Juvenile crabs were bought from Baiyangdian Lake, Hebei Province, China.

Took two tubes, one was blank, and the other was test, then added 0.04% soluble starch buffer 1 mL, reacting 2 - 5 min at 37˚C, and then added 0.02 mL test fluid in to test tube but not blank tube, blending, and then kept them in a 37˚C water bath for 7.5 min, then, we added iodine diluent 1 mL separately, at last, we added deionized water 6 mL in to test tube, and 6.02 mL in to blank tube, reading absorbance at 660 nm wavelength with the deionized water absorbency of 0.

The formula of Amylase activity:

One unit of amylase activity was defined as 10 mg starch that was hydrolysed by the reaction of 30 min between 100 mL enzyme filtrate and starch at 37˚C.

For male crabs, no matter unilateral eyestalk ablation or bilateral eyestalk ablation, with the increase of days, amylase activity presented a trend with gradually rise, and the maximum was at 20th days and they have significant difference with the control group (P < 0.05) (Figure 1). As for female crabs (Figure 2), no matter unilateral eyestalk ablation or bilateral eyestalk ablation,

compared with control group, amylase activity did not have obvious change (P > 0.05). Through experimental results we found that, after eyestalks were cut down, both pepsin and trypsin activity did not change significantly (P > 0.05) (Figures 3-6).

From the experiment results (Figure 7), we could found, male crabs’ serum total lipids down significantly in the first 5 - 10 days after eyestalks were cut off (P < 0.05), then with the extension of the days, the serum total lipids restored to its original level gradually. As for female crabs, no matter unilateral or bilateral eyestalk ablation, the serum lipids was higher than the control group, maximum peak appeared in the middle of experiment (Figure 8).

Amylase was a kind of digestive enzyme that mainly digested starch and carbohydrate. After male crabs’ eyestalks were cut off, the activity of amylase kept increasing. The possible reason of it was that eyestalk ablation speeded up the development of testis, and spermatic development needed to consume large amounts of energy. And monosaccharide was the most directly using form of animals, with the extension of days, spermatic development continued to mature, and the requirements to glucose also constantly increased, while amylase was the main digestive enzyme that hydrolyzed carbohydrate, so the amylase activity also increased accordingly. And the amylase activity was higher in bilaterally ablated male crabs, the reason was that with bilateral eyestalk ablation, X-organ-sinus gland complex was totally broken, and this removed the inhibition of the spermatic development, and more energy was needed with the faster development. But when female crabs’ eyestalks were cut off, amylase activity declined at first, but with not significant change, then recovered to a normal level. The possible reason was that ovarian development needed more energy, but the energy stored in hepato-pancreas was not able to meet the needs, meanwhile, female crabs may had another regulating system, when the eyestalks were ablated, this regulating system played a role to make amylase activity recover to initial level. So if we cut off crabs’ eyestalk, we need to feed some food with higher starch to fulfill their growth and development especially to male crabs.

Crustaceans’ pepsin and trypsin possessed an important portion in vivio [14], but they did not have obvious content with each other [15]. With the ablation of eyestalk, gonadal development speeded up, and gonad needed to accumulate vast nutrition and energy before its development. The hepatopancreas was the main place for accumulating energy, and this accumulation was affected by environmental factors especially the forage. Because gonadal development required a lot of energy, the nutrition and energy which were stored in hepatopancreas were often consumed soon. 

No matter male crabs or female crabs, with the ablation of eyestalk, the effects on pepsin and trypsin did not have obvious change compared with the control, which meant eyestalk ablation did not have distinct effects on the two kinds of digestive enzymes. The possible reason was that enzyme activity was influenced by various elements, and just ablation of eyestalk could not make the enzyme activity produce very big change. Or we could say that animals themselves had a self control system, they could able to maintain their own metabolism in a relatively stable level. 

In this experiment, we could find that amylase activity was the highest in three digestive enzymes. We guessed that the demand of saccharides was higher than other material, so we should pay attention to the proportion of nutrients of forage to meet the growth of crabs. 

The existing researches showed that the lipids played a very important role in growth reproduction of many crustaceans. The main organ of crustacean absorption, machining and storage of lipids was hepatopancreas, and studies have found that the lipids in hepatopancreas were transferred to the ovary through the blood [16]. Crustaceans needed to consume larges of energy in the reproduction period, and the demand for lipids would also increase in gonadal development period. The lipids in hepatopancreas could not meet the needs of the gonadal development, and soon the lipids were processed by hepatopancreas from forage and were transported to the ovary. 

Serum total lipids were the general call of all sorts of lipids component in serum, including triglycerides (TG), phospholipids, cholesterol (TC) and a small amount of free fatty acid and so on, but the main was total cholesterol and triglyceride. Cholesterol as a precursor of hormone synthesis of steroids [17] was a kind of neutral fat. It played an important role in reproduction and secretion of crustaceans [18]. Because crustaceans couldn’t synthesize cholesterol in the body, they stored cholesterol in hepatopancreas and muscles before laying eggs, in the periods of gonad development, the cholesterol was assembled and transported to the ovary. The research has found that cholesterol in ovary of P. duorarum was gradually increasing in the periods of sexual maturity [19], the cholesterol in the blood of china shrimp kept increasing in the early periods of gonad maturation, and down with no obvious change in the later periods [20]. 

Another kind of neutral fat was triglycerides, which was the main energy of shrimp, eggs and larvae before feeding [21]. Lipids, especially triglycerides, were not only the main energy material in the process of crustaceans’ growth, and at the same time, it also had the function of energy store [22]. 

In this experiment, when female crabs’ eyestalks were cut off, the total lipids of the serum were higher than control group. It could be concluded that after the ablation of female crabs’ eyestalk, triglyceride and cholesterol were sent to ovary through blood continually, this could provide energy for ovarian growth; meanwhile, it also was a kind of way that parent crabs saved energy for their off-springs. And after the ablation of male crabs’ eyestalk, the contents of the lipids decrease significantly in the early stage, and gradually increase with the extension of time. We inferred that spermary needed more lipids during its development, the lipids stored in heaptopancreas was not able to meet the needs of gonadal development of male crabs, so the lipids of blood which as a transportation passageway were also reduced. So in the production of male crabs with eyestalk ablation, we should pay more attention to adding some food which contained abundant lipids to guarantee a smooth growth.