A method for selecting suitable individuals for transplantation of porous star corals after cutting.

Abstract

This invention relates to the field of coral reef ecological restoration technology, specifically to a method for selecting suitable individuals for transplantation of *Porous Hermitia* corals after cutting, comprising: culturing *Porous Hermitia* corals after cutting until the polyp formation stage; selecting individuals whose average daily surface area growth during the polyp formation stage is significantly higher than that during the bud formation stage, and whose antioxidant enzyme activity during the polyp formation stage has recovered to the level of uncut healthy corals as suitable transplantation individuals. This invention discloses and utilizes *Porous Hermitia* corals for the first time... Astreopora sp. The unique species-specific recovery pattern of "late-mover advantage" – that is, the tissue regeneration rate reaches its peak during the polyp formation period and is significantly higher than in the early stage – provides a more scientific basis for accurately determining the transplantation window period of Porous Coral.

Description

Technical Field

[0001] This invention relates to the field of coral reef ecological restoration technology, specifically to a method for selecting suitable individuals for transplantation of porous corals after cutting. Background Technology

[0002] Coral reef ecosystems possess extremely high biodiversity and ecosystem service value, earning them the title of "rainforests of the ocean." However, due to climate change and human activities, global coral reefs are severely degraded, urgently requiring ecological restoration. Among numerous restoration methods, transplantation restoration is one of the most widely used and effective techniques. This technique involves cutting corals into smaller coral seedlings (i.e., asexually propagated seedlings) and transplanting them to degraded reef areas to rapidly restore coral cover. In current transplantation practices, to achieve high coverage rates in the short term, restoration projects generally tend to use cladoceran corals (such as those in the genus *Tamarix*). Acropora (spp.) This type of coral grows rapidly, has large branch spacing, and the damaged area after cutting is small. It also exhibits significant morphological changes, and its recovery process can be tracked relatively accurately with the naked eye or conventional measurements.

[0003] However, as the restoration goal shifts from "single colony building" to "constructing stable and highly resilient ecosystems," the introduction of resilient, turbidity-tolerant, and structurally stable clump-like corals has become an inevitable trend. However, due to the specific nature of coral growth, traditional macroscopic observation methods for "survival / bleaching" are difficult to apply to other corals and are prone to misjudgment. Summary of the Invention

[0004] To develop a method for screening suitable individuals for transplantation of *Porous Myostar* corals, this invention provides a method for screening suitable individuals for transplantation of *Porous Myostar* corals after cutting. This invention discloses and utilizes *Porous Myostar* corals (…) for the first time… Astreopora (sp.) The unique species-specific recovery pattern of 'late-onset first'—that is, the tissue regeneration rate reaches its peak during the polyp formation period and is significantly higher than in the previous period—is established. Based on this pattern, the criterion of 'significantly higher' provides a more scientific basis for accurately determining the transplantation window period of Porous Coral.

[0005] This invention provides a method for screening suitable individuals for transplantation of porous star corals after cutting, comprising the following steps: Porous unicorn corals were cut and then cultured to the polyp formation stage, which included the symbiotic healing stage, the bud base formation stage, and the polyp formation stage. During the recovery and culture process, the daily average surface area growth and antioxidant enzyme activity of porous coral were measured. Individuals who meet the criteria are selected as suitable transplant recipients; The aforementioned indicator meets the following conditions simultaneously: (a) The daily average surface area growth during the polyp formation period was higher than that during the bud base formation period, and there was a statistically significant difference between the daily average surface area growth during the polyp formation period and the daily average surface area growth during the bud base formation period. (b) The activity of antioxidant enzymes during polyp formation has recovered to the level of uncut healthy corals.

[0006] This invention discloses and utilizes porous unicorn coral (for the first time) Astreopora The unique species-specific recovery pattern of *Sp.*'s 'late-onset first'—that is, the tissue regeneration rate reaches its peak during the polyp formation period and is significantly higher than in the early stage—provides a more scientific basis for accurately determining the transplantation window of *Sp.*'s porosiformis*, and enables the scientific selection of suitable individuals for transplantation after *Sp.*'s cutting procedure.

[0007] Furthermore, the antioxidant enzyme activity includes superoxide dismutase activity, catalase activity, and glutamine synthase activity.

[0008] Furthermore, the criteria for determining the symbiotic healing period, bud formation period, and polyp formation period are as follows: The healing phase of commensal tissue: New commensal tissue gradually covers the damaged area, and by the end of this phase, the commensal tissue completely covers the damaged area; Bud formation stage: The symbiotic tissue continues to proliferate and thicken, and bud bases with nipple-like protrusions begin to appear on the surface; Polyp formation stage: The bud base develops into a mature coral polyp.

[0009] Furthermore, the calculation process for the average daily surface area increase is as follows: the net increase in the area covered by the meat during this stage is divided by the number of days that the stage lasts.

[0010] Furthermore, the quantitative measurement of the co-flesh coverage area includes: taking pictures of the coral cutting area using a ruler reference, and calculating the co-flesh coverage area at each time point using image analysis software.

[0011] Furthermore, the image analysis software is ImageJ software.

[0012] Furthermore, the method for determining antioxidant enzyme activity involves measuring antioxidant enzyme activity at at least two time points: at the end of the sarcophyll healing period, at the end of the bud base formation period, and at the end of the polyp formation period.

[0013] Furthermore, the steps for determining antioxidant enzyme activity were as follows: coral samples were selected at 29-30 minutes, 1-2 days, 11-12 days, 15-16 days, and 21-22 days after cutting for antioxidant enzyme activity determination.

[0014] Furthermore, the determination of suitability for transplantation also includes: the density of symbiotic zooxanthellae and / or the chlorophyll a content of the coral have recovered to a level that is not significantly different from that of uncut healthy corals.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to propose the use of porous star coral ( Astreopora (sp.) is used for transplantation and repair. In view of the technical problem that traditional macroscopic observation methods for "survival / bleaching" are completely ineffective for *Porous Myristic Coral*, this invention proposes a screening method for suitable individuals for transplantation of *Porous Myristic Coral*.

[0016] This invention discloses and utilizes porous unicorn coral ( Astreopora The unique species-specific recovery pattern of *Sp.*—that is, the tissue regeneration rate reaches its peak during polyp formation and is significantly higher than in the early stages—is observed. Based on this pattern, the criterion of "significantly higher than" provides a more scientific basis for accurately determining the transplantation window of *Sp.* porphyria*. This enables the scientific selection of suitable individuals for transplantation after *Sp. porphyria* cutting surgery.

[0017] This invention enables the quantitative measurement of tissue regeneration rate. By taking daily photographs and analyzing them using ImageJ software, the quantitative measurement of the area covered by the commensal tissue is achieved, transforming the vague concept of "wound healing speed" into a specific numerical value (daily average growth), providing an objective indicator for assessing the repair capacity of porous corals.

[0018] This invention reveals the dynamic changes in oxidative stress. By monitoring the dynamic changes of antioxidant indicators such as SOD activity, CAT activity, GSH content, and / or LPO level and / or GS activity, the entire process of oxidative stress after cutting of coral masses—from occurrence to peak to relief—is elucidated for the first time, providing a physiological basis for determining whether corals have survived the stress period. Among these, GSH, as an important non-enzymatic antioxidant in cells, directly reflects the antioxidant capacity and redox balance of coral tissue. LPO (lipid peroxidation) level indicates the degree of damage to cell membrane lipids from reactive oxygen species, and its changes indicate that lipid repair is a relatively slow process. GS (ammonia assimilate) activity reflects the synergistic response of nitrogen metabolism and the antioxidant system in corals. The combined use of these multiple indicators can more comprehensively reflect the multidimensional characteristics of oxidative stress after cutting damage.

[0019] This invention is the first to combine tissue regeneration rate with the degree of oxidative stress recovery, establishing a scientific and comprehensive evaluation standard. Compared with existing technologies, it no longer simply judges "life or death," but can accurately answer key questions such as "to what extent has it recovered" and "is it suitable for transplantation."

[0020] This invention addresses the challenges of assessing the recovery of clump-shaped corals by employing precise morphological observation and physiological indicator monitoring. It provides technical support for the protection of species diversity in coral restoration. Attached Figure Description

[0021] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The morphological changes of porous corals from 30 min to 30 d postoperatively; In the figure, a shows the morphological changes of the porous coral 30 minutes after surgery; b shows the morphological changes of the porous coral 11 days after surgery; c shows the morphological changes of the porous coral 15 days after surgery; Image d shows the morphological changes of the porous coral 21 days after surgery; The red box shows the damaged area, the red arrow in c points to the new shoot, and the red arrow in d points to the new polyp.

[0023] Figure 2 The graph shows the changes in enzyme activity at key time points (30 min to 21 d) after surgery in the porous coral *Coralia micrantha*. In the figure, a represents the change in SOD activity of the porous coral from 30 min to 21 d after surgery; b shows the changes in CAT activity of porous corals from 30 min to 21 d post-surgery; c represents the changes in GS activity of porous coral from 30 min to 21 d post-surgery.

[0024] Figure 3 The daily average surface area increase and polyp density in the later stage of regeneration of porous corals at different stages after surgery. In the figure, (a) represents the daily average surface area increase at different stages after surgery for the porous coral; (b) The density of polyps in the later stages of regeneration at different stages after surgery for porous cosmophora.

[0025] Figure 4 Changes in the density and chlorophyll a content of symbiotic zooxanthellae during the postoperative healing of porous coral; In the figure, (a) shows the change in density of zooxanthellae symbiotic algae during the postoperative healing of porous symbiotic corals; (b) Changes in chlorophyll a content in the symbiotic parasites of the porous coral after postoperative healing.

[0026] Figure 5 The image shows the changes in tissue morphology of the shield-shaped gyrocoral 30 min to 30 d after surgery; In the figure, a is a tissue morphology diagram of the shield-shaped gyrocoral 30 minutes after surgery; b is a tissue morphology diagram of the shield-shaped gyrocoral 7 days after surgery; c is a tissue morphology diagram of the shield-shaped gyrocoral 15 days after surgery; Image d shows the tissue morphology of the shield-shaped gyrocoral 30 days after surgery; The red box shows the damaged area, the red arrow in c points to the new shoot, and the red arrow in d points to the new polyp.

[0027] Figure 6 The daily average surface area increase at each stage after surgery for shield-shaped gyrocorals.

[0028] Figure 7 This is a graph showing the changes in enzyme activity at key time points (30 min to 30 d) after surgery for *Gyropoda shieldii*. In the figure, a represents the change in SOD activity of the shield-shaped gyrocoral from 30 min to 30 d after surgery; b represents the changes in CAT activity in shield-shaped gyroscope corals from 30 min to 30 d post-surgery; c represents the change in GSH content of shield-shaped gyroscope coral from 30 min to 30 d after surgery; d represents the change in LPO levels from 30 min to 30 d after surgery for the shield-shaped gyrocoral. Detailed Implementation

[0029] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0030] Example 1: Evaluation of the recovery status after cutting porous star coral.

[0031] This embodiment uses porous star coral (… Astreopora The method described in this invention is used to evaluate the recovery status of a specimen after surgery (sp.), with the following specific steps: 1. Sample preparation and experimental setup Healthy mother corals of the porous star coral were selected and cut into uniformly sized pieces using bone shears, each piece approximately 10 cm³. These pieces were then fixed onto ceramic bases and placed in an indoor recirculating aquaculture system for recovery and cultivation. The cultivation conditions were: water temperature maintained at 27℃, salinity 33 ppt, light intensity approximately 200 μmol photons·m⁻²·s⁻¹, pH 8.1, and a photoperiod of 12 h:12 h (light:dark). Uncut healthy porous star corals were used as a control group.

[0032] 2. Daily morphological observation and recovery phase division Starting from the day the cutting surgery was completed (recorded as post-operative day 0), photographs of the coral fragments were taken daily at set times. During photography, the ceramic base was carefully rotated using tweezers to capture multiple views of the coral fragment (at least one clear digital photograph of each view) to ensure a complete record of the morphological changes in the cut area and surrounding tissue. A right-angle ruler of known dimensions was placed in each photograph as a reference for subsequent dimensioning and area calculations.

[0033] Through continuous observation, the postoperative recovery process of porous star corals was divided into the following three characteristic stages: (1) Co-suture healing period (days 0 to 11 postoperatively): In the early postoperative period, the co-suture tissue at the edge of the wound begins to proliferate and expand towards the center, and the new tissue is yellowish-brown. By day 11, the co-suture tissue completely covers the edge of the damaged area, and the wound changes from the original cut surface to a state where it is wrapped by new tissue.

[0034] (2) Sprouting stage (11-15 days post-surgery): After the co-flesh covering is completed, the coral tissue continues to proliferate, and cells aggregate and differentiate to form sprouts (papillary protrusions). By day 15 post-surgery, all coral seedlings had sprouted sprouts, which were approximately 2 mm in size and appeared as translucent vesicles.

[0035] (3) Polyp formation period (15 to 21 days after surgery): On the 21st day after surgery, the coral bud bases have further differentiated to form new polyps. The new polyps are light in color and semi-transparent.

[0036] Postoperative morphological changes of porous star corals such as Figure 1 As shown: Figure 1 Figure a shows the cut surface of the coral block 30 minutes after surgery; Figure 1 b shows that 11 days post-surgery, the lesion had been covered by tissue and the edges had healed; Figure 1 The CT scan showed that new buds appeared on the tissue surface 15 days after surgery; Figure 1 The results showed that new polyps had formed 21 days after the operation.

[0037] 3. Quantitative measurement of the area covered by the co-meat The ImageJ image analysis software (developed by the National Institutes of Health, version 1.53 or later) was used to perform quantitative analysis on the photos taken at each time point in step 2. The specific steps are as follows:

[0038] (1) Open ImageJ software and import the coral photos to be analyzed; (2) Use the “Straight Line tool” to draw a line segment of known length along the known scale of the right angle ruler in the photo. Use the “Analyze → Set Scale” function to calibrate the pixels and actual size, and input the known actual distance and unit. (3) Use the "Polygon Selections tool" or "Freehand Selections tool" to carefully outline the boundary of the co-flesh tissue along the cut area on the side of the coral, and select the area covered by the co-flesh; (4) Measure the area of ​​the selected area using the “Analyze → Measure” function. The software will automatically output the area value in actual units (cm²). (5) Measure and sum the multiple photos taken at the same time point to obtain the total fleshy coverage area of ​​the coral piece at that time point; (6) Group the total surface area data of the meat cover at each time point according to the recovery stage, and calculate the daily average surface area growth of each stage.

[0039] The method for calculating the daily average surface area increase is: the net increase in the area covered by meat during the period divided by the number of days the period lasts.

[0040] The measurement results are as follows Figure 3 As shown: During the healing period of the sarcophagus (0–11 days): the average daily surface area increase was 0.209 ± 0.034 cm². During the bud formation period (11–15 days): the average daily surface area increase was 0.173 ± 0.042 cm². Polyp formation period (15–21 days): The average daily surface area increase is 0.274 ± 0.054 cm².

[0041] Statistical analysis showed that the average daily surface area increase of porous corals during the polyp formation period was significantly higher than that during the co-medullary healing period and the bud base formation period (p<0.05).

[0042] 4. Periodic sampling and enzyme activity index determination Sampling was conducted at the following key postoperative time points: 30 minutes postoperatively, 1 day postoperatively, 11 days postoperatively (end of the co-healing period), 15 days postoperatively (end of the budding period), and 21 days postoperatively (mature stage of polyp formation). At each time point, three coral samples (n=3) were randomly selected from the experimental group and the control group for antioxidant index determination.

[0043] The specific operating method is as follows: (1) Tissue fluid extraction: Take coral samples, rinse the surface with pre-cooled 0.86% physiological saline to remove seawater residue, and then blot the surface moisture with filter paper. Accurately weigh about 0.5 g of coral tissue, add 9 times the volume of pre-cooled physiological saline (i.e., at a weight-to-volume ratio of 1:9), and homogenize using a tissue homogenizer under ice bath conditions (2500 rpm, 10 seconds each time, 30 seconds interval, repeated 5 times until the tissue is fully homogenized). Centrifuge the homogenate at 4℃ and 3000 rpm for 10 minutes, and take the supernatant as the tissue fluid to be tested.

[0044] (2) Protein content determination: The total protein content in the tissue fluid was determined by the Bradford method. The supernatant to be tested was mixed with Coomassie Brilliant Blue G-250 staining solution in a certain proportion, and the absorbance value was measured at a wavelength of 595 nm. A standard curve was plotted using bovine serum albumin (BSA) as a standard, and the protein concentration of the sample was calculated.

[0045] (3) SOD (superoxide dismutase) activity assay: An SOD activity assay kit (e.g., Beijing Box Biotechnology Co., Ltd., catalog number AKAO001M) was used, following the kit instructions. The basic principle is that superoxide anion radicals (O2⁻·) are generated through the xanthine and xanthine oxidase reaction system. These radicals can reduce nitroblue tetrazolium to formazan, and SOD can inhibit this reaction. The absorbance value was measured at 560 nm. One unit of SOD activity (U / mg prot) was defined as the amount of SOD corresponding to a 50% inhibition rate of nitroblue tetrazolium reduction per milligram of tissue protein.

[0046] (4) CAT (catalase) activity assay: Use a CAT activity assay kit (e.g., Beijing Box Biotechnology Co., Ltd., catalog number AKAO003M) and follow the kit instructions. The basic principle is that CAT can catalyze the decomposition of H2O2 into H2O and O2. CAT activity is quantified by measuring the rate of decrease in absorbance at a wavelength of 240 nm. One unit of CAT activity (U / mg prot) is defined as the amount of H2O2 decomposed per milligram of tissue protein per minute.

[0047] (5) GS (glutamine synthase) activity assay: Use the GS activity assay kit and follow the instructions. The basic principle is that GS (glutamine synthase) catalyzes the reaction of ammonia and glutamate to produce glutamine. The activity of GS is indirectly quantified by measuring the release of inorganic phosphorus or by measuring the consumption of NADH using a coupled enzyme reaction.

[0048] It should be noted that GSH and LPO were not measured in this embodiment, but GS was used instead to monitor the synergistic response between coral nitrogen metabolism and antioxidant system.

[0049] The measurement results are as follows Figure 2 As shown: SOD activity such as Figure 2 As shown in Figure a: SOD activity increased sharply from 30 min to 11 days postoperatively, reaching a peak on day 11, which was significantly higher than the level at 30 min postoperatively (p<0.05). It began to decline on day 15 postoperatively, and by day 21 postoperatively, SOD activity had decreased to a level that was not significantly different from the preoperative normal level.

[0050] CAT activity such as Figure 2 As shown in b, CAT activity exhibits a similar dynamic trend to SOD, increasing sharply from 30 min to 11 days post-surgery, peaking on day 11, declining from day 15, and returning to normal levels by day 21 post-surgery.

[0051] GS activity such as Figure 2 As shown in c, GS activity remained at a high level during the acute injury period, the co-mucosa healing period, and the bud base formation period. It began to decrease during the polyp formation period and gradually dropped to a level that was not significantly different from the normal state.

[0052] 5. Recovery Status Assessment and Judgment By combining morphological observation results, the growth rate of the co-flesh coverage area, and antioxidant enzyme activity characteristics, the postoperative recovery status of porous corals was comprehensively evaluated according to the following correspondence: Table 1. Postoperative recovery status assessment and determination of *Porous stellaria* Judgment criteria: A coral is deemed "fully restored and suitable for transplantation" when it simultaneously meets the following three conditions: (1) It has entered the polyp formation stage (15-21 days after surgery), and mature coral polyps have appeared; (2) Increased tissue regeneration rate - the average daily surface area growth during the polyp formation period (0.274±0.054 cm²) was higher than the average daily growth during the previous stage of bud formation period (0.173±0.042 cm²). (3) Key antioxidant indicators (SOD activity, CAT activity, GS activity) have recovered to levels that are not significantly different from those of the uncut healthy control group (p>0.05).

[0053] For porous corals, all of the above conditions are usually met 21 days after the operation, and they are considered suitable for transplantation.

[0054] 6. Changes in the regenerated surface area and the number of newly formed polyps of the porous coral *Symplocos macrantha* Experimental methods: Starting from day 0 post-surgery, photos of all four sides of each coral fragment were taken daily between 16:00 and 17:00 using a filter, with a right-angle ruler used for dimensional comparison during the photos. Image processing software was used to analyze the photos, calculate the regenerated surface area of ​​the coral tissue, and statistically analyze the daily average surface area growth at different stages. In the later stages of regeneration, the number of newly formed polyps (including bud bases and polyps) of each coral fragment was directly counted, and the polyp density in the later stages of regeneration was calculated per unit of regenerated area to analyze the morphological regeneration characteristics of the porous coral.

[0055] result: like Figure 3 As shown, in terms of the increase in regenerated surface area, the growth rate tends to stabilize during the co-healing period (0-11 days) and the bud formation period (11-15 days), which represent the initial stage of coral post-operative repair of damaged areas. The average daily increase in surface area during the co-healing period was 0.1985 ± 0.0504 cm². 2 The average daily surface area increase during the bud formation period was 0.1755 ± 0.066 cm². 2 The growth rate during the polyp formation period (15-21 days) was significantly higher than in other stages, representing a period of rapid increase in coral regeneration area, with the average daily increase in surface area reaching 0.2922 ± 0.095 cm². 2 .

[0056] The density changes of polyps in the later stages of regeneration were statistically analyzed for the regenerated polyps of *Coral spp.* from day 14 to day 30. On day 14, 6 out of 10 observed corals showed budding, with a polyp regeneration density ranging from 0 to 3.626473 units / cm². On day 18, the density ranged from 0.881834 to 6.077606 units / cm². On day 22, the density ranged from 3.843382 to 9.950249 units / cm². On day 26, the density ranged from 8.008154 to 10.61882 units / cm². On day 30, the density ranged from 9.630147 to 12.14815 units / cm².

[0057] 7. Changes in the density of zooxanthellae and their chlorophyll a content in different stages of the porous coral *Coral simonii*. Methods for determining zooxanthellae density and chlorophyll a content: Take 6 aliquots of 12 mL each from the aforementioned tissue homogenate, centrifuge at 4000 rpm for 10 min, remove the supernatant, and resuspend the remaining algae in 5 mL of formaldehyde for zooxanthellae density determination. The number of symbiotic algae is counted under a microscope using a hemocytometer, and the unit is the coral regeneration area (cell / cm2). Add 8 mL of methanol to the other 3 aliquots and extract overnight at 4 ℃ for chlorophyll a content determination. Then centrifuge at 4000 rpm for 10 min, and take the supernatant to scan the 250-750 nm band in a UV-Vis spectrophotometer.

[0058] The formula for calculating chlorophyll a (Chl a) in the extract is as follows: ; Where A630, A664, and A750 represent the absorbance values ​​at wavelengths of 630, 664, and 750 nm, respectively. Chlorophyll a content is expressed in units of regeneration area (g / cm²). 2 ).

[0059] The results are as follows Figure 4 As shown in (a), the density of zooxanthellae continued to increase with the regeneration process, and the chlorophyll a content increased significantly during the co-healing period.

[0060] like Figure 4 As shown, during the postoperative healing process of the porous coral, the changes in zooxanthellae density and chlorophyll a content were consistent, with the highest zooxanthellae density and chlorophyll a content in the normal control group (Con). From 11 to 21 days after surgery, the zooxanthellae density and chlorophyll a content showed a gradual upward trend, indicating that the symbiotic algal population and chlorophyll a content were gradually recovering and expanding in the later stage of coral regeneration, but there were still significant differences compared with the normal control group (P<0.05).

[0061] Comparative Example 1: Assessment of postoperative recovery status of shield-shaped gyroscope coral after cutting.

[0062] This comparative example uses shield-shaped gyroscope coral ( Turbinariapeltata The following steps are taken as the object and the recovery status after the resection is evaluated using the method described in this invention: 1. Sample preparation and experimental setup Healthy shield-shaped gyroscope coral mothers were selected and cut into uniform coral pieces using bone shears, each piece approximately 10 cm³. The coral pieces were then fixed onto ceramic bases and placed in an indoor recirculating aquaculture system for recovery and cultivation. The cultivation conditions were: water temperature maintained at 27℃, salinity 33 ppt, light intensity approximately 200 μmol photons·m⁻²·s⁻¹, pH 8.1, and a photoperiod of 12 h:12 h (light:dark). Uncut healthy shield-shaped gyroscope corals served as a control group; these samples underwent no cutting treatment and were cultivated under the same conditions.

[0063] 2. Daily morphological observation and recovery phase division Starting from the day the cutting surgery was completed (recorded as post-operative day 0), photographs of the coral fragments were taken daily at set times. During photography, the ceramic base was carefully rotated using tweezers to capture multiple views of the coral fragment (at least one clear digital photograph of each view) to ensure a complete record of the morphological changes in the cut area and surrounding tissue. A right-angle ruler of known dimensions was placed in each photograph as a reference for subsequent dimensioning and area calculations.

[0064] Based on daily continuous morphological observation, the postoperative recovery process of shield-shaped gyroscope corals was divided into the following three characteristic stages: (1) Co-suture healing period (days 0-7 post-surgery): In the early post-operative period, the coral-cut surface tissue is exposed, and the bone cross-section is visible. As time progresses, new co-suture tissue proliferates from the edge of the wound and expands towards the center. The new tissue is translucent milky white, which is clearly different in color from the surrounding original tissue. By day 7, the new co-suture tissue has completely covered the damaged area, and the exposed bone is no longer visible to the naked eye.

[0065] (2) Sprouting stage (7-15 days post-surgery): After the sarcoma tissue completely covers the damaged area, it continues to proliferate and thicken, and papillary protrusions—sprouting bases—begin to appear on the tissue surface. The sprouting bases are about 2 mm in size and are semi-transparent vesicles. These sprouting bases are the precursor structures for subsequent polyp development.

[0066] (3) Polyp formation period (15 to 30 days post-surgery): Pigmentation begins to appear in the new tissue, and the color gradually deepens, becoming consistent with the surrounding normal tissue. The bud base further develops and differentiates, forming a mature coral polyp with complete tentacle structure and oral disc. The newly formed polyp is usually slightly lighter in color than the surrounding original polyps.

[0067] Postoperative morphological changes of gyroscope corals, such as Figure 5 As shown: Figure 5 Figure a shows the initial cut surface of the coral block 30 minutes after surgery, with obvious damage area visible; Figure 5b shows that sclerotherapy tissue completely covered the damaged area 7 days after surgery; Figure 5 The CT scan showed papillary new shoots on the tissue surface 15 days post-surgery. Figure 5 The results showed that mature new polyps had formed 30 days after the operation.

[0068] 3. Quantitative measurement of the area covered by the co-meat The measurement results are as follows Figure 6 As shown: During the symphysis healing period (0-7 days): the average daily surface area growth was 0.535±0.136 cm²; during the bud formation period (7-15 days): the average daily surface area growth was 0.075±0.04 cm²; during the polyp formation period (15-30 days): the average daily surface area growth was 0.103±0.033 cm².

[0069] Statistical analysis showed that the average daily surface area increase during the healing period of the sarcophylloides was significantly higher than that during the bud formation and polyp formation periods (p<0.05), indicating that the early postoperative period is a critical stage for the rapid proliferation of sarcophylloid tissue and its coverage of the damaged surface, and the tissue regeneration rate is most active during this stage.

[0070] 4. Periodic sampling and enzyme activity index determination Sampling was conducted at the following key postoperative time points: 30 minutes postoperatively, 1 day postoperatively, 7 days postoperatively (end of the co-healing period), 15 days postoperatively (end of the bud formation period), and 30 days postoperatively (mature stage of polyp formation). At each time point, three coral samples (n=3) were randomly selected from the experimental group (cutting group) and the control group (uncutting group) for antioxidant index determination.

[0071] The specific operating method is as follows: (1) Tissue fluid extraction: Take coral samples, rinse the surface with pre-cooled 0.86% physiological saline to remove seawater residue, and then blot the surface moisture with filter paper. Accurately weigh about 0.5 g of coral tissue, add 9 times the volume of pre-cooled physiological saline (i.e., at a weight-to-volume ratio of 1:9), and homogenize using a tissue homogenizer under ice bath conditions (2500 rpm, 10 seconds each time, 30 seconds interval, repeated 5 times until the tissue is fully homogenized). Centrifuge the homogenate at 4℃ and 3000 rpm for 10 minutes, and take the supernatant as the tissue fluid to be tested.

[0072] (2) Protein content determination: The total protein content in the tissue fluid was determined by the Bradford method. The supernatant to be tested was mixed with Coomassie Brilliant Blue G-250 staining solution in a certain proportion, and the absorbance value was measured at a wavelength of 595 nm. A standard curve was plotted using bovine serum albumin (BSA) as a standard, and the protein concentration of the sample was calculated.

[0073] (3) SOD activity assay: The SOD activity assay kit (e.g., Beijing Box Biotechnology Co., Ltd., catalog number AKAO001M) was used, and the procedure was performed according to the kit instructions. The basic principle is that superoxide anion free radicals (O2⁻·) are generated through the xanthine and xanthine oxidase reaction system. The latter can reduce nitroblue tetrazolium to formazan, and SOD can inhibit this reaction. The absorbance value was measured at a wavelength of 560 nm. The amount of SOD corresponding to a 50% inhibition rate of nitroblue tetrazolium reduction per milligram of tissue protein was defined as one unit of SOD activity (U / mg prot).

[0074] (4) CAT activity assay: Use a CAT activity assay kit (e.g., Beijing Box Biotechnology Co., Ltd., catalog number AKAO003M) and follow the kit instructions. The basic principle is that CAT can catalyze the decomposition of H2O2 into H2O and O2. CAT activity is quantified by measuring the rate of decrease in absorbance at a wavelength of 240 nm. One unit of CAT activity (U / mg prot) is defined as the amount of H2O2 decomposed per milligram of tissue protein per minute.

[0075] (5) GSH content determination: The reduced glutathione (GSH) assay kit was used, and the operation was performed according to the kit instructions. The basic principle is that GSH reacts with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to generate a yellow product. The absorbance value was measured at a wavelength of 412 nm. A standard curve was plotted using GSH standards of known concentrations, and the GSH content of the sample (μmol / gprot) was calculated.

[0076] (6) LPO level determination: The LPO level was reflected by measuring the malondialdehyde (MDA) content using a lipid peroxidation detection kit. The basic principle is that MDA reacts with thiobarbituric acid (TBA) under high temperature and acidic conditions to generate a red product. The absorbance value was measured at a wavelength of 532 nm. A standard curve was plotted using tetraethoxypropane as a standard, and the result was expressed as μmol / g prot.

[0077] The measurement results are as follows Figure 7 As shown: SOD activity such as Figure 7 As shown in Figure a: SOD activity increased sharply from day 0 to day 7 post-surgery, reaching a peak on day 7, and the peak value was significantly higher than that at 30 minutes post-surgery (p<0.05). It began to decline on day 15 post-surgery, and by day 30 post-surgery, SOD activity had recovered to a level that was not significantly different from the initial level at 30 minutes post-surgery (p>0.05).

[0078] CAT activity such as Figure 7As shown in b, CAT activity increased sharply within 7 days post-surgery, peaking on day 7. At 15 and 30 days post-surgery, CAT activity decreased slightly but remained at a high level, showing an overall downward trend.

[0079] GSH content such as Figure 7 As shown in c, GSH levels increased sharply within 7 days post-surgery, peaking on day 7 and significantly higher than the level at 30 minutes post-surgery (p<0.05). GSH levels began to decline on day 15 post-surgery, returning to levels not significantly different from those at 30 minutes post-surgery by day 30 (p>0.05).

[0080] LPO levels as Figure 7 As shown in d, LPO levels significantly increased and reached a peak within 7 days post-surgery. Although they decreased during the subsequent recovery period, they remained at a high level. By day 30 post-surgery, there was still a significant difference from the initial level at 30 minutes post-surgery (p<0.05), suggesting that the repair of lipid peroxidation damage is a relatively slow process.

[0081] 5. Recovery Status Assessment and Judgment By combining the morphological observation results of step 2, the growth rate of the co-flesh coverage area of ​​step 3, and the antioxidant enzyme activity characteristics of step 4, the postoperative recovery status of the shield-shaped gyroscope coral was comprehensively evaluated according to the following correspondence, and the suitability of the coral seedlings for transplantation was determined.

[0082] Table 2. Postoperative recovery status assessment and determination of *Gyrococcus pluvialis* Judgment criteria: A coral is deemed "fully restored and suitable for transplantation" when it simultaneously meets the following three conditions: (1) It has entered the polyp formation stage (15-30 days after surgery), and mature coral polyps have appeared; (2) The tissue regeneration rate tends to stabilize - the daily average surface area growth is not significantly different from the previous stage (bud formation period) (p>0.05); (3) Key antioxidant indicators (SOD activity, GSH content) have recovered to levels that are not significantly different from those of the uncut healthy control group (p>0.05).

[0083] For shield-shaped gyroscope corals, all of the above conditions are usually met 30 days after the operation, and they are judged to be suitable for transplantation.

[0084] As shown in Example 1 and Comparative Example 1, the postoperative recovery process of different species of nodular corals has the following common characteristics: all undergo a three-stage recovery process: the co-healing stage → the bud formation stage → the polyp formation stage; the antioxidant enzyme activity reaches its peak during the co-healing stage and then gradually declines to normal levels. Meanwhile, there are also significant differences between different species: for *Gyrococcus pterosa*, the co-healing stage is 0–7 days (average daily growth of 0.535 cm²), the bud formation stage is 7–15 days, and the polyp formation stage is 15–30 days, with the peak of tissue regeneration occurring in the first stage; while for *Gyrococcus porphyria*, the co-healing stage is 0–11 days (average daily growth of 0.209 cm²), the bud formation stage is 11–15 days, and the polyp formation stage is 15–21 days, with the peak of tissue regeneration occurring in the third stage. The recovery pattern of *Coral porphyria* in Example 1 differs from that of *Coral scutellaria* in Comparative Example 1 (where growth is fastest during the co-healing phase, followed by a significant slowdown). *Coral porphyria* exhibits a different recovery pattern—the polyp formation phase is the stage of fastest tissue expansion, indicating species-specific postoperative tissue regeneration patterns among different species of nodular corals. This suggests that different species of nodular corals possess species-specific recovery patterns and optimal transplantation timing.

[0085] Compared with Comparative Example 1 (Shield-shaped Gyratory Coral), the average daily surface area growth of Porous Coral during the polyp formation period (15-21 days) (0.274±0.054 cm²) not only did not decrease, but was significantly higher than that during its budding period (0.173±0.042 cm²) (p<0.05). This difference indicates that for Porous Coral, the transplantation criterion of 'regeneration rate tending to stabilize' in Comparative Example 1 cannot be mechanically applied; instead, 'significantly increased regeneration rate' must be used as the core indicator that it has entered the suitable transplantation period.

[0086] Therefore, the 'postoperative recovery assessment method for porous star coral' proposed in this invention is a non-obvious technical solution specifically designed for this species and based on its unique physiological regeneration patterns.

[0087] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0088] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for selecting suitable individuals for transplantation of porous star coral (Astreopora sp.) after cutting, characterized in that, Includes the following steps: Porous unicorn corals were cut and then cultured to the polyp formation stage, which included the symbiotic healing stage, the bud base formation stage, and the polyp formation stage. During the recovery and culture process, the daily average surface area growth and antioxidant enzyme activity of porous coral were measured. Individuals who meet the criteria are selected as suitable transplant recipients; The aforementioned indicator meets the following conditions simultaneously: (a) The daily average surface area growth during the polyp formation period was higher than that during the bud base formation period, and there was a statistically significant difference between the daily average surface area growth during the polyp formation period and the daily average surface area growth during the bud base formation period. (b) The activity of antioxidant enzymes during polyp formation has recovered to the level of uncut healthy corals.

2. The method according to claim 1, characterized in that, The antioxidant enzyme activities include superoxide dismutase activity, catalase activity, and glutamine synthase activity.

3. The method according to claim 1, characterized in that, The criteria for determining the symbiotic healing period, bud base formation period, and polyp formation period are as follows: The healing phase of commensal tissue: New commensal tissue gradually covers the damaged area, and by the end of this phase, the commensal tissue completely covers the damaged area; Bud formation stage: The symbiotic tissue continues to proliferate and thicken, and bud bases with nipple-like protrusions begin to appear on the surface; Polyp formation stage: The bud base develops into a mature coral polyp.

4. The method according to claim 1, characterized in that, The calculation process for the average daily surface area increase is as follows: the net increase in the area covered by the meat during this period divided by the number of days that the period lasts.

5. The method according to claim 4, characterized in that, The quantitative measurement of the co-flesh coverage area includes: taking pictures of the coral cutting area using a ruler reference object, and calculating the co-flesh coverage area at each time point using image analysis software.

6. The method according to claim 5, characterized in that, The image analysis software is ImageJ.

7. The method according to claim 1, characterized in that, The method for determining antioxidant enzyme activity is as follows: antioxidant enzyme activity is determined at at least two time points, namely, at the end of the sarcophyll healing period, at the end of the bud base formation period, and at the end of the polyp formation period.

8. The method according to claim 7, characterized in that, The steps for determining antioxidant enzyme activity were as follows: coral samples were selected at 29-30 minutes, 1-2 days, 11-12 days, 15-16 days, and 21-22 days after cutting for antioxidant enzyme activity determination.

9. The method according to claim 1, characterized in that, The determination of suitability for transplantation also includes: the density of symbiotic zooxanthellae and / or chlorophyll a content of the coral have recovered to the level of uncut healthy corals.

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