A rotifer micro-flow water steady-state continuous expansion culture method based on density-egg rate-ammonia nitrogen linkage regulation

Abstract

The present application relates to a kind of rotifer microflow water steady-state continuous expansion culture methods based on density-oviposition rate-ammonia nitrogen linkage control, suitable for the continuous supply of live bait in fry opening stage.The method is based on rotifer inoculation, microflow water change and a small amount of multiple feeding, and takes rotifer density, oviposition rate, total ammonia nitrogen and density variation coefficient as operation judgment index.When the total ammonia nitrogen in culture system increases, the oviposition rate decreases or the rotifer density fluctuates significantly in a short time, adjust the water change proportion, feeding frequency and harvesting proportion respectively.After the culture system reaches the preset steady-state condition, it is semi-continuous harvesting according to a certain proportion, and equal volume of new water and corresponding bait are added.The method can reduce the problems such as water quality deterioration, slow density recovery after harvesting and unstable continuous supply in fixed parameter culture.

Description

Technical Field

[0001] This invention belongs to the field of live feed cultivation technology in aquaculture, specifically involving a method for steady-state continuous propagation of rotifers in micro-flow water based on the linkage regulation of rotifer density, egg-bearing rate, total ammonia nitrogen and density variation coefficient, which is particularly suitable for the continuous and stable production of live rotifer feed during the fry feeding stage. Background Technology

[0002] Rotifers are a common live food source for many fish fry during their initial feeding stage. In fish breeding farms, rotifer cultivation is typically carried out using batch or semi-batch methods, meaning feeding, water changes, and harvesting are based on water color, rotifer density, and experience. This method is simple to operate, but when a continuous supply is required, fluctuations in culture density and short-term feed shortages can easily occur.

[0003] In actual cultivation, excessive feeding at a single time can lead to the accumulation of uneaten feed and metabolic products, resulting in increased total ammonia nitrogen. Insufficient water exchange reduces the egg-carrying rate of rotifers and weakens the population's recovery ability. Excessive water exchange or improper drainage methods can cause rotifers to be lost with the drainage. Existing methods mainly rely on fixed water exchange ratios and fixed feeding amounts, lacking a correlation assessment of the relationship between rotifer density, egg-carrying rate, total ammonia nitrogen, and population fluctuations, making it difficult to identify the true cause of the decline in rotifer density. Therefore, it is necessary to establish a control method suitable for continuous expansion cultivation, enabling the rotifer cultivation process to be dynamically adjusted according to population status and water quality changes. Summary of the Invention

[0004] The purpose of this invention is to provide a steady-state continuous propagation method for rotifers based on the linkage regulation of density, egg-bearing rate, and ammonia nitrogen. By establishing a linkage regulation mechanism of density, egg-bearing rate, ammonia nitrogen, and density variation coefficient, the traditional empirical rotifer propagation method is transformed into a steady-state production method with data reference.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for steady-state continuous propagation of rotifers in a microfluidic system based on density-egg-carrying rate-ammonia nitrogen linkage regulation includes the following steps: S1, Select rotifer germplasm for short-term pre-culture; S2, Inoculate the pre-cultured rotifers into a micro-flow culture container; S3, a micro-flow culture container maintains culture conditions and provides continuous micro-aeration; S4 sets the daily water exchange volume and daily feeding frequency; S5, rotifer density, egg-bearing rate and total ammonia nitrogen were detected at 9:00 every day, and the density variation coefficient during continuous culture was calculated. The density variation coefficient was calculated as the ratio of the standard deviation of the daily rotifer density to the average density during the stable culture phase. S6. Based on the changes in rotifer density, egg-bearing rate, total ammonia nitrogen, and density variation coefficient, the daily water exchange volume, feeding frequency, and harvesting ratio are adjusted accordingly. S7. Once the culture system reaches the steady-state determination condition, semi-continuous harvesting is carried out, and an equal volume of fresh water and corresponding feed are added to maintain the steady-state expansion of the rotifer population.

[0006] As a preferred option, S1 should be selected from rotifer sources that are vigorous, in normal reproductive condition, and have low levels of contamination by miscellaneous organisms.

[0007] Preferably, the rotifer is Brachycercus calycifolium (…). Brachionus calyciflorus The initial inoculation density is 80-120 ind. / mL, preferably 100 ind. / mL.

[0008] As a preferred embodiment, the S3 microfluidic culture container maintains a culture water temperature of 24-30 ℃, dissolved oxygen > 5.0 mg / L, and pH 7.3-8.0.

[0009] Preferably, S4 controls the daily water exchange volume to 15%-25% of the culture water volume and divides the total daily feeding into 4-6 feedings; the daily water exchange volume is preferably 20% of the culture water volume and the feeding frequency is preferably 4 times a day.

[0010] Preferably, the steady-state determination conditions include: rotifer density maintained at 250-350 ind. / mL for 3 consecutive days, egg-carrying rate >25%, total ammonia nitrogen <0.5 mg / L, and density variation coefficient <0.06; The density variation coefficient was calculated as the ratio of the standard deviation of the daily rotifer density to the average density during the stable culture phase.

[0011] As a preferred option, in S6... When the total ammonia nitrogen is 0.5-0.6 mg / L, adjust the daily water exchange rate to 25%. When the total ammonia nitrogen is >0.6 mg / L, adjust the daily water exchange rate to between 25% and 30%, and reduce or suspend feeding within 1-2 days.

[0012] As a preferred option, in S6... When the egg-carrying rate is <25% and the total ammonia nitrogen is <0.5 mg / L, the feeding frequency should be adjusted to 5-6 times per day; When the egg-carrying rate is <25% and the total ammonia nitrogen is >0.5 mg / L, increase the water exchange ratio and reduce the residual feed load.

[0013] As a preferred option, in S6... When the density of rotifers decreases by 15%-30% within 24 hours, suspend harvesting for 1-2 days, adjust the daily water exchange rate to 25%-30%, and reduce the total daily feeding amount by 10%-20%. When the rotifer density decreases by more than 30% within 24 hours, it is considered a culture instability and an abnormal treatment is initiated.

[0014] As a preferred option, in S7, when the rotifer density reaches 280-350 ind. / mL, the egg-bearing rate is >25%, and the total ammonia nitrogen is <0.5mg / L, 10%-25% of the culture water volume is harvested daily. After harvesting, an equal volume of fresh water that has been aerated and temperature-controlled is added, and the corresponding feed is supplemented according to the remaining rotifer density and the volume of water added to maintain the culture system in a stable state.

[0015] The present invention has the following advantages: 1. A steady-state assessment criterion suitable for continuous expansion culture has been established. This invention uses rotifer density, egg-bearing rate, total ammonia nitrogen, and density variation coefficient as indicators for judging the stable operation of the system, rather than relying solely on water color, daily density, or production experience to determine the culture status. By simultaneously observing population size, reproductive status, water metabolic load, and density fluctuations, it is possible to more accurately determine whether the rotifer system is suitable for entering the continuous harvesting stage.

[0016] 2. This invention enables tiered regulation based on the causes of anomalies. It adjusts the water exchange ratio, feeding frequency, feed composition, and harvesting ratio according to specific circumstances such as increased total ammonia nitrogen, decreased egg-bearing rate, and short-term decrease in rotifer density. This method helps reduce culture instability caused by untimely or inappropriate adjustments.

[0017] 3. Facilitates a stable and continuous supply of rotifers. This invention combines low-intensity micro-flow water, a drainage structure to prevent rotifer loss, semi-continuous harvesting, equal-volume water replenishment, and appropriate supplemental feeding. This reduces the accumulation of uneaten feed and metabolic products while minimizing the risk of rotifer loss during water changes. Once the culture system reaches steady state, continuous harvesting can be carried out while maintaining a certain baseline density and egg-bearing rate, suitable for the continuous supply of live rotifers during the fry feeding stage.

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the microfluidic culture container structure according to an embodiment of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] The present invention discloses a method for steady-state continuous propagation of rotifers in a microfluidic system based on density-egg-carrying rate-ammonia nitrogen linkage regulation, comprising the following steps: S1, Experimental Facilities and Seed Source Preparation. Select easy-to-operate incubation containers ( Figure 1 (As shown) is used as a container for rotifer propagation. Before use, the culture container is cleaned and disinfected, and rinsed thoroughly with clean water. The culture water is aerated in advance and adjusted to near the culture temperature. Rotifers with active swimming, a high proportion of egg-bearing individuals, and low contamination by miscellaneous organisms are selected as the germplasm. Pre-culture is carried out before formal inoculation to ensure that the germplasm is in a relatively consistent state.

[0022] S2, Inoculation. Before inoculation, thoroughly mix the rotifer stock and determine the stock density using a microscopic counting method, counting in triplicate and taking the average value as the basis for calculation. Calculate the inoculation amount based on the effective water volume of the hatching tank and the target initial density, ensuring the initial inoculation density is controlled at 80-120 ind. / mL, preferably 100 ind. / mL. After inoculation, maintain slight aeration to ensure even distribution of rotifers in the water and avoid localized high-density aggregation.

[0023] S3, Environmental Control. During cultivation, maintain a water temperature of 24-30 ℃, dissolved oxygen > 5.0 mg / L, and pH 7.3-8.0. Use weak artificial light and continuous micro-aeration. The intensity of micro-aeration should be such that the water body slowly and evenly agitates, rotifers do not locally settle, and there is no violent rolling.

[0024] S4, Micro-flow water exchange and feeding. During the initial stage of cultivation, the daily water exchange volume should be 15%-25% of the cultivation water volume, preferably 20%. Feed should be yeast-based, 4-6 times daily, preferably 4 times daily. Before feeding, thoroughly mix the feed and evenly sprinkle it along the hatching tank.

[0025] S5, Routine Monitoring and Steady-State Assessment. Rotifer density, egg-carrying rate, total ammonia nitrogen, pH, dissolved oxygen, and water temperature are monitored daily at 9:00 AM. Rotifer density, egg-carrying rate, total ammonia nitrogen, and density coefficient of variation are used as indicators for steady-state assessment. Rotifer density is used to determine population size, egg-carrying rate reflects reproductive status, total ammonia nitrogen reflects the metabolic load on the water body, and density coefficient of variation is used to evaluate the degree of population fluctuation during continuous culture.

[0026] S6, Anomaly Detection and Coordinated Control. Based on monitoring results, the water exchange ratio, feeding frequency, and harvesting ratio are adjusted in a coordinated manner. Specifically, this includes: (1) if total ammonia nitrogen is 0.5-0.6 mg / L, increase the daily water exchange rate to 25%; (2) if total ammonia nitrogen is >0.6 mg / L, increase the daily water exchange rate to 25%-30%, and reduce or suspend feeding for 1-2 days; (3) if the egg-bearing rate is <25% and total ammonia nitrogen is <0.5 mg / L, adjust the feeding frequency to 5-6 times per day; (4) if the egg-bearing rate is <25% and total ammonia nitrogen is >0.5 mg / L, increase the daily water exchange rate to 25%, and promptly remove any remaining feed; (5) if the rotifer density decreases by 15%-30% within 24 hours, suspend rotifer harvesting, increase the daily water exchange rate to 25%-30%, and reduce the total daily feeding amount by 10%-20%; (6) if the rotifer density decreases by >30% within 24 hours, determine that the culture system is unstable, and stop harvesting. When the rotifer density is 250-350 ind. / mL for 3 consecutive days, the egg-bearing rate is >25%, the total ammonia nitrogen is <0.5 mg / L, the density variation coefficient is <0.06, and the density decrease is <30% in 24 h, the culture system is considered to have entered the steady-state expansion stage.

[0027] S7. Once the culture system reaches the steady-state criteria, semi-continuous harvesting is carried out. Rotifer density is 280-350 ind. / mL, egg-bearing rate >25%, and total ammonia nitrogen <0.5 mg / L. Harvest 10%-25% of the culture water volume daily, replenishing with an equal volume of aerated and temperature-controlled fresh water. Supplement with appropriate feed based on the remaining rotifer density and the replenished water volume to maintain the culture system in a steady state.

[0028] General test conditions In the following examples, unless otherwise specified, rotifer culture was conducted using hatching tanks of the same size, with three replicates for each treatment. Continuous micro-aeration was maintained during culture, with a water temperature of 24-30 ℃, dissolved oxygen > 5.0 mg / L, and pH 7.3-8.0.

[0029] Rotifer density was determined using a microscopic counting method. The culture water was thoroughly mixed before sampling, and each sample was counted three times in parallel; the average value was taken as the sample density. The egg-bearing rate was calculated as the proportion of egg-bearing rotifers to the total number of observed rotifers. Total ammonia nitrogen was measured using a water quality analyzer (CIMA AR8407).

[0030] The density variation coefficient during the stable period was calculated as the ratio of the standard deviation of the daily rotifer density to the average density during the stable culture phase. Daily and cumulative yields were calculated based on the daily harvest volume, rotifer density at harvest, and the number of consecutive days of operation.

[0031] Example 1: Screening of microfluidic water exchange intensity Brachiopoda calyx ( Brachionus calyciflorusUsing rotifers as the experimental subject, four treatment groups were set up: no water change, 10% daily water change, 20% daily water change, and 30% daily water change. Each group had three replicates, and the experimental period was 10 days. The detection indicators included rotifer density, coefficient of variation of density during the stable period, average egg-carrying rate, total ammonia nitrogen, daily yield, cumulative yield, and number of unstable records. The results are shown in Table 1.

[0032] Table 1. Effects of different water exchange ratios on rotifer propagation. Table 1 shows that different water exchange ratios have a significant impact on the stability of rotifer populations. In the no-water-change treatment, total ammonia nitrogen increases rapidly during cultivation, rotifer density fluctuates greatly, and there are many records of instability.

[0033] When the daily water exchange rate was 10%, water quality and population stability improved compared to the no-water-exchange treatment, but the average egg-carrying rate and daily yield remained low. Increasing the daily water exchange rate to 20% resulted in higher rotifer density, egg-carrying rate, and daily yield, while TAN remained at a low level. Further increasing the daily water exchange rate to 30% did not lead to further increases in rotifer density and yield, indicating that excessively high water exchange rates do not necessarily result in higher propagation effects.

[0034] Therefore, under the conditions of this experiment, a daily water exchange rate of 20% can achieve a good balance between water quality control and rotifer population maintenance, and is suitable as the basic water exchange condition for subsequent experiments.

[0035] Example 2: Initial inoculation density screening Under the 20% daily water exchange condition selected in Example 1, three treatment groups were set up with initial inoculation densities of 50, 100, and 150 ind. / mL, with three replicates in each group, and the experimental period was 10 days. The results are shown in Table 2.

[0036] Table 2. Effects of different initial inoculation densities on the propagation efficiency of rotifers. Table 2 shows that the initial inoculation density affects the speed at which the rotifer population enters the stable expansion phase and the subsequent fluctuations. The initial population size of the 50 ind. / mL treatment was low, and although the TAN level was low, the average density and cumulative yield during the culture period were also low.

[0037] The 150 ind. / mL treatment provides a high basal density in the early stages, but the TAN level increases in the later stages, the CV level increases during the stationary phase, and the oocyte-carrying rate decreases, which is not conducive to long-term stable expansion culture.

[0038] The 100 ind. / mL treatment showed good performance in terms of rotifer density, egg-bearing rate, TAN, and yield, ensuring a certain initial population size without increasing water load. Therefore, this invention preferably controls the initial inoculation density at around 100 ind. / mL.

[0039] Example 3: Feeding Frequency Screening Under conditions of 20% daily water exchange and an inoculation density of 100 ind. / mL, three treatment groups were set up for feeding twice, four times, and six times a day. The total daily feed amount was the same for all groups, and only the feeding frequency was adjusted. The results are shown in Table 3.

[0040] Table 3. Effects of different feeding frequencies on the propagation effect of rotifers. As shown in Table 3, when feeding twice a day, the TAN level is relatively high, while the egg-bearing rate and daily yield are low.

[0041] The data from feeding 4 times a day and 6 times a day are not significantly different. However, considering that 6 times a day requires more manpower, feeding 4 times a day is more suitable as a routine operating parameter. When the egg-bearing rate decreases or the water body is underutilized, the frequency can be temporarily increased to 5-6 times a day.

[0042] Example 4: Continuous Culture Comparison Experiment The optimal parameter combination obtained from Examples 1 to 3, namely 20% daily water exchange, 100 ind. / mL initial inoculation density, and 4-6 feedings per day, was compared with the existing conventional rotifer culture method at the station for 30 days of continuous culture. The results are shown in Table 4.

[0043] Table 4 Results of the 30-day continuous culture comparative experiment As shown in Table 4, compared with the conventional rotifer culture method in the station, the average density, egg-bearing rate, daily yield and cumulative yield of rotifers were improved by adopting a steady-state expansion culture process with 20% daily water change, an initial inoculation density of 100 ind. / mL and feeding 4-6 times a day.

[0044] Example 5: Comparative Experiment of Fixed Parameter Method and Dynamic Linkage Control Method This embodiment included a fixed-parameter group and a dynamic control group, with continuous culture for 45 days. The fixed-parameter group used a 20% daily water change, an initial inoculation density of 100 ind. / mL, and feeding four times a day throughout the process. The dynamic control group, based on the above, adjusted the water change ratio, feeding frequency, feed composition, and harvesting ratio according to total ammonia nitrogen, egg-bearing rate, rotifer density, and density variation coefficient. The experimental results are shown in Table 5.

[0045] Table 5 Comparison of Fixed Parameter Method and Dynamic Linkage Control Method As shown in Table 5, under the same basic parameter conditions, the average density, egg-bearing rate, average daily yield and cumulative harvest of the dynamic control group were higher than those of the fixed parameter group, while the peak TAN, stable period CV and number of instabilities were lower than those of the fixed parameter group.

[0046] The fixed parameter group used 20% daily water changes, an inoculation density of 100 ind. / mL, and feeding 4 times a day. However, after the continuous culture time was extended, TAN levels increased and density fluctuations increased, indicating that fixed water changes and fixed feeding were difficult to adapt to changes in the rotifer population status in a timely manner.

[0047] The dynamic control group adjusted the water exchange ratio, feeding frequency, and harvesting ratio in a timely manner based on changes in TAN, egg-bearing rate, and rotifer density. When the water load increased, the water exchange ratio was increased first, and the high-load feed was reduced; when the egg-bearing rate decreased but TAN did not increase, the feeding frequency was changed; when the density decreased, harvesting was reduced or suspended. This treatment method reduced the time of continuous deterioration caused by a single factor, thus resulting in better culture stability.

[0048] The specific control methods of the dynamic control group include: (1) Total ammonia nitrogen 0.5-0.6 mg / L, daily water exchange rate increased to 25%; (2) If total ammonia nitrogen > 0.6 mg / L, increase the daily water exchange rate to 25%-30% and reduce or suspend feed feeding for 1-2 days; (3) If the egg-bearing rate is <25% and the total ammonia nitrogen is <0.5 mg / L, the feeding frequency should be adjusted to 5-6 times per day; (4) If the egg-carrying rate is <25% and the total ammonia nitrogen is >0.5 mg / L, the daily water exchange rate should be increased to 25%, and feed residue should be removed in a timely manner; (5) If the density of rotifers decreases by 15%-30% within 24 hours, rotifer harvesting should be suspended, the daily water exchange should be increased to 25%-30%, and the total daily feed should be reduced by 10%-20%. (6) If the density of rotifers decreases by more than 30% within 24 hours, the culture system is deemed unstable and harvesting should be stopped.

[0049] Example 6: Test on the effectiveness of drainage structure for preventing rotifer runoff Both a regular drainage system and a runoff prevention filter system were installed, with a 20% daily water change rate for 10 consecutive days. The runoff prevention filter system had a removable filter screen with a pore size of 50-100 μm at the drainage end and a bottom drain outlet for discharging sediment. The test results are shown in Table 6.

[0050] Table 6 Comparison of the effectiveness of drainage structures for preventing rotifer runoff As shown in Table 6, after installing a filter to prevent rotifer loss at the drainage end, the rotifer loss rate per water change and the estimated cumulative loss rate over 10 days were significantly reduced, and the density recovery rate after water change was improved. Under normal drainage methods, some rotifers will be directly discharged with the water change, resulting in some cumulative losses.

[0051] Example 7: Screening Test of Semi-Continuous Harvesting Ratio After the rotifer culture system reached steady state, daily harvesting volumes of 10%, 15%, 20%, 25%, and 30% of the culture water were set, respectively. After harvesting, an equal volume of fresh water was added, and the remaining feed was replenished according to the density. This process was repeated for 30 days, and the cumulative harvest was an estimated value calculated based on the average density, daily harvesting ratio, and number of consecutive days of operation. The experimental results are shown in Table 7.

[0052] Table 7. Effects of different semi-continuous harvesting ratios on steady-state propagation. Table 7 shows that the semi-continuous harvesting ratio affects both the stability of the rotifer population and the cumulative harvest. When 10% is harvested daily, the culture system has a higher average density and a lower CV, but the cumulative harvest is relatively low.

[0053] When 15%-20% of the rotifers are harvested daily, the average density, egg-bearing rate, TAN, and CV during the stationary phase are relatively balanced, and the cumulative harvest is significantly increased.

[0054] When the daily harvest rate increases to 25% or more, the harvest volume continues to increase in the short term, but the average density of rotifers and the egg-bearing rate decrease, the CV during the stable period increases, and the number of instabilities increases.

[0055] in conclusion The above examples demonstrate that a single fixed parameter is insufficient to ensure the long-term stability of the culture system during continuous rotifer propagation. Appropriate daily water exchange, initial inoculation density, and feeding frequency can improve the basic conditions for rotifer propagation, but as the cultivation time increases, the rotifer population will still be affected by factors such as water load, feed utilization, harvesting intensity, and drainage loss.

[0056] This invention uses 20% daily water exchange, an initial inoculation density of approximately 100 ind. / mL, and feeding 4-6 times daily as basic operating conditions. Furthermore, it introduces rotifer density, egg-bearing rate, total ammonia nitrogen (TAN), and density variation coefficient as indicators for steady-state assessment. When the culture system exhibits elevated TAN levels, decreased egg-bearing rate, or a short-term decrease in density, the water exchange ratio, feeding frequency, and harvesting ratio are adjusted according to the specific causes of the anomalies.

[0057] Using the above method, the rotifer culture system can achieve semi-continuous harvesting under conditions of low water quality load and minimal density fluctuations. This method is suitable for production scenarios in fish breeding stations where there is a continuous supply of live rotifer feed.

Claims

1. A method for steady-state continuous propagation of rotifers in a microfluidic environment based on density-egg-carrying rate-ammonia nitrogen linkage regulation, characterized in that, Includes the following steps: S1, Select rotifer germplasm for short-term pre-culture; S2, Inoculate the pre-cultured rotifers into a micro-flow culture container; S3, a micro-flow culture container maintains culture conditions and provides continuous micro-aeration; S4 sets the daily water exchange volume and daily feeding frequency; S5, rotifer density, egg-bearing rate and total ammonia nitrogen were detected at 9:00 every day, and the density variation coefficient during continuous culture was calculated. The density variation coefficient was calculated as the ratio of the standard deviation of the daily rotifer density to the average density during the stable culture phase. S6. Based on the changes in rotifer density, egg-bearing rate, total ammonia nitrogen, and density variation coefficient, the daily water exchange volume, feeding frequency, and harvesting ratio are adjusted accordingly. S7. Once the culture system reaches the steady-state determination condition, semi-continuous harvesting is carried out, and an equal volume of fresh water and corresponding feed are added to maintain the steady-state expansion of the rotifer population.

2. The method for steady-state continuous propagation of rotifers in a microfluidic environment based on density-egg-carrying rate-ammonia nitrogen linkage regulation according to claim 1, characterized in that, S1 requires selecting rotifer strains that are vigorous, in normal reproductive condition, and have low levels of contamination from other organisms.

3. The method for steady-state continuous propagation of rotifers in a microfluidic environment based on density-egg-carrying rate-ammonia nitrogen linkage regulation according to claim 1, characterized in that, The rotifer is Brachycephala calyx ( ). Brachionus calyciflorus The initial inoculation density is 80-120 ind. / mL, preferably 100 ind. / mL.

4. The method for steady-state continuous propagation of rotifers in a microfluidic environment based on density-egg-carrying rate-ammonia nitrogen linkage regulation according to claim 1, characterized in that, The S3 microfluidic culture vessel maintains a culture water temperature of 24-30 ℃, dissolved oxygen > 5.0 mg / L, and pH 7.3-8.

0.

5. The method for steady-state continuous propagation of rotifers in a microfluidic environment based on density-egg-carrying rate-ammonia nitrogen linkage regulation according to claim 1, characterized in that, S4 controls the daily water exchange volume to 15%-25% of the culture water volume and divides the total daily feeding into 4-6 feedings; the daily water exchange volume is preferably 20% of the culture water volume and the feeding frequency is preferably 4 times a day.

6. The method for steady-state continuous propagation of rotifers in a microfluidic environment based on density-egg-carrying rate-ammonia nitrogen linkage regulation according to claim 1, characterized in that, The steady-state determination conditions include: rotifer density maintained at 250-350 ind. / mL for 3 consecutive days, egg-carrying rate >25%, total ammonia nitrogen <0.5 mg / L, and density variation coefficient <0.06; The density variation coefficient was calculated as the ratio of the standard deviation of the daily rotifer density to the average density during the stable culture phase.

7. The method for steady-state continuous propagation of rotifers in a microfluidic environment based on density-egg-carrying rate-ammonia nitrogen linkage regulation according to claim 1, characterized in that, In S6, When the total ammonia nitrogen is 0.5-0.6 mg / L, adjust the daily water exchange rate to 25%. When the total ammonia nitrogen is >0.6 mg / L, adjust the daily water exchange rate to between 25% and 30%, and reduce or suspend feeding within 1-2 days.

8. The method for steady-state continuous propagation of rotifers in a microfluidic environment based on density-egg-carrying rate-ammonia nitrogen linkage regulation according to claim 1, characterized in that, In S6, When the egg-carrying rate is <25% and the total ammonia nitrogen is <0.5 mg / L, the feeding frequency should be adjusted to 5-6 times per day; When the egg-carrying rate is <25% and the total ammonia nitrogen is >0.5 mg / L, increase the water exchange ratio and reduce the residual feed load.

9. The method for steady-state continuous propagation of rotifers in a microfluidic environment based on density-egg-carrying rate-ammonia nitrogen linkage regulation according to claim 1, characterized in that, In S6, When the density of rotifers decreases by 15%-30% within 24 hours, suspend harvesting for 1-2 days, adjust the daily water exchange rate to 25%-30%, and reduce the total daily feeding amount by 10%-20%. When the rotifer density decreases by more than 30% within 24 hours, it is considered a culture instability and an abnormal treatment is initiated.

10. The method for steady-state continuous propagation of rotifers in a microfluidic environment based on density-egg-carrying rate-ammonia nitrogen linkage regulation according to claim 1, characterized in that, In S7, when the rotifer density reaches 280-350 ind. / mL, the egg-bearing rate is >25%, and the total ammonia nitrogen is <0.5 mg / L, 10%-25% of the culture water volume is harvested daily. After harvesting, an equal volume of fresh water that has been aerated and temperature-controlled is added. Appropriate feed is added according to the remaining rotifer density and the volume of water added to maintain the culture system in a stable state.

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