Nippon Suisan Gakkaishi
`
`57(4), 681-694 (1991)
`
`Efiective Components in Cuttlefish Meal and Raw
`Krill for Improvement of Quality of Red
`Seabream Pagrus major Eggs“
`
`Takeshi Wataiiabe,“ Min-Jer Lee,“ Jun Miziitani,“
`Tetsuo Yamada,“ Shuichi Satoh,“ Toshio Takeuchi,*‘l
`Noriaki Yoshida,*" Tetsuo Kitada,“ and Toshihisa Arakawa“
`(Received August 3, 1990)
`
`Red seabream broodstock were fed various diets of different nutritional quality for either 26
`days or shortly before spawning to clarify the effective components in cuttlelish meal and raw krill
`which aid in quality egg production.
`The percentage of buoyant eggs was lowest in the control group receiving the white fish meal
`diet, and was elevated by the addition of 200 mg Di.-a-tocopheryl acetate. The value was also
`etlectively improved by replacement of white fish meal with dcfatted or intact cuttlefish meal as a
`protein source. Feeding broodstock with frozen raw krill after previously being fed control diet
`resulted in elevation of the percentage of buoyant eggs and normal larvae. Equally good results
`were obtained by substitution of cuttlefish liver oil in the control diet with 2.5 ‘X, krill polar lipid or
`2.5% krill nonpolar lipid. However, neither defatted krill meal nor fat-soluble fraction of cut-
`tlefish meal showed the good effect on the egg quality.
`Consequently, the superior quality of cuttlefish meal to the white fish meal as a protein source
`for red seabrcarn broodstock diets was reconfirmed. And the effective components in raw krill,
`aiding the reproduction of red seabream. are suggested to be the polar and nonpolar lipid fractions.
`In addition, vitamin E was also found to have the same efficiency for improvement of the egg
`quality.
`
`In the series of studies on red scabream Pagrur
`major broostock nutrition,“ 6’
`it was found that
`spawning and egg quality were always greatly
`improved by feeding the broodstock on a diet
`containing cuttlefish meal as protein source or on
`frozen raw krill shortly before spawning or during
`spawning. Supplementation of diets with
`/3-
`carotene and canthaxanthin or with krill oil extract
`
`containing astaxanthin was also found to improve
`egg quality. This promoting role of raw krill on
`reproduction of red seabream may be due to the
`carotenoid pigments in krill. The productivity
`of viable larvae from the total eggs produced by
`one female ranged from 24 to 39°/,, in the brood-
`stock fed the control diet containing white fish
`meal as protein source in this series of experiments
`during 8 years. The viability increased to 704
`90% by replacement of white fish meal with cut-
`tlefish meal and to 68-80% by feeding frozen raw
`krill. One of the major chemical difference be-
`
`tween white fish meal and cuttlefish meal is 21 high
`content of calcium and phosphorus in the former,
`derived mainly from tricalcium phosphate (hy-
`droxyapatite) in the bones. This probably sug-
`gests the ill effect of a large amount of tricalcium
`phosphate on reproduction of
`red seabream.
`The supplementation of a high quantity of «-
`tocophciol was also found to be effective in im-
`proving spawning and egg quality.
`to clarify
`These experiments were carried out
`the elfectivc components in cuttlefish meal and
`raw krill which aid in aquality egg production by
`red seabream. For this purpose cuttlefish meal
`and raw krill were separated into lipid and non-
`lipid fractions. The lipid fraction of raw krill
`was further fractionated into polar and nonpola1',
`the latter containing astaxanthins. Etfect of these
`fractions, together with vitamin E, on egg quality
`were compared by feeding diets to broodstock red
`scabream shortly before spawning. The effect of
`
`“ Nutritional Studies iniithe Seed Prodiictioriiiofdiiishl-XX.
`*2 Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan, Minato, Tokyo 108, Japan
`(fiifi
`fit‘.
`45
`B)’l’f§l:
`7l<2§
`It u
`‘il'a’?l. Lllfllfililti
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`i"Ii’i@El5_3 Fii;fi7l<i‘J”E’j\‘$"£§3,fi‘éi‘fi3Z§51=‘l«).
`Nagasaki Prcfectural Institute of Fisheries, Nomozaki. Nagasaki 851-05, Japan (E El llziiflt,
`;lt{l]'-tljik,
`fiiiinnz t?<ili€iL'+’=7l<i$i’ii’.%%).
`
`000001
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`
`ENZYMOTEC - 1039
`
`000001
`
`

`
`682
`
`Watana be at al.
`
`a supplement diet with tricalciuni phosphate was
`also examined.
`
`As already demonstrated in the previous ex-
`periments,“-") quality of eggs of the red seabream is
`quite easily influenced by nutritional quality of
`the diets given to broodstock even during the
`spawning period. Therefore, one group of brood-
`stock was fed alternatively every three days, on a
`low and a high nutritive diets,
`to correlate the
`daily egg quality with the dietary composition.
`
`Materials and Methods
`
`FeedI'IIg of Red Seabrcam Broodstock
`Broodstock were developed from juvenile red
`seabream by feeding them on a commercial diet
`and minced fish meat for about 3 years, according
`to the same procedures described previously," at
`the Aquaculture Research Laboratory of Naga-
`saki Prefectural
`Institute of Fisheries. These
`
`broodstock weighing about 700g were kept on
`the control diet (diet 1, Table 4) for 60 clays from
`January 20 to March 26,
`in floating net cages
`(4 >:4>-:2m)
`in the Nomo Inlet. Later about
`50 fish were randomly selected and stocked in
`each of the 5 floating nets (3 X3 X2 rn; diets I
`to
`5) in the Inlet for 26 days from March 26 to April
`20, I984. Twelve to sixteen males and eleven to
`fourteen females from each lot were then transfer-
`ed to 6t concrete tanks in the aquarium of the
`laboratory for the investigation of spawning and
`egg quality (Fig. 1). Furthermore,
`the broods-
`Lock, which had been fed on the control diet from
`January 20 to April 20, were divided into 7 lots
`and fed on raw krill (diet 6) and diets containing
`
`200 mg of aw-tocopherol (diet 7), 5 ‘X, krill oil extract
`(diet 8), 2.5% krill oil extract (diet 9), 2.5% polar
`lipid fraction of krill oil (diet 10) and 2.5”‘, non-
`polar lipid fraction (astaxanthin fraction; diet 11),
`respectively. The broodstock, which had been
`fed on the cuttlefish meal diet from March 26 to
`April 20, were divided into two groups, one con-
`tinued to be fed the same diet (diet 3) and the
`other was fed on diet 3 and a diet containing corn
`oil
`in place of Cuttlefish liver oil, alternatively
`every three days. The broodstock on diet
`5
`were also divided into two groups from April 20,
`and one of the groups fed on a diet containing the
`lipid fraction of cuttlefish meal. The broodstock
`on diets 1-5 and 8 were kept in 6 t tanks and those
`on diets 6, 7. and 9-13 in I
`t polycarbonate thanks
`for spawning (Fig. l).
`Each test diet was given twice daily and there
`was no marked difference in the total amount
`consumed by fish during the feeding experiment,
`ranging from 250 to 300 g per broodstock except
`for raw krill 900-I200 g of which was accepted by
`one broodstock shortly before and during spawn-
`ing. Water temperature increased gradually from
`13°C in March to 21°C in May during the experi-
`mental period. Othcr experimental conditions
`were as described in a previous paper.“
`
`FracrI'ana!I'0n of Cuttlefish Men] and Krill A/lea!
`Cuttlefish meal was extracted with 20 folds of
`a hexane-ethanol mixture (71 : 29) and separated
`into lipid and nonlipid fractions. Krill meal was
`also fractionated into lipid and non-lipid fractions
`by hexane and the lipid fraction was then separat-
`ed into polar and nonpolar components by
`
`r DIET 1 :coNTnoI. —~
`WHITE FISH MEAL
`
`--——--
`DIET 1 —
`DIET 6 (RAW KRILL)
`DIET 7 (+ VTTAIIIIIN E)
`DIET e (5%. KRILL OIL)
`‘DIET 9 (2.5% KRILL OIL)
`‘ DIET 10(2.5% POLAR LIPIDS
`mom KRILL OIL)
`LDIET 11(2.59e NONPOLAR
`LIPIDS CONTAINING
`CAROTENOID PIGMENTS)
`
`----_ a TON
`1 TON
`1 ToN
`6 ToN
`1 ToN
`1 TON
`
`1ToN
`
`DIET 2 ---- —-————~—~_s TDN
`
`RED SEABHEAM
`BRooDsTocIt OF-CONTROL
`3 YEARS OLD
`DIET
`(m._.,0og,
`
`i
`
`DIET 2 : DEFATTED
`KRILL MEAL +
`CUTTLEFISH LIVER OIL
`I-DIET 3 2 CUTTLEFISH
`MEAL
`
`6 TON
`[~ DIET 3 ———~—-————~~—--
`DIET 12(ALTERNATlVELY DIET 3 1 ToN
`AND CORN OIL DIET)
`HDIET 4 : currLEFIsH g-—DII»:T 4 — ——— ——
`-~——--—~————»-6 ToN
`MEAL 0- Ca
`
`Wwmr ‘ GTON
`"DIET 5 ’‘
`'D|ET 5 =L|%l:-__f§FMEEA;_’.—"'*'
`“Er ‘3¢‘:‘:',l’,‘Lfi_',f:’}‘,’s':,“;"E’}f,’_',“
`‘ 1°"
`UTTLEFISH LIVER OIL
`MAR.26__
`,_ , 4APR.2O _
`__
`,_
`MAY 30
`_
`JAN.2o_~
`-7
`26 DAYS
`60 DAYS -—— 1934
`19“
`1984
`1984
`is-—— FLOATING NET CAGES IN THE sEA——-—
`—-
`—- SPAWNING TANKS IN AQUARIUM ———
`The feeding schedule for red seabream broodstock.
`Fig. 1.
`
`000002
`
`000002
`
`

`
`Effective Components for Red Seabream Egg Quality Improvement
`
`683
`
`Table 1.
`Lipid classes of four kinds of oils used in the experimental diets for red seabream
`broodstock* (%)
`
`
`. .. .
`.... W
`Total lipid
`
`..,
`
`_._.K“_“._9.“
`Polar lipid
`
`,
`
`,_,
`
`_,_
`
`Nonpolar lipid
`
`Nonpolar lipid
`SE
`TG
`FFA
`FS
`DG
`MG
`
`Polar lipid
`PE
`PC
`SPM
`PLC
`
`~
`4 .0
`L5
`1.8
`2.]
`1.4
`
`3 2
`37 4
`2 6
`——
`
`—
`0.5
`01
`tr
`tr
`—
`
`4.6
`84.9
`8.0
`_
`
`——
`80,9
`1.9
`9,5
`2.3
`3,8
`
`-
`
`-
`1.1
`0.1
`__
`
`extracted from
`Cuttlefish meal
`WW,‘ K177“
`0.5
`3,0
`45
`259
`.._
`_
`
`4.1
`40.7
`--
`16.0
`
`'1
`
`[Values expressed as percentage in ihtal lipid.
`Abbreviation:
`SE, slerol esters; TG, triglycerides; FFA, free fatty acids; FS,frees1erols; DG, diglycerides; MG, monoglycerides;
`PE, phusphatidylellianolamina; PC, phosphatidyleholine; SPM, sphigomyelin; LPC, lysophusphaiidylchulinc,
`
`Table 2. Fatty acid compositions of six kinds of oils used for red seabre-am broodstock diets
`(area ‘X,)
`
`Fatty
`acid
`
`14:0
`15:0
`16:0
`16:1
`1622
`17:0
`16:3
`16:4
`18:0
`18:]
`18:2n-6
`18:31)-6
`18:3n—3
`18:4n-3
`20:0
`20:1
`20:2n—6
`202311-6
`20:4n-6
`20:4n-3
`20:5n-3
`22:0
`22:1
`22:4n-6
`22:5n-6
`22:5n—3
`22:6n-3
`‘Totalwn-6
`Totaln-3
`SW01
`n-31-[UFA
`
`r_w»h
`Total
`lipid
`14.9
`0.2
`20.6
`8.1
`0.6
`-—-
`—
`1.6
`1.8
`19.6
`2.2
`0.1
`0.9
`4.1
`—»-
`1.0
`0.1
`0.3
`0.5
`0.5
`12.4
`~~~
`0.8
`0.1
`0.1
`0.2
`8.0
`3.4"
`26.1
`21.1
`I
`
`"_— Krill 011 _j
`Polar
`Nonpolar
`lipid
`lipid
`5.7
`21.1
`0.3
`0.1
`30.4
`15.1
`2.7
`9.9
`0.4
`0.8
`—
`~«~
`0.4
`0.2
`0.2
`2.3
`0.9
`1.0
`13.6
`21.8
`2.3
`2.9
`0.1
`0.1
`1.4
`1.0
`4.7
`9.8
`——
`0.1
`0.9
`1.7
`0.1
`—-
`0.3
`0.8
`0.6
`0.2
`0.5
`0.2
`20.1
`5.}
`—
`—
`1.0
`0.4
`0.3
`~
`0.7
`0.1
`0.5
`0.1
`7 S
`4.0
`4.4
`4.3
`34.7
`20.2
`28.6
`9.8
`
`7
`
`1
`
`O1l%)i(t)1;;:tlcted
`Cuttlefish
`meal
`4.3
`0.9
`29.3
`3.8
`0.7
`1.6
`0.3
`0.2
`9.9
`7.0
`2 4
`——
`1.3
`0.4
`V
`4.7
`0.4
`4
`4.]
`———
`12.7
`0-4
`1.0
`0'3
`0.4
`0.6
`11.1
`7.6
`26.1
`24.4
`
`V
`
`/
`
`A
`
`Cmluefish
`hgiclr
`
`6.6
`0.7
`17.3
`6.7
`1.2
`0.6
`0.7
`1.3
`2.0
`16.7
`1.6
`~—
`1.2
`2.6
`—-
`7.4
`0.5
`0.1
`1.0
`1.0
`11.4
`~
`5.0
`0-1
`0.3
`0.9
`
`3.7
`27.7
`23.9
`
`V‘
`
`Com
`oil
`
`——
`—
`10.1
`0.2
`0.1
`~—
`
`~-
`2.0
`28.9
`55.6
`0.5
`1.7
`—-
`0.5
`0.5
`
`——
`——
`——
`—
`—
`H
`—
`——
`»_
`_ --—
`6.1
`1.7
`2
`
`000003
`
`000003
`
`

`
`684
`
`Watanabe et al.
`
`Table 3. Proximate and mineral compositions of four kinds of meals for red seabream broodstock
`diets
`
`‘ A"
`
`_ A
`
`C
`
`White
`fish meal
`
`Defatted
`krill meal
`
`Dcfatted
`cuttlefish meal
`
`Cuttlefish "5 '
`meal
`
`(%)
`Moisture
`Crude protein (%)
`Crude lipid
`(%)
`Crude ash
`C’/6)
`
`Ca
`P
`Mg
`K
`Na
`Fe
`Zn
`Mn
`Cu
`
`(mg/2)
`(mg/g)
`(mg/8)
`(mg/g)
`(mg/g)
`(,ug/g)
`(,ug/g)
`(ug/g)
`(pg/s)
`
`10.9
`63.2
`9.7
`15.9
`
`49.12
`25.15
`2.02
`4.92
`8.60
`129.0
`74.7
`10.1
`4. 3
`
`9.8
`70.6
`5.0
`12.7
`
`24.31
`13.05
`7. 32
`3. 34
`14.15
`134.0
`71.2
`5.64
`61.7
`
`7.0
`83 .4
`1.8
`9,1
`
`7.63
`7.80
`3.80
`5 .40
`15.31
`246.2
`125.9
`5.74
`40. 8
`
`10.0
`747
`13.1
`5.6
`
`3.15
`7.22
`3 _()2
`2 . 59
`10.21
`201 .4
`90.6
`3.59
`25.3
`
`Table 4. Composition of the experimental diets for red seabream broodstock (%)
`
`
`Diet no.
`
`Ingredient
`
`—/-~~~—~
`
`1
`
`-~--— ~~
`
`White fish meal
`Defattcd krill meal
`Cuttlefish meal
`Defatted cuttlefish meal
`
`67
`——
`~~
`~—-
`
`15
`Alpha-starch
`5
`Mineral mixture
`2
`Vitamin mixture
`1
`Choline chloride
`4*
`Cuttlefish liver oil
`6
`Celluose
`»—
`Tri-calcium phosphate
`"W ‘All lhejiets contain ahouti50vmg V175/i100 1; W
`
`A
`
`2
`
`.
`64
`——
`.-
`
`15
`5
`2
`1
`7*
`6
`~ -
`
`3
`
`.
`.——
`61
`.—.
`
`15
`5
`2
`1
`2*
`14
`— ~
`
`————
`4
`
`-.
`~.
`61
`._.
`
`15
`5
`2
`1
`2*
`6
`8
`
`~
`
`-
`
`..~-.
`
`5
`
`_ " 7
`._
`-...
`55
`
`'
`
`1 5
`‘ 5
`2
`1
`9*
`13
`__
`
`Table 5. Composition of the experimental diets for red seabream broodstock just before spawning
`(0/0)
`
`I >:v_7"7?—V‘_____‘77
`ngre ient
`—-:——~--—-—-~
`6
`7
`67
`15
`5
`
`V
`V
`White fish meal
`Alpha-starch
`Mineral mixture
`
`_n
`3
`5;;
`3
`
`‘:
`
`Vitamin mixture
`Choline chloride
`Cuttlefish liver 611
`Cellulose
`Krill oil
`
`Krill polar lipid
`Krill nonpolar lipid
`n—3 HUFA
`Corn oil
`Oil extracted from
`Cuttlefish meal
`
`2
`1
`4*
`6
`—
`
`-
`<~~
`....
`—
`—— »
`
`8
`67
`15
`5
`
`2
`1
`
`5
`5*
`
`——
`~—
`..--
`—
`———
`
`W‘ Diet no.
`9
`10
`67
`67
`15
`15
`5
`5
`
`2
`l
`25*
`5
`2.5
`
`~—~
`—
`—
`M
`——~
`
`2
`1
`25*
`6
`~—
`
`2.5
`—-
`-~—
`—.
`—-
`
`: 7
`
`U 7
`—
`
`—
`
`v
`
`—.
`
`ll
`67
`15
`5
`
`2
`1
`2 0*
`5
`_
`
`—
`2,5
`0.5
`_.
`—
`
`12
`67
`1 5
`5
`
`2
`1
`
`4
`_.
`
`-_
`_.
`.~
`6*
`._
`
`- All the diets except diet 7 contain 50 mg ve/71-00 g 012:1. The 01:: 7 co1iiia71ll:s"2o0 mg \7/7i.«'/Itmng 1112-1.
`
`000004
`
`7
`‘P 7:
`,,,,,,4--
`13
`67
`15
`5
`
`C l
`
`"
`
`2
`1
`1=-
`5
`
`_.
`._
`__
`__
`4
`
`000004
`
`€
`

`
`Effective Components for Red Scabrearn Egg Quality Improvement
`
`685
`
`acetone." Astaxanthins in the krill meal were
`transferred to the nonpolar lipid fraction. Lipid
`
`class and fatty acid compositions of each lipid
`fraction from cuttlefish meal and krill meal are
`shown in Tables 1 and 2. The main component
`of the total lipid from krill meal was triglycerides,
`whereas that of residual oil from cuttlelish meal
`was free sterol (cholesterol). Phosphatidyl choline
`was the main component in the polar lipid frac-
`tion of both cuttlefish meal and krill meal. Ly-
`sophosphatidyl choline was also high in the latter.
`Each lipid fraction from both the meals was high
`in the concentration of n-3 highly unsaturated
`fatty acids (n-3 HUFA) such as 20: 5n—3 and 22:
`6n-3, except for the nonpolar lipid of krill oil.
`The lipid from the cuttlcfish meal showed a similar
`fatty acid distribution to that of the cuttlefish
`liver oil.
`Proximate and mineral compositions of dcfatted
`cuttlefish meal and krill meal are shown in Table
`3, together with white fish meal and intact cut-
`tlefish meal for comparison. White fish meal was
`characteristically high in the content of calcium
`and phosphorus due to tricalcium phosphate in
`fish bones and low in the copper content.
`
`Experimental Diets
`The composition of the experimental diets is
`shown in Tables 4 and 5. Diet 1 was a control
`diet containing white fish meal as a protein source
`and the composition was the same as that used in
`the previous experiments.’-3-“’ White fish meal
`was replaced by defatted krill meal
`indict 2 to
`examine the effect of the nonlipid fraction of krill
`on egg quality and by cuttlefish meal in diets 3 and
`4; diet 4 being supplemented with tricalcium
`phosphate at a level equivalent
`to the calcium
`level of white fish meal diet, to compare the dietary
`value of the two protein sources and examine sup-
`plemental eflect of calcium on egg quality.
`In
`diet 5 white fish meal was also substituted by
`defatted cuttlefish meal
`to verify effective com
`ponents in the meal
`for
`imporvement of egg
`quality. Diet 6 was frozen raw Antarctic krill
`Euphausia superba. Diets 7 to 13 were all modifi-
`cation of the control diet. Diet 7 was essentially
`the same as diet
`l except for a supplement of
`200 mg of DL-a-tocopheryl acetate, which has
`already proved to be effective for improvement
`of egg quality. Diets 8 to 12 were arranged to
`clarify the effective fraction of raw krill for re-
`production of red seabream, containing respective-
`ly 5% krill oil (diet 8), 2.5% krill oil (diet 9),
`2.5% polar lipids (diet 10) and 2.5% nonpolar
`
`In diets 12 and 13, cuttlefish
`ll).
`lipids (diet
`liver oil was substituted by 4% of lipid fraction
`from cuttliefish meal, the level being comparable
`to that contained in the cut tlefish meal diet and
`corn oil. The broodstock on diet 12 were fed on
`both a high quality diet (diet 3) and a low quality
`diet (the essential fatty acid (EFA)-deficient corn
`oil diet) alternatively every three days; to examine
`how the quality of eggs produced changes every
`day.
`The protein and lipid levels were adjusted to
`approximately 45 and 10"/O, respectively, the same
`levels as
`those used in the previous experi-
`inents.1-W’
`The analytical data on the test diets and raw
`krill are shown in Tables 6-8. There was no
`marked difference in proximate composition among
`the test diets except for slightly low contents of
`crude lipid in diets l and 4, and of crude ash in
`diets 3 and 5, both containing euttlefish meal as a
`protein source. The level of crude ash together
`with calcium and phosphorus of diet 4 was elevated
`to almost the same level as the control diet by a
`supplement of tricalcium phosphate in the diet.
`The mineral composition of white
`fish meal
`based diets indicated higher content of calcium
`and phosphorus, and lower level of copper. The
`values for diets and raw krill are all expressed on
`a dry basis. All the diets contained 40-50 mg of
`vitamin E except for diet 7 which had about 130 mg
`of the vitamin,
`the value being lower than that
`actually added to the diet (200 mg).
`Among the lipid classes, the proportion of polar
`lipids was lower in diets 2 and 5, containing dc-
`fattcd krill meal and cuttlefish meal respectively
`and was highest
`in diet
`l() supplemented with
`2.5% krill polar lipid. Cholesterol was high in
`diets 3 and 4 containing Cuttlefish meal and diet
`13 containing the lipid fraction of krill meal.
`As shown in Table 8, all
`the diets contained
`suflicient amount of n-3 H UFA, the EFA for red
`seabream, derived from Cuttlefish liver oil, krill
`oil and white fish meal or Cuttlefish meal to satisfy
`its requireinent“ ; except for the corn oil diet (diet
`12) which was rich in 18: Zn-6 and deficient
`in
`n-3 HUFA.
`
`Inverligatiorz of Sprlwnzflg and Evaluation of Egg
`Quality
`The eggs produced naturally by female brood-
`stock, given each test diet, were collected every
`day from 16:00 to 09:00 next morning during
`the experimental period until May 30 in both 6t
`and 1 t tanks in the aquarium. The method of
`
`000005
`
`000005
`
`

`
`686
`
`Watanabe er al.
`
`Table 6. Proximate and mineral compositions of the experimental diets for red seabream brood-
`
`13
`711
`10
`9
`6*”7m 8
`5
`4
`3
`2
`1
`Dietno.
`7.1
`8.1
`8.0
`7.8 4.011.812.0106 7.6
`81610.5 10.7
`7.1
`1%)
`Moisture
`44.2 46.2 44.9 44.7 46.6 54.1
`43.2 44.8 44.8 43.5 44.2 44.6 44.3
`Crudeprote1'n(%)
`8.7
`9.6
`9.1
`8.2
`9.2 19.4 10.0
`11.5
`10.
`10.6 10.5
`11.4
`13.2
`Crudelipid
`(“A1
`14.9
`11.0
`9.0 15.9
`7.8 21.1
`15.8
`16.6
`16.
`16.3
`16.4 16.3
`17.3
`Crucleash
`("/,,)
`31.5
`32.1
`31.7 31.9
`120.2 37.9”40.1 93971
`30.8 “14.3”M5.4 30.4“ 6.51
`Ca
`(111;/g)
`40.5
`38.1
`40.7 37.5
`33.9
`18.3
`15.6 33.4 33.9
`7.6 33.2
`33.9 25.1
`P
`(mg/g)
`2.5
`2.4
`2.5
`2.4
`2.3
`4.9
`3.0
`3.1
`2.9
`4.4
`2.2
`2.5
`2.4
`Mg
`(mg/g)
`8.3
`7.8
`7.9
`7.8
`8.4
`7.4
`7.7
`7.9
`7.3
`3.7
`7.4
`8.0
`8.4
`K
`(mg/g)
`8.1
`7.8
`80
`7.7
`89 11.6
`11.1
`11.1
`11.5
`39.7
`9.6 10.0
`9.9
`Na
`(mg/g)
`156
`158
`154
`149
`122
`143
`127
`133
`191
`tr
`64
`64
`65
`Fe
`(,.g/g)
`46.9 46.4 55.0 46.7
`46.2 45.9 49.3 52.6 45.7 43.1
`40.2 41.3 36.1
`Zn
`(,ug/g)
`26.5 23.7 25.4 28.7 25.5
`16.8
`24.8 27.0 24.8 21.7 23.0 22.4 26.6
`Mn
`(pg/g)
`__
`_‘ . _
`.__._
`Cu
`(pg/g)
`7.9 52.3 35.7 36.1
`30.9
`17.4
`7.4 10.5
`10.2
`7.1
`8.3
`6.8
`8.2
`
`26
`
`,_
`
`—
`V1'taminE
`43.6 41.9 53.0 54.7 35.0 37.2 129.4 43.3 42.0 36.]
`(mglloog)
`* The values of frozen raw krill are expressed on a dry basis.
`
`38.4
`
`Table 7. Lipid classes of the experimental diets for red scabream broodstock
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`Dictno.
`
`1
`
`13
`12
`711‘
`33.4 62.7 28.8 23.5 32.4
`43.3
`4.2 15.7 29.1
`11.4 42.0 56.3
`(%) 32.2
`Polarlipid
`66.6 37.3 71.2 76.5
`67.6
`88.6 58.0 43.7 95.8 84.3 70.9 65.7
`(%) 67.8
`Nonpolar lipid
`0.7
`0.7
`0.8
`0.7
`1.1
`0.8
`0.9
`0.6
`1.1
`1.0
`0.8
`0.6
`Cho1estero1estcrs(%) 1.3
`Triglycerides
`(%)62.8 77.5 28.3 21.0 94.0 21.1
`65.1
`47.0 58.8 32.9 60.2 71.2 40.3
`Freesterols
`(%) 2.3
`5.5 22.4 20.2
`01
`5.1
`3.0
`6.2
`4.3
`2.3
`6.8
`3.2 22.8
`
`Table 8. Fatty acid compositions of the experimental diets for red scabream broodslock (area ‘%,)
`
`_
`Fatty acid
`
`14:0
`16:0
`16:1
`18:0
`18:1
`18:2n—6
`18:3n-3
`181411-3
`20:1n-9
`20:2n-6
`20:3n-6
`
`2O:3n—3}
`2o:4n_6
`202411-3
`20:5n-3
`22:1
`22:4n-6
`22:5n-6
`22:5n-3
`222611-3
`
`—~ —-'—---5
`
`2
`3
`5
`7.3
`3.9
`4.6
`5.2 11.5
`17.8
`16.1
`21.8 24.2
`16.6 21.3
`7.6 10.6
`4.3
`4.5
`6.3
`7.8
`3.2
`3.2
`7.6
`6.9
`3.2
`2.2
`18.1
`15.4
`12.3
`11.7
`16.3
`17.0
`1.7
`6.2
`2.8
`1.9
`2.0
`2.5
`1.0
`2.4
`2.0
`1.3
`1.6
`(1.6
`1.6
`3.5
`1.6
`1.2
`2.4
`2.5
`8.5
`4.7
`6.2
`5.2
`7.1
`0.8
`0.2
`-—
`0.3
`0.3
`0.2
`0.1
`—-
`——
`—
`—
`0.1
`0.4
`
`Dick no.
`V ~-——-fi—-—-*4
`7
`8
`9
`10
`5.0 10.0
`7.2
`5.0
`17.2 20.0
`18.9
`19.8
`7.3
`8.5
`7.7
`7.1
`3.1
`2.7
`2.8
`2.7
`18.6 21.2
`19.7
`17.9
`1.2
`1.7
`1.4
`1.4
`1.2
`0.8
`2.5
`1.3
`1.7
`2.4
`2.3
`2.0
`8.9
`5.8
`7.5
`7.3
`0.2
`0.1
`0.3
`0.1
`———
`——
`
`0.1
`
`~~—~———»-— —
`12
`11
`1.6
`7.9
`13.4
`15 3
`2.8
`7 8
`2.9
`2.7
`31.3
`19.2
`1.8 29.5
`0.9
`1.5
`4.4
`0.4
`6.8
`3.8
`0.1
`0.1
`0.3
`~—~
`
`——
`13
`3.8
`19.0
`6.0
`5.()
`16.3
`1.8
`0.9
`1.0
`8.8
`0.2
`0.1
`
`31
`28
`06
`08
`.
`.
`.
`.
`0.5
`0.4
`0.7 ~
`9.4 11.2
`10.3
`8.9
`2.6
`2.3
`5.1
`3.8
`0.2
`0.2
`0.1
`«~
`0.3
`0.4
`0.2
`——~
`0.7
`0.8
`0.9
`v—~
`11.9
`8.9 12.6 14.1
`
`07
`12
`.
`.
`0.6
`0.9
`11.9 17.5
`4.0
`0.8
`0.1
`().1
`0.3
`0.1
`0.9
`0.4
`13.2
`8.7
`
`O9
`.
`0.7
`10.8
`5.4
`0.1
`0.2
`().9
`11.5
`
`05 0808 07 0219
`.
`.
`.
`.
`.
`.
`0.3
`0.4
`0 6
`0.6
`0.1
`0.4
`9.3
`9.4 12.5
`10.5
`3.6 11.5
`3.6
`4.2
`4.5
`3.9
`2.2
`4.9
`——
`0.1
`——
`——
`0.1
`-
`—
`0.2
`0.1
`-
`~
`0.2
`0.1
`0.7
`0.7
`0.8
`0.3
`().9
`0.4
`7.2 12.6
`10.6
`11.2
`4.2 13.0
`
`26.6 26.9 27.2
`23.1
`17.8
`23.8
`Sug_13(gUFA
`8.3
`9.2 19.4*
`9.8
`8.3W9.5
`1.1pid(%)
`‘ The value of frozen raw krill is expressed on a dry basis.
`
`8.0 25.8
`17.2 23.1 24.4 23.1
`23.9
`10.0 113 10.2W10.6i10.5 111.4 13.2
`
`000006
`
`000006
`
`

`
`Effective Components for Red Seabream Egg Quality Improvement
`
`687
`
`egg collection and quality evaluation by percent-
`age of buoyant eggs (normal eggs floating on water
`surface), deposited eggs
`(abnormal eggs going
`down to the bottom of the tank), number of oil
`globules in an egg, rate of hatching and percentage
`of normal larvae hatched from buoyant eggs have
`all been described earlier."
`
`into lipid classes and quantified by using an
`Iatroscan
`(latron ’l‘l-l—l0).” Cholesterol
`and
`vitamin E (LI-t0COphef()l) were measured using
`GLC,”’ and high speed liquid chroniatographyf“
`respectively.
`
`Results and Discussion
`
`Analytical Methods
`Both buoyant and deposited eggs produced by
`each experimental broodstock were washed with
`distilled water, stored at —--20°C, water on the
`surface of eggs was wiped off with filter paper
`before analysis. Analytical procedures such as
`lipid extraction, separation of polar and non-
`polar
`lipids, preparation of methyl esters and
`GLC operating conditions were all
`the same as
`reported in the previous papers.‘’»“’’ Mineral
`distribution was determined by atomic absorp-
`tion spectrometry,” and that of phosphorus by
`the method of Lowery and Lopez“) Lipids ex-
`tracted from eggs and the test diets were separated
`
`Results of Spawning and Egg Quality
`The size and number of broodstoclc used for
`
`spawning is shown in Table 9 and the results of
`natural spawning by these female broodstock fed
`different diets containing various
`fractions of
`Cuttlefish meal and krill meal are shown in Table
`10 and Fig. 2. As indicated therein the average
`number of eggs produced by one female during
`the experimental period ranged from 22.7><lU‘
`to ll0.9><l0‘,
`the lowest
`in the broodstock led
`diet 10 containing 2.5%, hrill polar lipid and the
`higherst in those on diet 3 with cuttleiish meal as
`a protein source. These numbers of eggs pro-
`duced were quitc lower than those obtained in
`
`Table 9. Average body length (mm), body weight (g) and number of red seabrcarn broodstock
`used for spawning
`
`Diet
`“°'
`
`Sex
`
`wee“
`Body length
`
`282.1j;24.3*
`285.1:t2l.4
`286.8:l:14.7
`286.5-_l_‘13.5
`282.1:l:]9.4
`287.9:L2l.2
`284.7:i:23.8
`296.6;Ll8.7
`289.3;l;_18.l
`282.9;l;20.5
`299.7:t10.3
`292.o_-t 7.8
`290.3;l:22.9
`284.0::ll.2
`274.4:t22.0
`283.9~_]:23.]
`283.0:t29.4
`282.0:L' 7.3
`286.7:l:l8.9
`298.3::Il9.9
`273.3;l:l8.4
`287.3:l;l1.5
`309.7:l;25.9
`295.7_Ll5.4
`310.3-l:17.2
`287.5;L- 3.5
`
`W“ 'm'
`Body weight
`
`771.4-_L243.5
`794-.3,'l:l62.6
`8l7.l':l:l32.6
`302.7::l;l5l.9
`8l0.0:l;ll9.l
`8Z0.9:i:l73.l
`791.3:l:202.3
`857.9:t1l4.0
`8l6.2_-f;l66.4
`780.0:l:l53.9
`956.7:tl56.3
`783.3;t 9.4
`3l6.7_-ltl67.8
`753.3_-l: 62.4
`7ll.4j:l73.4
`763.63-_l75.4
`776.7:£-197.5
`773.3i 45.0
`726.7:l:l40.6
`853.3,i:l34.7
`703.3:l;ll0.9
`763.3'i:1l0.9
`963.3::l:295.8
`870.03: 58.9
`960.0-_l~]29.3
`740.0:L 20.0
`
`'
`
`" ‘re
`Body length
`
`296.3152 Z
`273.7;l:l3 6
`275.7}; 6 5
`290.01-36 4
`285.7;i:l3 l
`276.7_l_l5 5
`227.3.."l: 4 6
`295.0124 3
`300.3122 9
`234 it 9 2
`302.3.l.l6 6
`292..7¢11t7
`268.0_~l-:32.0
`275.7:l;l4-.0
`272.7j:22.9
`287.0:l:l3.l
`230.3:t35 5
`279.7~_j_— 9 0
`298.03: 3 5
`230.0:l:46 7
`277.7:l:20.6
`292.0:l:l0.8
`339.3j:32.9
`295.U'i:l9.7
`3l2.0:i-22.5
`306.7;h34.4
`
`--
`W“
`Body weight
`
`884.3j_443.3
`769.71: 93.6
`687.7,}: 19.9
`369.0:l:3l1.0
`302.0;l..l83.8
`72l.3j_»l47.6
`657.0;l‘_ 67.6
`794.0j_l.l5.9
`855.0il94.l
`795.0;'rl83.8
`926.7,i-184.3
`832-3:}; 21.1
`755.O_-13226.3
`699.71 72.]
`67S.0,—i:223..l
`740.3_“~l4-7.8
`679.0; 217.7
`724.07; 57.8
`783.3: 66.6
`80.5.0_~_;360.6
`638.3_+l00_6
`775.3;:l35.l
`878.3;t34l.0
`808.3-3103.2
`898.0 _l75.l
`982.2*l_366.l
`
`Number
`
`14
`14
`14
`13
`l2
`ll
`l6
`l4
`l3
`12
`3
`3
`3
`3
`14
`l4
`3
`3
`3
`3
`3
`3
`3
`3
`3
`3
`
`000007
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`Male
`Female
`Male
`Female
`Male
`Female
`Male
`Female
`Male
`Female
`Male
`Female
`Male
`Female
`Male
`Female
`Male
`Female
`Male
`Female
`Male
`Female
`Male
`Female
`Male
`Female
`-7 ivie;:i;§.5:
`
`ll
`
`12
`
`13
`
`000007
`
`

`
`688
`
`Watanabc at al.
`
`
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`
`Effective Components for Red Seabream Egg Quality Improvement
`
`639
`
`.\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\v.\\\\‘
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`
`8
`
`§
`
`NO
`
`
`
`
`
`S
`
`Eggs0!larvaeproduced/flnh(x10“) NoS
`
`Dlel no.
`
`Effect of the nutrltlenal quallly of broodstock
`
`diets on the spawning oi red seabream.
`
`Eggs produced
`
`Buoyant eggs
`
`
`
`Larvae hntched out
`Normal Larvae
`
`Fig. 2. Effect of the nutritional quality of broodstock diets on the spawning of red scabrcam.
`
`the previous experiments due to a shorter spawn-
`ing period of 21--26 days. However, these values
`are doubtful because number of female brood-
`
`stock in each test lot which spawned could not be
`detected. The percentage of buoyant eggs is very
`important for evaluation of egg quality as de-
`scribed previously.” When the results were com-
`pared, among the broodstock fed on diets 1 to 5
`for the same period from March 26 to May 30 in
`6t
`tanks,
`the percentage of buoyant eggs was
`lowest
`in the group on the control diet (diet 1).
`The replacement of white fish meal by defatted
`krill meal was not effective in improving egg
`quality, although the value of 65.6% in the control
`group was slightly elevated to 69.2 "4, suggesting
`that the non—fat-soluble fraction is not an effective
`
`component of raw krill on reproduction of red
`seabream. On the other hand,
`the value was
`almost 100% for the eggs produced by the brood-
`stock fed on the diets containing defatted or
`in tact cuttlefish meal as a protein source. Sup-
`plementation of the Cuttlefish meal diet with Ca
`at a level equivalent to the white fish meal diet ex-
`erted no ill effect on the percentage of buoyant
`eggs, suggesting that the difference of nutritional
`quality between white fish meal and cuttlefish
`meal is not due to a high Ca content in the former
`meal. The rate of hatching was also high in
`
`these eggs, the value being over 80 ‘X, in comparison
`to less than 60 ‘X, in the control group and those on
`the defatted krill meal diet. More than 97% of
`the hatched larvae were normal, resulting in high
`production of healthy seedlings. Thus the su-
`perior quality of cuttlelish meal
`to white fish
`meal as a protein source for red scabream brood-
`stock diets was reconfirmed by this experiment.
`The addition of the fat-soluble fraction of cut-
`tlefish meal to the control diet (diet 13) resulted in
`no marked improvement in final productivity of
`normal larvae, nevertheless an increase from 33.4
`to 41.2% was brought about by the treatment.
`This suggests that the high dietary value of cut-
`tlefish meal
`is mainly due to the non-fat-soluble
`fraction in the meal. The high egg quality ob-
`tained in the group fed the cuttlefish meal deter-
`iorated on feeding them the corn oil diet al-
`ternatively every three days (diet 12). The per-
`centage of buoyant eggs was reduced from 99.3 ‘)4’,
`to 72.1%, and the percentage of abnormal eggs
`with more than 2 oil globules especially incre-
`ased when fed the corn oil diet. The productivity
`of normal larvae was better than that on the corn
`oil diet alone,“-‘V5’ but
`lower
`than that on the
`cuttlefish meal diet. Thus quality of red seabream
`eggs was found to be greatly influenced by the
`quality of diets given to brooclstock even during
`
`000009
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`000009
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`

`
`690
`
`Watanabe er al.
`
`spawning. The effectiveness of vitamin E on the
`reproduction of red seabream was confirmed again
`by this experiment. The addition of 200 mg of
`oi.-a-tocopheryl acetate to the control diet elevat-
`ed the rate of normal
`larvae production from
`33.4%, to 72.4%, although only about 130 mg of
`the vitamin was detected in the diet.
`The results obtained in the broodstock fed diet
`6 (raw krill) and diets 8-11, containing each frac-
`tion of raw krill clearly indicate effective com-
`ponents in krill for reproduction of red seabream.
`Feeding broodstock with frozen raw krill
`(diet
`6) after previously being fed the control diet re-
`sulted in elevation of the percentage of buoyant
`eggs and their hatchability together with the rate
`of normal larvae, lead to a high productivity of
`seed available for mass propagation of juvenile
`fish. The productivity was elevated to 75.0%
`from 33.4 "/3 by feeding frozen raw krill. Equally
`good results, at times even better than forzen raw
`krill, were obtained by substitution of Cuttlefish
`liver oil in the control diet with 5.0% (diet 8) or
`2.5“/,, (diet 9) of krill oil extract. These results,
`together with those obtained in the broodstock
`fed diet 2 containing the non—fat-soluble fraction
`of krill, indicate that the fat-soluble fractions are
`effective for reproduction of red seabream. The
`addition of 2.5% krill polar lipid or 2.5% non-
`polar lipid containing astaxanthins to the control
`diet effectively improved egg quality in terms of
`percentage of buoyant eggs and hatchability.
`The productivity of seed increased from the
`original value of 33.4 '7; to 76.4 %, with polar lipid
`and 83.0",, with nonpolar lipid. Thus,
`the ef-
`fective components in raw krill, aiding the re-
`production of red seabream, are suggested to be
`the polar and nonpolar lipid fractions. As shown
`in Table l, the specific component is phosphatidyl
`
`choline in the polar lipid fraction and astaxanthins
`in
`the nonpolar
`lipid fraction.
`In addition,
`vitamin E was also found to have the same ef-
`
`ficiency for imporvement of egg quality. These
`facts
`suggest
`that
`the seabream are common
`factors between phospholipids and astaxanthins
`or vitamin E such as free radical scavengers.
`Further experiments will be necessary to re-
`confirm the effectiveness of these components on
`the reproduction of red seabream.
`
`Chemical C0/Izponmts aft/ze Eggs
`Buoyant eggs produced on May 3, 5, 6, 8 and
`10 by all
`the groups of broodstock, and the
`deposited eggs obtained on May 10, 13 and 15
`from the broodstock fed on diets 1 and 2,
`those
`obtained on May 18, 20 and 22 from the fish fed
`on diet 12, and those on May 13, 15 and 17 from
`the broodstock fed on diet 13 were analysed for
`proximate, mineral and fatty acid compositions,
`lipid classes and vitamin E. The values are in-
`dicated by the average of five determinations.
`Proximate composition of buoyant eggs and
`deposited eggs is shown in Table 11. There was
`no marked diflerence in proximate composition of
`buoyant eggs produced by each experimental
`broodstock as observed previously,” except for
`a slight decrease of crude ash content in the groups
`fed diets 6 (raw krill) and 7 (vitamin E). The
`crude ash content was higher in deposited eggs
`than buoyant eggs as noted in the previous ex-
`periment.” This may be caused by permeation of
`seawater into eggs due to the loss of membrane
`potential of the deposited eggs. This is also sup-
`ported by mineral composition of deposited eggs
`which were high in Na, Mg, and Mn,
`rich in
`seawater
`(Table
`12). The higher ash content
`reflected a lower protein content in the deposited
`
`Table 11. Proximate composition of both the buoyant and deposited eggs from each experimental
`broodstock ( %)
`
`Diet no.
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`Buoyant egg“
`Moisture
`Crudeprotein
`Crudclipid
`Crudeash
`
`90.3