Application of LOC100858984 protein as biomarker for freezing resistance of cock sperms

Abstract

The invention discloses an application of LOC100858984 protein as a biomarker for the freezing resistance of cock sperms. According to the invention, the key differential protein LOC100858984 of cock sperm anti-freezing difference individuals is identified by adopting a 4D-label free technology, and the expression quantity of the protein in highly anti-freezing individuals is found to be obviously reduced. According to the invention, a classification model is constructed by adopting the protein content and corresponding anti-freezing performance groups, and an ROC curve is drawn to evaluate the effectiveness of the biomarker as the anti-freezing performance of the cock sperms, and the result shows that AUC is 0.9531; therefore, the LOC100858984 protein is determined to be used as a biomarker for screening or predicting the freezing resistance of the sperms of the cocks to be applied to breeding of the breeding cocks with high freezing resistance of the sperms. The method is beneficial for improving the cryopreservation efficiency of the chicken sperms, and has application prospects in the aspects of promotion of protection of chicken genetic resources, efficient utilization of excellent cocks and the like.

Description

Technical Field

[0001] The present invention relates to a new use of the LOC100858984 protein, and in particular to a use of the LOC100858984 protein as a biomarker for the antifreeze performance of rooster sperm, belonging to the field of new uses of the LOC100858984 protein. Background Art

[0002] Semen cryopreservation uses liquid nitrogen (-196°C) or dry ice (-79°C) as a cold source. After the semen is diluted, balanced, cryoprotectants are added, and rebalanced, it is quickly cooled to inhibit the metabolism of sperm, allowing the sperm to be stored at ultra-low temperatures for a long time. After thawing and warming, the sperm can regain its fertilization ability. Ultra-low temperature preservation technology of semen is an important means of preserving the reproductive capacity of male animals in the long term, and has broad application prospects in germplasm resource preservation and variety improvement. However, the structural characteristics of chicken sperm with a small head and a long tail result in its poor antifreeze ability. Establishing a stable and efficient chicken semen cryopreservation technology has always been a difficult problem that people in this field have been working hard to solve.

[0003] Currently, much research is focused on screening semen cryopreservation procedures, while research on the mechanisms of cryoinjury is limited, with no breakthroughs to date. Sperm are highly differentiated cells lacking endoplasmic reticulum, Golgi apparatus, catabolites, peroxisomes, or ribosomes. Consequently, transcription and translation are very limited in mature spermatids. Recent technological advances have enabled researchers to uncover the mechanisms of cryoinjury by examining changes in the expression of relevant genes and proteins in sperm cells before and after freezing. Among the available methods for studying the molecular basis of male fertility, proteomics is particularly well-established. The sperm freezing process results in the expression of numerous cryoproteins, which play an essential role in maintaining sperm function. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a biomarker for predicting or identifying the antifreeze performance of rooster sperm;

[0005] The second object of the present invention is to provide a method for breeding roosters with high sperm antifreeze performance;

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] One aspect of the present invention provides the use of LOC100858984 protein as a biomarker for the antifreeze performance of rooster sperm; wherein the amino acid sequence of the LOC100858984 protein is shown in SEQ ID No. 1:

[0008] MDEQKAVIKDTDMLEEMQQQAVQCAVLAIEKYSVEREIAALIKRE FEKKYSPTWHCVVGRKFGSYVSHETKHFIFFLVRGLNVLLFKAG (SEQ ID No. 1).

[0009] In a preferred embodiment of the present invention, the use of LOC100858984 protein as a biomarker for the antifreeze performance of rooster sperm includes: (1) detecting the expression level of LOC100858984 protein in the rooster to be detected; (2) if the expression level of LOC100858984 protein in the rooster sperm is high, the antifreeze performance of the rooster sperm is low; if the expression level of LOC100858984 protein in the rooster sperm is low, the antifreeze performance of the rooster sperm is high; more preferably, if the expression level of LOC100858984 protein in the rooster sperm is greater than 1988 ng / ml, the antifreeze performance of the sperm of the rooster to be detected is low; if the expression level of LOC100858984 protein in the rooster sperm is less than 1988 ng / ml, the antifreeze performance of the rooster sperm to be detected is high.

[0010] Another aspect of the present invention provides a method for breeding roosters with high sperm antifreeze performance using the LOC100858984 protein as a biomarker, comprising: detecting the expression level of the LOC100858984 protein in the sperm of the rooster to be detected, and screening to obtain roosters with low expression levels of the LOC100858984 protein, that is, obtaining roosters with high sperm antifreeze performance; preferably, if the expression level of the LOC100858984 protein in the rooster sperm is less than 1988 ng / ml, then the roosters with high sperm antifreeze performance are screened.

[0011] Those skilled in the art can use various conventional methods to detect the expression level of LOC100858984 protein in rooster sperm; as a specific embodiment of the present invention, Western blot can be used to identify the expression level of LOC100858984 protein in rooster sperm.

[0012] The present invention also provides a detection kit for predicting or identifying the antifreeze performance of rooster sperm, wherein the detection kit contains LOC100858984 protein antibody.

[0013] The present invention uses 4D-label-free technology to identify the key differential protein LOC100858984 in the cryoprotection of rooster sperm, and this protein is significantly downregulated in individuals with high cryoprotection. The present invention further uses the LOC100858984 protein content and the corresponding antifreeze performance grouping to construct a classification model and draws an ROC curve to evaluate its effectiveness as a biomarker for the antifreeze performance of rooster sperm. The results showed an AUC of 0.9531, demonstrating that this protein can be used as a biomarker for screening the antifreeze performance of rooster sperm. The present invention is beneficial to improving the efficiency of chicken sperm cryopreservation and is of great significance for promoting the protection of chicken genetic resources and the efficient utilization of high-quality roosters. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Western blot analysis was used to verify the differential expression of LOC100858984 protein in the high and low antifreeze groups of rooster sperm.

[0015] Figure 2 This is a correlation analysis between the LOC100858984 protein content in rooster sperm and the changes in sperm motility parameters before and after freezing.

[0016] Figure 3 ROC curve for evaluating the effectiveness of LOC100858984 protein content as a biomarker for the antifreeze performance of rooster sperm. DETAILED DESCRIPTION

[0017] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0018] Example 1 Screening for Sperm Differential Proteins Related to Cock Sperm Freeze Resistance

[0019] (1) Screening individuals with high and low frost resistance

[0020] Semen was collected from 160 healthy, 30-week-old Beijing Oily Roosters using dorsal and abdominal massage. Immediately, 200 μL of semen was aspirated and gently mixed with 200 μL of diluent preheated at 37°C, then equilibrated at 4°C for 30 min. 400 μL of diluent containing glycerol was added, gently mixed, and equilibrated at 4°C for 10 min. Semen was packaged into 0.5 mL straws, frozen in a programmable freezer, and then stored in liquid nitrogen. Upon thawing, the straws were removed from liquid nitrogen and quickly immersed in a 5°C water bath until completely thawed (approximately 3 min). Sperm motility was assessed using CASA before and after freezing. Semen diluent was prepared by slowly mixing 10 μL of fresh semen with 990 μL of 37°C preheated DMEM or 10 μL of thawed semen with 240 μL of 37°C preheated DMEM. 10 μL of each of the above semen dilutions was dropped onto a CASA-specific counting chamber, placed on a phase-contrast microscope stage at 37°C, and image data from five different fields of view were collected and analyzed using CASA. The change in sperm motility (%) was calculated as |post-freezing -pre-freezing| × 100. The change in sperm motility reflects the cryoprotection of sperm. Ten individuals were ultimately selected, with 5 in each of the high and low cryoprotection groups (Table 1).

[0021] Table 1 Comparison of sperm motility before and after freezing in individuals with high and low cryoprotection

[0022]

[0023] (2) Extraction and concentration determination of sperm protein from high and low cryoprotective individuals

[0024] Semen from individuals with high and low cryoprotective capacity was collected using dorsal and abdominal massage. Semen was centrifuged at 12,000 g for 10 minutes at 4°C. The seminal plasma was removed, washed twice with PBS, and then lysed in 2 volumes of lysis buffer containing protease inhibitors. The mixture was plunged into liquid nitrogen and stored at -80°C. For the final experiment, fresh semen samples from individuals with high and low cryoprotective capacity were removed from -80°C and lysed by sonication using 4 volumes of lysis buffer (1% Triton X-100, 1% protease inhibitors, 50 μM PR-619, 3 μM TSA, 50 mM NAM). Semen was centrifuged at 12,000 g for 10 minutes at 4°C to remove cell debris. The supernatant was transferred to a fresh centrifuge tube, and protein concentration was determined using a BCA assay.

[0025] (3) Screening for differential proteins in sperm from individuals with different antifreeze properties

[0026] Sperm protein samples were quantitatively analyzed using 4D-label-free technology to screen for differentially expressed sperm proteins associated with the cryoprotective properties of rooster sperm. The specific procedure is as follows: Equal amounts of each sample protein were enzymatically digested, and the volume was adjusted to a uniform level using lysis buffer. TCA was slowly added to a final concentration of 20%, vortexed, and precipitated at 4°C for 2 hours. The pellet was centrifuged at 4500g for 5 minutes, the supernatant discarded, and the pellet washed two to three times with pre-chilled acetone. After air drying, the pellet was added with TEAB to a final concentration of 200mM and sonicated. Trypsin was then added at a ratio of 1:50 (protease:protein, m / m) and digested overnight. Dithiothreitol (DTT) was added to a final concentration of 5mM, and the pellet was reduced at 56°C for 30 minutes. Iodoacetamide (IAA) was then added to a final concentration of 11mM, and the mixture was incubated at room temperature in the dark for 15 minutes. Liquid chromatography-mass spectrometry analysis was performed, with peptides resolved in LC mobile phase A and separated using a NanoElute ultra-high performance liquid chromatography system. Mobile phase A consisted of 0.1% formic acid and 2% acetonitrile in water; mobile phase B consisted of 0.1% formic acid and 100% acetonitrile. The gradient was as follows: 6% to 24% B (0-92 min); 24% to 35% B (92-112 min); 35% to 80% B (112-116 min); and 80% B (116-120 min). The flow rate was maintained at 450 nL / min. Peptides were separated using an ultra-high performance liquid chromatography (UPLC) system and injected into a Capillary ion source for ionization and analysis on a timsTOF Pro mass spectrometer. The ion source voltage was set to 1.7 kV. Peptide precursor ions and their secondary fragments were detected and analyzed using a high-resolution TOF. The secondary mass spectrometer scan range was set to 100–1700. Data acquisition was performed in parallel accumulation serial fragmentation (PASEF) mode. After acquiring a primary mass spectrometer, 10 secondary spectra were acquired in PASEF mode with parent ion charge numbers ranging from 0 to 5. The dynamic exclusion time for the tandem mass spectrometry scan was set to 30 seconds to avoid repeated scanning of parent ions. A sample-specific protein database was constructed based on the sample source, and the database was searched using analytical software.

[0027] The results showed that a total of 2,309 proteins were identified, of which 1,699 were quantified. Proteins were considered differentially expressed when the fold difference in abundance was >1.2 and P < 0.05. A total of 42 differentially expressed proteins were identified, accounting for 2.5% (42 / 1,699) of the total quantified proteins. Twenty proteins were upregulated and 22 proteins were downregulated in the high-freeze group compared to the low-freeze group (Table 2).

[0028] The results of this experiment showed that the relative expression of LOC100858984 protein in sperm from roosters in the high-freeze tolerance group was significantly lower than that in the low-freeze tolerance group (P < 0.05). Therefore, it is speculated that the high expression of LOC100858984 protein in rooster sperm may lead to reduced sperm motility and freezing tolerance.

[0029] Table 2 Differential protein screening results

[0030]

[0031]

[0032] Experimental Example 1: Western blot technology was used to verify the expression difference of LOC100858984 protein in high and low antifreeze groups of chicken sperm

[0033] 1 Materials and Methods

[0034] 1.1 Experimental animals and husbandry

[0035] This experiment was conducted at the Changping Experimental Base of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. Two hundred healthy, 46-week-old Beijing Oily roosters were selected for the study. Semen was collected every other day using dorsal and abdominal massage before the formal experiment. The chickens were housed individually in the same laying house, with free access to feed and water. The dietary nutritional level followed the standard for chicken husbandry (NY / T 33-2004), and a 16L:8D light cycle with a light intensity of 20 lx was used.

[0036] 1.2 Screening individuals with high and low frost resistance

[0037] Semen was collected from 200 healthy Beijing oily chicken roosters aged 46 weeks using the dorsal and abdominal massage method. 200 μL of semen was immediately drawn out and gently mixed with 200 μL of 37°C preheated diluent, and then equilibrated at 4°C for 30 minutes. 400 μL of diluent containing glycerol was added, gently mixed, and equilibrated at 4°C for 10 minutes. The semen was packaged in 0.5 mL straws, placed in a programmed freezer for cooling and freezing, and then stored in liquid nitrogen. When thawing, the straw was removed from the liquid nitrogen and quickly immersed in a 5°C water bath until completely thawed (approximately 3 minutes). CASA was used to assess sperm motility before and after freezing. The specific operations were as follows:

[0038] Prepare semen diluents by slowly mixing 10 μL of fresh semen with 990 μL of DMEM preheated at 37°C, or by slowly mixing 10 μL of thawed semen with 240 μL of DMEM preheated at 37°C. 10 μL of each of these diluents was dropped onto a CASA-designed counting chamber, placed on a 37°C thermostated stage of a phase-contrast microscope, and image data from five different fields of view were collected and analyzed using CASA. The change in sperm motility (%) was calculated as |post-freezing -pre-freezing| × 100. The change in sperm motility reflects the cryoprotection of sperm. Six individuals were ultimately screened, with three each in the high and low cryoprotection groups (Table 3).

[0039] Table 3 Comparison of sperm motility before and after freezing in individuals with high and low cryoprotection

[0040]

[0041] 1.3 Western blot technology verification LOC100858984

[0042] Western blotting (immunoblotting) is a technique that uses specific antibodies to stain cells or tissue samples after gel electrophoresis. The location and depth of the staining are analyzed to determine the expression of a specific protein in the analyzed cells or tissues.

[0043] The specific procedure was as follows: 4 volumes of lysis buffer (1% Triton X-100, 1% protease inhibitors, 50 μM PR-619, 3 μM TSA, 50 mM NAM) were added to each sample from high- and low-freeze-resistant individuals and lysed by sonication. The cells were centrifuged at 12,000 g for 10 min at 4°C to remove cell debris. The supernatant was transferred to a fresh centrifuge tube and the protein concentration was determined using a BCA kit. Based on the protein concentration results, an equal amount of protein (20 μg) was transferred to each centrifuge tube. After electrophoresis, transfer to a membrane, and blocking, an antibody (LOC100858984 Rabbit mAb) was added for primary antibody incubation. After the primary antibody incubation, a secondary antibody was added. After rinsing, a chemiluminescent HRP substrate was added for incubation for 2 minutes. Signal capture was performed according to the operating instructions of the chemiluminescent imaging system.

[0044] 1.4 Data Statistical Analysis

[0045] GraphPad Prism statistical software was used for data analysis. * indicates P < 0.05, indicating statistically significant difference.

[0046] 2 Test results

[0047] Western blot results are as follows Figure 1 .according to Figure 1As can be seen, there are certain differences in the target protein bands between the two groups. The HF group has a lower abundance of the protein near 10 kD, while the LF group has a relatively higher level of the target protein near 10 kD. Grayscale value statistics show that the average expression level of LOC100858984 protein in the antifreeze group (HF group) is significantly lower than that in the non-antifreeze group (LF group) (P < 0.05).

[0048] Experimental Example 2: Validation of LOC100858984 Protein as a Biomarker for the Antifreeze Performance of Rooster Sperm

[0049] 1 Materials and Methods

[0050] 1.1 Experimental animals and husbandry

[0051] This experiment was conducted at the Changping Experimental Base of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. Twenty healthy 50-week-old Beijing Oily roosters were selected for the study. Semen was collected every other day using dorsal and abdominal massage before the formal experiment. The chickens were housed in individual cages in the same laying house, with free access to feed and water. The dietary nutritional level followed the standard for chicken husbandry (NY / T 33-2004), and a 16L:8D light cycle with a light intensity of 20 lx was used.

[0052] 1.2 Determination of sperm freezing resistance

[0053] Sperm cryoprotection was determined according to the method described in Example 1, as follows: rooster semen was collected using the dorsal and abdominal massage method. 200 μL of semen was immediately drawn and gently mixed with 200 μL of 37°C preheated diluent, then equilibrated at 4°C for 30 min. 400 μL of glycerol-containing diluent was added, gently mixed, and equilibrated at 4°C for 10 min. The semen was packaged in 0.5 mL straws, placed in a programmable freezer for cooling and freezing, and then stored in liquid nitrogen. When thawing, the straws were removed from the liquid nitrogen and quickly immersed in a 5°C water bath until completely thawed (approximately 3 min). Sperm motility was assessed before and after freezing using CASA, as follows: 10 μL of fresh semen was slowly mixed with 990 μL of 37°C preheated DMEM, or 10 μL of thawed semen was slowly mixed with 240 μL of 37°C preheated DMEM to prepare semen diluent. 10 μL of each of the above semen dilutions was dropped onto a CASA-designed counting chamber, placed on a phase-contrast microscope's 37°C thermostatted stage, and image data from five different fields of view were collected and analyzed using CASA. The change in sperm motility (%) was calculated as |post-freezing -pre-freezing| × 100. The change in sperm motility reflects the freezing resistance of sperm.

[0054] 1.3 Determination of sperm LOC100858984 protein content

[0055] Individual semen of roosters (n=20) was collected by dorsal and abdominal massage, and the LOC100858984 protein ELISA detection kit (Beijing Ruida Henghui Technology Development Co., Ltd.) was used to determine the LOC100858984 protein content. Referring to the instructions, the specific steps are as follows: First, the washed sperm pellet was crushed and centrifuged at 3000g for 10 minutes to obtain the supernatant. Subsequently, the specimen, standard, and HRP-labeled detection antibody were added to the coated microwells pre-coated with the LOC100858984 antibody in sequence, incubated at 37°C and thoroughly washed. The substrate TMB was used for color development. TMB was converted to blue under the catalysis of peroxidase and converted to the final yellow under the action of acid. The depth of the color was positively correlated with the LOC100858984 protein content in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using an enzyme reader to calculate the sample concentration.

[0056] 1.4 Data Statistical Analysis

[0057] GraphPad Prism statistical software was used for data analysis. P < 0.01 was considered statistically significant. Pearson correlation analysis was used to investigate the relationship between cryoprotection and sperm LOC100858984 protein content. Receiver operating characteristic (ROC) curves were constructed using GraphPad Prism statistical software. ROC curves are a tool used to evaluate the performance of binary classification models and are commonly used in medical diagnosis or biomarker validation.

[0058] In this application, a classification model is constructed based on the LOC100858984 protein content of individual roosters and the corresponding antifreeze performance groups (high antifreeze group and low antifreeze group), wherein the LOC100858984 protein content is used as a predictor variable and the antifreeze performance group is used as the actual classification result. The ROC curve is drawn by the true positive rate (TPR) and false positive rate (FPR) under different thresholds. It is necessary to select a suitable threshold range, usually within the range of 0 to the maximum protein content, gradually adjust the threshold, and calculate the TPR and FPR under each threshold. For each possible threshold:

[0059] True positive (TP): The sample is actually in the high-freeze resistance group and the protein content is lower than the threshold.

[0060] False positive (FP): The sample is actually in the low antifreeze group but the protein content is lower than the threshold.

[0061] True negative (TN): The sample is actually in the low antifreeze group and the protein content is higher than the threshold.

[0062] False negative (FN): The sample is actually in the high-freeze group but the protein content is higher than the threshold.

[0063] TPR=TP / (TP+FN), FPR=FP / (FP+TN)

[0064] Connect all the calculated (FPR, TPR) points to form the ROC curve. Calculate the area under the curve (AUC) to evaluate the overall performance of the model. The closer the AUC value is to 1, the better the classification performance of the model.

[0065] 2 Test results

[0066] The results of the measurement of LOC100858984 protein content in sperm and sperm motility parameters before and after freezing of 20 roosters are shown in Tables 4 and 5. The results showed that the correlation coefficients between LOC100858984 protein content in sperm and the changes in motility and VAP before and after freezing were 0.49 and 0.54, respectively, and both showed significant positive correlations (P < 0.05) (Table 6, Figure 2 ), indicating that the higher the content of rooster sperm protein LOC100858984, the lower the antifreeze performance, that is, the greater the change in sperm movement parameters before and after freezing.

[0067] Sperm cryoprotection, as reflected by the magnitude of change in sperm motility before and after freezing, was evaluated. Eight roosters were selected as the low cryoprotection group (individual numbers: 1, 2, 4, 5, 6, 9, 11, and 17) and eight as the high cryoprotection group (individual numbers: 3, 8, 10, 12, 13, 15, 16, and 19) (Table 4). Significant differences in LOC100858984 protein content and the magnitude of change in sperm motility parameters were examined between the high and low cryoprotection groups. The results are shown in Table 7. The LOC100858984 protein content in sperm from the high cryoprotection group was significantly lower than that in the low cryoprotection group. Furthermore, the magnitude of change in sperm motility before and after freezing was significantly lower in the high cryoprotection group than in the low cryoprotection group (P < 0.01).

[0068] Furthermore, the LOC100858984 protein content (ng / ml) of the high and low groups of roosters and their antifreeze performance groups 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 0.9531, close to 1, indicating that the model has a strong ability to distinguish between the high and low antifreeze groups ( Figure 3). The 95% confidence interval is 0.8498 to 1.000, indicating that there is a 95% probability that the true AUC is within this range. P = 0.0023, indicating that the discriminatory ability of the model is statistically significant. Sensitivity% refers to the sensitivity at different cutoff values, that is, the true positive rate. For example, when the cutoff value is greater than 1338ng / ml, the sensitivity is 100%, which means that all low-resistance individuals can be correctly identified, but the specificity is low at this time. Specificity%: Specificity at different cutoff values, that is, the true negative rate. For example, when the cutoff value is greater than 2150ng / ml, the specificity reaches 100%, which means that no high-resistance individual will be misjudged as a low-resistance individual, but the sensitivity will decrease. In summary, based on the Youden index (= sensitivity + specificity - 1), 1988ng / ml can be used as a better choice. When breeding roosters with high frost resistance, the LOC100858984 protein content threshold can be set to 1988ng / ml to efficiently screen target individuals. The design of the detection kit (containing the LOC100858984 antibody) should optimize the detection sensitivity based on this threshold.

[0069] Table 4 Results of sperm LOC100858984 protein content determination

[0070]

[0071] Table 5 Results of sperm motility parameters before and after freezing of rooster sperm

[0072]

[0073]

[0074] Table 6 Correlation analysis between sperm LOC100858984 protein content and the change range of sperm motility parameters before and after freezing

[0075]

[0076] Table 7 Comparison of the average content of LOC100858984 protein in sperm of roosters in the high and low freezing resistance groups and the changes in sperm motility parameters before and after freezing

[0077]

[0078] Note: When comparing the same group, different lowercase superscript letters indicate significant differences (P<0.01).

[0079] Table 8 ROC sensitivity and specificity analysis results

[0080]

Claims

1. Use of LOC100858984 protein as a biomarker for the antifreeze properties of rooster sperm.

2. The use according to claim 1, characterized in that The amino acid sequence of the LOC100858984 protein is shown in SEQ ID No.

1.

3. The use according to claim 1, characterized in that include: (1) Detect the expression level of LOC100858984 protein in the rooster sperm to be tested; (2) If the expression level of LOC100858984 protein in the rooster sperm is high, the antifreeze performance of the rooster sperm to be tested is low; if the expression level of LOC100858984 protein in the rooster sperm is low, the antifreeze performance of the rooster sperm to be tested is high.

4. The use according to claim 3, characterized in that If the expression level of LOC100858984 protein in rooster sperm is greater than 1988 ng / ml, the antifreeze performance of the rooster sperm to be tested is low; if the expression level of LOC100858984 protein in rooster sperm is less than 1988 ng / ml, the antifreeze performance of the rooster sperm to be tested is high.

5. A method for breeding roosters with high sperm antifreeze performance, characterized in that: include: The expression level of LOC100858984 protein in the sperm of the roosters to be tested was detected, and roosters with low expression levels of LOC100858984 protein were screened to obtain breeding roosters with high antifreeze performance of sperm.

6. The method according to claim 5, characterized in that The expression level of LOC100858984 protein in the sperm of the rooster to be tested is detected. If the expression level of LOC100858984 protein in the sperm of the rooster to be tested is less than 1988 ng / ml, a breeding rooster with high antifreeze performance of sperm is screened.

7. The method according to claim 5, characterized in that The amino acid sequence of the LOC100858984 protein is shown in SEQ ID No.

1.

8. A test kit for identifying the antifreeze properties of rooster sperm, characterized in that: The detection kit contains LOC100858984 protein antibody.

9. The detection kit according to claim 8, characterized in that The amino acid sequence of the LOC100858984 protein is shown in SEQ ID No. 1.

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