~ . This paper not to be cited without prior reference to the authors
International Council for the Mariculture Committee
Exploration of the Sea C.M. 1985/F:41/Ref. K.
Evaluation of Two Formulations for Use as a Standard Reference Diet for
Crustacean Nutrition Research
by
J.D. Casteil, J.C. Kean, L.R. D'Abramo l and D.E. Conklin2
Department of Fisheries and Oceans
Fisheries Research Branch , Scotia-Fundy Region
Fisheries and Environmental Sciences Division
Dlsease and Nutrition Section
Halifax Fisheries Research Laboratory
P.O. Box 550 • Rallfax, Nova Scotia Canada, B3J 2S7.
IMississippi State University, Dept. Wildlife and Fisheries, P.O. Drawer LW,
Mississippi State, Mississippi 39762 2University of California, Bodega Marine Laboratory, P.O. Box 247, Bodega
Bay, CA 94923 2
Abstract
An international feeding trial to evaluate two possible Standard
Reference Diets (SRD) for crustaceans was initiated in 1984. This report
at an informal crustacean nutrition session during the 1984 World Mariculture
Society (WMS) meeting in Vancouver, B.C. One diet was deve10ped at the
Bodega Marine Laboratory, University of California, Davis and is based upon
casein, egg-albumin and lecithin (BML 81 S). The other HFX CRD 84 was
developed at the Department of Fisheries and Oceans of Canada's Halifax
laboratory using purified rock crab, Cancer irroratus, protein. Both of
these diets had been used to culture lobster, Homarus sp. These two diets
are being compared with the usual control diet of 19 research teams from 10
different countries in feeding trials with 21 different species of
crustaceans including freshwater and marine shrimp, prawn, lobster, crayfish
and crab.
Details of the formulation, preparation and proximate composition of the
diets used in this study are;also included. The pre1iminary growth and
~ survival data for Penaeus monodon, Pandalus danae, Penaeus stylirostris,
~. vannamei, ~. brasiliensis, ~. ~etiferus and Homarus americanus all suggest
that either BML 81 S or HFX CRD 84 would be acceptable as a SRD for these
species. The on1y species that would not accept these diets, to date, was
Panu1irus argus.
Introduction
The field of crustacean nutrition research has had a re1atively short
history. The first published semipurifled dlet proposed for a crustacean was perhaps that of Kanazawa et al. (1970) for Penaeus japonicus. This diet was 3
based on soybean protein but was later modified by using the milk protein,
casein (Kanazawa et ale 1976). The rapid development of commercial shrimp
aquaculture has triggered a flood of shrimp feed and nutrition publications.
In his shrimp nutrition review paper, New (1976) criticized the lack of
standardization in experimental design, culture conditions, and analytical
techniques which limited the value of published information and made
comparison of research results from different laboratories difficult or • impossible. In response to New's criticisms, the World Mariculture Society (WMS) established a Nutrition Task Force in 1977 to propose guidelines for
standardizing aquaculture nutrition research methodology. The EIFAC, IUNS
and lCES Working Group on Standardization of Methodology in Fish Nutrition
Research whlch met in Hamburg, Federal Republic of Germany, 21 - 23 March
1979 supported and expanded this discussion of standardization (EIFAC Tech.
Rep. 36, 1980). These working groups advocated the establishment of a standard reference
diet (or diets) as one of the most important steps in facilitating direct
~ comparison of results among laboratories, experiments and species. They
recommended that the following fa~tors must be considered in the development
of a SRD:
nutrient balance
nutrient positive control reproducibility between lots and between laboratories
availability of components
standard processing
lot identification of diet and ingredients
form of nutrients availability and utilization of nutrients by the animals
stability or shelf life 4
The concept of standardization has been a priority at informal nutrition
discussions held each year in conjunction with the WMS annual meetings. In
1984 this discussion group decided to initiate an international feeding trial
to evaluate two possible SRDs for crustacean research (BML 81 Sand
HFX CRD 84). Both of these purified diet formulations were developed for use
with the lobster, Homarus sp. The following will be areport of events
leading to the development of the BML 81 Sand HFX CRD 84 formulations as
weIl as some preliminary results of the international study.
Development of Lobster Reference Diets
The first semipurified diets for lobster (Homarus sp.) research used
casein as the protein (Castell and Budson 1974, Schlesser and Gallager 1974)
and was similar in composition to that used by Kanazawa et al. (1970) for
prawn. The casein based diets resulted in poor growth and low survival of
juvenile lobsters associated with a "malt death" syndrom (Bowser and
Rosenmark 1981). Conklin et al. (1980) found that the molt death syndrom
could be prevented by addin~;6-8% soy lecithin to the casein or 4It casein/albumin based diets. Their discovery led to the deve10pment of the BML 81 S formulation (Conk1in et ~~. 1983).
A pure protein, other than casein, was sought at Halifax for 10bster
nutrition studies. Many commercial1y availab1e proteins end severa1 proteins
purified from 10ca1ly available marine organisms were tested. It was found
that protein from rock crab (Cancer irroratus) resulted in superior growth
and surviva1 (Boghen et a1., 1982).
When the crab protein replaced casein, the addition of soy lecithin was
unnecessary (Kean et al., 1985). Optimum growth was obtained with a crab protein level of 20-30% compared with 50% of the dry weight of diet if casein
was used (Castell et a1., 1985). The HFX CRD 84 contalns crab protein rather
than casein and is based upon the work at Halifax. ------
5
In the past we have used a Bodega Marine Laboratory Diet as a reference
diet for comparing results at Halifax. Moncton and Bodega Bay. As a result
of discussions at the informal crustacean nutrition discussion sessions at
the WMS meetings in Washington 1983. and Vancouver 1984. it was decided that
we would prepare about 30 kg each of the BML 81 Sand HFX CRD 84 at the
Halifax Laboratory and make them available for a collaborative international
crustacean nutrition study. These diets would be compared with the various
control diets at the participating laboratories. Both reference diets were
prepared as dry pellets in 1/16". 1/8" and 1/4" or 5/16" pellet sizes. were
nitrogen flushed. and vacuum sealed at 500 g/bag. The diets were stored at
-40°C until shipped. It was advised that upon receipt each group repackage
the feed in quantities equa1 to one weeks' feeding supp1y and store between
-20 and -40°C to reduce degradation.
This paper presents a summary report of the research resu1ts 3 availab1e
to date. In addition. the merits and applications of a standard reference
diet will be' discussed.
Participants In the Study • Co-operating scientists. nattonality and species studied are 1isted in Tab1e 1.
Diet Information
The formulations for BML 81 S. HFX CRD 84 and vitamin premixes are
listed in Tab1e 2. Some of the analytical data for the BML 81 Sand HFX CRD
84 as weIl as analysis of crab protein are provided in Tables 3-7. Since the Formula in Tab1e 2 indicated 16.5% lipid in BML 81 S. the 12.9% probably
reflects incomplete extraction and recovery of the 10% soy lecithin. The
3A complete presentation of results from all collaborators has been
tentatively scheduled as a "special session" for the 1986 WMS meeting to
held in Reno. Nevada. 6
10.5% lipid analysed in Table 3 for HFX CRD 84 corresponds we11 with the
10.2% indicated in Table 2. The Kjeldahl estimates for protein in the two
diets are almost equal. The nitrogen content of pure crab protein based on
amino acid analysis is on1y 14.3% compared to 16% in casein. Thus the
estimate would be 42.6% protein in the HFX CRD 84, when nitrogen is
multiplied by 6.99 rather than 6.25.
Some analyses of the 1984 lot of crab protein used for the diet in this • study are presented in Table 4. The ash content of 10.5% probably reflects the level of shell material that was included with the flesh in the Baader
deboning process. Depending on the tightness of the pressure belt in the
deboner, the ash content of crab protein concentrate has varied in past lots
from 1 to 18%. Finally, as the ash content is relatively high in the crab protein,
Dr. A. Boghen of the University of Moncton has provided the analysis of the
major elements (Table 5, Boghen and Yezina 1983) •
• , 7
Table 1
List of Researehers Who Have Been Sent BML 81 Sand HFX CRD 84.
Name Country Speeies
Wtlfredo Blaneo Cuba Panulirus argus John Castell Halifax, Canada Homarus amerICänus John Clark United Kingdom Homarus gammarus Douglas Conklin California, U.S.A. Homarus sp Gerard Cuzon Tahiti, Freneh Polynesia Penaeus monodon P. vannamei P. indieus • P. orientalis P. stylirostris Anthomy D'Agostino New York, U.S.A. Homarus amerieanus Louis D'Abramo Miss., U.S.A. Macrobraehium rosenbergii Jorge Fenueei Argentina Artemesia longinaris Pleoticus muelleri Kim Harrison Halifax, Canada Homarus americanus Akio Kanazawa Japan Penaeus japonieus Paul Margerelli Hawaii, U. S. A. P. japonicus P. stylirostris P. vannamei P. marginatus P. mono don Jeffrey Marliave Vaneouver B.C., Canada Pandalus danae Edgar Mason Victoria, P.E.I., Canada Homarus americanus Lori Barek Möore Hawai i; U. S. A. Macrobraehium rosenbergii Noel Morrissy Perth, Aus tralia Cherax tenuimanus (crayfis Yeh-Shen Peng Arizona, U.S.A. Penaeus stylirostris Gordon Sanford Antigua,'West Indies Penaeus stylirostris • " P. vannamei P. setiferous P. mono don Panulirus argus Mareelo Seelzo Venezuela Penaeus bräSIliensis P. notialis P. sehmitt A. Venkataramlah Miss., U.S.A. Penaeus aztecus or P. setiferus 8
Table 2 a
CRAB PROTEIN REFERENCE DIET (HFX CRD 84)
Ingredient Percent Suppl1er La
Crab Protein 40.0 Purified '84 Halifax Lab Wheat Gluten 5.0 Dover Mills t Halifax Corn Starch 15.0 ICN 1 Dextrin 5.0 U.S. Biochemieals 26 Alpha cellulose 17 .8 U. S. Biochemicals 23 Mineral Mix (Modified 4.0 U.S. Biochemicals CON.No. 30 Bernhart-Tomarelli) Vitamin Mix (See Table for 2.0 (HL '84) vitamin premixes) Vitamin E (DL alpha tocopherol) 0.2 U.S. Biochemieals 34 Cod Li ver Oil 6.0 Vitahealth 3H Corn Oil 3.0 Mazola Corn Oil 3 Choline Chloride 1.0 U.S. Biochemieals 37 Cholesterol (MP 144-146) 1.0 Anachemia 080 Total 100
• 9
BODEGA BAY DIET 81S
Ingredient Percent Suppl1er
Vitamin Free Casein 31.0 U.S. Biochemieals Corn Starch 24.0 ICN Wheat Gluten 5.0 Dover Mills, Halifax Refined Soy Lecithin 10.0 The Bean Sprout, Halifax Lipid Premix 6.0 Vitahealth* (2% Mazola Corn Oil and 4% Cod Liver Oil*) Spray Dried Egg White 4.0 Nutritional Biochemieals Vitamin Premix BML-2 (See Table 4.0 vitamin premixes) Mineral Premix (Modified 3.0 U.S. Bioehemieals Bernhart Tomarelli) • Cholesterol (MP 144-146) 0.5 Anaehemia Ltd. Vitamin E (DL alpha tocopherol) 0.2 U.S. Biochemieals Alpha cellulose 12.1 U.S. Biochemicals Vitamin A (50,000 IU/g) 0.1 ICN Vitamin 03 (400,000 IU/g) 0.1 Nutritional Biochemie
Total 100
• .. 10
Table 2 b
VITAMIN PREMIXES Percent f Ingredient Supplier Lot No. BML-2 HL '84
Thiamin (HCl) ICN 11980 0.5 0.32 Riboflavin ICN 19264 0.8 0.72 Niacinamide ICN 3448 2.6 2.60 D-Biotin U.S. Biochemieals 36648 0.1 0.008 Ca-Pantothenate U. S. Biochemieals 20704 1.5 1.43 Pyridoxine HCl ICN 13291 0.3 0.24 Folie Acid U.S. Biochemieals 22699 0.5 0.097 Menadione U.S. Biochemieals 5700 0.08 • ICN 8042 0.1 0.27 Cobolamine rnositol rCN 11972 18. 1 12.7 Cholecalciferol (850,000 rU/g) rCN 101196 0.002 Vit. A Acetate (500,000 rU/g) U.S. Biochemieals 3531 0.51 Ascorbic Acid rCN 0430 12.1 6.1 Nutr. Biochem. 5390 0.1 0.076 BHA 0.076 BHT rCN 1011 62 2.02 PABA Nutr. Biochem. 6340 3.0 Cellufil U.S. Biochemieals 23303 60.3 72.77
• ,,
Tab1e 3. Proximate composition of reference diets being used in 1984 study.
Ingredient BML 81 S HFX CRB 84
Moisture 5.3±0.1 1.4±0.3 Dry Weight Analysis 94.1±0.1 92.6±0.3 Crude Protein (Dry Wt)a 38.8±0.3 38.1±0.3 Lipid 12.9±0.3 10.5±0.6 a-Cellu1oseb 12.2 11.8 Carbohydrate (starch & dextrose)b 24.0 20.0 Ash 3.7±0.1 6.5±0.001 • Gross Energy Calories/g 5.2±0.2 5.0±0.6 a Crab protein amino acid analysis shows nitrogen content of 14.3 rather than 16.0. If Kje1dahl nitrogen is mu1tip1ied by 6.99 for HFX CRd 84 rather than 6.25, the protein content is estimated as 42.6%. b Values from formula (Table 2a) not analyzed •
• , 12
Table 4. Proximate analysis of crab protein used in HFX CRD 84.
Ingredient
Moisture 6. 8±1. 8 Dry Matter 93.2±1.8 Crude Protein 19.0 (88.4 based upon 14.3% N) Lipid 0.02±0.005 G1ycogen Not ana1yzed. Ash 10.5±O.6 • Crude Fiber Not ana1yzed •
• 13
Table 5. Analysis of Crab Protein Concentrate from A.D. Soghen and B.P. Vezina "A Series of Studies of the American Lobster (Homarus . americanus) and Rock Crab (Cancer irroratus) in the Maritime Provinces" Canadian Department of Supply and Services contract 08sc FP 101-1-0186.
Proximate Analysis
Protein 91.1 Glycogen 0.29 Lipid 0~47 Ash 9.92 • Total 101.78 Analysis of Ash
Element
P 28.2 Ca 25.3 Cl 21.3 Na 9.8 Mg 4.9 K 3.1 Si02 0.30 Fe 0.12 Zn 0.095 Cu q.035 . Al 0.027 Mn 0.023 0.010~(Uthe and Chou. pers. comm.) • Cd ... 14
Research Results to Date
Results of feeding trials have only been received from a few of the 19
research teams involved in this year's international study. It would not
have been unexpected if a reference diet developed for one speeies should
prove unsatisfaetory for a seeond species because of differenees in
nutritional requirements. For example. the Oregon Test Diet developed for
~ rainbow trout. (Salmo gairdneri). proved to be unsatisfaetory for juvenile
lobsters due to sterol defieieneies (Castell et alt 1975). With the possible
exeeption of Panulirus argus (Sanford. personal eommunication), both BML 81S
and HFX CRD 84 have been weIl aecepted by and given good growth and survival
in all speeies of crustaeeans reported to date.
The first non-lobster aquaculturist to participate in our referenee diet
study was Dr. Gerard Cuzon of Tahiti, Freneh Polynesia. In 1983 Dr. Cuzon
fed the HFX CRD 83 diet to 4 replicate groups of 15 shrimp.
2 (Penaeus monodon). at 27°C. density of 301m • feeding rate 10% body • weight/day for aperiod of 26 days (Table 6) • Table 6. Growth and survival of Penaeus monodon fed HFX CRD 83.
Group Initial Final %Weight % Weight Weight Gain Survival g g
1 1.89 4.25 125 100 2 1. 92 4.40 129 93.3 3 1. 93 4.30 123 100 4 1.87 4.60 146 100 15
Although his results for the 1984 comparison of HFX CRD 84 with BML 81 S
have not yet been reeeived, his eomments with regard to the response of P.
monodon to the 1983 lot of referenee diet were very encouraging.
Dr. Jeffery Marliave, of the Publie Aquarium in Vancouver, British
Columbia, Canada compared the two reference diets with frozen krill
(Euphausia paeifiea) in a 5 month feeding trial with cold-water marine
shrimp, Pandalus danae (Table 7).
Table 7. Growth and survival of Pandalu~ danae fed BML 81S, HFX CRD 84 or krill.
Instantaneous Diet Initial Final %Weight % N Growth Weight Weight Gain Survival K
BML 81 S 1.4±0.4g 2.81±0.61g 100.7 55.7 61 0.0046 g/day HFX CRD 84 1. 4±0. 4g 3.15±1.01g 125.0 60.7 61 Krill 1.4±0.4g 2.98±0.55g 112.9 57.4 61
The relatively high mortality was caused by cannibalism, as each test
group was held communally. Both reference diets appear to be at least equal
to froze~ krill as food for 'this cold-water shrimp• Dr. Gordon Sanford, of the. Antigua.. Shrimpery Ltd, has reported good • acceptance and growth of Penaeus "monodon, ~ stylirostris and ~ vannamei with both of the reference diets. He noted, however, that Panulirus argus
would not accept either diet. None of these data has yet been received from
Dr. Sanford.
Dr. Mareilo Scelzo of the University de Orient in Venezuela has run two
feeding trials with Penaeus brasiliensis eomparing our two reference
diets with their control GSCA diet (Table 8). 16
Table 8. Growth and survival of Penaeus brasiliensis fed BML 81 S, HFX CRD 84 or GSCA control.
Diet Initial Final Days %Weight % N Weight Weight Gain/day Survival g g
BML 81 S 1. 81 2.56 21 1.97 40 15
BML 81 S 1.65 3.16 33 2.77 73.4 15 (duplicate)
HFX CRD 84 1.80 6.21 76 3.22 63.4 15 • HFX CRD 84 1.27 1.88 32 1.50 100 15 (duplicate)
Control GSCA 1.76 5.64 76 2.90 33.3 15
The standard deviations on weight data were gi yen to us by Dr Scelzo but are
omitted from this page to save space. In this study the HFX CRD 84 seems to
be giving the best growth and survival of P. brasiliensis wi th the control GSCA being intermediate and BML 81 S the poorest. Dr. A. Venkataramiah of the Gulf Coast Research Laboratory in Ocean
Springs, Mississippi, compared our two reference diets wlth his patented GCRL • 75 shrimp dlet in feeding triais ~ith the white shrimp, Penaeus setiferus (Table 9). He fed each diet to dupllcate groups of 35 shrimp (initial weight
19) for six weeks at 25.5 C, 15 C salinity in recirculated fiberglass aquaria
with 150 gal artificial sea water (Instant Ocean). Each tank was equlpped
wlth a biological filter, and ammonla, nitrite and nitrate concentrations in
the water were recorded over the course of the experiment. 17
Table 9. Growth, survival, feed conversion and water ammonia levels of Penaeus setiferus fed BML 81 S, HFX CRD 84 or GCRL 75.
Diet Growth Rate Survival Feed Ammonia mg/day/shrimp Conversion mg/day
BML 81 S 10 68 4.6 0.10 HFX CRD 84 20 61 5.3 0.06 GCRL 15 23 82 3.9 0.09
Although the GCRL 15 diet based on natural raw feed stuffs gave better growth
• and survival than either of our reference diets, Dr. Venkataramiah feIt that both of these diets performed suffieiently weIl with P. setiferus to be
considered as a Standard Referenee Diet for this speeies.
Douglas E. Conklin and Nancy Baum, partieipants in the initiation of
this project and developers of the BML 815 Diet eondueted in 153 d feeding
trial. Thirty-six 4th stage animals were started on eaeh diet, fed once
every day after removal of any uneaten food from the day before • . "0,' eJ- Initial Final % Inital Final Diet ..Me- tt> Sl:1r .'i'(8:},., ....S~". wt I'\a -, I ot-<. • + BML 815 36 21 58 -40 mg 1580 630 HFX CRD84 36 21 58 -4Cmg 1300 ~ 520
The final weights ranged from 590-2307 ng on the HFX CRD 84 and 673-2720
ng one BML 815. The survival of lobster on HFX eRD 84 had been eonsiderably
higher than BML 815 until the 4th month of the experiment.
Discussion and Conelusions The purpose of a standard referenee diet 1s to provide neither a feed
for eommereial eulture of various speeies nor the optimal nutritional
------.. _------l1lI 18
formulation for all speeies eoneerned. Rather, the objeetive is to provide a
reprodueible, readily available, nutritionally balaneed, defined diet of
known nutrient eomposition, that ean be used as a referenee for eomparing
results among laboratories, speeies and experiments. Although both BML 81 S and HFX CRD 84 are promising for (suggested) use as a Standard Referenee Diet
for erustaeean nutrition studies, we have not yet eolleeted and analyzed
suffieient data to make meaningful eomparisons among speeies and
laboratories. It is being proposed that final reports and interpretations of
all the results of these studies"be presented as a special session at the
World Marieulture Soeiety Meetings in Reno, Nevada in January, 1986.
Applieation example
An example of the value of a Standard Referenee Diet is provided by the
results of two separate feeding trials with juvenile lobsters, Homarus
amerieanus, done by Edgar Mason of Marine Lobster Farms 1ne., Vietoria,
Prinee Edward Island, and twd other feeding trials eondueted at different ~ times at the Halifax Fisheries .Research..... Laboratory (Table 10). In eaeh ease HFX CRD 84 was fed as a referenee diet. At both loeations the praetice is to
obtain gravid female lobsters in the fall and control time to hatching of
eggs by manipulating temperature of the seawater. Thus it is possible to
obtain larval lobsters at almost any time of the year. Beeause the referenee diet was used for all treatments, any differenees in growth must be
attributed to non-dietary effects. One possible explanation for differences
observed at eaeh site involves the lipid levels of the larvae at the time of
hatching. GIen Sasaki, (PhD thesis Woods Hole Oceanography Institute)
demonstrated that the reserves of lipid at the time of hatchlng were
inversely related to the length of time before hatehing. In both locations, 19
The juveniles produeed from Fall hatching (and consequently longer time at
low temperatures for the eggs) exhibited slower growth on the HFX CRD 84 than
those hatched in the Spring. This poorer growth may have resulted from the
lower lipid levels of larvae whose eggs underwent delayed hatching.
Table 12. Growth and survival of Homarus americanus fed HFX CRD 84 at two sites and different seasons.
Initial 8 week 14 week % N Weight Weight Weight Survival
MLF Spring-summer 150mg 675mg 1348mg 96.6 30 MLF Fall-winter 121 420 continuing 100 27 HFRL Summer-fall 37 231 527 92.5 40 HFRL Fall-winter 37 145 350 82.5 40
The Marine Lobster Farms (MLF) lobsters seemed to grow faster than those at
Halifax. The growth differences between sites may reflect genetic
adaptations of two different stocks. The MLF experiments were done with
lobsters captured in the Northumberland Strait while the Halifax experiments
used stock captured off Eastern Passage near Halifax in the Atlantie Ocean.
The thesis work of Brian Mackenzie, an MSc candidate at Dalhousie University, • has shown that larvae from diife~nt areas of the Maritime Provinces fishery have different optimal developmental temperatures. The Northumberland larvae
having the highest optimal temperature (about 20°C) of all stocks tested.
The faster growth rate of MLF lobsters fed HFX CRD 84 might be the result of
genetie differenees. these juveniles having a preference for the standard
20°C experimental temperatures used by both study groups. Other factors such
as water quality might also account for differences in growth rate (for
example the MLF salinity tends to be about 27% salinity compared with the 32% at Halifax). Whatever the reasons for the differences. the benefits of using
a standard referenee diet are apparent. By keeping the nutritional control 20
constant, other factors such as season, stocks, environment, species, etc., can be more accurately assessed. We are looking forward to comparing results with the rest of our collegues participating in this international multi-speeies feeding trial.
While either BML 81 S or HFX CRD 84 might not prove to be applicable to all marine and freshwater crustaeeans, they do appear to hold promise as a
Standard Reference Diet for many species. The acceptance of a reference diet for use among many crustaeean nutrition research laboratories around the world will be but a first step in applying the many reeommendations and suggestions of the EIFAC, IUNS and lCES working Group on Standardization of
Methodology in Fish Nutrition Research. lt is hoped that improvements in presenting data on composition and analysis of experimental diets, improved experimental designs and more appropriate proeedures for data analyses will also be incorporated into future crustacean nutrition research.
Acknowledgements
We are grateful to many coworkers who, over the past few years, have , participated experiments that have lead to the development of the HFX CRD.
These inelude: Nancy Kay, Eric Pass, Michael Jackson. Rose Featherstone,
Margaret Schenk, Andrew Boghen, Dave McCann and Santosh Lall.
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