Simple, convenient and efficient cryopreservation and resuscitation method for rumen ciliates

Abstract

The invention belongs to the technical field of protozoa preservation. Relates to a cryopreservation and resuscitation method of rumen ciliates. Comprising the following steps: preparing a special cryopreservation culture medium; gathering insect bodies through centrifugation; mixing the cryopreservation protection liquid with the enriched polypide according to a ratio, and slowly and preliminarily freezing in a refrigerator at the temperature of 80 DEG C below zero; transferring into liquid nitrogen for long-term cryopreservation after staying overnight; during resuscitation, preparing a resuscitation culture medium, taking out the cryopreservation tube from the liquid nitrogen, and quickly placing the cryopreservation tube in a water bath at 39 DEG C for unfreezing; centrifugally washing to remove the cryopreservation protective agent and re-enriching the polypide; and inoculating the polypide into a resuscitation culture medium, and culturing in a constant-temperature incubator at 39 DEG C. A standardized cryopreservation resuscitation process of the internal hair rumen ciliates is established, and the operation is simple, convenient and rapid; the survival rate of the resuscitated ciliates is remarkably improved, and the highest survival rate of the mixed species can reach 58.1% through verification; particularly, the highest survival rate of a single species of incarnaeus incarnaeus can reach 66.4%; the resuscitated ciliates can quickly recover and proliferate; the method is simple, rapid and good in repeatability.

Description

Technical Field

[0001] This invention belongs to the field of protozoan preservation technology. Specifically, this invention relates to a simple and efficient method for cryopreservation and revival of rumen ciliates. Background Technology

[0002] Rumen ciliates are an important component of rumen microorganisms, accounting for 50% of the total rumen microbial biomass. Rumen ciliates interact with rumen prokaryotic microorganisms such as bacteria and archaea, playing a vital role in degrading plant cellulose, regulating rumen fermentation rates, stabilizing the rumen environment, and promoting rumen methane emissions.

[0003] Rumen ciliates inhabit the complex rumen microenvironment. Their demanding growth requirements—strict anaerobic conditions, specific temperature and pH levels, dependence on symbiotic prokaryotic microorganisms, and slow growth—make maintaining their in vitro culture in the laboratory extremely difficult. Currently, in vitro culture of rumen ciliates mainly employs subculturing. However, long-term studies have found that the in vitro subculturing time for rumen ciliates typically does not exceed 24 months. After 12-18 months of culture, a significant decline in ciliate viability is observed, followed by an irreversible decline until death, making long-term stable subculturing difficult. Furthermore, prolonged in vitro subculturing is not only tedious and time-consuming, but also prone to contamination, functional degradation, and even the accumulation of mutations during multiple subculturing processes, leading to alterations in the original biological and metabolic characteristics of the worms and making long-term stable preservation impossible.

[0004] Cryopreservation is an effective method for the long-term stable preservation of various cell types, and it has also been used for the long-term preservation of rumen ciliates. Currently, cryopreservation methods for rumen ciliates are mainly divided into one-step cryopreservation and two-step cryopreservation. As early as 1989, Marcin used a one-step cryopreservation method for the rumen ciliate *Eutheropoda simplex* (Eutheropoda simplex). Entodinium simplex The cells were cryopreserved using 0.38 mol / L DMSO as the cryoprotectant, with a cryopreservation concentration of 80,000 cells / mL. The cells were incubated at 39°C for 30 minutes to equilibrate and allow the cryoprotectant to fully interact with the cells. The cells were then slowly cooled to -100°C at a rate of 2–3°C / min, and finally transferred directly to liquid nitrogen for long-term storage. The optimal survival rate of the *E. simpleta* after thawing and culturing was 15%. Subsequently, Marcin et al. used a one-step cryopreservation method for *E. acuminata* (…). Entodinium caudatumWhile cryopreservation of this type of cell was attempted, the survival rate was only 7.5%, significantly lower than the cryopreservation results of other cell types. Further, Kišidayová et al. (1995) improved the one-step cryopreservation method for *Uterus acuminata*, establishing a two-step cryopreservation method. In this method, the cryopreserved *Uterus acuminata* cells were incubated in a water bath at 25°C for 5 minutes, then slowly cooled to approximately -25°C at a rate of 1.5–3°C / min and maintained at this temperature for 45 minutes. Finally, the cells were rapidly immersed in liquid nitrogen for preservation. This two-step method successfully increased the cell survival rate significantly from 7%–15% in the one-step method to 36%, and demonstrated that the cells could still recover their growth activity after one year of storage in liquid nitrogen. However, although the two-step cryopreservation method significantly improved the survival rate of *Urinaria spicata* after cryopreservation, it was found in the subsequent culture process that although a large number of active *Urinaria spicata* could be observed after thawing, the number of *Urinaria spicata* that could proliferate after resuscitation culture was extremely small, resulting in a decrease in the number of worms in the early stage of resuscitation culture. Nsabimana et al. (2003) further optimized a two-step cryopreservation method for rumen ciliates. This method uses a specific species of rumen ciliates from the rumen of a single protozoan rumen as cells, incubates them with the cryoprotectant DMSO at 25°C for 5 minutes to equilibrate, then slowly cools them at a rate of 1.2-2.5°C / min until extracellular ice crystals form, maintains them at -30°C for 45 minutes, and finally places them in liquid nitrogen for preservation. During thawing, fresh rumen fluid is used as the thawing medium for rapid thawing at 39°C, and after thawing, the ciliates are re-inoculated into the rumen of a protozoan-free rumen for culture. Although this method ultimately increased the cell survival rate of cryopreserved rumen ciliates to 60%, it is not strictly speaking a cryopreservation and thawing method for in vitro cultured rumen ciliates.

[0005] Although previous studies have explored methods for the cryopreservation and resuscitation of rumen ciliates cultured in vitro, both one-step and two-step cryopreservation methods suffer from extremely low survival rates after cryopreservation. Furthermore, precise control of the cooling rate during freezing relies on specialized programmed freezing equipment, which is expensive, complex, time-consuming, and requires a high level of technical expertise, significantly limiting its widespread adoption and large-scale application in ordinary laboratories. Therefore, there is a need in this field to establish a simple and efficient cryopreservation and resuscitation method for rumen ciliates cultured in vitro to solve the technical challenge of long-term stable preservation of rumen ciliate resources. Summary of the Invention

[0006] The purpose of this invention is to provide a method for cryopreservation and revival of rumen ciliates. This method significantly improves the survival rate and continuous proliferation and growth capacity of rumen ciliates after cryopreservation and revival, and can preserve live rumen ciliate resources for a long time.

[0007] This invention provides a method for cryopreservation and thawing of rumen ciliates, wherein cryopreservation includes the following steps: ① The source is a single or mixed ciliate in vitro culture system in the logarithmic growth phase; the ciliates in the culture system are filtered using a nylon filter to remove culture medium residue and other large particulate impurities to obtain a ciliate enrichment solution; the source is fresh rumen fluid, the fresh rumen fluid is settled through a separatory funnel and then filtered using a nylon filter to obtain a ciliate enrichment solution. ② Centrifuge the ciliate enrichment solution obtained in step ①, remove the supernatant, retain the precipitate, and obtain the enriched ciliates; ③ Add the pre-prepared and pre-cooled cell cryopreservation solution to the enriched worms obtained in step ② and resuspend them to obtain a cryopreserved ciliate suspension. Then, dispense the cryopreserved ciliate suspension into cell cryopreservation tubes. ④ Place the cell cryovials containing the cryopreserved ciliate suspension obtained in step ③ in a programmed cooling box and freeze overnight at -80°C.

[0008] The specific operation of step ④ is as follows: 1) Place the cell cryovials containing the cryopreserved ciliate suspension into the programmed cooling box; 2) Store the frozen cells in a programmed cooling box with a cooling rate of 1℃ / minute and freeze them in a -80℃ freezer for 12-24 hours; 3) Transfer the cell cryopreservation tubes containing the cryopreserved ciliate suspension in the programmed cooling box to liquid nitrogen for preservation.

[0009] The cell cryopreservation solution was prepared by adding the following components to a modified SP medium: 5-10% fetal bovine serum, 5-10% horse serum, 4-6% dimethyl sulfoxide, and 0.2-0.8M trehalose, all based on final concentrations; stored at 4°C for later use.

[0010] Each 10 mL of modified SP medium contains the following components: 1 mL of protozoan-free rumen fluid, 200 μL of 1% cysteine ​​hydrochloride solution, 200 μL of antibiotic solution, and 8.6 mL of SP salt solution.

[0011] ⑤ Place the cell cryopreservation tubes in liquid nitrogen for cryopreservation.

[0012] In step ①, the mesh size of the nylon filter screen is 125-300 mesh.

[0013] In step ②, the centrifugation speed is 500-800 r / min and the centrifugation time is 8-10 min.

[0014] In step ③, the ciliates were resuspended and enriched using cell cryopreservation solution, and the ciliate density was adjusted to ≥1×10⁻⁶. 6 per mL.

[0015] The following steps are included in the resuscitation method for the cryopreserved rumen ciliates: ⑥ Remove the cell cryopreservation tube containing the cryopreserved ciliate suspension from the liquid nitrogen, place it in a water bath and shake it rapidly to thaw, and obtain the thawed cryopreserved ciliate suspension. ⑦ Centrifuge the frozen ciliate suspension, remove the supernatant, retain the precipitate, and obtain the enriched ciliates; ⑧ The enriched worms were added to a resuscitation medium preheated to 39°C and resuspended to obtain a resuscitated ciliate suspension. ⑨ The revived ciliate suspension was transferred to a test tube containing revival culture medium and revived in an incubator at 39°C.

[0016] In step ⑥, the temperature of the water bath is 38-41℃.

[0017] In step ⑦, the centrifugation speed is 500-800 r / min and the centrifugation time is 8-10 min.

[0018] The specific operation of step 9 is as follows: Transfer the revived ciliate suspension to a 10-15 mL test tube containing revival culture medium; Add 100-200 μL of substrate suspension to the test tube; Slowly introduce CO2 gas into the headspace of the test tube for 5-10 seconds, then quickly seal the test tube with a rubber stopper. Subsequently, place it in a 39℃ constant temperature incubator for recovery culture for 2-3 days. During the recovery culture period, add 100-200μL of fresh substrate suspension every day.

[0019] The resuscitation medium was prepared by adding sucrose to the supernatant of protozoan-free rumen fluid to a final sucrose concentration of 0.04%.

[0020] The substrate suspension is prepared by taking 1-1.5g of rice starch and 1-1.5g of whole wheat flour, adding distilled water to a final volume of 50mL, and mixing well.

[0021] After 2-3 days of recovery culture in step 9, switch to long-term culture. The long-term culture method is as follows: add 200 μL of fresh substrate suspension daily and change the medium every 3-4 days.

[0022] The procedure for changing the solution is as follows: 1) Before changing the medium, let the test tube stand vertically for 15-30 minutes to allow most of the ciliates to settle to the bottom of the test tube. 2) Replace 40-60% of the original medium with modified SP medium, purge with carbon dioxide for 5-10 seconds, then quickly seal the test tube with a rubber stopper and incubate in a 39℃ constant temperature incubator.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The cryopreservation method of this invention features a stable and controllable freezing rate, simple and convenient operation, and uses common and relatively few instruments and equipment. It does not require a professional and expensive programmed cooling device and can be cultured under ordinary laboratory conditions. The entire process is relatively short. Compared with one-step and two-step cryopreservation methods, it is more efficient and suitable not only for routine experiments but also for rapidly processing large numbers of samples.

[0024] 2. The key steps of the original cryopreservation and resuscitation method for rumen ciliates are as follows: (1) 5% DMSO is used as the cryoprotectant in combination with 0.2-0.8M trehalose; (2) 5% fetal bovine serum and 5% horse serum are added to the cryoprotectant; (3) The cooling method is to put the sample into a programmed cooling box with a cooling rate of 1℃ / minute and then put it into a -80℃ freezer for cooling. The cooling method is simple and controllable, and the equipment requirements are not high; (4) The resuscitation medium is 0.04% sucrose added to the supernatant of protozoan-free rumen fluid; (5) The survival rate of the worms is significantly improved after cryopreservation.

[0025] 3. This application has successfully established a cryopreservation and thawing method for mixed species or single species of the genus *Urechis* suitable for in vitro culture. The thawing culture method in this application follows a standardized protocol that has been successfully established in the laboratory, and for the first time, a cryopreservation method more suitable for the genus *Urechis* has been discovered.

[0026] 4. In the cryopreservation and thawing method of this application, trehalose, used alone or in combination with 5% DMSO, showed good cryopreservation protection for mixed-cultured rumen ciliates. The protection effect of the combination was significantly better than that of the single use, specifically reflected in the number of ciliates surviving in the early stage of culture, the population density after one week of culture, and the growth rate. This advantage remained consistent under different cryopreservation times, including short-term (3 days), medium-term (7 days), and long-term (30 days). The highest survival rate of mixed-cultured rumen ciliates reached 58.1%.

[0027] 5. The cryopreservation and thawing method of this application, which uses 0.3M trehalose in combination with 5% DMSO as a cryoprotectant, showed the best protective effect on monocultured *Urechis formosanus*. The survival rate of ciliates was highest immediately after thawing and 24 hours after thawing, with the highest survival rate reaching 66.4% after 24 hours of thawing. After 3 days of culture, the density was greater than 30,000 individuals / mL, and the ciliate vigor was no different from that of normal culture, and the growth was stable, with the ability to be continuously passaged, which proved that its functional integrity was well maintained. Attached Figure Description

[0028] Figure 1 This is the growth curve of freshly sampled rumen ciliates after cryopreservation and thawing in Example 1 of the present invention; Figure 2 shows the recovery survival rate and growth curve of the *Enteroceratops* rumen ciliates in vitro co-cultured in Example 2 of this invention, and the recovery survival rate after 3 days of cryopreservation. Figure 2-a ) and growth curve ( Figure 2-b ); Survival rate results after 7 days of cryopreservation (Figure) Figure 2-c ) and growth curve ( Figure 2-d ); Survival rate results after 30 days of cryopreservation (Figure) Figure 2-e ) and growth curve ( Figure 2-f ); Figure 3 shows the survival rate of 13 groups of single species of Formosanus worms cultured in vitro after 3 days of cryopreservation in Example 3 of the present invention. Figure 3-a ), growth curve ( Figure 3-b ); Figure 4 shows the survival rate of a single species of Formosanus worm cultured in vitro after 3 days of cryopreservation using trehalose in combination with 5% DMSO as a cryoprotectant in Example 3 of the present invention. Figure 4-a ) and growth curve ( Figure 4-b ); Figure 5 shows the survival rate of a single species of Formosanus worm cultured in vitro after 7 days of cryopreservation using trehalose in combination with 5% DMSO as a cryoprotectant in Example 3 of the present invention. Figure 5-a ) and growth curve ( Figure 5-b ); Figure 6 shows the survival rate of a single species of Formosanus worm cultured in vitro after 30 days of cryopreservation using trehalose in combination with 5% DMSO as a cryoprotectant in Example 3 of the present invention. Figure 6-a ) and growth curve ( Figure 6-b ); The cryopreservation time refers to the total time from when the sample is immersed in liquid nitrogen until it is removed for subsequent experiments; statistical analysis showed that P≤0.01, which means that the observed differences were significant and statistically significant. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] The sources, components, and concentrations of the reagents used in the examples are as follows: Fresh rumen fluid: After being sterilized by autoclaving, the rumen fluid collection catheter is inserted into the rumen through the cow's mouth and esophagus to collect rumen fluid. The collected rumen fluid is filtered through four layers of medical gauze, sealed, and stored at 39°C.

[0031] Protozoan-free rumen fluid: The collected fresh rumen fluid was sterilized at 121℃ under high temperature and high pressure for 20 min. After cooling, it was centrifuged at 1000×g for 10 min. The supernatant was collected to obtain the protozoan-free rumen fluid supernatant and stored at 4℃.

[0032] Double antibiotic solution: 300 mg penicillin G sodium, 250 mg streptomycin sulfate, 10 mL distilled water.

[0033] SP salt solution: Weigh 5.5g of dipotassium hydrogen phosphate trihydrate, 4.0g of potassium dihydrogen phosphate, 0.07g of magnesium sulfate heptahydrate, and 0.5g of sodium chloride, and add distilled water to a final volume of 800mL to prepare solution A; weigh 0.04g of calcium chloride, and add distilled water to a final volume of 100mL to prepare solution B; weigh 6.0g of sodium bicarbonate, and add distilled water to a final volume of 100mL to prepare solution C; sterilize solutions A, B, and C separately by high-temperature autoclaving, and store at room temperature for later use; before use, mix solutions A, B, and C thoroughly, and purge with carbon dioxide to obtain the SP salt solution, then seal and store for later use.

[0034] Example 1: Effects of cryoprotectants on cryopreservation and thawing of freshly sampled rumen ciliates 1.1 Preparations before freezing: ① Preparation of worms: Fresh rumen fluid collected from the rumen was selected, and after sedimentation through a separatory funnel, it was filtered through a 125-mesh nylon mesh to obtain a ciliate enrichment solution. The enrichment solution contained rumen ciliates from multiple genera, including Inner Trichoderma, Thick Trichoderma, Protrichoderma, Isotrichoderma, Cephalotrichoderma, and Polyceratops.

[0035] ② Preparation of special cryopreservation solution: Prepare modified SP culture medium in advance, which consists of: 1 mL of protozoan-free rumen fluid, 200 μL of 1% cysteine ​​hydrochloride solution, 200 μL of double antibiotic solution, and 8.6 mL of SP salt solution.

[0036] Based on this, cell cryopreservation solutions containing different concentrations of trehalose (A, B, C) and cell cryopreservation solutions containing different concentrations of trehalose and 5% dimethyl sulfoxide (DMSO) (D, E, F, G) were prepared, and the distribution of each group is as follows: Cryopreservation solution A (final trehalose concentration 0.2M): Take 700μL of modified SP medium, add 50μL of fetal bovine serum, 50μL of horse serum, and 200μL of 1M trehalose stock solution, and mix well.

[0037] Cryopreservation solution B (final trehalose concentration 0.3M): Take 600μL of modified SP medium, add 50μL of fetal bovine serum, 50μL of horse serum, and 300μL of 1M trehalose stock solution, and mix well.

[0038] Cryopreservation solution C (final trehalose concentration 0.4M): Take 500μL of modified SP medium, add 50μL of fetal bovine serum, 50μL of horse serum, and 400μL of 1M trehalose stock solution, and mix well.

[0039] Cryopreservation solution D (5% DMSO + trehalose final concentration 0.2M): Take 650μL of modified SP medium, add 50μL fetal bovine serum, 50μL horse serum, 50μL dimethyl sulfoxide, and 200μL 1M trehalose stock solution, and mix well.

[0040] Cryopreservation solution E (5% DMSO + trehalose final concentration 0.3M): Take 550 μL of modified SP medium, add 50 μL of fetal bovine serum, 50 μL of horse serum, 50 μL of dimethyl sulfoxide, and 300 μL of 1M trehalose stock solution, and mix well.

[0041] Cryopreservation solution F (5% DMSO + trehalose final concentration 0.4M): Take 450 μL of modified SP medium, add 50 μL fetal bovine serum, 50 μL horse serum, 50 μL dimethyl sulfoxide, and 400 μL 1M trehalose stock solution, and mix well.

[0042] Cryopreservation solution G (5% DMSO): Take 850 μL of modified SP medium, add 50 μL of fetal bovine serum, 50 μL of horse serum, and 50 μL of dimethyl sulfoxide, and mix well.

[0043] Mix the prepared cryopreservation solution thoroughly and refrigerate at 4°C for later use.

[0044] 1.2 Preparation and dispensing of cryopreserved insect suspension: ① Centrifuge the filtered ciliate suspension obtained in step 1.1, remove the supernatant, retain the precipitate, and obtain enriched ciliates; add the above 7 cryopreservation solutions (AG) to the enriched ciliate precipitate, gently resuspend by pipetting, count, and adjust the ciliate density to 1.0 × 10⁻⁶. 6 cells / mL. Divided into 7 groups, each group had 3 replicates, using the aforementioned 7 cryopreservation solutions (AG); ② Dispense the resuspended worm suspensions from 7 groups into 2mL cell cryopreservation tubes, 0.2mL per tube, and tighten the caps.

[0045] 1.3 Slow freezing: ① Immediately place the pre-packaged cryovials containing the insect suspension into a programmable cooling box at 1℃ / minute, and then place the programmable cooling box in an ultra-low temperature freezer at -80℃; ② Slowly freeze the insect body at -80℃ for 12 to 24 hours; ③ The next day, the cryovials were removed from the programmed cooling box and quickly transferred to liquid nitrogen for long-term preservation.

[0046] 1.4 Preparations before recovery: ① Preparation of resuscitation medium: Take the supernatant of protozoan-free rumen fluid, add sucrose to make a final concentration of 0.04% (w / v), mix well, and preheat in a 39℃ water bath for at least 30 minutes; ② Preparation of substrate suspension: Weigh 1g of rice starch and 1g of whole wheat flour, add distilled water to make up to 50mL, mix well, and prepare substrate suspension; ③ Equipment preparation: 39℃ constant temperature water bath, centrifuge, 15mL sterile test tubes, 15mL centrifuge tubes.

[0047] 1.5 Quick defrosting: Remove the cryovial from the liquid nitrogen and quickly place it in a 39°C constant temperature water bath, gently shaking it continuously until the suspension is completely thawed.

[0048] Remove protective agent: ① Transfer the thawed suspension to a 15 mL centrifuge tube containing resuscitation medium; ② Centrifuge at 500 r / min for 8 minutes, carefully discard the supernatant to remove the cryoprotectant, and retain the accumulated worms at the bottom of the tube.

[0049] 1.6 Resuscitation and Cultivation: ① Gently resuspend the enriched worms in 1 mL of preheated resuscitation medium; ② Transfer the entire resuspension to a 15mL sterile test tube containing 9mL of resuscitation medium; ③ Add 200 μL of substrate suspension to the test tube as a nutrient source for the insects; ④ Slowly introduce CO2 gas into the headspace of the test tube for 5-10 seconds to create an anaerobic environment, and then quickly seal the test tube with a sterile rubber stopper. ⑤ Immediately place the test tube in a 39℃ constant temperature incubator for incubation; ⑥ Count and observe the changes in insect density and activity every 24 hours; usually after 24-48 hours of resuscitation culture, the insects can be observed to regain motility and begin to divide and proliferate under a microscope.

[0050] ⑦ In step ⑤: Resuscitation culture is performed for the first 3 days of culture. During the resuscitation culture, 100-200 μL of fresh substrate suspension is added daily. After 3 days of resuscitation culture, the culture was changed to long-term culture. The long-term culture method was to add 200 μL of fresh substrate suspension every day and change the medium every 3 days.

[0051] ⑧ The procedure for changing the solution is as follows: 1) Before changing the medium, let the test tube stand vertically for 15-30 minutes to allow most of the ciliates to settle to the bottom of the test tube. 2) Replace 50% of the original medium with modified SP medium, purge with carbon dioxide for 5-10 seconds, then quickly seal the test tube with a rubber stopper and incubate in a 39℃ constant temperature incubator.

[0052] 1.7 Growth Curve Plotting Starting from the second day after resuscitation, the cultured and resuscitated mixed worms were counted and observed at fixed times each day. For the counting operation, the culture medium was first thoroughly mixed. Then, three 10 μL samples were randomly selected and added to clean glass slides. The number of active worms in each sample was counted under an optical microscope. The average of the three counts was multiplied by 100 to calculate the population density of the mixed worms (worms / mL). A growth curve of the mixed worms was plotted with culture time (d) on the x-axis and worm density (worms / mL) on the y-axis.

[0053] result: This experiment is the first to evaluate the cryopreservation effects of trehalose alone, trehalose in combination with DMSO, and traditional DMSO as a cryoprotectant on rumen ciliates. To minimize the interference of worm condition and viability on the cryopreservation results, freshly collected rumen ciliate samples were used for cryopreservation.

[0054] See the growth curves based on continuous culture after resuscitation. Figure 1 The results showed that the traditional protectant DMSO group had poor protective effects, with all worms dying within 3 days after resuscitation, and the highest density during culture was only 367 worms / mL. In contrast, the experimental groups using trehalose alone and those using it in combination with DMSO showed better worm survival and proliferation in the early stages of resuscitation than the DMSO-treated group.

[0055] Although both the trehalose group and the trehalose-DMSO combination group showed a decrease in ciliate numbers after one week of culture, we analyzed the possible reasons as follows: First, the samples used for this cryopreservation were mixed rumen ciliate populations containing multiple genera, and the culture conditions after thawing were based on the *Enterotrichum* system successfully established in vitro in vitro, which may not be suitable for the growth requirements of other ciliate species. Second, there are significant differences in body size among different genera of ciliates. For example, *Isotrichum*, *Cephalotrichum*, and *Polycarpus* can reach approximately 180 μm in length, while *Enterotrichum*, *Dichotomus*, and *Monocetus* are mostly 20-80 μm in length. Therefore, in mixed culture, larger ciliates may have an advantage in feeding competition, leading to smaller individuals having limited growth due to insufficient nutrient intake. Furthermore, after some ciliates die during culture, their decomposition in the system may cause bacterial proliferation and fermentation, further affecting the normal growth environment of surviving ciliates. This cryopreservation method is suitable for in vitro culture of mixed species of *Urechis* ciliates, as well as single species of *Urechis* such as *Forkedtail*.

[0056] Although the long-term culture results after this resuscitation were not ideal, the experimental results indicate that trehalose, as a cryoprotectant, is significantly more effective than traditional DMSO in the cryopreservation of rumen ciliates, and has certain application potential. Future studies will use rumen ciliates of the genus *Enteroceratops* that have been successfully cultured in our laboratory for cryopreservation to eliminate interference from factors such as competition in mixed populations and culture mismatch, further improving the reliability and reproducibility of the experiments.

[0057] Example 2: Effects of cryoprotectants on cryopreservation and thawing of in vitro co-cultured rumen ciliates. 2.1 Preparations before freezing: ① Preparation of the worms: Take 50 mL of a mixed in vitro culture system of laboratory-cultured internal caterpillars (long-nuclear internal caterpillars, forked-tail internal caterpillars, and pointed-tail internal caterpillars) in the logarithmic growth phase, with a worm density of approximately 1.5 × 10⁻⁶. 4 –2.5×10 4 per mL.

[0058] ② Preparation of special cryopreservation solution: Prepare modified SP culture medium in advance, which consists of: 1 mL of protozoan-free rumen fluid, 200 μL of 1% cysteine ​​hydrochloride solution, 200 μL of double antibiotic solution, and 8.6 mL of SP salt solution.

[0059] Based on this, cell cryopreservation solutions containing different concentrations of trehalose (A, B, C) and cell cryopreservation solutions containing different concentrations of trehalose and 5% dimethyl sulfoxide (D, E, F, G) were prepared. The distribution of each group is as follows: Cryopreservation solution A (final trehalose concentration 0.2M): Take 700μL of modified SP basal medium, add 50μL fetal bovine serum, 50μL horse serum, and 200μL 1M trehalose stock solution, and mix well.

[0060] Cryopreservation solution B (final trehalose concentration 0.3M): Take 600μL of modified SP basal medium, add 50μL fetal bovine serum, 50μL horse serum, and 300μL 1M trehalose stock solution, and mix well.

[0061] Cryopreservation solution C (final trehalose concentration 0.4M): Take 500μL of modified SP basal medium, add 50μL fetal bovine serum, 50μL horse serum, and 400μL 1M trehalose stock solution, and mix well.

[0062] Cryopreservation solution D (5% DMSO + trehalose final concentration 0.2M): Take 650 μL of modified SP basal medium, add 50 μL fetal bovine serum, 50 μL horse serum, 50 μL dimethyl sulfoxide, and 200 μL 1M trehalose stock solution, and mix well.

[0063] Cryopreservation solution E (5% DMSO + trehalose final concentration 0.3M): Take 550 μL of modified SP basal medium, add 50 μL fetal bovine serum, 50 μL horse serum, 50 μL dimethyl sulfoxide, and 300 μL 1M trehalose stock solution, and mix well.

[0064] Cryopreservation solution F (5% DMSO + trehalose final concentration 0.4M): Take 450 μL of modified SP basal medium, add 50 μL fetal bovine serum, 50 μL horse serum, 50 μL dimethyl sulfoxide, and 400 μL 1M trehalose stock solution, and mix well.

[0065] Cryopreservation solution G (5% DMSO): Take 850 μL of modified SP basal medium, add 50 μL of fetal bovine serum, 50 μL of horse serum, and 50 μL of dimethyl sulfoxide, and mix well.

[0066] Mix the prepared cryopreservation solution thoroughly and refrigerate at 4°C for later use.

[0067] ③ Equipment preparation: Prepare 125 mesh nylon mesh, 50mL centrifuge tubes, programmed cooling box, 2mL cell cryopreservation tubes, and centrifuge.

[0068] 2.2 Accumulation of parasites: ① Filter the ciliates in the culture system through a 125-mesh nylon mesh to remove culture medium residue and large particulate impurities, and collect the filtrate in a 50mL centrifuge tube; ② Centrifuge the filtrate at 500 r / min for 8 minutes. After centrifugation, discard the supernatant and retain the accumulated insect bodies precipitated at the bottom of the tube.

[0069] This stage is defined as "before cryopreservation." Because rumen ciliates are anaerobic, they are exposed to oxygen during filtration and centrifugation, and centrifugation enrichment also damages the worms. These operations cause a small percentage of ciliates to die. After separate testing, the ciliate survival rate at this stage is defined as 95%-100%. Figure 2-a , Figure 2-c , Figure 2-e The first column of each group is shown in the image.

[0070] 2.3 Preparation and dispensing of cryopreserved insect suspension: ① Centrifuge the filtered ciliate suspension obtained in step 2.2, remove the supernatant, retain the precipitate, and obtain enriched ciliates; add the above 7 cryopreservation solutions (AG) to the precipitate of enriched ciliates, gently resuspend by pipetting, count, and the ciliate density is approximately 1×10⁻⁶. 6 -1.5×10 6 The experiment consisted of 7 treatment groups, each corresponding to one of the 7 cryopreservation solutions (AG), with 3 replicates in each group; and each cryopreservation solution was used for 3 days, 7 days and 30 days of cryopreservation.

[0071] ② Dispense the resuspended worm suspensions from 7 groups into 2mL cell cryopreservation tubes, 0.2mL per tube, and tighten the caps.

[0072] 2.4 Slow freezing: ① Immediately place the pre-packaged cryovials containing the insect suspension into a programmable cooling box at 1℃ / minute, and then place the programmable cooling box in an ultra-low temperature freezer at -80℃; ② Slowly freeze the insect body at -80℃ for 12 to 24 hours; ③ The next day, the cryovials were removed from the programmed cooling box and quickly transferred to liquid nitrogen for long-term preservation.

[0073] 2.5 Preparations before recovery: ① Preparation of resuscitation medium: Take the supernatant of protozoan-free rumen fluid, add sucrose to it and make the final concentration 0.04% (w / v), mix well and preheat in a 39℃ water bath for at least 30 minutes; ② Preparation of substrate suspension: Weigh 1g of rice starch and 1g of whole wheat flour, add distilled water to make up to 50mL, mix well, and prepare substrate suspension; ③ Equipment preparation: 39℃ constant temperature water bath, centrifuge, 15mL sterile test tubes, 15mL centrifuge tubes.

[0074] 2.6 Quick defrosting: Remove the cryovial from the liquid nitrogen and quickly place it in a 39°C constant temperature water bath, gently shaking it continuously until the suspension is completely thawed.

[0075] 2.7 Remove the protective agent: ① Transfer the thawed suspension to a 15 mL centrifuge tube containing resuscitation medium; ② Centrifuge at 500 r / min for 8 minutes, carefully discard the supernatant to remove the cryoprotectant, and retain the accumulated worms at the bottom of the tube.

[0076] 2.8 Resuscitation and Cultivation: ① Gently resuspend the enriched worms in 1 mL of preheated resuscitation medium; ② Transfer the entire resuspension to a 15mL sterile test tube containing 9mL of resuscitation medium; ③ Add 200 μL of substrate suspension to the test tube as a nutrient source for the insects.

[0077] ④ Slowly introduce CO2 gas into the headspace of the test tube for 5-10 seconds to create an anaerobic environment, and then quickly seal the test tube with a sterile rubber stopper.

[0078] ⑤ Immediately place the test tube in a 39℃ constant temperature incubator for incubation.

[0079] ⑥ Count and observe the changes in worm density and activity every 24 hours. Usually, after 24-48 hours of resuscitation culture, the worms can be observed to regain motility and begin to divide and proliferate under a microscope.

[0080] ⑦ In step ⑤: Resuscitation culture is performed for the first 3 days of culture. During the resuscitation culture, 100-200 μL of fresh substrate suspension is added daily. After 3 days of resuscitation culture, the culture was changed to long-term culture. The long-term culture method was to add 200 μL of fresh substrate suspension every day and change the medium every 3 days.

[0081] ⑧ The procedure for changing the solution is as follows: 1) Before changing the medium, let the test tube stand vertically for 15-30 minutes to allow most of the ciliates to settle to the bottom of the test tube. 2) Replace 50% of the original medium with modified SP medium, purge with carbon dioxide for 5-10 seconds, then quickly seal the test tube with a rubber stopper and incubate in a 39℃ constant temperature incubator.

[0082] 2.9 Growth Curve Plotting Starting from the second day after resuscitation, the resuscitated mixed worms in the culture medium were counted and observed at fixed times each day. Before counting, the culture medium was thoroughly mixed. Then, three 10 μL samples were randomly selected and added to clean glass slides. The number of active worms in each sample was counted under an optical microscope. The average of the three counts was multiplied by 100 to calculate the population density of the mixed worms (worms / mL). A growth curve of the mixed worms was plotted with culture time (d) on the x-axis and worm density (worms / mL) on the y-axis.

[0083] result: Growth curve results showed that trehalose, whether used alone or in combination with 5% DMSO, exhibited good cryopreservation protection for in vitro cultured rumen ciliates.

[0084] Results of survival rate after thawing after 3 days of cryopreservation ( Figure 2-a ) and growth curve ( Figure 2-b The results showed that, compared with the groups using trehalose and DMSO alone, the three experimental groups using trehalose combined with 5% DMSO performed better in terms of initial density on the first day of culture, growth rate of ciliates, and final number after one week of culture. The highest survival rate of the trehalose combined with 5% DMSO was 41.9%; the highest survival rate of the groups using trehalose and DMSO alone was 20.5%.

[0085] Results of survival rate after 7 days of cryopreservation ( Figure 2-c ) and growth curve ( Figure 2-dThe results showed that by day four, the ciliate population in the three experimental groups using trehalose combined with 5% DMSO had recovered to normal culture levels (25,000–35,000 individuals / mL), with a maximum survival rate of 41.5%. In contrast, the groups using trehalose or DMSO alone had densities below 6,000 individuals / mL, with a maximum survival rate of 17.35%. An operational error during medium change caused a temporary decrease in ciliate population on day five, but the population remained around 20,000 individuals / mL and rapidly recovered to normal levels in subsequent cultures. Results of survival rate after 30 days of cryopreservation ( Figure 2-e ) and growth curve ( Figure 2-f The results showed that the experimental group with trehalose and 5% DMSO (highest survival rate 58.1%) was significantly better than the group with trehalose and DMSO alone (highest survival rate 20.5%) in terms of initial density and growth rate. Its population size had recovered to normal level (>25,000 individuals / mL) by the seventh day of culture.

[0086] In summary, the protective effect of using trehalose and DMSO in combination is significantly better than using either alone, specifically reflected in the number of ciliates surviving in the early stages of culture, the population density after one week of culture, and the growth rate. This advantage remained consistent across different cryopreservation times (cryopreservation time refers to the total time from when the sample is immersed in liquid nitrogen until it is removed for subsequent experiments) including short-term (3 days, highest survival rate 41.9%), medium-term (7 days, highest survival rate 41.5%), and long-term (30 days, highest survival rate 58.1%).

[0087] The results of the continued culture after resuscitation are shown in Figure 2. The final density of ciliates in each experimental group can be restored to the normal range of 25,000–35,000 individuals / mL. The cell viability is not significantly different from that of ciliates in conventional subculture. This indicates that trehalose combined with 5% DMSO as a protectant can effectively maintain the normal growth and reproductive capacity of ciliates after resuscitation, with a maximum survival rate of 58.1%.

[0088] Although the above results confirm the effectiveness of trehalose combined with DMSO, the experimental subjects were laboratory-cultured *Urechis acuminata*, *Urechis longinus*, and *Urechis forkedtail* ciliates. Differences in cryopreservation tolerance among different species make it difficult to determine the optimal trehalose concentration based on mixed populations. To more accurately screen for the optimal concentration, in Example 3 of this application, *Urechis forkedtail*, a single-species cultured within the *Urechis* genus, will be used as the research subject, and a more refined trehalose concentration gradient will be established in combination with 5% DMSO as a cryoprotectant to further clarify its optimal concentration.

[0089] Example 3: Effects of cryoprotectants on cryopreservation and thawing of in vitro cultured Forktail caterpillars 3.1 Preparations before freezing: ① Preparation of the insect body: Take 50 mL of the in vitro culture system of Forktail Internal Caterpillar in the logarithmic growth phase, with an insect density of approximately 1.5 × 10⁻⁶. 4 –2.5×10 4 per mL.

[0090] ② Preparation of specialized cryopreservation solutions: A modified SP medium was prepared in advance, consisting of: 1 mL of protozoan-free rumen fluid, 200 μL of 1% cysteine ​​hydrochloride solution, 200 μL of antibiotic solution, and 8.6 mL of SP salt solution. Based on this, cell cryopreservation solutions containing different concentrations of trehalose (A, B, C, D, E, F) and different concentrations of trehalose and 5% DMSO (G, H, I, J, K, L, M) were prepared. The distribution of each group is as follows: Cryopreservation solution A (final trehalose concentration 0.2M): Take 700μL of modified SP medium, add 50μL fetal bovine serum, 50μL horse serum, and 200μL 1M trehalose stock solution, and mix well.

[0091] Cryopreservation solution B (final trehalose concentration 0.3M): Take 600μL of modified SP medium, add 50μL fetal bovine serum, 50μL horse serum, and 300μL 1M trehalose stock solution, and mix well.

[0092] Cryopreservation solution C (final trehalose concentration 0.4M): Take 500μL of modified SP medium, add 50μL of fetal bovine serum, 50μL of horse serum, and 400μL of 1M trehalose stock solution, and mix well.

[0093] Cryopreservation solution D (final trehalose concentration 0.5M): Take 400μL of modified SP medium, add 50μL fetal bovine serum, 50μL horse serum, and 500μL 1M trehalose stock solution, and mix well.

[0094] Cryopreservation solution E (final trehalose concentration 0.6M): Take 300μL of modified SP medium, add 50μL fetal bovine serum, 50μL horse serum, and 600μL 1M trehalose stock solution, and mix well.

[0095] Cryopreservation solution F (final trehalose concentration 0.8M): Take 100μL of modified SP medium, add 50μL fetal bovine serum, 50μL horse serum, and 800μL 1M trehalose stock solution, and mix well.

[0096] Cryopreservation solution G (5% DMSO + trehalose final concentration 0.2M): Take 650 μL of modified SP medium, add 50 μL fetal bovine serum, 50 μL horse serum, 50 μL dimethyl sulfoxide, and 200 μL 1M trehalose stock solution, and mix well.

[0097] Cryopreservation solution H (5% DMSO + trehalose final concentration 0.3M): Take 550 μL of modified SP medium, add 50 μL of fetal bovine serum, 50 μL of horse serum, 50 μL of dimethyl sulfoxide, and 300 μL of 1M trehalose stock solution, and mix well.

[0098] Cryopreservation solution I (5% DMSO + trehalose final concentration 0.4M): Take 450 μL of modified SP medium, add 50 μL fetal bovine serum, 50 μL horse serum, 50 μL dimethyl sulfoxide, and 400 μL 1M trehalose stock solution, and mix well.

[0099] Cryopreservation solution J (5% DMSO + trehalose final concentration 0.5M): Take 350 μL of modified SP medium, add 50 μL fetal bovine serum, 50 μL horse serum, 50 μL dimethyl sulfoxide, and 500 μL 1M trehalose stock solution, and mix well.

[0100] Cryopreservation solution K (5% DMSO + trehalose final concentration 0.6M): Take 250μL of modified SP medium, add 50μL fetal bovine serum, 50μL horse serum, 50μL dimethyl sulfoxide, and 600μL 1M trehalose stock solution, and mix well.

[0101] Cryopreservation solution L (5% DMSO + trehalose final concentration 0.8M): Take 50 μL of modified SP medium, add 50 μL of fetal bovine serum, 50 μL of horse serum, 50 μL of dimethyl sulfoxide, and 800 μL of 1M trehalose stock solution, and mix well.

[0102] Cryopreservation solution M (5% DMSO): Take 850 μL of modified SP medium, add 50 μL of fetal bovine serum, 50 μL of horse serum, and 50 μL of dimethyl sulfoxide, and mix well.

[0103] Mix all the prepared cryopreservation solution components thoroughly and store at 4°C for later use.

[0104] ③ Equipment preparation: Prepare 300-mesh nylon mesh, 50mL centrifuge tubes, programmed cooling box, 2mL cell cryopreservation tubes, and centrifuge.

[0105] 3.2 Enrichment of parasites: ① Filter the forktail caterpillars in the culture system through a 300-mesh nylon mesh to remove culture medium residue and large particulate impurities, and collect the filtrate in a 50mL centrifuge tube; ② Centrifuge the filtrate at 500 rpm for 8 minutes. After centrifugation, discard the supernatant and retain the accumulated ciliates at the bottom of the tube. This is defined as "before cryopreservation." Because rumen ciliates are anaerobic, they are exposed to oxygen during filtration and centrifugation, and centrifugation also damages the ciliates. These operations will cause a small portion of the ciliates to die. After separate testing, the ciliate survival rate at this point is defined as 95%-100%. Figure 3-a , Figure 4-a , Figure 5-a , Figure 6-a The first column of each group is shown in the image.

[0106] 3.3 Preparation and dispensing of cryopreserved insect suspension: ① Add the 13 pre-cooled cryopreservation solutions (AM) prepared above to the precipitate rich in worms, gently pipette to resuspend, count, and the worm density is approximately 1×10⁻⁶. 6 -1.5×10 6 The experiment consisted of 13 treatment groups, each corresponding to one of the 13 cryopreservation solutions (AG), with three replicates in each group; and each cryopreservation solution was used for three different cryopreservation times: 3 days, 7 days, and 30 days.

[0107] ② Dispense the resuspended worm suspension into 2 mL cell cryopreservation tubes, 0.2 mL per tube, and tighten the caps.

[0108] 3.4 Ciliate survival rate determination before cryopreservation: The survival rate of ciliates before cryopreservation was determined using the trypan blue staining method. 10 μL of enriched ciliate suspension was mixed with 10 μL of 0.4% trypan blue solution, allowed to stand for 3 minutes, and then observed and counted under a microscope. Unstained, transparent ciliates were considered live, while those stained blue were considered dead. The experiment was repeated three times, and the average survival rate was calculated.

[0109] 3.5 Slow freezing: ① Immediately place the pre-packaged cryovials containing the insect suspension into a programmable cooling box at 1℃ / minute, and then place the programmable cooling box in an ultra-low temperature freezer at -80℃; ② Slowly freeze the insect body at -80℃ for 12 to 24 hours; ③ The next day, the cryovials were removed from the programmed cooling box and quickly transferred to liquid nitrogen for long-term preservation.

[0110] 3.6 Preparations before recovery: ① Preparation of resuscitation medium: Take the supernatant of protozoan-free rumen fluid, add sucrose to it and make the final concentration 0.04% (w / v), mix well and preheat in a 39℃ water bath for at least 30 minutes; ② Preparation of substrate suspension: Weigh 1g of rice starch and 1g of whole wheat flour, add distilled water to make up to 50mL, mix well, and prepare substrate suspension; ③ Equipment preparation: 39℃ constant temperature water bath, centrifuge, 15mL sterile test tubes, 15mL centrifuge tubes.

[0111] 3.7 Quick defrosting: Remove the cryovial from the liquid nitrogen and quickly place it in a 39°C constant temperature water bath, gently shaking it continuously until the suspension is completely thawed.

[0112] 3.8 Remove protective agent: ① Transfer the thawed suspension to a 15 mL centrifuge tube containing resuscitation medium; ② Centrifuge at 500 r / min for 8 minutes, carefully discard the supernatant to remove the cryoprotectant, and retain the accumulated worms at the bottom of the tube.

[0113] 3.9 Determination of ciliate survival rate after thawing: The survival rate of revived worms was determined using the trypan blue staining method. 10 μL of the revived and washed worm suspension was mixed with 10 μL of 0.4% trypan blue solution, allowed to stand for 3 minutes, and then observed and counted under a microscope. Unstained, transparent worms were considered live, while those stained blue were considered dead. The experiment was repeated three times, and the average survival rate was calculated.

[0114] 3.10 Resuscitation and Cultivation: ① Gently resuspend the enriched worms in 1 mL of preheated resuscitation medium; ② Transfer the entire resuspension to a 15mL sterile test tube containing 9mL of resuscitation medium; ③ Add 200 μL of substrate suspension to the test tube as a nutrient source for the insects.

[0115] ④ Slowly introduce CO2 gas into the headspace of the test tube for 5-10 seconds, and then quickly seal the test tube with a sterile rubber stopper.

[0116] ⑤ Immediately place the test tube in a 39℃ constant temperature incubator for incubation.

[0117] ⑥ Count and observe the changes in worm density and activity every 24 hours. Usually, after 24-48 hours of resuscitation culture, the worms can be observed to regain motility and begin to divide and proliferate under a microscope.

[0118] ⑦ In step ⑤: Resuscitation culture is performed for the first 3 days of culture. During the resuscitation culture, 100-200 μL of fresh substrate suspension is added daily. After 3 days of resuscitation culture, the culture was switched to long-term culture. The long-term culture method was as follows: 200 μL of fresh substrate suspension was added daily, and the medium was changed every 3 days. The medium change procedure was as follows: 1) Before changing the medium, let the test tube stand vertically for 15-30 minutes to allow most of the ciliates to settle to the bottom of the test tube. 2) Replace 50% of the original medium with modified SP medium, purge with carbon dioxide for 5-10 seconds, then quickly seal the test tube with a rubber stopper and incubate in a 39℃ constant temperature incubator.

[0119] 3.11 Growth Curve Plotting Starting from the second day after resuscitation, the resuscitated Forktail caterpillars were counted and observed at fixed times each day. For the counting procedure, the culture medium was first thoroughly mixed. Then, three 10 μL samples were randomly selected and added to clean glass slides. The number of active caterpillars in each sample was counted under an optical microscope. The average of the three counts was multiplied by 100 to calculate the population density of Forktail caterpillars (caterpillars / mL). A growth curve of Forktail caterpillars was plotted with culture time (d) on the x-axis and caterpillar density (caterpillars / mL) on the y-axis.

[0120] 3.12 Ciliate survival rate determination after 24 hours of culture The survival rate of worms after 24 hours of resuscitation and culture was determined using the trypan blue staining method. 10 μL of worm suspension after 24 hours of resuscitation was mixed with 10 μL of 0.4% trypan blue solution, allowed to stand for 3 minutes, and then counted under a microscope. Unstained, transparent worms were considered live, while worms stained blue were considered dead. The experiment was repeated three times, and the average survival rate was calculated.

[0121] result Forktail caterpillars using trehalose alone as a cryoprotectant showed improved cryopreservation after 3 days of freezing (e.g. Figure 3-a As shown), the survival rate of *Forktail* worms after 24 hours of resuscitation was zero, indicating that most of the worms had lost their activity in the early stages of resuscitation. Subsequent cultures (such as...) Figure 3-b As shown in the figure, the 0.6M trehalose group exhibited a brief period of proliferation, with a maximum density of only 300 individuals / mL, far below normal culture levels. Further culture failed to produce growth, the density rapidly decreased, and all individuals eventually died. The other concentration groups showed no proliferation and all individuals died within 4 days. After extending cryopreservation to 7 and 30 days, no individuals from any concentration group survived upon revival. In conclusion, trehalose alone cannot provide effective protection for the cryopreservation of *Enterocera candida*.

[0122] Furthermore, when trehalose is used in combination with DMSO, it shows good cell protection effects in short-term, medium-term and long-term cryopreservation for 3 days (Figure 4), 7 days (Figure 5) and 30 days (Figure 6).

[0123] Growth curve results of the recovery of Forktail caterpillars after 3 days of cryopreservation (e.g.) Figure 4-b As shown in the figure, the recovery effect of the trehalose combined with DMSO group was significantly better than that of the DMSO group, specifically manifested in higher initial density and faster growth rate. Among them, 0.3M Tre + 5% DMSO had the best protective effect. Although the population density was too high (>35,000 organisms / mL) on the 5th day of culture due to limited culture space, and the number of organisms was not separated, the density was still maintained at about 30,000 organisms / mL, which did not affect the overall recovery effect.

[0124] Growth curve results of the recovery of Forktail caterpillars after 7 days of cryopreservation (e.g.) Figure 5-b As shown in the figure, the population density of the 0.2M–0.4M trehalose combined with DMSO groups approached or reached normal culture levels by the mid-stage of culture. However, in comprehensive comparison, the 0.3M Trehalose + 5% DMSO group showed the most outstanding effect among all groups.

[0125] Growth curve results of the recovery of Forktail caterpillars after 30 days of cryopreservation (e.g.) Figure 6-b As shown in the figure, even after prolonged cryopreservation, the 0.2M–0.4M trehalose combined with DMSO group effectively maintained ciliate viability, and the population density eventually recovered to 30,000 individuals / mL after thawing and culture. By comprehensively comparing the number of ciliates on the first day of culture with the subsequent growth rate, it can be confirmed that 0.3M Trehalose + 5% DMSO remains the optimal cryopreservation method.

[0126] In summary, the combined use of trehalose and DMSO demonstrated good cell protection effects during short-term to long-term cryopreservation ranging from 3 to 30 days. The experimental group using 0.3M trehalose combined with 5% DMSO as the cryoprotectant showed the best protective effect against *Enterocera canaliculata*. Specifically, the immediate survival rates after thawing were 33.15% (3 days cryopreservation), 20.85% (7 days cryopreservation), and 66.4% (30 days cryopreservation), respectively. The survival rates after 24 hours were 52.65% (3 days cryopreservation), 50.45% (7 days cryopreservation), and 78.1% (30 days cryopreservation), respectively.

[0127] It can be seen that the survival rate immediately after resuscitation can reach up to 66.4%. The ciliates in this group recovered rapidly, and their density exceeded 30,000 individuals / mL after 3 days of culture. Moreover, the ciliate viability was not significantly different from that of normal culture, and their growth was stable, demonstrating the ability to be continuously passaged.

Claims

1. A method for cryopreservation and thawing of rumen ciliates, characterized in that, Includes the following steps: ① The source is a single or mixed ciliate in vitro culture system in the logarithmic growth phase. The ciliates in the culture system are filtered with a nylon filter to remove culture medium residue and other large particulate impurities to obtain a ciliate enrichment solution. The source is fresh rumen fluid. After the fresh rumen fluid settles through a separatory funnel, it is filtered through a nylon filter to obtain a ciliate enrichment solution. ② Centrifuge the ciliate enrichment solution obtained in step ①, remove the supernatant, retain the precipitate, and obtain the enriched ciliates; ③ Add the pre-prepared and pre-cooled cell cryopreservation solution to the enriched ciliates obtained in step ② and resuspend them to obtain a cryopreserved ciliate suspension. Then, dispense the cryopreserved ciliate suspension into cell cryopreservation tubes. ④ Place the cell cryovials containing the cryopreserved ciliate suspension obtained in step ③ in a programmed cooling box and freeze overnight; ⑤ Place the cell cryopreservation tubes in liquid nitrogen for cryopreservation; ⑥ Remove the cell cryopreservation tube containing the frozen rumen ciliate suspension obtained in step ⑤ from the liquid nitrogen, place it in a water bath and shake it rapidly to thaw, and obtain the thawed frozen ciliate suspension. ⑦ Centrifuge the frozen ciliate suspension obtained in step ⑥, remove the supernatant, retain the precipitate, and obtain the enriched ciliates. ⑧ The enriched ciliates obtained in step ⑦ were added to a resuscitation medium preheated at 38-41℃ and resuspended to obtain a resuscitation ciliate suspension. ⑨ Transfer the revived ciliate suspension obtained in step ⑧ to a test tube containing revival culture medium and revive it in an incubator at 38-41℃.

2. The method for cryopreservation and thawing of rumen ciliates according to claim 1, characterized in that, In step ①, the mesh size of the nylon filter screen is 125-300 mesh.

3. A method for cryopreservation and resuscitation of rumen ciliates according to claim 1 or 2, characterized in that, In step ②, the centrifugation speed is 500-800 r / min and the centrifugation time is 8-10 min.

4. The method for cryopreservation and thawing of rumen ciliates according to claim 3, characterized in that, In step ③, the ciliates were resuspended and enriched using cell cryopreservation solution, and the ciliate density was adjusted to ≥1×10⁻⁶. 6 per mL.

5. A method for cryopreservation and resuscitation of rumen ciliates according to claim 1, 2, or 4, characterized in that, The specific operation of step ④ is as follows: 1) Place the cell cryovials containing the cryopreserved ciliate suspension into the programmed cooling box; 2) Store the frozen cells in a programmed cooling box with a cooling rate of 1℃ / minute and freeze them in a -80℃ freezer for 12-24 hours; 3) Transfer the cell cryopreservation tubes containing the cryopreserved ciliate suspension in the programmed cooling box to liquid nitrogen for preservation.

6. The method for cryopreservation and thawing of rumen ciliates according to claim 5, characterized in that, The cell cryopreservation solution was prepared by adding the following components to a modified SP medium: 5-10% fetal bovine serum, 5-10% horse serum, 4-6% dimethyl sulfoxide, and 0.2-0.8M trehalose, all based on final concentrations; stored at 4°C for later use. Each 10 mL of the modified SP medium contains the following components: 1 mL of protozoan-free rumen fluid, 200 μL of 1% cysteine ​​hydrochloride solution, 200 μL of double antibiotic solution, and 8.6 mL of SP salt solution.

7. The cryopreservation and thawing method according to claim 1, characterized in that, In step ⑥, the temperature of the water bath is 38-41℃.

8. The cryopreservation and thawing method according to claim 1, characterized in that, In step ⑦, the centrifugation speed is 500-800 r / min and the centrifugation time is 8-10 min.

9. The cryopreservation and thawing method according to claim 1, characterized in that, The resuscitation medium described in step ⑧ is made by adding sucrose to the supernatant of protozoan-free rumen fluid to a final sucrose concentration of 0.04%.

10. The cryopreservation and thawing method according to claim 1, characterized in that, Step 9 is as follows: 1) Transfer the revived ciliate suspension to a 10-15 mL test tube containing revival culture medium; 2) Add 100-200 μL of substrate suspension to the test tube; 3) Slowly introduce CO2 gas into the headspace of the test tube for 5-10 seconds, then quickly seal the test tube with a rubber stopper, and then place it in a 39℃ constant temperature incubator for recovery culture for 2-3 days. During the recovery culture period, add 100-200μL of fresh substrate suspension every day. 4) After 2-3 days of resuscitation culture, the culture is transferred to long-term culture. The long-term culture method is to add 100-200 μL of fresh substrate suspension every day and change the medium every 3-4 days. The medium change method is to replace 40-60% of the original medium with modified SP medium and purge carbon dioxide for 5-10 seconds. The substrate suspension is prepared by mixing 1-1.5g of rice starch and 1-1.5g of whole wheat flour with distilled water to a final volume of 50mL.

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