Method for preserving cattle sex-controlled frozen sperms

Abstract

The invention relates to the technical field of frozen sperms, in particular to a cattle sex control frozen sperm preservation method which comprises the following steps: collecting bull semen, and purifying the bull semen to obtain a sperm suspension so as to determine the qualification of the quality of the semen; performing sperm sorting on the sperm suspension by using a flow cytometry to separate target sperms based on a determination result that the quality of the sperm is qualified; uniformly mixing the target sperms with a refrigerating fluid, standing for a preset period of time, and freezing; obtaining a particle size distribution curve and an ROS concentration curve of the target sperm in the freezing process, and determining the qualification of the temperature drop rate according to the distribution proportion of the particle size distribution curve; adjusting the temperature drop rate according to a comparison result of an ROS fluctuation index of the ROS concentration curve and a preset index on the basis of a judgment result that the temperature drop rate is unqualified; and unfreezing and resuscitating the frozen target sperms to determine the sperm malformation rate so as to adjust the preset activity or the preset index. According to the invention, the quality of frozen sperms is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of frozen sperm, and in particular to a method for preserving sex-controlled frozen sperm of cattle. Background Art

[0002] Although existing semen freezing technology has been widely used in the fields of animal husbandry and human reproduction, there is a general lack of monitoring of the freezing process. The traditional freezing process only evaluates the quality of sperm preservation through pre-freezing semen analysis (such as sperm concentration and motility detection) and post-freezing recovery rate, while ignoring the dynamic changes in the physiological state of sperm during the freezing process. The freezing damage mechanism of sperm is complex. During the cooling stage (especially the ice crystal formation period of -5°C to -15°C), it is easy to cause membrane structure destruction and DNA damage due to intracellular ice crystal growth, osmotic pressure imbalance and oxidative stress. The existing technology lacks real-time monitoring of the morphological integrity, motility decay curve and metabolic activity of sperm in various stages of freezing (such as temperature shock period and ice crystal latent heat release period). As a result, key parameters such as the amount of protective agent added and the cooling rate can only be set based on experience, which makes it difficult to cope with individual differences.

[0003] Chinese Patent Publication No. CN108575989A discloses a method for cryopreservation of sexed semen of breeding cattle, comprising the following steps: collection, testing, filtration, addition of antibiotics, staining, water bath, addition of egg yolk, glycerol emulsification, suspension and mixing of sperm sediment, counting, sampling, filling, freezing, random inspection, and liquid nitrogen storage. In the production of frozen bovine semen, fresh semen is first placed in a 33°C water bath after collection. After preliminary testing, it is directly placed in a 19°C environment and finally placed in a low-temperature operating cabinet for cooling and equilibrium, and then packaged and printed. Through this step, the sperm can effectively gradually adapt to the lowering temperature, slowing energy loss and reducing mortality.

[0004] The existing technology also has the following problems: due to the lack of real-time monitoring of the status of the sperm freezing process, the existing technology cannot be adjusted according to the real-time status of the sperm, resulting in low quality of frozen sperm and high deformity rate after sperm recovery. Summary of the Invention

[0005] To this end, the present invention provides a method for preserving sex-controlled frozen sperm of cattle, which is used to overcome the problem in the prior art that there is a lack of real-time monitoring of the status of the sperm freezing process, resulting in an inability to adjust according to the real-time status of the sperm, thereby resulting in low quality of the frozen sperm and a high deformity rate after sperm recovery.

[0006] To achieve the above object, the present invention provides a method for preserving sex-controlled frozen cattle sperm, comprising:

[0007] collecting bull semen, purifying the bull semen to obtain a sperm suspension, and determining the eligibility of the semen quality based on a comparison result of sperm motility of the sperm suspension with a preset motility;

[0008] Based on the result of judging that the semen quality is qualified, the sperm suspension is subjected to sperm sorting using a flow cytometer to separate target sperm;

[0009] The target sperm is mixed evenly with the freezing solution and then allowed to stand for a preset time for freezing;

[0010] Obtain the particle size distribution curve and ROS concentration curve of the target sperm during the freezing process to determine the eligibility of the temperature drop rate based on the distribution ratio of the particle size distribution curve;

[0011] Based on the determination result that the temperature drop rate is unqualified, adjusting the temperature drop rate according to the comparison result of the ROS fluctuation index of the ROS concentration curve and the preset index;

[0012] The frozen target sperm are thawed and revived to determine the sperm deformity rate so as to adjust the preset motility or preset index.

[0013] Furthermore, sperm motility tests include:

[0014] Acquiring a plurality of images of the sperm suspension at preset time intervals, and determining a plurality of sperm in the images using an image algorithm;

[0015] Adjacent images are compared to determine the position information of corresponding sperms to determine the linear distance of corresponding sperms;

[0016] For a single sperm, determining a ratio of the straight-line distance to the preset time interval as a sperm velocity, and determining a plurality of sperm velocities based on the image;

[0017] determining a velocity variance of each of the sperm velocities and comparing the velocity variance with a preset variance;

[0018] Determining that the sperm motility is qualified based on a comparison result that the velocity variance is greater than or equal to a preset variance;

[0019] The percentage of sperm with qualified motility to the total number of sperm is determined as sperm motility.

[0020] Furthermore, the process of determining the eligibility of semen quality based on the sperm motility includes:

[0021] comparing the sperm motility with a predetermined motility;

[0022] The semen quality is determined to be qualified based on the comparison result that the sperm motility is greater than or equal to the preset motility.

[0023] Furthermore, the process of determining the distribution ratio includes:

[0024] Overlapping the particle size distribution curve with the standard distribution curve based on the coordinate origin;

[0025] The ratio of the area of the overlapping portion to the area of the standard distribution curve is determined as the distribution ratio.

[0026] Furthermore, the process of determining the eligibility of the temperature drop rate based on the distribution ratio includes:

[0027] Comparing the distribution ratio with a preset distribution ratio;

[0028] The temperature drop rate is determined to be unqualified based on the comparison result that the distribution ratio is less than the preset ratio.

[0029] Furthermore, the process of determining the ROS fluctuation index includes:

[0030] Determine several peaks of the ROS concentration curve, and connect each peak to the coordinate origin;

[0031] determining a plurality of slopes of the connecting line, and comparing the slopes with a slope mean to determine a slope standard deviation;

[0032] The slope standard deviation is determined as the ROS fluctuation index;

[0033] Wherein, the slope mean is determined according to the standard ROS concentration curve.

[0034] Furthermore, the process of adjusting the temperature drop rate based on the ROS fluctuation index includes:

[0035] Subtracting the ROS fluctuation index from a preset index to obtain an index difference;

[0036] A plurality of rate adjustment coefficients corresponding to the index differences are set to reduce the temperature decrease rate based on the rate adjustment coefficients.

[0037] Furthermore, the process of determining the adjustment parameter based on the sperm deformity rate includes:

[0038] comparing the sperm deformity rate with a preset deformity rate;

[0039] Determining to increase the preset index based on a comparison result that the sperm deformity rate is greater than a first preset deformity rate and less than a second preset deformity rate;

[0040] The determination of increasing the preset motility is based on a comparison result that the sperm deformity rate is greater than the second preset deformity rate.

[0041] Furthermore, the process of adjusting the preset index includes:

[0042] subtracting the sperm deformity rate from the first preset deformity rate to obtain a first difference;

[0043] A plurality of index adjustment coefficients corresponding to the first difference are set to reduce the preset index based on the index adjustment coefficients.

[0044] Furthermore, the process of adjusting the preset vitality includes:

[0045] subtracting the sperm deformity rate from the second preset deformity rate to obtain a second difference;

[0046] A plurality of vitality adjustment coefficients corresponding to the second difference are set to increase the preset vitality based on the vitality adjustment coefficients.

[0047] Compared with the existing technology, the beneficial effect of the present invention is that when determining sperm motility, compared with the existing technology CASA which relies on frame-by-frame tracking of individual sperm trajectories, the present invention replaces a single speed indicator with speed variance, which can comprehensively reflect the movement consistency of the sperm group. The smaller the variance, the higher the sperm speed convergence and the more stable the group motility; the larger the variance, the more significant the heterogeneity of motility, which may indicate that the number of sperm with functional defects is greater. Through the dynamic indicator of speed variance, the stability of sperm movement can be more sensitively reflected, effectively avoiding the misjudgment caused by CASA due to sperm swinging in place or nonlinear movement, thereby improving the detection accuracy of sperm motility and further improving the quality of frozen sperm.

[0048] Furthermore, the present invention collects the particle size distribution curve of sperm in real time during the sperm freezing process. The particle size distribution curve indirectly reflects the formation of ice crystals and the degree of cell damage during the freezing process of sperm. If the temperature drop rate of sperm is too fast, the ice crystal size will be too large, causing the particle size distribution curve of sperm to become more dispersed. If the temperature drop rate of sperm is too slow, it will cause lipid peroxidation and protein denaturation of sperm cell membrane, thereby causing the release of cell contents, causing the particle size to first decrease and then increase. The smaller the area overlap between the particle size distribution curve and the standard distribution curve, the greater the degree of deviation of the particle size distribution curve, and the worse the qualification of the temperature drop rate. Determining the qualification of the temperature drop rate based on the particle size distribution curve can eliminate the influence of individual differences between samples, thereby further improving the quality of frozen sperm.

[0049] Furthermore, the present invention determines the adjustment range of the temperature drop rate based on the ROS fluctuation index. An unqualified temperature drop rate will lead to an increased oxidative stress response of sperm cells, resulting in large fluctuations in ROS concentration. The temperature drop rate is accurately adjusted according to the ROS fluctuation index, thereby further improving the quality of frozen sperm.

[0050] Furthermore, the present invention thaws and revives the frozen sperm and determines the deformity rate, and adjusts the freezing process according to the deformity rate. A high deformity rate increases the criterion for determining sperm motility, thereby improving the motility of the frozen sperm. A low deformity rate increases the criterion for determining the temperature drop rate, thereby improving the temperature control accuracy, thereby further improving the quality of the frozen sperm. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a flow chart of a method for preserving sex-controlled frozen sperm of cattle according to an embodiment of the present invention;

[0052] Figure 2 This is a flow chart of determining whether semen quality is qualified according to an embodiment of the present invention;

[0053] Figure 3 A flow chart for determining the eligibility of a temperature drop rate according to an embodiment of the present invention;

[0054] Figure 4 This is a flow chart of determining adjustment parameters according to an embodiment of the present invention. DETAILED DESCRIPTION

[0055] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0056] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0057] See also Figures 1-4 As shown, Figure 1 This is a flow chart of a method for preserving sex-controlled frozen sperm of cattle according to an embodiment of the present invention; Figure 2 This is a flow chart of determining whether semen quality is qualified according to an embodiment of the present invention; Figure 3 A flow chart for determining the eligibility of a temperature drop rate according to an embodiment of the present invention; Figure 4 This is a flow chart of determining adjustment parameters according to an embodiment of the present invention.

[0058] The embodiment of the present invention provides a method for preserving sex-controlled frozen cattle sperm, comprising:

[0059] Step S1, collecting bull semen, purifying the bull semen to obtain a sperm suspension, and determining the eligibility of the semen quality based on a comparison result of sperm motility of the sperm suspension with a preset motility;

[0060] Step S2, based on the result of judging that the semen quality is qualified, performing sperm sorting on the sperm suspension using a flow cytometer to separate target sperm;

[0061] Step S3, mixing the target sperm with the freezing solution evenly and then standing for a preset time to freeze;

[0062] Step S4, obtaining a particle size distribution curve and a ROS concentration curve of the target sperm during the freezing process, and determining the eligibility of the temperature drop rate based on the distribution ratio of the particle size distribution curve;

[0063] Step S5, adjusting the temperature drop rate according to a comparison result of the ROS fluctuation index of the ROS concentration curve with a preset index based on the determination result that the temperature drop rate is unqualified;

[0064] Step S6: thawing and resuming the frozen target sperm to determine the sperm deformity rate so as to adjust the preset motility or preset index.

[0065] Specifically, the bull semen collection method includes a pseudo-vagina method or an electric stimulation semen collection method, and the bull semen processing method includes:

[0066] The collected bull semen is immediately placed in a constant temperature environment at 37°C and diluted and liquefied with a basic diluent at a volume ratio of 1:1-3 for 30-60 minutes, wherein the basic diluent is 20% egg yolk Tris-A solution or Tris-glucose buffer;

[0067] Centrifuge the diluted liquefied bull semen at low speed, discard the supernatant and retain the precipitate, repeat the above operation 1-2 times to obtain the precipitate which is the sperm suspension;

[0068] The sperm suspension is subjected to an automatic sperm counter to determine the sperm concentration;

[0069] Determining to dilute the sperm suspension based on a comparison result that the sperm concentration is greater than a target concentration;

[0070] The sperm suspension is concentrated based on the comparison result that the sperm concentration is less than the target concentration.

[0071] It is understood that the dilution process is to add a basic diluent to dilute the sperm suspension to the target concentration, and the concentration method is to concentrate it to the target concentration by density gradient centrifugation using Percoll or PureSperm gradient medium, wherein the target concentration is 1×10 6 -5×10 6 Sperm / mL.

[0072] Specifically, the Tris-A working solution is prepared as follows: Dissolve 35.32g of Trizma IBase, 17.21g of citric acid, and 12.65g of D-fructose in 750mL of double-distilled water. Stir for 30 minutes, then add double-distilled water to bring the solution to 1000mL. Adjust the pH of the solution to 6.8, filter sterilize using a 0.22μm filter, and store at 5°C ± 1°C. The expiration date is 14 days.

[0073] Preparation of 20% egg yolk Tris-A working solution: Add 51 mL of egg yolk solution to 199 mL of Tris-A working solution, stir for 15 minutes, and let stand at 5°C ± 1°C for 12 hours. Remove the precipitate, aspirate the surface egg fat, and centrifuge at 18,500g. Sterilize the solution by filtration using a 0.22 μm filter, add antibiotics, and store at 5°C ± 1°C. The solution is valid for 7 days. The antibiotics should include at least one of penicillin G, streptomycin, gentamicin, and ciprofloxacin.

[0074] Specifically, when using a flow cytometer to sort target sperm, a separator buffer (Tris-sheath) is used as a sheath fluid to wrap the sperm suspension for sorting. The preparation method of the separator buffer is as follows: prepare 20L of Tris-sheath buffer: take 477.6g of Trizma Base, 232.6g of citric acid, and 171.0g of D-fructose and dissolve them in 3.0L of double-distilled water and stir for 30 minutes, and add hydrochloric acid to adjust the pH to 6.8, then add antibiotics and filter sterilize with a 0.22μm filter, then add double-distilled water to 20L capacity, adjust the pH to 6.8, the osmotic pressure to 290mOsm±10mOsm, and store at 5℃±1℃. The shelf life is 14d.

[0075] Specifically, when testing sperm motility, it is necessary to stain the sperm to show the morphological structure of the sperm for the purpose of motility testing. The preparation method of sperm staining stock solution is as follows: 100 mL of sperm staining stock solution is prepared, and 0.925 g of HEPES, 0.008 g of magnesium chloride (MgCl2·6H20), 0.5518 g of sodium chloride (NaCl), 0.0224 g of potassium chloride (KCl), and 0.0224 g of disodium hydrogen phosphate (Na2HPO4) are weighed in sequence. 4) 0.004g, sodium bicarbonate (NaHCO 3) Dissolve 0.084 g of dapoxetine, 0.022 g of sodium pyruvate, 0.09 g of glucose, 0.361 mL of sodium lactate, and 0.3 g of BSA in 100 mL of double-distilled water, adjust the pH to 7.4, add 0.25 mL of gentamicin (10 mg / mL), filter sterilize through 0.22 μm, and store at 5°C ± 1°C. The shelf life is 7-10 days.

[0076] Specifically, before sorting the target sperm by flow cytometry, the sperm needs to be fluorescently labeled using 0.5% fluorescent dye solution. The specific preparation method is: 10 mg of fluorescent dye Hochest33342 is diluted with 2 mL of double-distilled water and stored in the dark at 5°C±1°C. The shelf life is 90 days.

[0077] Specifically, the freezing solution is a 12% glycerol Tris-B solution, prepared as follows: first, prepare 23% (volume fraction) egg yolk solution with Tris-B working solution, stir for 15 minutes, and then place at 5°C ± 1°C for 12 hours to precipitate. After standing, the surface egg fat is aspirated and the precipitate is further centrifuged at 18,500g. The solution is then sterilized by filtration using a 0.22μm filter. Glycerol is then added to a final glycerol concentration of 12% (volume fraction). The solution pH is adjusted to 6.8, antibiotics are added, and the solution is stored at 5°C ± 1°C with a shelf life of 7 days.

[0078] Specifically, the preparation method of the Tris-B solution is as follows: take 35.746g of Trizma Base, 19.980g of citric acid, and 14.712g of D-fructose and dissolve them in 750mL of double-distilled water, stir for 30 minutes, then add double-distilled water to 1000mL, adjust the pH of the solution to 6.8, then filter and sterilize with a 0.22μm filter, store at 5℃±1℃, and have a shelf life of 14 days.

[0079] Specifically, the mixing ratio of freezing solution and target sperm is 1:1-3, and the preset time is 30-60 minutes.

[0080] Specifically, sperm suspension with qualified sperm concentration is left to stand for 30-60 minutes before sperm motility test is performed. It is understandable that the mitochondrial function of sperm is temporarily suppressed after the purification process, and it takes time to restore energy metabolism.

[0081] Specifically, the sperm motility detection method includes:

[0082] Acquiring a plurality of images of the sperm suspension at preset time intervals, and determining a plurality of sperm in the images using an image algorithm;

[0083] Adjacent images are compared to determine the position information of corresponding sperms to determine the linear distance of corresponding sperms;

[0084] For a single sperm, determining a ratio of the straight-line distance to the preset time interval as a sperm velocity, and determining a plurality of sperm velocities based on the image;

[0085] determining a velocity variance of each of the sperm velocities and comparing the velocity variance with a preset variance;

[0086] Determining that the sperm motility is qualified based on a comparison result that the velocity variance is greater than or equal to a preset variance;

[0087] The percentage of sperm with qualified motility to the total number of sperm is determined as sperm motility.

[0088] Specifically, the value range of the preset time interval is set to [20ms, 60ms], and 40ms is preferred in the embodiment of the present invention.

[0089] Specifically, the value range of the preset variance is set to [0.2, 0.5], and 0.4 is preferred in the embodiment of the present invention.

[0090] Specifically, the image algorithm can use YOLO5 or YOLO8 to optimize the anchor frame for the slender structure of sperm, or use Attention U-Net to perform pixel-level segmentation to accurately extract the boundary of sperm.

[0091] It is understandable that healthy and vigorous sperm mainly moves forward in a fast, straight or slightly swinging manner, showing regular curved motion, and the speed can reach more than 25μm / s. However, in the existing technology, when using the automated system CASA (computer-assisted sperm analysis) to judge the proportion of forward-moving sperm (PR value), the irregular movement of sperm with severe head deformities due to unstable center of gravity may be classified as non-forward motion, thereby causing the PR value to increase. In this application, whether the sperm is continuously moving regularly is determined based on the dynamic changes of the sperm, thereby improving the accuracy of judging the movement state of the sperm.

[0092] Specifically, the process of determining the eligibility of semen quality based on the sperm motility includes:

[0093] comparing the sperm motility with a predetermined motility;

[0094] Determining that the semen quality is qualified based on the comparison result that the sperm motility is greater than or equal to the preset motility;

[0095] The semen quality is determined to be unqualified based on the comparison result that the sperm motility is less than the preset motility.

[0096] Specifically, the value range of the preset vitality is set to [40%, 70%], and 50% is preferred in the embodiment of the present invention.

[0097] Specifically, if the semen quality is judged to be unqualified, the sperm suspension is discarded.

[0098] The process of determining the distribution ratio includes:

[0099] Overlapping the particle size distribution curve with the standard distribution curve based on the coordinate origin;

[0100] The ratio of the area of the overlapping portion to the area of the standard distribution curve is determined as the distribution ratio.

[0101] Specifically, the process of determining the eligibility of the temperature drop rate based on the distribution ratio includes:

[0102] Comparing the distribution ratio with a preset distribution ratio;

[0103] Determining that the temperature drop rate is unqualified based on a comparison result that the distribution ratio is less than a preset ratio;

[0104] The temperature drop rate is determined to be qualified based on a comparison result that the distribution ratio is greater than or equal to the preset ratio.

[0105] Specifically, the value range of the preset proportion is set to [70%, 90%], and 80% is preferred in the embodiment of the present invention.

[0106] Specifically, the process of determining the ROS fluctuation index includes:

[0107] Determine several peaks of the ROS concentration curve, and connect each peak to the coordinate origin;

[0108] determining a plurality of slopes of the connecting line, and comparing the slopes with a slope mean to determine a slope standard deviation;

[0109] The slope standard deviation is determined as the ROS fluctuation index;

[0110] Wherein, the slope mean is determined according to the standard ROS concentration curve.

[0111] Specifically, the standard ROS concentration curve is the ROS concentration curve during the freezing process of sperm whose motility meets the standard after thawing of frozen sperm, and is determined based on the ROS concentration range of all frozen sperm that meet the standard.

[0112] Specifically, the process of adjusting the temperature drop rate based on the ROS fluctuation index includes:

[0113] Subtracting the ROS fluctuation index from a preset index to obtain an index difference;

[0114] A plurality of rate adjustment coefficients corresponding to the index differences are set to reduce the temperature decrease rate based on the rate adjustment coefficients.

[0115] Specifically, the index difference is compared with a preset index difference;

[0116] Determining to reduce the temperature drop rate by a first rate adjustment coefficient based on a comparison result that the index difference is greater than a preset index difference;

[0117] Based on the comparison result that the index difference is less than or equal to the preset index difference, it is determined to reduce the temperature decrease rate by a second rate adjustment coefficient.

[0118] Specifically, the preset index is determined according to the standard ROS concentration curve according to the above method. The value range of the embodiment of the present invention is set to [0.4, 0.7], preferably 0.6; the value range of the preset index difference is set to [0.1, 0.3], preferably 0.2 in the embodiment of the present invention; the value range of the first rate adjustment coefficient is set to [0.86, 0.92], preferably 0.9 in the embodiment of the present invention; the value range of the second rate adjustment coefficient is set to [0.921, 0.96], preferably 0.94 in the embodiment of the present invention.

[0119] Specifically, the rate adjustment coefficient is adjusted in such a way that the product of the corresponding adjustment coefficient and the initial temperature drop rate is the adjusted temperature drop rate. For example, in this embodiment, the initial temperature drop rate is set to 1°C / min, and the first rate adjustment coefficient is used for adjustment, then the adjusted temperature drop rate is 0.9°C / min.

[0120] Specifically, the process of determining the adjustment parameters based on the sperm deformity rate includes:

[0121] comparing the sperm deformity rate with a preset deformity rate;

[0122] Determining to increase the preset index based on a comparison result that the sperm deformity rate is greater than a first preset deformity rate and less than a second preset deformity rate;

[0123] The determination of increasing the preset motility is based on a comparison result that the sperm deformity rate is greater than the second preset deformity rate.

[0124] Specifically, the value range of the first preset deformity rate is set to [4%, 6%], and 5% is preferred in the embodiment of the present invention; the value range of the second preset deformity rate is set to [7%, 13%], and 8% is preferred in the embodiment of the present invention.

[0125] Specifically, the process of adjusting the preset index includes:

[0126] subtracting the sperm deformity rate from the first preset deformity rate to obtain a first difference;

[0127] A plurality of index adjustment coefficients corresponding to the first difference are set to reduce the preset index based on the index adjustment coefficients.

[0128] Specifically, comparing the first difference with a preset difference;

[0129] Determining to reduce the preset index by a first index adjustment coefficient based on a comparison result that the first difference is greater than the preset difference;

[0130] Based on a comparison result that the first difference is less than or equal to the preset difference, it is determined to reduce the preset index by a second index adjustment coefficient.

[0131] Specifically, the value range of the preset difference is set to [1%, 4%], and 3% is preferred in the embodiment of the present invention; the value range of the first exponential adjustment coefficient is set to [0.8, 0.9], and 0.85 is preferred in the embodiment of the present invention; the value range of the second exponential adjustment coefficient is set to [0.91, 0.98], and 0.95 is preferred in the embodiment of the present invention.

[0132] Specifically, the process of adjusting the preset vitality includes:

[0133] subtracting the sperm deformity rate from the second preset deformity rate to obtain a second difference;

[0134] A plurality of vitality adjustment coefficients corresponding to the second difference are set to increase the preset vitality based on the vitality adjustment coefficients.

[0135] Specifically, comparing the second difference with a preset difference;

[0136] determining to increase the preset vitality by a first vitality adjustment coefficient based on a comparison result that the second difference is greater than the preset difference;

[0137] Based on a comparison result that the second difference is less than or equal to the preset difference, it is determined to increase the preset vitality by a second vitality adjustment coefficient.

[0138] Specifically, the value range of the first vitality adjustment coefficient is set to [1.3, 1.6], and preferably 1.4 in the embodiment of the present invention; the value range of the second vitality adjustment coefficient is set to [1.1, 1.29], and preferably 1.2 in the embodiment of the present invention.

[0139] In an embodiment of the present invention, after the target sperm is frozen, it is sent to the Bovine Frozen Semen Quality Supervision and Inspection Center for thawing and recovery, and then quality inspection is performed. The inspection results are shown in the table, where the quality inspection items include appearance, dosage, sperm motility, number of progressively motile sperm, sperm deformity rate, and bacterial colony count.

[0140] The quality test results of sex-controlled frozen sperm

[0141]

[0142]

[0143] As can be seen from the table, the performance of the sperm frozen using the cattle sex-controlled frozen sperm preservation method provided by the embodiment of the present invention after thawing and recovery is better than the standard.

[0144] Thus far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art may make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will fall within the scope of protection of the present invention.

Claims

1. A method for preserving sex-controlled frozen cattle sperm, characterized in that: include: collecting bull semen, purifying the bull semen to obtain a sperm suspension, and determining the eligibility of the semen quality based on a comparison result of sperm motility of the sperm suspension with a preset motility; Based on the result of judging that the semen quality is qualified, the sperm suspension is subjected to sperm sorting using a flow cytometer to separate target sperm; The target sperm is mixed evenly with the freezing solution and then allowed to stand for a preset time for freezing; Obtain the particle size distribution curve and ROS concentration curve of the target sperm during the freezing process to determine the eligibility of the temperature drop rate based on the distribution ratio of the particle size distribution curve; Based on the determination result that the temperature drop rate is unqualified, adjusting the temperature drop rate according to the comparison result of the ROS fluctuation index of the ROS concentration curve with a preset index; The frozen target sperm are thawed and revived to determine the sperm deformity rate so as to adjust the preset motility or preset index.

2. The method for preserving sex-controlled frozen cattle sperm according to claim 1, characterized in that: The method for detecting sperm motility includes: Acquiring a plurality of images of the sperm suspension at preset time intervals, and determining a plurality of sperm in the images using an image algorithm; Adjacent images are compared to determine the position information of corresponding sperms to determine the linear distance of corresponding sperms; For a single sperm, determining a ratio of the straight-line distance to the preset time interval as a sperm velocity, and determining a plurality of sperm velocities based on the image; determining a velocity variance of each of the sperm velocities and comparing the velocity variance with a preset variance; Determining that the sperm motility is qualified based on a comparison result that the velocity variance is greater than or equal to a preset variance; The percentage of sperm with qualified motility to the total number of sperm is determined as sperm motility.

3. The method for preserving sex-controlled frozen cattle sperm according to claim 2, characterized in that: The process of determining the acceptability of semen quality based on the sperm motility includes: comparing the sperm motility with a predetermined motility; The semen quality is determined to be qualified based on the comparison result that the sperm motility is greater than or equal to the preset motility.

4. The method for preserving sex-controlled frozen cattle sperm according to claim 3, characterized in that: The process of determining the distribution ratio includes: Overlapping the particle size distribution curve with the standard distribution curve based on the coordinate origin; The ratio of the area of the overlapping portion to the area of the standard distribution curve is determined as the distribution ratio.

5. The method for preserving sex-controlled frozen cattle sperm according to claim 4, characterized in that: The process of determining the eligibility of the temperature drop rate based on the distribution ratio includes: Comparing the distribution ratio with a preset distribution ratio; The temperature drop rate is determined to be unqualified based on the comparison result that the distribution ratio is less than the preset ratio.

6. The method for preserving sex-controlled frozen cattle sperm according to claim 5, characterized in that: The process of determining the ROS volatility index includes: Determine several peaks of the ROS concentration curve, and connect each peak to the coordinate origin; determining a plurality of slopes of the connecting line, and comparing the slopes with a slope mean to determine a slope standard deviation; The slope standard deviation is determined as the ROS fluctuation index; Wherein, the slope mean is determined according to the standard ROS concentration curve.

7. The method for preserving sex-controlled frozen cattle sperm according to claim 6, characterized in that: The process of adjusting the temperature drop rate based on the ROS fluctuation index includes: Subtracting the ROS fluctuation index from a preset index to obtain an index difference; A plurality of rate adjustment coefficients corresponding to the index differences are set to reduce the temperature decrease rate based on the rate adjustment coefficients.

8. The method for preserving sex-controlled frozen cattle sperm according to claim 7, characterized in that: The process of determining the adjustment parameters based on the sperm deformity rate includes: comparing the sperm deformity rate with a preset deformity rate; Determining to increase the preset index based on a comparison result that the sperm deformity rate is greater than a first preset deformity rate and less than a second preset deformity rate; The determination of increasing the preset motility is based on a comparison result that the sperm deformity rate is greater than the second preset deformity rate.

9. The method for preserving sex-controlled frozen cattle sperm according to claim 8, characterized in that: The process of adjusting the preset index includes: subtracting the sperm deformity rate from the first preset deformity rate to obtain a first difference; A plurality of index adjustment coefficients corresponding to the first difference are set to reduce the preset index based on the index adjustment coefficients.

10. The method for preserving sex-controlled frozen cattle sperm according to claim 8, characterized in that: The process of adjusting the preset vitality includes: subtracting the sperm deformity rate from the second preset deformity rate to obtain a second difference; A plurality of vitality adjustment coefficients corresponding to the second difference are set to increase the preset vitality based on the vitality adjustment coefficients.

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