Cryogenic preservation solution for sperms of mystus nemurus as well as preparation method and application of cryogenic preservation solution

Abstract

The invention provides an ultralow-temperature preservation solution for sperms of mystus nemurus as well as a preparation method and application of the ultralow-temperature preservation solution, and belongs to the technical field of artificial breeding of mystus nemurus. The cryopreservation solution for sperms of mystus nemurus comprises a diluent and acetamide, wherein the final concentration of the acetamide is 80-150g / L; the diluent comprises the following components: sodium chloride, calcium chloride, magnesium sulfate, potassium chloride, glucose, astragalus polysaccharide and taurine. The ultra-low temperature preserving fluid effectively keeps the original pH of semen, maintains the osmotic pressure of the semen and inhibits sperm activation, the addition of acetamide reduces the freezing point of cytoplasm and prevents the formation of ice crystals, and the addition of astragalus polysaccharide and taurine improves the mitochondrial function of sperm and enhances the toughness of cell membranes. The survival rate and the functionality of the frozen sperms are effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of artificial breeding technology of silktail catfish, and particularly relates to a cryopreservation solution for silktail catfish sperm, its preparation method and application. Background Technology

[0002] The silky-tailed catfish (Hemibagrus wyckioides), also known as the white-whiskered male or long-whiskered fish, is a medium-to-large-sized, warm-water fish endemic to the Lancang River basin, valued for both its edible and ornamental qualities. Its flesh is tender, it grows rapidly, and has no intramuscular bones, making it an important economic fish species in Xishuangbanna, Yunnan. However, due to overfishing, habitat destruction, and reliance on wild parent fish for artificial breeding, wild silky-tailed catfish resources have drastically declined, leading to its listing as a critically endangered species. Traditional methods of artificial breeding require the dissection of wild males to obtain testes, resulting in resource waste and risks of inbreeding and genetic degradation.

[0003] Due to significant differences in the structure, physiological characteristics, freezing sensitivity, and suitability of cryoprotectants among different fish sperm, specific cryopreservation solutions are required for each species to ensure sperm survival and fertilization rates during freezing and thawing. However, no reports have been found regarding cryopreservation solutions for *Gnaphalium affine* sperm. Therefore, developing efficient cryopreservation technology for *Gnaphalium affine* sperm is of great significance for the conservation of *Gnaphalium affine* germplasm resources, optimization of artificial breeding, and genetic breeding. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a cryopreservation solution for sperm from the silky-tailed wrasse, its preparation method, and its application. This invention uses acetamide as an antifreeze agent, which lowers the freezing point of the sperm cytoplasm, prevents ice crystal formation, and effectively improves the survival rate and functionality of the sperm after freezing.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a cryopreservation solution for sperm from the silky-tailed fish, the cryopreservation solution comprising a diluent and acetamide, wherein the final concentration of acetamide is 80-150 g / L;

[0007] The diluent comprises the following components at the following concentrations: sodium chloride 4–5.5 g / L, calcium chloride 0.05–0.15 g / L, magnesium sulfate 0.07–0.15 g / L, potassium chloride 1.8–2.8 g / L, glucose 20–30 g / L, astragalus polysaccharide 0.5–1.3 g / L, and taurine 1–3 g / L.

[0008] Preferably, the diluent comprises the following components at the following concentrations: sodium chloride 4.5–5 g / L, calcium chloride 0.08–0.13 g / L, magnesium sulfate 0.09–0.13 g / L, potassium chloride 2–2.5 g / L, glucose 22–28 g / L, astragalus polysaccharide 0.7–1.2 g / L, and taurine 1.5–2.8 g / L.

[0009] Preferably, the diluent comprises the following components at the following concentrations: sodium chloride 4.8 g / L, calcium chloride 0.11 g / L, magnesium sulfate 0.12 g / L, potassium chloride 2.2 g / L, glucose 25 g / L, astragalus polysaccharide 1.1 g / L, and taurine 2.6 g / L.

[0010] This invention provides a method for preparing the cryopreservation solution for sperm of the silky-tailed wrasse, comprising the following steps:

[0011] (1) Dissolve sodium chloride, calcium chloride, magnesium sulfate, potassium chloride, and astragalus polysaccharide in water to obtain an initial diluted solution;

[0012] (2) Add glucose and taurine to the initial diluent and mix to obtain the diluent;

[0013] (3) Mix acetamide with diluent to obtain cryopreservation solution for sperm of wrasse.

[0014] Preferably, the initial diluent is stored at 2–6°C.

[0015] Preferably, the diluted solution is stored in an environment protected from light at 2–6°C.

[0016] The present invention also provides a method for cryopreservation of sperm from the silky-tailed fish. The method involves mixing the aforementioned cryopreservation solution for silky-tailed fish sperm with the sperm of the silky-tailed fish to obtain a mixture. The mixture is then placed on ice for equilibration for 20 to 30 minutes, and then cooled to -80°C at a rate of 12 to 18°C / min. Finally, the mixture is placed directly into liquid nitrogen for preservation.

[0017] Preferably, the volume ratio of the cryopreservation solution for *Cyprinus spp.* sperm to the semen of *Cyprinus spp.* is 1 to 3:1.

[0018] The present invention also provides the application of the cryopreservation solution for sperm of *Gnaphalium affine* or the cryopreservation solution for sperm of *Gnaphalium affine* obtained according to the preparation method, in preserving sperm of *Gnaphalium affine* or in in vitro artificial insemination of *Gnaphalium affine*.

[0019] Compared with the prior art, the present invention has the following beneficial effects: the ultra-low temperature preservation solution of the present invention effectively maintains the original pH of the semen, maintains the osmotic pressure of the semen, and inhibits the activation of sperm. The addition of acetamide lowers the freezing point of the cytoplasm and prevents the formation of ice crystals. The addition of astragalus polysaccharide and taurine enhances the function of sperm mitochondria and strengthens the cell membrane toughness, effectively improving the survival rate and functionality of sperm after freezing. Detailed Implementation

[0020] This invention provides a cryopreservation solution for sperm from the silky-tailed fish, the cryopreservation solution comprising a diluent and acetamide, wherein the final concentration of the acetamide is 80-150 g / L, preferably 90-140 g / L, and more preferably 110 g / L;

[0021] The diluent comprises components of the following concentrations:

[0022] Sodium chloride concentration: 4–5.5 g / L, preferably 4.5–5 g / L, more preferably 4.8 g / L;

[0023] The concentration of calcium chloride is 0.05–0.15 g / L, preferably 0.08–0.13 g / L, and more preferably 0.11 g / L;

[0024] Magnesium sulfate is 0.07–0.15 g / L, preferably 0.09–0.13 g / L, and more preferably 0.12 g / L;

[0025] The potassium chloride concentration is 1.8–2.8 g / L, preferably 2–2.5 g / L, and more preferably 2.2 g / L;

[0026] The glucose concentration is 20–30 g / L, preferably 22–28 g / L, and more preferably 25 g / L;

[0027] The astragalus polysaccharide concentration is 0.5–1.3 g / L, preferably 0.7–1.2 g / L, and more preferably 1.1 g / L;

[0028] Taurine concentration is 1–3 g / L, preferably 1.5–2.8 g / L, and more preferably 2.6 g / L.

[0029] In this invention, there are no special limitations on the source of the acetamide and diluent components; conventional commercially available products can be used. In this invention, the solvent for the diluent is water, preferably ultrapure water.

[0030] This invention provides a method for preparing the cryopreservation solution for sperm of the silky-tailed wrasse, comprising the following steps:

[0031] (1) Dissolve sodium chloride, calcium chloride, magnesium sulfate, potassium chloride, and astragalus polysaccharide in ultrapure water to obtain an initial diluted solution;

[0032] (2) Add glucose and taurine to the initial diluent and mix to obtain the diluent;

[0033] (3) Mix acetamide with diluent to obtain cryopreservation solution for sperm of wrasse.

[0034] In this invention, the initial diluent serves to maintain the pH and osmotic pressure of the semen and is stored at 2–6°C, preferably 3–5°C, and more preferably 4°C. Glucose, as a non-permeable cryoprotectant, can reduce the extracellular solute concentration, dehydrate the cells, reduce intracellular ice crystal formation, and reduce solute damage. Astragalus polysaccharides and taurine provide nutrients for the sperm. Acetamide, as a permeable cryoprotectant, can penetrate into the sperm, lower the cytoplasmic freezing point, and prevent ice crystal formation. The combination of glucose and acetamide improves the cryopreservation effect of *Meretrix meretrix* semen, ensuring that the rewarmed sperm still retains high motility.

[0035] In this invention, the diluent is stored in an environment protected from light at 2–6°C, preferably at 3–5°C, and more preferably at 4°C.

[0036] The present invention also provides a method for cryopreservation of sperm from the silky-tailed fish. The method involves mixing the aforementioned cryopreservation solution for silky-tailed fish sperm with the sperm of the silky-tailed fish to obtain a mixture. The mixture is then placed on ice for equilibration for 20 to 30 minutes, and then cooled to -80°C at a rate of 12 to 18°C / min. Finally, the mixture is placed directly into liquid nitrogen for preservation.

[0037] In this invention, the volume ratio of the cryopreservation solution for *Ceratos spp.* sperm to the semen of *Ceratos spp.* is 1–3:1, preferably 1.5–2.5:1, and more preferably 2:1; the mixture is preferably placed in a cryovial, and then the cryovial containing the mixture is placed on ice for equilibration for 20–30 minutes, preferably 22–28 minutes, and more preferably 25 minutes; the cooling rate is preferably 13–17°C / min, and more preferably 15°C / min.

[0038] The present invention also provides the application of the cryopreservation solution for sperm of *Gnaphalium affine* or the cryopreservation solution for sperm of *Gnaphalium affine* obtained according to the preparation method, in preserving sperm of *Gnaphalium affine* or in in vitro artificial insemination of *Gnaphalium affine*.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] Preparation of cryopreservation solution for sperm of the silky-tailed mandarin fish:

[0042] (1) Weigh 0.48g sodium chloride, 0.011g calcium chloride, 0.012g magnesium sulfate, 0.22g potassium chloride, and 0.11g astragalus polysaccharide and dissolve them in 90mL of ultrapure water to obtain the initial dilution solution;

[0043] (2) Add 2.5g glucose and 0.26g taurine to the initial dilution solution and mix. Make up to 100mL with ultrapure water to obtain the dilution solution. Store in a refrigerator at 4℃ away from light.

[0044] (3) Mix 11g of acetamide (antifreeze protectant) with diluent to obtain cryopreservation solution for sperm of wrasse.

[0045] Example 2

[0046] Preparation of cryopreservation solution for sperm of the silky-tailed mandarin fish:

[0047] (1) Weigh 0.4g sodium chloride, 0.015g calcium chloride, 0.015g magnesium sulfate, 0.18g potassium chloride, and 0.13g astragalus polysaccharide and dissolve them in 90mL of ultrapure water to obtain the initial dilution solution;

[0048] (2) Add 3g of glucose and 0.1g of taurine to the initial dilution solution and mix. Make up to 100mL with ultrapure water to obtain the dilution solution. Store in a refrigerator at 4℃ away from light.

[0049] (3) Mix 8g of acetamide (antifreeze protectant) with diluent to obtain cryopreservation solution for sperm of wrasse.

[0050] Example 3

[0051] Preparation of cryopreservation solution for sperm of the silky-tailed mandarin fish:

[0052] (1) Weigh 0.55g sodium chloride, 0.005g calcium chloride, 0.007g magnesium sulfate, 0.28g potassium chloride, and 0.05g astragalus polysaccharide and dissolve them in 90mL of ultrapure water to obtain the initial dilution solution;

[0053] (2) Add 2g of glucose and 0.3g of taurine to the initial dilution solution and mix. Make up to 100mL with ultrapure water to obtain the dilution solution. Store in a refrigerator at 4℃ away from light.

[0054] (3) Mix 15g of acetamide (antifreeze protectant) with diluent to obtain cryopreservation solution for sperm of wrasse.

[0055] Comparative Example 1

[0056] The cryopreservation solution for sperm of the silky-tailed wrasse was prepared using the method described in Example 1, except that glucose was removed and the amount of glucose was supplemented with astragalus polysaccharide.

[0057] Comparative Example 2

[0058] The cryopreservation solution for sperm of the silky-tailed mandarin fish was prepared using the method described in Example 1, except that the cryoprotectant acetamide was replaced with dimethyl sulfoxide.

[0059] Comparative Example 3

[0060] The cryopreservation solution for sperm of the silktailed mandarin fish was prepared using the method described in Example 1, except that glucose was replaced with trehalose.

[0061] Comparative Example 4

[0062] The cryopreservation solution for sperm of the silky-tailed mandarin fish was prepared using the method described in Example 1, except that taurine was not added, and the amount of taurine was supplemented with astragalus polysaccharide.

[0063] Comparative Example 5

[0064] The cryopreservation solution for sperm of the silky-tailed mandarin fish was prepared using the method described in Example 1, except that the concentrations of sodium chloride, calcium chloride, magnesium sulfate, potassium chloride, glucose, astragalus polysaccharide, taurine, and acetamide in the cryopreservation solution were 3 g / L, 0.03 g / L, 0.05 g / L, 1.5 g / L, 15 g / L, 0.3 g / L, 0.5 g / L, and 50 g / L, respectively.

[0065] Comparative Example 6

[0066] The cryopreservation solution for sperm of the silky-tailed mandarin fish was prepared using the method described in Example 1, except that the concentrations of sodium chloride, calcium chloride, magnesium sulfate, potassium chloride, glucose, astragalus polysaccharide, taurine, and acetamide in the cryopreservation solution were 7 g / L, 0.3 g / L, 0.3 g / L, 3.5 g / L, 35 g / L, 2 g / L, 5 g / L, and 200 g / L, respectively.

[0067] Experimental Example 1

[0068] Sperm from *Meretrix meretrix* were cryopreserved using cryopreservation solutions obtained in Examples 1-3 and Comparative Examples 1-6, respectively. After one month of preservation, the cryopreservation tubes were removed from liquid nitrogen and the mixture containing semen was completely thawed in a 37°C water bath. The sperm viability and motility in the thawed semen were then assessed. Freshly activated sperm motility was used as a control. The results are shown in Table 1.

[0069] Cryopreservation method: Select 5 male threadfin bream with mature testes, clean the area around their vents, and gently and continuously squeeze the abdomen from behind the pectoral fins toward the genital opening to collect semen. Mix the semen from the 5 fish, and then mix the threadfin bream semen with ultra-low temperature preservation solution at a volume ratio of 1:2. Add 1 mL of the mixture to each cryopreservation tube, equilibrate on ice for 25 min, and then place them in a programmed cooling device to cool to -80°C at a rate of 15°C / min. Immediately afterward, immerse them in liquid nitrogen for preservation.

[0070] Sperm motility test: 1 μL of thawed semen was pipetted and an equal volume of sperm activation solution (using ultrapure water as solvent, sodium chloride 2.8 g / L, potassium chloride 0.4 g / L) was added. Sperm motility was then tested using CASA.

[0071] Sperm survival rate = (number of surviving sperm / total number of sperm) * 100%;

[0072] Sperm motility = (number of motile sperm / number of surviving sperm) * 100%.

[0073] Table 1. Effects of different cryopreservation solutions on sperm survival rate and motility.

[0074] sample Survival rate (%) Sperm motility (%) sample Survival rate (%) Sperm motility (%) Comparison 91.1 91.4 Comparative Example 2 41.1 56.1 Example 1 76.2 84.7 Comparative Example 3 37.3 45.9 Example 2 72.8 82.2 Comparative Example 4 46.7 63.8 Example 3 73.4 83.6 Comparative Example 5 22.9 39.1 Comparative Example 1 32.8 42.4 Comparative Example 6 28.3 46.4

[0075] Table 1 shows that the cryopreservation solution for *Meretrix meretrix* sperm obtained in this application can effectively protect the sperm. Changing the ratio of the cryopreservation solution significantly affects sperm survival rate and motility. Compared with Comparative Examples 1 and 3, glucose can be used as a non-permeable cryoprotectant in the cryopreservation solution, effectively protecting *Meretrix meretrix* sperm, and its effect is superior to using trehalose. Compared with Comparative Example 2, using acetamide as a cryoprotectant results in a significantly higher sperm survival rate after thawing than dimethyl sulfoxide. A comparison of Example 1 and Comparative Example 4 shows that using astragalus polysaccharide and taurine together as sperm nutrients is significantly more effective than using astragalus polysaccharide alone.

[0076] Example 4

[0077] Methods for cryopreserving semen from threadfin bream:

[0078] Five male threadfin bream with mature testes were selected. After cleaning the area around their vents, the abdomen was gently and continuously squeezed from behind the pectoral fins toward the genital opening to collect semen. The semen from the five bream was mixed and then mixed with the cryopreservation solution obtained in Example 1 at a volume ratio of 1:2. The mixture was then transferred to cryopreservation tubes, with 1 mL of the mixture added to each tube. After equilibration on ice for 25 minutes, the tubes were placed in a programmed cooling apparatus and cooled to -80°C at a rate of 15°C / min. The tubes were then immediately placed in liquid nitrogen for preservation.

[0079] Example 5

[0080] Methods for cryopreserving semen from threadfin bream:

[0081] Five male threadfin bream with mature testes were selected. After cleaning the area around their vents, the abdomen was gently and continuously squeezed from behind the pectoral fins toward the genital opening to collect semen. The semen from the five fish was mixed and then mixed with the cryopreservation solution obtained in Example 1 at a volume ratio of 1:1. The mixture was then transferred to cryovials, with 1 mL of the mixture added to each cryovial. After equilibration on ice for 20 minutes, the cryovials were placed in a programmed cooling apparatus and cooled to -80°C at a rate of 18°C / min. The cryovials were then immediately placed in liquid nitrogen for preservation.

[0082] Example 6

[0083] Methods for cryopreserving semen from threadfin bream:

[0084] Five male threadfin bream with mature testes were selected. After cleaning the area around their vents, the abdomen was gently and continuously squeezed from behind the pectoral fins toward the genital opening to collect semen. The semen from the five bream was mixed and then mixed with the cryopreservation solution obtained in Example 1 at a volume ratio of 1:3. The mixture was then transferred to cryovials, with 1 mL of the mixture added to each cryovial. After equilibration on ice for 30 minutes, the cryovials were placed in a programmed cooling apparatus and cooled to -80°C at a rate of 12°C / min. The cryovials were then immediately placed in liquid nitrogen for preservation.

[0085] Comparative Example 7

[0086] The semen of the silky-tailed fish was cryopreserved using the method described in Example 4. The difference was that the semen of the silky-tailed fish was mixed with an ultra-low temperature preservation solution and then immediately placed in liquid nitrogen for preservation.

[0087] Comparative Example 8

[0088] The semen of the silktailed mullet was cryopreserved using the method described in Example 4, except that the cooling rate of the programmed cooling device was adjusted from 15°C / min to 5°C / min.

[0089] Experimental Example 2

[0090] Semen preserved using the cryopreservation methods described in Examples 4-6 and Comparative Examples 7 and 8 was used as samples. After one month of preservation, the cryovials were removed from liquid nitrogen and the mixture containing semen was completely thawed in a 37°C water bath. The sperm viability and sperm motility in the thawed semen were then tested (the testing method is the same as in Example 1). Freshly activated sperm motility was used as a control. The results are shown in Table 2.

[0091] Table 2. Effects of different cryopreservation methods on sperm survival rate and motility.

[0092] sample Survival rate (%) Sperm motility (%) sample Survival rate (%) Sperm motility (%) Example 4 76.2 84.7 Comparative Example 7 35.8 44.4 Example 5 74.3 81.1 Comparative Example 8 30.7 38.7 Example 6 71.9 80.6 Comparison 91.1 91.4

[0093] As shown in Table 2, both excessively fast and slow cooling rates reduce sperm survival rate and motility. The reason for this may be that excessively fast or slow freezing rates can lead to an increase in the formation of ice crystals in sperm cells, damaging the cell membrane and acrosome structure, resulting in a significant decrease in sperm survival rate and motility after thawing.

[0094] Experimental Example 3

[0095] The fertilization rate and hatching rate of the cryopreserved silktail semen obtained in Example 4 were tested, and the fresh activated sperm was used as the control group. The results are shown in Table 3.

[0096] Artificial insemination: Cryopreservation tubes frozen for 1 month were taken out of liquid nitrogen, thawed in the manner described in Example 1, and then the sperm and eggs were mixed at a volume ratio of sperm:egg = 1:1000. The sperm were activated by adding room temperature activation solution, and the cells were incubated at room temperature. The fertilization rate and hatching rate were then recorded.

[0097] Fertilization rate = (Number of embryos developed to the 4-16 cell stage / Total number of eggs) * 100%;

[0098] Hatching rate = (Number of newly hatched fry / Number of fertilized embryos) * 100%.

[0099] Table 3 Sperm fertilization rate and hatching rate

[0100] sample Fertilization rate (%) Hatching rate (%) sample Fertilization rate (%) Hatching rate (%) Example 4 66.5 72.4 control group 93.1 79.8

[0101] As shown in Table 3, the hatching rate of the cryopreservation solution for sperm of the silky-tailed mullet in this application is close to that of fresh sperm, indicating that the cryopreservation solution and preservation method of this application can effectively protect the sperm of the silky-tailed mullet and can meet the requirements for artificial breeding.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cryopreservation solution for sperm of the silky-tailed wrasse, characterized in that, The cryopreservation solution for sperm from the silky-tailed mullet includes a diluent and acetamide, wherein the final concentration of acetamide is 80–150 g / L. The diluent comprises the following components at the following concentrations: sodium chloride 4–5.5 g / L, calcium chloride 0.05–0.15 g / L, magnesium sulfate 0.07–0.15 g / L, potassium chloride 1.8–2.8 g / L, glucose 20–30 g / L, astragalus polysaccharide 0.5–1.3 g / L, and taurine 1–3 g / L.

2. The cryopreservation solution for sperm of the silky-tailed wrasse according to claim 1, characterized in that, The diluent comprises the following components at the following concentrations: sodium chloride 4.5–5 g / L, calcium chloride 0.08–0.13 g / L, magnesium sulfate 0.09–0.13 g / L, potassium chloride 2–2.5 g / L, glucose 22–28 g / L, astragalus polysaccharide 0.7–1.2 g / L, and taurine 1.5–2.8 g / L.

3. The cryopreservation solution for sperm of the silky-tailed wrasse according to claim 2, characterized in that, The diluent comprises the following components at the following concentrations: sodium chloride 4.8 g / L, calcium chloride 0.11 g / L, magnesium sulfate 0.12 g / L, potassium chloride 2.2 g / L, glucose 25 g / L, astragalus polysaccharide 1.1 g / L, and taurine 2.6 g / L.

4. The method for preparing the cryopreservation solution for *Gnaphalium affine* sperm according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Dissolve sodium chloride, calcium chloride, magnesium sulfate, potassium chloride, and astragalus polysaccharide in water to obtain an initial diluted solution; (2) Add glucose and taurine to the initial diluent and mix to obtain the diluent; (3) Mix acetamide with diluent to obtain cryopreservation solution for sperm of wrasse.

5. The preparation method according to claim 4, characterized in that, The initial diluent was stored at 2–6°C.

6. The preparation method according to claim 4, characterized in that, The diluted solution should be stored in a dark environment at 2–6°C.

7. A method for cryopreservation of sperm from the silky-tailed wrasse, characterized in that, The cryopreservation solution for sperm of the silky-tailed fish as described in claim 1 is mixed with the sperm of the silky-tailed fish to obtain a mixture. The mixture is placed on ice for equilibration for 20 to 30 minutes, and then cooled to -80°C at a cooling rate of 12 to 18°C / min. After that, it is directly placed in liquid nitrogen for preservation.

8. The method according to claim 7, characterized in that, The volume ratio of the cryopreservation solution for silkworm sperm to the semen of silkworms is 1 to 3:

1.

9. The cryopreservation solution for *Gnaphalium affine* sperm according to any one of claims 1 to 3, or the cryopreservation solution for *Gnaphalium affine* sperm obtained by the preparation method according to any one of claims 4 to 6, for the preservation of *Gnaphalium affine* semen or for in vitro artificial insemination of *Gnaphalium affine*.