阅读说明：本技术 一种鸭精液冷冻保存稀释液 (Duck essence cryopreservation diluent ) 是由 照那木拉 李迎春 苏瑛 刘渊博 陈进军 陈志宝 康丹菊 于 2021-09-28 设计创作，主要内容包括：本发明公开了一种鸭精液冷冻保存稀释液,包括不含甘油的Tris-卵黄冷冻保存稀释液A和含甘油的Tris-卵黄冷冻保存稀释液B,每100mL Tris-卵黄冷冻保存稀释液B中含有：Tris-碱0.85g、乳糖0.35g、柠檬酸三钠二水0.5g、肌醇0.02g、鸭卵黄10-25g、甘油1.5-12mL和2mg/mL的硫酸阿米卡星200μL,余量为水。本发明的Tris-卵黄冷冻保存稀释液用于雷州黑鸭或其他品种鸭精子冷冻保存能大大提升保存的鸭精液品质,配制及操作简单易行,能有效的提高冻解精子活力,为雷州黑鸭乃至禽类精液冷冻保存、种质资源保存和利用等领域研究提供技术支撑。(The invention discloses a duck semen cryopreservation diluent, which comprises a Tris-yolk cryopreservation diluent A without glycerol and a Tris-yolk cryopreservation diluent B with glycerol, wherein each 100mL of the Tris-yolk cryopreservation diluent B contains: 0.85g of Tris-base, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 10-25g of duck egg yolk, 1.5-12mL of glycerol, 200 mu L of amikacin sulfate at 2mg/mL, and the balance of water. The Tris-yolk cryopreservation diluent is used for cryopreservation of sperms of black rey ducks or other varieties of ducks, can greatly improve the quality of the preserved duck sperms, is simple and easy to prepare and operate, can effectively improve the activity of the frozen sperms, and provides technical support for researches in the fields of cryopreservation of sperms, germ plasm resource preservation and utilization and the like of black rey ducks and even poultry.)

1. The diluent for cryopreservation of duck semen is characterized by comprising a Tris-yolk cryopreservation diluent A and a Tris-yolk cryopreservation diluent B, wherein each 100mL of the Tris-yolk cryopreservation diluent A contains: 0.85g of Tris-base, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 10-25g of duck egg yolk, 200 mu L of 2mg/mL amikacin sulfate and the balance of water; the Tris-yolk cryopreservation diluent B comprises per 100 mL: 0.85g of Tris-base, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 10-25g of duck egg yolk, 1.5-12mL of glycerol, 200 mu L of amikacin sulfate at 2mg/mL, and the balance of water.

2. The duck semen cryopreservation diluent as claimed in claim 1, wherein said Tris-yolk cryopreservation diluent a comprises per 100 mL: 0.85g of Tris-base, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 15-20g of duck egg yolk, 200 mu L of 2mg/mL amikacin sulfate and the balance of water.

3. The duck semen cryopreservation diluent as claimed in claim 1, wherein said Tris-yolk cryopreservation diluent B comprises per 100 mL: 0.85g of Tris-base, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 15-20g of duck egg yolk, 3-6mL of glycerol, 200 mu L of amikacin sulfate at 2mg/mL, and the balance of water.

4. A duck semen freezing and preserving method is characterized by comprising the following steps:

a. collecting duck semen, and preheating semen qualified in quality inspection to 35-37 deg.C for use;

b. diluting the semen by 4 times with Tris-yolk cryopreservation diluent A at 35-37 deg.C, loading into a centrifuge tube, placing the centrifuge tube into 35 deg.C water, and slowly cooling and balancing at 5 deg.C for 2-3 h; after equilibration to 5 ℃ the diluent was diluted in two portions with Tris-yolk cryopreservation diluent B: diluting semen and Tris-yolk cryopreservation diluent B according to the volume ratio of 2:1 for the first time, and then balancing for 5-10 min; diluting the semen and the Tris-yolk cryopreservation diluent B according to the volume ratio of 2:1 for the second time, and then balancing for 5-10 min; then subpackaging the diluent into freezing tubules and sealing, fumigating with liquid nitrogen for 10-15min, and storing in liquid nitrogen.

5. The method of claim 4, wherein the duck is a black Leizhou duck, a white duck or a sheldrake.

6. The method of claim 4, wherein the centrifuge tube is a 15mL gauge centrifuge tube.

7. The method of claim 4, wherein the freezing tubule is a 0.25mL freezing tubule.

8. The method of claim 4 wherein Tris-yolk cryopreservation diluent A and Tris-yolk cryopreservation diluent B are maintained at 5 ℃ prior to use.

Technical Field

The invention belongs to the technical field of animal breeding, and particularly relates to a diluent for freezing and preserving duck semen.

Background

The semen preservation of poultry is a key link in the artificial insemination technology, and the economic benefit in the production process is directly influenced by the level of the technology. The semen can be frozen and stored without being limited by time and space, the utilization rate of excellent breeding cock is improved, and the variety improvement is promoted. By using cryopreservation technology, sperm, ovum, and embryo can be placed in-196 deg.C environment to inhibit their metabolism, so as to be preserved, and thawed for use when necessary. At present, the fertility rate is extremely unstable due to low vitality of poultry frozen semen, and only a few countries establish poultry sperm freezers. The poultry semen cryopreservation research at home and abroad mainly focuses on chickens, and the research on ducks is less and related reports are less. The duck semen can be preserved for a long time by freezing, so that the semen is used, selected and hybridized to be improved, the time-space limitation of the semen use is broken, and excellent genes are rapidly transmitted. The Rigzhou black duck is widely distributed in a Rigzhou peninsula area, belongs to a local duck with black feather, egg and meat, and is one of local excellent varieties with the germplasm characteristics of delicious meat, high egg yield, strong disease resistance, high green shell rate, coarse feeding resistance and the like. The preservation of the black duck semen in the Riezhou by freezing has important significance for variety resource protection, and the preservation cost can be reduced. At present, the artificial insemination of ducks uses fresh semen or semen preserved for 2-3 days at 15 ℃, the fertilization rate is high, but the artificial insemination cannot be widely used due to time limitation. Compared with chickens and geese, the duck semen is sensitive to low-temperature stimulation, the sperm activity is reduced after the duck semen is frozen and stored, and further the artificial insemination cannot be popularized and utilized due to the problems of low fertilization rate and the like.

The semen quality of the breeding drakes is influenced by a plurality of factors. The male duck age, health condition, seasonal climate, semen collection mode and the like have great influence on semen quality, and further influence the laying rate and the hatching rate. Research on the aspects of the frozen diluent and the frozen preservation technology of the black duck semen in Rezhou is not blank.

Disclosure of Invention

In view of the above problems in the prior art, the present invention is directed to a diluent for cryopreservation of duck semen to improve the viability of frozen-thawed sperm, the integrity of plasma membrane and the survival time.

The first purpose of the invention is to provide a diluent for cryopreservation of duck semen.

The duck semen cryopreservation diluent comprises a Tris-yolk cryopreservation diluent A and a Tris-yolk cryopreservation diluent B, wherein each 100mL of the Tris-yolk cryopreservation diluent A contains: 0.85g of Tris-base, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 10-25g of duck egg yolk, 200 mu L of 2mg/mL amikacin sulfate and the balance of water; the Tris-yolk cryopreservation diluent B comprises per 100 mL: 0.85g of Tris-base, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 10-25g of duck egg yolk, 1.5-12mL of glycerol, 200 mu L of amikacin sulfate at 2mg/mL, and the balance of water.

Preferably, the Tris-yolk cryopreservation diluent A comprises per 100 mL: 0.85g of Tris-base, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 15-20g of duck egg yolk, 200 mu L of 2mg/mL amikacin sulfate and the balance of water.

Preferably, the Tris-yolk cryopreservation diluent B comprises per 100 mL: 0.85g of Tris-base, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 15-20g of duck egg yolk, 3-6mL of glycerol, 200 mu L of amikacin sulfate at 2mg/mL, and the balance of water.

In the diluent formula, Tris-alkali has high buffering capacity, so that the structure and physiological integrity of sperm cells are guaranteed, and the pH value of the diluent is maintained and adjusted; lactose provides energy for sperm metabolism; trisodium citrate dihydrate has good pH regulation and buffering performance; inositol has effects of promoting sperm metabolism and promoting development; the duck egg yolk has the effects of protecting sperm cell membrane and providing energy; the glycerol is used as a permeability protective agent, so that the formation of ice crystals is reduced, the damage of free radicals to cells is relieved, and the permeability of cell membranes is changed; amikacin sulfate has the function of sterilization, thereby prolonging the survival time of sperms.

The second purpose of the invention is to provide a duck semen freezing and preserving method, which comprises the following steps:

a. collecting duck semen, and preheating semen qualified in quality inspection to 35-37 deg.C for use;

b. diluting the semen by 4 times with Tris-yolk cryopreservation diluent A at 35-37 deg.C, loading into a centrifuge tube, placing the centrifuge tube into 35 deg.C water, and slowly cooling and balancing at 5 deg.C for 2-3 h; after equilibration to 5 ℃ the diluent was diluted in two portions with Tris-yolk cryopreservation diluent B: diluting semen and Tris-yolk cryopreservation diluent B according to the volume ratio of 2:1 for the first time, and then balancing for 5-10 min; diluting the semen and the Tris-yolk cryopreservation diluent B according to the volume ratio of 2:1 for the second time, and then balancing for 5-10 min; then subpackaging the diluent into freezing tubules and sealing, fumigating with liquid nitrogen for 10-15min, and storing in liquid nitrogen.

Preferably, the duck is a black Leizhou duck, a white duck or a sheldrake. The duck semen cryopreservation diluent and the duck semen cryopreservation method can also be applied to cryopreservation of the semen of other poultry.

The centrifuge tube is a 15mL centrifuge tube.

The freezing tubule is a 0.25mL freezing tubule.

The Tris-yolk cryopreservation diluent A and the Tris-yolk cryopreservation diluent B are kept at 5 ℃ before use.

1. The influence of the Tris-yolk cryopreservation diluent on the sperm motility of the frozen-thawed Rigzhou black duck

The invention develops and utilizes the freezing preservation of Tris-yolk freezing preservation diluent to carry out screening and proportion optimization on main components in the basic diluent.

The influence of lactose, fructose, glucose and sucrose with the same concentration added into the Tris-yolk diluent on the sperm motility of the frozen-thawed Rigzhou black duck is as follows: the sugar can provide energy for the sperm to live in vitro and plays an important role. The results of adding the same amount of different saccharides into the basic diluent show that the lactose has the best effect and has obvious difference (P is less than 0.05) with the fructose group, and the comparison of the glucose group and the sucrose group has no obvious difference. The lactose effect was significant and likely dependent on the base diluent.

Effect of different concentrations of duck egg yolk on sperm motility after thawing: compared with a control group (without yolk), the sperm activity (P <0.05) of the duck yolk group is obviously improved by adding the yolk group, and compared with a low-dose group and a high-dose group, the sperm activity of the frozen duck is obviously improved by 15 percent of the yolk group. The yolk has the protective effect on sperms as a nutrient supply and a surfactant, the protective effect can not be achieved at a low dose, and the osmotic pressure can be improved to influence the life span or the movement capacity of the sperms.

The effect of the anti-freezing agent glycerol on the sperm motility of the freeze-thaw Rigzhou black duck: the results show that the effect of 3-6% of glycerol is optimal, the excessive concentration of glycerol can increase osmotic pressure and generate toxic effect, and the dehydration effect is insufficient at low concentration to cause the formation of ice crystals in sperm cells, thereby damaging the cell structure. The application of freezing and preserving semen in artificial insemination is of great importance by optimizing the freezing diluent to improve the sperm motility and maintain the fertilization capability and the hatchability.

2. Influence of yolk and yolk-free diluent on semen quality of frozen-Jiezhou black duck

Tris-yolk diluent is commonly used for cryopreservation of mammalian semen, and the application report of the Tris-yolk diluent in freezing of poultry semen is less. The invention develops and utilizes a new Tris-yolk cryopreservation diluent, screens a yolk-free diluent (group II) with the optimal effect on poultry for verifying the effect of the diluent, performs a comparison experiment, and analyzes the influence on the sperm motility, the cytoplasmic membrane integrity rate and the teratogenesis rate of the frozen-thawed Rigzhou black duck. The results of the study showed that the frozen group significantly reduced sperm motility compared to fresh sperm motility, with sperm motility significantly higher in group I (Tris-egg yolk cryopreservation diluent) than in group II (P < 0.05). The different diluents have no obvious difference on the integrity and the aberration rate of the plasma membrane of the sperm of the freeze-thaw black duck among various freeze-preservation groups.

Influence of preservation in a 38 ℃ water bath for 6h after thawing on sperm motility: the results demonstrate that the Tris-yolk cryopreservation diluent effectively maintains sperm motility.

The experiment results verify that the quality of the preserved duck semen can be greatly improved when the Tris-yolk cryopreservation diluent is used for cryopreservation of the semen of the Rigzhou black duck or other varieties of ducks.

The invention has the beneficial effects that:

(1) the invention carries out screening test on the types of sugar, duck egg yolk concentration and glycerin concentration in Tris-egg yolk cryopreservation diluent (component I) to select the optimal suitable sugar, duck egg yolk concentration and glycerin concentration. The diluent prepared by the preferable scheme of the invention is used for freezing and storing the Rigzhou black duck, so that the activity of the frozen-thawed sperms of the Rigzhou black duck is improved.

(2) The invention also provides a refrigerated preservation diluent (component II) of the black rey duck semen in Rigzhou with low price and obvious benefit, which is compared with the refrigerated preservation diluent (component I) of Tris-yolk, and the result shows that the sperm motility and the cell membrane integrity are effectively maintained.

(3) The semen dilution and balancing method of the present invention has the advantages of: the diluted semen is put into a glass cup filled with warm water and is placed in a refrigerator with the temperature of 5 ℃ for slowly cooling and balancing for 2-3h, so as to avoid the increase of the death rate of the semen caused by the low-temperature stimulation of the semen caused by too fast cooling and the stress effect.

(4) After the temperature is balanced to 5 ℃, the sperm is diluted for two times by using the glycerol-containing diluent, so that the sperm is in a dormant state at a low temperature, the movement of the sperm is inhibited, the metabolism speed is reduced, the energy consumption is reduced, and the service life of the sperm is prolonged. Secondly, glycerol acts as a permeability protectant, reducing the formation of ice crystals, thereby protecting the sperm. However, premature addition or one-time addition of glycerol can cause rapid increase of osmotic pressure of the diluent, which leads to severe dehydration or death of the sperm, so that the survival rate of the sperm is improved by two times of dilution.

(5) When semen is frozen, a floating boat is made in a liquid-filled nitrogen foam box (-20-30 ℃), and a semen-filled freezing thin tube is placed on a boat for fumigation for 15min, so that the slow cooling can be kept to avoid the death of the semen caused by rapid cooling.

The Tris-yolk cryopreservation diluent developed by the invention is simple and easy to prepare and operate, can effectively improve the activity of frozen sperms, and provides technical support for research in the fields of cryopreservation of the sperm of black ducks and even poultry in Rizhou, preservation and utilization of germplasm resources and the like.

Drawings

FIG. 1 is a route chart of the semen cryopreservation experiment technique.

FIG. 2 shows the effect of different sugars (diluents: group I) on the sperm motility of freeze-thawed Rigzhou black ducks; significant differences (P <0.05) are indicated between different letters.

FIG. 3 shows the effect of different concentrations of duck egg yolk (diluent: group I) on the sperm motility of frozen-thawed Rigzhou black ducks; significant differences (P <0.05) are indicated between different letters.

FIG. 4 is a graph showing the effect of different concentrations of glycerol (diluent: group I) on the sperm motility of freeze-thawed Rigzhou black ducks; significant differences (P <0.05) are indicated between different letters.

FIG. 5 is a graph showing the effect of different dilutions on sperm motility of freeze-thaw ducks (Rezhou black duck, white duck, sheldrake); significant differences (P <0.05) are indicated between different letters.

FIG. 6 is a graph showing the effect of different dilutions on different indicators of semen quality of a frozen-thawed Rizhou black duck, with significant differences (P <0.05) between letters; FIG. A: sperm motility, panel B: survival, panel C: sperm cell membrane integrity, panel D: distortion rate.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1

1 materials and methods

1.1 materials and laboratory animals

The starting materials referred to in the examples are all from the common commercial products.

The black duck used in the test is provided by the constant-growing professional cooperative society of Guangdong university of oceans and the sloping field of Zhanjiang city. Selecting male ducks with the age of more than 12 months for semen collection training, and finally screening 13 black female ducks in Renzhou, 5 white ducks and 6 sheldrakes for testing.

1.2 methods

A semen cryopreservation technique roadmap is shown in FIG. 1.

1.2.1 preparation of Diluent

Tris-yolk cryopreservation dilutions (group I):

tris-yolk frozen stock diluent A, does not contain glycerin; taking 0.85g of Tris-alkali, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 10-25g of duck egg yolk and 200 mu L of 2mg/mL amikacin sulfate, and adding ultrapure water to supplement the solution to 100 mL.

Tris-yolk cryopreservation diluent B containing glycerol; taking 0.85g of Tris-alkali, 0.35g of lactose, 0.5g of trisodium citrate dihydrate, 0.02g of inositol, 10-25g of duck egg yolk, 1.5-12mL of glycerol and 200 mu L of 2mg/mL amikacin sulfate, and adding ultrapure water to make up to 100 mL.

Yolk-free dilutions (group ii):

yolk-free diluent A, no glycerol; taking 0.8g of fructose, 0.2g of potassium citrate, 1.1g of sodium glutamate, 0.06g of inositol, 0.88g of monopotassium phosphate and 200 mu L of 2mg/mL amikacin sulfate, and adding ultrapure water to make up to 100 mL.

Yolk-free diluent B containing glycerol; taking 0.8g of fructose, 0.2g of potassium citrate, 1.1g of sodium glutamate, 0.06g of inositol, 0.88g of monopotassium phosphate, 5mL of glycerol and 200 mu L of 2mg/mL amikacin sulfate, and adding ultrapure water to make up to 100 mL.

Precautions for preparing the diluent: the used instruments must be cleaned and disinfected; preparing the diluent for 2-3 days before use, storing in a refrigerator at 5 deg.C, and preheating at 37 deg.C for 30min before use; the medicine components for preparing the diluent are pure, accurately weighed and fully dissolved; diluent A (without glycerol) and diluent B (with glycerol: used after equilibration to 5 ℃) were prepared separately.

1.2.2 Collection of seminal fluid

The Leizhou black duck is provided by the constant-growing professional cooperative society in the Hill-Jiang city slope head area of Guangdong ocean university, male ducks with the age of more than 12 months are selected to collect semen by adopting a back massage method and an attraction method, the semen quality inspection is carried out on the collected semen without abnormity in color and smell, and the semen qualified in the quality inspection is mixed and preheated to 35 ℃ for later use.

1.2.3 dilution and temperature reduction equilibration of semen

The diluent is preheated to 35 ℃ in advance, and then mixed with the original semen (mixed with qualified semen) for further dilution, wherein the original semen is diluted by each diluent A (without glycerol) according to the ratio of 1: 4 (original semen: diluent A) are diluted in a 15mL centrifuge tube respectively. Then 300mL of 35 ℃ warm water is filled in a 500mL glass cup, the centrifuge tube filled with the diluted semen is placed in the glass cup, and then the glass cup is placed in a 5 ℃ refrigerator for slowly cooling and balancing; after balancing to 5 ℃, diluting the mixture by using diluent B (containing glycerol) at 5 ℃ in two times; first dilution according to semen: the diluent B is 2:1, and then balancing for 5 min; then, carrying out second dilution according to the proportion of semen: the diluent B is 2:1, and then equilibrated for a further 5 min.

1.2.4 semen cryopreservation and thawing

Then sucking the balanced semen into 0.25mL freezing tubule, sealing, fumigating with liquid nitrogen for 15min on a prepared liquid nitrogen floating boat (floating boat is made on liquid nitrogen poured into 2/3 part in foam box), and storing in liquid nitrogen. The semen quality is detected by thawing after being stored in liquid nitrogen for 1 week, the thin tube is taken out from the liquid nitrogen rapidly during thawing and is immediately thawed in warm water at 37 ℃ for 20s, and the semen quality is evaluated.

1.2.5 semen quality examination

Sperm motility: placing the thawed semen in a water bath kettle at 37 ℃ for thawing and reviving for 10min, taking 10 mu L of semen sample, placing the semen sample on a preheated glass slide, covering the glass slide, and selecting 3 visual fields in different directions under a microscope to detect the sperm motility. Repeating the detection for 3 times, wherein the number of the sperms counted each time is not less than 100, respectively calculating the sperm motility in 3 visual fields, and taking the average value, wherein the percentage is the sperm motility value of the test. The formula for sperm motility is: sperm motility ═ 100w +75x +50y +25z) × 100%. Wherein w is the percentage of fast moving sperm to total sperm, x is the percentage of slow moving sperm to total sperm, y is the percentage of gyrating or non-continuous moving sperm to total sperm, and z is the percentage of in-situ swinging sperm to total sperm.

Sperm survival rate: evaluation was carried out by LIVE/DEAD sphere viability kit (SYBR-14/PI) staining. 5 mul of semen is added into 45 mul of DPBS and is kept at the constant temperature (37 ℃) for 5min, and then 3 mul of SYBR-14 is added and is mixed evenly and kept at the constant temperature for 5 min. After the constant temperature, PI3 μ L is added into the incubator and placed for 5 min. Placing the sample under a fluorescence microscope equipped with a 37 ℃ constant temperature table for amplifying and observing by 400 times, and synchronously obtaining fluorescence and phase difference microscope images by using a DFC300 image acquisition system, wherein not less than 100 sperms are observed in each sample. The sperm heads in the images were stained red as dead sperm and green as live sperm.

Sperm plasma membrane integrity: using hypotonic expansion method, mixing 10 μ L semen with 90 μ L hypotonic solution, placing in 38 deg.C water bath for 10min, sucking 10 μ L mixed solution, placing on glass slide, covering with cover glass, and observing sperm plasma membrane integrity under microscope. The number of sperm per field can not be less than 100, and the integrity of the plasma membrane of sperm is expressed as the percentage of sperm with curled tail (intact plasma membrane) to the total number of sperm.

Sperm teratogenesis rate: using the giemsa staining method, 10 μ L of semen is sucked and placed on a glass slide for smearing, absolute ethyl alcohol is used for fixing for 15min, giemsa staining is carried out for 1.5h, and then washing, drying and detection are carried out from the back. The number of sperms in each visual field can not be less than 100, and the sperm aberration rate is expressed as the percentage of the sperm with abnormal shape to the total number of sperms.

1.2.6 data analysis

After the data are processed by EXCEL software, Duncan's in SPSS 19.0 software are used for mean significance test and one-factor analysis of variance, and the result is represented by mean plus or minus standard deviation (mean plus or minus SEM); p <0.05 indicates significant difference.

2 results and analysis

2.1 influence of different kinds of sugars in Tris-yolk cryopreservation diluent on sperm motility of frozen-thawed Rizhou black duck

Adding different kinds of sugar (lactose, fructose, glucose or sucrose) into a Tris-yolk cryopreservation diluent (wherein the concentration of duck yolk in the diluent is 20g/100mL, and the concentration of glycerol in the diluent B is 6mL/100mL) until the final concentration is 0.35g/L, and analyzing the influence of the different kinds of sugar on the sperm motility of the black ducks in Freeze-thaw states. The results are shown in FIG. 2: the effect of lactose addition significantly improved (P <0.05) sperm motility compared to fructose; the effect of comparing glucose and sucrose improved sperm motility but there was no significant difference (P > 0.05).

2.2 influence of different concentrations of Duck yolk in Tris-yolk cryopreservation dilution on sperm motility of frozen-thawed Rigzhou Black Duck

Adding duck egg yolk into Tris-egg yolk cryopreservation diluent (wherein the concentration of lactose in the diluent is 0.35g/100mL, and the concentration of glycerol in the diluent B is 6mL/100mL) to the final content of 0 (control), 10g, 15g, 20g and 25g per 100mL respectively, and analyzing the influence of the duck egg yolk with different concentrations on the sperm motility of the frozen-thawed Rigzhou black duck. The results are shown in FIG. 3: the sperm motility (P <0.05) of all groups added with the duck egg yolk is remarkably improved compared with the control group; the effect of adding the duck egg yolk group with the final concentration of 15% by mass is the best, and the sperm motility is obviously improved (P is less than 0.05) compared with the low concentration (10% by mass) and the high concentration (25% by mass); the duck egg yolk low-concentration group (mass fraction of 10%) has no significant difference with the high concentration (mass fraction of 25%) (P > 0.05).

2.3 influence of Glycerol with different concentrations in Tris-yolk cryopreservation dilution on sperm motility of frozen-thawed Rizhou black duck

Adding glycerol into a Tris-yolk cryopreservation diluent (wherein the concentration of lactose in the diluent is 0.35g/100mL, the content of duck yolk is 15g/100mL) to the final concentration of 0 (control), 1.5%, 3%, 6%, 9% and 12% by mass respectively, and analyzing the influence of the glycerol with different concentrations on the sperm motility of the black ducks in Leizhou. The results are shown in FIG. 4: addition of glycerol significantly improved sperm motility compared to the control group (P < 0.05). The final concentration of the glycerol is 3 percent of the mass fraction and 6 percent of the group, the activity is obviously higher than that of other addition groups (P <0.05), and the mass fractions are 1.5 percent, 9 percent and 12 percent without significant difference (P > 0.05).

2.4 Effect of different dilutions on sperm motility of different varieties of frozen-thawed ducks (black Leizhou duck, white duck, sheldrake)

The influence of different diluents (Tris-yolk diluent, group I; no-yolk diluent, group II) on the sperm motility of the semen of different varieties of ducks (black duck, white duck and sheldrake) in freezing and thawing is used. As shown in fig. 5: for the semen of the same duck variety, the sperm motility of different dilutions has obvious difference (P < 0.05); the sperm motility difference of the same diluent between the sperms of different varieties of ducks is not obvious.

2.5 Effect of different dilutions on the quality of frozen-thawed Rizhou Black Duck semen

As shown in fig. 6: influence of different diluents (Tris-yolk cryopreservation diluent (group I) and no yolk diluent (group II) on the quality of the frozen-thawed black duck semen. The effect of different diluents on the sperm motility and survival rate of the freeze-thaw black duck is shown in fig. 6A and 6B: compared with fresh semen, sperm motility and survival rate of the freezing preservation groups (groups I and II) are obviously reduced (P is less than 0.05); but sperm motility (62.5% vs 45.8%), survival (49.0% vs 37.8%) was significantly higher in group i than in group ii (P < 0.05). The effect of different diluents on the plasma membrane integrity and the teratogenicity of the frozen-thawed black duck sperm is shown in fig. 6C and 6D: there was no significant difference between the cryopreserved groups compared to the control group.

2.6 influence of freeze-thaw and preservation at 38 ℃ for 6h on activity of Leizhou black duck semen

The influence of the frozen-thawed (contrast: fresh semen) different diluents on the vitality of the semen of the Rigzhou black duck at 38 ℃ for 6h (detection: 0, 0.5, 1, 3 and 6h time points) is detected. The results are shown in table 1: sperm motility decreased significantly with prolonged storage at 38 ℃. Sperm motility was significantly reduced (P <0.05) at each storage time period in the freeze-thaw group compared to the control group (fresh semen). Group I sperm motility was significantly improved compared to group II at various time periods (P <0.05) during 6h storage at 38 ℃.

TABLE 1 Effect of the preservation time (38 ℃) after freezing-thawing on the vitality of the semen of Rigzhou Black Duck

Note: each experiment was repeated at least 5 times, all results expressed as mean. + -. standard error (mean. + -. SEM),^a-cwith significant differences in representation between different letters in the same column (P)<0.05)。