Dietary Klebsormidium sp. Supplementation Improves Growth Performance, Antioxidant and Anti-Inflammatory Status, Metabolism, and Mid-Intestine Morphology of Litopenaeus Vannamei

Filamentous microalga Klebsormidium sp. has huge potential to become a natural and healthy additive in aquatic feed since it contains various bioactive nutrients, such as linoleic acid (LA), carotenoids, and chlorophylls. Therefore, an eight-week feeding experiment was performed to evaluate the effects of dietary Klebsormidium sp. on the growth performance, antioxidant and anti-inflammatory status, metabolism, and mid-intestine morphology of Litopenaeus vannamei. Two isonitrogenous and isolipid diets supplemented with and without 5% Klebsormidium sp. were prepared. Results showed that L. vannamei fed with Klebsormidium sp. had better growth performance and feed utilization by optimizing mid-intestine morphology and improving the carbohydrate metabolism. In addition, Klebsormidium sp. also enhanced the antioxidant capacity of L. vannamei by downregulating antioxidant parameters (hepatopancreas T-SOD, hepatopancreas GSH-PX, hemolymph T-SOD, hemolymph MDA) and RNA expression levels of antioxidant genes (gsh-px and cat). Furthermore, the supplementations of dietary Klebsormidium sp. significantly improved hepatopancreas health by downregulating RNA expression levels of pro-inflammatory related genes (relish and rho). Therefore, a dose of 5% Klebsormidium sp. is recommended for the daily diet of L. vannamei to improve the growth performance, antioxidant and anti-inflammatory status, metabolism, and mid-intestine morphology of shrimp.


Microscale Reagent 3 preparation in microscale operation procedure:
(a) Prepare MDA Reagent 3 according to normal operation procedure: Pour 1 vial of MDA Reagent 3 in flask, add 64ml* 90℃～100℃ hot double distilled water, dissolve completely (you can heat solution properly during dissolving). Add 60ml glacial acetic acid after cooling, mix sufficiently.
(b) Dilute prepared MDA Reagent 3 with 50%** glacial acetic acid at ratio of 2:1, consider solution volume according to you need.
For example, if you need 15ml Reagent 3, then you can prepare 10ml Reagent 3 according to procedure above, then add 5ml 50% glacial acetic acid, mix sufficiently. Prepared can be stored at 4℃ in fridge away from light (you should get glacial acetic acid***).
Note: * Double distilled water will bulge when it becomes hot, and evaporation can not be ignored during heating process. So you need to add 64ml hot double distilled water to make sure 60ml double distilled water (after cooling) is add in reaction system . ** Mix 50ml double distilled water & 50ml glacial acetic acid together in order to prepare 50% glacial acetic acid.
*** Glacial acetic acid is also named as ethanoic acid, you can buy it from pharmaceuticals company or medicament company generally. It is better to buy analytical pure (AR) ethanoic acid,

CH3COOH>99%.
This kit can be stored by cold preservation for at least 1 year.

Hyperlipidemia sample MDA assay procedure:
This method can be used to measure MDA content in hyperlipidemia blood or lipids. For example, you can use this method to measure MDA content in soybean oil, salad oil or rubsen seed oil, etc.
(1) Slight hyperlipidemia blood MDA assay: Slight hyperlipidmia blood serum (or plasma) appears less limpid, you can also measure MDA according to common operation method or midrange hyperlipidemia operation method. Calculate as follows: (2) Midrange hyperlipidemia blood MDA assay: Midrange hyperlipidmia blood serum (or plasma) appears quite turbid.
② Operation 9. Erythrocyte MDA assay procedure: (1) Microscale erythrocyte MDA assay procedure: Please use the method when sample volume is quite small.

① Sample pretreatment:
You can use one of these two methods below: a. Take whole blood by slide to measure MDA content: If you blood sample is very small & very precious, such as neonate finger blood or mouse tail blood, then you can add 1~2 droplets heparin on slide, mix sufficiently, dry it by stoving (<60℃) or blowing naturally. Add smallscaled blood sample on "heparin slide", use micropipet to take heparin anticoagulated blood of volume you need, prepare 1:99 hemolysate.

b. Take erythrocytes by glass microtainer and hemolysate preparation:
(a) Take 20l heparin anticoagulated whole blood by 20l glass microtainer. Put glass microtainer at horizontal position, remove suction bulb quickly, put the terminal with longer vacant space in alcohol burner flame (at 1/3 site of flame, increase temperature until glass becomes red glow) until the terminal becomes globular clogging. Measure and record length of blood column (a cm). Pack microtainer by paper, insert microtainer in centrifuge tube, centrifugate at 1500rpm for 5~10 minutes. Take microtainer out of centrifuge tube, use small ;grinding wheel to carve at the dividing line of blood plasma and erythrocytes (you can also shear microtainer by scissors directly

Hb calculation:
Hb content (gHb/L) = OD × 367.7 If your sample volume is very small, then you can halve sample and reagent (in fact, you can reduce volumes of sample and reagent as you want, but please keep the right ratio. (2) Erythrocyte MDA assay procedure in whole blood: Please use the method when sample volume is large enough.
① Sample pretreatment: a. Blood cell preparation: Take 0.15ml heparin anticoagulated whole blood, add 2~3ml physiological saline, centrifugate at 2500~3000rpm for 5~10 minutes (mouse erythrocytes are fragile, so it is better to centrifugate at 1000~1500rpm for 5~10 minutes), remove supernatant and keep sediment of erythrocytes. Please remove supernatant completely or it will disturb results.
b. Hemolysate preparation: Take sediment after centrifugation, add double distilled water of 4 times whole blood volume (0.6ml), mix by vortex for 1 minute in order to lyze cells completely.
c. Extraction: Add dehydrated alcohol (or 95% alcohol) of 2 times whole blood, mix sufficiently