In ovo sex determination

We, who are active in the world of poultry, all know it very well, but are you sure that the consumer is also aware that his roast chicken isn’t the brother of the hen laying his eggs for breakfast?

A highly negative correlation prevents breeding improvements in both laying performance and growth at the same time. This is how specialized lines came about as time went by, either for efficient production of high quality eggs or efficient production of poultry meat. Whilst both males and females are fattened in broiler production, it is not possible for males of layers to be reared economically. The result? Routine culling of day-old male chicks in the hatchery which is a great ethical problem and finding alternative solutions to this is a big challenge for breeding companies, science and hatcheries.

In this context, several projects are discussed and underway:

1. Spring chickens: despite their reduced growth performance, males of layer lines are still reared and fattened. Dependent on feeding and breed, the birds can be slaughtered at an approximate weight of 650 g after 49 days.

2. Dual purpose breeds: a cross of meat and layer lines which result in a „compromise“ that naturally comes along with both a lower egg production and growth performance and higher production costs (feed consumption).

3. Sex determination before eggs are incubated: with the aid of optical methods in which light is used for analysis, the germinal disc is examined to determine the size of the chromosome which indicates the sex of the embryo.

4. Sex determination of incubated eggs: male and female embryos can be detected according to different hormone levels in the allantoic fluid.

LOHMANN TIERZUCHT is involved in each and every one of these projects. In one research project, LTZ reared spring chickens and initiated marketing strategies for this niche product. The combined breeding for egg production and growth performance at the same time, resulted in „LOHMANN DUAL“, a dual purpose bird that received a lot of attention at the Franchise Distributor Meeting 2012 in Berlin.

The third and fourth topics – possibilities and applications of in ovo sex determination prior to or during incubation, are subjects of a research project supported by the Federal Institute of Agriculture and Nutrition (BLE), where aside from LOHMANN TIERZUCHT scientists of the universities of Leipzig, Dresden and Jena are involved. The endocrinological approach, i.e. in ovo sex determination by hormone levels, will be described in this article.

For several years now, there are techniques which are known to be able to determine the hormonal status of embryos in the second half of the incubation period (i.e. days 13 – 17). Second phase particles such as sex determining hormones and related substances are accumulated in the allantois – the urinary bladder of the embryo – in different amounts, which can be used for gender differentiation. However, analyses at this stage are relatively late and must also be critically considered from an ethical point of view. According to today’s knowledge, the embryo’s perception of pain begins on around the 10th day of incubation, so culling / removing eggs from the incubator at a later stage cannot be seen as an alternative to the culling of day-old chicks.

In numerous trials with several hormones at different stages of development, the working group of the Leipzig University led by Prof. Einspanier in the Endocrinology Department found out that hormones and their derivates can be detected in allantoic fluid as early as the seventh day of incubation. The amount of estrone sulfate (a form of oestrogene) has been proven to be the best indicator for gender differentiation. Whilst there were no differences on day 7, the amount of estrone sulfate on day 9 was three times higher in female embryos than in males (Weißmann et al., 2013). Hatching eggs containing male embryos could therefore be easily recognised and removed making it possible for only female chicks to hatch after 21 days.

Figure 1: Experimental set-up in the hatchery

Figure 2: Withdrawal of allantoic fluid

How does this work practically?
Essentially:

1. Eggs are incubated for nine days, as usual.

2. Eggs are candled on day 10.

3. The egg shell of fertilized eggs is per forated (e.g. with a drill) close to the air cell.

4. A small amount of allantoic fluid is withdrawn with a syringe.

5. Hormone content is measured with the aid of an ELISA-test in the lab.

6. The amount of estrone sulfate is an indicator for the sex of the embryo.

With much more eggs than in laboratory trials, the practicability of this approach was tested in the hatchery. In three different trials, allantoic fluid was obtained from more than 4000 LB eggs either on day 9 or 10 of incubation. Almost the same number of eggs was used for each test as control group. All the eggs were labelled and hatched separately under a cover so that the predicted and actual gender could be assigned individually. Furthermore, chicks were weighed individually after each hatch. In these trials, sex could be correctly determined in nearly 95 % of the chicks, with only less than 3 % reduction in hatchability. Chick weight was not significantly different ranging from 37.9 – 38.6 g.

Have all problems been solved now?

For starters: Yes, sex determination with this technique is indeed possible with sufficient accuracy and only small losses in hatchability. But (and this is a big But!): At the moment, one person is able to take 250 samples on one day in the hatchery. And again one person per day is needed to analyse these 250 samples in the lab since analyses are not possible in the hatchery. Considering that 45 million female chicks are hatched in Germany per year, around 2.3 million eggs would have to be analysed per week!

So although the results are promising, inovo sex determination still seems to be far off from daily business. The automation of the technical processes, egg handling and the implementation of short-term analytics in the hatchery are challenges we have to deal with in the near future.

Dr. Anke Förster
Genetics