Method for creating sterile heterologous heptaploid culter crucian carp with herpes virus resistance

Abstract

The invention belongs to the technical field of fish genetic breeding, and particularly relates to a method for creating sterile heterologous heptaploid culter crucian carp with herpes virus resistance. The method comprises the following steps: breeding double-triploid female crucian carp and double-diploid male crucian carp, detecting ploidy of an obtained filial generation G1, selecting the double-triploid female crucian carp with the filial generation G1 being double tetraploid as a required female parent, controlling the double-triploid female crucian carp to lay eggs again, fertilizing the obtained eggs and double-diploid crucian carp sperms inactivated by ultraviolet rays, and hatching to obtain a double-triploid maintenance line filial generation G2; female fishes in the filial generation G2 serve as female parents, diploid culter alburnus serve as male parents, filial generation G3 is obtained through breeding, and the filial generation G3 is the sterile heterologous heptaploid culter crucian carp with the anti-herpes virus capacity. The breeding route provided by the invention can be used for creating a new sterile crucian carp strain with remarkable crucian carp blister disease virus resistance, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of fish genetic breeding, and particularly relates to a method for creating sterile allopolyploid culter crucian carp with the ability to resist herpes virus. Technical Background

[0002] As a classic and effective cell engineering breeding technology, polyploid breeding has been widely applied in the breeding of animals and plants, including the variety selection of cultured fish and shellfish. Allopolyploids are produced by the integration and doubling of the genomes of two different parental species, and they often combine the advantages of both hybridization and polyploidization, showing better growth advantages and stronger stress resistance than their diploid species. Optimizing or developing new and efficient ploidy manipulation technologies to create excellent germplasms and provide core breeding materials for new variety cultivation is one of the important directions in the genetic breeding of aquatic animals.

[0003] Crucian carp belongs to the genus Carassius in the family Cyprinidae. The genus Carassius is a complex polyploid system, mainly including the diploid crucian carp (Carassius auratus) (AABB) with 100 chromosomes that reproduces sexually and the triploid silver crucian carp (C. gibelio) (AAABBB) with more than 150 chromosomes that reproduces parthenogenetically by gynogenesis. At the same time, crucian carp is also one of the important aquaculture fish. However, crucian carp is generally susceptible to the crucian carp herpesvirus (CaHV), and the acute gill hemorrhage disease caused by it has caused extremely serious damage to the industry. The acute gill hemorrhage disease has a very fast onset, and the affected population generally dies within a week, and there is still no effective treatment method at present. During the breeding process, prevention is still the main measure, but the effect is limited.

[0004] Topmouth culter (Culter alburnus) belongs to the genus Culter in the subfamily Cultrinae of the family Cyprinidae, and is a diploid (CC) with 48 chromosomes that reproduces sexually. Previous studies have shown that when the eggs (AB) of diploid crucian carp are hybridized with the sperm (C) of diploid topmouth culter, due to the very distant genetic relationship between the two fish species, distant hybridization is incompatible, and the formed hybrid offspring (ABC) can hardly survive. The applicant's team has tried to fertilize the sperm of topmouth culter with the eggs (AAABBBB) of triploid silver crucian carp. However, because silver crucian carp reproduces parthenogenetically by gynogenesis and the sperm does not fuse with the egg nucleus, the sperm of topmouth culter only stimulates the activation of the egg to initiate embryonic development, and the offspring bred in this way is still silver crucian carp (AAABBBB), rather than a hybrid.

[0005] Cultivating allopolyploids with the integration of the genomes of crucian carp and topmouth culter is expected to become an effective way to create improved varieties with multiple traits. However, there is currently no successful case of forming normal offspring after integrating the two genomes. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention provides a method for creating a sterile allopolyploid blunt snout bream - crucian carp with anti - herpes virus ability.

[0007] The present invention adopts the following technical solutions:

[0008] A method for creating a sterile allopolyploid blunt snout bream - crucian carp with anti - herpes virus ability, comprising the following steps:

[0009] S1. Screening the female parent: Breed the female fish of double - triploid crucian carp with the male fish of double - diploid crucian carp to obtain offspring G1, and detect the ploidy of offspring G1. If all offspring G1 are double - tetraploids, then select the corresponding female fish of double - triploid crucian carp as the required female parent;

[0010] S2. Creating the maintenance line: The female parent screened in step S1 lays eggs again. Fertilize the obtained eggs with the sperm of double - diploid crucian carp inactivated by ultraviolet light. After hatching, obtain the offspring G2 of the double - triploid maintenance line;

[0011] S3. Creating the allopolyploid blunt snout bream - crucian carp: Use the female fish in offspring G2 as the female parent and diploid top - mouth culter as the male parent to breed and obtain offspring G3, and the offspring G3 is the sterile allopolyploid blunt snout bream - crucian carp with anti - herpes virus ability.

[0012] Preferably, the female parent of gynogenetic double - triploid silver crucian carp in step S1 is the "Zhongke No. 3" of hybrid silver crucian carp.

[0013] Preferably, in steps S1 and S3, dry fertilization is adopted to obtain fertilized eggs and then hatch them.

[0014] Preferably, in step S1, randomly select 50 tails from offspring G1 to detect the ploidy. If all the 50 selected tails are double - tetraploids, then all offspring G1 are double - tetraploids.

[0015] Preferably, in step S1, the method for detecting the ploidy of offspring G1 is as follows: Cut the fin rays of the fry to take a small amount of blood, add it to the CyStain DNA1Step solution and mix well, and then use a flow cytometer to detect the DNA content of the blood cells of each individual.

[0016] Preferably, in step S2, the operation of ultraviolet inactivation is as follows: Take the semen of double - diploid crucian carp, dilute the semen with a sperm preservation solution at a volume ratio of 1:50, spread a layer of the diluted semen in a culture dish, place the culture dish on an ice box, and irradiate it at a distance of 20 cm under a 15w ultraviolet lamp for 6 - 8 minutes to obtain ultraviolet - inactivated sperm.

[0017] Compared with the prior art, the progress of the present application lies in:

[0018] Traditional methods involve directly crossing the sexual reproduction amphidiploid crucian carp (AABB) with the diploid topmouth culter (CC) or bighead carp (CC), and all the offspring (ABC) are lethal. In previous research, the inventor's team created a new double triploid population by integrating the genomes of gynogenetic silver crucian carp and sexual reproduction crucian carp, and found that most of the females among them restored the ability of parthenogenetic gynogenesis, while a few females acquired a unique reproductive mode (see Lu M, Zhang Q C, Zhu Z Y, et al. An efficient approach to synthesize sterile allopolyploids through the combined reproduction mode of ameiotic oogenesis and sperm-egg fusion in the polyploid Carassius complex[J]. Science Bulletin, 2023, 68(10):1038 - 1050. and Lu Meng, Zhou Li, Gui Jianfang. Discovery of the "amitotic fusion" reproductive mode and its breeding practical significance[J]. Chinese Science Bulletin, 2024, 69(19):2704 - 2706.). These new double triploid females not only inherit the ability to produce non-reduced eggs through amitotic division from parthenogenetic gynogenesis Crucian carp but also inherit the ability to form fertilized eggs through the fusion of egg nucleus and sperm nucleus from sexual reproduction crucian carp. The inventor's team named this unique reproductive mode "amitotic fusion" reproduction. The double triploid eggs (AAABBB) produced by double triploid crucian carp with this characteristic have a higher ploidy than the double haploid eggs (AB) formed by amphidiploid crucian carp. These eggs have higher compatibility with the genomes of distant male parents. Therefore, this characteristic can be used to create allopolyploid populations. However, current reports related to "amitotic fusion" reproduction mainly focus on the fertility control of offspring.

[0019] 1) In this application, based on previous research, the inventor successfully achieved the integration of the genomes between crucian carp and topmouth culter for the first time, creating the allotetraploid culter-crucian carp (AAABBBBC), breaking through the problem of incompatibility in distant hybridization between crucian carp and topmouth culter in the past.

[0020] 2) The inventor discovered for the first time that topmouth culter is not infected by crucian carp herpesvirus. The crucian carp herpesvirus infection experiment shows that topmouth culter not only has extremely strong resistance to crucian carp herpesvirus, but also can inherit this resistance to the created allotetraploid culter-crucian carp, confirming that topmouth culter is the key male parent for creating new antiviral crucian carp strains. The anti-herpesvirus ability of the allotetraploid culter-crucian carp created in this application is increased by 96.97% compared to "Zhongke No. 3" and hardly gets infected by the virus.

[0021] 3) The allopolyploid blunt snout bream-crucian carp created in this application is sterile. Even if it escapes during cultivation, it will not cause potential ecological risks. At the same time, since its female parent "maintenance line" and male parent "topmouth culter" are both fertile, effective control of fertility in seedling production can be achieved, which is conducive to protecting the intellectual property rights of the aquaculture seed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the creation roadmap of the sterile allopolyploid blunt snout bream-crucian carp with anti-herpesvirus ability provided by this application.

[0023] Figure 2 They are the external appearance photos of the allopolyploid blunt snout bream-crucian carp and allopolyploid bighead carp-crucian carp created in this application.

[0024] Figure 3 They are the results of distinguishing the chromosomes from the female parent crucian carp and the male parent topmouth culter or bighead carp in the metaphase of lymphocytes of the allopolyploid blunt snout bream-crucian carp and allopolyploid bighead carp-crucian carp by the whole genome in situ hybridization technique.

[0025] Figure 4 They are the experimental results of the allopolyploid blunt snout bream-crucian carp against crucian carp herpesvirus CaHV.

[0026] Figure 5 They are the external appearance and sections of the gonads of the allopolyploid blunt snout bream-crucian carp in the breeding season. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] For ease of understanding, the technical solutions of the present invention will be described in more detail below in conjunction with specific embodiments.

[0028] As Figure 1 shown, a method for creating a sterile allopolyploid blunt snout bream-crucian carp with anti-herpesvirus ability includes the following three stages:

[0029] The first stage: screening the female parent of the "amitotic fusion" reproductive double triploid crucian carp

[0030] Cross-breed the double-triploid female crucian carp with the double-diploid male crucian carp to obtain the offspring G1. Detect the ploidy of the offspring G1. If the offspring G1 is double-tetraploid, select the corresponding double-triploid female crucian carp as the required female parent. Specifically, during the breeding season, select a batch of sexually mature female parents from the double-triploid crucian carp population. After artificial induction of spawning, collect the eggs (AAABBB) and fertilize them with the sperm (AB) of the double-diploid crucian carp. Incubate the fertilized eggs into fry separately according to different female parents to obtain the offspring G1. Randomly detect the ploidy of 50 tails from each group of fry. If the ploidy of all fry is double-tetraploid (AAAABBBB), it is determined that the corresponding double-triploid female parent of this batch of fry has the "ameiotic fusion" reproductive ability. If the ploidy of all fry is double-triploid (AAABBB), it is determined that the corresponding double-triploid female parent of this batch of fry has gynogenetic ability. Select the double-triploid female fish with the "ameiotic fusion" reproductive ability as the required female parent.

[0031] The specific operation steps of this stage are as follows:

[0032] S1. Selection of male and female parents: During the breeding season, select double-triploid female crucian carp with enlarged and soft abdomens. By gently squeezing the cloaca, if the eggs discharged are plump and dispersed, select this female fish as the sexually mature female parent. Similarly, by gently squeezing the cloaca of the double-diploid male crucian carp, if a large amount of thick and milky white semen flows out, select this male fish as the sexually mature male parent.

[0033] S2. Artificial dry fertilization: After artificial induction of spawning, obtain mature eggs from the double-triploid female crucian carp selected in S1. Mix the eggs with the semen of the double-diploid male crucian carp in a plastic dish and then pour it into a porcelain dish filled with water for fertilization. During this period, stir with a feather to make the fertilized eggs evenly adhere to the bottom of the porcelain dish.

[0034] S3. Detection of the ploidy of offspring fry: Incubate the fertilized eggs into fry. After 1 month of feeding, when the body length reaches 2.5 cm, randomly select 50 tails from each group. Cut the tail to take 0.1 - 0.5 μL of trace blood, add it to the CyStain DNA1Step (source: Partec, Germany) solution and quickly mix well. Use this solution to quickly fix and stain the blood cells, and then measure the DNA content of the blood cells of each individual through a flow cytometer.

[0035] In this stage, referring to the above steps, a total of 13 sexually mature double-triploid female crucian carp and 1 sexually mature double-diploid male crucian carp were selected for breeding experiments. Among them, the offspring of 11 female fish were all double-triploid after ploidy detection, and the offspring of the other 2 female fish were all double-tetraploid after ploidy detection, belonging to female fish with "ameiotic fusion" reproduction, accounting for 15.4%.

[0036] The second stage: Create the "maintenance line" of the double-triploid with "ameiotic fusion" reproduction

[0037] By controlling water temperature, light, and nutrients, the double triploid crucian carp females with "amitotic fusion" reproduction screened in the first stage were induced to lay eggs again. Their eggs were fertilized with ultraviolet-inactivated sperm of double diploid crucian carp, and artificial gynogenesis was carried out to expand the population, creating the "maintenance line" G2 of double triploid with "amitotic fusion" reproduction.

[0038] The specific operation steps in this stage are as follows:

[0039] S1. Controlling the female parent to lay eggs again: The double triploid crucian carp females with "amitotic fusion" reproduction screened in the first stage were cultured in a farming system with a water temperature of 20 - 23 °C and sufficient light. High-protein crucian carp feed was fed 2 times a day, and natural animal protein baits such as bloodworms were additionally fed 1 time. The feeding cycle was 15 - 20 days. By gently squeezing the female cloaca, if the discharged egg grains were plump and scattered, it was determined that the female fish could lay eggs again.

[0040] S2. Ultraviolet inactivation treatment of double diploid crucian carp semen: Take double diploid crucian carp semen, dilute the semen with sperm preservation solution at a volume ratio of 1:50. Add 4 ml of the diluted semen into a plastic petri dish with a diameter of 10 cm, shake it and spread it evenly on the bottom of the petri dish. Then place a horizontal shaker under a 15 w ultraviolet lamp tube, place the petri dish on the shaker, pad an ice box in the middle to keep it at a low temperature, and at the same time make the sperm liquid surface 20 cm directly below the lamp tube, irradiate the semen for 6 - 8 min to obtain ultraviolet-inactivated sperm.

[0041] S3. Creating the double triploid "maintenance line" with "amitotic fusion" reproduction: Artificially induce ovulation of the double triploid crucian carp females with "amitotic fusion" reproduction cultured in S1, mix the eggs with the sperm treated in S2 in a plastic dish and then pour them into a porcelain dish with water for fertilization. After the obtained fertilized eggs were hatched, fry G2 of the double triploid "maintenance line" with "amitotic fusion" reproduction were obtained.

[0042] On the basis of the first-stage experiment, one female fish was selected from the 2 screened double triploid crucian carp females with "amitotic fusion" reproduction, and it was fertilized with ultraviolet-inactivated sperm of double diploid crucian carp to obtain about 2000 artificial gynogenesis "maintenance line" G2. These offspring of the double triploid "maintenance line" still had the ability of "amitotic fusion" reproduction.

[0043] The third stage: Creating a sterile allopolyploid population

[0044] Using the new double triploid "maintenance line" with "amitotic fusion" reproduction as the female parent and diploid topmouth culter as the male parent to create allopolyploids. The specific operation is as follows:

[0045] The G2 obtained in the second stage was cultured until sexual maturity. After artificially inducing spawning in sexually mature individuals, their eggs were fertilized with Culter alburnus sperm by dry fertilization. The obtained fertilized eggs were hatched to obtain the offspring G3, and the allotetraploid Culter alburnus - Carassius auratus gibelio was obtained.

[0046] In this stage, 20 individuals were randomly selected from the artificial gynogenesis "maintenance line" G2 prepared in the second stage, and their eggs were fertilized with the sperm of 3 Culter alburnus. The average fertilization rate and embryo hatching rate of fertilization with Culter alburnus sperm were 90.47% ± 1.79% and 97.23% ± 0.68% respectively.

[0047] Control

[0048] The distantly related diploid bighead carp was selected as the male parent. According to the steps of the above - mentioned third stage, with the new double - triploid "maintenance line" produced by "amitotic fusion" reproduction as the female parent, its eggs were fertilized with bighead carp sperm by dry fertilization. The obtained fertilized eggs were hatched to obtain the offspring G3, and the allotetraploid bighead carp - Carassius auratus gibelio was obtained.

[0049] 20 individuals were randomly selected from the artificial gynogenesis "maintenance line" G2 prepared in the second stage, and their eggs were fertilized with the sperm of 3 bighead carp. The average fertilization rate and embryo hatching rate of fertilization with bighead carp sperm were 85.62% ± 2.46% and 97.49% ± 0.19% respectively.

[0050] See Figure 2 , the allotetraploid Culter alburnus - Carassius auratus gibelio and the allotetraploid bighead carp - Carassius auratus gibelio basically maintain the overall shape of Carassius auratus gibelio, and only the body color and mouth shape are affected by the male parent.

[0051] Experiment

[0052] 1. Chromosome analysis

[0053] The chromosomes from the female parent Carassius auratus gibelio and the male parent Culter alburnus / bighead carp in the metaphase of lymphocytes of the G3 - generation allotetraploid Culter alburnus - Carassius auratus gibelio and the allotetraploid bighead carp - Carassius auratus gibelio were distinguished by the whole - genome in - situ hybridization technique. The results are shown in Figure 3 . From Figure 3 A in it, it can be seen that the total number of chromosomes of the allotetraploid Culter alburnus - Carassius auratus gibelio is about 174. The thin arrows in the figure indicate the chromosomes from the female parent double - triploid Carassius auratus gibelio, and these chromosomes only have strong green fluorescence signals at the centromere part, with a total of about 150; the thick arrows indicate the chromosomes from the male parent diploid Culter alburnus, and these chromosomes are evenly covered with red fluorescence signals from head to tail, with a total of 24. This result confirms that the chromosome set (AAABBBC) of the allotetraploid Culter alburnus - Carassius auratus gibelio is composed of the complete chromosome set (AAABBB) of the female parent double - triploid Carassius auratus gibelio and a set of chromosome sets (C) of the male parent Culter alburnus. Similarly, from Figure 3As shown in Figure B, the chromosomes of the maternal crucian carp and paternal bighead carp in the metaphase of lymphocytes of the alloheptaploid bighead carp and crucian carp were from both parents, indicating that the experiment successfully created alloheptaploid bighead carp and alloheptaploid bighead carp.

[0054] 2. Carassius auratus Herpesvirus Infection Experiment

[0055] The obtained G3 generation allogeneic heptaploid crucian carp and allogeneic heptaploid bighead carp were artificially infected with herpes virus CaHV to verify the antiviral properties. The experimental process is as follows:

[0056] S1. Preparation of fish for infection experiment: Allogeneic heptaploid crucian carp, allogeneic heptaploid bighead carp, and the control group of heterogeneous silver crucian carp "Zhongke No. 3", double triploid "maintenance line", Culter alba and bighead carp fry were raised in the same environment to a size of about 20g. After being brought back to the laboratory, the breeding water temperature was gradually transitioned to 23℃ (±1℃), and the experimental fish were raised for two weeks and observed to confirm that there were no abnormalities.

[0057] S2. Intraperitoneal injection of virus: The four experimental fish groups were randomly divided into three parallel groups, a total of 18 groups of fish, each group of about 20 to 30 fish, and injected simultaneously. The amount of CaHV filtrate injected was 0.1 ml per 10 g body weight, and the amount of virus in the filtrate was 2.9×10 8 Herpesvirus / mL, injected into the abdominal cavity from the base of the pectoral fin.

[0058] S3. Evaluation of disease resistance of each clone: ​​After virus injection, the culture water temperature was maintained at 23°C (±1°C), and the water was changed every day to keep the water clean. Dead individuals were removed in time. The survival rate of 18 groups of fish after intraperitoneal virus injection was counted every day, and the infection period was 21 days. The average survival rate of the three parallel groups corresponding to each experimental group was calculated.

[0059] The results are as follows Figure 4 shown. Figure 4 Figure A is the experimental result of the resistance of the alloheptaploid carp to the crucian carp herpes virus (CaHV) created in this application, in which the control group of heterogeneous silver carp "Zhongke No. 3" and the double triploid "maintenance system" all died within 9 days after being injected with the virus (the average survival rate was 0%), while the Culter albicans did not die after being injected with the virus, and the survival rate of the alloheptaploid carp after being injected with the virus was as high as 96.97%, indicating that the integration of the Culter albicans genome has significantly improved the disease resistance of the alloheptaploid carp. Figure 4 In B, the experimental results of the allogeneic heptaploid bighead carp and crucian carp created in this application are against the crucian carp herpes virus CaHV. The bighead carp in the control group did not die after being injected with the virus, while allogeneic heptaploid bighead carp and crucian carp died after being injected with the virus, indicating that bighead carp is not infected with crucian carp herpes virus, but the integration of the bighead carp genome cannot improve the disease resistance of allogeneic heptaploid bighead carp and crucian carp. In other words, the bighead carp's ability to resist herpes virus cannot be inherited by allogeneic heptaploid bighead carp and crucian carp, and allogeneic heptaploid bighead carp and crucian carp are extremely susceptible to herpes virus. Therefore, Culter albus is the key paternal parent for creating a new strain of antiviral crucian carp.

[0060] 3. Fertility Detection Experiment

[0061] As Figure 5 shown, during the breeding season, the gonads of 100 allopolyploid heptaploid Culter alburnus×Carassius auratus G3 were dissected and histologically examined. The results showed that there were no mature eggs in the ovaries of all the tested individuals, nor were there any mature sperm in the testes, confirming that the allopolyploid heptaploid Culter alburnus×Carassius auratus is sterile.

[0062] The above experiment proves that the breeding route provided by this application can create a new strain of crucian carp with significant resistance to crucian carp pox virus and sterility, and thus is an important way for the cultivation of new crucian carp varieties.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the present invention; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for producing a sterile alloheptaploid crucian carp with anti-herpes virus ability, characterized in that: The following steps are involved: S1. Screening the mother parent: breeding a double triploid crucian carp female with a double diploid crucian carp male to obtain offspring G1, detecting the ploidy of the offspring G1, and if all offspring G1 are double tetraploid, selecting the corresponding double triploid crucian carp female as the desired mother parent; S2. Creation of maintenance line: The female parent selected in step S1 lays eggs again, and the eggs are fertilized with ultraviolet-inactivated amphidiploid crucian carp sperm, and after hatching, a double triploid maintenance line progeny G2 is obtained; S3. Creation of allogeneic heptaploid carp: using the female fish in the offspring G2 as the female parent and the diploid Culter albus as the male parent, breeding to obtain offspring G3, which is the infertile allogeneic heptaploid carp with the ability to resist herpes virus.

2. The method for producing a sterile alloheptaploid crucian carp with anti-herpes virus ability as claimed in claim 1, characterized in that: The female parent of the gynogenetic triploid silver crucian carp in step S1 is the allogynogenetic silver crucian carp "Zhongke No. 3".

3. The method for producing a sterile alloheptaploid crucian carp with anti-herpes virus ability as claimed in claim 1, characterized in that: In the step S1 and step S3, dry insemination is used to obtain fertilized eggs and hatch them.

4. The method for producing a sterile alloheptaploid crucian carp with anti-herpes virus ability as claimed in claim 1, characterized in that: In step S1, 50 offspring G1 are randomly selected to detect ploidy. If the selected 50 offspring are all ditetraploid, then all offspring G1 are ditetraploid.

5. The method for producing a sterile alloheptaploid crucian carp with anti-herpes virus ability as claimed in claim 1, characterized in that: In step S1, the method for detecting the ploidy of the offspring G1 is to cut the fin rays of the fry to obtain a small amount of blood, add CyStain DNA 1Step solution and mix well, and then detect the DNA content of each individual blood cell by flow cytometry.

6. The method for producing a sterile alloheptaploid crucian carp with anti-herpes virus ability as claimed in claim 1, characterized in that: In step S2, the ultraviolet inactivation operation is to take amphidiploid crucian carp semen, dilute the semen with sperm preservation solution at a volume ratio of 1:50, spread a layer of the diluted semen in a culture dish, place the culture dish on an ice box, and irradiate it at a distance of 20 cm under a 15W ultraviolet lamp for 6 to 8 minutes to obtain ultraviolet inactivated sperm.

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