1. Introduction

2. Non-optical in ovo sexing methods

2.2. Immunosensing

Hormones play an essential role in chickens' sexual differentiation, with concentrations changing during incubation and growth of the fetus. A simplified cascade of these main hormones is depicted in Figure 2. These hormones are mainly gauged via competitive enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) due to the high accuracy and sensitivity of these assays. Competitive ELISA or RIA assays are based on the competition between the ssample target and the same labeled target for specific antibodies, whereas the label for a target can be either a fluorescent enzyme (ELISA) or radioisotope (RIA). Table 1 gives a brief overview of papers and patents with the hormones analyzed using immunoassays.

The first paper regarding the use of immunoassays dates back to 1979 when Tanabe et al. reported detecting hormones from embryos (day 17 and 20) [23]. Moreover, a paper from Gill et al. [24] showed the possibility of detecting several hormones from day 10 and 14 (Table 1), while showing the hormonal level increase during the incubation stages. Furthermore, in 1998, Patricia Phelps protected [25] and published [11] a method measuring estrogens and androgens in extra-embryonic fluid (e.g., AF). Tyczkowski [26] patented another method in 2006, describing an assay type to detect estrogenic steroid compounds using microparticles and a specific fluorescently labeled binding protein.

Furthermore, Butt and Tran [27] patented, in 2002, a new yeast-based assay type for detecting E2 in AF on day 17, claiming higher hormone concentrations in females. Results supporting this patent's claims can be found in a paper from 2010 [28]. However, in 2017 Einspanier [29] protected a method that would allow in ovo sexing based on the E1S concentration in AF extracted from the egg on days 9 and 10. The patent was corroborated by Weissman et al. [30], which showed significant concentration differences for E1S at day 9 and E2 at day 10. This scientific report is currently considered a standard golden method for AF extraction and sex determination through hormone concentration measurement in the AF sample.

Some other studies show the presence of hormones in yolk [31,32] and serum [33]. First, in 2002, Müller et al. [31] studied the relationship between maternal social status and the deposited concentrations of androgens (see Table 1) in freshly laid eggs, concluding that the androgens concentrations in yolk were insufficient for sex differentiation. Second, in 2013, Aslam et al. [32] showed that hormone yolk concentrations were correlated with glucose concentration and unincubated egg dimensions and weight. Finally, Wang et al. [33] showed differences from days 8 to 16, concluding that T, dihydrotestosterone (DHT) and E1 were correlated with sex, and the differences between hormone concentrations varied with the incubation days.

Moreover, a strategy that stands out from the standard ELISA or RIA, while using antibodies, was used by Decuypere and Fey [34], who filed a US patent related to fluorescently labeled antibodies inserted in the egg albumen before incubation. The antibody was specific for several genes encoded proteins from the W-chromosome (e.g., CHDIW, ASW), or the Z-chromosome (e.g., DMRT-1), or hormones, e.g., anti-Mullerian hormone. However, as discussed in the genetic engineering section, the consumers and industry still question approaches that need the introduction of extra components in consumption products [35].

In summary, the present section showed that day 9 was the earliest day on which it was possible to make this differentiation with E1S (the most promising hormonal biomarker) [21,30]. Moreover, the company Seleggt (Seleggt Gmbh, Cologne, Germany) applies ELISA for in ovo sexing. Considering market application, the two challenges for this technology are the considerable cost of ELISA or RIA (~4 €/day-old-chick) and the duration of analysis (> 45 min), excluding egg handling and sample extraction.

Table 1. Overview of papers and patents related to immunosensing strategies for in ovo sexing with hormones.

Table 1. Overview of papers and patents related to immunosensing strategies for in ovo sexing with hormones.

Hormone	Sample	Detection (days)	Remarks	Reference
Papers
Luteinizing hormone, T, E2, P4	Blood	17 and 20	Detection with RIA Progesterone and estradiol with the bigger differences between males and females	Tanabe et al. [23]
E1, E2-17β, E2-17αsulphate, E1, E2-17β, E2-17αglucuronide	AF	8 to 17	Detection with RIA	Gill et al. [24]
T, A4	Yolk		Differences were not sufficient for sex determination	Müller et al. [31]
E2	AF	17	Detection with RIA Significant differences	Phelps et al. [11]
E2-17ss	AF	17	Use of a yeast transactivation assay Significant differences	Tran et al. [28]
T, E2, A4, P4, DHT	Yolk		Differences were not sufficient for sex determination	Aslam et al. [32]
E1S, E2, T	AF	9 and 10	Detection with ELISA Significant differences for E1S on day 9 and for E2 on day 10	Weissmann et al. [30]
E2, T, E1, A4, DHT	Serum	8 to 16	Detection with ELISA Significant differences with T, DHT, and E1	Wang et al. [33]
Patents
E2, E2-17ss, estriol, E1, T, DHT	AF	11 to 19		Phelps [25]
E2, E2-17ss, estriol, E1, T, DHT	AF	11 to 19		Tyczkowski et al. [26]
E2	AF	17		Butt and Tran [27]
E1S	AF	9 and 10		Einspanier [29]

A4, androstenedione; DHT, dihydrotestosterone; E1, estrone; E2, estradiol; E1S, estrone sulfate; P4, progesterone; T, testosterone; RIA, radioimmunoassay; ELISA, enzyme-linked immunosorbent assay; AF, allantoic fluid.

2.6. Other techniques

This section groups different in ovo sexing approaches described in the literature with a unique methodology or without a defined category. Remarkably, the oldest patents were defined within this group, such as measuring magnetic polarity [51,52,53,54]s or using a galvanometer [55]. Furthermore, no paper was reported on these pioneering in ovo sexing technologies, and their patent protection has expired or is inactivated due to non-payment [56].

The following two concepts are more recent but did not provide experimental data. The first approach utilized a plaster with a needle inserted through a hole in the eggshell. At the tip of the needle, a fluorescent marker signal could be read once in contact with a sex-specific molecule such as T or E2 [57]. Although the inventors did not specify the marker, it is expected that it might be related to an immunosensing approach. Second, with an electrode close to the shell, a passive recording of the dynamic electromagnetic spectrum on the egg surface would allow sexual sorting prior to incubation. According to the inventors, this electromagnetic field was intrinsically formed through the egg's biological, biochemical, and electrochemical processes [58]. Whereas the patent with the plaster approach was granted, the second one is still under examination. These techniques have not been explored and might require more study. To conclude this section, an overview of all non-optical in ovo sexing techniques is presented in Table 2.

Table 2. Overview of non-optical in ovo sexing techniques.

Table 2. Overview of non-optical in ovo sexing techniques.

Category	Target	Incubation day Accuracy Throughput	Technology (patents)/ research gaps (papers)	Advantages (+)/ Drawbacks (-)	References
DNA analysis	gDNA (e.g., CHD, XhoI, SWIM, HINTW) from different samples (e.g., AF, blood, feathers, brain)	Days 8 to 13 100 % Slow (slower in case of need for DNA extraction), PCR can take more than 60 minutes	Technology > Research Lack of papers using AF Several possible female-specific sequences are not reported in the literature	+ High accuracy Early in incubation (> day 2.5) - Lengthy processes and expensive Can be highly invasive if not using AF	Patents ⊗ Griffiths [14], ⊗ Clinton [19], ∅ Dalton et al. [59], ∅ Arie and Danielli [60], ∅ Molina and Espeut [61] Papers Li et al. [15], Haunshi et al. [17], Clinton et al. [18], He et al. [20]
Immunosensing	Sexual hormones (e.g., E2 or estrone-3-sulfate) in AF or blood	Minimum day 8 Reported as 99 % on day 9 Relatively slow using ELISA/RIA, which can take up to 90 minutes.	Technology ≅ Research Well defined in both papers and patents.	+ Applicable in AF - Limited to after day 8 of incubation. Lower accuracies than DNA analysis Expensive and lengthy testing.	Patents ∅ Tyczkowski et al. [26], ⊕ Butt and Tran [27], ∅ Einspanier [29], ⊕ Phelps [25] Papers Phelps et al. [11], Tanabe et al. [23], Weissmann et al. [30,62], Müller et al. [31], Aslam et al. [32], Wang et al. [33]
IMS and MS	Metabolites in AF	Days 9 to 10 90 % to higher than 95 % Relatively fast	Technology > Research Only defined by two different groups of inventors in the patent literature No work has been published in the scientific literature	+ Quick sampling and relatively fast analysis - No scientific literature on physiological background	Patents ∅ Daum and Atkinson [37], ∅ Bruins and Stutterheim [38,63], ∅ Stutterheim [64] Papers -
Genetic engineering	gDNA	Days 0 to 2.5 100 % High if using labels	Technology > Research Only one short communication in papers, mainly present in patents.	+ High accuracies at an early incubation stage. - Not accepted in several countries (see text) Invasive injection of a labeled molecule (antibody/DNA sequence)	Patents ∅ Decuypere and Fey [34], ∅ Offen [41], ⊗ Beisswanger [42] Papers Doran et al. [40]
VOC analysis	VOCs released from the egg	Day 0 or during incubation Accuracy not defined Seconds to minutes	Technology > Research Patents use real-time analysis systems such as THz spectroscopy, SIFT- or PTR-MS Research papers use conventional SPME-GC-MS analysis and more research is required for generating robust sexing models	+ Non-invasive Potential for early-stage application - Contamination risk of egg odor by environment or other eggs	Patents ∅ Rivers [45], ∅ Gabbai [48], ∅ Knepper et al. [65], ⊕ Tongli et al. [66] Papers Webster et al. [43], Costanzo et al. [44], Xiang et al. [49]
Other	1) Electrical and magnetic polarity of the egg 2) Dynamic electromagnetic spectrum 3) Sex-specific compounds close to the eggshell	Day 0 Accuracies unknown No throughputs defined	Technology > Research Only defined in different patents and no scientific literature	+ Before incubation - No scientific evidence	Patents ⊗ Young [51], ⊗ Williams [52,53], ⊗ Chadfield [55], ⊗ Pacala et al. [56], ⊕ Visser [57], ∅ Rastoutsau et al. [58] Papers -
Abbreviations: d, day; gDNA, genomic DNA; AF, allantoic fluid; E2, estradiol; ELISA, enzyme-linked immunosorbent assay; RIA, radioimmunoassay; THz, terahertz; SPME-GC-MS, solid phase micro extraction–gas chromatography-mass spectrometry; SIFT-MS, selected ion flow tube-mass spectrometry; PTR-MS, proton transfer reaction-mass spectrometry; IMS and MS, ion mobility and mass spectrometry; ⊕, granted; ∅, pending; ⊗, ceased/rejected/discontinued.

3. Optical in ovo sexing methods

3.5. Morphometric studies

In the following category, egg and embryo morphometric studies were considered since both use images from industrial cameras. The first approach was patented by Taniguchi, who published different applications from 2001 to 2021 in which the egg shape was used to determine the sex (121–126). Although applicable before incubation, sex differences in egg dimensions are likely to be minimal, and ratios of these parameters will be required to improve the accuracy. The described concept relied on the idea that male eggs would generally be less spherical and more pointed than female eggs [121]. Images were used to measure the shape by quantifying the egg length, width, and distance from the equator to the blunt end. The protection for this patent expired in 2019, and more recently, a follow-up invention included egg distortion [126].

Concerning the concept of the more spherical-shaped female eggs and more pointed male eggs, similar indications have been published in a study on white-layer eggs [127]. The researchers measured the width and length and calculated a shape index that would discriminate the sex from this. However, the measured overlap between the sexes was considerable and did not allow a highly accurate sexual separation.

The second approach used machine vision imaging of embryonic blood vessels around day 4 (128–132). This technique is assumed to be low-cost and requires a light source, an industrial camera, and a computer. After egg illumination, an asymmetry in the blood vessel branching was observed for the female embryos and was linked to the asymmetric gonadal development in which the right gonad regresses, and less blood is assigned (Figure 7A). On the other hand, the blood vessel branching would be symmetric for the equally developing testes in the males (Figure 7B). The authors obtained an accuracy of 89.74 % on the prediction set using white eggs [128]. However, it is questioned whether this technique would also work for brown eggs, given that the brown shell might absorb more light and complicate the blood vessels' visualization through the shell.

Table 3. Overview of optical in ovo sexing techniques.

Table 3. Overview of optical in ovo sexing techniques.

Category	Target	Incubation day Accuracy Throughput	Technology (patents)/ research gaps (papers)	Advantages (+)/ Drawbacks (-)	References
Raman and fluorescence spectroscopy	Blood, eggshell, germinal disk, or other embryonic structures.	Day 3.5 91 % accuracy on blood Day 6 92.3 % on embryo and blood Low to high throughput depending on the invasiveness	Technology ≅ Research The applicability on blood vessels is well reported in academic literature and is protected by patents Technology > Research A scientific gap exists in the analysis of the eggshell, germinal disk, or other embryonic structures since these are only reported in patent literature	+ Early applicable in incubation No liquid or tissue sampling required - Intensive sampling procedure and relatively invasive process	Patents ⊗ Baron et al. [71], ⊕ Steiner et al. [72], ⊕ Galli et al. [77], ⊕ Schortgen [78], ⊕ Opitz et al. [79], ⊕ Popp et al. [81], ⊕ Schart [133], ∅ Herzog and Hurlin [134], ∅ Steiner et al. [135], ⊕ Hurlin et al. [136] Papers Galli et al. [69,74,75], Gokdag et al. [137]
IR and THz spectroscopy	Germinal disk	Day 0 Accuracy not defined Average throughput	Technology ≅ Research From the same group of authors, both patents and papers are reported for the IR approach Technology > Research Only a patent is reported for the non-invasive THz approach	+ Applicable in an early stage - IR approach infringes hatchability	Patents ∅ Steiner et al. [85], ∅ May et al. [86] Papers Steiner et al. [83,84]
VIS-NIR spectroscopy	Heartbeat, blood hemoglobin, movement, feather color, yolk ratio, or growth (opacity)	Days 0 to 14 depending on the technology High accuracies on color sexing 99 % Blood sexing or other spectral features range from 80 to 90 % High throughput	Technology ≅ Research Color sexing in the egg is commercially available and is described in both patent and paper literature Blood detection through the eggshell is described in paper and patent literature Technology > Research Other VIS-NIR spectroscopy on specific egg regions on specific days is only described in patents and lacks scientific background	+ Has the potential for non-invasive implementation Equipment is affordable Early application potential - Lack of physiological background for sex determining spectral features described in patents	Patents ⊕ McKay [91], ∅ Preusse [96], ⊕ Fujitani [95,101], ⊗ Rams and Toellner [100], ∅ Hebrank [97], ∅ Wang et al. [98], ∅ Colvin [102], ⊕ Ngadi et al. [104,106], ⊗ Zhao et al. [105,113], ⊕ Rozenboim and Ben Dor [107], ⊗ Pan et al. [108], ∅ Green [109], ⊕ Zhu [110], ∅ Fischer and Meissner [111,112] Papers Gohler et al. [90], Corion et al. [92], Khaliduzzaman et al. [93], Rahman et al. [94], Li et al. [103], Pan et al. [138]
NMR spectroscopy	Gonads, hormones, or metabolites	Days 4 to 11 for hormones and metabolites, accuracy not defined and relatively fast Day18 gonads, accuracy not defined and relatively slow	Technology > Research Almost only patent literature has been reported A single research paper reports the difficulty of detecting gonads	+ Non-invasive Early incubation - No prediction accuracies are reported or known Gonad sexing relatively late	Patents ∅ Bruins and Stutterheim [63], ⊕ Reynells and Flegal [116], ∅ Haase et al. [118], ∅ Hergenroder et al. [119], ⊕ Sewiolo and Ziroff [120] Papers Davenel et al. [117]
Morphometric studies	Eggshell morphology or blood morphology	Day 0 for eggshell, ~93 to 100 % Relatively fast (using imaging technique) Day 4 for blood 89.74 % Relatively high throughput expected, yet equilibrium time needed in a horizontal position	Technology > Research Numerous patents are filed and a few papers One paper is using the formulas from a patent Not all papers did find sexual differences Using the shape index, there was a trend for female eggs to be more spherical, although the overlap was big with male eggs Blood vessel morphology sexing is described both in patent and paper literature, and it might require more research to verify the robustness	+ Non-invasive Before or early incubation - Not enough scientific evidence that proves this technique works robustly nor an explanation of why there are sexual differences in the egg shape Blood vessel imaging requires a 2 minutes equilibration time in a horizontal position	Patents Taniguchi ⊕ [121], ∅ [122], ⊕ [123], ∅ [124], ∅ [125], ⊕ [126], ⊕ Xiaohui et al. [129], ⊗ Li et al. [130], ⊗ Li [131], ⊕ Chen et al. [132], ∅ [139] Papers Yilmaz-Dikmen et al. [127], Rutkowska et al. [140]
Abbreviations: d, day; IR, infrared; THz, terahertz; VIS-NIR, visible-near-infrared; ⊕, granted; ∅, pending; ⊗, ceased/rejected/discontinued.

This approach received increased interest due to the current higher performance of cameras and software for image analysis and has been explored to study eggshells non-invasively and blood vessel morphometry, enabling fast screening. Advantageous is that the first method was applicable before incubation, whereas the second method required blood vessel development in early incubation. However, whether the prediction accuracy is high enough to use morphometric differences between sexes is questioned. Nevertheless, the technique is described in both patents and papers and is expected to hold potential for early and non-invasive in ovo sexing. An overview of all the optical in ovo sexing techniques is presented in Table 3.

4. Trends in in ovo sexing techniques

5. Conclusion

Abbreviations

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