Effects Of Environmental Factors On The Embryonic Development Of The Fertilized Eggs Of Pharaoh Cuttlefish (Sepia Pharaonis) And The Hemostasis Effect Of The Cuttlebone

The species of Cephalopods have always been important economic marine animals in China.Due to its fast growth,high value of nutrition and high market price,pharaoh cuttlefish(Sepia pharaonis)has become a promising aquaculture species.However,the lower the embryo hatching rate and the lower survival rate of its breeding have become the major factors which hinder the cultured scale of pharaoh cuttlefish.The knowledge about the propagation and physiological features of pharaoh cuttlefish will pave a new way for its artificial breeding.In this study,we observed the embryonic development of the fertilized egg of pharaoh cuttlefish.In addition,we explored the effect of temperature,salinity and p H value on the hatching rate of the embryo of pharaoh cuttlefish.Furthermore,the morphological features of the haemocytes of pharaoh cuttlefish were observed by microscopy.The major results are following:1.Observation of embryonic developmement.By microimaging system,the embryonic stages of pharaoh cuttlefish have been divided into eight different stages as follows: Stage I:Newborn fertilized eggs.The animal pole is located at the opposite of yolk.Stage II: Cleavage I t th.The fertilized egg begins to divide and enters the gastrula.Stage III: Protrusion stage.The embryo protrudes from the yolk.Stage IV:Appearance of eyes and organs start to differentiate.Stage V:The head and wrist begin to differentiate,the formation of torso.Stage VI:The organs differentiated obviously,the wrist appeared sucker and red colour appeared in the eyes.Stage VII: The structures of the embryo are basically the same with the adult,the ink sac appears in the back,black colour appeared in the eyes.Stage VIII:Hatching stage.The larvae has been hatched from the fertilized egg.2.The effects of temperature,salinity and p H value on the embryonic development.The ranges of temperature were set as 18 ?,21 ?,24 ?,27 ?,30 ? and 33 ?,respectively.While,the ranges of the salinity were set as 15‰,18‰,21‰,24‰,27‰,30‰,33‰ and 36‰,respectively.In addition,the ranges of the p H value were set as 7.0,7.5,8.0 and 8.5,respectively.The hatching rates of the fertilized eggs of pharaoh cuttlefish were observed under the above conditions.The results showed that different temperatures had significant effects on the hatching rate(P < 0.05).The fertilized eggs could be hatched at the temperature of 21-30 ?,and the optimal temperature was 27 ? with the hatching rate of 72.2%.The effect of salinity on the hatching rates was significant(P < 0.05).The fertilized eggs could be hatched at the salinity of 27~36 ‰,and the optimal salinity was 30-33 ‰ with the hatching rate of 70%~80%.By contrast,the effect of p H value on the hatching rate was not significant(P < 0.05),and the hatching rate was all greater than 70% under the p H values of 7-8.5.Taken together,the artificially hatching conditions for fertilized egg of pharaoh cuttlefish could be set as 24?-27?,30-33‰ and p H value of 7-8.5.3.Observation of haemocytes.The microscopic imaging system was used to observe and take pictures of haemocytes stained with Giemsa staining solution.According to the size,shape and granules present in the cytoplasm,the haemocytes could be divided into four types,named granular hemocyte,small granular hemocyte,U-shaped nuclear hemocyte,and hyaline hemocyte.Cytoplasm of granular hemocyte contains a large number of granules,accounting for about 30% of the total number of haemocytes.Hyaline hemocyte has no granules in the cytoplasm.It has the largest size and the smallest amount,account for about 15% of the total number of haemocytes.Small granular hemocyte has a small number of granules in the cytoplasm.It has the largest number of the total number of haemocytes,accounting for about 50% of the total number of haemocytes.The U-shaped nuclear hemocyte is irregular with horseshoe shape,account for about 5% of the total number of haemocytes.4.The main components and the hemostatic effect of the cuttlebone of pharaoh cuttlefish.The chemical elements and calcium carbonate content in cuttlebone were determined.The results showed that the main chemical composition in the cuttlebone was Ca CO3,which accounted for 81.4%.The chemical elements in cuttlebone mainly included Ca,Na,K,P,Mg,Zn,Fe,Gu and Mn.Among of them,Ca accounted about 34.99%,P accounted 0.029%.The concentrations of the other main chemical elements were as follows: Na: 1.04×104mg/kg,Mg: 1.91×103mg/kg,K: 334 mg/kg,Zn: 299 mg/kg,Fe: 171 mg/kg,Cu: 5.43 mg/kg and Mn: 10.2mg/kg,respectively.At the same time,there were 15 kinds of amino acids in the cuttlebone,It's sorted by content:Met>Asp>Glu=Tyr>Leu>Ala>Ser>Val>Gly>Lys>His>Ile>Thr>Phe>Arg.The hemostatic effect of the cuttlebone was compared with those of other common hemostatic materials using mouse as model.The effect of cuttlebone powder on hemostasis was not significant different from that of yunnan baiyao,but it was significantly different from those of the natural hemostasis group,the calcium carbonate group and the starch group,indicating that the cuttlebone had a good hemostasis effect on the bleeding of mice.