A diluent for improving the quality and fertilization ability of frozen bovine semen

Abstract

The present invention provides a diluent for improving the quality and fertilization ability of frozen bovine semen, including chlorogenic acid and a calcium chelator. Compared with the prior art, the present invention adds antioxidant chlorogenic acid or caffeic acid and a calcium chelator to the semen cryopreservation diluent, effectively improves the vitality, plasma membrane integrity, acrosome integrity and fertilization ability of frozen-thawed semen, significantly improves the antioxidant performance, reduces the ROS level, inhibits premature capacitation at low temperature, and significantly improves the cryopreservation effect of bovine sperm. It is suitable for long-term cryopreservation of bovine semen in liquid nitrogen and artificial insemination. The diluent preparation method is simple, the cryopreservation operation method is simple, and it is easy to promote, and significantly improves the quality and fertilization ability of bovine semen after freezing and thawing.

Description

Technical Field

[0001] The invention belongs to the technical field of artificial insemination of cattle, and in particular relates to a diluent for improving the quality and fertilization ability of frozen and preserved cattle semen. Background Art

[0002] The popularization of artificial insemination technology for cattle can give full play to the genetic potential of male animals, and semen freezing is an important part of artificial insemination, providing a sperm bank for the implementation of artificial insemination technology for cattle. With the widespread application of cattle semen cryopreservation technology, the use of frozen semen is not limited by time and space, and has the advantages of increasing the utilization rate of excellent breeding bulls, reducing economic costs, improving the benefits of animal husbandry, and promoting breed improvement. Semen freezing technology has important practical significance for the sustainable development of my country's animal husbandry and the satisfactory supply of livestock products.

[0003] During the freezing process, sperm is susceptible to factors such as ice crystal formation and oxidative stress, which can lead to intracellular metabolic disorders, DNA damage, protein structure changes, mitochondrial dysfunction, cytoplasmic instability, and excessive reactive oxygen species (ROS) free radical generation. As a result, the viability, vitality, plasma membrane integrity, acrosome integrity, mitochondrial activity, and DNA integrity of frozen-thawed bovine sperm significantly decrease, resulting in an inability to maintain fertilization ability, thereby reducing pregnancy and delivery rates. Currently, domestic and foreign surveys have shown that the fertilization rate after artificial insemination with frozen sperm has been declining year by year. This is because the permeability and fluidity of the plasma membrane have changed under low temperature stimulation, causing a large amount of Ca 2+ Entering the sperm, it causes premature capacitation, which in turn affects the fertilization ability of semen after freezing and thawing. Therefore, in order to improve the quality of frozen and thawed sperm and improve fertilization ability, it is very necessary to improve the basic cryopreservation diluent and inhibit peroxidation damage and premature capacitation during cryopreservation by certain means. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention is directed to a diluent for improving the quality and fertilization ability of frozen bovine semen.

[0005] The first object of the present invention is to provide a diluent for improving the quality and fertilization ability of frozen bovine semen, characterized in that it is composed of any of the following:

[0006] a. 2 g / L fructose, 22 g / L Tris-base, 12 g / L citric acid, 20% egg yolk, 6% glycerol, 1% sodium amino-dodecyl sulfate, 0.004 g / L amikacin sulfate, 100 µM chlorogenic acid, 6 µM calcium chelator, and the balance is water;

[0007] b. 2 g / L fructose, 22 g / L Tris-base, 12 g / L citric acid, 20% egg yolk, 6% glycerol, 1% sodium amino-dodecyl sulfate, 0.004 g / L amikacin sulfate, 100 µM caffeic acid, 6 µM calcium chelator, and the balance is water.

[0008] Preferably, the calcium chelator is EGTA or BAPTA.

[0009] A second object of the present invention is to provide a method for freezing and preserving bovine semen, comprising the following steps:

[0010] S1. The diluent and bovine semen were balanced for 2-3 hours and then slowly cooled to 5°C;

[0011] S2. Dilute the bovine semen by mixing the diluent with the bovine semen in a volume ratio of 1:1 and equilibrate for 10 minutes;

[0012] S3. After the balance is completed, suck it into the frozen straw and seal it;

[0013] S4. Place the frozen vials containing bovine semen at -30°C or below for 15 minutes and then store them in liquid nitrogen.

[0014] Preferably, the cattle are Leiqiong cattle.

[0015] Preferably, the freezing tube is a freezing tube with a specification of 0.25 mL.

[0016] Beneficial effects of the present invention:

[0017] The present invention adds an antioxidant (chlorogenic acid or caffeic acid) and a calcium chelator (EGTA or BAPTA) to a diluent for the cryopreservation of bovine semen, effectively improving the activity, plasma membrane integrity, acrosome integrity and fertilization ability of frozen-thawed semen, significantly improving the antioxidant performance, reducing the ROS level, inhibiting premature capacitation at low temperature, and significantly improving the cryopreservation effect of bovine sperm. The invention is suitable for long-term cryopreservation of bovine semen in liquid nitrogen and artificial insemination. The diluent preparation method is simple, the cryopreservation operation method is simple and easy to promote, and significantly improves the quality and fertilization ability of bovine semen after freeze-thaw. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Experimental technology roadmap for semen cryopreservation.

[0019] Figure 2The effects of different cryoprotectants on the semen motility of Leiqiong cattle stored at -196℃, -80℃ and -30℃, respectively; (A) is the total motility of sperm after being stored at -196℃ for one week with different cryoprotectants, (B) is the forward motility of sperm after being stored at -196℃ for one week with different cryoprotectants, (C) is the motility of sperm after being stored at -80℃ for 1 week, 2 weeks and 1 month with different cryoprotectants, (D) is the motility of sperm after being stored at -80℃ for 1 week, 2 weeks and 1 month with different cryoprotectants, (E) is the motility of sperm after being stored at -30℃ for 1 week, 2 weeks and 1 month with different cryoprotectants, (F) is the motility of sperm after being stored at -30℃ for 1 week, 2 weeks and 1 month with different cryoprotectants; different letters indicate significant differences ( P<0.05 ); NO.1-3 represent three tested bulls respectively, F is fresh bull semen, A is bull semen frozen and preserved by diluent A, B is bull semen frozen and preserved by diluent B, and C is bull semen frozen and preserved by diluent C.

[0020] Figure 3 Effects of sugars (A), different concentrations of egg yolk (B), glycerol (C) and OEP (D) on the motility of frozen-thawed bovine sperm; CT (control) in AD is no sugar, no egg yolk, no glycerol, no OEP, and different letters indicate significant differences ( P<0.05 ).

[0021] Figure 4 is the effect of CGA on the quality of frozen-thawed bovine semen; A is the effect of CGA on the sperm motility in frozen-thawed bovine semen, B is the effect of CGA on the sperm vitality in frozen-thawed bovine semen, C is the effect of CGA on the integrity of sperm plasma membrane in frozen-thawed bovine semen, D is the effect of CGA on the integrity of sperm acrosome in frozen-thawed bovine semen, and E is the effect of CGA on the sperm survival rate in frozen-thawed bovine semen; different letters indicate significant differences ( P<0.05 ).

[0022] Figure 5 is the effect of CA on the quality of frozen-thawed bovine semen; A is the effect of CA on sperm motility in frozen-thawed bovine semen, B is the effect of CA on sperm vitality in frozen-thawed bovine semen, C is the effect of CA on sperm plasma membrane integrity in frozen-thawed bovine semen, D is the effect of CA on sperm acrosome integrity in frozen-thawed bovine semen, and E is the effect of CA on sperm survival rate in frozen-thawed bovine semen; different letters indicate significant differences ( P<0.05 ).

[0023] Figure 6The effects of CGA and CA on antioxidant enzymes in frozen-thawed bovine semen; A is the effect of CGA and CA on CAT enzyme activity in frozen-thawed bovine semen, B is the effect of CGA and CA on SOD enzyme activity in frozen-thawed bovine semen, and C is the effect of CGA and CA on GSH-PX enzyme activity in frozen-thawed bovine semen; different letters indicate significant differences ( P<0.05 ).

[0024] Figure 7 Effects of CGA and CA on MDA (A), ROS content (B) and MMP (C) in frozen-thawed bovine sperm; different letters indicate significant differences ( P<0.05 ).

[0025] Figure 8 is the effect of calcium chelator on the quality of frozen-thawed semen; A is the effect of calcium chelator on sperm motility in frozen-thawed bovine semen, B is the effect of calcium chelator on sperm motility in frozen-thawed bovine semen, C is the effect of calcium chelator on sperm plasma membrane integrity in frozen-thawed bovine semen, D is the effect of calcium chelator on sperm acrosome integrity in frozen-thawed bovine semen, E is the effect of calcium chelator on sperm survival rate in frozen-thawed bovine semen; different letters indicate significant differences ( P<0.05 ).

[0026] Fig. 9 Calcium chelators are added to sperm Ca 2+ A is the effect of calcium chelator added before freezing on sperm Ca 2+ concentration, B is the effect of adding calcium chelator after freezing and thawing on the Ca 2+ concentration; CT represents the optimized diluent C (control), CGA represents the optimized diluent C with 100 µM CGA added, CGA+EGTA represents the optimized diluent C with 100 µM CGA and 6 µM EGTA-AM added, and CGA+BAPTA represents the optimized diluent C with 100 µM CGA and 6 µM BAPTA-AM added; different letters indicate significant differences ( P<0.05 ).

[0027] Fig.10 Adding calcium chelator to diluent C before freezing has an effect on capacitation-related genes Catsper1 , Catsper2 and PKACT represents the optimized diluent C (control), CGA represents the optimized diluent C with 100 µM CGA added, CGA+EGTA represents the optimized diluent C with 100 µM CGA and 6 µM EGTA-AM added, and CGA+BAPTA represents the optimized diluent C with 100 µM CGA and 6 µM BAPTA-AM added; different letters represent significant differences ( P< 0.05 ). DETAILED DESCRIPTION

[0028] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0029] Example 1

[0030] 1. Test Materials

[0031] 1.1 Experimental animals

[0032] The experimental bulls were provided by the Agricultural Machinery Technology Promotion Station of Zhanjiang City, Guangdong Province. Ten 4-6-year-old Leiqiong cattle were selected, which were healthy and strong, with good physique and appearance and vigorous reproductive function.

[0033] 2. Test methods

[0034] 2.1 Preparation of diluent

[0035] 2.1.1 Formula of diluent C

[0036] Each 100 mL of diluent C contains: 0.2 g fructose, 2.2 g Tris-base, 1.2 g citric acid, 12 mL egg yolk, 9 mL glycerol, 2 mL OEP (sodium amino-dodecyl sulfate) and 200 µL of 2 mg / mL amikacin sulfate, and the balance is water.

[0037] 2.1.2 Dilution formula for control

[0038] (1) Dilution A

[0039] Each 100 mL of dilution solution A contains: 8.8 g sucrose, 0.85 g trisodium citrate, 20 mL egg yolk, 3 mL glycerol, 0.5 mL OEP, and the rest is ultrapure water.

[0040] (2) Dilution B

[0041] Each 100 mL of dilution solution B contains: 1.35 g glucose, 3.5 g Tris base, 1.0 g citric acid, 0.65 g EDTA-2Na, 18 mL egg yolk, 6 ml glycerol, and the balance is ultrapure water.

[0042] 2.1.3 Screening of dilution component concentrations

[0043] (1) Screening of carbohydrate substances: Sugar in the diluent provides energy for sperm metabolism. Glucose, fructose, lactose, sucrose and no sugar (control) were replaced in the basic diluent to explore the effect on the cryopreservation of bovine semen.

[0044] (2) Screening of yolk concentration: Yolk protects the sperm cell membrane and provides energy. Different concentrations of yolk (0%-25%) were added to the basal diluent to explore the improvement effect on frozen bovine semen. No yolk was added (i.e., yolk concentration was 0%) as a control.

[0045] (3) Screening of glycerol concentration: Glycerol acts as an osmotic protective agent that reduces the formation of ice crystals, alleviates the damage of free radicals to cells, and changes the permeability of cell membranes. In order to further improve the effect of semen cryopreservation, the glycerol concentration was screened and adjusted, and 0%-12% was added respectively. The control was no glycerol added (i.e., 0% glycerol concentration).

[0046] (4) Screening of OEP concentration: OEP is a surfactant that protects the sperm plasma membrane. Different concentrations of OEP (0-2%) were added to the basal diluent to explore the effect on sperm motility after semen freezing and thawing. No OEP was added (i.e., OEP concentration was 0%) as a control.

[0047] 2.1.4 Screening of antioxidants

[0048] Effects of adding antioxidant chlorogenic acid (CGA) or caffeic acid (CA) to the basic diluent after composition optimization in 2.1.3 (i.e., each 100 mL of optimized diluent C contains: 0.2 g fructose, 2.2 g Tris-base, 1.2 g citric acid, 20% egg yolk, 6% glycerol, 1% OEP, and 200 µL of 2 mg / mL amikacin sulfate, and the balance is water) on the quality and fertilization ability of frozen bovine semen. CGA or CA was added to a final concentration of 0 μM, 50 μM, 100 μM, 200 μM, and 400 μM.

[0049] 2.1.5 Screening of calcium chelators

[0050] 6 μM calcium chelator (EGTA-AM or BAPTA-AM) was added to the optimized diluent C, and the effects of the added calcium chelator on the quality and fertilization ability of bovine frozen semen were determined.

[0051] Furthermore, after screening out the optimal CGA concentration, the optimal concentration of CGA and 6 μM calcium chelator (EGTA-AM or BAPTA-AM) were added to the optimized diluent C at the same time to determine the effect of the diluent with the addition of calcium chelator on the quality and fertilization ability of bovine frozen semen.

[0052] 2.2 Semen collection and processing

[0053] Semen was collected using a pseudo-vagina method and stored in a 33°C thermos cup and brought to the laboratory for testing within 1 hour. Semen motility was above 0.80 and density was 10×10 8 Samples with SP / mL or above are used for testing. The technical roadmap for the bovine semen cryopreservation test is as follows: Figure 1 shown.

[0054] 2.2.1 Dilution and storage of bovine semen

[0055] Place the diluent and bovine semen in a 5℃ refrigerator in advance for 2-3 hours to cool and balance, and then slowly cool to 5℃. Then, mix the diluent with the bovine semen in a volume ratio of 1:1, dilute the bovine semen, and absorb it into a 0.25mL frozen capillary after balancing for 10 minutes, seal it, and place it on a pre-prepared liquid nitrogen floating boat (make a floating boat by pouring liquid nitrogen into 2 / 3 of the foam box). After liquid nitrogen fumigation and balance for 15 minutes, store it in liquid nitrogen. After one week of cryopreservation, thaw the bovine semen and evaluate its quality and in vitro fertilization ability.

[0056] 2.3 Quality assessment of frozen bovine semen

[0057] 2.3.1 Sperm motility and vitality test

[0058] After the frozen bovine semen was thawed (thawed in warm water at 37°C for 10 s), 10 μL of semen sample was dropped on a glass slide heated to 37°C, and covered with a cover glass to evaluate sperm motility and vitality under a 200× microscope. At least 3 different fields of view were observed each time, and the vitality was evaluated by visual observation under a microscope. Different indexes (100%, 75%, 50%, 25%, 0%) were assigned to sperm of different motility types. The specific calculation method of sperm motility is shown in the formula: sperm motility = (A×100%+B×75%+C×50%+D×25%+E×0%) / 100;

[0059] In the formula, A is the number of sperm moving forward in a straight line vigorously, B is the number of sperm moving forward slowly, C is the number of sperm moving in a circle in place, D is the number of sperm moving in place by tail rotation, and E is the number of non-moving or dead sperm. Repeat the experiment 5 times.

[0060] 2.3.2 Sperm plasma membrane integrity test

[0061] The integrity of the plasma membrane was determined by the hypotonic swelling method. A hypotonic solution containing 9 mg / mL fructose and 4.9 mg / mL sodium citrate dihydrate was prepared. Then, the sperm was mixed with the hypotonic solution at a volume ratio of 1:9 and placed in a constant temperature water bath at 37°C for 30 minutes. Each sample was mixed with eosin staining solution at a volume ratio of 1:1 and smeared. After natural air drying, the sperm tail was observed under a 400× microscope, and the tail rate was counted. More than 200 sperm were observed each time. The test was repeated 5 times.

[0062] 2.3.3 Sperm acrosome integrity detection

[0063] The Coomassie Brilliant Blue G250 staining method was used for determination: 100 μL of semen was taken, centrifuged at 2000 r / min for 3 min, the supernatant was discarded, and 100 μL of DPBS was added to wash twice. The washed sperm was mixed with 1 mL of 4% paraformaldehyde aqueous solution and allowed to stand for 10 min, then centrifuged at 2000 r / min for 10 min, the supernatant was discarded, and it was washed once with 1 mL of DPBS, and finally resuspended with 20 μL of DPBS, smeared, and soaked in Coomassie Brilliant Blue dye solution for 30 min, rinsed and dried naturally, and the acrosome staining was observed under a 400× microscope, and more than 200 sperm were observed each time. If the acrosome region is blue and not damaged, the acrosome is intact, and if the acrosome region is lightly stained or damaged, the acrosome is incomplete. Repeat the test 5 times.

[0064] 2.3.4 Sperm survival rate test

[0065] Using the SYBR-14 / PI fluorescent staining method, take 200 μL of semen, centrifuge at 2000 r / min for 3 min, discard the supernatant and wash once with DPBS, then resuspend with 1 mL DPBS, add 5 μL of 50-fold diluted SYBR-14 fluorescent dye, incubate in a 37℃ incubator for 10 min, add 5 μL of PI fluorescent dye and incubate at 37℃ for another 10 min, finally perform flow cytometry and observe using a fluorescence microscope. Sperm heads marked with red are dead sperm, and heads marked only with green are live sperm. Repeat the experiment 5 times.

[0066] 2.4 Assessment of IVF capacity

[0067] The ovaries of healthy cattle (from Leichong cattle or hybrid cattle) were collected from the slaughterhouse, washed with saline several times, and then placed in saline containing double antibiotics (penicillin-streptomycin), moved into a thermos cup and maintained at 30℃~33℃, and brought back to the laboratory within 2 hours. A disposable syringe was then used to extract the fluid from the follicles with a diameter of 2 to 8 mm on the surface of the ovary, and the mixture was placed in a centrifuge tube for 10 min. The supernatant was removed and transferred to a culture dish. The cumulus-oocyte complexes (COCs) with more than three layers of cumulus cells were carefully selected under a stereomicroscope, and the COCs were washed with maturation medium (TCM199 + 0.6 mM cysteine ​​+ 0.02 AU follicle-stimulating hormone (FSH) + 5% fetal bovine serum (FBS) + 50 μg / mL gentamicin). 500 μL of culture medium (i.e., maturation medium) was added to each well of a four-well culture dish to culture 50 COCs. The culture medium was covered with mineral oil and cultured in a humidified incubator at 38.5°C and containing 5% CO2 for 21 h. After maturation culture, bovine frozen semen was used for in vitro fertilization (IVF). The bovine frozen semen was thawed in warm water at 37°C for 10 s and washed with in vitro fertilization medium (IVF100) by centrifugation at 500×g for 5 min. The supernatant was removed and IVF100 was added to adjust the sperm concentration to 1×10 6 Sperm / mL, the mature cultured oocytes were added to a humidified incubator at 38.5℃ and 5% CO2 for co-culture for 5 h. After co-culture, the oocytes were repeatedly washed more than 3 times in an in vitro culture medium (TCM199+5% FBS+5 μg / mL insulin+50 μg / mL gentamicin) to remove residual cumulus cells and sperm, and cultured in a three-gas incubator at 38.5℃, 100% humidity, 5% CO2, 5% O2, and 90% N2 for 7 days. Half of the culture medium was replaced on the 3rd and 5th days of culture, and the blastocyst formation rate was detected on the 7th day. Five repeated experiments were performed.

[0068] 2.5 Evaluation of antioxidant properties of frozen-thawed bovine semen

[0069] 2.5.1 ROS content detection

[0070] After the frozen sperm tubes were thawed in a 37°C water bath, 10 6 -10 7 The sperm cells were washed 1-2 times with DPBS, and DCFH-DA staining solution was added. The cells were incubated at 37°C in the dark for 30 min, and the fluorescence intensity was detected using the FITC channel of the flow cytometer. The experiment was repeated 5 times.

[0071] 2.5.2 Antioxidant enzyme activity detection

[0072] CAT enzyme content detection: The CAT enzyme content in sperm was processed according to the instructions of the catalase (CAT) test kit, and a standard curve was drawn. Finally, the absorbance at 405 nm was detected by an enzyme marker. The test was repeated 5 times.

[0073] T-SOD enzyme content detection: According to the instructions of the T-SOD kit, the sample was processed and a standard curve was drawn. Finally, the absorbance at 450 nm was detected by an ELISA reader. The test was repeated 5 times.

[0074] GSH-PX enzyme content detection: The GSH-PX enzyme content in sperm was processed according to the instructions of the glutathione peroxidase (GSH-PX) test kit, and a standard curve was drawn. Finally, the absorbance at 412 nm was measured after standing at room temperature for 5 minutes. The test was repeated 5 times.

[0075] 2.5.3 MDA content detection

[0076] The MDA content in sperm was processed according to the instructions of the malondialdehyde (MDA) test kit and a standard curve was drawn. Finally, 0.25 mL was taken and the absorbance at 532 nm was detected by an enzyme marker. The test was repeated 5 times.

[0077] 2.5.4 Mitochondrial membrane potential (MMP) detection

[0078] After the frozen sperm tubes were thawed in a 37°C water bath, 10 6 -10 7 Sperm cells were washed 1-2 times with DPBS and mixed with JC-1 staining solution. After incubation at 37°C in the dark for 30 min, they were washed 1-2 times with DPBS. Finally, data were obtained using the FITC / PE channel of the flow cytometer. The size of MMP was indicated by the ratio of red fluorescence to green fluorescence. The experiment was repeated 5 times.

[0079] 2.6 Sperm Ca 2+ Detection of concentration and capacitation genes

[0080] 2.6.1 Sperm Ca 2+ Concentration detection

[0081] Before sperm freezing and after sperm thawing, 100 μM CGA and 6 μM EGTA or 6 μM BAPTA were added to diluent C at the same time. Sperm cells were collected according to the instructions of the calcium ion detection kit and washed twice with DPBS. 1 mL Fluo-4 staining solution was added and resuspended into a single cell suspension. Incubated at 37°C in the dark for 30 min. After incubation, flow cytometry was performed directly. The flow cytometer had an excitation wavelength of 488 nm and an emission wavelength of 525 nm to detect the intensity of fluorescence. The experiment was repeated 5 times.

[0082] 2.6.2 Sperm capacitation gene testing

[0083] The RNA in semen was extracted using a total semen RNA kit. First, BufferSP and proteinase K were added to the frozen-thawed semen, mixed and placed in a 56°C water bath for 5-10 min. Next, the mixture was cleaned up by adding Buffer L9 and centrifuging. Then, the supernatant was transferred to a centrifuge tube containing anhydrous ethanol, and the mixture was transferred to a nucleic acid purification column for purification. Finally, the eluted RNA concentration and OD value were measured by a UV spectrophotometer and stored at -80°C for future use.

[0084] The extracted cell RNA was reverse transcribed using the Novozyme HiScript III RT SuperMix for qPCR kit to obtain cDNA samples. The specific steps are to add 4 μL 4×gDNA wiperMix, 1 μg template RNA and RNase-free ddH2O to an RNase-free centrifuge tube to make up to a total system of 16 μL, gently mix and incubate at 42°C in a PCR instrument for 2 min to obtain a reaction solution. Then, 5×HiScript III qRT SuperMix was directly added to the reaction solution, gently mixed with a pipette, and reverse transcription reaction was performed in a PCR instrument. The reaction conditions were 37°C for 15 min and 85°C for 5 s. Finally, the product was stored in a -20°C refrigerator for use. The experiment was repeated 5 times.

[0085] Fluorescence quantitative PCR detection of capacitation gene expression:

[0086] The relative expression of capacitation genes was detected by dye method combined with qRT-PCR technology. Fluorescence quantitative primers were designed according to the CDs region sequences of the genes to be screened provided on the NCBI website, and primer design was performed using primer 5.0 software. During the experiment, GAPDH was selected as the internal reference, and the corresponding quantitative system was set up. The PCR reaction conditions were set as: 95 ℃ 30 s; 95 ℃ 10 s, 60 ℃ 30 s, 40 cycles. Each sample was repeated 3 times to ensure the reliability of the experimental results. Fluorescence quantitative primers and PCR reaction systems are shown in Tables 1 and 2, respectively.

[0087] Table 1 Fluorescence quantitative primers

[0088]

[0089] Table 2 Real-time fluorescence quantitative PCR reaction system

[0090]

[0091] 2.7 Data Statistics and Analysis

[0092] GraphPad 8.0 was used to analyze the data. SPSS 26.0 was used to perform analysis of variance (ANOVA) on all data according to LSD (Least significance difference) to test the significance of the results, and Tukey's method was used for multiple comparisons of each group of data. Data are expressed as mean ± standard error (mean ± SEM). Different letters indicate significant differences ( P <0.05, the same letters indicate no significant difference ( P >0.05).

[0093] 3. Results and Analysis

[0094] 3.1 Effects of different diluents and cryopreservation temperatures on bovine sperm motility and fertilization ability

[0095] Storage temperature is also one of the most critical factors in sperm preservation. Cryopreservation can inhibit sperm activity, reduce energy consumption and metabolic activity, and temporarily keep life in a static state, so as to achieve the purpose of extending the storage time of sperm. Semen cryo-diluent can protect sperm during the freezing process, and different diluent formulas have different effects on sperm protection. Carbohydrates are essential as energy supply substances for semen cryo-diluents, which can supplement the consumption caused by physiological activities such as sperm glycolysis and oxidative phosphorylation, and extend the storage time of sperm. Egg yolk is also crucial for sperm to avoid freezing damage. Studies have shown that lecithin and unsaturated fatty acids in egg yolk can reduce the effects of free radicals, stabilize cell membranes, promote lipoprotein synthesis, and improve sperm metabolism. Glycerol and OEP, as permeability protectants, reduce the formation of ice crystals, reduce the damage of free radicals to cells, and change the permeability of cell membranes. This experiment explored the cryopreservation temperature, diluent, sugar and antifreeze agent to screen out the most effective combination to improve the effect of cryopreservation of bovine semen.

[0096] The results are as follows Figure 2 As shown in Figure A, diluent C improved the motility of bovine sperm after freeze-thaw compared with commercial diluents (A and B) (after the semen of three bulls was frozen and stored for one week with diluents A, B, and C, the average total motility was 15.2%, 58.9%, and 67.6%, respectively). Figure 2 The effects of freezing at different temperatures for one week are shown in Table 3. The order of preservation effects from best to worst is -196℃ (liquid nitrogen) > -80℃ > -30℃. As shown in Table 3, the fertilization rate and blastocyst formation rate of bovine semen cryopreserved with diluent C under different freezing temperature conditions for IVF are the best at -196℃ (liquid nitrogen) ( P<0.05 ).

[0097] Table 3 Effects of cryopreservation at different temperatures on in vitro fertilization rate and blastocyst formation rate in cattle

[0098]

[0099] 3.2 Effects of sugars and different concentrations of egg yolk, glycerol, and OEP on bovine sperm motility

[0100] The results are as follows Figure 3 As shown, the sugar screening test showed that glucose, fructose, and sucrose all significantly increased ( P<0.05 ) frozen-thawed bovine sperm motility, fructose was the best ( Figure 3 The results of the effects of different concentrations of egg yolk, glycerol and OEP on sperm motility showed that the optimal concentrations were 20% ( Figure 3 B in), 6% ( Figure 3 C) and 1% ( Figure 3 D in.

[0101] 3.3 Effects of CGA and CA on cryopreservation of bovine semen

[0102] During the cryopreservation of semen, the drastic changes in the internal and external environment of sperm cells cause excessive production of ROS. ROS carry unpaired electrons, which can cause lipid peroxidation in the sperm plasma membrane, reduce axonemal protein phosphorylation, damage intracellular tissues, and reduce sperm motility and motility. In addition, the activity of antioxidant enzymes decreases under freezing conditions, causing the ROS clearance rate to be lower than the production rate, resulting in excessive accumulation of ROS in sperm cells. Adding antioxidants can reduce the production of excessive ROS, alleviate oxidative stress damage, and improve the quality of frozen semen. In this experiment, plant extracts CGA and CA were selected as antioxidants to determine the protective effect of adding these two to semen freezing diluent on the quality of bovine semen.

[0103] 3.3.1 Effects of CGA and CA on the quality and fertilization ability of frozen-thawed bovine semen

[0104] CGA and CA at final concentrations of 0, 50, 100, 200, and 400 μM were added to the above-mentioned preferred cryopreservation diluent (optimized diluent C), and the effects of different concentrations of CGA and CA on the quality and fertilization ability of bovine semen after freezing and thawing were determined. Figure 4 and Figure 5 As shown in Figure 2, compared with the control group (CT), 100 μM CGA and 100 μM CA significantly increased sperm motility, viability, plasma membrane integrity, and survival rate ( P<0.05 ), but there was no significant difference in acrosome integrity compared with the other groups ( P>0.05). The results of in vitro fertilization are shown in Table 4. The fertilization rate and blastocyst formation rate of the 100 μM CGA and 100 μM CA groups were significantly higher than those of the control group ( P <0.05).

[0105] Table 4 Effects of CGA and CA on fertilization rate and blastocyst formation rate after bovine frozen-thawed sperm in vitro fertilization (IVF)

[0106]

[0107] Note: Fresh means fresh bovine semen that has not been cryopreserved, Control means cryopreserved in optimized diluent C, CGA 100 μM means cryopreserved in diluent C supplemented with 100 μM CGA, CA 100 μM means cryopreserved in diluent C supplemented with 100 μM CA.

[0108] 3.3.2 Effects of CGA and CA on the antioxidant properties of frozen-thawed bull sperm

[0109] like Figure 6 As shown in Figure 2, 100 μM CGA and 100 μM CA significantly increased the activities of CAT, SOD, and GSH-PX enzymes in bovine sperm compared with the control group (CT). P <0.05), there was no significant difference between 100 μM CGA and 100 μM CA ( P >0.05). Effects of 100 μM CGA and 100 μM CA on MDA, ROS content and MMP in frozen-thawed bovine sperm Figure 7 As shown in Figure 2, 100 μM CGA and 100 μM CA could significantly reduce the MDA in bovine sperm compared with the control group. Figure 7 A) and ROS ( Figure 7 B) content ( P <0.05, and can significantly increase the level of MMP in bovine sperm ( P <0.05), and the effect of 100 μM CGA was superior to that of 100 μM CA.

[0110] 3.4 Effect of calcium chelators on cryopreservation of bovine semen

[0111] 3.4.1 Effects of calcium chelators EGTA and BAPTA on the quality and fertilization ability of frozen-thawed bovine semen

[0112] EGTA-AM and BAPTA-AM are both cell-permeable calcium chelators that can act inside cells to regulate intracellular Ca 2+EGTA-AM or BAPTA-AM was added to diluent C to determine the quality and fertilization ability of frozen-thawed bovine semen. Figure 8 As shown in Table 5, EGTA and BAPTA can significantly improve sperm motility, viability, plasma membrane integrity, survival rate, fertilization rate and blastocyst formation rate after thawing compared with the control group (CT / control) ( P <0.05).

[0113] Table 5 Effects of calcium chelators on fertilization rate and blastocyst formation rate after frozen-thawed sperm in vitro fertilization (IVF)

[0114]

[0115] Note: Control means cryopreserved in optimized diluent C (control), EGTA means cryopreserved in diluent C supplemented with 6 μM EGTA-AM, and BAPTA means cryopreserved in diluent C supplemented with 6 μM BAPTA-AM.

[0116] 3.4.2 Effects of calcium chelators EGTA and BAPTA on Ca2+ in frozen-thawed bovine sperm 2+ Effect of concentration

[0117] The results are as follows Fig. 9 Shown: Addition of CGA during freezing resulted in a higher post-thaw Ca 2+ The fluorescence signal increased significantly ( P <0.05, and 6 μM calcium chelator was added to 100 μM CGA ( Fig. 9 A) will briefly cause Ca 2+ Fluorescence signal increased ( P <0.05). After 1 h and 3 h of incubation, the calcium chelator group Ca 2+ The fluorescence signal was significantly lower than that of the CGA group ( P <0.05). Add calcium chelator after thawing ( Fig. 9 (B) In the calcium chelator group, Ca 2+ The fluorescence signal was significantly higher than that of the CGA group ( P <0.05), Ca in CGA+EGTA group at 3 h 2+ There was no significant difference in fluorescence signal between the CGA group and the CGA+BAPTA group ( P >0.05).

[0118] 3.4.3 Effects of calcium chelators EGTA-AM and BAPTA-AM on capacitation gene expression in frozen-thawed bovine sperm

[0119] To detect the relative mRNA expression of sperm capacitation-related genes, bovine semen was frozen and stored for 1 week with diluent C (CT, control), diluent C with 100 µM CGA (CGA), diluent C with 100 µM CGA and 6 µM EGTA-AM (CGA+EGTA), and diluent C with 100 µM CGA and 6 µM BAPTA-AM (CGA+BAPTA), and then thawed for quantitative PCR detection. The results are shown in Figure 2. Fig.10 Shown: CGA+EGTA group Catsper1 There was no significant difference in the relative expression of gene mRNA compared with the control group (CT) ( P >0.05, but the relative mRNA expression levels in the CGA group and CGA+BAPTA group were significantly decreased compared with the control group ( P <0.05). And CGA group Catsper2 and PKA There was no significant difference in the relative mRNA expression level between the control group and the control group ( P >0.05, while the CGA+EGTA group and CGA+BAPTA group were significantly lower than the control group ( P <0.05).

[0120] The ion channels on the sperm plasma membrane are an important component, which are divided into voltage-gated channels and second messenger-gated channels, such as Na + , K + , Ca 2+ These ion channels are responsible for regulating membrane potential, osmotic pressure balance, intracellular pH balance, and ion concentration. The series of events that occur in sperm cells are largely dependent on Ca 2+ Channel, Ca 2+ It will affect sperm capacitation, acrosome reaction, etc. Studies have shown that Ca 2+ The disruption of homeostasis will lead to reduced sperm motility, reduced ATP production, and reduced fertility. During the cryopreservation process, the permeability and fluidity of the sperm membrane change under low temperature stimulation, causing a large amount of Ca 2+ Entering the sperm, leading to premature capacitation and thus affecting the fertilization ability of semen after freezing-thawing. 2+ In order to inhibit sperm capacitation during thawing, Ca 2+ The CatSper channel is a highly evolutionarily conserved calcium ion channel that exists only in male gametes and regulates Ca 2+ Entering the intracellular environment helps to achieve super-activated sperm motility and fertilization ability, which was detected in frozen-thawed bovine sperm in existing studies Catsper1 and 2 mRNA expression levels were significantly downregulated, but in this experiment, the CGA group and the calcium chelator group Catsper1 and Catsper2 The mRNA levels were all decreased to a certain extent, which indicated that Ca 2+ Influx does not completely depend on CatSper channels. PKA Gene mRNA expression levels were measured and it was found that calcium chelators could block the cAMP / PKA pathway and inhibit sperm capacitation.

[0121] In summary, the optimal freezing temperature for bovine semen is -196℃, and the best diluent formula is diluent C (Tris base-citric acid-egg yolk). Adding 100 μM CGA and 100 μM CA as antioxidants to the freezing diluent can relieve oxidative stress and protect sperm. Adding 6 μM calcium chelators (EGTA-AM, BAPTA-AM) while adding 100 μM CGA can improve semen quality and fertilization ability, inhibit premature sperm capacitation, prolong in vitro survival time, and improve its application value in artificial insemination.

Claims

1. A diluent for improving the quality of frozen bovine semen, characterized in that: Composition: 2 g / L fructose, 22 g / L Tris-base, 12 g / L citric acid, 20% egg yolk, 6% glycerol, 1% amino-sodium dodecyl sulfate, 0.004 g / L amikacin sulfate, 100 µM chlorogenic acid, 6 µM calcium chelator, and the balance is water; The cattle are Leiqiong cattle; The calcium chelating agent is EGTA or BAPTA.

2. A method for freezing and preserving bovine semen, characterized in that: The steps include: S1. The diluent according to claim 1 and the bovine semen were balanced for 2-3 h and slowly cooled to 5°C; S2. Dilute the bovine semen by mixing the diluent with the bovine semen in a volume ratio of 1:1 and equilibrate for 10 minutes; S3. After the balance is completed, suck it into the frozen straw and seal it; S4. Place the frozen vials containing bovine semen at -30°C or below for 15 minutes and then store them in liquid nitrogen.

3. The method according to claim 2, characterized in that The freezing capillary tube is a freezing capillary tube with a specification of 0.25 mL.

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