`
`57(4), 6817694 (1991)
`
`Effective Components in Cuttlefish Meal and Raw
`Krill for Improvement of Quality of Red
`Seabream Pagrus major Eggs’“L
`
`Takeshi Watanabe,"'2 Min—Jer Lee,“ Jun Mizutanifi‘2
`Tetsuo Yamadaft2 Shuichi Satoh,“ Toshio Takeuchi,“
`Noriaki Yoshida,“ Tetsuo Kitada,M and Toshihisa Arakawat‘a
`(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
`effectively improved by replacement of white fish meal with defatted 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 if, 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 scabrcam 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 seabream 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 cuttlcfish meal is a 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 a—
`tocophc1ol was also found to be effective in im-
`proving spawning and egg quality.
`to clarify
`These experiments were carried out
`the effective 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 nonpolar,
`the latter containing astaxanthins. Effect 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
`
`*nr
`“ Nutritional Studies in the Seed Production of Fish-XX.
`Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan, Minato, Tokyo 108, Japan
`li‘a’il,
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`l‘fl’ifitilli fiifi7l<ifl§jt$§fifififiit$fil
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`71MB:
`Nagasaki Prefectural Institute of Fisheries, Nomozaki. Nagasaki 851—05, Japan (=5 El tan,
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`Petition for Inter Partes Review
`
`Of U.S. Patent 8,278,351 Exhibit
`
`ENZYMOTEC - 1039
`
`000001
`
`
`
`682
`
`Watana be et al.
`
`a supplement diet with tricalcium 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
`
`Feeding of Red Seabream 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 days 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 Tn; diets l
`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-
`tock, 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 a-tocopherol (diet 7,), 5 % 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"0 non~
`polar lipid fraction (astaxanthin fraction; diet 11),
`respectively. The broodstock, which had been
`fed on the cuttlefish meal dict 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 l~5 and 8 were kept in 6 t tanks and those
`on diets 6, 7. and 913 in l
`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 900-4200 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 pcriod. Other experimental conditions
`were as described in a previous paper.“
`
`Fractionation of Cuttlefish Men] and Krill rMea/
`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 :CONTROL —~
`WHITE FISH MEAL
`
`,___,,-
`DIET 1 —
`DIET 6 (RAW KRILL)
`DIET 7 (+ VITAMIN E)
`DIET a (5% KRILL OIL)
`DIET 9 (2.5% KRILL OIL)
`‘ DIET 10(2.5% POLAR LIPIDS
`L
`FROM KRILL OIL)
`DIET 1 “2.5% NONpoLAn
`LIPIDS CONTAINING
`CAROTENOID PIGMENTS)
`
`~~»~~ a TON
`1 TON
`1 TON
`a TON
`1 TON
`1 TON
`
`1 TON
`
`
`
`DIET 2 : DEFATTED
`Eflii'L'I‘EIi-éigfuvm OIL
`
`DIET 2 ”a ——————~-~_e TON
`
`
`RED SEABHEANI
`BROODSTOCK OF-CONTROL
`3 YEARS 0"”
`mgr
`(WTJOOg)
`
`i
`
`
`
`t- DIET a : CUTI'LEFISH
`MEAL
`
`6 TON
`[- DIET 3 m~—w~*~~~~
`DIET 12(ALTERNATWELY DIET 3 1 TON
`AND CORN OIL DIET)
`l-DIET 4 : currLEFISN g-EDIET 4 , __,
`~ ~-«——-——-—-we TON
`MEAL '- Ca
`
`
`>DIET 5 :ngréHfiIAEEAio— DIET 5 7— W1 TON
`UTTLEFISH LIVER OIL
`D'Er laétfit'géfiim'éfi'f
`‘ To"
`_.__MAR.26__
`g , 4APR.20 _
`'_ if? ”7
`MAY 30
`JAN.2o_fi__
`26 DAYS
`1934
`1934
`1984
`60 mm
`1984
`kT—ir— FLOATING NET CAGES IN THE SEA.. V.
`7-
`)fé ~ SPAWNING TANKS IN AQUARIUM W )l
`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" (%)
`
`
`
`2.. 2.
`. ... .
`.22
`_.,,KI 111311
`
`Total lipid
`Polar lipid
`
`.-...--2 .~.2.,..,..
`Nonpolar lipid
`
`Oil extracted from
`cuttlefish meal
`
`Nonpolar lipid
`0.5
`—-
`—
`——
`SE
`3.0
`80 9
`0.5
`4 .0
`TG
`4.5
`1 .9
`0.1
`] .5
`FFA
`26.9
`9.6
`tr
`1.8
`FS
`DG
`2.1
`tr
`2 1
`,._
`MG
`1.4
`7
`’5 8
`_
`
`
`Polar lipid
`4.1
`-
`4.6
`3.2
`PE
`40.7
`1.1
`84.9
`37.4
`PC
`_.
`0.1
`8.0
`2.6
`SPM
`16.0
`_-
`_
`——
`PLC
`Values expressed as percentage in total lipid.
`Abbreviation: SE, slerol esters; TG, triglycerides; FFA, free fatty acids; FS, free sterols; DG, diglyceridcs; MG, monoglycerides;
`PE, phosphatidyletlianolamina; PC, phosphatidylcholinE; SPM. sphigOmyelin; LPC, lymphusphfllidylchulinc.
`
`~
`
`,2
`_.
`10.1
`0.2
`01
`-—
`
`Table 2. Fatty acid compositions of six kinds of oils used for red seabrcam broodstock diets
`
`(area %)
`.
`'
`'
`Oil
`x
`‘
`Com
`Cuttlefish
`.__
`_-___K:“_m.l.
`-2---
`Fatty
`irgfiaed
`acid
`Total
`Polar
`Nonpolar
`cuttleflsh
`hV-Clr
`oil
`
`lipid
`lipid
`lipid
`meal
`"1
`14.9
`5.7
`21.1
`4.3
`6.6
`14:0
`0.2
`0.3
`0.1
`0.9
`0,7
`15:0
`20.6
`30.4
`15.1
`29.3
`17.3
`16:0
`8.1
`2.7
`9.9
`3.8
`6.7
`16:1
`06
`04
`03
`OJ
`12
`1&2
`—-
`——
`~-
`1.6
`06
`U:0
`—
`0.4
`0.2
`0.3
`0.7
`16:3
`1.6
`0.2
`2.3
`0.2
`1.3
`16:4
`1.8
`0.9
`1.0
`9.9
`2.0
`18:0
`19.6
`13.6
`21.8
`7.0
`16.7
`18:]
`2.2
`2.3
`2.9
`2.4
`1.6
`18:2n-6
`0.1
`0.1
`0.1
`—
`4-
`18:3n-6
`0.9
`1,4
`1.0
`1.3
`1.2
`18:3n—3
`4.1
`4.7
`9.8
`0 4
`2.6
`18:4n-3
`_._
`——
`0.1
`.
`2.
`20:0
`L0
`09
`L7
`47
`74
`201
`01
`OJ
`——
`04
`on
`2m2m6
`2.
`0.3
`0.3
`0.8
`2
`0.1
`20:311-6
`fl
`0.5
`0.6
`0.2
`4.1
`1.0
`20:411-6
`_»
`0.5
`0.5
`0.2
`,2
`1.0
`20:4n43
`_
`12.4
`20.1
`5.1
`12.7
`11.4
`20:5n-3
`—
`~—~
`—
`~
`0.4
`—
`22:0
`.“
`08
`10
`04
`L0
`50
`2&1
`_
`0.1
`0.3
`~
`0.3
`0.1
`22:4n-6
`.—
`0.1
`0.7
`0.1
`0.4
`0.3
`22:5n-6
`225m3
`02
`05
`OJ
`06
`09
`._
`
`22:6n-3
`8.0
`7 S
`4.0
`11.1
`10.6
`.._
`
`._
`2.0
`28.9
`55.6
`0,5
`1.7
`2.
`0_5
`05
`
`Toiéiiii-s
`Total n-3
`SUH‘Of
`n-SHUFA
`
`3.4
`26.1
`21.1
`i
`
`4.4
`34.7
`28.6
`
`1
`
`4.3
`20.2
`9.8
`
`7.6
`26.1
`24.4
`
`3.7
`27.7
`23.9
`
`5,1
`1.7
`.4
`
`000003
`
`000003
`
`
`
`684
`
`Watanabe et a].
`
`Table 3. Proximate and mineral compositions of four kinds of meals for red seabream broodstock
`diets
`
`
`‘ A"
`_ A
`.
`White -7
`Defatted
`Defatted
`Cuttlefish 2- '
`fish meal
`krill meal
`cuttlefish meal
`meal
`
`
`(%)
`Moisture
`Crude protein (%)
`Crude lipid
`(%)
`Crude ash
`(‘3/0)
`
`10.0
`7.0
`9.8
`10.9
`7417
`83 .4
`70.6
`63.2
`9.7
`5.0
`1.8
`]3_1
`
`15.9
`12.7
`9.1
`5.6
`
`Ca
`P
`Mg
`K
`Na
`Fe
`Zn
`Mn
`Cu
`
`(mg/g)
`(mg/g)
`(mg/g)
`(mg/g)
`(mg/g)
`(,ug/g)
`(,ug/g)
`(ug/g)
`
`(pg/g)
`
`3.15
`7.63
`24.31
`49.12
`7.22
`7.80
`13.05
`25.15
`3.02
`3.80
`7.32
`2.02
`2 . 59
`5 .40
`3. 84
`4.92
`10.21
`15.31
`14.15
`8.60
`201 .4
`246.2
`184.0
`129.0
`90.6
`125.9
`71.2
`74.7
`3.59
`5.74
`5.64
`10.1
` .8
`4. 3
`61.7
`40. 8
`25
`
`
`
`Table 4. Composition of the experimental diets for red seabream broodstock (%)
`Diet no.
`
`__.__, 2,.
`A
`2,.
`~
`—
`Ingredient
`2-5m”.
`l
`2
`3
`4
`5
`
`
`w
`
`White fish meal
`Defatted krill meal
`Cuttlefish meal
`Defatted cuttlefish meal
`
`67
`——
`~~
`—
`
`~
`64
`~—
`w
`
`——
`61
`W
`
`—
`~
`61
`H
`
`_ fl 2
`_
`4 ._
`55
`
`1 5
`15
`15
`15
`15
`Alpha—starch
`‘ 5
`5
`5
`5
`5
`Mineral mixture
`2
`2
`2
`2
`2
`Vitamin mixture
`1
`1
`l
`1
`1
`Choline chloride
`9*
`2*
`2*
`7*
`4*
`Cuttlefish liver oil
`13
`6
`14
`6
`6
`Celluose
`
`Tri-calcium phosphate __ _ — — ~ 8
`
`
`
`
`" All the diets contain about 50 mg VIE/100 g diet.
`
`'
`
`Table 5. Composition of the experimental diets for red seabream broodstock just before spawning
`(%)
`
`Diet n0.
`-
`
`Ingredient
`_.___..2.W__._. ~-
`,
`.
`2.
`........2
`9
`10
`11
`12
`13
`6
`7
`8
`
`White fish meal
`67
`67
`67
`67
`67
`67
`67
`Alpha-starch
`15
`15
`15
`15
`15
`1 5
`15
`Mineral mixture
`5
`5
`5
`5
`5
`5
`5
`
`Vitamin mixture
`Choline chloride
`Cuttlefish liver oil
`Cellulose
`Krill oil
`
`‘11
`3
`5
`B
`é
`
`2
`1
`4*
`6
`—-
`
`2
`1
`.—
`5
`5*
`
`2
`l
`25*
`5
`2.5
`
`2
`1
`2. 5*
`6
`»—
`
`2
`l
`20*
`5
`—
`
`2
`1
`
`4
`—.
`
`2
`1
`1*
`5
`
`E
`:
`
`Krill polar lipid
`Krill nonpolar lipid
`n—3 HUFA
`Corn 011
`Oil extracted from
`
`cuttleflsh meal
`
`_.
`-_
`—
`2.5
`-22
`——
`.—
`fl
`2.5
`—-
`—
`~—
`——
`2
`0.5
`-»—
`—
`2
`——
`6*
`——
`—
`25
`—
`4
`.4
`—
`—
`——
`2 2
`
`- All the diets except diet 7 contain 50 mg VEl’lOO g diet. The diet 7 winning-200 mg {til/100}; diet.
`
`-
`—
`w
`—
`—
`
`000004
`
`000004
`
`
`
`Effective Components for Red Scabream 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 cuttlefish 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 euttlefish
`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 experimental-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 effect 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
`lipids (diet 11).
`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)—deficicnt 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%, respectively, the same
`levels as
`those used in the previous experi-
`InCfltS.l-3rn)
`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 cuttlefish 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 exprCSsed on
`a dry basis. All the diets contained 4050 mg of
`vitamin 13 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 de-
`fattcd krill meal and cuttlcfish meal respectively
`and was highest
`in diet 10 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
`seabrcam, derived from cuttlefish liver oil, krill
`oil and white fish meal or cuttlefish meal to satisfy
`its requirements) ; except for the corn oil diet (diet
`12) which was rich in 18: 2n-6 and deficient
`in
`n-3 HUFA.
`
`Investigation of Spawning 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 l t tanks in the aquarium. The method of
`
`000005
`
`000005
`
`
`
`686
`
`Watanabe et al.
`
`Table 6. Proximate and mineral compositions of the experimental diets for red seabream brood~
`stock
`
`
`13
`12
`11
`10
`,
`Diet—110
`1
`2
`3
`4
`5
`6*
`7
`8
`9
`71
`8.1
`8.0
`7.6
`(36)
`Ldobture
`7.1
`8.6 10.5
`10.7
`7.8 4 0 11.8
`12.0
`10.6
`Crude protein (%)
`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
`Crude lipid
`(Z)
`8. 7
`9.6
`9.1
`8.2
`9.2 19.4 10.0
`11.5
`10.2
`10.6 10.5
`11.4
`13.2
`
`Crude ash
`(%)
`14. 9
`11.0
`9.0 15. 9
`7.8 21.1
`15.8
`16.6
`16.6
`16. 3
`16. 4
`16. 3
`17.3
`Ca
`7 (mg/g)
`30.8 14.3 5. 4
`30. 4
`6.1
`20.2
`37.9 40.1
`39.1 31.5 32.1
`31. 7 31. 9
`P
`(mg/g)
`33.9 18.3156 33.4 33.9
`7.6 33.2
`33.9 25.1
`40.7 37.5
`38.1
`40.5
`Mg
`(mg/g)
`2.3
`4.9
`3.0
`3.1
`2.9
`4.4
`2.2
`2.5
`2.4
`2.5
`2,4
`2,4
`2.5
`K
`(mg/g)
`8.4
`7.4
`7.7
`7.9
`7.3
`3.7
`7.4
`8.0
`8.4
`7.9
`7.8
`7.8
`8.3
`Na
`(mg/g)
`8.9
`11.6
`11.1
`11.1
`11.5
`39.7
`9.6 10.0
`9.9
`8.0
`7.7
`7.8
`8.1
`Fe
`(,Lg/g)
`122
`143
`127
`133
`191
`tr
`64
`64
`65
`154
`149
`158
`156
`Zn
`(,ug/g)
`46.2 45.9 49.3 52.6 45.7 43.1
`40.2 41.3 36.1
`46.9 46.4 55.0 46.7
`Mn
`(pg/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
`
`I
`
`(pg/g)
`
`6. 8 8.2
`8.3
`7.1
`10.2
`7.4 10.5
`17.4
`30.9
`7.9 52.3 35.7 36.1
`Cu
`V‘mminfi
`43.6 41.9 53.0 54.7 35.0 37.2
`129.4 44.2 52.0 43.3 42.0 36.1
`38.4
`
`(mg/100g)
`7* The values offrozen raw kr11lare expregsjad-onaerQsis.
`7
`4..
`
`
`
`Table 7. Lipid classes of the experimental diets for red seabream broodstock
`13
`12
`Diet no
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`Folarlipid
`(%) 32. 2
`11. 4 42.0 56. 3
`4.2 15. 7 29.1
`43.3
`33.4 62.7 28.8 23.5 32.4
`Nonpolar l1p1d
`‘7) 67 8
`88. 6 58.0 43. 7 95.8 84.3 70.9 65.7
`66.6 37.3 71.2 76.5
`67.6
`Cholesterol esters (/) 1. 3
`0.8
`0.9
`0.6
`1.1
`1.0
`0.8
`0.6
`0.7
`0. 7
`0.8
`0.7
`1.1
`Trig1ycerides
`(%) 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
`
`Free sterols
`(X) 2.3 5.5 22.4 20.2
`0.1
`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 seabream broodstock (area “/)
`D101 no
`’d
`F
`
`2
`3
`7
`8
`9
`10
`at” a“
`4.
`14:0
`7.3
`3.9
`5.0 10.0
`7.2
`5.0
`5
`
`21.8 24.2
`.
`.
`16:0
`16.1
`17.2 20.0
`18.9
`19.8
`17.8
`
`4.5
`6.3
`7.8
`16:1
`7.6 10.6
`4.3
`7.3
`8.5
`7.7
`7.1
`18:0
`3. 2
`3.2
`7.6
`6.9
`3.2
`2.2
`3.1
`2.7
`2.8
`2.7
`18:1
`1.81
`15.4
`12.3
`11.7
`16.3
`7.0
`18.6 21.2
`19.7
`17.9
`18:2n-6
`1.7
`6.2
`2.8
`1.9
`2.0
`2.5
`1.2
`1.7
`1.4
`1.4
`18:3n-3
`10
`2.4
`2.0
`1.3
`1.6
`0.6
`1.2
`0.8
`2.5
`1.3
`18240—3
`1.6
`3.5
`1.6
`1.2
`2.4
`2.5
`1.7
`2.4
`2.3
`2.0
`20: 1n-9
`8.5
`4.7
`6.2
`5.2
`7.1
`0.8
`8.9
`5.8
`7.5
`7.3
`20:2n-6
`0 2
`-—
`0.3
`0.3
`0.2
`0.1
`0.2
`0.1
`0.3
`20:311-6
`—-
`——
`—
`—
`0.1
`0.4
`0.1 m
`——
`
`1
`13
`12
`11
`7.9 1.6 3.8
`15.3
`13.4
`19.0
`7.8
`2.8
`6.0
`2.7
`2.9
`5.0
`19.2
`31.3
`16.3
`1.8 29.5
`1.8
`0.9
`1.5
`0.9
`4.4
`0.4
`1.0
`6.8
`3.8
`8.8
`0.1
`0.1
`0.2
`0.3
`~—-
`0.1
`
`0.1
`
`05 0808 O7 0219
`O9
`07
`12
`31
`28
`06
`08
`20:3{1—3}
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`20:4n-6
`0.3
`0.4
`0.6
`0.6
`0.1
`0.4
`0.7
`0.6
`O. 9
`0.5
`0.4
`0.7 ~
`20:411-3
`9.3
`9.4 12.5
`10.5
`3.6 11.5
`10.8
`7.5
`11.9
`9.4 11.2
`10.3
`8.9
`20: 511—3
`3.6
`4.2
`4.5
`3.9
`2.2
`4.9
`5.4
`0.8
`4.0
`2.6
`2.3
`5.1
`3.8
`2221
`74
`0.1
`~-
`——
`0.1
`0.1
`0.1
`0.1
`0.2
`0.2
`0.1
`8*
`22:4n-6
`-
`.
`0 2
`0.1
`-
`»
`0.2
`0.1
`0.2
`0.1
`0.3
`0. 3
`0.4
`0.2
`~9
`22: 511-6
`22: 511-3
`0.9
`0. 7
`0.8
`0.9
`0.4
`0.9
`0.4
`0.7
`0. 7
`0.8
`0.3
`0.9
`22:611-3
`11.9
`8. 9
`l2. 6 141
`13.2
`8.7
`11.5
`7.2 12.6
`10.6
`11.2
`4.2 13.0
`
`
`Bug-13(13UFA
`23.8
`17.8
`
`
`23.1
`
`8.0 25.8
`17.2 23.1 24.4 23.1
`23.9
`26.6 26.9 27.2
`
`8.3
`9. 8
`9 5
`8.3
`Lipid (%)
`9.2 19.4”“
`10.0 11. 5
`10.2 10. 6
`10.5 114 13.2
`The value of frozen raw krillIs expressed on a dry basis.
`
` .
`
`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 lid—10).” Cholesterol
`and
`vitamin E (a-tocopherol) were measured using
`GLCJ“ and high speed liquid chromatography,”
`respectively.
`
`Results and Discussion
`
`Analytical Met/rods
`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 papersfiA") 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 broodstoek 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 22.7xlU‘
`to 110.9x10‘,
`the lowest
`in the broodstock led
`diet 10 containing 2.5%, hrill polar lipid and the
`higherst in those on diet 3 with cuttlensh meal as
`a protein source. These numbers of eggs pro-
`duced were quite lower than those obtained in
`
`Table 9. Average body length (mm), body weight (g) and number of red seabrearn broodstock
`used for spawning
`
`Initial
`7
`.
`Final
`”774‘?“
`VWF‘W'E— "/ “A V”"
`V
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`7777*
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`
`Body length
`Body weight
`Body length
`Body weight
`
`Diet
`"0'
`
`Sex
`
`Number
`
`M 1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`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
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`296.3i52.2
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`282.1i24.8*
`278.7;{313 6
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`285.1:t21.4
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`817.1:t132.6
`286.8:hl4.7
`290.0i-36 4
`802.7:t151.9
`28651186
`285.7;t;18 1
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`282.1:t19.4
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`2680712320
`816.7:17167.8
`290.3:lz22.9
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`276.7j;l4.0
`753.371: 62.4
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`272.7j;22.9
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`230.3;t35 5
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`28301294
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`295.0}:197
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`295.7_L15.4
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`9600-11293
`312.0:l:22.5
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`287.5;t 3.5
`740.0:L 20.0
`306.7i34.4
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`884.33.443.24
`769.7;fi 93.6
`687.7,l: 19.9
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`657.01: 67.6
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`Effective Components for Red Seabream Egg Quality Improvement
`
`689
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`5
`
`Dlal no.
`
`Effect of the nutritional quallly of broodstock
`
`dleta on the spawning oi red seabream.
`
`Eggs produced
`
`Buoyant eggs
`
`
`
`55% Larvae hatched 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 Ely-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 defattcd
`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 "/0, 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 dict 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 31, in comparison
`to less than 60 X in the control group and those on
`the defatted krill meal diet. More than 973/,J of
`the hatched larvae were normal, resulting in high
`production of healthy seedlings. Thus the su-
`perior quality of euttlclish meal
`to white fish
`meal as a protein source for red seabream brood—
`stock diets was reconfirmed by this experiment.
`The addition of the fat-soluble fraction of cut-
`tlcfish 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 ‘34“,
`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 aloncf-U’ but
`lower
`than that on the
`cuttlefish meal dict. Thus quality of red seabrcam
`eggs was found to be greatly influenced by the
`quality of diets given to broodstock even during
`
`000009
`
`000009
`
`
`
`690
`
`Watanabe e! a].
`
`spawning. The effectiveness of vitamin E on the
`reproduction of red seabream was confirmed again
`by this experiment. The addition of 200 mg of
`DL-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 8711, containing each frac-
`tion 0f 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‘7O 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 ”/3 with polar lipid
`and 83.0”, with nonpolar lipid. Thus,
`the ef-
`fective components in raw krill, aiding the re-
`production of red seabrearn, are suggested to be
`the polar and nonpolar lipid fractions. As shown
`in Table 1, 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 imporvemcnt 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 efl'ectiveness of these components on
`the reproduction of red seabream.
`
`Chemical Components aft/re 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 difi'erence 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
`ll
`12
`
`13
`
`7
`k 7
`_
`-
`—
`BUOYant egg'“l
`90.7
`90.3 90.6 90.2 90.3 90.4 90.3 90.8
`90.3 90.3 90.4 90.5
`Moisture
`5.0
`5.2
`5.1
`5.4
`5.1
`5.3
`5.2
`5.1
`5.2
`5.2
`5.1
`5.2
`Crudeprotein
`Crudellpid
`2.1
`2 3
`2.2
`2.2
`2.3
`2.2
`2.1
`2.2
`2.1
`2.0
`2.0
`2.0
`
`Crude ash
`2.0
`19
`2.0
`1.9
`1.7
`1.7
`1.6
`1.8
`1.9
`1.8
`1.8
`1.8
`
`9
`
`7
`90.4
`5.1
`2.3
`19
`
`Tn”.
`Deposited egg”
`91.1
`90.4
`91.0 90.9
`Moist