Suma's method of producing ovulated eggs

Abstract

To provide a technique capable of efficiently collecting ovulated eggs of mackerel tuna.SOLUTION: A production method of ovulated eggs of mackerel tuna includes: a spawning time specifying step of specifying a spawning time of the mackerel tuna; a mackerel tuna transfer step of putting the mackerel tuna into a closed space, the mackerel tuna determined to be in the later stage of a final maturation period based on the spawning time; and an ovulated egg collection step of slaughtering the mackerel tuna when no spawning behavior of the mackerel tuna is observed after the mackerel tuba transfer step and acquiring ovulated eggs of the mackerel tuna at a time later than spawning time, where a diameter in the horizontal direction in the closed space is 2 times or more and 4 times or less of a minimum rotation diameter of the mackerel tuna.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing ovulated eggs of a small stag beetle. [Background technology]

[0002] Artificial insemination has been used for breeding fish with superior traits and ensuring a stable supply.

[0003] Various techniques have been proposed in relation to artificial insemination of fish. For example, Patent Document 1 describes a technique for culturing fish stem cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2021-126055 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the unfertilized eggs (ovulated eggs) used in artificial insemination have the problem of being difficult to secure in sufficient quantities when needed, due to the difficulty in controlling fish spawning. In particular, the present inventors have focused on the fish species, the barracuda, and have conducted research into a technique for efficiently obtaining barracuda ovulated eggs.

[0006] The present invention has been made in consideration of the above-mentioned circumstances, and an object of the present invention is to provide a technology that can efficiently collect ovulated eggs from smallmouth bass. [Means for solving the problem]

[0007] The present inventors discovered that ovulated eggs can be efficiently collected by utilizing the phenomenon that egg-laying behavior stops when small oocytes in the late final maturation stage are temporarily housed in a specified closed space, and thus completed the present invention. Specifically, the present invention provides the following.

[0008] (1) a spawning time determination step for determining the spawning time of barracuda; A suma transfer process in which suma determined to be in the late final maturation stage based on the spawning time are placed in a closed space; an ovulated egg recovery step of slaughtering the barracuda after the barracuda transfer step when no egg-laying behavior of the barracuda is observed and at a time later than the egg-laying time, to obtain ovulated eggs; Including, The horizontal diameter in the closed space is 2 to 4 times the minimum rotation diameter of the suma, How Suma produces ovulated eggs.

[0009] (2) The manufacturing method described in (1), wherein the minimum rotation diameter of the suma is 2.5 to 3 times the body length of the suma.

[0010] (3) A manufacturing method described in (1) or (2), in which, in the step of transferring the stag beetles, the transfer of the stag beetles is carried out at a time that is at least 1 hour and not more than 3 hours earlier than the spawning time.

[0011] (4) A manufacturing method according to any one of (1) to (3), wherein in the ovulated egg collection step, the barracuda is slaughtered within one hour from the time of egg laying.

[0012] (5) A manufacturing method described in any one of (1) to (4), wherein the age of the chick is 12 months or more and 60 months or less. [Effects of the Invention]

[0013] According to the present invention, a technique is provided that can efficiently collect ovulated eggs from smallmouth bass. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a diagram showing the relationship between the developmental stage of a fertilized suma egg and the time elapsed since fertilization. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not particularly limited thereto.

[0016] <Suma's ovulated egg manufacturing method> The method for producing ovulated eggs from bigfin reef sharks (hereinafter also referred to as the "production method of the present invention") according to the present invention comprises the steps of: determining the egg-laying time of bigfin reef sharks; transferring bigfin reef sharks that are determined to be in the late final maturation stage based on the egg-laying time; and slaughtering the bigfin reef sharks after the egg-laying time in a closed space; and recovering the ovulated eggs by slaughtering the bigfin reef sharks at a time after the egg-laying time when no egg-laying behavior is observed. The horizontal diameter of the closed space is between two and four times the minimum rotational diameter of the bigfin reef sharks.

[0017] For example, in the waters off Ainan Town, Ehime Prefecture, the spawning season for barracudas is from June to September, with the peak season being from July to August. Mature females are thought to ovulate and spawn almost daily in the middle of this spawning season.

[0018] The outline of oogenesis in the smallmouth bass is as follows. After storing enough nutrients (yolk) inside for several months, the oocyte gradually becomes an egg (a fertilizable female gamete). This process is called "final maturation" and is completed within 24 hours in the case of the spermatheca. At final maturation, nuclear changes occur and the oocyte is released from the follicular layer (this process is called "ovulation") and stored in the ovarian cavity within the ovary until egg laying. The unfertilized egg in the ovarian cavity that can be fertilized is called an "ovulated egg."

[0019] Because ovulated eggs have a high fertilization rate through artificial insemination, securing a sufficient number of high-quality ovulated eggs enables efficient artificial insemination, etc. To do this, it was previously necessary to collect eggs from the spermatheca after ovulation but before spawning. However, in the case of barracudas, the time from ovulation to spawning is extremely short (estimated to be within a few tens of minutes). Furthermore, females that have ovulated have been observed fleeing at high speed while being aggressively pursued by males (spawning behavior), making them difficult to capture. Furthermore, final maturation is thought to be a mechanical event that is initiated by oocyte maturation-inducing hormones and cannot be stopped midway, making it extremely difficult to control the timing of ovulation. For these reasons, it has traditionally been difficult to collect ovulated eggs.

[0020] Therefore, the present inventors focused on the fact that spawning is an act that involves spawning behavior by males and females, and is an event that can be stopped midway. They discovered that by allowing the eggs to progress to final maturation while preventing egg laying, they could efficiently collect ovulated eggs from the bodies of barracudas. Specifically, if a stag beetle in the late stage of final maturation is subjected to moderate stress, i.e., moved to a small, closed space, it will cease to spawn. However, because final maturation is a mechanical event as mentioned above, final maturation will continue and ovulation will occur even if spawning behavior is stopped. If the barracuda that has stopped spawning in this way is slaughtered after the time of spawning, the ovulated eggs can be collected from its body. The collected ovulated eggs can be used for artificial insemination either directly or after storage under appropriate conditions.

[0021] As described above, the present invention is based on the novel finding that by allowing the final maturation of the smallmouth to proceed while halting egg-laying behavior, ovulated eggs are stored in the ovaries, allowing the ovulated eggs to be efficiently collected.

[0022] The production method of the present invention will be described in detail below.

[0023] (1) Smartphone In the present invention, "Sma" refers to "Euthynnus affinis", a fish of the family Scombridae, subfamily Scombridae, order Perciformes.

[0024] The chicks used in the production method of the present invention are not particularly limited as long as they are female, but from the viewpoint of obtaining sufficient ovulated eggs, their age is preferably 12 months or more and 60 months or less, more preferably 24 months or more and 48 months or less.

[0025] The barracuda used in the production method of the present invention preferably has a body length of 45 cm or more and 75 cm or less, more preferably 50 cm or more and 70 cm or less, from the viewpoint of obtaining a sufficient number of ovulated eggs.

[0026] In the present invention, "body length" means fork length (the distance from the tip of the upper jaw to the outer edge of the central depression where the caudal fin forks).

[0027] The weight of the small foal used in the production method of the present invention is preferably 2 kg or more and 8 kg or less, more preferably 2.5 kg or more and 6.5 kg or less, from the viewpoint of obtaining a sufficient number of ovulated eggs.

[0028] Until they are subjected to the suma transfer process described below, the suma are grown without stress in a regular fish pen (a space in the sea area surrounded by bamboo fences, netting, etc.) that is large enough for them to swim freely.

[0029] The number of individuals of the scallops used in the production method of the present invention is not particularly limited. Since male and female scallops are usually kept together in the fish tank, it is preferable to use a plurality of individuals (for example, 5 to 6 fish) in order to more reliably obtain scallops in the late final maturation stage described below.

[0030] (2) Spawning time identification process The spawning time specifying step is a step of specifying the spawning time of the barracuda.

[0031] During the spawning season, barracudas ovulate and lay eggs at a nearly regular cycle every day. For example, the interval between spawning is usually about 24 hours, and the interval between spawning and the next final maturation completion time (particularly, ovulation time) is thought to be 23 hours or more. Once the final stage of maturity is reached and ovulation occurs, egg-laying behavior is observed. Furthermore, by observing the developmental stages of the laid eggs, it is possible to estimate the time elapsed since fertilization. Therefore, the time of spawning can be determined by visually observing the spawning behavior of the barracuda (intense chasing behavior, etc.) and by checking the developmental stage of the laid eggs.

[0032] The time of ovulation can be determined, for example, by the method described in the Examples, in which the time of ovulation was determined by estimating the time elapsed since fertilization based on the developmental stage of the egg. In addition, during the peak spawning season, barracudas usually spawn between 5:30 pm and 6:30 pm.

[0033] The time of ovulation is usually considered to be before the time of egg laying and within one hour of the time of egg laying.

[0034] From the viewpoint of specifying the spawning time more accurately, the spawning time specifying step is preferably carried out the day before the suma transfer step described below (for example, 20 to 24 hours before the start of the suma transfer step). However, in environments such as heavy rain, excessive stress may be placed on the barracudas, disrupting their ovulation and egg-laying cycles. Therefore, it is preferable to avoid stormy weather and perform the egg-laying time specification step and the subsequent barracuda transportation step on a day with stable weather (preferably sunny).

[0035] (3) Smart transfer process The suma transferring step is a step of placing the suma determined to be in the late final maturation stage based on the spawning time identified in the spawning time identifying step into a closed space. This process stops egg-laying behavior while the final maturation of the spermatheca progresses, allowing the ovulated eggs to be stored in the ovaries, making it possible to efficiently collect the ovulated eggs.

[0036] Whether the suma are in the late final maturity stage or not can be determined from the spawning conditions and spawning time at the time of the spawning time specification step (usually the day before the suma transfer step). Specifically, since the interval between egg laying and the completion of final maturation (particularly, the time of ovulation) is usually about 23 hours, a stag beetle that has been present for 18 to 22 hours (particularly, 21.5 to 22 hours) since egg laying can be determined to be in the late stage of final maturation.

[0037] Those deemed to be in the late final maturity stage are transferred to a closed space.

[0038] In order to minimize the impact on the eggs, the transfer of the stag beaks is preferably carried out at a time point that is at least 1 hour and at most 4 hours, more preferably at least 1 hour and at most 3 hours, before the time of spawning.

[0039] When transferring the sardines from the fish pen to the enclosed space, they are usually caught using fishing gear, etc. However, care must be taken not to cause excessive stress to the sardines (such as injuries from protrusions or swallowing of fishing hooks).

[0040] (Details of the enclosed space) Based on the results of previous research, the inventors have found that, in the case of barracuda weighing 2.5 to 4.0 kg, if they are caught and kept in a circular fish pen with a diameter of 10 m or more (approximately 8 m deep) or a square fish pen with a side length of 10 m or more (approximately 8 m deep), they will spawn as normal. On the other hand, the inventors also found that spawning was not observed when the fish were caught and kept in a square fish pond with sides of about 5 m (about 4 m deep). The reasons for this are thought to be that the fish are unable to fully perform the tracking behavior that precedes spawning, and the stress caused by the change in environment due to being kept in a small space. Based on the above, the present inventors have discovered that the egg-laying behavior of bluefin tuna can be controlled by housing the bluefin tuna in a specified closed space.

[0041] In the present invention, the term "closed space" means a space whose horizontal diameter is between two and four times the minimum rotation diameter of the suma. As a result of the inventors' research, they found that if the space is larger than the above-mentioned closed space, the spawning behavior of the barracudas will not stop, and if the space is smaller than the above-mentioned closed space, the barracudas will be killed or injured due to friction with the walls or suffocation. In other words, the closed space of the present invention is a space that can apply appropriate stress to the bluefin tuna, allowing it to progress to its final maturation while also stopping its spawning behavior.

[0042] In the present invention, the "horizontal direction (of the closed space)" means the direction parallel to the water surface. In the present invention, the "horizontal diameter (of the closed space)" includes the length of one side or the diameter of a circle, depending on the shape of the closed space.

[0043] In the present invention, the "minimum rotation diameter of a sablefish" means the minimum diameter of the circular motion orbit of a sablefish when it swims while breathing and without rubbing its body against the wall (net, sheet, etc.) of a closed space.

[0044] In the closed space, at least one horizontal diameter constituting the space is set to be between two and four times the minimum rotation diameter of the smart device. However, from the viewpoint of reliably stopping spawning behavior, it is preferable that all horizontal diameters of the closed space are between two and four times the minimum rotation diameter of the barracuda.

[0045] The minimum rotation diameter of a barracuda can be determined depending on the body length (fork length) and weight of the barracuda, but is preferably between 2.5 and 3 times the body length of the barracuda. For example, for a shark with a body length of 50 cm (usually weighing about 2.5 kg), the minimum turning diameter can be set to 125 to 150 cm. In this case, the horizontal diameter in the closed space can be set to 250 to 600 cm.

[0046] When multiple individual stag beetles are subjected to the stag beetle transport process, it is preferable to set the horizontal diameter in the closed space to near the upper limit (for example, 3 to 4 times the minimum rotation diameter of the stag beetle) in order to avoid excessive stress on the stag beetles.

[0047] The shape of the closed space of the present invention is not particularly limited, and may be rectangular (square, cubic, etc.), circular (cylindrical, etc.), or the like.

[0048] The depth (diameter perpendicular to the water surface) of the closed space of the present invention is not particularly limited as long as the entire body of the small shark is sufficiently immersed in water. For example, the lower limit of the depth of the enclosed space of the present invention may be five times or more the body height of the barracuda (the maximum distance from the abdomen to the back (excluding the fins)). The upper limit of the depth of the closed space of the present invention may be 25 times or less the height of the barracuda, from the viewpoint of applying an appropriate amount of stress to the barracuda.

[0049] When the closed space of the present invention is rectangular, it may have a length of 2.5 to 6 m, a width of 2.5 to 6 m, and a depth of 1 to 5 m. When the closed space of the present invention is circular, it may have a diameter of 2.5 to 6 m and a depth of 1 to 5 m.

[0050] The material that constitutes the closed space is not particularly limited, and may be the same as the material used for ordinary fish cages (netting, vinyl, etc.).

[0051] (4) Ovulated egg collection process The ovulated egg collection step is a step in which, after the stag beak transport step, the stag beak is not observed to be laying eggs and is slaughtered at a time later than the egg laying time to obtain the ovulated eggs. In the suma transfer step, while the final maturation of the suma progresses, egg-laying behavior stops, so the ovulated eggs stored in the ovaries are collected in the ovulated egg collection step.

[0052] The time from the completion of the transportation of the barracudas to the time they are removed from the enclosed space for slaughter, i.e., the time the barracudas stay in the enclosed space, is the time after the egg-laying time has passed and up to any point during which egg-laying behavior of the barracudas is not observed. However, from the viewpoint of reliably collecting ovulated eggs, the shorter the time from the time of egg laying, the better.

[0053] From the viewpoint of ensuring the collection of ovulated eggs, it is preferable to slaughter the barnyard mackerel within one hour of the time of egg laying.

[0054] The method of slaughtering barracuda is not particularly limited, and examples include slaughtering by slaughtering the barracuda while it is still alive. The slaughtered stag beaks are appropriately stored on ice and dissected to allow the collection of ovulated eggs from the ovaries.

[0055] The obtained ovulated eggs can be stored by any method as needed and used for artificial insemination or the like. The shorter the storage time of ovulated eggs (for example, within 5 hours), the higher the fertilization rate tends to be. [Example]

[0056] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0057] <Test 1> Ovulated eggs were obtained from the smallmouth bass according to the following method. In all of the following steps, the seawater temperature was maintained at approximately 24°C.

[0058] In this example, female barracudas of approximately 24 months of age, 50 to 56 cm in body length and 2.1 to 3.2 kg in weight were used.

[0059] (1) Confirmation of egg laying First, to determine the spawning time, we observed the spawning behavior of the barracuda in the fish pen. At the time of spawning, the barracudas exhibited vigorous chasing behavior, followed by a loud splashing sound near the water surface (this splashing sound was caused by the barracudas' movements associated with spawning). In this example, the time when the sound of water was heard was determined to be the approximate time of spawning.

[0060] The fish pen was circular (diameter: approximately 10 m, made of polyvinyl) and large enough for the barracudas to swim freely, and the inside was surrounded by a vinyl sheet with a water depth of approximately 3 m. This is to prevent the eggs from scattering, as the specific gravity of barracuda eggs (equivalent to a salt concentration of approximately 3.3%) is slightly lighter than that of seawater and they tend to float near the surface of the sea.

[0061] (2) Spawning time identification process Based on the approximate spawning time determined in (1) above, eggs were collected from the cage after spawning behavior had finished. A soft net (such as a dip net for insects) was used for collection to avoid damaging the eggs. The developmental stage of the collected eggs was identified by microscopic observation, and the time of egg laying (around 6 p.m. in this case) was determined.

[0062] The rate of development depends on the water temperature. As shown in Figure 1, for example, 16-cell eggs were spawned 95 to 110 minutes after fertilization in water at 24°C.

[0063] (3) Smart transfer process The day after the spawning time was determined (spawning time determination step), the barracudas were caught and placed in an enclosed space.

[0064] (3-1) Catching a Spanish mackerel The time period for catching the spotted barracudas was chosen to be close to the time of spawning in order to minimize the impact on the eggs. In this example, taking into account the preparation time for the artificial insemination test (approximately 2 hours), the barracudas were caught 2 to 2.5 hours before the time of spawning. The barracudas at the time of catching corresponded to barracudas judged to be in the late stage of the final maturation period (approximately 21.5 to 22 hours after spawning). Commercially available fishing hooks were used for catching the fish, except that the barbs had been scraped off to avoid damaging the fish, and a vinyl cable tie was attached to prevent the fish from swallowing the hook.

[0065] The caught barracuda were hauled into a water net (a 5m square net with a vinyl bottom) and the hooks were removed within 10 seconds, taking care not to cause excessive stress to the fish.

[0066] (3-2) Confinement in a closed space (small fish tank) The caught Spanish mackerel were placed in a small rectangular fish cage. The accommodation (transfer of the barracuda) was carried out 2 to 2.5 hours before the time of spawning. The body length of the barracuda used in this example was approximately 50-56 cm, so the minimum turning diameter of the barracuda was calculated to be approximately 125-170 cm (2.5-3 times the body length of the barracuda). Based on this value, the horizontal length of the small fish pen was set to 5 m length x 5 m width (3-4 times the minimum turning diameter of the barracuda), and a depth of 4 m.

[0067] (4) Ovulated egg collection process The barracudas housed in a closed space and showing no evidence of egg-laying behavior (chasing behavior, etc.) were removed, slaughtered, and the ovulated eggs were collected. The stag beaks were slowly collected in a dip net (a dip net about 50 cm in diameter with a vinyl bottom). The collection took place after spawning, but within one hour of spawning. The stag beaks at the time of collection correspond to "stag beaks that were not showing spawning behavior and were collected after the spawning time." The harvested barracuda was quickly killed by cutting the spine and draining the blood, and then immediately placed on ice in a cooler box to cool. Next, the abdomen of the barracuda was opened, and the ovaries were removed after clamping the genital opening behind the ovaries to prevent the ovulated eggs from leaking out. After removing blood and body fluids from outside the ovaries, the ovulated eggs were collected in a plastic container. The collected ovulated eggs were placed in a plastic test tube (50 ml) in an amount of 10 to 15 g and stored in a constant temperature incubator at 20°C for 0 to 12 hours.

[0068] (5)Artificial insemination To examine whether the obtained ovulated eggs could be fertilized, they were inseminated using the following method (dry induction method). The sperm used for the slaughter was diluted 100-fold with physiological saline solution (Hank's solution) and stored in a constant temperature incubator at 10°C. 0.1 to 0.2 g of ovulated eggs (150 to 300 eggs) were weighed out and placed in a plastic cup (450 ml), and 0.1 ml of semen was poured on top. Immediately afterwards, 50 ml of filtered seawater (24°C) was added, gently mixed and left to stand for 5 minutes, after which 250 ml of filtered seawater was added and the mixture was stored in a constant temperature incubator at 24°C.

[0069] (6) Results The fertilization rate of the ovulated eggs obtained was extremely high, nearly 100%. In particular, the shorter the storage time of the ovulated eggs (the interval between the ovulated egg collection process and artificial insemination), the higher the fertilization rate. Specifically, when the ovulated eggs were stored for 5 hours or less, the fertilization rate was consistently high.

[0070] Furthermore, the hatching rate of the ovulated eggs and the rate of normal fry were both high, around 90% and 80%, respectively.

[0071] In this example, the fertilization rate, hatching rate, and normal fry rate have the following meanings, respectively. Fertilization rate (%) = number of fertilized eggs that developed into embryos ÷ total number of ovulated eggs used × 100 Hatching rate (%) = number of hatched larvae / total number of ovulated eggs used × 100 Normal larvae rate (%) = number of normal larvae without abnormalities ÷ total number of hatched larvae × 100

[0072] From the above, it has been found that the present invention makes it possible to efficiently obtain high-quality ovulated eggs of Japanese stag beetles, mass-produce seedlings through planned crossbreeding of superior individuals, and increase the number of microinjection treatments for sterilization in surrogate parent production.

Claims

1. a spawning time specifying step of specifying the spawning time of the barracuda; A suma transfer process in which suma determined to be in the late final maturation stage based on the spawning time are placed in a closed space; an ovulated egg recovery step of slaughtering the barracuda after the barracuda transfer step when no egg-laying behavior of the barracuda is observed and at a time later than the egg-laying time, to obtain ovulated eggs; Including, The horizontal diameter in the closed space is 2 to 4 times the minimum rotation diameter of the suma, How Suma produces ovulated eggs.

2. The method of claim 1, wherein the minimum rotation diameter of the suma is 2.5 to 3 times the body length of the suma.

3. 3. The method of claim 1, wherein in the step of transferring the stag beetles, the transfer of the stag beetles is carried out at a time that is at least one hour and at most three hours earlier than the spawning time.

4. The method according to claim 1 , wherein in the ovulated egg collection step, the barracuda is slaughtered within one hour from the time of egg laying.

5. The method according to any one of claims 1 to 4, wherein the age of the stag beetle is between 12 months and 60 months.

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