TWI806760B - Coral culturing method - Google Patents

Abstract

The present invention relates to a method for culturing coral, comprising: fixing a healthy coral fragment in a miniature transparent container; culturing the healthy coral fragment in a seawater environment with a constant temperature of 23-25°C; and providing a light intensity ranging from 60 to 120 μmol/m ²/s. The coral culturing method of the present invention can reduce the costs and provide an observation environment at the coral cell level.

Description

Coral culture method

The present invention relates to a coral cultivation method, in particular to a coral cultivation method using a micro system.

Coral reefs are colorful and vibrant ecosystems that are very important to marine life and resources. However, corals are very sensitive and vulnerable to changes in seawater, so coral restoration and related research on corals can be used as a reference for marine resource planning.

In order to conduct related research on corals, researchers need to culture corals in order to conduct various experimental tests, such as testing corals' responses to different components to understand the pollution status of seawater. However, the cultivation of corals is very difficult. The traditional coral cultivation is to raise corals in an aquarium to simulate the growth environment of corals in the ocean, so as to raise living corals. The experimental test is also carried out in the whole aquarium. Once the coral dies, it needs to be recultivated before the experiment can be carried out again.

Because of the difficulty of coral cultivation, the cost of coral research cannot be reduced. In addition to the cost of maintaining the aquarium, the cost of space, the cost of consumables, etc. are also quite high, and the time-consuming cost is also relatively high. There is an urgent need for more effective and cost-effective alternatives. .

The invention provides a coral cultivation method, comprising: fixing a healthy coral segment in a miniature transparent container; cultivating in a seawater environment with a constant temperature of 23-25°C; and providing an illumination intensity ranging from 60 to 120 μmol/m ² /s.

In some embodiments, the light source used includes at least blue light and red light.

In some embodiments, the light source is irradiated for 10 to 14 hours per day.

In some embodiments, the coral cultivation method further comprises feeding protein twice a week.

In certain embodiments, the protein is brine shrimp.

In some specific embodiments, the amount of the brine shrimp is 5-11 per milliliter of seawater.

In some embodiments, the water is changed more than 2-5 hours after each feeding.

In some embodiments, the salinity of the seawater is between 32 and 38.

In some embodiments, the light intensity is in the range of 60-80 μmol/m ² /s.

In some embodiments, the miniature transparent container is a Petri dish.

The coral cultivation method provided by the invention enables the coral to grow stably in the micro system, can significantly increase the growth speed of the coral, and can be cultivated for more than three months. Since corals can survive in the micro system, space cost, cost of cultivation consumables (such as sea water, container, food, etc.) are saved. When conducting coral experiments, only a small amount of reagents are needed to meet the test conditions, which reduces the cost of reagents and the consumption of samples. More importantly, corals and micro-systems can be directly moved to the microscope for observation, and changes at the cell level can be observed, which is significantly more helpful to research than the traditional cultivation method using aquariums.

Other technical contents, features and effects of the present invention will be clearly presented in the following detailed description of preferred embodiments with reference to the drawings.

The invention provides a coral cultivation method, comprising: fixing a healthy coral segment in a miniature transparent container; cultivating it in a seawater environment with a constant temperature of 23-25°C; and providing an illumination intensity of 60-120 μmol/m ² /s. In certain embodiments, the temperature is about 23°C, 24°C, 25°C. In some specific embodiments, the light intensity can be 60 μmol/m ² /s, 65 μmol/m ² /s, 70 μmol/m ² /s, 75 μmol/m ² /s, 80 μmol/m ² /s, 85 μmol/m ² /s, 90μmol/m ² /s, 95μmol/m ² /s, 100μmol/m ² /s, 105μmol/m ² /s, 110μmol/m ² /s, 115μmol/m ² /s, 120μmol/m ² /s. In a preferred specific embodiment, the light intensity is in the range of 60 to 80 μmol/m ² /s, for example, the light intensity is 60 μmol/m ² /s, 62 μmol/m ² /s, 64 μmol/m ² /s, 66 μmol/ m ² /s, 68 μmol/m ² /s, 70 μmol/m ² /s, 72 μmol/m ² /s, 74 μmol/m ² /s, 76 μmol/m ² /s, 78 μmol/m ² /s, 80 μmol/m ² /s.

In some embodiments, the light source used includes at least blue light and red light. The light source may further contain violet or green light. In some specific embodiments, the light source can use LED lamps, and the number of blue LEDs is greater than that of red LEDs, for example, about 3 times, about 4 times, about 5 times, about 6 times the number. In a specific embodiment, the LED lamp includes purple LEDs, blue LEDs, green LEDs, and red LEDs. In a preferred embodiment, the ratio of the number of purple LEDs, blue LEDs, green LEDs, and red LEDs is 4:4:1:1.

In some specific embodiments, the light source is irradiated for 10 to 14 hours per day, such as 10 hours, 11 hours, 12 hours, 13 hours, 14 hours.

In some embodiments, the coral cultivation method further comprises feeding protein twice a week.

In certain embodiments, the protein is brine shrimp.

In some specific embodiments, the amount of the brine shrimp is 5-11/ml seawater, such as 5/ml seawater, 6/ml seawater, 7/ml seawater, 8/ml seawater, 9/ml Milliliter seawater, 10/ml seawater, 11/ml seawater.

In some specific embodiments, the water is changed after more than 2-5 hours after each feeding, such as 2 hours, 3 hours, 4 hours, 5 hours.

In some specific embodiments, the salinity of the seawater is between 32 and 38, for example, the salinity is 32, the salinity is 33, the salinity is 34, the salinity is 35, the salinity is 36, the salinity is 37, The salinity is 38.

In some embodiments, the miniature transparent container is a Petri dish.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the articles "a", "an" and "any" refer to one or more than one (ie, at least one) of the grammatical items of the item. For example, "an element" means one element or more than one element.

As used herein, the term "about", "approximately" or "approximately" means that the stated value or range is within 20%, preferably within 10%, and more preferably within 5%. Numerical quantities provided herein are approximations and are intended to be inferred if the terms "about", "approximately" or "approximately" are not used.

As used herein, the term "salinity" refers to the total grams of dissolved substances in one kilogram of seawater.

Example Coral culture experiment

As shown in Figure 1, select a healthy stony coral or soft coral group, cut out about 5 * 5 mm coral fragments (step A), fix the coral fragments on the petri dish with underwater instant glue (step B), and then Add seawater (salinity about 34~36) to cover the coral fragments (step C). The volume of seawater required for the petri dish is about 60ml, and the lid should be covered to prevent water from evaporating. Put the petri dish into a constant temperature incubator and cultivate it at a constant temperature of about 23 ° C ~ 25 ° C (step D).

LED lights (Illumagic, Taiwan) suitable for coral growth were installed in the constant temperature incubator, and the light was illuminated for 12 hours every day. As shown in the LED light spectrum diagram in Figure 2, the main wavelength of the LED light source is blue light of 370nm, 400nm, 420nm and 450nm and a small part of red light of 620nm. The relative intensity refers to the light corresponding to each wavelength. The intensity value (unit: mW/m ² ) is divided by the light intensity value of the highest peak in the entire spectrum and multiplied by 100%; that is, the highest peak in the spectrum is regarded as relative intensity 100%, and other wavelengths (nm) are converted relative strength value. The highest peak in this figure is 470nm, which means its relative intensity is 100%. Based on this, the corresponding intensity of 620nm red light wavelength is about 20-30%. Blue light in different wavelength ranges can be used by corals.

Corals absorb calcium ions and bicarbonate ions from the water as they grow to form calcium carbonate skeletons. Coral and other microorganisms in the petri dish will also produce nutrients such as ammonia, nitrite, nitrate, and phosphate during the metabolic process. However, corals are very sensitive to the concentration of nutrients in the water, so the nutrients should be controlled at an extremely low level. Concentrations are very important culturing details. After research, the water quality conditions suitable for coral growth are approximately: salinity 34-36 ppt, alkalinity 7-10 KH, calcium ion concentration 380-450 ppm, phosphate <0.03 ppm, nitrate <0.2 ppm, nitrite < 0.2 ppm, ammonia <0.1 ppm. The embodiment of the present invention was tested by feeding 7 to 9 freshly hatched brine shrimp per milliliter of seawater twice a week, removing the seawater in the container and adding new seawater after each feeding for 3 hours to provide a suitable growth environment for corals.

In order to understand the effect of different light intensities on coral fragments, the growth of coral fragments under three different light intensities was tested. Applying the above culture conditions, three coral colonies were used to cut coral fragments required for three different light intensity groups for cultivation experiments. Each group cut 9 coral fragments from three coral colonies respectively, so each group of experimental corals The number of samples is 27. The first group was given a light intensity of 20-40 μmol/m ² /s for cultivation and testing, the second group was given a light intensity of 60-80 μmol/m ² /s for cultivation and testing, and the third group was given a light intensity of 100-120 μmol /m ² /s light intensity for cultivation and experiment.

Coral Fragment Observation

As shown in Figure 3A to Figure 3F, on the 0th day, use a digital camera to photograph and observe the tissue state of coral fragments from the first group to the third group placed under a dissecting microscope to obtain photos 3A to 3C, and in Take pictures in the 4th week to get photo 3D to photo 3F. From the progress of the photos, it can be confirmed that after 4 weeks of cultivation, the range of corals in the second group (60~80μmol/m ² /s) was obviously expanded by three times, and the range of corals in the third group (100~120μmol/m ² /s) The range of corals has obviously expanded by more than two times, and they all grow faster than the first group.

As shown in Figure 4A, observe and record the number of surviving coral fragments in each group on day 0, week 2, and week 4. Coral fragments with tissue or polyps on them are considered living coral fragments. The calculation method for the survival rate of coral fragments is as follows: survival rate = (number of surviving coral fragments/number of original coral fragments) × 100%. After 4 weeks of cultivation, the survival rate of coral fragments in each group was still 100%, showing that under the aforementioned culture conditions but different light intensities, the survival rate of coral fragments was not affected.

As shown in Figure 4B, observe and record whether the coral fragments of each group of coral fragments on day 0, week 2, and week 4 are abnormal. Anomalous coral fragments are defined as: (a) fragments exhibiting loss of flesh or detachment or fragments of tissue that have disappeared together with the skeleton; and (b) that have lost more than 15% of the original area of the coral fragment. The calculation method of abnormal rate of coral fragments is as follows: abnormal rate = (number of abnormal coral fragments/number of original coral fragments) × 100%. After 4 weeks of cultivation, the abnormal rate of coral fragments in each group was 0%, showing that under the aforementioned culture conditions but different light intensities, the abnormal rate of coral fragments would not increase.

As shown in Figure 5, observe and record the number of coral tissue growth and attachment to the petri dish in the 0th day, the 2nd week, and the 4th week of each group of coral fragments. The definition of attachment is that the newly grown coral tissue is attached to Petri dish, and grow on the Petri dish along with the tissue. The calculation method of the attachment rate of coral fragments is as follows: attachment rate = (number of attached coral fragments/number of original coral fragments) × 100%. After 4 weeks of cultivation, it can be seen that the attachment rate of coral fragments in the second group exceeded 20% in the second week, which is faster than other groups. However, by the 4th week, the coral fragment attachment rates of the three groups were almost between 80% and 100%.

As shown in Figure 6A, use a digital camera (SONY DSC-RX100M2, Japan) to take photos of coral fragments in each group on the 0th day, 2nd week, and 4th week under a dissecting microscope. J software calculates the area size of the coral fragments on the photo, and calculates the area growth rate of the coral fragments. The calculation method is as follows: area growth rate = (the area of the coral fragments on the nth day / the area of the coral fragments on the 0th day), n=0, 14 , or 28, the statistical method is one-way ANOVA (p<0.05). After 4 weeks of cultivation, there was no difference between the three groups in the first stage of cultivation, but in the 4th week, it can be clearly found that the growth rate of the area of coral fragments in the second group is the highest, followed by the third group, and the first group The area growth rate of other coral fragments is the lowest. Therefore, using a light intensity of 60-120 μmol/m ² /s under the aforementioned culture conditions can increase the growth rate of corals, and it is better to use a light intensity of 60-80 μmol/m ² /s, which can greatly shorten the time required for coral cultivation .

Coral fragment weights for each group were measured at day 0, week 2, and week 4 as shown in Figure 6B. Pour out the water in the petri dish before weighing, wipe off the remaining liquid with the lens paper, place the petri dish and the coral fragments growing on it on a precision balance for three times, and calculate the weight of the coral fragments after taking the average of the three weights Weight changes. The weight growth of coral fragments is calculated as follows: weight growth = [(petri dish weight on day n-petri dish weight on day 0)/3], n=0, 14, or 28. During the 4-week cultivation process, it can be found that the weight growth of coral fragments in the second group is higher than that of the other two groups, indicating that the use of light intensity of 60-80 μmol/m ² /s has a better effect on the growth of corals. Facilitate the effect.

Photochemical Efficiency Experiment of Symbiotic Algae

In the past research, we have known that corals will live in symbiosis with symbiotic algae, and symbiotic algae provide the main source of nutrition for corals. Chemical efficiency (Fv/Fm) is the most commonly used measure of coral health (Warner et al., 1999; Jones et al., 2000).

Chlorophyll fluorescence analysis method was used to measure and record the change of photochemical efficiency of Symbiodinium photosystem II (PSII) in coral fragment tissue of three groups on day 0 and week 4. The measurement method is to place the petri dish to be measured in a dark environment for 30 minutes, and then use the Junior PAM Teaching Chlorophyll Fluorometer (JUNIOR-PAM Teaching ChlorophyII Fluorometer) (Heinz Walz GmbH, Germany) to randomly select coral fragments in a dark environment. The organized position was measured first, and the measured Ft value (steady-state value) was fixed at 240±10, and the photochemical efficiency of photosystem II (PSII photochemical efficiency) was measured at two organized positions randomly selected in each coral segment. efficiency, Fv/Fm), where F0 is the difference between the lowest and highest fluorescence, and Fm is the highest value of fluorescence after irradiating a saturated light source in a dark state, so it is measured according to the calculation formula Fv/Fm=(Fm-F0)/Fm Get the photochemical efficiency value. The Ft value is the real-time fluorescence output. According to previous studies, the Ft value of corals is mostly between 200-300, and the average Ft value of corals in our experiments is 240±10. This value is fixed to measure light accurately. chemical efficiency.

As shown in Figure 7, the photochemical efficiency of the symbiotic algae in the coral fragments of the first group averaged about 0.689 ± 0.002 in the fourth week, and the photochemical efficiency of the symbiotic algae in the coral fragments of the second and third groups The mean values at week 4 were 0.627 ± 0.002 and 0.605 ± 0.003, respectively. The values of the above photochemical efficiency are all within the normal range, which can show that the symbiotic algae in the coral cultured with the light intensity of 20~120μmol/m ² /s are healthy. Although the photochemical efficiency of symbiotic algae is slightly lower under the light intensity of 60-120 μmol/m ² /s, it can be known from the aforementioned research on the area growth rate of coral fragments and the weight growth of coral fragments that the light intensity of 60-80 μmol/m ² /s The growth rate is the fastest under the light intensity, and the slight difference in photochemical efficiency value does not affect the growth of corals.

In previous studies, the calcification efficiency of corals reared in aquarium systems was higher under stronger light (300 μmol/m ² /s, measured on coral colonies) than under weaker light (150 μmol/m ² /s , measured on coral colonies) cultured corals, accelerated skeletal formation, and the density of symbiotic algae in weaker light cultures was higher than in stronger light cultures (Dobson et al., 2021).

However, the light intensity used in the coral cultivation method of the embodiment of the present invention is weaker than that used in previous studies, the maximum light intensity is only 120 μmol/m ² /s, and it is found that the light intensity is between 20~40 μmol/m ² /s, 60 Among the three light intensities of ~80μmol/m ² /s and 100~120μmol/m ² /s, coral fragments grew faster under the light of 60~120μmol/m ² /s. More particularly, the coral fragments grow fastest under the light of 60-80 μmol/m ² /s, and there are statistical differences with the coral fragments cultured in the other two groups of light. It can be seen that the coral fragments provided by the embodiments of the present invention Cultivate methods to overcome past technical biases and produce unanticipated efficacy.

As shown in Figure 8A and Figure 8B, the coral cultivation method of the embodiment of the present invention can continuously cultivate coral fragments for up to three months, and it can be seen from the photos that the coral fragments can continue to grow steadily. In addition, during the cultivation process, the alkalinity (bicarbonate concentration) and calcium ion concentration in the water are both consumed and reduced, and it can also be confirmed that the coral absorbs nutrients and forms skeletons. It can be seen that the cultivation method of the embodiment of the present invention can provide various growth requirements of corals, and is suitable as a cultivation method for corals for research or breeding.

The present invention has been disclosed through the above-mentioned embodiments, which are only part of the preferred embodiments of the present invention, but they are not intended to limit the present invention. Any person familiar with this technical field who has ordinary knowledge can understand the aforementioned technology of the present invention Features and embodiments, and equivalent changes or modifications made without departing from the spirit and scope of the present invention still fall within the scope of the present invention, and the scope of patent protection of the present invention must be defined by the appended claims of this specification Whichever prevails.

A: Step A B: Step B C: Step C D: Step D

Fig. 1 is a schematic flow chart of the cultivation method of the embodiment of the present invention.

Fig. 2 is a spectrum diagram of the LED lamp used in the embodiment of the present invention.

Figure 3A to Figure 3F are photos of coral fragments in the embodiment of the present invention, Figure 3A, Figure 3B, and Figure 3C are respectively the first group at the initial stage of cultivation (light intensity is 20-40 μmol/m ² /s), the second group The photographs of group (60-80 μmol/m ² /s) and the third group (100-120 μmol/m ² /s), Figure 3D, Figure 3E, Figure 3F are respectively the first and fourth weeks of culture. Photos of the first group, the second group, and the third group.

Fig. 4A is a line graph of the survival rate of coral fragments in different groups according to the embodiment of the present invention (one-way ANOVA, p<0.05). Fig. 4B is a line chart of the abnormal rate of coral fragments in different groups according to the embodiment of the present invention (one-way ANOVA, p<0.05).

Fig. 5 is a line chart of the attachment rate of coral fragments of different groups in the embodiment of the present invention (one-way ANOVA, p<0.05).

Fig. 6A is a line graph of area growth rate of coral fragments in different groups according to the embodiment of the present invention (one-way ANOVA, p<0.05). Fig. 6B is a line graph of weight growth of coral fragments in different groups according to the embodiment of the present invention (one-way ANOVA, p<0.05).

Fig. 7 is a bar graph of the photochemical efficiency of different groups of coral fragments in the embodiment of the present invention at the beginning of culture and the 4th week (one-way ANOVA, p<0.05).

Fig. 8A is a photograph of coral fragments at the initial stage of cultivation according to an embodiment of the present invention. Fig. 8B is a photograph of coral fragments in the third month of cultivation according to the embodiment of the present invention.

Claims (10)

A method for cultivating corals, comprising: fixing a healthy coral fragment in a miniature transparent container; cultivating in a seawater environment with a constant temperature of 23-25°C; and providing light intensity ranging from 60 to 120 μmol/m ² /s. The coral cultivation method as described in Claim 1, wherein the light source used at least includes blue light and red light. The coral cultivation method as described in Claim 2, wherein the light source is irradiated for 10 to 14 hours per day. The coral cultivation method as described in Claim 1, which further comprises feeding protein twice a week. The coral cultivation method as described in claim 4, wherein the protein is brine shrimp. The method for cultivating corals as described in claim 5, wherein the amount of the brine shrimp is 5-11 per milliliter of seawater. The coral culture method as described in claim item 4, wherein the water is changed after feeding more than 2 to 5 hours each time. The coral cultivation method as described in Claim 1, wherein the salinity of the seawater is between 32 and 38. The coral cultivation method according to claim 1, wherein the light intensity is in the range of 60 to 80 μmol/m ² /s. The method for cultivating coral as described in Claim 1, wherein the miniature transparent container is a petri dish.

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