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. 2021 Nov 29;101(3):101619. doi: 10.1016/j.psj.2021.101619

Nondestructive characterization gender of chicken eggs by odor using SPME/GC-MS coupled with chemometrics

Xiao-le Xiang *,†, Gan Hu ⁎, Yong-guo Jin *, Guo-feng Jin *, Mei-hu Ma *,1
PMCID: PMC8741610  PMID: 34995877

Abstract

It's a difficult task for researchers to identify the gender of chicken eggs by nondestructive approach in the early of incubation, which not only could reduce the cost of incubation, but also could improve the welfare of chicks. Therefore, SPME/GC-MS has been applied to investigate its potential as a nondestructive tool for characterizing the differences of odor between male and female chicken eggs during early of incubation and even before hatch. The results showed that more volatiles were found in female White leghorn eggs during early of incubation and 6,10-dimethyl-5,9-undecadien-2-one, 6-methyl-5-hepten-2-one, nonanal, decanal, octanal, 2-nonen-1-ol, etc. were important for the distinction of male and female White leghorn eggs during E1-E9 of incubation. 2-ethyl-1-hexanol; octanal, nonanal, 2,2,4-trimethyl-3-carboxyisopropyl pentanoic acid isobutyl ester; 2-nonen-1-ol, cyclopropanecarboxamide, heptadecane were correlated with gender of unhatched White leghorn, Hy-line brown and Jing fen eggs, respectively. Moreover, sex-related volatiles have been strongly influenced by incubation process and egg breed, and to be related to steroid hormone biosynthesis. What's more, this study enables us to develop a new visual for ovo sexing of chicken eggs and advances our understanding of the biological significance behind volatiles emitted from chicken eggs.

Key words: chicken eggs, sex-related volatiles, ovo sexing, nondestructive characterization

INTRODUCTION

The increasing specialization of chicken lines for meat and egg production has made male and female chicks are used for broiler and layer strains, respectively (Galli et al., 2017). More than 7.0 billion freshly hatched cockerels with unwanted gender, therefore, were culled globally annually, especially for male day-old chicks in commercial hatcheries (Alin et al., 2019). Which not only cause significant economic losses but also raise serious ethical issues (Galli et al., 2017). Under these pressure, there is urgent need for new techniques of sex determination “in ovo” during early of incubation (Galli et al., 2017, 2018).

Nowadays, many minimally invasive and/or nondestructive techniques have been used to detect the gender of embryo in ovo during early of incubation or even in unhatched fertilized eggs (Alin et al., 2019). For example, the concentration of hormonal (estrogen) in allantoic fluid (Weissmann et al., 2013) and reflectance spectroscopy both provided good sexing results at the mid period of incubation (Rozenboim and Ben Dor, 2001). Infrared and optical spectroscopy have been applied for sexing of unhatched eggs by addressing the DNA content extracted from blastoderm cells (Steiner et al., 2011; Galli et al., 2018; Wu et al., 2019). Raman and fluorescence spectral information of blood from embryo through eggshell membrane at day 3.5 of incubation (E3.5) for ovo sexing with a correct rate up to 90 and 93%, respectively (Galli et al., 2018). In addition, the shape and color of eggs have been proposed related to their sex (Aviles et al., 2011; Yilmazdikmen and Dikmen, 2013).

However, all of the above methods require hatched eggs to be opened with a shell windowing, which will strongly affects hatching rate and chick health in future, and not easy to be exploited in practice (Galli et al., 2018). Therefore, many researchers have attempted to apply nondestructive strategies to solve this problem and spectroscopy have been considered as the most promising technologies until now. For example, hyperspectral spectroscopy has been successfully used to identify the gender of unhatched eggs(Ngadi et al., 2018). But the spectral features acquired from unhatched eggs could not provide enough valid information to characterize the differences between male and female fertilized eggs.

Fortunately, an increasing number of researches have focused on the roles of odor or olfaction for sex recognition in avian (Caro et al., 2015; Costanzo et al., 2016). More importantly, it was surprisingly found that there were certain differences in odor profiles between male and female Japanese quail eggs both at E8 and E1 (Webster et al., 2015). What's more, sex-related variation in odor of eggs were also found in wild barn swallow at E10-11 (Costanzo et al., 2016). Hence, it could suspected that there may be certain difference in volatiles between unhatched male and female fertilized eggs. In ovo sexing of chicken eggs by odor not only has the potential to enables nondestructive testing but also provides more detailed information for mechanism (Caro et al., 2015; Costanzo et al., 2016).

It is widely accepted that sex-specific differences in metabolites between male and female embryos were existed in the middle and later of incubation, due to sex differentiation (Smith and Sinclair, 2004; Weissmann et al., 2013). However, it is difficult to understand sex differences in violates and spectral characteristics of hatched eggs were existed at E1 or even before hatched, except for genetic information (Webster et al., 2015; Ngadi et al., 2018). In fact, as far as we know, the starting point of sex determination and/or differentiation for avian embryo could be advanced to meiosis I (Uller and Badyaev, 2009), cell-autonomous mechanisms of somatic sex identity and sex-based differences in steroid hormones derived from maternal investment could support it indirectly (Radder, 2007).

From the above, sex-specific volatiles and spectral features have begun to be realized in quail, barn swallow eggs and fertilized chicken eggs, respectively. However, to our knowledge, no researches have reported on the sex-specific volatiles of chicken eggs till now. The present study, thus, was designed to characterize the composition and differences of odor emitted from male and female chicken eggs during early of incubation and then to further evaluate the variation between unhatched male and female chicken eggs. Gender detection of unhatched fertilized chicken eggs by odor would improve productivity of hatcheries, beneficial for animal welfare and offers the potential for industrial exploitation in future.

MATERIALS AND METHODS

Fertilized Eggs Storage and Incubation

Freshly fertilized chicken eggs, including white Leghorn (W), Hy-line brown (H), and Jing fen (J), were obtained from a commercial supplier (Wuhan, Hubei province, China) and stored in room temperature until hatch at 38°C and 60% humidity in an incubator (Fuhui Tech Co., Wuhan, China).

SPME-GC-MS

Acquisition of volatiles from hatched eggs were performed using 50/30 μm DVB/CAR/PDMS (Supelco, Bellefonte, PA) following the protocol in Xiang (Xiang et al., 2019).

  • •

    GC: The VOCs enriched from chicken eggs were desorbed in GC injector at 250°C for 5 min in a splitess mode with a helium (99.99%) flow rate of 1.0 mL/min and separated on a HP-5MS capillary column (30 m × 0.25 mm × 0.25 mm film thickness) using 7890B-5977A GC-MS instrument (Agilent Technologies, Santa Clara, CA). GC oven was programmed from 30°C for 2 min, increased to 45°C at 2°C min−1, and increased to 120°C at 3°C min−1 (hold 2 min), finally increased to 230°C at 6°C min−1 and maintained for 5 min. Quantitative datas of VOCs were semiquantified by peak areas of in the selected ion monitor (SIM).

  • •

    MS: Temperatures of ion source and quadrupole were 230°C and 150°C, respectively. Quadrupole mass spectrometer was operated in EI mode at 70 eV and scan range was set at m/z 35–450. Tentative VOC identification was performed by NIST 11.0 Mass-Spectral Search Library and RI (Xiang et al., 2019).

Molecular Sexing

DNA from embryos in each egg were extracted using DNA tissue Kit (Sangon Biotech, Shanghai, China) following manufacturers’ protocols. PCR amplification was run using primers SF (5’-GTGCATTGCAGAAGCAATATT-3’) and SR (5’-GCCTCCTGTTTATTATAGAATTCAT-3’). About 25 μL systems were used: 1.5 μL (10 μmol/L) of both primers, 8.5 μL Red Master Mix (Sangon Biotech), 1.5 μL extracted DNA, and 1.5 μL H2O. PCR assay conditions were set at 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 40 s and a final extension step of 72°C for 7 min. PCR reactions were performed using a T100 Thermal Cycler PCR (Bio-Rad, Hercules, CA). PCR products were separated on 1.8% agarose gels at (120 V, 15 mA) and visualized with 4S Green Nucleic Acid Stain and UV light. One and two band indicated male and female egg, respectively (Galli et al., 2018).

Statistical and Bioinformatics Analyses

All statistical and bioinformatics analysis were performed by IBM SPSS 24 and Metabo Analyst 4.0, respectively.

RESULTS AND DISCUSSION

Differences in VOCs Between (Hatched) Male and Female Eggs (W) During E0–E9

Fouteen fertilized eggs were used for data acquisition during E0–E7, 5 eggs were identified as male and female eggs, respectively and the rest 4 eggs were infertile or sex were not sure. Thirteen embryo eggs (6 male and 7 female) were used for data acquisition during E9 (Figure S1). The weight of these fertilized eggs had no significant difference.

Comparison Analysis

A total of 18 VOCs were identified in hatched eggs (Figure 1 and Table 1), including: 7 aldehydes (hexanal, heptanal, octanal, nonanal, decanal, undecanal, dodecanal); 3 alcohols (2-ethyl-1-hexanol, 1-nonanol, 2-nonen-1-ol); 2 ketones (6-methyl-5-Hepten-2-one, 6,10-dimethyl-5,9-Undecadien-2-one); 2 alkanes (n-hexane, 2-fluoro-7-hydroxybicyclo[2.2.1] heptane), 1-heptadecanamine, 3-(bromomethyl)-piperidine, cedrene and carbon dioxide. Most of these VOCs have been reported in hatched quail, barn swallow eggs (Webster et al., 2015; Costanzo et al., 2016) and fertilized chicken eggs (Xiang et al., 2019). The abundance of almost VOCs emitted from female eggs were higher than that from male eggs during early of incubation and obvious difference in VOCs were obtained at E1 and E5 (Figures 1, S2 and Table 1). Similar results were obtained in barn swallow and quail hatched eggs (Webster et al., 2015; Costanzo et al., 2016) and which might be due to sex difference in embryonic metabolism or selective utilization of egg components (Martins, 2004).

Figure 1.

Figure 1

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Sex difference in concentration of VOCs emitted from chicken eggs during E0–E9 of incubation (Mean ± SE; M: male, F: female; left: area, right: percentage).

Table 1.

Mean levels of VOCs between male and female eggs for W breed during E0–E9 of incubation (mean ± SE).

E0 RT min Volatile compounds M (n = 5) F (n = 5) M (n = 5) F (n = 5)
2.67 n-Hexane 707,289 ± 314,503 248,720 ± 86,168 2.84 ± 1.56 0.76 ± 0.25
7.50 Hexanal 1,919,745 ± 1,586,849 1,293,390 ± 959,108 2.66 ± 1.24 2.81 ± 1.90
12.56 Heptanal 1,191,947 ± 670,059 1,073,318 ± 471,558 2.51 ± 1.06 2.55 ± 0.99
17.31 6-methyl-5-Hepten-2-one 1,376,781 ± 521,867 1,418,052 ± 511,202 3.79 ± 1.26 4.15 ± 1.19
18.08 Octanal 1,225,262 ± 395,234 1,127,781 ± 201,276 3.39 ± 0.37 3.14 ± 0.24
19.59 2-ethyl-1-Hexanol 491,138 ± 177,650 108328 ± 108328 1.45 ± 0.64 0.22 ± 0.22
23.37 Nonanal 22,245,370 ± 6,172,755 22,751,195 ± 3,261,170 64.77 ± 4.16 64.42 ± 1.90
26.63 2-Nonen-1-ol 655,662 ± 195328 699,271 ± 107,457 1.85 ± 0.28 2.01 ± 0.19
28.35 Decanal 5,131,160 ± 1,492,170 5,695,401 ± 463,820 14.37 ± 1.18 17.21 ± 2.47
39.52 6,10-dimethyl-5,9-Undecadien-2-one 424,690 ± 223,201 640,914 ± 290,067 1.16 ± 0.75 1.88 ± 0.69
43.83 Cedrene 330,705 ± 131,457 245,510 ± 101,300 1.04 ± 0.48 0.64 ± 0.22
E1 RT min Volatile compounds M (n = 5) F (n = 5) M (n = 5) F (n = 5)
2.67 n-Hexane 159,031 ± 100,907 103,603 ± 103,603 0.90 ± 0.75 0.45 ± 0.45
7.50 Hexanal 211,689 ± 145,803 240,690 ± 91,426 0.40 ± 0.31 0.31 ± 0.14
12.56 Heptanal 579,731 ± 356,533 1,511,187 ± 227,597 0.91 ± 0.57 2.02 ± 0.47
17.31 6-methyl-5-Hepten-2-one 1,401,011 ± 444,627 10,177,035 ± 6,259,544 2.70 ± 0.73 6.31 ± 2.54
18.08 Octanal 2,276,164 ± 998,216 4,567,335 ± 1,858,499 4.44 ± 0.71 3.82 ± 0.45
19.59 2-ethyl-1-Hexanol 1,744,661 ± 1,681,903 550,671 ± 487,749 2.82 ± 2.75 0.55 ± 0.36
21.55 1-Nonanol 76,388 ± 76,388 286,406 ± 137,757 0.09 ± 0.09 0.19 ± 0.08
23.37 Nonanal 21,234,819 ± 4,173,891 36,532,779 ± 6,215,023 53.04 ± 6.43 48.21 ± 10.53
26.63 2-Nonen-1-ol 2,059,868 ± 1,016,308 6,217,979 ± 3,264,844 3.75 ± 0.81 4.38 ± 1.17
28.35 Decanal 15,499,005 ± 6,221,383 38,023,189 ± 1,673,7899 29.59 ± 3.93 29.7 ± 5.61
31.69 1-Heptadecanamine 52,249 ± 52,249 306637 ± 165,645 0.06 ± 0.06 0.20 ± 0.08
33.03 Undecanal 127,234 ± 83,870 517,315 ± 285,712 0.17 ± 0.11 0.33 ± 0.14
39.52 6,10-dimethyl-5,9-Undecadien-2-one 546,348 ± 194,957 5,769,678 ± 4,001,630 1.06 ± 0.32 3.32 ± 1.67
43.83 Cedrene 0 ± 0 127,584 ± 53,664 0.00 ± 0.00 0.12 ± 0.07
E3 RT min Volatile compounds M (n = 5) F (n = 5) M (n = 5) F (n = 5)
7.50 Hexanal 136,108 ± 136,108 8,573 ± 8,573 0.21 ± 0.21 0.02 ± 0.02
12.56 Heptanal 22,306 ± 13,693 161,951 ± 161,951 0.06 ± 0.04 0.36 ± 0.36
17.31 6-methyl-5-Hepten-2-one 1,946,654 ± 480774 2,260,972 ± 806,622 6.11 ± 1.59 7.87 ± 2.66
18.08 Octanal 1,961,132 ± 330,221 1,840,817 ± 598,321 5.86 ± 0.32 5.01 ± 0.44
19.59 2-ethyl-1-Hexanol 184,280 ± 119,468 0 ± 0 0.86 ± 0.61 0.00 ± 0.00
21.55 1-Nonanol 0 ± 0 56,443 ± 56,443 0.00 ± 0.00 0.09 ± 0.09
23.37 Nonanal 13,939,047 ± 4,144,008 12,186,148 ± 3,664,919 38.3 ± 3.75 37.78 ± 7.25
26.63 2-Nonen-1-ol 1,754,216 ± 315,068 2,101,424 ± 992,929 5.22 ± 0.25 5.33 ± 1.16
28.35 Decanal 13,695,073 ± 2,566,530 14,279,402 ± 4,950,148 40.73 ± 1.89 39.54 ± 4.24
31.69 1-Heptadecanamine 0 ± 0 50313 ± 50,313 0.00 ± 0.00 0.08 ± 0.08
33.03 Undecanal 47,313 ± 47,313 78,244 ± 78,244 0.07 ± 0.07 0.13 ± 0.13
39.52 6,10-dimethyl-5,9-Undecadien-2-one 711,989 ± 109,937 1,046,693 ± 377,237 2.36 ± 0.54 3.67 ± 1.30
E5 RT min Volatile compounds M (n = 5) F (n = 5) M (n = 5) F (n = 5)
1.71 Carbon dioxide 189,757 ± 76,733 432,268 ± 105,489 0.2 ± 0.08 0.35 ± 0.05
2.67 n-Hexane 607,043 ± 419,218 451,448 ± 146,844 0.54 ± 0.39 0.27 ± 0.10
7.50 Hexanal 1,143,704 ± 756,053 2,140,233 ± 1,235,922 1.05 ± 0.70 1.11 ± 0.65
12.56 Heptanal 1,783,603 ± 525,191 2,541,099 ± 866,840 1.69 ± 0.46 1.54 ± 0.47
17.31 6-methyl-5-Hepten-2-one 4,967,512 ± 2,026,371 11,723,078 ± 5,384,954 4.57 ± 1.42 7.37 ± 2.49
18.08 Octanal 6,711,175 ± 1,712,218 9,216,594 ± 2,420,535 7.32 ± 0.46 6.66 ± 0.31
18.52 2-Fluoro-7-hydroxybicyclo[2.2.1] heptane 154,326 ± 94,957 65,748 ± 65,748 0.13 ± 0.08 0.04 ± 0.04
19.59 2-ethyl-1-Hexanol 124,393 ± 76,295 65,800 ± 56,879 0.10 ± 0.06 0.04 ± 0.03
21.55 1-Nonanol 644,152 ± 214,165 1,096,920 ± 365,662 0.59 ± 0.17 0.64 ± 0.18
23.37 Nonanal 22,932,633 ± 3,518,122 30,722,671 ± 7,490,784 28.97 ± 3.59 25.86 ± 4.3
26.63 2-Nonen-1-ol 7,519,158 ± 2,034,952 12,056,957 ± 3,435,050 7.82 ± 0.84 8.11 ± 0.80
28.35 Decanal 38,283,684 ± 8,082,834 55,841,642 ± 14,470,657 44.03 ± 1.16 41.46 ± 1.1
31.69 1-Heptadecanamine 361,857 ± 149,036 957,676 ± 336,104 0.32 ± 0.13 0.55 ± 0.15
33.03 Undecanal 639,752 ± 214,755 1,553,224 ± 550,442 0.59 ± 0.18 0.9 ± 0.24
35.97 Piperidine, 3-(bromomethyl)- 0 ± 0 194,289 ± 133,152 0.00 ± 0.00 0.09 ± 0.06
37.76 Dodecanal 49,621 ± 49,621 246,576 ± 112,988 0.05 ± 0.05 0.14 ± 0.06
39.52 6,10-dimethyl-5,9-Undecadien-2-one 2,052,258 ± 896,894 7782,720 ± 3,599,126 1.97 ± 0.64 4.81 ± 1.71
43.83 Cedrene 88,889 ± 54,605 114,224 ± 114,224 0.08 ± 0.05 0.06 ± 0.06
E7 RT min Volatile compounds M (n = 5) F (n = 5) M (n = 5) F (n = 5)
1.71 Carbon dioxide 192,127 ± 72,367 110,692 ± 63,363 0.35 ± 0.15 0.23 ± 0.13
2.67 n-Hexane 84,345 ± 84,345 47,631 ± 40,581 0.17 ± 0.17 0.09 ± 0.08
7.50 Hexanal 48,193 ± 48,193 0 ± 0 0.08 ± 0.08 0.00 ± 0.00
12.56 Heptanal 577,464 ± 216,950 286,001 ± 263,128 0.90 ± 0.36 0.35 ± 0.31
17.31 6-methyl-5-Hepten-2-one 2,334,341 ± 857,860 3,369,616 ± 1,098,751 3.68 ± 1.31 5.76 ± 1.52
18.08 Octanal 4,665,762 ± 739,104 4,045,272 ± 755,074 7.69 ± 0.33 6.35 ± 0.40
19.59 2-ethyl-1-Hexanol 214,313 ± 111,207 185,777 ± 125,744 0.38 ± 0.19 0.32 ± 0.25
21.55 1-Nonanol 217,082 ± 90,686 170,146 ± 106,019 0.32 ± 0.13 0.20 ± 0.13
23.37 Nonanal 1,5638,615 ± 1,447,539 14,833,732 ± 2,656,896 26.9 ± 1.91 23.74 ± 2.04
26.63 2-Nonen-1-ol 4,256,260 ± 730,177 4,668,091 ± 912,702 7.00 ± 0.37 7.23 ± 0.43
28.35 Decanal 3,0409,966 ± 4,495,900 32,828,397 ± 4,585,357 50.57 ± 1.74 52.48 ± 0.91
31.69 1-Heptadecanamine 100,277 ± 62,097 118,989 ± 72,902 0.13 ± 0.08 0.14 ± 0.09
33.03 Undecanal 260,347 ± 71,743 328,412 ± 49,381 0.39 ± 0.1 0.53 ± 0.03
39.52 6,10-dimethyl-5,9-Undecadien-2-one 966,082 ± 283,944 1,543,351 ± 553,621 1.43 ± 0.39 2.58 ± 0.76
E9 RT min Volatile compounds M (n = 6) F (n = 7) M (n=6) F (n = 7)
1.71 Carbon dioxide 442,090 ± 123,026 474,897 ± 175,688 0.70 ± 0.31 0.90 ± 0.39
7.50 Hexanal 259,849 ± 120,719 121,785 ± 53,356 0.21 ± 0.10 0.16 ± 0.08
12.56 Heptanal 706,175 ± 354,736 329,054 ± 216,066 0.60 ± 0.29 0.34 ± 0.23
17.31 6-methyl-5-Hepten-2-one 3,748,807 ± 1,596,740 3,992,474 ± 1,206,013 3.57 ± 0.74 5.78 ± 1.74
18.08 Octanal 8,105,079 ± 3,076,097 4,420,044 ± 1,112,527 7.60 ± 0.61 5.97 ± 0.61
19.59 2-ethyl-1-Hexanol 74,164 ± 74,164 380,747 ± 183,959 0.06 ± 0.06 0.60 ± 0.30
21.55 1-Nonanol 545,431 ± 287,063 196,731 ± 110,800 0.40 ± 0.13 0.21 ± 0.10
23.37 Nonanal 18,990,678 ± 4,810,172 14,688,386 ± 2,985,200 21.00 ± 2.33 20.55 ± 3.03
26.63 2-Nonen-1-ol 8,988,117 ± 3,970,659 5,424,999 ± 1,539,190 7.79 ± 0.72 7.10 ± 0.55
28.35 Decanal 55,721,327 ± 1,896,6737 38,823,219 ± 8,226,992 55.68 ± 1.91 54.07 ± 2.59
31.69 1-Heptadecanamine 342,342 ± 214,986 165,149 ± 98,250 0.21 ± 0.10 0.17 ± 0.08
33.03 Undecanal 676,199 ± 330,365 456,383 ± 140,090 0.56 ± 0.13 0.60 ± 0.06
39.52 6,10-dimethyl-5,9-Undecadien-2-one 1,584,791 ± 594,151 2,337,356 ± 684,490 1.64 ± 0.40 3.54 ± 1.09
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Bold: 0.05 < P < 0.1; bold and italic: P < 0.05.

It's clear that the average levels of 6,10-dimethyl-5,9-undecadien-2-one, 6-methyl-5-hepten-2-one, undecanal, heptadecanamine, 2-nonen-1-ol, nonanal, etc. emitted from female eggs were higher than that from male eggs and the opposite result was obtained for 2-ethyl-1-hexanol, hexane, etc (Figure 1). But the difference of VOCs between male and female eggs was not statistically significant, except for the abundance of heptanal, nonanal and cedrene at E1 and the percentage of octanal at E7 and E9 (Table 1). As well known, saturated aldehydes have usually been considered as the derivative of lipid oxidation degradation and Strecker reaction of amino acid (Mir et al., 2017; Xiang et al., 2019; Jia et al., 2020).

Multivariate Analysis

Trend of sex differences in odor emitted from eggs has been preliminarily discovered during early of incubation and multivariate analysis was then used to visualize the differences between male and female eggs (Xiang et al., 2019). As expected, the VOCs emitted from male and female eggs during early of incubation (E1–E9) were separated in 2D score plots of OPLS-DA model, except for E0 and E5 (Figure 2). However, a clear difference (or trend) of VOCs between eggs with either sex were shown in 3D score plots (Figure S3). It suggests that there are indeed some subtle differences between VOCs emitted from male and female chicken eggs. Minor differences between VOCs profile for male and female eggs was obtained at E5 may be caused by the surge in metabolic activity of embryo. Which may expanded the variation of VOCs emitted from eggs and then the difference between VOCs emitted from male and female eggs has been relatively concealed (Bruggeman et al., 2002; Ayers et al., 2013).

Figure 2.

Figure 2

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Sex difference of VOCs emitted from male and female chicken eggs by OPLS-DA during E0–E9 of incubation.

Six,10-dimethyl-5,9-undecadien-2-one, 6-methyl-5-hepten-2-one, nonanal, decanal, octanal, 2-nonen-1-ol, etc. were important for the distinction of male and female eggs during E1-E9 (Figure 2). Moreover, most of these VOCs were more abundant in female eggs, except for 2-ethyl-1-hexanol and hexane. Similarly, many ketones, acids, alcohols and aldehydes have been reported more abundant in female eggs (Webster et al., 2015; Costanzo et al., 2016). For instance, methylheptenone has been reported to be more abundant in female organisms and been considered as biological relevant odors for rats’ erection (Curran et al., 2007; Nielsen et al., 2013). What's more, female ostriches has been reported more sensitive to 6-methyl-5-hepten-2-one than male ostriches (Sole et al., 2010) and 2,2,6-trimethylcyclohexanone was identified as female-specific compounds (Li and Zhang 2018). On the contrary, 2-heptanone and 6,10-dimethyl-5,9-undecadien-2-one have been reported as male-specific pheromone compounds (Ayers et al., 2013; Mayo et al., 2013). It can be inferred that sex-pheromone ketones may be affected by many factors, including species, environment and so on.

Moreover, aldehydes have been identified as the main pheromone volatiles for chicken eggs (Xiang et al., 2019) and sex difference in aldehydes have been found in rabbit meat, Parasitoid, Bracon hebetor Say and olive fly (Botsi et al., 1995; Dweck et al., 2010; Xie et al., 2016). For example, nonanal has been considered as a minor sex-pheromone for olive fly (Botsi et al., 1995) and been proved to exert higher influence in females during oviposition period (Malheiro et al., 2015). Unsaturated nonen-1-ol has been reported produced by male Anastrepha ludens to atteact conspeific females (Nation, 1983).

Discriminant and Correlation Analysis

VOCs emitted from male and female eggs during early of incubation (E0–E9) were well separated in canonical discriminant (CD), except for one unhatched male egg was misjudged as female egg. In other words, the accuracy of (W) egg sexing during E1 –E9 of incubation was almost 100% (Figure 3 and Table S1). It was very interesting and lucky that VOCs emitted from female eggs were always located at the upper or left of male eggs (Figure 3). Furthermore, hexanal, heptanal, octanal, 6-methyl-5-hepten-2-one, 1-nonanol, etc. and octanal, nonanal, 2-ethyl-1-hexanol, 1-nonanol, etc. were greater contribution on the distinction of male and female eggs during E1 and E3 (Table S2). Hexanal showed a significant difference between male and female Leptolossus zobatus (Inoue et al., 2019) and heptanal could reduce its sensitivity to the peripheral and central olfactory level independently of mating status (Deisig et al., 2012). Hexanal, heptanal, and nonanal were also reported have the potential to attract female T. infestans (Fontan et al., 2002).

Figure 3.

Figure 3

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Scatter plot of VOCs emitted from male and female chicken eggs during E0-E9 of incubation by canonical discriminant analysis.

While carbon dioxide, heptanal, 2-fluoro-7-hydroxybicyclo[2.2.1] heptane, etc. carbon dioxide, hexanal, decanal, undecanal, etc. and octanal, 6,10-dimethyl-5,9-undecadien-2-one, 2-ethyl-1-hexanol, 1-nonanol, etc. were greater contribution on the distinction of male and female eggs during E5, E7, and E9, respectively (Table S2). It is well accepted that the difference of carbon dioxide between eggs from both sexes may be resulted from differential metabolism of male and female embryo in eggs (Martins, 2004). Coincidentally, more alcohols were also detected in male starlings during mating and breeding (Amo et al., 2012), such as nonanol was only found in male Trupanea vicina abdomen and released from pleural glands to influence the female's receptivity for mating attempts (Kosi et al., 2013).

In addition, the relationship between VOCs emitted from hatched eggs with their sex was further assessed by correlation analysis. Nonanal, cedrene (area, 0.01 < P < 0.05), heptanal (area, 0.05 < P < 0.1) and carbon dioxide (area, 0.01 < P < 0.05), heptadecanamine, undecanal (area, 0.05 < P < 0.1) were significantly positively correlated with gender of eggs during E1 and E5, respectively. Six,10-dimethyl-5,9-undecadien-2-one (area, 0.05 < P < 0.1), octanal (percentage, 0.01< P < 0.05), and octanal (percentage, 0.05 < P < 0.1) were significantly negatively correlated with gender of eggs during E7 and E9, respectively (Table 2). Cedrene could be selectively bonded and transported by CmedPBP4, which exhibited different expression levels and showed obvious antenna-specific expression patterns between sexes (Sun et al., 2016).

Table 2.

Correlation between sex and VOCs emitted from chicken eggs (W) during E0–E9.

E0
 E1
 E3
RT min Volatile compounds Area Percentage Area Percentage Area Percentage
R p R p R p R p R p R p
2.67 n-Hexane −0.453 0.189 −0.453 0.189 −0.129 0.723 −0.214 0.552
7.50 Hexanal −0.104 0.774 −0.104 0.774 0.244 0.496 0.175 0.629 −0.050 0.892 −.050 .892
12.56 Heptanal 0.070 0.848 −0.070 0.848 0.594 0.070 0.384 0.273 −0.129 0.723 −0.129 0.723
17.31 6-methyl-5-Hepten-2-one 0.104 0.774 0.105 0.773 0.313 0.378 0.314 0.376 0.035 0.924 0.433 0.244
18.08 Octanal 0.174 0.631 −0.035 0.924 0.313 0.378 −0.244 0.497 −0.174 0.631 −0.383 0.275
19.59 2-ethyl-1-Hexanol −0.557 0.094 −0.631 0.050 0.000 1.000 0.000 1.000 −0.497 0.144 −0.497 0.144
21.55 1-Nonanol 0.431 0.213 0.279 0.435 0.333 0.347 0.333 0.347
23.37 Nonanal 0.313 0.378 −0.174 0.631 0.661 0.037 0.104 0.774 −0.174 0.631 −0.035 0.924
26.63 2-Nonen-1-ol 0.035 0.924 0.174 0.631 0.313 0.378 0.035 0.924 −0.244 0.497 −0.383 0.275
28.35 Decanal 0.313 0.378 0.244 0.497 0.313 0.378 −0.035 0.924 −0.174 0.631 −0.244 0.497
31.69 1-Heptadecanamine 0.510 0.132 0.431 0.213 0.333 0.347 0.333 0.347
33.03 Undecanal 0.409 0.241 0.334 0.345 0.050 0.892 0.050 0.892
39.52 6,10-dimethyl-5,9-Undecadien-2-one 0.176 0.626 0.176 0.626 0.349 0.323 0.349 0.323 0.244 0.497 0.244 0.497
43.83 Cedrene −0.349 0.323 −0.140 0.700 0.643 0.045 0.643 0.045
E5 E7 E9
1.71 Carbon dioxide 0.661 0.037 0.419 0.228 −0.247 0.492 −0.247 0.492 0.000 1.000 0.000 1.000
2.67 n-Hexane 0.106 0.771 0.106 0.771 0.129 0.723 0.129 0.723
7.50 Hexanal 0.000 1.00 −0.070 0.848 −0.333 0.347 −0.333 0.347 −0.195 0.523 −0.065 0.833
12.56 Heptanal 0.140 0.700 −0.245 0.495 −0.317 0.372 −0.388 0.268 −0.259 0.394 −0.188 0.538
17.31 6-methyl-5-Hepten-2-one 0.313 0.378 0.244 0.497 0.279 0.434 0.419 0.228 0.082 0.789 0.247 0.415
18.08 Octanal 244 0.497 −0.313 0.378 −0.174 0.631 −0.731 0.016 −0.330 0.271 −0.536 0.059
18.52 2-Fluoro-7-hydroxybicyclo[2.2.1] heptane −0.300 0.400 −0.300 0.400
19.59 2-ethyl-1-Hexanol −0.157 0.665 −0.157 0.665 −0.037 0.919 −0.111 0.759 0.353 0.237 0.353 0.237
21.55 1-Nonanol 0.140 0.700 0.000 1.000 −0.111 0.759 −0.224 0.535 −0.347 0.245 −0.390 0.187
23.37 Nonanal 0.313 0.378 −0.174 0.631 −0.174 0.631 −0.313 0.378 −0.289 0.339 −0.165 0.590
26.63 2-Nonen-1-ol 0.383 0.275 0.000 1.000 0.035 0.924 0.104 0.774 −0.082 0.789 −0.103 0.737
28.35 Decanal 0.383 275 −0.453 0.189 0.104 0.774 0.313 0.378 −0.124 0.687 −0.082 0.789
31.69 1-Heptadecanamine 0.599 0.067 0.424 0.222 0.157 0.665 0.118 0.745 −0.179 0.558 −0.112 0.715
33.03 Undecanal 0.559 0.093 0.419 0.228 0.104 0.349 0.419 0.228 −0.041 0.894 0.000 1.000
35.97 3-(bromomethyl)-Piperidine 0.497 0.144 0.497 0.144
37.76 Dodecanal 0.431 0.213 0.394 0.260
39.52 6,10-dimethyl-5,9-Undecadien-2-one 0.383 0.275 0.349 0.323 −0.301 0.055 0.419 0.228 0.247 0.415 0.372 0.211
43.83 Cedrene −0.129 0.723 −0.129 0.723
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R: Spearman's correlation coefficient, p: significance value.

Bold: 0.05 < P < 0.1; bold and italic: P < 0.05.

More importantly, it is noticed that the variation and correlation between VOCs and sex of chicken eggs were strongly influenced by incubation time. Hexane, 2-ethyl-1-hexanol, decanal, cedrene, etc. were greater contribution on the distinction of unhatched male and female eggs and 2-ethyl-1-hexanol (area, 0.05 < P < 0.1; percentage, P = 0.05) were significantly negatively correlated with gender of unhatched fertilized eggs. Furthermore, the potential role of other sex-related pheromone alcohols, including 2-ethyl-1-hexanol, 1-octanol, etc., are not yet clear (Levi-Zada et al., 2013; Webster et al., 2015).

Differences in VOCs Between Unhatched Male and Female Eggs for W, H, J Breed

Based on the above findings, 69 H and 60 J unhatched fertilized eggs were used for datas acquisition to explore the difference between VOCs from unhatched male and female eggs together. Thirty five and 29 fertilized eggs (H) were identified as male and female eggs, the rest 5 H eggs were infertile or sex were not sure; 26 fertilized eggs (J) were both identified as male and female eggs, the rest 8 J eggs were infertile or sex were not sure (Figure S1). The weight of fertilized eggs for H and J breed had no significant difference.

Comparison Analysis

A total of 27 VOCs were identified in unhatched fertilized eggs, among them, 11, 14, and 20 VOCs in W, H, and J eggs, respectively (Figure 4 and Table 3). There were certain variation in absolute abundance and relative content of each VOC between male and female eggs and no significant difference in common VOCs (both for area and percentage) were found between male and female eggs for 3 breeds (W, H, and J) (Figure 4 and Table 3). But, some sex-specific VOCs were found in eggs for each breed, for instance, the concentration (area and percentage) of nonanal was significant different between unhatched male and female H eggs (0.05 < P < 0.1); the percentage of pentanoic acid, 2,2,4-trimethyl-3-carboxyisopropyl, isobutyl ester and 6,10dimethyl-5,9-undecadien-2-one emitted from female H eggs were found to be higher than that from male H eggs (0.05 < P < 0.1). While the percentage of octanal, 2-nonen-1-ol, decanal (0.05 < P < 0.1) and cyclopropanecarboxamide, heptadecane (P < 0.05) were different between male and female J eggs and the absolute abundance (area) of undecanal (0.05 < P < 0.1) from female J eggs was higher than that from male J eggs.

Figure 4.

Figure 4

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Sex difference in concentration of VOCs emitted from unhatched chicken eggs (W, H, J). (Mean ± SE; M: male; F: female). Asterisk *: 0.05< P < 0.1; **: P < 0.05 above each bar indicates significant difference (P < 0.05/0.01) (NWM = 5, NWF = 5; NHM = 35, NHF = 29; NJM=26, NJF = 26). (Hxe: Hexane, Hea: Hexanal, Hpa: Heptanal, MHO: 6-methyl-5-Hepten-2-one, Ota: Octanal, EHL: 2-ethyl-1-Hexanol, Noa: Nonanal, NL: 2-Nonen-1-ol, Dea: Decanal, DMUO: 6,10-dimethyl-5,9-Undecadien-2-one, Ce: Cedrene, OtL: 1-Octanol, HDA: Heptadecanamine, Uda: Undecanal, DMUO: 6,10-dimethyl-5,9-Undecadien-2-one, TCPE: Pentanoic acid,2,2,4-trimethyl-3-carboxyisopropyl,isobutyl ester, FAA: 2-fluoro-Acetamide, BB: butyl-Benzene, UA1: Unknown amines-1, OO:9-oxabicyclo[6.1.0]nonan-4-One, MA: N-methyl-1,3-Propanediamine, FD: 1-fluoro-Dodecane, CPA: Cyclopropanecarboxamide, HPD: Heptadecane, CA: Cyclopropanecarboxamide, MHA: 5-methyl-2-Hexanamine, UA2:Unknown amines-2, PCE: Phthalic acid,4-cyanophenyl nonyl ester).

Table 3.

Mean levels of VOCs between unhatched male and female eggs for W, H, and J breeds (mean ± SE).

Peak area
 Relative percentage
W RT (min) Volatile compounds M (5) F (5) M (5) F (5)
2.67 n-Hexane 707,289 ± 314,503 248,720 ± 86,168 2.84 ± 1.56 0.76 ± 0.25
7.50 Hexanal 1,919,745 ± 1,586,849 1,293,390 ± 959,108 2.66 ± 1.24 2.81 ± 1.90
12.56 Heptanal 1,191,947 ± 670,059 1,073,318 ± 471,558 2.51 ± 1.06 2.55 ± 1.10
17.31 6-methyl-5-Hepten-2-one 1,376,781 ± 521,867 1,418,052 ± 511,202 3.79 ± 1.26 4.15 ± 1.19
18.08 Octanal 1,225,262 ± 395,234 1,127,781 ± 201,276 3.39 ± 0.37 3.14 ± 0.24
19.59 2-ethyl-1-Hexanol 491,138 ± 177,650 108,328 ± 108,328 1.45 ± 0.64 1.09 ± 0.00
23.37 Nonanal 22,245,370 ± 6,172,755 2,2751,195 ± 326,1170 64.77 ± 4.16 64.42 ± 2.12
26.63 2-Nonen-1-ol 655662 ± 195,328 699,271 ± 107,457 1.85 ± 0.28 2.01 ± 0.21
28.35 Decanal 5,131,160 ± 1,492,170 5,695,401 ± 463,820 14.37 ± 1.18 17.21 ± 2.76
39.52 6,10-dimethyl-5,9-Undecadien-2-one 424,690 ± 223,201 640,914 ± 290,067 1.16 ± 0.75 2.35 ± 0.65
43.83 Cedrene 330,705 ± 131,457 245,510 ± 101,300 1.04 ± 0.48 0.64 ± 0.24
H RT (min) Volatile compound M (35) F (29) M (35) F (29)
2.82 n-Hexane 1,344,555 ± 577,628 999,085 ± 394,616 5.01 ± 2.1 3.61 ± 1.48
7.60 Hexanal 2,138,657 ± 3,00,294 1,571,211 ± 311,178 5.87 ± 0.95 3.95 ± 0.74
12.62 Heptanal 398,762 ± 86,116 280,835 ± 85,806 0.74 ± 0.15 0.53 ± 0.16
17.33 6-methyl-5-Hepten-2-one 2,628,461 ± 450,410 3,304,721 ± 834,025 6.76 ± 0.67 8.09 ± 1.16
18.11 Octanal 1,783,573 ± 242,790 1,568,451 ± 247,404 4.82 ± 0.24 4.43 ± 0.32
21.57 1-Octanol 51,470 ± 22,393 37,121 ± 21,386 0.07 ± 0.03 0.04 ± 0.02
23.39 Nonanal 10,018,213 ± 1,002,809 7,567,484 ± 1,039,759 31.74 ± 1.90 23.5 ± 1.33
26.64 2-Nonen-1-ol 1,967,318 ± 320745 1,771,096 ± 318,926 4.96 ± 0.36 4.69 ± 0.31
28.38 Decanal 12,962,365 ± 1,939,274 11,827,630 ± 1,964,846 34.83 ± 2.06 32.58 ± 1.85
31.72 1-Heptadecanamine 38,494 ± 18,716 34,333 ± 19,362 0.05 ± 0.02 0.04 ± 0.02
33.05 Undecanal 104,955 ± 32,775 77,739 ± 31,926 0.16 ± 0.05 0.11 ± 0.04
39.53 6,10dimethyl-5,9-Undecadien-2-one 1,271,976 ± 233,460 1,617,635 ± 365,640 3.23 ± 0.34 4.21 ± 0.65
43.79 Pentanoic acid, 2,2,4-trimethyl-3-carboxyisopropyl,isobutyl ester 69,292 ± 22,000 101,251 ± 31,307 0.19 ± 0.08 0.63 ± 0.25
43.85 Cedrene 353,624 ± 66,068 334,235 ± 85,153 1.45 ± 0.4 1.76 ± 0.57
J RT(min) Volatile compound M (26) F (26) M (26) F (26)
4.32 2-fluoro-Acetamide 89,612 ± 49,903 97,020 ± 50,797 0.80 ± 0.55 0.32 ± 0.14
17.31 6-methyl-5-Hepten-2-one 174,959 ± 38,928 172,731 ± 42,658 1.27 ± 0.28 1.19 ± 0.33
18.10 Octanal 232,916 ± 27,542 284,891 ± 66,766 1.87 ± 0.13 1.44 ± 0.20
20.73 butyl-Benzene 34,630 ± 14,274 38,886 ± 16,178 0.22 ± 0.11 0.12 ± 0.05
23.40 Nonanal 4,242,659 ± 609,424 4,521,948 ± 91,3521 34.36 ± 1.85 32.34 ± 2.07
26.27 Unknown amines-1 11,027 ± 7,670 24,854 ± 12,116 0.04 ± 0.03 0.08 ± 0.04
26.64 2-Nonen-1-ol 420,446 ± 44,638 656,148 ± 142,147 3.57 ± 0.24 4.19 ± 0.28
28.37 Decanal 4,810,998 ± 516,709 7,162,462 ± 14,65934 41.73 ± 2.31 46.91 ± 1.61
33.03 Undecanal 39,338 ± 15,266 102,362 ± 29,554 0.18 ± 0.07 0.36 ± 0.09
37.77 9-Oxabicyclo[6.1.0]nonan-4-one 4,334 ± 4,334 28,449 ± 14,076 0.01 ± 0.01 0.08 ± 0.04
39.52 6,10-dimethyl-5,9-Undecadien-2-one 845,923 ± 169,633 829,030 ± 17,3031 6.72 ± 0.77 6.13 ± 0.95
41.10 N-methyl-1,3-Propanediamine 31,791 ± 13,382 44,538 ± 19,718 0.16 ± 0.07 0.16 ± 0.07
43.84 Cedrene 602,349 ± 82,750 576,509 ± 101,946 5.42 ± 0.66 5.19 ± 0.69
44.97 1-fluoro-Dodecane, 68,623 ± 28,688 59,430 ± 28,449 0.34 ± 0.14 0.21 ± 0.09
46.08 Cyclopropanecarboxamide 167,133 ± 31,618 116,582 ± 35,191 1.37 ± 0.18 0.48 ± 0.12
46.22 Heptadecane 180,208 ± 40,670 125,787 ± 41,306 1.32 ± 0.22 0.52 ± 0.14
48.08 2-cyano-Acetamide 14,744 ± 8,737 15,242 ± 8,606 0.07 ± 0.04 0.05 ± 0.03
48.24 5-methyl-2-Hexanamine 247,63 ± 12,251 20,491 ± 11,816 0.11 ± 0.06 0.06 ± 0.04
48.83 Unknown amines-2 14,114 ± 6,781 13,787 ± 7,863 0.10 ± 0.06 0.04 ± 0.02
49.38 Phthalic acid, 4-cyanophenyl nonyl ester 36,206 ± 9,541 35,250 ± 10,052 0.30 ± 0.09 0.14 ± 0.04
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Meanwhile, average concentrations of hexane emitted from male eggs was found higher than that from female eggs both for W and H breed; mean concentrations of 6-methyl-5-hepten-2-one, 6,10-dimethyl-5,9-undecadien-2-one and cedrene, octanal emitted from female eggs were higher and lower than that from male eggs for all 3 breed, respectively (Figure S4).

Discriminant and Correlation Analysis

As might be expected, VOCs emitted from unhatched male and female eggs for W, H, and J breed were well separated in CD model, the accuracy of egg sexing were almost 90% (90–100%), except for 68.8% (area)-76.6% (Percentage) of H eggs (Table S3) and VOCs of female eggs were all trend to the upside of the male eggs (Figure 5 and Table S3). So it is verified that there were some difference between unhatched male and female eggs for 3 breeds. Hexane, 2-ethyl-1-hexanol, 2-nonen-1-ol, decanal, cedrene, etc., nonanal, hexanal, pentanoic acid, 2,2,4-trimethyl-3-carboxyisopropyl, isobutyl ester, etc., decanal, undecanal, cedrene, cyclopropanecarboxamide, etc. mostly contributed to differentiate unhatched (E0) male and female eggs for W, H, and J breed, respectively. Moreover, nonanal, decanal, 2-nonen-1-ol may contribute greater on the distinction of unhatched male and female eggs and cedrene may contributed greater for W and J eggs (Table S4).

Figure 5.

Figure 5

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Scatter plots of VOCs emitted from unhatched male and female W, H and J eggs by canonical discriminant analysis. (A: W, B: H, C: J; A (left): area, P (right): percentage; 105/6 = magnification of VOC percentage).

Furthermore, spearman's correlation was used to assess the intrinsic connection between VOCs emitted from unhatched eggs with their sex. Two-ethyl-1-hexanol (area, 0.05 < P < 0.1; percentage, P = 0.05) were found significantly negatively correlated with gender of unhatched W eggs. Octanal (percentage, 0.05 < P < 0.1), nonanal (area, 0.05 < P < 0.1; percentage, P < 0.01) and 2,2,4-trimethyl-3-carboxyisopropyl pentanoic acid, isobutyl ester (percentage, 0.05 < P < 0.1) were found significantly negatively and positively correlated with sex of unhatched H eggs, respectively. 2-Nonen-1-ol (percentage, 0.05 < P < 0.1) and cyclopropanecarboxamide (area, 0.05 < P < 0.1; percentage, P < 0.01), heptadecane (percentage, 0.05 < P < 0.1) were found significantly positively and negatively correlated with sex of unhatched J eggs (Table 4). Heptadecane has been reported as sex pheromones in 3 species of female moths (Wakamura et al., 2001; Minaeimoghadam et al., 2017).

Table 4.

Correlation between sex and VOCs emitted from unhatched chicken eggs (W, H, J).

RT min Breeds W (M = 5; F = 5)
 H (M = 35; F = 29)
 J (M = 26; F = 26)
Volatile compounds Area
 Percentage
 Area
 Percentage
 Area
 Percentage
R p R p R p R p R p R p
2.67 n-Hexane −0.453 0.189 −0.453 0.189 0.132 0.297 0.140 0.270
4.32 2-fluoro-Acetamide 0.086 0.546 0.078 0.581
7.50 Hexanal −0.104 0.774 −0.104 0.774 −0.167 0.187 −0.183 0.149
120.56 Heptanal 0.070 0.848 −0.070 0.848 −0.132 0.299 −0.146 0.251
170.31 6-methyl-5-Hepten-2-one 0.104 0.774 0.105 0.773 −0.009 0.942 0.120 0.345 −0.016 0.909 −0.068 0.632
180.10 Octanal 0.174 0.631 −0.035 0.924 −0.076 0.548 −0.212 0.092 −0.062 0.664 −0.150 0.287
19.59 2-ethyl-1-Hexanol −0.557 0.094 −0.631 0.050 .
20.73 butyl-Benzene 0.020 0.885 −0.035 0.803
21.57 1-Octanol −0.061 0.634 −0.075 0.554
23.40 Nonanal 0.313 0.378 −0.174 0.631 −0.223 0.076 −0.385 0.002 −0.115 0.416 −0.094 0.510
26.27 Unknown amine-1 0.120 0.396 0.115 0.415
26.64 2-Nonen-1-ol 0.035 0.924 0.174 0.631 −0.009 0.946 −0.121 0.339 0.027 0.850 0.279 0.045
28.37 Decanal 0.313 0.378 0.244 0.497 −0.033 0.795 −0.133 0.293 0.005 0.971 0.217 0.123
31.72 1-Heptadecanamine −0.020 0.873 −0.030 0.815
33.03 Undecanal −0.072 0.571 −0.081 0.523 0.221 0.115 0.215 0.126
37.77 9-Oxabicyclo[6.1.0]nonan-4-one 0.200 0.154 0.205 0.144
39.52 6,10-dimethyl-5,9-Undecadien-2-one 0.176 0.626 0.176 0.626 0.055 0.664 0.159 0.209 −0.041 0.773 −0.145 0.306
41.10 N-methyl-1,3-Propanediamine 0.017 0.903 −0.003 0.981
43.79 2,2,4-trimethyl-3-carboxyisopropyl, Pentanoic acid, isobutyl ester 0.123 0.333 0.228 0.070
43.84 Cedrene −0.349 0.323 −0.140 0.700 −0.052 0.683 −0.063 0.622 −0.123 0.385 −0.083 0.557
44.97 1-fluoro-Dodecane −0.089 0.533 −0.095 0.501
46.08 Cyclopropanecarboxamide −0.238 0.089 −0.508 0.000
46.22 Heptadecane −0.207 0.142 −0.393 0.004
48.08 2-cyano-Acetamide 0.002 0.987 −0.012 0.935
48.24 5-methyl-2-Hexanamine −0.052 0.715 −0.069 0.626
48.83 Unknown amines-2 −0.039 0.784 −0.069 0.626
49.38 Phthalic acid, 4-cyanophenyl nonyl ester −0.006 0.967 −0.122 0.390
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R: Spearman's correlation coefficient, p: significance value.

Bold: 0.05 < P < 0.1; bold and italic: P < 0.05.

What's more, hexanal and 6,10-dimethyl-5,9-undecadien-2-one were found to be negatively and positively correlated with sex for W and H eggs, while nonanal was negatively correlated with sex for H and J eggs. More importantly, cedrene was found to be negatively correlated with eggs sex for all breeds, namely, the concentrations of which from unhatched male eggs were higher than female eggs for W, H, and J breed. The relation between most of these sex-related VOCs with sex has been discussed in detail during early of incubation and we won't reiterate it here. It should be stressed that, however, sex-related VOCs emitted from unhatched eggs may be mainly due to differential maternal allocation of resoures other than differential metabolism of embryo, such as estradiol, dihydrotestosterone and so on (Petrie et al., 2001; Kölliker et al., 2012). Fortunately and coincidentally, sex-specific VOCs emitted from chicken eggs were found to be related with steroid hormone biosynthesis in KEGG by enrichment analysis using Metabo Anlyst 4.0 (Figure 6).

Figure 6.

Figure 6

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Pathway of sex-related VOCs emitted from unhatched fertilized eggs in KEGG.

CONCLUSIONS

Difference in the composition (or content) of VOCs between (hatched) male and female chicken eggs (W) during E0-E9 and between unhatched male and female chicken eggs (W, H, and J) were confirmed in this research for the first time. Sex-specific VOCs were strongly influenced by incubation process and egg breed and have been found related with steroid hormone biosynthesis in KEGG. These results will be helpful for understanding the mechanisms of sex identity by cell-autonomous and maternal sex allocation in chicken eggs. More importantly, this study provide a new potential to identify the gender of unhatched fertilized chicken eggs by nondestructive way, although we have neglected the ecological roles of odor emitted from chicken eggs for a long time. Therefore, further works are necessary to investigate the formation mechanism of sex-related VOCs and to put it into practice.

ACKNOWLEDGMENTS

This work was supported by the Special Fund for the Modern Agro-Industry Technology Research System (Project code No. CARS-40-K24); Educational Science Research Project of Hunan Province (19C0080); Start-up fee for doctoral research in Changsha University of Science and Technology (097/000301521)

Authorship contribution statement: Xiaole Xiang: Writing-Original Draft, Methodology, Data Curation, Resources; Gan Hu: Methodology, Data Curation; Formal analysis; Guofeng Jin: Methodology, Resources, Writing-Review & Editing; Yongguo Jin: Resources, Software, Validation; Meihu Ma: Conceptualization, Supervision.

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent: Informed consent is not applicable for this study.

DISCLOSURES

The authors declare that they have no conflict of interest.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2021.101619.

Appendix. Supplementary materials

mmc1.docx (4.4MB, docx)

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