Application of VDAC1 protein as biomarker of freezing resistance of cock sperms

Abstract

The invention discloses application of VDAC1 protein as a biomarker for determining the freezing resistance of cock sperms. The key differential protein VDAC1 of cock sperm anti-freezing difference individuals is identified by adopting a 4D-label free technology, and the expression of the protein in highly anti-freezing individuals is remarkably up-regulated; the VDAC1 protein content and corresponding anti-freezing performance groups are further adopted to construct a classification model, an ROC curve is drawn to evaluate the effectiveness of the VDAC1 protein serving as the biomarker for screening the anti-freezing performance of the cock sperms, an ROC curve evaluation result shows that AUC is 0.9898, and the reliability of the VDAC1 protein serving as the biomarker for screening the anti-freezing performance of the cock sperms is proved. The method is beneficial for improving the cryopreservation efficiency of the chicken sperms, and has application prospects in the aspects of promotion of chicken genetic resource protection, efficient utilization of excellent cocks and the like.

Description

Technical Field

[0001] The present invention relates to a new use of VDAC1 as a biomarker, in particular to the use of VDAC1 protein as a biomarker for the cryotolerance of rooster sperm, and belongs to the field of new uses of VDAC1 protein as a biomarker. Background Art

[0002] Semen cryopreservation is a process in which semen is diluted, equilibrated, cryoprotectant is added, and then re-equilibrated, followed by programmed cooling to inhibit sperm metabolism, enabling sperm to be stored at ultra-low temperature in liquid nitrogen (-196°C) for a long time. After thawing and warming, the sperm can recover their fertilization ability. Semen cryopreservation technology is an important means for long-term preservation of male animal reproductive ability and has broad application prospects in germplasm resource preservation and variety improvement. Establishing a stable and efficient chicken semen cryopreservation technology has always been a research hotspot and difficulty in this field.

[0003] Currently, the research of many scholars focuses on the screening of semen cryopreservation procedures, and there is less research on the mechanism of cryodamage, and no breakthrough has been made so far. Sperm are highly differentiated cells without endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes or ribosomes. Therefore, in mature sperm cells, transcription and translation are very limited. In recent years, with the development of technology, people can detect the changes in the expression levels of related genes and proteins in sperm cells before and after freezing through technical means such as transcriptomics and proteomics to reveal the mechanism of cryodamage.

[0004] There may be a close correlation between the protein composition and content of sperm and the cryotolerance of sperm. These proteins play an indispensable role in maintaining sperm function during sperm freezing and preservation. By detecting the content of specific sperm proteins, non-destructive detection of the cryotolerance of individual sperm can be achieved, which helps to quickly screen cryotolerant individuals. Summary of the Invention

[0005] One object of the present invention is to provide the use of VDAC1 protein as a biomarker for the cryotolerance of rooster sperm;

[0006] Another object of the present invention is to provide a method for selecting rooster sperm with high cryotolerance by using VDAC1 protein;

[0007] A further object of the present invention is to provide a detection kit for identifying the cryotolerance of rooster sperm.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] One aspect of the present invention is to provide the use of VDAC1 protein as a biomarker for the high or low cryotolerance of rooster sperm; wherein, the amino acid sequence of the VDAC1 protein is shown as SEQ ID No.1.

[0010] A preferred specific embodiment of the present invention includes: identifying the expression level of VDAC1 protein in rooster sperm; if the expression level of VDAC1 protein in rooster sperm is high, then the cryotolerance of the rooster sperm is high; if the expression level of VDAC1 protein in rooster sperm is low, then the cryotolerance of the rooster sperm is low; more preferably, if the expression level of VDAC1 protein in rooster sperm is greater than or equal to 3688 ng / ml, then the cryotolerance of the rooster sperm is high; if the expression level of VDAC1 protein in rooster sperm is less than 3688 ng / ml, then the cryotolerance of the rooster sperm is low.

[0011] Another aspect of the present invention is to provide a method for breeding roosters with high cryotolerance sperm by applying the VDAC1 protein, including: detecting the expression level of VDAC1 protein in rooster sperm, and screening to obtain roosters with high expression level of VDAC1 protein, that is, obtaining roosters with high cryotolerance sperm.

[0012] A preferred specific embodiment of the present invention is that if the expression level of VDAC1 protein in rooster sperm is greater than or equal to 3688 ng / ml, then the cryotolerance of the rooster sperm is high, that is, obtaining roosters with high cryotolerance sperm.

[0013] Those skilled in the art can use various conventional methods to identify the expression level of VDAC1 protein in roosters. As a specific embodiment of the present invention, Western blot can be used to identify the relative expression level of VDAC1 protein in roosters.

[0014] Another aspect of the present invention is to provide a detection kit for identifying the high or low cryotolerance of rooster sperm, including: an antibody, a standard product, an HRP-labeled detection antibody, and a chromogenic solution, wherein the antibody is a VDAC1 protein antibody.

[0015] The present invention identified the key differential protein VDAC1 of rooster spermatozoa with different cryotolerance by 4D-label free technology, and this protein was significantly up-regulated in individuals with high cryotolerance. The PRM technology and Western blot technology were used to verify that the VDAC1 protein can be used as a biomarker for screening cryotolerant individuals. The present invention further constructed a classification model by grouping according to the VDAC1 protein content and the corresponding cryotolerance performance and drew an ROC curve to evaluate the effectiveness of VDAC1 as a biomarker for the cryotolerance performance of rooster spermatozoa. The evaluation result of the ROC curve showed that the AUC was 0.9898, close to 1, proving that the VDAC1 protein can be used as a biomarker for screening the cryotolerance performance of rooster spermatozoa, and the VDAC1 protein can be used as a biomarker to breed roosters with high cryotolerance performance of spermatozoa. The present invention is beneficial to improving the cryopreservation efficiency of chicken spermatozoa and has important significance for promoting the protection of chicken genetic resources and the efficient utilization of excellent roosters. Brief Description of the Drawings

[0016] Figure 1 Verify the differential expression of VDAC1 in high and low cryotolerance groups of rooster spermatozoa by PRM analysis.

[0017] Figure 2 Verify the differential expression of VDAC1 in high and low cryotolerance groups of rooster spermatozoa by Western blot analysis.

[0018] Figure 3 For the correlation analysis between the VDAC1 protein content of spermatozoa and the change range of sperm motility parameters before and after freezing.

[0019] Figure 4 ROC curve for evaluating the effectiveness of VDAC1 protein content as a biomarker for the cryotolerance performance of rooster spermatozoa. Detailed Embodiments

[0020] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0021] Example 1 Screening of sperm differential proteins related to the cryotolerance performance of rooster spermatozoa

[0022] (1) Screening of individuals with high and low cryotolerance differences:

[0023] The semen of 160 healthy 30-week-old Beijing Fatty Chicken roosters was collected by dorsal and abdominal massage method. Immediately, 200 μL of semen was aspirated and gently mixed with 200 μL of pre-warmed diluent at 37 °C, and then placed at 4 °C for 30 min of equilibration. 400 μL of diluent containing glycerol was added, gently mixed and placed at 4 °C for 10 min of equilibration. It was packaged with 0.5 mL thin tubes, put into a programmed freezer for cooling and freezing, and then stored in liquid nitrogen. During thawing, the thin tubes were taken out from liquid nitrogen and quickly immersed in a water bath at 5 °C until completely thawed (about 3 min). CASA was used to evaluate sperm motility before and after freezing. The specific operation was as follows: 10 μL of fresh semen was slowly mixed with 990 μL of pre-warmed DMEM at 37 °C or 10 μL of thawed semen was slowly mixed with 240 μL of pre-warmed DMEM at 37 °C to prepare semen diluent. 10 μL of the above semen diluent was respectively dropped onto a CASA special counting plate, placed on a 37 °C constant temperature stage of a phase contrast microscope, and image data of 5 different fields of view were collected by CASA and analyzed. Calculate the change range of sperm motility (%) = |after freezing - before freezing| × 100. The cryoresistance of sperm was reflected by the change range of sperm motility. Finally, 10 individuals were screened out, with 5 in each of the high and low cryoresistance groups (Table 1);

[0024] Table 1 Comparison results of sperm motility before and after freezing in high and low cryoresistance individuals

[0025]

[0026]

[0027] (2) Sperm protein extraction and protein concentration determination of high and low cryoresistance individuals:

[0028] The semen of high and low cryoresistance individuals was collected by dorsal and abdominal massage method, centrifuged at 12,000 g for 10 min at 4 °C to remove seminal plasma, washed twice with PBS, added with 2 volumes of lysis buffer containing protease inhibitor, put into liquid nitrogen, and then stored at -80 °C. During the formal experiment, the fresh semen samples of high and low cryoresistance individuals were taken out from -80 °C, and 4 volumes of lysis buffer (1% Triton X-100, 1% protease inhibitor, 50 μM PR-619, 3 μM TSA, 50 mM NAM) were respectively added for ultrasonic lysis. Centrifuged at 12,000 g for 10 min at 4 °C to remove cell debris, and the supernatant was transferred to a new centrifuge tube, and the protein concentration was determined using a BCA kit.

[0029] (3) Screening differential proteins of sperm in cryoresistance differential individuals:

[0030] Protein quantification detection of sperm protein samples was carried out by 4D-label free technology to screen for differential sperm proteins related to the cryopreservation performance of rooster sperm. The specific process is as follows: Equal amounts of proteins from each sample were digested with enzymes, and the volume was adjusted to be the same with the lysis buffer. 20% TCA was slowly added to a final concentration, vortexed thoroughly, and precipitated at 4°C for 2 h. Centrifuged at 4500g for 5 min, the supernatant was discarded, and the precipitate was washed with pre-cooled acetone 2 - 3 times. After air-drying the precipitate, TEAB with a final concentration of 200 mM was added, the precipitate was dispersed by sonication, and trypsin was added at a ratio of 1:50 (protease:protein, m / m) for overnight digestion. Dithiothreitol (DTT) was added to a final concentration of 5 mM and reduced at 56°C for 30 min. Then iodoacetamide (IAA) was added to a final concentration of 11 mM and incubated in the dark at room temperature for 15 min; Liquid chromatography-mass spectrometry was used for analysis. The peptide segments were dissolved in mobile phase A of liquid chromatography and separated using a NanoElute ultra-high performance liquid system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; Mobile phase B was a solution containing 0.1% formic acid and 100% acetonitrile. The liquid phase gradient was set as follows: 0 - 92 min, 6% - 24% B; 92 - 112 min, 24% - 35% B; 112 - 116 min, 35% - 80% B; 116 - 120 min, 80% B, and the flow rate was maintained at 450 nL / min. After the peptide segments were separated by the ultra-high performance liquid system, they were injected into the Capillary ion source for ionization and then analyzed by timsTOF Pro mass spectrometry. The ion source voltage was set to 1.7 kV, and both the peptide segment parent ions and their secondary fragments were detected and analyzed using a high-resolution TOF. The secondary mass spectrometry scan range was set to 100 - 1700. The data acquisition mode used parallel accumulation serial fragmentation (PASEF) mode. After one primary mass spectrometry acquisition, 10 PASEF mode acquisitions were performed to collect secondary spectra with the parent ion charge number in the range of 0 - 5. The dynamic exclusion time for tandem mass spectrometry scanning was set to 30 s to avoid repeated scanning of parent ions. A sample-specific protein database was constructed according to the source of the samples, and then an analysis software was used for database search.

[0031] (4) Test results

[0032] The results showed that a total of 2309 proteins were identified, of which 1699 were quantified proteins. When the difference multiple of protein abundance > 1.2 and P < 0.05, they were regarded as differential proteins. Finally, 42 differential proteins were identified, accounting for 2.5% (42 / 1699) of the total number of quantified proteins. There were 20 proteins with up-regulated expression and 22 proteins with down-regulated expression in the high cryotolerance group compared with the low cryotolerance group (Table 2).

[0033] The results of this experiment showed that, compared with the expression level of VDAC1 in sperm of the low anti-freezing group, the relative expression level of VDAC1 in sperm of the high anti-freezing group was significantly increased (P<0.05). Therefore, the decrease in the expression of VDAC1 may lead to a decrease in sperm motility and anti-freezing ability.

[0034] Table 2 Results of differential protein screening

[0035]

[0036]

[0037] Experimental Example 1 Verification of the accuracy of the detection results of the 4D-label free technique using the PRM technique 1 Materials and methods

[0038] 1.1 Experimental animals and feeding

[0039] This experiment was conducted at the Changping Experimental Base of the Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences; 30-week-old healthy Beijing Youji roosters were selected as experimental subjects, and semen collection was performed once every other day using the dorsal and abdominal massage method before the formal experiment. The experimental chickens were housed in the same laying house, in individual cages, with free access to food and water. The nutritional level of the diet was referred to the chicken feeding standard "NY / T33 - 2004 Chicken Feeding Standard", and the light rhythm was 16L:8D, with a light intensity of 20 lx.

[0040] 1.2 Screening of individuals with high and low anti-freezing differences

[0041] Semen from 160 30-week-old healthy Beijing Youji roosters was collected using the dorsal and abdominal massage method. Immediately, 200 μL of semen was gently mixed with 200 μL of pre-warmed diluent at 37 °C and then placed at 4 °C for 30 min of equilibration. 400 μL of diluent containing glycerol was added, gently mixed, and placed at 4 °C for 10 min of equilibration. It was packaged in 0.5 mL thin tubes, placed in a programmed freezer for cooling and freezing, and then stored in liquid nitrogen. During thawing, the thin tubes were taken out of liquid nitrogen and quickly immersed in a 5 °C water bath until completely thawed (about 3 min). CASA was used to evaluate sperm motility before and after freezing. The specific operation was as follows: 10 μL of fresh semen was slowly mixed with 990 μL of pre-warmed DMEM at 37 °C or 10 μL of thawed semen was slowly mixed with 240 μL of pre-warmed DMEM at 37 °C to prepare semen diluent. 10 μL of the above semen diluent was respectively dropped on a CASA special counting plate, placed on a 37 °C constant temperature stage of a phase contrast microscope, and image data of 5 different fields were collected using CASA and analyzed. Calculate the change range of sperm motility (%) = |after freezing - before freezing| × 100. The anti-freezing property of sperm was reflected by the change range of sperm motility. Finally, 10 individuals were selected, with 5 in each of the high and low anti-freezing groups (Table 1).

[0042] 1.3 Sperm protein extraction and protein concentration determination of high and low freeze-resistant individuals

[0043] The semen of high and low freeze-resistant individuals was collected by dorsal and abdominal massage method, centrifuged at 12,000 g for 10 min at 4 °C to remove seminal plasma, washed twice with PBS, added with 2 volumes of lysis buffer containing protease inhibitor, put into liquid nitrogen, and then stored at -80 °C. During the formal experiment, the fresh sperm samples of high and low freeze-resistant individuals were taken out from -80 °C, and 4 volumes of lysis buffer (1% Triton X-100, 1% protease inhibitor, 50 μM PR-619, 3 μM TSA, 50 mM NAM) were added respectively, and sonicated for lysis. Centrifuge at 12,000 g for 10 min at 4 °C to remove cell debris, transfer the supernatant to a new centrifuge tube, and use the BCA kit to determine the protein concentration.

[0044] 1.4 PRM verification

[0045] PRM (parallel reaction monitoring) is a targeted proteomics technology based on high-resolution and high-precision mass spectrometry, which can selectively detect target proteins and target peptides, so as to achieve the relative / absolute quantification of target proteins / peptides, and can be used to verify the results of quantitative proteomics. First, use the selective detection ability of the quadrupole mass analyzer to selectively detect the precursor ion information of the target peptide in the first-level mass spectrometry. Subsequently, fragment the precursor ions in the collision cell; finally, use the high-resolution and high-mass-accuracy analyzer to detect the information of all fragments within the selected precursor ion window in the second-level mass spectrometry. In this way, accurate and specific analysis of target proteins / peptides in complex samples can be carried out.

[0046] Equal amounts of proteins from each sample were digested with enzymes, and the volume was adjusted to be consistent with the lysis buffer. 20% TCA was added slowly to a final concentration, vortexed thoroughly, and precipitated at 4 °C for 2 h. Centrifuged at 4500 g for 5 min, the supernatant was discarded, and the precipitate was washed with pre-cooled acetone 2-3 times. After air-drying the precipitate, TEAB with a final concentration of 200 mM was added, and the precipitate was dispersed by sonication. Trypsin was added at a ratio of 1:50 (protease: protein, m / m) and digested overnight. Dithiothreitol (DTT) was added to a final concentration of 5 mM and reduced at 56 °C for 30 min. Then iodoacetamide (IAA) was added to a final concentration of 11 mM and incubated in the dark at room temperature for 15 min. The peptide segments were dissolved in mobile phase A of liquid chromatography and separated using an EASY-nLC 1000 ultra-high performance liquid system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient was set as follows: 0-16 min, 6% - 20% B; 16-22 min, 20% - 30% B; 22-26 min, 30% - 80% B; 26-30 min, 80% B, and the flow rate was maintained at 500 nL / min. After separation by the ultra-high performance liquid system, the peptide segments were injected into the NSI ion source for ionization and then analyzed by a mass spectrometer. The ion source voltage was set at 2.1 kV, and both the peptide segment precursor ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set at 400-1700 m / z, and the scan resolution was set at 70000; the Orbitrap scan resolution for secondary mass spectrometry was set at 17500. The data acquisition mode used a data-independent scanning (DIA) program, and the fragmentation energy of the HCD collision cell was set at 27. The automatic gain control (AGC) for primary mass spectrometry was set at 3E6, and the maximum ion injection time (Maximum IT) was set at 50 ms; for secondary mass spectrometry, the automatic gain control (AGC) was set at 1E5, the maximum ion injection time (Maximum IT) was set at 214 ms, and the isolation window was set at 1.6 m / z. The secondary mass spectrometry data was searched using Maxquant (v1.6.15.0).Retrieval parameter settings: The database is Blast_Bombus_terrestris_30195_PR_20201214.fasta (17,032 sequences). A reverse library was added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library was added to the database to eliminate the influence of contaminant proteins in the identification results. The digestion method was set to Trypsin / P; the maximum number of missed cleavage sites was set to 2; the minimum peptide length was set to 7 amino acid residues; the maximum number of peptide modifications was set to 5; the mass error tolerances for the first search and main search of precursor ions were set to 20 ppm and 4.5 ppm, respectively, and the mass error tolerance for product ions was 20 ppm. Carbamidomethyl (C) of cysteine was set as a fixed modification, and variable modifications were oxidation of methionine and acetylation of the protein N-terminus. The FDRs for protein identification and PSM identification were both set to 1%.

[0047] 1.5 Data processing and statistical analysis

[0048] The data was processed using Skyline 21.1. Peptide parameters: The protease was set to Trypsin[KR / P], the maximum number of missed cleavage sites was set to 0, the peptide length was set to 7 - 25 amino acid residues, and cysteine alkylation was set as a fixed modification. Transition parameters: The precursor ion charge was set to 2, 3, the product ion charge was set to 1, and the ion type was set to b, y. Fragment ions were selected starting from the third to the last one, and the mass error tolerance for ion matching was set to 0.02 Da. GraphPad Prism statistical software was used for data analysis.

[0049] 2 Experimental results

[0050] The PRM detection results are as Figure 1 shown. The changing trend of VDAC1 is completely consistent with the detection results of the 4D-label free technology, indicating that the detection results of the 4D-label free technology are highly reliable.

[0051] Experimental example 2 used Western blot technology to determine the expression difference of VDAC1 in the high and low cryotolerance groups of chicken sperm

[0052] 1 Materials and methods

[0053] 1.1 Experimental animals and feeding

[0054] This experiment was conducted at the Changping Experimental Base of the Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences. Healthy Beijing Fatty Chicken roosters at 46 weeks of age were selected as experimental subjects. Before the formal experiment, semen collection was performed once every other day using the dorsal-abdominal massage method. The experimental chickens were raised in the same laying house, in individual cages, with free access to food and water. The nutritional level of the diet was referred to the chicken feeding standard "NY / T 33-2004 Chicken Feeding Standard". A 16L:8D light rhythm was adopted, and the light intensity was 20 lx.

[0055] 1.2 Screening of individuals with high and low freezing resistance differences

[0056] Semen was collected from 200 healthy Beijing Fatty Chicken rooster individuals at 46 weeks of age using the dorsal-abdominal massage method. Immediately, 200 μL of semen was aspirated and gently mixed with 200 μL of diluent preheated at 37 °C, and then placed at 4 °C for 30 min of equilibration. 400 μL of diluent containing glycerol was added, gently mixed, and placed at 4 °C for 10 min of equilibration. It was packaged with 0.5 mL thin tubes, put into a programmed freezer for cooling and freezing, and then stored in liquid nitrogen. During thawing, the thin tubes were taken out of liquid nitrogen and quickly immersed in a 5 °C water bath until completely thawed (about 3 min). CASA was used to evaluate sperm motility before and after freezing. The specific operation was as follows:

[0057] 10 μL of fresh semen was slowly mixed with 990 μL of DMEM preheated at 37 °C, or 10 μL of thawed semen was slowly mixed with 240 μL of DMEM preheated at 37 °C to prepare semen diluent. 10 μL of the above semen diluent was respectively dropped on a CASA special counting plate, placed on a 37 °C constant temperature stage of a phase contrast microscope, and 5 different field image data were collected using CASA and analyzed. Calculate the change range of sperm motility (%) = |after freezing - before freezing| × 100. The freezing resistance of sperm was reflected by the change range of sperm motility. Finally, 6 individuals were screened out, with 3 in each of the high and low freezing resistance groups (Table 3);

[0058] Table 3 Comparison results of sperm motility before and after freezing in high and low freezing resistance individuals

[0059]

[0060] 1.3 Determination of VDAC1 by Western blot technique

[0061] The basic principle of Western Blot (immunoblotting test) is to stain cell or biological tissue samples treated by gel electrophoresis with specific antibodies. Information on the expression of specific proteins in the analyzed cells or tissues is obtained by analyzing the staining position and staining depth.

[0062] The specific operation is as follows: Samples of high- and low-freezing-tolerance individuals are respectively added with 4 volumes of lysis buffer (1% Triton X-100, 1% protease inhibitor, 50 μM PR-619, 3 μM TSA, 50 mM NAM) and lysed by sonication. Centrifuge at 12,000 g for 10 min at 4 °C to remove cell debris. Transfer the supernatant to a new centrifuge tube, and use a BCA kit to measure the protein concentration. According to the protein concentration measurement results, take an equal amount of protein (20 μg) from each sample into a centrifuge tube. After electrophoresis, membrane transfer, and blocking, add an antibody (VDAC1 Rabbit mAb) for primary antibody incubation. After the primary antibody incubation is completed, add a secondary antibody for incubation. After rinsing, add a chemiluminescent HRP substrate and incubate for 2 minutes, and capture the signal according to the operation instructions of the chemiluminescent imaging system.

[0063] 1.4 Data statistical analysis

[0064] In this experiment, GraphPad Prism statistical software was used for data analysis. * indicates P < 0.05, with statistical differences.

[0065] 2 Experimental results

[0066] The results of Western blot are shown in Figure 2 ; According to Figure 2 It can be seen that there are certain differences in the target protein bands between the two groups. Among them, the protein abundance near 31 kD in the HF group is relatively high, while the level of the target protein near 31 kD in the LF group is relatively low. Through gray value statistics, it can be seen that the average expression of VDAC1 in the HF group is significantly higher than that in the LF group (P < 0.05).

[0067] Experimental example 3 Verification experiment of VDAC1 protein as a biomarker for the high and low anti-freezing performance of rooster sperm

[0068] 1 Materials and methods

[0069] 1.1 Experimental animals and feeding

[0070] This experiment was carried out at the Changping Experimental Base of the Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences; 20 healthy Beijing Youji roosters at 50 weeks of age were selected as experimental subjects, and semen collection was performed once every other day by the dorsal and abdominal massage method before the formal experiment. The experimental chickens were raised in the same laying house, in single cages, with free access to food and water. The dietary nutrient level referred to the agricultural industry standard "NY / T 33 - 2004 Chicken Feeding Standard", and the light rhythm was 16L:8D, and the light intensity was 20 lx.

[0071] 1.2 Determination of sperm cryotolerance

[0072] According to the method described in Example 1, the measurement of sperm cryotolerance was carried out as follows: The semen of individual roosters was collected by the dorsal-abdominal massage method. Immediately, 200 μL of semen was aspirated and gently mixed with 200 μL of pre-warmed diluent at 37 °C, and then placed at 4 °C for 30 min for equilibration. 400 μL of diluent containing glycerol was added, and gently mixed and placed at 4 °C for 10 min for equilibration. It was packaged with a 0.5 mL capillary tube, put into a programmed freezer for cooling and freezing, and then stored in liquid nitrogen. During thawing, the capillary tube was taken out of liquid nitrogen and quickly immersed in a water bath at 5 °C until completely thawed (about 3 min). CASA was used to evaluate sperm motility before and after freezing. The specific operation was as follows: 10 μL of fresh semen was slowly mixed with 990 μL of pre-warmed DMEM at 37 °C, or 10 μL of thawed semen was slowly mixed with 240 μL of pre-warmed DMEM at 37 °C to prepare semen diluent. 10 μL of the above semen diluent was respectively dropped on a CASA special counting plate, placed on a 37 °C constant temperature stage of a phase contrast microscope, and image data of 5 different fields of view were collected using CASA and analyzed. Calculate the change range of sperm motility (%) = |after freezing - before freezing| × 100. The cryotolerance of sperm is reflected by the change range of sperm motility.

[0073] 1.3 Determination of sperm VDAC1 protein content

[0074] The semen of individual roosters (n = 20) was collected by the dorsal-abdominal massage method, and the VDAC1 protein content was determined using a Chicken Voltage Dependent Anion Channel 1 (VDAC1) ELISA kit (Beijing Ruida Henghui Technology Development Co., Ltd.). Referring to the instruction manual, the specific steps were as follows: First, the washed sperm precipitate was broken, and the supernatant was taken after centrifugation at 3000 g for 10 min. Subsequently, the specimen, standard product, and HRP-labeled detection antibody were successively added to the coated microplate pre-coated with the antibody against Voltage Dependent Anion Channel 1 (VDAC1), incubated at 37 °C and thoroughly washed. TMB was used as the substrate for color development. TMB was converted into blue under the catalysis of peroxidase and into the final yellow under the action of acid. The color depth was positively correlated with the Voltage Dependent Anion Channel 1 (VDAC1) in the sample. The absorbance (OD value) was measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm, and the sample concentration was calculated.

[0075] 1.4 Data statistical analysis

[0076] In this experiment, GraphPad Prism statistical software was used for data analysis. Pearson correlation analysis was performed to study the relationship between frost resistance and the sperm VDAC1 protein content. P<0.01 was considered to have statistically significant differences. GraphPad Prism statistical software was used to plot the receiver operating characteristic curve (ROC). The ROC curve is a tool used to evaluate the performance of binary classification models and is commonly used for the effectiveness evaluation of medical diagnoses or biomarkers.

[0077] In this application, grouping according to the VDAC1 protein content and the corresponding frost resistance performance of individual roosters (high frost resistance group and low frost resistance group) can be used to construct a classification model, where the protein content is used as the predictive variable and the frost resistance performance grouping is used as the actual classification result. The ROC curve is plotted based on the true positive rate (TPR) and false positive rate (FPR) at different thresholds. A suitable threshold range needs to be selected, usually within the range from 0 to the maximum protein content. The threshold is adjusted step by step, and the TPR and FPR at each threshold are calculated. For each possible threshold:

[0078] True positive (TP): Actually in the high frost resistance group and the protein content is higher than the threshold.

[0079] False positive (FP): Actually in the low frost resistance group but the protein content is lower than the threshold.

[0080] True negative (TN): Actually in the low frost resistance group and the protein content is higher than the threshold.

[0081] False negative (FN): Actually in the high frost resistance group but the protein content is lower than the threshold.

[0082] TPR = TP / (TP + FN), FPR = FP / (FP + TN)

[0083] All the calculated (FPR, TPR) points are connected to form the ROC curve. The area under the curve (AUC) is calculated to evaluate the overall performance of the model. The closer the AUC value is to 1, the better the classification performance of the model.

[0084] 2 Experimental results

[0085] The measurement results of the sperm VDAC1 protein content and sperm motility parameters before and after freezing of 20 individual roosters are shown in Tables 4 and 5. The results show that the correlation coefficients between the VDAC1 protein content in sperm and the change ranges of motility, curvilinear velocity VCL, straight-line velocity VSL, average path velocity VAP, amplitude of lateral head displacement ALH, and beating frequency BCF before and after freezing are -0.69, -0.77, -0.63, -0.70, -0.77, and -0.57, respectively, and all show significant negative correlations (P<0.01) (Table 6, Figure 3)It is shown that the higher the content of VDAC1 protein in rooster sperm, the higher the anti-freezing performance, that is, the smaller the change range of sperm motility parameters before and after freezing.

[0086] The anti-freezing property of sperm is reflected by the change range of sperm motility before and after freezing. Seven roosters were selected as the low anti-freezing group (individual numbers: 1, 4, 5, 7, 6, 8, 10), and seven roosters were selected as the high anti-freezing group (individual numbers: 14, 15, 16, 17, 18, 19, 20) (Table 4). It was tested whether there were significant differences in the content of VDAC1 protein and the change range of sperm motility parameters between the high and low anti-freezing groups. The test results are shown in Table 7: The content of VDAC1 sperm protein in the high anti-freezing group was significantly higher than that in the low anti-freezing group, and the change range of sperm motility parameters before and after freezing in the high anti-freezing group was significantly lower than that in the low anti-freezing group (P<0.01).

[0087] Furthermore, the VDAC1 protein content (ng / ml) of roosters in the high and low groups and their anti-freezing performance grouping were extracted from Table 4, and the TPR and FPR at each threshold were calculated. The results are shown in Table 8. The ROC analysis results showed that the AUC was 0.9898, close to 1, indicating that the model had a strong ability to distinguish between the high and low anti-freezing groups ( Figure 4 ). The 95% confidence interval was from 0.8498 to 1.000, indicating that there was a 95% probability that the true AUC was within this range. P = 0.0022, indicating that the discrimination ability of the model was statistically significant. Sensitivity% refers to the sensitivity at different cut-off values, that is, the true positive rate. Specificity%: The specificity at different cut-off values, that is, the true negative rate. When the critical value was 3688 ng / ml, the sensitivity reached 100%, and the specificity was 85.71%. At this time, the Youden index (= sensitivity + specificity - 1) = 100% + 85.71% - 1 = 0.8571, which was relatively large among these data combinations. Therefore, judging from the data in this table, the optimal threshold might be 3688 ng / ml. When breeding roosters with high anti-freezing performance, the VDAC1 protein content threshold can be set to 3688 ng / ml to efficiently screen target individuals. The design of the detection kit (containing VDAC1 antibody) should be optimized based on this threshold for detection sensitivity.

[0088] Table 4 Determination results of sperm VDAC1 protein content

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[0091] Table 5 Determination results of sperm motility parameters of rooster sperm before and after freezing

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[0094] Table 6 Correlation analysis of sperm VDAC1 protein content and the change range of sperm motility parameters before and after freezing

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[0097] Table 7 Comparison of the average sperm VDAC1 protein content and the change range of sperm motility parameters before and after freezing in roosters of high and low freeze-resistant groups

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[0099] Note: For comparison in the same row, different lowercase superscript letters indicate significant differences (P<0.01).

[0100] Table 8 Results of ROC sensitivity and specificity analysis

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Claims

Use of VDAC1 protein as a biomarker for the cryotolerance of rooster sperm.

2. The use according to claim 1, characterized in that, The amino acid sequence of the VDAC1 protein is as shown in SEQ ID No.

1.

3. The use according to claim 1, characterized in that, Including: Identifying the expression level of VDAC1 protein in rooster sperm; if the expression level of VDAC1 protein in rooster sperm is high, the cryotolerance of the rooster sperm is high; if the expression level of VDAC1 protein in rooster sperm is low, the cryotolerance of the rooster sperm is low.

4. The use according to claim 3, wherein, If the expression level of VDAC1 protein in rooster sperm is greater than or equal to 3688 ng / ml, the cryotolerance of the rooster sperm is high; if the expression level of VDAC1 protein in rooster sperm is less than 3688 ng / ml, the cryotolerance of the rooster sperm is low.

5. A method for breeding roosters with high sperm cryoresistance, characterized in that, Including: Detecting the expression level of VDAC1 protein in rooster sperm, screening for roosters with high expression levels of VDAC1 protein, and obtaining breeding roosters with high cryotolerance of sperm.

6. The method according to claim 5, wherein Detecting the expression level of VDAC1 protein in rooster sperm, if the expression level of VDAC1 protein in roosters is greater than or equal to 3688 ng / ml, then screening for breeding roosters with high cryotolerance of sperm.

7. The method according to claim 5, wherein The amino acid sequence of the VDAC1 protein is as shown in SEQ ID No.

1.

8. A detection kit for identifying the high or low anti-freezing performance of rooster sperm, comprising: An antibody, a standard product, an HRP-labeled detection antibody, and a chromogenic solution, characterized in that the antibody is a VDAC1 protein antibody.

9. The detection kit according to claim 8, wherein The amino acid sequence of the VDAC1 protein is as shown in SEQ ID No. 1.

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