (green) laser excitation. Other excitation wavelengths including 490 nm (blue), 600 nm, 635 nm (red) and 580 nm (yellow) were used subsequently. Finally, lifetimes of all egg layers were measured at 580 nm excitation.Green laser excitation

[0185] Individual components

[0186] As indicated in Stage 1 results, all egg components, outer shell, inner membrane, albumen, yolk and blastoderm, are fluorescent and the main challenge is to address and read out the signal from blastoderm while keeping other parts of the egg intact. “Reconstructing” fertilized egg from blastoderm to whole egg was done in stages. Therefore, the first lifetime of all individual components needs to be determined and compared. In Table 7, representative lifetimes for individual components measured at 532 nm excitation wavelength are shown.Table 7: Representative results for the two components of the lifetime (lifetime 1 and lifetime 2) of male and female blastoderm prior to incubation (day 0) and other egg components, measured at 532 nm excitation wavelength and power of 100 pW.

[0187] Note that only representative average lifetime values are shown in the table. However, yolk lifetime can vary for the first fluorescent component between 0.27 ns to 0.47 ns and for the second component between 3.0 ns to 4.3 ns, depending on colour of the yolk (yellow to orange). Also, the shell is not homogenous.

[0188] There are few conclusions to be drawn from this set of measurements. The most important finding is that there is no overlap in lifetime for any component, they are all distinct from each other, and, most importantly, they are distinct from both male and female blastoderm. The yolk is the most fluorescent component at 532 nm excitation, followed by the shell.

[0189] Fig. 12 shows schematic of lifetime of individual egg components indicating their relative position. Horizontal lines-yolk, dotted- shell, criss cross - male blastoderm, diagonal lines - inner membrane, white - thick albumen, black - female blastoderm. Excitation wavelength is 532 nm (green laser).

[0190] The intensity (brightness) of the most dominant fluorescence lifetime component can be denoted as: Intensity (brightness): Yolk > Shell > Male blastoderm > Inner membrane > Thick albumen > Female blastoderm and inverse order of lifetime shown in Table 7. Indeed, there is a universal inverse correlation between the most dominant fluorescence lifetime (T) and emission intensity (T) (Equation 1).1T~ - (Equation 1)

[0191] The experimental results are found to be consistent with the above equation. Yolk exhibited the shortest lifetime and the highest emission rate, whereas the female blastoderm revealed the highest value of the fluorescence lifetime and the lowest emission rate. The primary concern about the relative position of the blastoderm lifetime to other egg components is that fluorescent signals from the brighter shell and yolk may hinder the emission of the blastoderm. Hence, in some embodiments, before determining in-ovo gender and / or fertilisation status using numerical ranges, it may be better to first compare relative position of components, such as the relative positions shown in Fig. 12.

[0192] Shell - challenges

[0193] Eggs examine in this Stage were brown shell eggs. There are two main challenges regarding non-invasive optical imaging through the shell. The thick mineralized shell constitutes an optical barrier to the optical measurements. The measurements here showed that around 10% of incident visible light passes through the brown shell. Fig. 13 shows transmission of the shell (brown egg) as a function of wavelength. As shown in Fig. 13, transmission increases from 7.5% in the blue to 12% in the red part of the visible spectrum.

[0194] In addition to low transmission, brown eggshell begins to absorb at wavelengths shorter than 750 nm by excitation through the outer surface side. Red regions represent higher intensity fluorescence shell and blue is low intensity background (glass). The brown eggshell exhibits bright red fluorescence even at low excitation power (100 pW).

[0195] All layers

[0196] As previously discussed, the main obstacle in any optical measurement of an egg is the egg shell. For that reason, the lifetime of the shell in isolation and the lifetime of all layers included are compared. The question is how much of the optical input (laser excitation) and optical output (emission) can account for layers below the shell. Results shown in Fig. 14 are representative lifetimes of the shell only and the layers of the egg.

[0197] Fig. 14a shows the lifetime measurement of shell only. Fig. 14b shows the lifetime measurement of all eggs layers. Green laser excitation and 100 pW power. Even though the shell lifetime changes as all layers are included there is concern that the yolk, which is brighter than the blastoderm, may hinder the blastoderm signature. Fig. 15 shows a schematic of lifetime of yolk, shell, male blastoderm and all eggs layers indicating their relative position. Horizontal line-yolk, dotted- shell, criss cross- male blastoderm, vertical lines- all layers. The excitation wavelength is 532 nm (green laser).Individual components at other excitation wavelengths

[0198] With the supercontinuum source, various excitation wavelengths are filtered out using the emission filters. Excitation wavelengths used in this Stage were 490 nm (blue), 532 nm (green), 580 nm (yellow), 600 & 630 nm (red).

[0199] Excitation at 490 nm (blue), individual components and relative positionTable 8: Representative results for the first components of the lifetime (lifetime 1) of male blastoderm prior to incubation (day 0), yolk and shell, measured at 490 nm excitation wavelength (blue). Excitation power is 100 pW.

[0200] Fig. 16 shows a schematic of lifetime of individual egg components indicating their relative position, horizontal lines-yolk, dotted-shell, criss cross- male blastoderm. Excitation wavelength is 490 nm (blue laser).

[0201] As expected from the results of Table 8, for most of biomaterials excited closer to the UV region of the spectrum, the shell and male blastoderm become brighter, i.e., they haveshorter lifetimes. However, the yolk lifetime has not changed significantly. Consequently, the shell is the brightest egg component, followed by the yolk and male blastoderm.

[0202] These results indicate that the relative position of the lifetime change with a modification in excitation wavelength while maintaining distinct values. However, fluorescence of blastoderm is still hindered by yolk fluorescence. Therefore, other egg components were not further characterized, and longer wavelengths of excitation are considered.

[0203] Excitation at 600 nm

[0204] Red laser excitations are commonly preferred in many bioimaging applications. The light emission should be in the near-infrared region (NIR), 700-900 nm, as it penetrates centimetres into tissue, whereas visible light can only travel tens of microns to millimetres depending on the wavelength. These wavelengths would be also beneficial for light transmission through the shell. However, the shell itself becomes more fluorescent in the NIR region. Therefore, different wavelengths for laser excitation were considered.

[0205] Firstly, a 600 nm excitation wavelength was tested. The relative position of the yolk and male blastoderm change with the male blastoderm becoming more fluorescent than yolk for the first time. This is a preferred relative position of the egg components. Therefore, other components measurements were then investigated.Table 9: Representative results for the lifetime (lifetime 1) of male blastoderm prior to incubation (day 0), shell and yolk components, measured at 600 nm excitation wavelength. Excitation power is 100 pW.

[0206] Fig. 17 shows a schematic of lifetime of individual egg components indicating their relative position, horizontal lines-yolk, dotted- shell, criss cross- male blastoderm, diagonal- inner membrane, white - thick albumen, black- female blastoderm. Excitation wavelength is 600 nm (red laser).

[0207] Indeed, the red laser excitation enables better lifetime positioning of male blastoderm compared to all previous excitation attempts, as shown in Fig. 17. Capturing the fluorescent signature from male only blastoderm is sufficient for gender determination.

[0208] Excitation at 625 nm

[0209] Encouraging results from 600 nm excitation suggested a direction towards longer wavelengths. As such, the wavelength in slightly increased to 25 nm, i.e., 625 nm excitation wavelength.Table 10: Representative results for the lifetime (lifetime 1) of male and female blastoderm prior to incubation (day 0), shell and yolk components, measured at 625 nm excitation wavelength. Excitation power is 50 pW.

[0210] Even though, the relative ranking of the fluorescence lifetime of each egg components has not changed, it is apparent that the shell is becoming more fluorescent at 625 nm excitation compared to the 600 nm excitation wavelength, i.e. a shorter lifetime was measured. Additionally, it seems that the male blastoderm and the yolk are closer together and hence it would be harder to distinguish male and non-fertilized eggs at 625 nm excitation wavelength.

[0211] As mentioned earlier, there is a trade-off between the transparency and fluorescence of the shell. Higher wavelengths of excitation enable higher shell transparency but at the same time unwanted shell fluorescence may increase in the red and NIR region. In order to find an optimal excitation wavelength, a slightly shorter wavelength of excitation is Shell fluorescence should exhibit minimum around 580 to 600 nm.Table 11 : Representative results for the lifetime of brown eggshell at different excitation wavelengths.

[0212] Note that shell fluorescence is not linearly dependent on excitation wavelength as presented in Table 11. According to these results, 532 nm excitation would be beneficial in terms of shell autofluorescence but according to the results presented earlier, the yolk would hinder the male gender signature at this wavelength.

[0213] Excitation at 580 nm

[0214] As explained above, the minimum for the shell fluorescence should appear in the 580 to 600 nm excitation wavelength range. According to these results, the shell is most fluorescent, followed by the male blastoderm, then the yolk. The female blastoderm is the least fluorescent component compared to other parts of the egg.Table 12: Representative results for the lifetime (lifetime 1) of male and female blastoderm prior to incubation (day 0), shell and yolk components, measured at 580 nm excitation wavelength. Excitation power is 100 pW.

[0215] Those results are similar to the results obtained with a 600 nm excitation wavelength. This increases the operational wavelength range for future practical devices. Operating at a single wavelength would not be desirable.All layer at 580 excitation

[0216] First, blastoderms are extracted from fertilized eggs and deposited on a glass slide. Their fluorescent lifetime is measured at 10 to 20 individual spots, as described earlier. Then all egg layers are deposited, and lifetimes are measured again. Representative results for the blastoderm and all layers are shown in Table 13. The second column shows lifetime average values for the blastoderms only. Apart from the values in last two rows, all lifetimes clearly indicate the gender, shown in the third column. Note that only eggs with a female signature should be kept, or if more feasible, all eggs with a male signature should be extracted. The third column contains lifetimes after all layers are deposited on the blastoderms.

[0217] At first sight it appears that only measurements in the first and third row agree with the measurements for blastoderm only. For some of the eggs the gender signature has changedfrom female to male, i.e., the eggs in the fifth and seventh row. For majority of them there is uncertainty about their gender as commented in the last column of Table 13. Note that the lifetime of a non-fertilized egg should show values for yolk only. Non-hatching eggs include infertile eggs or fertile eggs in which the embryos had died during incubation that sometime can constitute up to 25% of all eggs. Uncertainty between male and non-fertilized eggs is irrelevant for a hatchery, as both should be extracted before incubation. On the other hand, any uncertainty between female and non-fertilized eggs should be minimized.Table 13: Lifetimes of blastoderm extracted from fertilized brown eggs (second column and all layers of fertilized eggs (fourth column) with comments on blastoderm gender (third column) and possible gender of all egg layers (last column). Excitation wavelength is 580 nm and power is 100 pW.

[0218] However, as explained previously, all of these values are average values obtained from 10 to 20 fluorescent spots measured on a single egg sample. The fact that the shell has the shortest lifetime (most fluorescent), implies that its value may appear in all measurements and suppress average values of all eggs. For that reason, individual spot measurements for each egg are now analysed, expecting to see in all sets of measurements some, if not most of individual measurements originating from the shell.

[0219] In Table 14 below, the most complicated case is shown where the gender signature has changed from female blastoderm to male as a result of including all layers, the seventh row from Table 13. Individual spots measurements are shown in the second column. The last row denotes average values of all individual spots. There are two steps in gender evaluation. First, all measurements that originate from the shell with the exclusion of lifetime valuesbelow 0.45 ns are excluded, as determined in Table 11. The resulting average lifetime has changed from 0.62 ns which could be the male blastoderm or yolk to 0.72 ns which could be the yolk or female according to results presented in Table 11. The next step should eliminate values smaller than 0.7 that originate from the yolk. The final result confirms that the egg has a female signature. It occurs that the female signature, that is least fluorescent, still can be read out by this methodology. With this approach, a 90% success rate in determining in-ovo gender was achieved.Table 14: Individual measurements of lifetimes of all layers (second column), values higher than 0.45 (third column), excluding shell measurements; and values higher than 0.7 (fourth column), excluding yolk measurements. The last row represents average values from individual spot measurements listed above. Excitation wavelength is 580 nm and power is 100 pW.Stage 3 methodology and results (whole egg)Aims

[0220] In Stage 1 above, it was demonstrated that the gender of blastoderm / embryonic tissue of fertilized chicken eggs can be determined accurately based on blastoderm / embryo intrinsic fluorescence. In Stage 2, the process of “reconstructing” a fertilized egg from blastoderm to whole egg was done in stages. These results constitute major elements for the Stage 3 project with the aim of investigating a whole fertilized egg, i.e., leaving the whole egg intact.Eggs supply

[0221] Fertilized eggs were supplied by Specialised Breeders Australia (SB A), Kamarooka hatchery, Victoria. The eggs supplied by SB A were Hy-line Brown with brown shell. All optical measurements were performed on fertilized eggs as received.Egg handling

[0222] On day 0 (before incubation) the fertilized eggs were subjected to optical measurements. After optical measurements, the eggs were cracked by hand, and the blastoderm were extracted using a razor blade to cut through the yolk and extract the blastoderm, spatula and tweezers were also used to assist the extraction. .All blastoderms were transferred to glass slides and washed with PBS prior to optical imaging to confirm measurements done on the whole egg.Confocal microscopy

[0223] The confocal systems and methods used herein are equivalent to the systems and methods described in Stage 1. Note that a single wavelength, low power laser is enough to determine the sex of a fertilized egg. According to the Stage 2 findings, a 580 nm excitation wavelength seems to be the optimal wavelength for brown shell eggs. Additionally, we have used a 532 nm excitation wavelength to demonstrate that it is possible to apply another excitation wavelength in the visible spectrum.Position of blastoderm

[0224] At the time of laying, the chick blastoderm measures about 5-8 mm in diameter. On the first day of the egg, the air gap is 4-5 mm, see Fig. 18 Two positions were selected after initial optical testing: blunt-end up (long axis perpendicular to the surface) and horizontal one (long axis parallel to the surface).

[0225] Blunt-end up position is preferred by hatcheries, but this position may be optically more challenging due to the existence of 4-5 mm air gap. The blastoderm from the vertical axis was found to be 26.8° ± 1.3 for non-incubated eggs in the blunt-end up position.Therefore, the blastoderm can be in any part of the edges of the cone denoted by the dashed lines, see Fig. 18 (showing shell 2301, blastoderm 2302, and yolk 2303). This blastoderm position may change with storage and incubation.

[0226] Eggs placed horizontally for long periods of time before scanning had significant changes in the positions of their blastoderm compared with those of eggs scanned minutes after positioning. To achieve the position of the blastoderm on top of the yolk, the eggs were turned using a recipe published in S. Klein et al, Localization of the fertilized germinal disc in the chicken egg before incubation, Poultry Science 81 :529-536, 2002. In particular, the egg is laid horizontally with its long axis parallel to the ground. This orientation facilitates the natural buoyancy of the disc. The eggs are then gently turned 90 degrees back and forth along the x-y plane twice per hour over a period of three hours, a motion that encourages the disc to migrate upward. Following this turning protocol, the eggs may rest undisturbed for at least two hours, allowing the germinal disc to stabilize in its topmost position on the yolk. In this case, the blastoderm 2302 is positioned close to the shell 2301 and therefore less challenging for optical scanning, see Fig. 19.Blastoderm targetingVertical Positioning (Blunt-End Up)

[0227] The egg is placed vertically on the microscope stage with the blunt end facing upward, i.e. the long axis is vertical and perpendicular to the ground. To account for the natural position of the blastoderm, the egg (i.e. the long axis) is manually tilted off vertical at an angle of roughly 25-30° relative to the central axis (from blunt to narrow end), as shown in Fig. 18. This adjustment aligns the blastoderm region within the optical path.Using micrometer-controlled rotatable knobs on the movable stage, the egg is brought closer to the objective lens in small increments. A tunable wavelength laser is set to 100-200 pW power, with a wavelength of 532 ± 20 nm or 580 ± 20 nm selected, and the laser is directed through the objective to illuminate the sample. The laser is placed above the egg pointing vertically downwardly to optically excite the egg. Further, the objective is also placed above the egg to measure the autofluorescence from above the egg. The distance between the objective and the egg is further reduced using coarse micrometer rotatable knobs until a distance of 1-2 mm is achieved. Fine rotational adjustments are made to focus until it reaches the working distance of the lOOx objective (0.12-0.18 mm). Maximum output counts recorded by the detector indicate optimal surface focus. The objective is mounted on an electrically controlled piezo stage and illuminates the eggs from above. The final focus adjustments are made using this piezo stage controlled via Lab VIEW software through aninterface, enabling micrometer-scale positioning of the objective at the point of best spatial resolution.

[0228] Optionally, a white light source can be used (directed through the objective) to illuminate the sample, and the output is monitored on the screen via a widefield camerato visually observe the scattered white light from the sample and achieve sharp focus. However, this step is optional. Once confirmed, the white light is switched off, and the laser (532 nm or 580 nm) is used for measurements.

[0229] To ensure comprehensive data collection in the vertical position, the egg is manually rotated 10-20 times about its long axis, allowing measurements to be taken around the cone (as shown in the Fig. 18) to locate the blastoderm. This defines a circle on the blunt side of the egg so that the circle constitutes the base of the cone and the opening angle of the cone is about 25-30 degrees.

[0230] Horizontal Positioning (Long Axis Parallel to Stage)

[0231] The egg is placed horizontally on the microscope stage with the blunt and narrow ends aligned laterally (left / right). In this orientation, yolk displacement naturally positions the blastoderm closer to the shell surface, eliminating interference from the 4-5 mm air gap characteristic of vertical positioning. The egg is manually adjusted slightly toward the blunt end (as shown in Fig. 19) to precisely locate the blastoderm. This horizontal configuration permits use of a lower magnification objective (<100x), providing both a larger working distance (0.2-0.6 mm) and greater clearance between sample and objective compared to vertical orientation. While this method offers optical advantages, the horizontal position demonstrates reduced stability - even minor disturbances can displace the egg and blastoderm’s position. This inherent instability may render the technique less suitable for high-throughput hatchery applications where consistent positioning is desirable.Results

[0232] The results presented here are for blunt-end up positioned eggs. Similar results are obtained for horizontally placed egg, once rotational turning is applied. As explained in Stage 2 results, individual spot measurements for each egg are analyzed, with shell -originatingsignals expected in all measurement sets. However, in a whole egg, any internal component may contribute to individual spot measurements.

[0233] Note that the value for each component within the range changes with egg’s freshness. Lower values (brighter) apply for fresh eggs and higher values (less bright) apply for stored eggs. We find it easier to determine the sex for older eggs and blastoderms. The older the egg, the more components can be detected.

[0234] Representative results on a whole egg with 580 nm excitation and no blastoderm targeting strategy applied are shown in Table 15. It is possible to record any of the values presented in Table 12. The data was recorded from 10-20 spots at a distance of 2-3 mm from the central axis within a horizontally positioned arc of 4-5 mm.

[0235] Table 15 Representative results for the first components of the lifetime (lifetime 1) of three whole eggs prior to incubation (day 0), measured at a 580 nm excitation wavelength. Excitation power is 200 pW. No blastoderm targeting strategy was used.

[0236] Those results are compared to the results presented in Table 12, identifying egg components. In addition, the blastoderm lifetime is checked for each egg after measurements on a whole egg were completed to confirm the sex of the blastoderm.• Egg 1 results indicate in 1 out of 20 measurements (only 10 shown here) that the egg contains a female blastoderm.• Egg 2 results indicate in 3 out of 20 measurements (only 10 shown here) that the egg contains a male blastoderm.• Egg 3 results measure only the shell component indicating that the egg is most likely non fertile.

[0237] The maximum success rate in determining sex without a blastoderm targeting strategy is 50 %. In at least 30 % cases the blastoderm is missed. It is easier to detect a male blastoderm as it is brighter (shorter lifetime) than a female blastoderm (longer lifetime). Similar results are obtained for the horizontally placed eggs.

[0238] When the blastoderm targeting strategy is applied, the success rate for both positions increases. The results are only shown for the blunt-end up position. Accuracy increases to 90% and 10% of blastoderms are missed. Representative results on a whole egg with 532 nm excitation are shown in Table 16. Results for each component are compared with the results presented in Table 7. The data was recorded from 10-13 spots.

[0239] Table 16 Representative results for the first components of the lifetime (lifetime 1) of three whole eggs prior to incubation (day 0), measured at a 532 nm excitation wavelength. Excitation power is 200 pW. Blastoderm targeting strategy was used.

[0240] Those results are compared to the results presented in Table 7, identifying egg components. In addition, the blastoderm lifetime is checked for each egg after measurements on whole eggs were completed to confirm the sex of the blastoderm.• Egg 1 has 3 out of 12 measurements (10 shown here) with signatures of a female blastoderm. This is confirmed by measuring the blastoderm lifetime.• Egg 2 has 3 out of 10 with signatures of a male blastoderm. Male sex is confirmed by measuring the lifetime of the blastoderm.• Egg 3 has only signatures from the shell and yolk indicating a non-fertile egg.

[0241] These results indicate that the in-ovo gender and / or fertilisation status can be determined from an egg optically while leaving the egg intact. That is, the results show that the egg can be excited optically with laser light, autofluorescence can be measured, the lifetime can be calculated and the gender and / or fertilisation status can be determined while leaving the egg intact (without breaking the eggshell).

[0242] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A non-invasive method of determining in-ovo gender and / or fertilisation status from an egg the method comprising: optically exciting the egg with light from a laser source; measuring autofluorescence of the egg over a period of time; calculating a fluorescence lifetime indicative of a decay of the autofluorescence over the period of time; and determining the in-ovo gender and / or fertilisation status based on the fluorescence lifetime.

2. The method of claim 1, wherein optically exciting the egg with light from the laser source and / or determining the in-ovo gender and / or fertilisation status occurs prior to incubation of the egg.

3. The method of claim 2, wherein the laser source is a low power laser source.

4. The method of claims 1, 2 or 3, wherein calculating the fluorescence lifetime comprises calculating the fluorescence lifetime for multiple ranges corresponding to multiple respective components of the egg, and determining the in-ovo gender and / or fertilisation status is based on the fluorescence lifetime corresponding to a blastoderm component.

5. The method of any one of the preceding claims, wherein the fluorescence lifetime is a shorter component of the lifetime of the autofluorescence of the egg.

6. The method of any one of the preceding claims, wherein determining the in-ovo gender is based on a difference in fluorescence lifetime between male and female blastoderm indicative of a difference in expression of proteins.

7. The method of any one of the preceding claims, wherein measuring the autofluorescence comprises detecting photons emitted from the egg over the period of time.

8. The method of claim 7, wherein detecting photons emitted from the egg over the period of time comprises time-tagging the photons emitted from the egg by collecting the photons arriving on a detector at multiple time intervals over the period of time.

9. The method of claim 8, wherein the calculating the fluorescence lifetime comprises fitting a double-exponential decay model to the photon collection at the multiple time intervals over the period of time, the fluorescence lifetime corresponding to a decay rate of the exponential decay model.

10. The method of any one of the preceding claims, wherein the period of time is between 10 and 30 seconds.

11. The method of any one of the preceding claims, wherein the laser source has a power below a photothermal damage threshold of the egg.

12. The method of claim 11, wherein the power of the laser source is between 50 pW to 0.5 mW.

13. The method of any one of the preceding claims, wherein the light from the laser source is visible or near infrared (NIR).

14. The method of claim 13, wherein the light from the laser source has an optimal wavelength of between 580 to 610 nm.

15. The method of any one of the preceding claims, wherein the measuring autofluorescence of the egg over a period of time comprises using one or more emission filters to filter desired wavelengths of the autofluorescence, the one or more emission filters being between 450 and 700 nm long-pass optical filters.

16. The method of any one of the preceding claims, wherein the light from the laser source comprises light pulses with a pulse width of between 1 to 10 ps and a pulse frequency of between 10 and 30 MHz.

17. The method of any one of the preceding claims, wherein the light from the laser source has a bandwidth of between 1 and 20 nm.

18. The method of any one of the preceding claims, wherein the in-ovo gender determined is: male upon the fluorescence lifetime being within a male range; or female upon the fluorescence lifetime being within a female range, wherein the male range and the female range are non-overlapping.

19. The method of any one of the preceding claims, wherein the in-ovo fertilisation status determined is: fertilised upon the fluorescence lifetime being within the male range or the female range; or unfertilised upon the fluorescence lifetime being outside of the male and the female range.

20. The method of claim 18 or 19, wherein the male range and the female range are dependent on the wavelength of the light from the laser source.

21. The method of claim 20, wherein the wavelength of the light corresponds to green light, wherein the male range is from 0.45 to 0.67 ns; and the female range is from 0.77 to 0.93 ns for brown eggs.

22. The method of claim 20, wherein the wavelength of the light corresponds to orange light, wherein the male range is from 0.53 to 0.65 ns; and the female range is from 0.77 to 0.

93. ns for brown eggs.

23. The method of any one of the preceding claims, wherein the method further comprises repeating the steps of optically exciting the egg with light from the laser sourceand measuring autofluorescence of the egg over a period of time for multiple locations on the egg, wherein the fluorescence lifetime is based on an average of the repeated measurements for egg components.

24. The method of claim 23, wherein the method further comprises excluding lifetimes originating from an egg shell of the egg, inner shell and white of the egg and / or a yolk of the egg from the repeated measurements before determining gender.

25. The method of any one of the preceding claims, wherein the egg is a chicken egg.

26. The method of any one of the preceding claims, wherein the method further comprises placing the egg in a horizontal position and rotating the egg to move the blastoderm of the egg towards the egg shell.

27. The method of any one of the preceding claims, wherein the method further comprises, placing an objective relative to the egg where a long axis of the egg is tilted off the optical path of the objective and repeatedly performing the steps of exciting, measuring, calculating and rotating the egg or the objective about its long axis to acquire multiple measurements.

28. The method of claim 26, wherein the egg is placed in a blunt-end up position and the multiple measurements are acquired along a circle on the blunt end of the egg centred in the long axis of the egg.

29. Software for non-invasive determining in-ovo gender and / or fertilisation status from an egg that, when installed on a computer and executed by the computer, causes the computer to perform the method of any one of the preceding claims, or part thereof.

30. A system of non-invasive determining in-ovo gender and / or fertilisation status from an egg, the system comprising: a laser source configured to emit light to optically excite the egg; a detector configured to measure autofluorescence of the egg over a period of time; and a processor configured to:calculate a fluorescence lifetime indicative of a decay of the autofluorescence over the period of time; and determine the in-ovo gender and / or fertilisation status based on the fluorescence lifetime.

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