Non-destructive egg breed separation using advanced VOC analytical techniques HSSE-GC-MS, PTR-TOF-MS, and SIFT-MS: Assessment of performance and systems' complementarity.

Over the past decade, advanced analytical techniques have been utilized to examine volatile organic compounds (VOCs) in eggs. These VOCs offer valuable insights into factors such as freshness, fertility, the presence of cracks, embryo sex, and breed. In our study, we assessed three mass spectrometry-based systems (headspace sorptive extraction gas chromatography-mass spectrometry; HSSE-GC-MS, proton transfer reaction time-of-flight-mass spectrometry; PTR-TOF-MS; and selected ion flow tube mass spectrometry; SIFT-MS) to analyze and identify VOCs present in intact hatching eggs from three distinct breeds (Dekalb white layer, Shaver brown layer, and Ross 308 broiler). The eggs were sampled on incubation days 2 and 8, to identify VOCs that distinguish breeds irrespective of incubation day. VOC measurements were conducted on 15 eggs per breed by placing them together with PDMS-coated stir bars inside inert Teflon® air sampling bags. After an accumulation period of 2 h, the headspace was analyzed using PTR-TOF-MS and SIFT-MS, while the VOCs adsorbed onto the stir bars were analyzed using GC-MS for additional compound identification. Partial least squares discriminant analysis (PLS-DA) models were constructed for breed differentiation, and variable selection was performed. As a result, 111 VOCs were identified using HSSE-GC-MS, with alcohols and esters being the most abundant. The PLS-DA models demonstrated the efficacy of breed discrimination, with the HSSE-GC-MS and the PTR-TOF-MS exhibiting the highest balanced accuracy of 95.5 % using a reduced set of 11 VOCs and 5 product ions, respectively. The SIFT-MS model had a balanced accuracy of 92.8 % with a reduced set of 11 product ions. Furthermore, complementarity was observed between HSSE-GC-MS, which primarily selected higher molecular weight VOCs, and PTR-TOF-MS and SIFT-MS. A higher correlation was found for compound abundances between the HSSE-GC-MS and the PTR-TOF-MS relative to the SIFT-MS, indicating that the PTR-TOF-MS was better suited to quantify specific compounds identified by the HSSE-GC-MS. Finally, the findings support the presence of VOCs originating from both synthetic and natural sources, highlighting the ability of the VOC analysis systems to non-destructively perform quality control and reveal differences in management practices or biological information encoded in eggs.

Allantoic Fluid-Based qPCR for Early Onset In Ovo Sexing.

The day-old male chick culling remains a welfare issue in the poultry industry. Several governments have prohibited this practice, pushing hatcheries to seek alternatives. Although different solutions exist for solving this problem, sex determination during the embryo's incubation (in ovo sexing) is considered the most suitable one among the consumers and industry. However, to be industrialized, in ovo sexing technologies must meet several requirements: compatibility with all egg colors and early developmental stages while maintaining a high hatchability rate and accuracy at low cost and high throughput. To meet these requirements, we studied the use of the sexual genes HINTW (female-specific) and DMRT-1 (both sexes) at incubation days 6-9. By utilizing the quantitative polymerase chain reaction in allantoic fluid (AF) samples, our study confirmed female-specific HINTW detection on all days without any significant detrimental effects on embryo development. We achieved 95% sexing accuracy using the HINTW cycle threshold (Ct) alone and 100% accuracy rate when using Δλ values (difference between the HINTW and DMRT-1 Ct). In conclusion, the developed assay can provide information about AF as a sample for in ovo sexing and open new industrial possibilities for faster and cheaper assays.

Noninvasive Assessment of Chicken Egg Fertility during Incubation Using HSSE-GC-MS VOC Profiling.

Volatile organic compounds (VOCs) carry crucial information about chicken egg fertility. Assessing the fertility before incubation holds immense potential for poultry industry efficiency. Our study used headspace sorptive extraction-gas chromatography-mass spectrometry to analyze egg VOCs before and during the initial 12 incubation days. A total of 162 VOCs were identified. Hexanal was significantly higher in unfertilized eggs, whereas compounds such as propan-2-ol, propan-2-one, and carboxylic acids were higher in fertilized eggs. Furthermore, the obtained multiple logistic regression model outperformed the partial least-squares-discriminant analysis (PLS-DA) model, demonstrating lower complexity and superior performance. Fertile eggs were accurately identified in the validation set in 68-75% of the cases during the initial 4 days, to 85 and 100% on days 6 and 8. Finally, hierarchical cluster analysis in fertilized eggs revealed the clustering of VOCs of the same chemical class, indicative of their shared biochemical origin. This suggests a promising direction for future research aimed at understanding the biological information embedded in VOCs and their relationship to biochemical processes during embryo development.

Non-Destructive Eggshell Strength Assessment Using Hertz Contact Theory Part I: Theory and Applicability.

In the egg industry, fast and highly reliable quality measurements are crucial. This study presents a novel method based on Hertz contact theory that allows for non-destructive determination of eggshell strength. The goal of the study was to evaluate the material strength (Young's Modulus) and structural strength (stiffness) of eggshells. To this end, an experimental setup was constructed to measure the collision of an eggshell with a small steel ball, which was recorded using a laser vibrometer. The study analyzed a sample of 120 eggs and found a correlation of 0.85 between the traditional static stiffness measured during quasi-static compression tests and the stiffness obtained from the Hertz contact theory. The results show that Hertz contact theory is valid for small steel spheres impacting eggshells, while a sensitivity analysis indicated that the most important factor in determining the strength of the eggshell is the contact duration between the egg and the impactor. These results open up the possibility of grading eggs based on their shell strength in a non-destructive manner.

Non-Destructive Eggshell Strength Assessment Using Hertz Contact Theory-Part II: Implementation and Validation.

Eggshell strength is a critical quality factor for consumption eggs as it affects the probability of breakage in practice. In this study, a fast and low-cost methodology for the non-destructive determination of eggshell strength is presented. The method utilized a small steel ball to impact the egg and a microphone to analyse the impact characteristics. Hertz contact theory was applied to relate the measured impact characteristics to the local stiffness of the eggshell. Therefore, a total of 150 eggs were studied on which eight consecutive measurements per egg were taken around the equator at equidistant places. The results showed a strong correlation of 0.93 between the traditional static stiffness measured during quasi-static compression tests and the average stiffness obtained from the new methodology. This paves the way towards fast, low-cost and non-destructive in-line shell strength measurements to reduce the number of cracked eggs reaching the consumer.

Trends in in ovo sexing technologies: insights and interpretation from papers and patents.

Numerous researchers and institutions have been developing in ovo sexing technologies to improve animal welfare by identifying male embryos in an early embryonic stage and disposing of them before pain perception. This review gives a complete overview of the technological approaches reported in papers and patents by performing a thorough search using Web of Science and Patstat/Espacenet databases for papers and patents, respectively. Based on a total of 49 papers and 115 patent families reported until May 2023 worldwide, 11 technology categories were defined: 6 non-optical and 5 optical techniques. Every category was described for its characteristics while assessing its potential for application. Next, the dynamics of the publications of in ovo sexing techniques in both paper and patent fields were described through growth curves, and the interest or actual status was visualized using the number of paper citations and the actual legal status of the patents. When comparing the reported technologies in papers to those in patents, scientific gaps were observed, as some of the patented technologies were not reported in the scientific literature, e.g., ion mobility and mass spectrometry approaches. Generally, more diverse approaches in all categories were found in patents, although they do require more scientific evidence through papers or industrial adoption to prove their robustness. Moreover, although there is a recent trend for non-invasive techniques, invasive methods like analyzing DNA through PCR or hormones through immunosensing are still being reported (and might continue to be) in papers and patents. It was also observed that none of the technologies complies with all the industry requirements, although 5 companies already entered the market. On the one hand, more research and harmony between consumers, industry, and governments is necessary. On the other hand, close monitoring of the market performance of the currently available techniques will offer valuable insights into the potential and expectations of in ovo sexing techniques in the poultry industry.

Increasing the Robustness of SIFT-MS Volatilome Fingerprinting by Introducing Notional Analyte Concentrations.

Selected ion flow tube-mass spectrometry (SIFT-MS) is an analytical technique for volatile detection and quantification. SIFT-MS can be applied in a "white box" approach, measuring concentrations of target compounds, or as a "black box" fingerprinting technique, scanning all product ions during a full scan. Combining SIFT-MS full scan data acquired from multibatches or large-scale experiments remains problematic due to signal fluctuation over time. The standard approach of normalizing full scan data to the total signal intensity was insufficient. This study proposes a new approach to correct SIFT-MS fingerprinting data. In this concept, all of the product ions from a full scan are considered individual compounds for which notional concentrations can be calculated. Converting ion count rates into notional analyte concentrations accounts for any changes in the instrument parameters. The benefits of the proposed approach are demonstrated on three years of data from both multibatches and long-term experiments showing a significant reduction of system-induced fluctuations providing a better focus on the changes of interest.

In ovo sexing of eggs from brown breeds with a gender-specific color using visible-near-infrared spectroscopy: effect of incubation day and measurement configuration.

The culling of day-old male chicks is an animal welfare issue within the laying hen industry that raises substantial ethical concern. Alternative methods are sought to pre-select males during embryonic development. This method is called in ovo sexing and allows more humane male culling. A robust and non-invasive in ovo color sexing technique was developed and validated in this research. To this end, visible-near-infrared (vis-NIR) point spectroscopy was used, which has advantages over state-of-the-art hyperspectral imaging in terms of accuracy and cost. Two independent experiments were each conducted on a batch of 600 Isa Brown eggs. These eggs were individually illuminated on d 8 to 14, and d 18 of incubation by a halogen lamp and the signal was measured in the vis-NIR range from 300 to 1,145 nm. Next, optimal preprocessing strategies were applied and partial least squares discriminant analysis (PLS-DA) models were built and further optimized after performing a forward interval partial least squares (FiPLS) for variable selection. Results demonstrated that d 12 is too early for vis-NIR in ovo sexing, resulting in a prediction accuracy of 86.49%. However, after 13 d of incubation, an accuracy of 97.78% was obtained, increasing to 99.52% on d 14. Furthermore, these accuracies were higher than earlier reported percentages from hyperspectral imaging and successful sexing was expedited from d 14 to d 13. Moreover, prediction improvement up to 99.05% was obtained on d 13 by correcting for the variability in eggshell properties using d 0 eggshell corrections. Applying the method on d 18 resulted in a lower accuracy of 94.62% due to stronger light attenuation by the growing embryos. Finally, a reduced spectral range of 749 to 861 nm was found to be sufficient for correct classification of 98.46% of the eggs. This paves the way for high-throughput and cost-efficient usage of smaller and cheaper spectrophotometers in commercial hatcheries.