1- PhD Student of Animal genetic and Breeding, Faculty of Animal Science and Food Technology, Agricultural Science and Natural Resources University of Khuzestan, Mollasani, Iran
2- Professor, Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Science and Natural Resources University of Khuzestan, Ahvaz, Iran.
3- 3. Associate Professor, Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Science and Natural Resources University of Khuzestan, Ahvaz, Iran.
4- 4. Associate Professor, Advanced Surface Engineering and Nano Materials Research Center Department of Electrical Engineering Ahvaz Branch, IAU, Ahvaz, Iran.
2- Professor, Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Science and Natural Resources University of Khuzestan, Ahvaz, Iran.
3- 3. Associate Professor, Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Science and Natural Resources University of Khuzestan, Ahvaz, Iran.
4- 4. Associate Professor, Advanced Surface Engineering and Nano Materials Research Center Department of Electrical Engineering Ahvaz Branch, IAU, Ahvaz, Iran.
Abstract: (39 Views)
Background:
Currently, determining the sex of poultry based on the physical differences between males and females is done by skilled technicians. This requires skilled labor and is also time-consuming. Raman spectroscopy is an analytical technique that relies on light scattering and the vibrational effects of molecules. It is recognized as a non-destructive tool for analyzing chemical structures and identifying various substances. This method was first discovered by Indian physicist Chandra Sekhar Venkata Raman in 1930, for which he was awarded the Nobel Prize. In this technique, samples are excited using a laser, and the light that is scattered from them provides valuable information about their structure and biochemical properties. One significant application of Raman spectroscopy is in determining the sex of chicks in eggs. Due to the biochemical differences between the cells of male and female birds, this technique can help identify the gender of eggs through blood cell analysis. This is particularly important in the poultry industry, Because chicks of the unwanted sex are usually killed after hatching., and employing Raman spectroscopy can help reduce this economic and ethical waste. As chick embryos develop, their blood composition undergoes significant changes, starting from the initial production of red blood cells to the full development of their circulatory system. Consequently, this study was conducted to determine the optimal time for identifying the sex of Iranian native chicken embryos through blood analysis using micro-Raman spectroscopy.
Methods:
Fertilized eggs were incubated at a temperature of 37.8°C and 53% humidity to facilitate embryonic development. Embryos at different ages—3.5, 5, 8, and 13 days—were evaluated separately using a UniRAM Microraman spectrometer with a wavelength of 785 nm. To visualize the embryonic vessels, a specialized camera system was developed that automatically identifies the relevant vessel and focuses the laser beam on it. The spectra obtained from the Raman spectrometer were plotted and analyzed using Origin software, with key indicators such as the intensity of Raman peaks and the intensity ratio of dominant peaks being utilized for analysis. Among the main indicators used to analyze the Raman spectrum were the intensity of the Raman peaks and the intensity ratio of the dominant peaks. After the analysis process was completed, in order to confirm the gender of the embryos, they were removed from the eggs and the DNA extraction process was carried out using the Animal Tissue DNA Isolation kit manufactured by the Denazist The quantity and quality of the extracted DNA were assessed using a NanoDrop device (Thermo Scientific NanoDROP 2000C, USA) and gel electrophoresis. The CHD gene was employed as a sex-determining marker, for which specific primers were designed. Polymerase chain reaction (PCR) was conducted to determine the sex of the samples using these primers, and the results were observed on a two-and-a-half percent agarose gel.
Results: The results of this study revealed significant differences in the average fluorescence intensity between male and female eggs, although there was considerable overlap. One of the charts examined in this research was the candlestick chart, this chart shows how the dominant peaks are distributed on different days of incubation for the tested eggs. Also, this chart shows the statistical distribution of the data, which includes information about the central indicators. Finally, this chart also provided a statistical distribution of the data, highlighting central indices. The most pronounced difference in fluorescence intensity was observed on day 3.5 of incubation, where the fluorescence spectra of male and female blood exhibited distinct shapes. After day 4, the fluorescence intensity remained higher in male chicks; however, the spectral shape diminished rapidly and was completely lost by day 5. These variations can be attributed to differences in the production of red blood cells and hemoglobin between the sexes, as well as the influence of sex chromosomes on gonadal structure. The difference in hemoglobin levels is related to the difference in the oxygen saturation of the blood of males and females, so that male blood is likely to have a higher oxygen affinity than female blood. Male chickens possess ZZ chromosomes, while female chickens have ZW chromosomes. This genetic distinction leads to differences in physiological and biochemical characteristics, as well as variations in gonadal structure. Male chickens have ZZ chromosomes and female chickens have ZW chromosomes. This difference in genetic structure leads to differences in physiological and biochemical characteristics and differentiation in the structure of the gonads. Polymerase chain reaction was used to confirm the Raman spectroscopy results. The results of agarose gel electrophoresis showed two bands with lengths of 461 and 322 base pair for female embryos (ZW) and one fragment with a length of 461 base pair for male embryos (ZZ).
Conclusion: According to the findings of this study, day 3.5 of incubation was identified as the optimal time for determining the gender of native chicken embryos using micro Raman spectroscopy. This method boasts several advantages, including high accuracy and a short analysis time, which are essential for developing industrial systems aimed at sexing eggs. By combining advanced techniques from Raman spectroscopy with genetic analysis, this approach allows for effective gender determination of chickens in the early stages of hatching, making it particularly beneficial for the poultry industry.
Currently, determining the sex of poultry based on the physical differences between males and females is done by skilled technicians. This requires skilled labor and is also time-consuming. Raman spectroscopy is an analytical technique that relies on light scattering and the vibrational effects of molecules. It is recognized as a non-destructive tool for analyzing chemical structures and identifying various substances. This method was first discovered by Indian physicist Chandra Sekhar Venkata Raman in 1930, for which he was awarded the Nobel Prize. In this technique, samples are excited using a laser, and the light that is scattered from them provides valuable information about their structure and biochemical properties. One significant application of Raman spectroscopy is in determining the sex of chicks in eggs. Due to the biochemical differences between the cells of male and female birds, this technique can help identify the gender of eggs through blood cell analysis. This is particularly important in the poultry industry, Because chicks of the unwanted sex are usually killed after hatching., and employing Raman spectroscopy can help reduce this economic and ethical waste. As chick embryos develop, their blood composition undergoes significant changes, starting from the initial production of red blood cells to the full development of their circulatory system. Consequently, this study was conducted to determine the optimal time for identifying the sex of Iranian native chicken embryos through blood analysis using micro-Raman spectroscopy.
Methods:
Fertilized eggs were incubated at a temperature of 37.8°C and 53% humidity to facilitate embryonic development. Embryos at different ages—3.5, 5, 8, and 13 days—were evaluated separately using a UniRAM Microraman spectrometer with a wavelength of 785 nm. To visualize the embryonic vessels, a specialized camera system was developed that automatically identifies the relevant vessel and focuses the laser beam on it. The spectra obtained from the Raman spectrometer were plotted and analyzed using Origin software, with key indicators such as the intensity of Raman peaks and the intensity ratio of dominant peaks being utilized for analysis. Among the main indicators used to analyze the Raman spectrum were the intensity of the Raman peaks and the intensity ratio of the dominant peaks. After the analysis process was completed, in order to confirm the gender of the embryos, they were removed from the eggs and the DNA extraction process was carried out using the Animal Tissue DNA Isolation kit manufactured by the Denazist The quantity and quality of the extracted DNA were assessed using a NanoDrop device (Thermo Scientific NanoDROP 2000C, USA) and gel electrophoresis. The CHD gene was employed as a sex-determining marker, for which specific primers were designed. Polymerase chain reaction (PCR) was conducted to determine the sex of the samples using these primers, and the results were observed on a two-and-a-half percent agarose gel.
Results: The results of this study revealed significant differences in the average fluorescence intensity between male and female eggs, although there was considerable overlap. One of the charts examined in this research was the candlestick chart, this chart shows how the dominant peaks are distributed on different days of incubation for the tested eggs. Also, this chart shows the statistical distribution of the data, which includes information about the central indicators. Finally, this chart also provided a statistical distribution of the data, highlighting central indices. The most pronounced difference in fluorescence intensity was observed on day 3.5 of incubation, where the fluorescence spectra of male and female blood exhibited distinct shapes. After day 4, the fluorescence intensity remained higher in male chicks; however, the spectral shape diminished rapidly and was completely lost by day 5. These variations can be attributed to differences in the production of red blood cells and hemoglobin between the sexes, as well as the influence of sex chromosomes on gonadal structure. The difference in hemoglobin levels is related to the difference in the oxygen saturation of the blood of males and females, so that male blood is likely to have a higher oxygen affinity than female blood. Male chickens possess ZZ chromosomes, while female chickens have ZW chromosomes. This genetic distinction leads to differences in physiological and biochemical characteristics, as well as variations in gonadal structure. Male chickens have ZZ chromosomes and female chickens have ZW chromosomes. This difference in genetic structure leads to differences in physiological and biochemical characteristics and differentiation in the structure of the gonads. Polymerase chain reaction was used to confirm the Raman spectroscopy results. The results of agarose gel electrophoresis showed two bands with lengths of 461 and 322 base pair for female embryos (ZW) and one fragment with a length of 461 base pair for male embryos (ZZ).
Conclusion: According to the findings of this study, day 3.5 of incubation was identified as the optimal time for determining the gender of native chicken embryos using micro Raman spectroscopy. This method boasts several advantages, including high accuracy and a short analysis time, which are essential for developing industrial systems aimed at sexing eggs. By combining advanced techniques from Raman spectroscopy with genetic analysis, this approach allows for effective gender determination of chickens in the early stages of hatching, making it particularly beneficial for the poultry industry.
Type of Study: Research |
Subject:
ژنتیک و اصلاح نژاد طیور
Received: 2025/01/18 | Accepted: 2026/02/21
Received: 2025/01/18 | Accepted: 2026/02/21
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