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)	d 8 to 13/ 100%/ Slow (slower in case of need for DNA extraction), PCR can take more than 60 min	Technology > Research Lack of papers using AF Several possible female-specific sequences are not reported in the literature	+  High accuracy Early in incubation (> d 2.5) - Lengthy processes and expensive Can be highly invasive if not using AF	Patents □ Griffiths [14], □ Clinton [15], ○ Dalton et al. [16], ○ Arie and Danielli [17], ◊ Molina and Espeut [18], ○ Weigel et al. [19] Papers Li et al. [20], Haunshi et al. [21], Clinton et al. [22], He et al. [23], Petitte and Kegelmever [24]
Immunosensing	Sexual hormones (e.g., E2 or estrone-3-sulfate) in AF or blood	Minimum d 8/ Reported as 99% on d 9/ Relatively slow using ELISA/RIA, which can take up to 90 min	Technology ≅ Research Well defined in both papers and patents	+  Applicable in AF - Limited to after d 8 of incubation Lower accuracies than DNA analysis Expensive and lengthy testing	Patents ◊ Tyczkowski et al. [25], ○ Butt and Tran [26], ○ Einspanier [27, 28], □ Phelps [29], ◊ Meter [30, 31] Papers Phelps et al. [12], Tanabe et al. [32], Weissmann et al. [33, 34], Müller et al. [35], Aslam et al. [36], Wang et al. [37]
IMS and MS	Metabolites in AF	d 9 to 10/ 90% to higher than 95%/ Relatively fast	Technology > Research Only defined by 2 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 [38], ○ Bruins and Stutterheim [39, 40], ◊ Stutterheim et al. [41–43] Papers -
Genetic engineering	gDNA	d 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 [44], ○ Offen [45], □ Beisswanger [46] Papers Doran et al. [47], Lee et al. [48]
VOC analysis	VOCs released from the egg	d 0 or during incubation/ Accuracy not defined/ s to min	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 [49], ○ Gabbai [50], ○ Knepper et al. [51], ○ Shi et al. [52] Papers Webster et al. [53], Costanzo et al. [54], Xiang et al. [55]
Other	1) Electrical and magnetic polarity 2) Dynamic electromagnetic spectrum 3) Sex-specific compounds close to the eggshell 4) Bioimpedance measurements	d 0 or d 9/ Accuracies unknown/ No throughputs defined	Technology > Research Only defined in different patents and no scientific literature	+  Before incubation Non-invasive - Little scientific evidence	Patents □ Young [56], □ Williams [57, 58], □ Chadfield [59], □ Pacala et al. [60], ○ Visser [61], ◊ Rastoutsau et al. [62] Papers Ching et al. [63]

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