In ovo sexing of chicken eggs by fluorescence spectroscopy
Culling of day-old male chicks in production of laying hen strains involves several millions of animals every year worldwide and is ethically controversial. In an attempt to provide an alternative, optical spectroscopy was investigated to determine nondestructively in ovo the sex of early embryos of the domestic chicken. The extraembryonic blood circulation system was accessed by producing a window in the egg shell and the flowing blood was illuminated with a near-infrared laser. The strong fluorescence and the weak Raman signals were acquired and spectroscopically analyzed between 800 and 1000 nm. The increase of fluorescence intensity between 3.5 and 11.5 days of incubation was found to be in agreement with the erythropoietic stages, thus enabling to identify hemoglobin as fluorescence source. Sex-related differences in the fluorescence spectrum were found at day 3.5, and principal component (PC) analysis showed that the blood of males was characterized by a specific fluorescence band located at ∼910 nm. Supervised classification of the PC scores enabled the determination of the sex of 380 eggs at day 3.5 of incubation with a correct rate up to 93% by combining the information derived from both fluorescence and Raman scattering.
KeywordsOptical spectroscopy Fluorescence Raman scattering Chicken embryo Sexing In ovo
The possibility to determine the sex of birds “in ovo” is driving increasing attention as a potential method to overcome the culling of day-old male chicks in poultry industry. Laying hen strains of modern breeding differ from broiler strains, so that male birds of the laying strain are not profitable for meat production. Therefore, day-old cockerels are culled directly in the hatchery. This practice involves a very large number of animals: approximately 370 million in North America and 420 million in Europe every year . Only in Germany, 40 millions of chicks are killed every year according to animal welfare legislation by asphyxiation with carbon dioxide or by grinding . Killing of day-old chicks is considered as problematic and ethical issues have triggered increasing research aimed to provide alternatives .
The optical spectrum contains information about the biochemical composition and/or the structure of a biological sample and can provide the information about the sex as well. Vibrational spectroscopic techniques have been applied to the sexing of birds, either of hatched birds as well as of incubated and unincubated eggs. UV resonance Raman spectroscopy  and Fourier-transform infrared absorption spectroscopy [5, 6] were used to retrieve the sex of hatched animals based on DNA differences by analyzing cell material extracted from the feather pulp. The use of Fourier-transform infrared spectroscopy was also reported earlier by our group for sex recognition based on the spectrum of germinal cells obtained from unincubated eggs .
Optical methods for in ovo sexing have the advantage of being applicable in situ without taking samples, and can provide real-time sexing by eliminating the need to wait for the results of chemical or genetic analyses performed on previously extracted samples. Therefore, optical methods have clear advantages toward an industrial exploitation when compared to other non-destructive approaches proposed for in ovo sexing that are based on measurement of hormone [8, 9, 10] and DNA analysis [11, 12, 13], which all require the extraction of egg material or fluids. Totally non-invasive methods, like the selection of unincubated chicken eggs based on morphometric parameters, can only bias to a low extent the ratio between sexes . There is also evidence that egg odor encodes sex information; however, this was not exploited for sexing .
We recently reported near-infrared Raman spectroscopy for in ovo sexing of incubated chicken eggs, showing that it provides correct sexing up to 90% without hindering embryo development . We demonstrated that, already during the fourth day of incubation (i.e., 84 ± 4 h), Raman measurements can be performed directly on the blood that flows in the extraembryonic vessels of the vitelline circulation avoiding all damage to the embryo. The Raman spectra delivered the biochemical fingerprint of the embryonic blood, from which the sex information was obtained by use of supervised classification algorithms. In this study, we observed that the fluorescence background intensity was significantly different between sexes, but this information was not exploited because of the very high overlap between sexes.
In Raman spectroscopy, the presence of an intense fluorescence background is normally considered a pitfall rather than a source of information and thus suppressed or removed during data processing . Fluorescence spectra of biological tissue are broad compared with Raman and FT-IR spectra, and they do not carry such detailed biochemical information. However, spectral fluorescence-based methods have continuously evolved and improved enabling to address new cellular features .
Here, we show that spectral analysis of the near-infrared fluorescence signal of the blood flowing in the extraembryonic vessels can indeed provide sex information of the domestic chicken eggs (Gallus gallus f. dom.). For this purpose, we analyzed spectroscopically the backscattered radiation of blood illuminated with a 785 nm laser, repeating the measurements at different time points of the incubation and comparing with the time course of erythropoiesis to identify the source of the fluorescence signal. Afterwards, we exploited the fluorescence spectral profile for sex determination and compared the results with the combination of fluorescence and Raman scattering.
Fertilized eggs of a white layer strain (LSL—Lohmann Selected Leghorn) were used in all experiments. They were obtained from Lohmann Tierzucht GmbH (Cuxhaven, Germany). The freshly laid eggs were inspected for any shell damage and then stored at approximately 14 °C. Immediately before starting the incubation, egg shells were windowed using a 30 W CO2 laser (Firestar v30, Synrad, Mukilteo, Washington, USA) equipped with scanning head (FH Flyer, Synrad, Mukilteo, Washington, USA). The shells were laser-scribed at the pointed end along a round path of 12 mm diameter, without removing the shell window. The incubation was performed in an automatic egg incubator (Favorit Olymp 192, Heka-Brutgeräte, Rietberg, Germany) at 37.8 °C and humidity of 53%, in vertical position with the pointed end downward. A ±45° tilting at an interval of 3 h was applied during the incubation time until day 3.5 (i.e., 84 h). At this time point, the eggs were subjected to the measurement.
The shell window was gently removed using a scalpel and the spectrum was acquired. After measurement, embryonic tissue samples were isolated for subsequent molecular sexing, or the shell windows were closed using a biocompatible adhesive tape (Leukosilk, BNS Medical GmbH, Hamburg, Germany) and further incubated to perform time-course experiments. Measurements were repeated at day 4.5, 5.5, 6.5, 7.5, 9.5, and 11.5. At each time, the tape was removed before the measurement and reapplied afterwards. The incubation between the measurements was performed with the egg in upright position to maintain the embryo and the main blood vessels optically accessible. Before measurement, the eggs were inspected and the spectrum was acquired only on blood vessels of vital embryos. Otherwise, samples of embryonic tissue were isolated and kept frozen at −80 °C for subsequent sex determination. After the last measurement at day 11.5, all embryos were isolated and kept frozen for sex determination as well.
Reference sexing was obtained with genetic analysis on embryonic tissue based on polymerase chain reaction (PCR). Alkaline extraction of DNA was performed as described elsewhere . The samples were treated in NaOH for 20 min at 75 °C and subsequently neutralized with Tris–HCl (pH = 7.5). Afterwards, the samples were centrifuged for 10 min at 14,000 rpm and the supernatants transferred to reaction tubes. The DNA content of the supernatant was measured using the Genesys 10 Bio UV–vis spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The amplification of the CHD-1 gene was performed in the PCR cycler T Gradient (Biometra GmbH, Göttingen, Germany). The primers and the temperature profile are described elsewhere [20, 21]. Finally, the amplified PCR products were separated by ethidium bromide agarose gel electrophoresis and visualized by UV light.
A Keplerian telescopic system was used as beam-expander to collimate the laser beam and to match the diameter to the objective pupil. The beam-expander was connected to a 45° filter cube that contained a short-pass dichroic mirror with edge at 670 nm (FF670-SDi01, Semrock Inc., Rochester, New York, USA). The mirror reflected to 90° the laser excitation in the microscope objective ×20/0.4NA Plan Apo NIR (Mitutoyo Corp., Kanagawa, Japan), which was also mounted on the filter cube. The laser spot in the focus had a diameter of ∼55 μm and the measured laser power was 160 mW. The light was collected in reflection mode by the objective. The collected near-infrared light with wavelength above 670 nm was reflected by the dichroic mirror back to the fiber probe and propagated to the spectrograph. The collected visible light was transmitted through the dichroic mirror and used to obtain the image of the sample. For this purpose, a CCD camera with 50 mm focal length lens and a NIR hot mirror were mounted on the aperture of the filter cube that faces the objective. In order to maximize the visibility of perfused blood vessels, side illumination with green LEDs was employed. A motorized x-y-z micrometer stage was used to move the egg and bring a blood vessel in the laser spot. Blood vessels with diameter larger than 100 μm were manually chosen for the measurement. An autofocus system based on blood flow detection in the camera images was used to set the laser focus inside the blood vessel. A tracking system remained active during the whole acquisition to compensate all movements of the blood vessel. The autofocus and tracking software modules are described elsewhere . An enclosure of the system provided rejection of ambient light and protection from reflected or scattered laser light during acquisition.
Total acquisition time was set to 40 s (20 accumulations of 2 s). The acquired spectral range was from 794 to 1054 nm (i.e., from 150 to 3250 rel. cm−1) and the spectral resolution was 0.3 nm (i.e., approximately 4 cm−1).
Spectroscopic data were analyzed using the MATLAB package (MathWorks Inc., Natick, USA) and statistics were calculated with Prism 6.0 (Graph Pad Software Inc., La Jolla, CA, USA).
The intensity of the fluorescence was calculated as sum area under the spectra in the range 820–1000 nm. Principal component analysis was performed on the raw spectra by using the MATLAB function princomp. Classification was performed using supporting vector machine. The MATLAB functions svmtrain was employed to train the classifier, by using a quadratic kernel and least-square method to find the separating hyperplane. Afterwards, the function svmclassify was employed in order to retrieve the classification.
Results and discussion
Spectral analysis of blood fluorescence
In the chicken egg, blood islands appear toward the end of the first day of incubation and already at day 2 of incubation the blood circulation starts driven by the primitive heart. Between day 3 and 4, the vascularized area of the yolk sac is roughly circular and reaches a diameter between 3 and 4 cm at day 4. The major blood vessels are the paired lateral vitelline arteries and veins, and the unpaired anterior and posterior vitelline veins. The branching pattern of arteries and veins is dichotomous, creating a treelike topology. Around day 8, the respiratory function is transferred to the blood vessels of the newly formed chorioallantoic membrane . When the egg is brought in vertical position at day 3.5 of incubation, the vascularized area moves on the top and, after opening of the shell window, it remains on the surface, so that the vessels can be optically sampled through the shell window (Fig. 1, inset). The position of measurement was always chosen on a large vessel (diameter larger than 100 μm), preferably in main lateral or anterior/posterior veins.
Different erythropoietic phases take place starting at day 2. The primitive erythrocytes undergo six rounds of mitosis until day 5. In the middle of day 5, the definitive erythrocytes start entering the circulation and gradually replace the primitive line by day 7. By day 8–9, the erythrocytes have completed their maturation . The time course of fluorescence is in agreement with the time course of hematocrit observed during erythropoiesis , and mimics the increase of hemoglobin measured in embryonic and mature erythrocytes . A linear increase of hemoglobin content was reported until day 5, followed by a plateau between day 5 and 6, corresponding to the transition from the proliferative to the post mitotic phase of primitive erythrocytes. This plateau can be seen also in the time course of fluorescence. Afterwards, the hemoglobin content increases again until day 9 and slightly decreases afterwards, when the erythrocyte maturation is concluded. Fluorescence intensity displays a similar trend at day 9.5 and 11.5 as well. The comparison between the time courses of fluorescence intensity and hemoglobin content indicates that hemoglobin is the main source of the observed NIR fluorescence.
Heme in red blood cells is a molecule that belongs to the family of porphyrin complexes. Porphyrins normally display strong reddish-orange fluorescence. However, heme constitutes an exception, as the fluorescence of the porphyrin ring is quenched by the coordinated iron . Steady state fluorescence of heme in the UV range is nevertheless observed and attributed to fluctuations in the protein structure that partly neutralize the quenching. The source of steady state fluorescence is identified in the tryptophan residues . Moreover, it was already shown in vitro that excitation at 785 nm of methemoglobin over the concentration range spanning the normal capillary blood range produces a fluorescence that increases linearly with increasing hemoglobin volume percent , although fluorescence detected in vivo was attributed to both plasma and hemoglobin .
In our experiments, the fluorescence was observed only during irradiation of blood (i.e., acquiring the signal from perfused blood vessels and from the heart chambers), while other embryonic tissue, egg components (egg white and yolk), as well as extraembryonic tissue (vitelline and corioallantoic membranes) did not display any fluorescence, but only Raman scattering. Moreover, blood taken from embryos at day 11.5 of incubation was centrifuged to separate erythrocytes from plasma and then the two components were subjected to spectroscopy. While the erythrocytes displayed strong fluorescence signal, plasma displayed Raman scattering only (Fig. 2c).
It is also worth to note that any bleaching of fluorescence during irradiation of perfused blood vessels is prevented by the blood flow itself. The blood flow velocity in the center of large vitelline vessels is around 1 mm/s already at early development stages , and therefore the exposure time of blood cells traveling through the laser spot is as short as 0.05 s.
The spectral differences between male and female blood fluorescence detected at day 3.5 may be used for in ovo sexing. We also have shown previously that at this time point, it is possible to measure in ovo without impairing embryo development .
In ovo sexing by means of fluorescence
Principal component analysis was applied in order to gain insights in the different spectral contributions that are related to the sex. Mathematically, principal component analyses enables extraction of the most important information from the observation data table, by performing a procedure of linear decomposition and computing new variables called principal components (PC). The values of these new variables are called scores, and can be interpreted as the projections of the observations onto the PCs . This approach allows discrimination among spectral groups using scatter plots of scores, and the loading vectors convey the spectral variations that differentiate the data .
The accuracy is limited by the variability of the data, which was likely related to intrinsic variability of blood composition. Both intensity and spectral shape of fluorescence depended from the development stage of the embryo. The measurements spanned over ∼8 h, and it is expected that developmental variations may occur in embryos even though all eggs are placed in the incubator at the same time. These differences exist inside the same setting of eggs depending from differences in the viability and vigor of embryos, size of individual eggs, or from local temperature gradients inside the incubation chamber . Experimental effects related to different volume sampling due to blood vessel dimensions or variation of the focal position did not affect the classification. This is proven by lack of any correlation between the score of PC #5 (representative of yolk) and the misclassified spectra. The mean score intensity calculated for wrongly classified spectra is −0.002 ± 0.062 (mean ± SD, n = 31) vs. 0.002 ± 0.050 calculated for the correctly classified ones (mean ± SD, n = 349). The mean score intensities are not statistically different (two-tailed t test, p = 0.7).
Compared to our previous research, the present study provides significant improvements. We first proposed infrared absorption spectroscopy of the feather pulp to distinguish the sex of hatched birds [5, 6]. The studies were performed on a small number of animals (turkeys and pigeons), as pilot studies aimed to demonstrate that optical spectroscopic methods can retrieve sex-related biochemical differences in birds. Furthermore, we investigated infrared spectroscopy of the germinal disk extracted from unincubated chicken eggs . Sexing was achieved with high accuracy, but the approach is not transferrable in the practice because windowing of unincubated eggs causes high occurrence of embryo mortality and morbidity [42, 43]. By analyzing the Raman spectra of embryonic blood, we achieved sex determination with an accuracy comprised between 88% and 90% . Moreover, the use of NIR laser excitation avoided phototoxic effects on embryos, so that healthy chicks hatched from the measured eggs. Here, we retained the experimental configuration with NIR excitation in order to rule out negative effects on embryos, but all spectral features of the backscattered spectra were used for sexing, thus exploiting the sex information conveyed by fluorescence too, and increasing the correct classification rate well over 90%.
The exploitation of multiple information contained in the backscattered light might open new possibilities for improvement of in ovo sexing accuracy. Fluorescence intensity, fluorescence spectral shape, and Raman scattering encode sex-related information that are associated to different blood components. Several approaches of spectral preprocessing could be applied in order to “amplify” a specific feature and afterwards a multi-parameter classification strategy developed to better comply with intrinsic data variability, thus further increasing the sexing accuracy. Moreover, the exploitation of fluorescence may contribute to overcome some disadvantages associated with Raman spectroscopy of biological samples, as Raman scattering is a low-intensity process which requires highly efficient laser sources, low-noise detectors, effective Rayleigh filters, and high-throughput optics . As fluorescence intensity is some orders of magnitude larger than the Raman scattering signal, it might enable to largely simplify the experimental setup and facilitate the future industrial deployment of optical techniques for in ovo sexing.
The results show that the sex information can be extracted from intensity and spectral shape of the near-infrared fluorescence of embryonic blood. Source of fluorescence was identified in the hemoglobin and the spectral features of fluorescence depend from the hematopoietic stage. The intrinsic variability of development stages affected the accuracy of sexing based exclusively on fluorescence. As the Raman scattering was simultaneously acquired with the fluorescence signal, it could be employed to further increase the sexing correct rate well over 90%, with a significant improvement compared to sex determination based on Raman spectra only.
The ideal criteria for sex determination have been defined since years and include lack of negative effects on embryo development and practicability on a large scale . In ovo sexing based of spectral analysis of the backscattered radiation satisfies these requirements, as it is non-invasive, does not require extraction of egg material, and does not use consumables. Moreover, the method is applicable during the fourth day of incubation, before onset of embryo sensitivity at day 7 , thus in agreement with animal welfare.
The exploitation of fluorescence offers the potential to develop industrial systems for egg sexing that are not based on expensive spectrometers, but just make use of few light detectors with suited bandpass filters to measure the signal intensity in selected spectral ranges. Afterwards, the sex information may be retrieved with simple calculations, such as ratio of intensities in order to make the method robust toward variations of laser power and detection efficiency. The availability of a cheap and easier approach alternative to spectroscopy might contribute to a broader diffusion of optical sexing in the hatchery practice. On an international scale, development of a practicable technique for in ovo sex determination has the potential to contribute to the prevention of annual culling of 7 billion male layer hybrids, whose female siblings produce the current global demand of about 68.3 million tons of eggs per year.
The authors gratefully acknowledge Andrea Büchner for performing molecular genetic analyses and Lohmann Tierzucht GmbH (Cuxhaven, Germany) for providing eggs. Special thanks to Prof. Rudolf Preisinger and Dr. Anke Förster (Lohmann Tierzucht GmbH, Cuxhaven, Germany) for the insightful discussions about hatchery practice.
Compliance with ethical standards
This work was financially supported by the German Federal Ministry of Food, Agriculture, and Consumer Protection (BMELV) through the Federal Office for Agriculture and Food (BLE), grant no. 511–06.01-28-1-33.010-07.
Conflict of interest
Patent applications for in ovo sexing with the methods described in this paper are pending.
- 1.Turner J. Animal reproduction, human control. In: Bekoff M, editor. Encyclopedia of animal rights and animal welfare. 2nd ed. Santa Barbara: ABC-CLIO, LLC; 2010. p. 30–6.Google Scholar
- 2.Tierschutz-Schlachtverordnung - TierSchlV (BGBl. I S. 2982). 20-12-2012.Google Scholar
- 9.Phelps P, Bhutada A, Bryan S, Chalker A, Ferrell B, Neuman S, et al. Automated identification of male layer chicks prior to hatch. World Poult Sci J. 2003;59(1):33–8.Google Scholar
- 14.Yilmaz-Dikmen B, Dikmen S. A morphometric method of sexing white layer eggs. Braz J Poult Sci. 2013;15(3):203–10.Google Scholar
- 19.Rudbeck L, Dissing J. Rapid, simple alkaline extraction of human genomic DNA from whole blood, buccal epithelial cells, semen and forensic stains for PCR. Biotechniques. 1998;25(4):588–90.Google Scholar
- 22.Schart C. Thesis: Entwiklung einer Steuerung für die Fokussierung und das Tracking embryionaler Blutgefäße im Hühnerei. Hochschule für Technik und Wirtschaft Dresden. 2015.Google Scholar
- 23.Baumann R, Meuer HJ. Blood-oxygen transport in the early avian embryo. Physiol Rev. 1992;72(4):941–65.Google Scholar
- 25.Johnston P. Hematocrit values for the chick embryo at various ages. Am J Physiol. 1955;180(2):361–2.Google Scholar
- 26.Campbell GL, Weintraub H, Mayhall BH, Holtzer H. Primitive erythropoiesis in chick embryogenesis. 2. Correlation between Hemoglobin Synthesis and the Mitotic History. J Cell Physiol. 1971;50(3):669–81.Google Scholar
- 33.Morita VS, Boleli IC, Cargnelutti A. Hematological values and body, heart and liver weights of male and female broiler embryos of young and old breeder eggs. Braz J Poultry Sci. 2009;11(1):7–15.Google Scholar
- 38.Cuadros MA, Coltey P, Carmen NM, Martin C. Demonstration of a phagocytic cell system belonging to the hemopoietic lineage and originating from the yolk sac in the early avian embryo. Development. 1992;115(1):157–68.Google Scholar
- 45.Aleksandrowicz E, Herr I. Ethical euthanasia and short-term anesthesia of the chick embryo. Altex-Altern Tierexp. 2015;32(2):143–7.Google Scholar