# In-ovo sexing

In poultry farming, in-ovo sexing is a chick sexing method carried out while chicks are still in ovo (Latin for "inside the egg"). There are various methods to determine a chick's sex in the 21-day incubation period before it hatches.
In-ovo sexing technology has branched into two categories, invasive and non-invasive. The first invasive technology to be successfully commercially introduced for poultry farming was bio-market detection through the Dutch–German company Seleggt in November 2018. Meanwhile, the non-invasive in-ovo sexing technologies have been introduced to the market by the German start-up Orbem and AAT, a subsidiary of the EW Group.

## History
### Background

A present-day ethical problem with egg production is chick culling of one-day-old male chicks, billions of male chicks that are killed as part of the production process each year. At the day the chicks hatch from their eggs the chicks are sexed. During chick sexing the day-old chicks are divided into male and female groups. Female chicks can be raised to become egg-laying hens, or broilers fed to be slaughtered for meat, both for human consumption; after the sexing, these female chicks are transported to the rearing farms where they are housed before they go to a laying hen farm or broiler farm. On the other hand, the male chicks are deemed to have much less economic value, as they cannot lay eggs and are usually less suited for meat production; most male chicks are therefore culled on the day they hatch after they have been sexed as male.

### Innovation in the poultry sector

In-ovo gender determination has the potential to bring an end to the killing of billions of male chicks. It is estimated that yearly around 7 billion day-old male chicks are killed. Implementing in-ovo sexing into the poultry industry results in a more animal friendly and more sustainable production: More animal friendly because the day-old male chicks no longer need to be culled, and more sustainable because less energy is used as only the female eggs need to be further incubated after sexing. The male eggs are sorted out and can be used for different purposes such as an alternative high-value protein source. When there are no longer males that hatch, other innovations are also available to egg producers, such as on-farm hatching and in-ovo vaccination.

### Male-chick culling ban

In January 2021, Germany was the first country to successfully outlaw the practice. A few months later, France also banned the one-day-old male chick slaughter. In 2022, the third country, Italy, followed the same steps, prohibiting the practice by 2026. Efforts have been done to push an EU-wide ban with standardized regulation for all countries. In 2024, the UK government's official Animal Welfare Committee recommended that the UK ban chick culling, although their recommendation is not legally binding.
In the US, male culling has received substantial attention from animal activists, although in-ovo sexing is not currently available. However, the egg industry has indicated that they will strongly support the adoption of this technology once it is available.
A number of state governments in India have mandated that in-ovo sexing be used once it becomes available.

### Early technological breakthroughs
#### Academic research

For a long time, it was held impossible to determine the sex of the hatching egg before or during the hatching process. The poultry sector has been working on this for years in order to be able to phase out chick culling in the interest of animal welfare.
Research has been conducted to achieve this goal. The first study on the matter, was published in 2013, for in-ovo sexing on day 9 of incubation. The researched procedure, later called bio-marker detection, used a hormonal test for the allantoic fluid of brown layers’ eggs. Further research was done by Prof. Dr. Einspanier following the same methodology. It is important to mention that hatchability is affected by this in-ovo sexing method, as reported by Dr. Einspeiner.
In 2016, the fluorescence spectroscopy methodology was developed, it analyzes the extraembryonic blood to determine the sex of the embryo through its blood wavelength. The supervised egg classification by a PC with a 93% error rate was able to determine the sex of 380 eggs at 3.5 incubation day. A variation of this methodology was explored in 2017. When the pattern analysis in hyperspectral images methodology was researched. First, the eggs would be candled with halogen lamps. Then, a hyperspectral camera would collect the transmitted light and the eggs would be classified using a linear discriminant analysis. This methodology could perform in-ovo sexing from 11-day up to 14-day embryos with a 97% accuracy.
In 2019, a new methodology was developed, AI-powered imaging. By combining AI and MRI together to perform in-ovo sexing of 12-day-old eggs with 95% accuracy level. Since 2007 MRI was used as a valuable tool for studying egg development in a contactless manner. Yet it would not be until it was combined with AI that it could be used for large-scale purposes.

#### First commercial application

In 2018, Seleggt was the first company to succeed in commercial in-ovo sexing. Seleggt managed to sex the hatching eggs on day 9 of the incubation process with a hormone test. The method is based on the fundamental research of Prof. Dr. Einspanier at Leipzig University. From 8 November 2018, consumption eggs that are laid by the hens that have been sexed with the Seleggt method are to be found on the shelves of the German supermarket REWE, in the Berlin region. The eggs that have been sexed with the Seleggt method are sold under the label "Respeggt". This label guarantees the promise "Free of chick culling". Since 2018, Respeggt eggs have been available in Germany, France, and the Netherlands.
European rollout
Since the launch of in-ovo sexing in 2018, multiple companies have had success in Europe, implementing different methodologies and technologies for in-ovo sexing. As of September 2023, it was estimated that 15 per cent of the layer flock in Europe used in-ovo sexing, from five different companies. An in-ovo sexed chick is estimated to cost around $3.80, compared to $0.95 for a conventional chick. Since a chick lays around 350 eggs over its life, this means an additional 0.8 cents per table egg sold. These eggs then sell for around 1-3 additional cents per table egg, the difference including the additional margins from each part of the supply chain.

## Sex determination methods

The in-ovo sexing technologies have evolved into two main categories: optical and non-optical (Fig. 1). Non-optical technologies are any techniques that requires extracting a sample from the egg through intrusive sampling and biochemical analysis for the in-ovo sexing process. Examples of these are biomarker detection, PCR and ELISA. The main drawbacks of invasive procedures is that they may affect the eggs’ hatchability and that the process implies a risk for bacterial infection. On the other hand, optical technologies, have a contactless in-ovo sexing procedure (e.g.y, spectroscopy, AI-powered imaging) are free of this previous risks.

### Non-optical technologies
#### Biomarker detection (Respeggt, In Ovo)

The Dutch–German company Respeggt measures a substance that is a 'biomarker' for the sex through a small hole in the eggshell on day 9 after fertilisation. After extraction of the fluid from the fertilized egg, the presence of a female-specific hormone is tested using an ELISA test. The test output indicates its presence: yellow a female during the absence of color a male, with a 98.5% accuracy rate. As of May 2019, Respeggt sexed one egg per second (3,600 an hour) and thus enabled 30,000 'no-kill' female chicks to hatch in Germany every week. The German Federal Ministry of Food and Agriculture (BMEL) has invested 5 million euros in the development of the Respeggt technology.
Another Dutch company is In Ovo, a spin-off of Leiden University, Netherlands. This company was founded in 2013 by students Wouter Bruins (biology) and Wil Stutterheim (biomedical sciences) and, just like Seleggt, uses a small amount of liquid from the hatching egg and determines whether it is a male or female hatching egg by means of biomarker detection on the 9th day. By January 2020, In Ovo was capable of sexing 1,500 eggs an hour (0.42 per second), but the Dutch poultry sector required 40,000 eggs an hour, so further innovation was necessary. In Ovo received millions of euros in research investments, mostly from German chemicals company Evonik, Singaporese venture capital company Visvires New Protein, and Rabobank Leiden-Katwijk.

#### PCR (Plantegg)

The German company Plantegg uses a PCR method, which uses DNA to determine whether the hatching egg is male or female. Like In Ovo and Seleggt, this method determines the sex on day 9 of the incubation process. This method is expected to be ready for use by the end of 2020.

### Non-invasive technologies
#### Spectroscopy (AAT, Project Soo, Hypereye)

Another German company, Agri Advanced Technologies (AAT), uses spectroscopy to determine the sex of the egg. The hatching egg is examined with by light beam, with a hyperspectral measuring technology the sex is determined on the basis of the calculated light spectrum. This method works for hatching eggs from sex-linked breeds where male and female birds have different color plumage, all males white-feathered and all females brown-feathered for example, and can take place from the 13th day of the hatching process. AAT's goal is to eventually be able to sex eggs at the 4th day. The German Agriculture Ministry has also invested in AAT's technological development.
The French company Tronico, based in La Roche-sur-Yon, collaborates with the French National Centre for Scientific Research (CNRS) on Projet Soo, which employs a mix of spectroscopy and the use of biosensors with the target of achieving 90% accuracy in ovo sexing at 9 days of incubation by the end of 2019. In 2017, French Agriculture Minister Stéphane Le Foll granted Projet Soo 4.3 million euros to finance its egg sexing research.
Hypereye is a Canadian spectroscopic technology that is being developed by the Egg Research Development Foundation (ERDF), initially funded by Poultry Industry Council in Ontario and later by Egg Farmers of Ontario (EFO). It aims to achieve a 99% accuracy rate and to process 30,000–50,000 eggs per hour (8.3–13.9 eggs per second). In 2018, Canadian Agriculture Minister Lawrence MacAulay announced an $844,000 investment in the research project to stimulate its development.

#### AI-powered imaging (Orbem)

Based on years of scientific research at the interface of AI, imaging technology and embryonic poultry development, Orbem was founded in 2019 as a spin-off from the Technical University of Munich. Orbem uses AI-powered MRI to conduct in-ovo sexing on day 11 - 12 of incubation, with a throughput of up to 24,000 eggs per hour. The solution applies equally to brown and white eggs. The solution is contactless and has no effect on the hatchability rate.
Genus Focus has different installations across Europe, including France, Germany, the Netherlands and soon Norway. Orbem has entered a strategic partnership with the Vencomatic Group (NL) for end-to-end automation of the process, which includes financing facilitated by the Vencomatic Group of up to 15 million Euros for equipment to be deployed at customer sites throughout Europe. This allows hatcheries to use the combined solution without any upfront investment just based on a performance fee per egg.