An in vitro storage method for improving the motility of sperm in non-human primates

Abstract

The present invention discloses an in vitro preservation method for improving the motility of sperm of non-human primates, and belongs to the field of biotechnology. The semen of non-human primates is allowed to stand and liquefy at room temperature, and then the semen is diluted 20-30 times, and then melatonin with a concentration of 0.1uM-0.5uM is added to the semen, mixed and stored at 37°C. The semen of non-human primates is placed in vitro at 37°C for at least 3 hours, and its utilization value is still not reduced, and the sperm motility and sperm acrosome integrity can be continuously maintained, the storage time is extended, and the utilization rate under the current collection and use situation is guaranteed. The in vitro preservation method of semen of non-human primates provided by the present invention has a good application effect, is worthy of promotion, reduces the stimulation and damage to animals, and also reduces material and labor, which has a certain significance for the preservation of germplasm resources.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an in vitro preservation method for improving sperm motility of non-human primates. Background Art

[0002] The most common non-human primates, crab-eating macaques and rhesus macaques, are highly similar to humans in genetics, physiology and anatomy, and are widely used in life science and medical research. In particular, crab-eating macaques are not restricted by the breeding season and have a menstrual cycle similar to that of humans in reproductive physiology. They are often used in the production of transgenic animal models and play an extremely important role in life science and medical research.

[0003] Compared with rhesus monkeys, sperm of non-human primates, especially crab-eating macaques, is more fragile. After separation from the body, as the storage time in vitro increases, it is affected by factors such as oxidative stress and changes in the water phase inside and outside the cells, which will increase the level of sperm cell apoptosis. In addition to the gradual consumption of its own nutrients, reactive oxygen free radicals (ROS) in the sperm will gradually accumulate. Excessive ROS will oxidatively damage the structure and function of the sperm, thereby leading to a decrease in semen quality, a slowdown in motility, and a weakening of sperm motility, seriously affecting its quality.

[0004] With the establishment and widespread use of transgenic and gene knockout animal models, the application of cynomolgus macaques has become more extensive. Whether the in vitro storage of their semen can maintain a certain vitality is becoming more and more important, because sperm vitality will seriously affect in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), greatly reducing the number of fertilized eggs. Efficient in vitro storage of non-human primate semen is an important technical prerequisite for the promotion and application of assisted reproductive technology.

[0005] Therefore, the research and development of in vitro preservation technology for non-human primates has important value and practical significance for improving sperm motility, and can also provide reference for the in vitro preservation of sperm of other non-human primates. In addition, it is also of great significance for the preservation of germplasm resources of non-human primates, avoiding the loss of manpower, material resources and resources caused by the collection of sperm from a second male monkey due to improper in vitro preservation.

[0006] The storage method of non-human primate semen has a great impact on the quality of semen. The main storage methods include long-term freezing and short-term storage at 37°C. Since non-human primate sperm is relatively fragile, sperm is very sensitive to temperature and freezing conditions during the freezing process. The freezing and thawing process forces sperm to suffer osmotic damage and oxidative stress. The quality of sperm seriously declines after recovery. The most intuitive manifestation is a significant decrease in motility, which leads to an extremely low subsequent fertilization rate.

[0007] In conclusion, excessively high temperatures or low environmental humidity may have a negative impact on sperm survival. The most suitable survival temperature for human sperm is 35 to 37°C. At this temperature, sperm metabolism and motility are the strongest, and the fertilization process can be completed better. Temperature is an important factor affecting sperm motility and survival time. Many studies have shown that simulating the body temperature (37°C), within the first 4 hours after semen is taken out, 37°C (directly placed in a 37°C incubator) is more conducive to protecting sperm motility than 26°C (normal temperature). In this regard, monkeys are similar to humans, but the volume of monkey semen is far less than that of human semen. Therefore, the in vitro storage of monkey semen currently requires the addition of conventional diluent HTF (human tubal fluid). However, when the conventional diluent is stored in vitro for more than 1.5 hours, the change in the environment will cause lipid peroxidation to produce reactive oxygen species (ROS), thereby causing a decrease in sperm motility and acrosome reaction disorders. There is currently no other diluent formula suitable for in vitro storage of monkey sperm.

[0008] Up to now, most assisted reproductive technologies use a method of preserving non-human primate semen by using it immediately after collection and storing it at 37°C. This method, after adding the conventional diluent HTF, has a storage time of more than 1.5 hours. Due to the decrease in sperm motility, it will affect the subsequent in vitro fertilization rate of oocytes and the quality of embryos, thereby reducing the pregnancy rate, resulting in a waste of resources. If a method can be found to extend the utilization time of semen by using it immediately after collection and storing it at 37°C, that is, a method to improve the quality of in vitro stored semen can be found, it will be helpful for assisted reproductive technology research, and at the same time reduce the number of semen collections, reduce the stimulation and damage to animals, and reduce material and labor. Summary of the invention

[0009] To solve the above problems, the present invention provides an in vitro storage method for improving the motility of sperm of non-human primates, by adding a certain amount of melatonin, the motility of sperm within 3 hours and the integrity of sperm acrosome are improved.

[0010] The present invention is achieved through the following technical solutions: an in vitro storage method for improving the motility of sperm of non-human primates, through the following operations:

[0011] The semen of non-human primates was allowed to stand at room temperature for 15-45 minutes to liquefy, and then diluted 20-30 times with normal saline. Then, melatonin at a concentration of 0.1uM-0.5uM was added to the semen so that the volume ratio of semen to melatonin was 300uL:100-200uL. After mixing, it was stored at 37°C.

[0012] The saline solution was replaced with HTF diluent.

[0013] The effects and advantages of the present invention are as follows:

[0014] The present invention provides a simple and effective in vitro preservation method. Compared with the prior art, the semen of non-human primates can be placed in vitro at 37°C for at least 3 hours without reducing its utilization value, and can continuously maintain sperm motility and sperm acrosome integrity, thereby extending the storage time and ensuring the utilization rate under the condition of immediate collection and immediate use. The in vitro preservation method of semen of non-human primates provided by the present invention has a good application effect and is worthy of promotion.

[0015] Furthermore, the method of the present invention can also significantly reduce the number of times semen is collected, reduce stimulation and damage to animals, and reduce material and labor, which is of certain significance to the preservation of germplasm resources. At the same time, it also provides basic data for in vitro preservation research of semen of other precious and endangered primates, which is convenient to carry out in ordinary laboratories and has wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the effect of different melatonin concentrations on sperm acrosome integrity when stored in vitro for 1.5 hours;

[0017] Figure 2 Schematic diagram of the effect of different melatonin concentrations on sperm acrosome integrity when stored in vitro for 3 hours.

[0018] Among them, “*” indicates p﹤0.05 compared with the control group. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below with reference to examples, but the protection scope of the present invention is not limited to the contents described above. The reagents and methods used in the examples are conventional reagents and methods unless otherwise specified. Example

[0020] Experimental animals: Two healthy, fertile male cynomolgus monkeys (aged 8-10 years old) were obtained from the Medical Primate Research Center, Institute of Medical Biology, Chinese Academy of Medical Sciences. They were kept in single cages, fed three meals a day, and given some clean fresh fruits. They were kept under 12h / 12h light and dark conditions and room temperature of 16-26℃.

[0021] Main instruments and equipment: Nikon microscope (Japan); Sw-cj-2f purification workbench (Suzhou Purification), CO2 incubator purchased from Thermo, USA; EP tube purchased from Corning, USA; sperm counting slide purchased from Leica, Germany (20uM-4 Chamberslide); Pasteur pipette purchased from FALCON, USA; pipette purchased from EPPENDORF, Germany. Animal electrical stimulation semen collection instrument purchased from Chengdu Huazhi Kaiwu Technology Co., Ltd.; sperm analyzer purchased from HAMILTON, USA.

[0022] Reagents: Physiological saline was purchased from Kunming Nanjiang Pharmaceutical Co., Ltd., melatonin was purchased from Sigma, USA. Human tubal fluid (HTF) was purchased from SAGE, and FITC-labeled peanut agglutinin (FITC-PNA) was purchased from Xi'an Qiyue.

[0023] An in vitro storage method for improving the motility of sperm of non-human primates, comprising the following steps:

[0024] (1) Semen of cynomolgus monkeys was collected by conventional methods. The semen was allowed to stand at room temperature for 30 minutes to liquefy. The monkey semen was diluted 20 times with physiological saline, and 300uL was dispensed into 1.5mL EP tubes. 200uL of melatonin with concentrations of 0.1uM, 0.5uM, 1uM, 10uM, 100uM, and 200uM was added to each tube. Four tubes were added for each concentration, mixed well, and stored in a 37°C incubator. At the same time, four tubes were dispensed without melatonin after dilution as control group A, which was also stored in a 37°C incubator.

[0025] (2) Semen of cynomolgus monkeys was collected by conventional methods. The semen was allowed to stand at room temperature for 30 minutes to liquefy. The semen was diluted 20 times with HTF, a common diluent for sperm of cynomolgus monkeys. 300uL of semen was dispensed into 1.5mL EP tubes. 200uL of melatonin with concentrations of 0.1uM, 0.5uM, 1uM, 10uM, 100uM, and 200uM was added to each tube. Four tubes were added for each concentration. The mixture was mixed and then stored in a 37°C incubator. At the same time, four tubes were dispensed with HTF diluent but without melatonin as control group B, which were also stored in a 37°C incubator.

[0026] The sperm motility test in this case includes sperm static characteristic indicators: total sperm density, active sperm density, active sperm percentage, forward-moving sperm density and forward-moving sperm percentage. Sperm motility grading indicators: fast-phase sperm count, medium-phase sperm count, slow-phase sperm count, stationary-phase sperm count, fast-phase sperm density and percentage, medium-phase sperm density and percentage, slow-phase sperm density and percentage, stationary-phase sperm density and percentage.

[0027] The World Health Organization divides sperm motility into three levels, namely progressive motility (PR), non-progressive motility (NP), and immotility (IM). The sperm motility index refers to the ratio of the number of sperm with progressive motility (PR) in the total number of sperm in semen, and is usually used as an important indicator for evaluating male reproductive capacity.

[0028] The South stipulates that the total sperm motility (PR+NP) ≥ 40% and the forward motility (PR) ≥ 32% are within the normal range.

[0029] Although there are some differences in the descriptions of semen indicators of non-human primates and humans, their meanings are the same. Both the percentage of forward-moving sperm in the static characteristic indicators and the percentage of fast phase density in the sperm motility grading indicators are over 40%, which is within the normal range.

[0030] 1. After 1.5 hours, take out 3uL of semen from each tube and put it into the sperm detection slide. Adjust the phase contrast ring of the sperm analyzer to the optimal position, and start to obtain sperm motility data after testing 10 fields of view for each sample.

[0031] At the same time, the sperm acrosome integrity test was performed: 10uL of semen smear was taken, dried in the air, anhydrous ethanol was added to cover the sample surface, fixed for 20 minutes, the fixative was carefully removed, and air dried again. 100μl 40 μg / ml FITC-labeled peanut agglutinin was covered on the smear, 37℃ away from light, incubated for 20 minutes, and then rinsed with PBS 5-8 times. Then, the acrosome fluorescence color was observed under a fluorescence microscope. Sperm with complete acrosomes were green, and sperm with incomplete acrosomes had no color or were light and uneven in color. Sperm with complete acrosomes were counted and the percentage of the total number of sperm was calculated. Three smears were made for each sample, and more than 200 sperm were observed in each smear, and the average of the three smears was taken.

[0032] Acrosome integrity rate (%) = number of sperm with intact acrosomes / total number of sperm × 100%.

[0033] The results obtained by saving method (1) are shown in Table 1:

[0034] Table 1

[0035] The results obtained by saving method (2) are shown in Table 2:

[0036] Table 2

[0037] As shown in Table 1 and Table 2, when the test results of the experimental group with melatonin added were compared with those of the control group after 1.5 hours of storage, the total sperm density, percentage of active sperm, and density and percentage of forward-moving sperm in the static characteristics of sperm were all improved (p﹤0.05); the number of fast-phase sperm, the number of medium-phase sperm, the number of slow-phase sperm, the density and percentage of fast phase, and the density and percentage of medium phase in the sperm motility grading index were also improved (p﹤0.05); while the number, density and percentage of static-phase sperm were significantly decreased (p﹤0.05). There was also a significant difference in sperm acrosome integrity between the two groups (p﹤0.05), see Figure 1 .

[0038] 2. At 3 hours, take out 3uL of semen from each tube and put it into the sperm test slide. Adjust the phase contrast ring of the sperm analyzer to the optimal position, and start acquiring data after testing 10 fields of view for each sample. At the same time, perform sperm acrosome integrity test, using the same method as above.

[0039] The results obtained by saving method (1) are shown in Table 3:

[0040] Table 3

[0041] As can be seen from Table 3, the total sperm density of 0.1uM and 0.5uM melatonin groups was significantly higher than that of control group A; the motile sperm density of 0.1uM and 0.5uM melatonin groups was significantly higher than that of control group A; the motile sperm percentage of 0.1uM and 0.5uM melatonin groups was significantly higher than that of control group A; the forward motile sperm density of 0.1uM and 0.5uM melatonin groups was significantly higher than that of control group A, and the 100uM group and 200uM group were significantly lower than those of control group A and 0.1uM group, and the forward motile sperm percentage of 0.1uM and 0.5uM melatonin groups was significantly higher than that of control group A (p﹤0.05); the fast phase density of 0.1uM and 0.5uM melatonin groups was significantly higher than that of control group A (p﹤0 .05); the fast phase density percentage of 0.1uM and 0.5uM melatonin groups was significantly higher than that of control group A (p﹤0.05); the medium phase density of 0.1uM melatonin group was significantly higher than that of control group A; there was no significant difference in the slow phase sperm density and percentage between each group and control group A; the stationary phase sperm density of 0.1uM and 0.5uM melatonin groups was significantly lower than that of control group A (p﹤0.05); the stationary phase density percentage of 0.1uM and 0.5uM groups was significantly lower than that of control group A (p﹤0.05); in addition, the fast phase density of 1uM group, 10uM group, 100uM group and 200uM group were lower than that of 0.1uM group and 0.5uM group (p﹤0.01 or p﹤0.05).

[0042] In conclusion, after 3 hours of storage, the total sperm density, active sperm density, active sperm percentage, forward motile sperm density and percentage in the static characteristics of sperm, the number of rapid phase sperm, the number of intermediate phase sperm, the density and percentage of rapid phase, and the density of intermediate phase in the sperm motility grading indicators were significantly improved compared with those of the control group A (p﹤0.01 or p﹤0.05). These results indicate that 0.1uM-0.5uM melatonin can significantly improve sperm motility; while the percentage of static phase sperm density was significantly reduced (p﹤0.05), indicating that 0.1uM-0.5uM melatonin can reduce the number of static sperm, increase the number of active sperm, and thus improve the motility of sperm.

[0043] from Figure 2 It can be seen that the sperm acrosome integrity in the 0.1uM and 0.5uM groups was higher than that in the 1uM, 10uM, 100uM and 200uM groups (p﹤0.05), indicating that adding 0.1uM-0.5uM melatonin has the best protection for the acrosome.

[0044] The results obtained by saving method (2) are shown in Table 4:

[0045] Table 4

[0046] As shown in Tables 3 and 4, when stored for 3 hours, there is no significant difference in sperm motility between diluting cynomolgus monkey semen with normal saline and diluting it with HTF. Normal saline is economical, so it can be used as the diluent under normal circumstances.

[0047] Example 2

[0048] The same as Example 1, except that the standing liquefaction time at room temperature was replaced with 15 minutes, the semen was diluted 30 times, and the volume ratio of semen to melatonin was 300uL:150uL.

[0049] Example 3

[0050] The same as Example 1, except that the standing liquefaction time at room temperature was replaced with 45 minutes, the semen was diluted 25 times, and the volume ratio of semen to melatonin was 300uL:100uL.

[0051] The test results of Example 2 and Example 3 were compared and recorded with Example 1, and the conclusions were the same as those of Example 1.

[0052] Therefore, the method of the present invention is simple and effective, and can significantly improve the in vitro sperm motility of non-human primates, thereby improving the quality of semen stored in vitro. The addition of melatonin at a concentration of 0.1-0.5uM can meet the sperm motility index requirement for at least 3 hours during the experiment of immediate collection and use (37°C).

Claims

1. An in vitro storage method for improving the motility of sperm of non-human primates, characterized in that After the following operations: The semen of non-human primates is liquefied at room temperature for 15-45 minutes, and then diluted 20-30 times with physiological saline, and then melatonin with a concentration of 0.1uM-0.5uM is added to the semen, so that the volume ratio of semen to melatonin is 300uL:100-200uL, and then mixed and stored at 37°C; The non-human primate is a cynomolgus monkey.

2. The in vitro storage method for improving the motility of sperm of non-human primates according to claim 1, characterized in that: The saline solution was replaced with HTF diluent.

Images