In-Ovo Sexing Technologies in Hatching eggs: Sex Determination of  Chicken Embryos before Hatch

In-Ovo Sexing Technologies in Hatching eggs: Sex Determination of  Chicken Embryos before Hatch

Recent advances in gene technology mean that it is now possible to differentiate between male and female chicks pre-hatch. This discovery provides an opportunity to improve animal production, reduce costs and eliminate ethical dilemmas in the egg laying and related industries.Recent advances in gene technology mean that it is now possible to differentiate between male and female chicks pre-hatch.Currently, culling male chicks post-hatch creates a major ethical dilemma for some countries. As a result, the poultry industry has invested in developing solutions to this issue.In some European countries, the need for a solution is urgent, following the call by some governments to introduce legislation to ban culling practices.

 In global poultry farming, the sex of chickens has a considerable impact on production performance and economic benefits. In terms of egg production, male birds cannot lay eggs and usually have a lower ratio of meat to feed than broilers. Male chicks are usually killed immediately after hatching since they are redundant in the industry and because male chicks will neither be suitable for egg production nor meat production. Ethical and animal welfare concerns about the culling of male chicks (approximately 7 billion each year have become an increasing issue in the egg industry; however, to date, no economically feasible in ovo sexing approaches for large-scale applications have been developed.

In large commercial hatcheries, sexing of newly born chicks is generally accomplished by three different methods according to the vent, color, or feathers of new hatching lines. Vent-based methods involve manual examinations of the reproductive organs and are not limited to specific breeds or crosses; however, these approaches require professional training to prevent disembowelment of the chick. In some breeds or strains, male and female chicks can be sexed according to their coat color or wing feather growth, which are determined by sex-linked genetic traits. These approaches are simpler and easier to explain to workers. However, these methods are still time- and labor-consuming. If sex can be identified at an early embryonic stage or even before incubation, male eggs could be used as feed components, which is a safe and good circular approach to nutrient use. Moreover, fewer eggs would need to be incubated, which would reduce feed space requirements, CO₂ emissions, and energy consumption, which are all economically beneficial to farmers and the environment.

In recent decades, scientists and researchers have used various in ovo sexing strategies in chicken eggs before hatching or incubation. These approaches can be divided into five major categories: (i) molecular-based techniques, (ii) spectral-based techniques, (iii) acoustic-based techniques, (iv) morphology-based techniques, and (v) volatile organic compound (VOC)-based techniques. Commercially applicable methods must be noninvasive, rapid enough for real-time applications, economically feasible, and ethically acceptable.

 It is common practice in the layer industry to cull male day-old-chicks directly after hatching. Male layers are unsuitable for egg production and too resource-inefficient for meat production. The practice, however, does raise concerns about animal welfare. An increasing number of initiatives are working on a solution for this challenge by developing technologies to determine the gender of a chicken while still in the egg.

In-ovo sexing

There is increasing resistance to the practice of culling male chicks in batches of day-old chicks for the laying sector. And it is financially advantageous to select out male embryos as early as possible. In addition to in-ovo vaccination, in-ovo sexing has also arrived. In-ovo sexing has made great progress in the last few years, and in some countries a ban on culling day-old male chicks is likely within the foreseeable future. The following information represents the situation in 2021. Various factors play a role in the choice of technology: • Invasive or non-invasive: making a tiny hole in the eggshell risks negatively affecting hatchability. So, the non-invasive option is preferable if reliability is comparable. • High reliability: the level of reliability must equal that of the current sexing method, used after hatching. • Timing of gender determination: the earlier, the better. Ideally, before being set in the incubator. • Speed of sexing: the volume of eggs handled at a hatchery is enormous, so a high capacity and throughput rate must be possible. This is important to prevent the eggs from cooling down too much. • Practical and robust technology: a method that works in laboratory conditions must be suitable for practical application at a hatchery.

There are a number of technologies under development:

• Spectroscopy: A beam of light is shone through the eggshell, or a tiny hole in the shell. The gender can be determined by the way the light scatters.

• Biomarkers: This method requires sampling fluid from the egg (allantoic fluid). These tests usually only work from day 7 or 8. Genetic modification will probably be an option in the future. So, either a marker will be developed that already shows a result on day 1, or the hens would only produce female offspring. The real question is whether this will be ethically acceptable for the consumer. Spectroscopy This non-invasive method has been ready for practical application since mid-2020, and has an accuracy of more than 95%. One machine can test more than 20,000 eggs an hour. No laboratory analysis is required, which makes this technique environmentally friendly (no chemicals required). Currently, the hyperspectral imaging technique only works with brown layer breeds, as of the 13th incubation day.

Biomarkers (SELEGGT and PLANTEGG)

These are two systems that work based on biomarkers:

• SELEGGT: marker reacts to the female hormone estrone-sulphate.

• PLANTEGG: uses the PCR-technique (DNA-analysis). On a carousel, lasers create a fine hole in the eggshell of an 8-10-day incubated egg. Then a minimal amount of allantoic fluid is extracted for analysis. The female hatching eggs are returned to the incubator. The inner membrane reseals on its own.

The carousel for collecting allantois fluid samples. A single laser beam is too weak to cause any damage, but the cross-point between two beams is powerful enough to bore through the eggshell. copyright protected The lasers make a tiny hole of 0.3 mm in diameter. A suction mechanism extracts allantoic fluid through this hole (PLANTEGG and SELEGGT).Colouring indicates if the fluid contains estrone sulphate (female) or does not (male) contain any (SELEGGT). The laser hole is 0.3 mm in diameter (PLANTEGG and SELEGGT).The PCR technology provides results within 1 hour (PLANTEGG).

Biomarkers: InOvo

The InOvo system works by indicating a specific metabolite in the allantoic fluid, as a biomarker around day 9. In this system, a tiny opening is pierced in the eggshell. The best location for this hole is determined for each egg. Then some allantoic fluid is extracted by a needle. The hole (< 1 mm) is resealed afterwards. The fluid is analysed very quickly using mass spectrometry. This analysis takes less than a second. The speed of the analysis and the lack of a need for chemical analysis make this concept viable for scaling up.Mass spectrometry can determine the gender of the embryo based on a small gender specific molecule in the allantoic fluid. After sexing, each egg is stamped with an additional identification code (male or female). The eggs with female embryos are then incubated and the males are removed.

The sampled eggs have a clearly visible seal-layer that closes the hole made during the process. Gender reversal Another option does not involve determining the gender but changing it. Gender is, of course, determined genetically. But suppressing the expression of the gene responsible for male gender will make an embryo develop into a female. An Israeli company is developing a technique based on subsonic sound, with the aim of achieving this. The exact mechanism involved in the influence of sound on embryonic development is still unknown. In early 2021, they achieved 63% females, and 70% in some parts of the incubator. This technique is still in an experimental stage, but the company expects commercially interesting results within a short time.copyright protected

 The potential of in-ovo sexing

Solving this challenge has the potential to both address animal welfare concerns and increase sustainability by saving resources. Hatching birds is an energy intensive process, so being able to identify the gender of a chick while it is still in the egg promotes both animal welfare and energy efficiency.

Expected speed of adoption

Our expectation is that as soon as a commercially viable solution becomes available, adoption will start. Germany is expected to be one of the first countries where the technology will be widely adopted. According to a recent court ruling, use of the technology will become mandatory as soon as it is commercially viable. Once that point is reached, other countries in Northwestern Europe may follow. Our current expectation is that the North American market may be slightly later in adopting the technology, but this can change quickly when large retailers and food companies decide to demand its use from their suppliers. We also know that not all markets will accept in-ovo sexing technologies, as the development of life is still disrupted (despite the earlier stage). We already observe a vast growth in the market of growing the male layer chicks for their meat. The organic market in Western Europe expected to adopt fully the growth of the male layer chicks for their chicken meat.

Differences between technologies

When looking at the in-ovo sexing technologies in this field it is important to have an eye for the following aspects:

• Invasive or non-invasive: Some technologies require making a hole in the egg. This is a concern, since it creates a risk for contamination and lower hatchability. Therefore, non-invasive technologies are preferred.
• High accuracy: The technology must be very accurate in determining the gender. The accuracy needs to be in line with existing levels of accuracy when sexing chicks after they hatch.
• The sooner the better: The sooner the sex can be determined in the egg the better. To address animal welfare concerns, eggs should be sexed before the age of pain sensation. Although scientists disagree about the exact number of days at which pain is felt, it is clear sooner is better. Obviously, the optimal solution, both for animal welfare and efficiency, would be to do in-ovo egg sexing pre-incubation.
• High speed of sexing eggs: To function in a commercial hatchery, the speed at which eggs need to be sexed must be very high. This is particularly important if sexing happens when eggs have already been in the incubator – to prevent them from losing too much heat. So, technologies need to operate at high speed.
• Small and robust equipment required (for hatchery environment): Going from a laboratory set-up to a commercial hatchery operation is a very difficult step to take. Successful integration in a hatchery requires a robust and easy to clean machine. The technology will have to be fitted in existing facilities.

Ongoing initiatives and their approaches

An ever-increasing number of initiatives are trying to determine the sex of a chicken before they hatch. Hendrix Genetics is actively following the most promising of these around the world. Several different approaches on in-ovo sexing are being tested:

• Spectroscopic detection: Spectroscopy is the technique of splitting light (or more precisely electromagnetic radiation) into its constituent wavelengths (a spectrum) in much the same way as a prism splits light into a rainbow of colors. It requires shining a light beam of laser through the eggshell or a hole in the egg. Via image analysis, the gender is determined. Although training the software to be sufficiently accurate requires tremendous amounts of data and therefore time, this technology does offer the prospect of early identification.
• Biomarker detection: This form of analysis requires taking a fluid from the egg, mixing it with a biomarker, and analyzing the resulting sample. In most cases, allantoic fluid is withdrawn. The fluid is then used for a test that allows to see the gender. There are several different tests being investigated or developed. Most of these tests work after day 7 or 8.
• Gene editing: A few projects are working on a solution using gene editing. The regulatory framework for this is still unclear as is consumer acceptance. On the other hand, if it were to be widely accepted, the benefits of such a technology would be that it is non-invasive and provides opportunity for pre-incubation egg sex detection.

Technologies closest to the market

This list is by no means exhaustive, but it is a selection to demonstrate the variety of approaches.

• Seleggt

The first company to reach the market was Seleggt. Since November 2018, eggs have been sold under the consumer brand Respeggt. The Respeggt label promises: “Free of Chick Culling”. They launched in 200+ supermarkets in the Berlin area and have since significantly expanded their coverage of the German market. Since the summer of 2019, the eggs are also available in Carrefour supermarkets in the Paris region in France. and since 2020 also in the Netherlands. The price is comparable to a free-range egg.

The in-ovo sexing technology has been developed in a joint venture between German retailer the REWE group, Dutch technology company HatchTech, and professor Einspanier from the University of Leipzig. On day 8 of the incubation, a laser beam burns a 0.3mm-wide hole in the shell. Then, air pressure is applied to the shell exterior, pushing a drop of fluid out of the hole. This fluid is analyzed using a proprietary biomarker, which allows them to determine the sex.

• In Ovo

The Dutch In Ovo company was founded in 2013 by biomedical scientist Wil Stutterheim and biologist Wouter Bruins, who met at the University of Leiden. The in-ovo sexing technology they use is based on biomarkers and is invasive. A sample is taken from each egg, mixed with a biomarker, and examined by mass spectrometry. At the end of 2018, Evonik and VisVires New Protein invested in In Ovo, along with the participation of Leiden University. With the investment capital, In Ovo plans to develop the technology to the stage where it can be applied on the commercial scale in hatcheries.

• PLANTegg

The aim of the PLANTegg innovative process is the gender determination in chicken eggs. This avoids the killing of hatched male day-old chicks in hatcheries during the production of laying hens. New technologies have been developed and combined for the so-called PLANTegg process – a PCR-based method for gender determination of hatching eggs. This molecular genetic method is based on the fact that the chickens have different gender chromosomes. The PCR technology makes the genetic differences, to be found in the DNA of the allantoic fluid, visible. The gender can thus be determined at an early stage of incubation with high precision and very quickly (within one hour). The eggs from which male chicks would hatch can then be sorted out early and used for other purposes. The method can be automated, is infinitely scalable and can therefore be used in both small and large hatcheries.

• Hypereye

Hypereye is a patented scanning in-ovo sexing technology that uses non-invasive hyperspectral imaging. This is done at day 0, pre-incubation. The technology was developed by Michael Ngadi at McGill University in Canada, with funding from Ontario Poultry Industry Council, Egg Farmers of Ontario and Livestock Research Innovation Corporation. They are currently testing prototypes to achieve the same accuracy and speed at a commercial scale as seen in the laboratory.

Potential source of new initiatives on in-ovo sexing

While ongoing initiatives on in-ovo sexing continue developing their technology further, from the laboratory to a commercial prototype, to eventually reach the market, new projects and initiatives keep popping up. In many cases, these new entrants are supported with funding from industry and governments.

One such source of new initiatives on in-ovo sexing is the competition organized by the Foundation for Feed and Agriculture Research (FFAR). They are awarding an Egg-Tech Prize to help organizations and individuals solve the scientifically and technologically complex issue of identifying the sex of a chick in the egg prior to certain embryo developmental milestones.

At the end of 2019, they announced that in Phase I of the competition, they awarded USD $1,056,957 to six organizations. Participating researchers are from around the globe and include academics, private research companies, and USDA Agriculture Research Service. The methods on in-ovo sexing these different groups will explore are diverse and include volatile organic compounds, reaction mass spectrometry, and magnetic resonance imagining. In Phase II of the prize competition, contestants will develop and validate a working prototype that meets the criteria that are set. FFAR will accept Phase II submissions in early 2021.

In Ovo Spectroscopy for Chicken Egg Sex Determination

In ovo spectroscopy is an emerging technology for the welfare of baby chicks. In the egg industry male chicks are considered flaws in production. Egg laying breeds do not grow enough meat for production, and males don’t produce eggs. Traditionally, male chickens were raised for meat and female chickens were kept for laying eggs. However, in the 1920’s-1930’s new breeds emerged that were each efficient in either fattening or egg laying. Thus, making male chick culling a commonplace action.

Today, day old male chicks are often grated, gassed, or even thrown in trash bags and are left to get crushed or suffocate to death. The toll amounts to about 7 billion worldwide per year according to the PoultrySite (about half of new born chicks). Some of the chicks are used in animal feed, fertilizer, or the pharmaceutical industry. However, this accounts for less than 2-3% of the toll. The culling of male chicks, especially on such a large scale raises ethical and animal welfare issues.

Efficiency Concerns

Additionally, the current process of sexing chicks is very labor intensive. Trained workers must sort out subtle differences in wing length or they may have to squeeze out a chick’s feces until its genitalia become visible to determine the chick’s gender. It would be much more convenient for the industry to be able to sort chicks before they hatch. This would save time and money going into incubation and making space for the eggs. Fortunately, new technologies are arising to pre-determine sex of each embryo before hatching. The male eggs can then be used either culled ethically or used in flu vaccines. Flu vaccines require about 100 million fertilized eggs per year.

In today’s article we will explore two different In Ovo Spectroscopy technologies and methods that can be used for chicken egg sex determination.

In Ovo Spectroscopy

Fourier Transform Infrared Spectroscopy

Process

Fourier Transform Infrared (FTIR) Spectroscopy can be used on newly laid chicken eggs. This form of spectroscopy relies on finding the blastoderm and inspecting the chemical makeup of its cells. To start the process of sexing, a device must shine infrared light on the blastoderm. However, the eggshell poses a barrier to optical inspection. Thus, most arising spectroscopic techniques require a small hole inside the eggshell. This can be done very precisely and under one second, with a CO2 laser. If the high-energy laser touches the blastoderm cells, they will be harmed and it could prevent hatching. In order to prevent this occurrence, the laser is directed to only penetrate the calcified shell. This leaves the inner membrane of the shell behind, protecting the future embryo.

Next in the FTIR Spectroscopy process, a detector will collect data on the reflected and transmitted light from the blastoderm cells. This data is a form of “molecular fingerprint” of the cells. Male and female blastoderms contain different chemical molecules, each producing a different light signature. Specifically, male blastoderm cells have more DNA than female cells. This whole sexing process with FTIR Spectroscopy takes only a few seconds.

Effects of In Ovo Spectroscopy- FTIR

In a study done in 2018, the smallest possible hole that would allow optical analysis was 12mm. This study also addressed the affect of FTIR light on hatching rates of inspected eggs. The researchers took a batch of 4,736  eggs and divided them into one control and several experimental group(s). Each of the groups incubation conditions were varied. Shell holes were opened by laser method described above.

The results showed a drastic difference between the hatching rates of non-incubated and incubated eggs post-FTIR Spectroscopy. The longer the eggs were incubated for (up to 72 hrs in this study), the higher the hatching rate. The non-incubated and freshly laid eggs saw a reduction in hatching rate by 6.6% compared to the lowest incubation time (24 hrs). [1] The FTIR process must be done with care as blastoderms are delicate and sensitive to their environment. Infrared light is strong enough to induce molecular changes in the blastoderms.

Raman Spectroscopy

Raman Spectroscopy is a similar method to FTIR In Ovo Spectroscopy. It is similar in that it utilizes the scattered light pattern of the early embryo cells to determine its biochemical composition. However, Raman Spectroscopy differs from FTIR in the method in which it identifies this composition. One difference is that the light used to illuminate the sample is in the near-infrared (NIR) light range which prevents damaging cells on contact.

Process

To start the Raman scattering process, a diode laser emitting a single wavelength in the NIR range (e.g. 785 nm) should be set up to point at a single blood vessel. It should connect to a microscope using optical fibers in the 100 micrometer range. There are cameras that have been in use that will automatically find and track good blood vessel specimens.

Once the scattering is collected by a detector, the pattern is analyzed through sophisticated methods. For example, chemometric techniques can be used with a superposition of high intensity fluorescent light and the weaker Raman signals. The reason why we cannot just analyze the intensity pattern directly is because there is a lot of scattering variation when Raman spectroscopy is involved. This would lead to inaccurate gender readings.

Analysis

Next, the superposition’s bands can be matched up to the hemoglobin, lipids, and nucleic acids in the blood cells of the embryo. Specifically, the hemoglobin provides the fluorescent light signals. Now we can determine the gender of the chick. As mentioned previously, the sex chromosomes and the amount of DNA in the blood cells of males are 2% larger. Further, after day 13 of incubation male embryos will have a stronger fluorescent signal than females. Additionally, the males have larger signals that correspond to C-C stretching modes and phosphodiester stretching vibrations of the DNA nucleic acids. On the other hand, females will exhibit larger amide III and CHx deformation modes.

Effects of In Ovo Spectroscopy using the Raman method

It should be noted that a 2016 study was done with Raman Spectroscopy where eggs were incubated for 80 to 88 hours. The shell hole width diameter was lasered to as low as 10 mm. Compared to the 12mm study group, the hatching rate was 4% higher. The minimum width of the hole that would allow adequate optical analysis is determined by the highest level of the numerical aperture of the microscope’s objective. However, in this particular study it was found the 12mm holes gave more reliable and consistent results. If the inner membrane covering the hole is punctured, bioadhesive tape may be used to seal it up.

Conclusion

As new technologies emerge utilizing In Ovo Spectroscopy, the mass culling of male baby chicks in cruel ways could soon come to an end. Some concerns about these technologies include the accuracy of sexing, cost-efficiency, and producing fast enough rates for the industry. Further, these techniques should be used before the chick embryo develops its sensory nervous system, which is around day 7, so that culling of the egg does not produce negative reactions. But the future of ethical egg farming is close in sight! The United Egg Producers and Human League are fine tuning these In Ovo Spectroscopic technologies for industrial usage.

Compiled  & Shared by- Team, LITD (Livestock Institute of Training & Development)

Image-Courtesy-Google

Reference-On Request.