Facultad de Ciencias Exactas y Naturales

ALKALINE PHOSPHATASE IN SHRIMP Artemesia longinaris: RESPONSE TO VITAMIN D IN DIET. Fenucci, J.L.*; Pereira,N.A.; FernndezGimenez, A.V. and Harn, N.S.

The techniques utilized in the production of peneidos shrimps have evolved due to the economic pressure and the environmental protection The development of methods for the formulation and elaboration of comercial food has reduced 30% to 40% the operating costs. Nevertheless, nutrition is still a limited economic factor. It is escential to know the nutritional needs of the cultived species in order to select the diet and the ingredients for its preparation.

 Pleoticus muelleri and Artemesia longinaris are the shrimp species exploited within the Argentinian sea. Both species are part of the benthic fauna and play an important role in the trophic chain as primary and secondary consummers.

PLEOTICUS MUELLERI
Distribution: from 20 S to 50S Temperature: between 6 and 24 C Salinity: between 31.5 and 35.5 ups. Feeding: Benthonic invertebrates, detritus, and organism particles. P.muelleri is omnivore-carnivore and can be cannibal under captivity conditions.

ARTEMESIA LONGINARIS
Distribution: from 23 S to 43S Temperature: between 6 and 24 C Salinity: between 31.5 and 35.5 ups. Feeding: Benthonic invertebrates, detritus, crustaceans remains, algae, and organism particles. A.longinaris is omnivore-carnivore and can be cannibal under captivity conditions.

 Between 1992 and 2007, the anual catches presented a varied range, from 8,000 to 80,000 tons of P. muelleri and 50 to 400 tons of A. longinaris. The exportation of these species produce an income of 60 to 100 million dollars per year! In Argentina are more than 90,000 acres to culture shrimps; Baha Blanca and San Blas (Provincia de Buenos Aires) and the coastal

The researching team called Acuicultura (Facultad de Ciencias Exactas y Naturales de la Universidad Nacional de Mar del Plata), has developed: Maduration and reproduction Massive culture of larvae Nutrition Pond growing

 The vitamins are one of the most important ingredients of the diet, and, in some way, they are essential.

 15 vitamins liposolubles: A, D, E y K; hydrosolubles, B1, B2, B6, B12, Pantothenic acid, nicotinic acid, biotine, folic acid, C, coline and inositol Within nutritional studies of shrimps, the requirement of vitamins is considered a less-studied area.

 Vertebrates: Vitamin D takes part in the calcium and phosphorus metabolism by promoting its intestinal absortion and stimulating bones mineralization. It also controls calcium levels in blood. Crustaceans: Vitamin D can also be involved in mineralization processes of exoskeleton. The formation of 1,25-OH-D3, catalized by specific enzymes present in vertebrates, has not been observed in crustaceans yet.

 Vitamin D is important for the survival of Marsupenaeus japonicus larvaes. Kanazawa (1985) The lack of vitamin D in the diet of Litopenaeus vannamei produced a loss in weight and appetite. He et al. (1992) Levels that are higher and lower than 0.1 mg/kg diet of vitamin D3 for Peneaus monodon, decreases the alkaline phosphatase activity. This enzyme would be involved in the calcium and phosphorus absorption and carrying. Shiau & Hwang, 1994

The development of biochemical and histological tools allows to evaluate in the midgut gland the nutritional state and the health of the organisms under cultivity conditions.

MATERIALS & MTHODS

EXPERIMENTAL CONDITIONS 6 weeks 108 prawns(1.5g intitialmeanweight); 108 shrimps(4.20ginitialmean weight) 150 l glass aquaria Density: 8 animals/m Environmentalconditions: 33salinity, 18C, pH7, 11:13hs photoperiod. Feedingregime:5%biomass. Eachdiet was tested in three replicate groups

COLLECTION OF SPECIMENS
Shrimps were obtained from a commercial fisherman and placed in 150 l glass aquaria (33 salinity, 20C, pH 7, 13:11 h photoperiod).

DIET ELABORATION
Five semi purified diets (vitamin free casein, manioc starch, squid protein concentrate and vitamin free fish oil) were designed containing 0 (D0); 8,000 (D8), 15,000 (D15); 30,000 (D30) and 40,000 (D40) UI vitamin D3/kg diet.

Diet PL4 was applied as a control treatment since it is considered nutritrional appropriated for the studied species.(Daz & Fenucci, 2002)

COMPOSITION OF SEMI PURIFIED DIETS

COMPOSITION OF DIET PL4

Ingredient
Harina de pescado Harina de soja Trigo Fcula de mandioca Protena de calamara Aceite de pescado Soluble de pescado Lecithin CHolesterol Vitamin

Porcentual %
48 17 8,5 20 1 2 2 0,5 0,5 0,5

HISTOLOGICAL ANALYSIS
At the end of trials,animals were anesthetized on ice and the midgut gland and haemolymph of all individuals, in intermolt, were aseptically removed. Tissues were fixed in Davidson fluid for 24h ,dehydrated in a progressive series of ethanol and embebbed in paraffin. Sections of 5 m were stained with hematoxylin-eosin.

ALKALINE PHOSPATASE
Alkaline phosphatase activity was determined in the protein extract of midgut gland and haemocytes (HLS) using p-nitrophenylphosphate (p-NPP) (Sigma N4645) as substrate.

RESULTS AND DISCUSSION

Treatmen t Mean weight Initial final (gS) (gS) Weight gain (gr.) Weight gain (%) Survival (%)

D0 D8000 D15000 D30000 D40000 PL4

Crecimiento y supervivencia en 2,88 Artemesia longinaris 21,5 3,50 0,62a 52a

0,564 1,50 0,238 2,85 0,570 1,49 0,266 2,92 0,647 2,52 0,325 0,688 1,60 0,206 3,70 0,745 1,71 0,353 3,96 0,829 2,90 0,431 0,10
b

6,7 29,8 14,4 35,6 15,1

71a 67a 75a 70a 60a

0,85 0,21
c

1,04 0,38
d

Histology of midgut gland of Artemesia longinaris

ARTEMESIA LONGINARIS15000 UI of vitamin D3/ kg diet

ARTEMESIA LONGINARIS 40000 UI of vitamin D3/ kg diet

Foamy cells!!

THE BEST.

Wild Prawn A longinaris 15000 UI of vitamin D3/ kg diet

THE WORST

Foamy cells!!

Artemesia longinaris D40

ALKALINE PHOSPHATASE ACTIVITY IN Artemesia longinaris.

Determinacin ptimo del pH

SUSTRATUM CONCENTRATION EFFECT ON ENZYMATIC ACTIVITY.

EFFECTS OF DIFFERENT LEVELS OF VITAMIN D3 IN THE DIET OF Artemesia longinaris ON ALKALINE PHOSPHATASE ACTIVITY.

A. longinaris The lower activity levels were obtained in wild animals, meanwhile the highest activity levels were registered in organisms feed on D40000 UI/kg diet

SOLUBLE PROTEINS AND ALKALINE PHOSPHATASE ACTIVITY IN MIDGUT GLAND OF Artemesia longinaris FEED DIFFERENT DIETS. Artemesia longinaris Soluble protein (mg/ml) 1,62+0,171a 9,99+1,697c 2,77+1,345a 8,17+0,318b 7,34+0,974b 8,62+0,415b 8,58 +0,184c 6,27+0,015e Alkaline phosphatase (Abs/min/mg prot.) 0,05+ 0,003e 0,250,009 0,140,007b 0,240,002 0,140,004b 0,180,009c 0,140,005b 0,300,016d

Wild PL4 Initial D0 D80000 D15000 D30000 D40000

ALKALINE PHOSPHATASE ACTIVITY IN MIDGUT GLAND OF AND ARTEMESIA LONGINARIS FEED ON DIFFERENT DIETS.

Alkaline phosphatase activity in the haemocytes of Artemesia longinaris

The present study shows the occurrence of different alkaline phosphatase activities in the midgut gland and haemocytes of Artemesia longinaris in response to varying levels of Vitamin D in diet.

THE DIETARY REQUIREMENT OF JUVENILE GRASS SHRIMP (PENAEUS MONODON) FOR VITAMIN D. SHIAU & HWANG

THE WORST!!!!

D 40000 UI de vitamina D3/ kg dieta TREATMENT IN Artemesia longinaris

Artemesia longinaris Soluble Alkaline protein phosphatase (mg/ml) (Abs/min/mg prot.) Wild PL4 1,62+0,171a 9,99+1,697c 0,250,009 0,05+ 0,003e

A longinaris 40000 UI of vitamin D3/ kg diet

Initial

2,77+1,345a 0,140,007b

Foamy cells!!

D0 8,17+0,318b 0,240,002 In human beings : cellular irreversible damages alkaline phosphatase activity. b D80000 7,34+0,974 0,140,004b

CONCLUSIONS
Vitamin D3 is not a assential nutrient for the survival of Artemesia longinaris in culture. The inclusion of 8000 and 30000 UI of vitamin D3/ kg diet in Artemesia longinaris acts in a negative way over the growing process.

CONCLUSIONS
In A. longinaris exists a relationship between vitamin D3 and the alkaline phosphatase activity, however, it is neccesary to do some extra researches in order to stablish its role in the calcium metabolism. The supply of 40000 UI of vitamin D3 /kg diet may be considered as hypervitaminosis in Artemesia longinaris.

CONCLUSIONS
15000 UI of vitamin D3 /kg diet, improve the sanitary conditions of the cultured A. longinaris. The occurrence of different alkaline phosphatase activities in the midgut gland and haemocytes of Artemesia longinaris in response to varying levels of Vitamin D in diet.

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