A culture method for regulating rotifer body size

Abstract

This application relates to the field of rotifer culture technology and discloses a method for regulating rotifer body size. This application successfully controls the body size of marine rotifers by regulating factors such as dissolved oxygen, temperature, salinity, and feed. Specifically, the conditions for large-scale rotifer culture include relatively low dissolved oxygen, temperature, and a larger feed, while the conditions for small-scale rotifer culture include relatively high dissolved oxygen, temperature, and a smaller feed. Certain cyanobacteria can also be used as supplementary feed. This application stably controls the environmental conditions for marine rotifer body size and selectively modifies the feed for the rotifers, achieving the goal of selectively regulating the body size of marine rotifers.

Description

Technical Field

[0001] This application relates to the field of rotifer culture technology, and more particularly to a culture method for regulating rotifer body size. Background Technology

[0002] In existing marine fish fry farming, fry generally go through the following developmental stages in sequence: hatching, initial feeding (rotifers), fry stage (artemia), and juvenile stage (formulated feed). The initial feeding stage is the most crucial: because the fry's digestive tract is not fully developed after hatching, they need to ingest exogenous nutrients to stimulate its development. Simultaneously, the supply of nutrients from the eggs is insufficient, requiring energy from exogenous nutrients to sustain life activities. The success of the initial feeding stage directly determines the fry's survival rate.

[0003] In existing marine rotifer farming, *Brachysaurus circumflexus* (commonly known as SS-type rotifers) and *Brachysaurus foldii* (commonly known as L-type rotifers) are the main farmed species. *Brachysaurus circumflexus* has a body size of about 120–220 μm during the egg-laying stage and is mainly used in the early stages of fry feeding, serving as the first exogenous nutrient intake for the fry. *Brachysaurus foldii* has a body size of about 240–320 μm during the egg-laying stage and is mainly used in the later stages of fry feeding, transitioning to the stage of feeding with brine shrimp.

[0004] The mouth size of fish fry determines the size of food they can ingest, and suitable food size is beneficial to fry development. The mouth size at the initial feeding stage is related to the fry species. Some species with smaller mouths at the initial feeding stage, such as grouper, typically have a mouth size of 80-140 μm, allowing them to only consume larvae of *Brachychophora*. However, the supply of *Brachychophora* larvae is often insufficient, and weaker grouper fry often have even smaller mouths, making it even more difficult for them to consume rotifers of suitable size. This results in a high hatching rate and low survival rate for grouper fry. Furthermore, since artichokes are already over 500 μm when they hatch, fry need to have a larger mouth before being fed artichokes. *Brachychophora* var. *placophora*, with a size of 240-320 μm, is too small for fry in the later stages of feeding. *Brachychophora* var. *placophora* itself tends to shrink during rearing. Inappropriate food size hinders energy accumulation in the fry, easily leading to decreased growth rate and reduced quality. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a method for regulating the body size of rotifers, so that the method can regulate the size of rotifers to match the size required by fish fry and adapt to the feed required at different stages of the fish fry's feeding period.

[0006] In order to solve the above-mentioned technical problems / achieve the above-mentioned objectives, or at least partially solve the above-mentioned technical problems / achieve the above-mentioned objectives, this application provides a method for regulating the body size of rotifers, including a method for miniaturizing rotifers and / or a method for enlarging rotifers.

[0007] The method for miniaturizing and culturing rotifers includes:

[0008] Provide the first seawater, adjust the salinity of the first seawater to 24‰-50‰, maintain the dissolved oxygen content above 6mg / L, and adjust the temperature to 30℃-40℃;

[0009] Rotifers are introduced into the first seawater for cultivation, fed with the first algae feed and the first algal density is maintained, and the salinity of the first seawater is increased by no more than 1‰ and / or the temperature of the first seawater is increased by no more than 1℃ per day, until the required salinity and temperature of the first seawater are reached for cultivation; wherein the salinity of the first seawater does not exceed 50‰ and the temperature does not exceed 40℃.

[0010] The method for large-scale rotifer culture includes:

[0011] Provide a second seawater, adjust the salinity of the second seawater to 10‰-16‰, maintain the dissolved oxygen content at no more than 6mg / L, and adjust the temperature to 15℃-20℃;

[0012] Rotifers are introduced into the second seawater for cultivation. They are fed with the second algae feed and the density of the second algae is maintained. The salinity of the second seawater is reduced by no more than 1‰ and / or the temperature of the second seawater is reduced by no more than 1℃ per day until the required salinity and temperature are reached. The second seawater salinity is not lower than 10‰ and the temperature is not lower than 15℃.

[0013] Optionally, the dissolved oxygen content of the first seawater is 6-16 mg / L, and the dissolved oxygen content of the second seawater is 5-6 mg / L.

[0014] Optionally, the first algae bait and the second algae bait are each independently selected from one or more of the following: *Chlorella vulgaris*, *Golden Algae*, *Chaetoceros*, *Phaeophyte Triangularis*, and *Microcystis aeruginosa*. More preferably, the first algae bait and the second algae bait are each independently selected from one or two of the following: *Chlorella vulgaris* and *Microcystis aeruginosa*.

[0015] Optionally, the diameter of the first algae bait is 1-4 μm, and the diameter of the second algae bait is 6-10 μm.

[0016] Optionally, the first algal feed further includes cyanobacteria with estrogen-like effects. More preferably, the cyanobacteria with estrogen-like effects include one or more of Anabaena, Nostoc, and Microcystis. More preferably, the algal density of the cyanobacteria with estrogen-like effects is maintained at 50 × 10⁻⁶.4 -100×10 4 cell / mL.

[0017] Optionally, the density of the first algae is 400 × 10⁻⁶. 4 -1200×10 4 cell / mL, the second algal density is 200×10 4 -400×10 4 cell / mL.

[0018] Optionally, the rotifers include Brachionus circumflex and Brachionus folded.

[0019] This application successfully controlled the body size of marine rotifers by regulating factors such as dissolved oxygen, temperature, salinity, and feed during their cultivation. Specifically, the conditions for cultivating large rotifers required relatively low dissolved oxygen and temperature, and a larger amount of feed, while the conditions for cultivating small rotifers required relatively high dissolved oxygen and temperature, and a smaller amount of feed. Certain cyanobacteria could also be used as supplementary feed. This application stably controlled the environmental conditions for the body size of marine rotifers and selectively modified the feed, thus achieving the goal of selectively controlling the body size of marine rotifers. Attached Figure Description

[0020] Figure 1 The image shown is a microscopic examination of small rotifers cultured using *Microcystis aeruginosa* in Example 2.

[0021] Figure 2 The image shown is a microscopic examination of small rotifers cultured using Micrococcus microcarpa and Anabaena in Example 3;

[0022] Figure 3 The image shown is a microscopic examination of large rotifers cultured using *Microcystis aeruginosa* in Example 4. Detailed Implementation

[0023] This application discloses a method for regulating the body shape of rotifers. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this application. The products, processes, and applications described in this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit, and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.

[0024] It should be noted that, in this document, relational terms such as "first" and "second," "step 1" and "step 2," and "(1)" and "(2)" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. Moreover, the embodiments and features described in this application can be combined with each other without conflict.

[0025] This application provides a method for regulating the size of marine rotifers, aiming to solve two major problems currently faced in this field: first, to address the problem that in the process of raising some marine fish fry, the fry have small mouths and the rotifers are too large due to the characteristics of the fish themselves, resulting in difficulty in the fry opening, leading to fry mortality and low breeding success rate; second, to address the problem that the size of medium and large rotifers becomes smaller during the breeding process, resulting in a mismatch between the size required by the fry and the fry size, which leads to a decrease in fry growth rate and reduced quality.

[0026] Previous research has shown that the size of marine rotifers is affected by dissolved oxygen, temperature, food type, and food size. Higher dissolved oxygen, higher temperature, smaller food size, and certain cyanobacteria can reduce rotifer size. This is likely because higher dissolved oxygen and smaller food reduce the accumulation of effective energy in rotifers, thus forcing them to become smaller. Some cyanobacteria (such as Anabaena) may contain regulatory substances that further reduce rotifer size. Conversely, lower dissolved oxygen, lower temperature, and larger food size can increase rotifer size. This is likely because, compared to the reduction in rotifer size under high dissolved oxygen, lower dissolved oxygen may cause less oxidative stress, allowing more energy to be stored rather than oxidized, thus increasing rotifer size. A larger body size reduces relative surface area, which effectively reduces heat loss, hence the tendency for rotifers to increase in size under low-temperature environments. Larger food with a suitable mouth diameter may reduce the energy rotifers expend when ingesting food, allowing more energy to be stored for body growth.

[0027] In actual marine rotifer culture, smaller rotifers are typically miniaturized, such as *Brachionus rotundiformis*, while larger rotifers are typically enlarged, such as *Brachionus plicatilis*, to improve culture efficiency. In some embodiments of this application, the initial density of *Brachionus rotundiformis* is 0.1–500 int / mL; the initial density of *Brachionus plicatilis* is 0.1–200 int / mL. Using the culture method of this application, the size of *Brachionus rotundiformis* can be stably reduced from the conventional 120–220 μm to 60–120 μm, or stably increased to about 260 μm; the size of *Brachionus plicatilis* can be stably increased from the conventional 240–320 μm to 280–400 μm, or stably reduced to about 160 μm.

[0028] The cultivation methods in this application include a method for miniaturizing rotifers and / or a method for enlarging rotifers, and the two cultivation methods can be carried out individually or simultaneously as needed.

[0029] In the first aspect, the method for miniaturizing rotifer culture includes:

[0030] The method for miniaturizing and culturing rotifers includes:

[0031] Provide the first seawater, adjust the salinity of the first seawater to 24‰-50‰, maintain the dissolved oxygen content above 6mg / L, and adjust the temperature to 30℃-40℃;

[0032] Rotifers are introduced into the first seawater for cultivation, fed with the first algae feed and the first algal density is maintained, and the salinity of the first seawater is increased by no more than 1‰ and / or the temperature of the first seawater is increased by no more than 1℃ per day, until the required salinity and temperature of the first seawater are reached for cultivation; wherein the salinity of the first seawater does not exceed 50‰ and the temperature does not exceed 40℃.

[0033] During the miniaturization process of rotifers, the salinity and temperature of the primary seawater can be increased simultaneously each day to rapidly reduce their size. However, daily changes exceeding 1‰ salinity and 1℃ will cause excessively rapid changes in salinity and temperature, negatively impacting rotifer health, hindering growth, and even causing death, thus leading to domestication failure. Increasing only one of these influencing factors can also achieve rotifer miniaturization, but it will take longer. The impact of temperature increases is greater than that of salinity increases. Salinity exceeding 50‰ and temperature exceeding 40℃ will be detrimental to rotifer growth.

[0034] In certain embodiments of this application, the salinity of the first seawater can be adjusted to any one of or any point between any two of the following: 24‰, 25‰, 26‰, 27‰, 28‰, 29‰, 30‰, 31‰, 32‰, 33‰, 34‰, 35‰, 36‰, 37‰, 38‰, 39‰, 40‰, 41‰, 42‰, 43‰, 44‰, 45‰, 46‰, 47‰, 48‰, 49‰, and 50‰. The temperature of the first seawater can be adjusted to any one of or any point between any two of the following: 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, and 40℃.

[0035] In some embodiments of this application, the dissolved oxygen content of the first seawater can be regulated by using nanostones or by introducing pure oxygen. Higher dissolved oxygen will promote further miniaturization of rotifers, but dissolved oxygen exceeding 16 mg / L begins to inhibit the growth of rotifers and affect the final rotifer yield. Therefore, this application controls the dissolved oxygen content of the first seawater to 6-16 mg / L, for example, any one or any two of the following values: 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, and 16 mg / L.

[0036] In some embodiments of this application, the first algal feed may be selected from one or more of *Chlorella vulgaris*, *Chlorella*, *Chaetoceros*, *Phaeolipophyta*, and *Microcystis*. In other embodiments of this application, normal culture conditions (without adjusting temperature, salinity, or dissolved oxygen) are used, focusing only on rotifer growth, with the final rotifer density under the same culture conditions used to reflect the nutritional differences among the algae. Results show that *Microcystis* and *Chlorella vulgaris* have higher nutritional value for rotifers and can be used as preferred microalgal feed for rotifer cultivation; therefore, the first algal feed is one or two of *Chlorella vulgaris* and *Microcystis*.

[0037] In some embodiments of this application, the diameter of the first algal bait is 1-4 μm, for example, any one of 1 μm, 2 μm, 3 μm, 4 μm, or any value between any two. A first bait diameter that is too low is not conducive to rotifer feeding, resulting in a low feed conversion rate; a first bait diameter that is too high will prevent rotifers from feeding, leading to rotifer death.

[0038] In some embodiments of this application, when feeding the first algal feed, the initial algal density is controlled at 200 × 10⁻⁶. 4 -800×10 4 cell / mL, then maintained at 400×10⁻⁶ 4 -1200×10 4cell / mL, which is the initial algal density maintained. Algal densities below the initial and maintenance levels are detrimental to rotifer growth, resulting in slow growth rates; algal densities above the initial and maintenance levels will inhibit rotifer growth and may even lead to death.

[0039] In some embodiments of this application, the first algal feed also includes cyanobacteria with estrogen-like effects. Cyanobacteria with estrogen-like effects can shorten the rotifer gestation period, reduce the diameter of rotifer eggs, and thus reduce the size of new-generation rotifers.

[0040] In some embodiments of this application, the cyanobacteria possessing estrogen-like effects include one or more of Anabaena, Nostoc, and Microcystis. In other embodiments of this application, using Chlorella marinea as microalgae feed, the effects of various cyanobacteria on rotifer growth and body size were investigated under normal culture conditions (without temperature, salinity, or dissolved oxygen control). The final size and density of rotifers under the same culture conditions were used to represent the effect of each algae on improving rotifer body size. The results showed that Anabaena was the best algae for improving rotifer body size (considering both final rotifer density and controlled body size).

[0041] In some embodiments of this application, the initial and maintenance algal cell density of the cyanobacteria with estrogen-like effects are both controlled at 50 × 10⁻⁶. 4 -100×10 4 A density of cell / mL below this level is not conducive to reducing the size of rotifers, while a density above this level inhibits rotifer population growth and may lead to an increase in the number of sexual generations of rotifers.

[0042] In the second aspect, the method for large-scale rotifer culture includes:

[0043] The method for large-scale rotifer culture includes:

[0044] Provide a second seawater, adjust the salinity of the second seawater to 10‰-16‰, maintain the dissolved oxygen content at no more than 6mg / L, and adjust the temperature to 15℃-20℃;

[0045] Rotifers are introduced into the second seawater for cultivation. They are fed with the second algae feed and the density of the second algae is maintained. The salinity of the second seawater is reduced by no more than 1‰ and / or the temperature of the second seawater is reduced by no more than 1℃ per day until the required salinity and temperature are reached. The second seawater salinity is not lower than 10‰ and the temperature is not lower than 15℃.

[0046] During the process of rotifer enlargement, the salinity and temperature of the second seawater can be decreased simultaneously each day to rapidly increase rotifer size. Daily changes exceeding 1‰ salinity and 1℃ will cause excessively rapid changes in salinity and temperature, negatively impacting rotifer health, hindering growth, and even causing death, leading to domestication failure. Decreasing only one of the influencing factors can also achieve rotifer enlargement, but it takes longer. The impact of temperature reduction is greater than that of salinity reduction. Salinity between 0-10‰ is detrimental to rotifer growth, especially below 5‰, where rotifers can die out within days; therefore, the salinity of the second seawater should not be lower than 10‰. At temperatures below 15℃, rotifer size may still tend to increase, but population growth will cease, meaning rotifer density will no longer increase.

[0047] In some embodiments of this application, the salinity of the second seawater can be adjusted to any one of 10‰, 11‰, 12‰, 13‰, 14‰, 15‰, and 16‰, or any value between any two. The temperature of the second seawater can be adjusted to any one of 15℃, 16℃, 17℃, 18℃, 19℃, and 20℃, or any value between any two.

[0048] In some embodiments of this application, the dissolved oxygen content of the second seawater can be regulated by using nanostone. Lower dissolved oxygen will promote the further growth of rotifers. Introducing pure oxygen will cause the dissolved oxygen to exceed 6 mg / L, which will cause the rotifers to shrink in size, which is not conducive to the growth of rotifers. Dissolved oxygen below 5 mg / L begins to inhibit the growth of rotifers. Therefore, this application controls the dissolved oxygen content of the second seawater at 5-6 mg / L.

[0049] In some embodiments of this application, the second algal bait may also be selected from one or more of the following: *Chlorella vulgaris*, *Golden Algae*, *Chaetoceros*, *Phaeophyta triangularis*, and *Microcystis aeruginosa*. Based on the effects of the aforementioned algal baits on the final density of rotifers, the second algal bait may also be one or two of *Chlorella vulgaris* and *Microcystis aeruginosa*.

[0050] In some embodiments of this application, the diameter of the second algal food is 6-10 μm, for example, any one or any value between 6 μm, 7 μm, 8 μm, 9 μm, and 10 μm. Too low a diameter of the second food will cause the rotifers to shrink in size, which is not conducive to their growth into larger sizes; too high a diameter of the second food will prevent the rotifers from feeding, leading to their death.

[0051] In some embodiments of this application, when feeding the second algae feed, the initial algal density is controlled at 100 × 10⁻⁶. 4 -300×10 4 cell / mL, then maintained at 200×10⁻⁶4 -400×10 4 cell / mL, or the maintenance second algal density. Algal densities below the initial and maintenance levels are detrimental to rotifer growth, resulting in slow growth rates; algal densities above the initial and maintenance levels will inhibit rotifer growth and may even lead to death.

[0052] In the study on rotifer enlargement and miniaturization in this application, the difference caused by changes in rotifer body size was not significant when the salinity was between 16‰ and 24‰; when the temperature was between 20℃ and 30℃, the body size tended to become smaller as the temperature increased, but the trend was relatively slow.

[0053] In the comparative experiments provided in this application, unless otherwise specified, all experimental conditions and materials remain consistent to ensure comparability. Furthermore, all materials used in this application are commercially available.

[0054] The following is a further explanation of the culturing method for regulating rotifer body shape provided in this application.

[0055] Example 1: Screening of algae feed

[0056] (1) Screening of bait microalgae

[0057] Marine Chlorella, golden algae, Chaetoceros, Browniana triangularis, and Micrococcus microcarpa were selected as nutrient sources for rotifers. The culture conditions were normal (no temperature, salinity, or dissolved oxygen control; natural culture in natural seawater). At this stage, only rotifer growth was considered, and the final rotifer density under the same culture conditions was used to reflect the nutritional differences among the algae. The results are shown in Table 1 below.

[0058] Table 1

[0059]

[0060]

[0061] According to the results in Table 1, Microcystis aeruginosa and Microcystis spp. have higher nutritional value for rotifers and can be used as feed algae for rotifer cultivation.

[0062] (2) Improved algae screening

[0063] Anabaena, Spirulina, Nostoc, and Microcystis, all exhibiting estrogen-like effects, were selected as algae for rotifer body size modification. Using Chlorella vulgaris as feed, the effects of these body size-modifying algae on rotifer growth and body size were investigated under normal culture conditions (no control of temperature, salinity, or dissolved oxygen; natural culture in natural seawater). The final size and density of rotifers under the same culture conditions were used to represent the effectiveness of each algae in improving rotifer body size. The results are shown in Table 2 below.

[0064] Table 2

[0065]

[0066] According to the results in Table 2, although Microcystis can reduce the size of rotifers to a lower 116 μm among the four algae, the final density of rotifers is significantly lower than that of the other three algae, so it is not given priority. Spirulina cannot reduce the size of rotifers, so it is not considered. Both Nostoc and Anabaena can reduce the size of rotifers and maintain a certain density, among which Anabaena is the most suitable algae for improving the size of rotifers.

[0067] Example 2: Cultivation of small rotifers using *Microcystis globulus*

[0068] Take a sample of natural seawater with a salinity of 30‰ and place it in a 300L rubber-lined water tank (76cm opening * 65cm bottom * 85cm height). Maintain the water level at approximately 60cm, at which point the salinity is suitable. Do not adjust the salinity. Introduce 120-220μm circular rotifers (Brachionus rotundiformis) to achieve a density of 122 int / mL. Maintain the water level at approximately 60cm throughout the process, increasing the salinity by 1‰ daily until it reaches 40‰. Maintain the salinity at 40‰.

[0069] Using nano air stones, air is introduced and the air output is adjusted to a slight boiling state. Pure oxygen is intermittently introduced to maintain the dissolved oxygen content in the water above 8 mg / L.

[0070] Inoculate with *Nannochloropsis* sp. (1-4 μm in diameter), mix well, and bring the algal density to 360 × 10⁻⁶. 4 The algal density was maintained at 400 × 10⁻⁶ cells / mL during the subsequent culture process. 4 -1200×10 4 cell / mL;

[0071] Place the heater in the tubing and maintain the water temperature at 30°C. Increase the temperature by 1°C each day until the water temperature reaches 38°C. Maintain the water temperature at 38°C for 28 days.

[0072] During the body size regulation period, which lasts for a long time, the water needs to be changed every 7 days. The specific water change method is as follows: put new water into a first-class large new bucket, adjust the temperature to the corresponding temperature of the original breeding bucket, stop the gas in the original breeding bucket for 2 hours, take out the live rotifers from the top layer, and put them into the new bucket. The remaining steps are carried out normally.

[0073] Rotifer size measurement method: Place 1 mL of rotifers in a 2 mL centrifuge tube, add 30-50 μL of Luger's reagent to fix the rotifers, wait 10 minutes for the rotifers to precipitate, and place the rotifers under an Osparing microscope. Rotifers with a length greater than or equal to the length of the smallest egg-bearing rotifer in the population are counted as adults. Measure and record the length of the adult rotifers to determine the rotifer size range. See [link to relevant documentation]. Figure 1 Ultimately, round Brachionus rotifers with an adult size of 75-108 μm were obtained.

[0074] Example 3: Cultivation of small rotifers using *Microcystis aeruginosa* and *Anabaena*

[0075] Take a quantity of natural seawater with a salinity of 30‰ and place it in a 300L rubber-lined water tank (76cm opening * 65cm bottom * 85cm height), maintaining the water level at approximately 60cm. At this point, the salinity is suitable and should not be adjusted. Introduce 120-220μm circular rotifers (Brachionus rotundiformis) to achieve a density of 114 int / ml. Maintain the water level at approximately 60cm throughout the process, increasing the salinity by 1‰ daily until it reaches 40‰, then maintain the salinity at 40‰.

[0076] Using nano air stones, air is introduced and the air output is adjusted to a slight boiling state. Pure oxygen is intermittently introduced to maintain the dissolved oxygen content in the water above 8 mg / L.

[0077] Inoculate with *Nannochloropsis* sp. with a diameter of 1-4 μm, mix well, and bring the algal density to 420 × 10⁻⁶. 4 The density of the *Microcystis aeruginosa* culture was maintained at 400*10⁻⁶ cells / mL during the subsequent culture process. 4 ~1200*10 4 cell / mL, simultaneously inoculated with 73×10 4 Anabaena sp. (cells / mL) was used to maintain the density of the algal solution at 50 × 10⁻⁶ cells / mL. 4 -100×10 4 cell / mL;

[0078] Place the heater in the water and maintain the water temperature at 30°C. Then increase the temperature by 1°C each day until the water temperature reaches 38°C. Maintain the water temperature at 38°C for 28 days.

[0079] During the body size regulation period, which lasts for a long time, the water needs to be changed every 7 days. The specific water change method is as follows: put new water into a first-class large new bucket, adjust the temperature to the corresponding temperature of the original breeding bucket, stop the gas in the original breeding bucket for 2 hours, take out the live rotifers from the top layer, and put them into the new bucket. The remaining steps are carried out normally.

[0080] Rotifer size measurement method: Place 1 mL of rotifers in a 2 mL centrifuge tube, add 30-50 μL of Luger's reagent to fix the rotifers, wait 10 minutes for the rotifers to precipitate, and place the rotifers under an Osparing microscope. Rotifers with a length greater than or equal to the length of the smallest egg-bearing rotifer in the population are counted as adults. Measure and record the length of the adult rotifers to determine the rotifer size range. See [link to relevant documentation]. Figure 2 Ultimately, round Brachionus rotifers with an adult size of 65-98 μm were obtained.

[0081] Example 3: Cultivation of large rotifers using *Microcystis globulus*

[0082] Take a quantity of natural seawater with a salinity of 30‰ and place it in a 5L beaker. Adjust the salinity to 16‰ using fresh water and adjust the water level to about 20cm. Introduce 240-320μm Brachionus plicatilis to achieve a density of 56 int / ml. Then maintain the water level at about 20cm and decrease the salinity by 1‰ each day until the salinity drops to 10‰. Maintain the salinity at 10‰.

[0083] Using nano air stones, air is introduced and the air output is adjusted to a slight boiling state to maintain the dissolved oxygen level in the water at 5-6 mg / L.

[0084] Inoculate with *Nannochloropsis* sp. (6-10 μm in diameter), mix well, and bring the algal density to 280 × 10⁻⁶. 4 The algal density was maintained at 200 × 10⁻⁶ cells / mL during the subsequent culture process. 4 -400×10 4 cell / mL;

[0085] Place the container in a constant temperature chamber and adjust the water temperature to around 20°C. Then, decrease the temperature by 1°C each day until it reaches 15°C and maintain this temperature for 18 days.

[0086] Due to the long duration of the body size regulation period, the water needs to be changed every 7 days. The specific water change method is as follows: put new water into a first-class large beaker, adjust the temperature to the corresponding temperature of the original beaker, stop the gas in the original beaker for 2 hours, take out the live rotifers from the top layer, and transfer them to the new bucket. The remaining steps are carried out normally.

[0087] Rotifer size measurement method: Place 1 mL of rotifers in a 2 mL centrifuge tube, add 30-50 μL of Luger's reagent to fix the rotifers, wait 10 minutes for the rotifers to precipitate, and place the rotifers under an Osparing microscope. Rotifers with a length greater than or equal to the length of the smallest egg-bearing rotifer in the population are counted as adults. Measure and record the length of the adult rotifers to determine the rotifer size range. See [link to relevant documentation]. Figure 3 Ultimately, rotifers with an adult size of 300-390 μm were obtained.

[0088] Example 4: Effects of different seawater salinity, temperature and dissolved oxygen on rotifer body size

[0089] (1) Rotifer miniaturization

[0090] Using Brachiopoda circularis (120-220μm) as the acclimatization target, natural seawater (initial seawater salinity 20‰, temperature 22-24℃) was selected and cultured according to the method of Example 2. The final temperature, salinity, dissolved oxygen and culture time parameters in Table 3 below were used. The average body size of the cultured rotifers is shown in Table 3.

[0091] Table 3

[0092]

[0093] As shown in Table 3, the higher the temperature, salinity, and dissolved oxygen, the smaller the rotifers become. The culture time is usually determined by the temperature, salinity, and dissolved oxygen levels, typically ranging from 15 to 30 days.

[0094] (2) Rotifer enlargement

[0095] Using Brachionus folds (240-320μm) as the acclimatization target, natural seawater (initial seawater salinity 20‰, temperature 22-24℃) was selected and cultured according to the method of Example 4. The final temperature, salinity, dissolved oxygen and culture time parameters in Table 4 are as follows. The average body size of the cultured rotifers is shown in Table 4.

[0096] Table 4

[0097]

[0098]

[0099] As can be seen from Table 4, the lower the temperature, salinity, and dissolved oxygen, the larger the rotifers become. The length of the culture time is usually determined by the temperature, salinity, and dissolved oxygen levels, typically ranging from 10 to 30 days.

[0100] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for regulating the body shape of rotifers, characterized in that, This includes methods for culturing rotifers in miniaturized and macroaturized forms; The method for miniaturizing and culturing rotifers includes: Provide the first seawater, adjust the salinity of the first seawater to 24‰-50‰, maintain the dissolved oxygen content at 6-16mg / L, and adjust the temperature at 30℃-40℃; Rotifers were introduced into the first seawater for cultivation, fed with the first type of algae feed, and the density of the first type of algae feed was maintained at 400 × 10⁶. 4 -1200×10 4 The cells / mL were increased daily, with the salinity of the first seawater not exceeding 1‰ and the temperature not exceeding 1℃, until the desired salinity and temperature of the first seawater were reached for cultivation. The salinity of the first seawater did not exceed 50‰ and the temperature did not exceed 40℃. The method for large-scale rotifer culture includes: Provide a second seawater, adjust the salinity of the second seawater to 10‰-16‰, maintain the dissolved oxygen content at 5-6 mg / L, and adjust the temperature to 15℃-20℃; Rotifers were introduced into the second seawater for cultivation, fed with a second type of algae feed, and the density of the second algae feed was maintained at 200 × 10⁶. 4 -400×10 4 The cells / mL of the second seawater are reduced by no more than 1‰ and the temperature of the second seawater is reduced by no more than 1℃ per day until the required salinity and temperature of the second seawater are reached. The second seawater salinity is not lower than 10‰ and the temperature is not lower than 15℃. The first and second algal feeds are independently selected from one or two of *Chlorella vulgaris* and *Microcystis aeruginosa*. The diameter of the first algal feed is 1-4 μm, and the diameter of the second algal feed is 6-10 μm. The first algal feed includes cyanobacteria with estrogen-like effects, which include one or more of *Anabaena*, *Nostoc*, and *Microcystis*. The thallus density of the cyanobacteria with estrogen-like effects is maintained at 50 × 10⁻⁶. 4 -100×10 4 cell / mL.

2. The cultivation method according to claim 1, characterized in that, The rotifers include Brachionus circumflex and Brachionus folded.

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