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Growth, carcass composition, and plasma growth hormone levels in cyclically fed rainbow trout, Oncorhynchus… Inglis, Susan D. 1992

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GROWTH, CARCASS COMPOSITION, AND PLASMA GROWTHHORMONE LEVELS IN CYCLICALLY FED RAINBOW TROUT,Oncorhynchus mykissBySUSAN DALE INGLISB.Sc.,^The University of British Columbia, 1984A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF ZOOLOGYWe accept this thesis as conformingto the r uired standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1992© Susan Dale Inglis, 1992(Signature)In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of ZoologyThe University of British ColumbiaVancouver, CanadaDate -PA) 30) icig'3DE-6 (2/88)ABSTRACTThe compensatory growth response (C.G.R.), a phase of rapid growthfollowing a period of feeding restriction, is investigated in rainbowtrout (Oncorhynchus mykiss). Underyearling rainbow trout wereindividually identified using coded tags and placed on either a cyclicfeeding regime of 3 weeks of starvation followed by 3 weeks ofrefeeding (to elicit a G.C.R.) or a daily feeding regime. The effects ofthis feeding regime on growth, body composition, and plasma growthhormone levels were recorded weekly for 24 weeks to include 4feeding cycles. Fish that were placed on a cyclic feeding regimeexhibited higher specific growth rates, protein levels and lower fatcontent than fish that were fed daily. The condition factor, which isan index of the fatness or leanness of a fish, was slightly lower thanthat of the control groups at the end of the experiment as a leanerfish was produced. The response to the starvation phase of thefeeding cycle (as far as weight and fat loss were concerned)decreased in intensity with subsequent feeding cycles, indicating thatthe fish were acclimating to the feeding regime. There was asignificant increase in variability of both the weight and length datawithin the cyclically fed population as the experiment progressedand they showed increased sensitivity to water quality. No "inphase" cycling of plasma growth hormone levels was observed forthe cyclically fed fish. The implications of these findings in the useof cyclic feeding regimes in fish culture are discussed.i iTABLE OF CONTENTSAbstract^ iiTable of Contents^ iiiList of Tables i vList of Figures^ vAcknowledgements v iChapter 1: Introduction^ 1Chapter 2: Compensatory Growth in Rainbow TroutA. Introduction^ 5B. Methods and Materials^ 7C. Results 15D. Discussion^ 42Chapter 3: Carcass CompositionA. Introduction 46B. Methods and Materials^ 49C. Results^ 54D. Discussion 65Chapter 4: Changes in Plasma Growth Hormone LevelsA. Introduction^ 68B. Methods and Materials^ 71C. Results 72D. Discussion^ 77Chapter 5: Conclusions and Fish Culture Implications^80References^ 85AppendicesAppendix 1: Mean weights and lengths of Groups A, B, and C 92Appendix 2: Mean weights and lengths of Groups A', B', and C' 93Appendix 3: Mean carcass composition values of Groups A, 94B, and CiiiLIST OF TABLESTable 1: Record of mortalities for experiment^3 4Table 2: Body and gonad weight of fish at week 24^4 0Table 3: Carcass composition of Groups A', B', and C'^5 5at week 24Table 4: Rainbow trout plasma growth hormone assay results 7 3ivLIST OF FIGURESFigure 1: Layout of experimental facility^ 9Figure 2: Experimental design^ 13Figure 3: Mean weight of Groups A, B, and C for weeks 0-24^1 7Figure 4: Mean weight of Groups A', B', and C' for weeks 0-24 1 9Figure 5: Mean length of Groups A, B, and C for weeks 0-24^2 1Figure 6: Mean specific growth rates for Groups A, B, and C^2 3for weeks 0-24Figure 7: Mean condition factor for Groups A, B, and C for^2 5weeks 0-24Figure 8: Peak mean weight loss of Group A for^2 7all four feed cyclesFigure 9: Peak mean weight loss of Group A' for^2 9all four feed cyclesFigure 10: Specific growth rate of Group A for week 1 of the^3 1refeeding phase of all four feed cyclesFigure 11: Peak mean specific growth rate for Group A^3 6all four feed cyclesFigure 12: Fluctuation in water temperature^3 8Figure 13: Sections of fish used to represent the whole^5 0fish for carcass composition analysisFigure 14: Mean moisture levels of Groups A, B, and C^5 7Figure 15: Mean lipid levels of Groups A, B, and C 5 9Figure 16: Mean protein levels of Groups A, B, and C^6 1Figure 17: Mean ash levels of Groups A, B, and C 6 3Figure 18: Mean plasma growth hormone levels of Groups A,^7 5B, and C.vACKNOWLEDGEMENTSI would like to thank my supervisor, Dr. R. Blake and themembers of my committe (Dr. Liley, Dr. Iwama, Dr. Donaldson, Dr.Wilimovsky) for their advice and guidance. There are many people Iwould like to thank from the West Vancouver Federal FisheriesResearch Branch. Dr. Higgs, and Bakshish Dosanjh supplied me withthe materials and training for the chemical analysis sections of thisresearch and Helen Dye trained and assisted me with the growthhormone assay. Unfortunately, we were unable to get the assay torun properly at that time and I am very grateful to Dr. Leatherlandwho generously agreed to run the samples at his facility. I am alsovery grateful to the other members of Dr. Blake's laboratory for theirsupport and encouragement and to Alister and Andrew who helpedme conquer the computer.v iCHAPTER 1GENERAL INTRODUCTIONGrowth in fish varies greatly throughout their lifespan andseasonal patterns of growth in temperate species are common(Dobson and Holmes, 1984). Generally, fish living in temperatewaters have high growth rates in the spring and summer months andlower or negative growth rates in autumn and winter. Theseseasonal changes in growth are correlated with changes in watertemperature, photoperiod, and food abundance (Weatherly and Gill,1987).Intrinsic biological rhythms of growth are common in fish(Weatherly and Gill, 1987). Brown (1946a) found cycling in thegrowth of brown trout (Salmo trutta). In this case, time was found tobe a significant determinant in growth rate even though themagnitude of this significance declined with declining watertemperature. Wagner and McKeown (1985), reported that thegrowth rate in juvenile rainbow trout (Oncorhynchus mykiss) alsocycles. These fish cycled every three to four weeks. They suggestthat the hypothalamus probably controls this cycling effect.In salmonids growth has also been associated with lunar cycles(Farbridge and Leatherland, 1991). In this study rainbow trout(Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch)showed bi-weekly patterns of food consumption, plasma L-thyroxine1and growth hormone levels that correlate with new and full moons.They found increased levels of plasma L-thyroxine and growthhormone associated with the full or new moon. An increase in foodconsumption was observed several days before the new or full moon.The compensatory growth response (C.G.R.) is anothercharacteristic of fish growth. Compensatory growth refers to thesignificant increase in growth that occurs upon refeeding an animalthat has previously been starved. Although just recently becomingimportant in the aquaculture industry, the phenomenon has beenknown to exist for a long time in agricultural animals. One of thefirst references made to this type of growth was by Waters (1908,1909) who looked at the effects of undernutrition on beef steers.Waters found that undernourished cattle could recover and reachnormal mature weights and heights. He felt that this was anessential ability of animals in the wild that often live through timesof severe food shortages.Clarke and Smith (1938) found that rats previously placed ona restricted diet when refed showed growth rates that surpassed theweights of the control animals. They termed this phenomenon "over-compensation". This observation is thought to be due to theadditional adipose tissue that develops in animals once they arerefed (Wilson and Osbourn, 1960).Wilson and Osbourn (1960) state that this ability of animals todisplay compensatory growth is a constant feature among 'higheranimals' and that high compensatory growth rates may result inlarger than normal animals being produced. Too severe a restrictionin diet, however, was found to result in a permanent reduction in2growth. Also food restriction in the earlier stages of growth wasmore detrimental to the animal than if starved at a more maturestage and consequently the ability of the animal to recover oncerefed was also reduced (Wilson and Osbourn, 1960).Wilson and Osbourn (1960) concluded that several factorscontrol the degree of compensatory growth. These factors are thecomposition of the restricted diet, the degree of severity andduration of starvation, the rate and level of maturity of the animal atthe time of starvation, and the refeeding pattern used. They foundthat the degree of ultimate recovery and diet restriction wereinversely related, but that the extent of compensatory growthobserved once the animal was refed increased the more severe thefood restriction was. They state that the ability of the animal toincrease its food conversion efficiency during the early stages ofrefeeding is important in the recovery of previously starved animals.Because an animal of equal size and weight to animals with aconstant feed source can be produced with significantly less feed, theC.G.R. is an important economic factor in livestock production. Feedcosts are a major component of the production cost in theaquaculture industry as well, and fisheries scientists are constantlylooking for improved optimal harvest strategies. Fish haverepeatedly been documented to exhibit compensatory growth (Biltonand Robins, 1973; Weatherly and Gill, 1981; Dobson and Holmes,1984; Miglays and Jobling, 1989a, b; Quinton and Blake, 1990;Farbridge, Flett, and Leatherland, 1991).The mechanism of compensatory growth is not wellunderstood. This study was initiated to gain further insight into this3economically important phenomenon. To accomplish this, rainbowtrout (Oncorhynchus mykiss) were cyclically fed to elicit a C.G.R.Changes in growth, carcass composition, and plasma growth hormonelevels associated with the response were recorded and possiblemechanisms underlying the C.G.R. are discussed.4CHAPTER 2COMPENSATORY GROWTH IN RAINBOW TROUT,ONCORHYNCHUS MYKISSINTRODUCTIONCompensatory growth in fish was first documented by Biltonand Robbins (1973) for sockeye salmon fry. They found that fishthat were treated with short term starvation of up to three weekscaught up in length and weight to the control group when refed.Periods of starvation longer than three weeks however, resulted inpermanent stunting and/or death. These findings are consistent withWilson and Osbourn's (1960) observation that prolonged starvationin mammals reduced the ability of the animal to recover.Weatherly and Gill (1981) investigated the C.G.R. in fingerlingrainbow trout (Oncorhynchus mykiss). They observed that whenthese fish were placed on restricted rations of 3% body weight (B.W.)/day, or starved for 3 week and 13 week intervals, they recoveredand produced a growth rate almost equal to that of the control groupwith respect to wet body weight and condition factor when refed tosatiation. The compensating fish had a higher percent dry weightthan the controls. They also reported that results from the fishmaintained on a restricted ration resembled more closely the prolongstarvation treatment group rather than the short term (3 week)treatment group.5Dobson and Holmes (1984) found similar results for farmedrainbow trout. After several periods of starvation and refeedingthey observed significant increases in weight gain and overall lengthin the experimental fish as compared to the control group that wasfed constantly. Due to the increase in overall length, the increase inweight can be considered growth and not just gut fat deposits orwater uptake.Quinton and Blake (1990) repeated the Dobson and Holmes'experiment and determined the effect of different feeding cycles andration levels on the compensatory growth response in rainbow trout.A feeding cycle was defined as the period from the onset ofstarvation to the end of the following feeding period. The controlgroup was fed constantly at a ration level of 5% body weight(B.W.)/day. The experimental groups were fed at 3% and 7%(B.W.)/day respectively and at 1, 2, or 3 week feeding cycles. Thelength, weight, length to weight relationship or general fitness of thefish (condition factor), and ratio of food intake to weight gain (feedconversion efficiency) were recorded and compared between thegroups. They found that there was no significant difference betweenthe length, weight and condition factor of fish on a 3 week starvationand 3 week refeeding cycle and those fish fed daily (controls). Thecyclically fed fish were also found to have a higher feed conversionefficiency.A second experiment compared length, weight, condition factorand feed conversion efficiency of a constantly fed control group withan experimental group on a 3 week feeding cycle at the same rationlevel of 5% B.W./day. These experiments indicated that the most6productive feeding cycle and ration level was a combination of a 3week feeding cycle with a 3% B.W./day ration level.Farbridge, Flett, and Leatherland (1991) fed rainbow trout tosatiation 5 days, 3 days, or 1 day a week for 6 weeks and then refedto satiety 5 days a week for 8 weeks. They observed a C.G.R, butonly in the fish fed 1 day a week. Therefore, a period of starvation isrequired before a C.G.R. becomes apparent. In this experimentincrease in growth was associated with an increase in foodconsumption. The C.G.R observed by Quinton and Blake (1990) wasnot associated with an increase in food consumption as the fish wereon restricted refeeding rations.The methods to be described for the following experiment arebased on the "preferred" feeding cycle of Quinton and Blake (1990).METHODS AND MATERIALExperimental AnimalsUnderyearling rainbow trout (Oncorhynchus mykiss), weighingapproximately 60-80 grams were purchased from Sun Valley TroutFarm in Mission, B.C. and delivered to the laboratory at the SouthCampus Research Facility of the University of British Columbia. Theywere held in circular fiberglass tanks 4 feet deep and 8 feet indiameter with water supplied through aerator bars. The water wassupplied from the City of Vancouver water system. Particulates andchlorine were removed by using two Triton Model TR-140 activatedcharcoal filters (capacity, 140 gallons/minute). A thiosulfate7injection system using a Mec-o-matic Powermatic II continuousinjection pump was used daily to further reduce chlorine levels. Thewater temperature fluctuated between 7-10 °C. A 12:12 light/darkphotoperiod was maintained using fluorescent lights. The fish werefed a commercial dry food pellet manufactured by the Moore- ClarkFeed Company. The layout of the experimental facility is shown inFig. 1.Experimental DesignThis experiment ran from October 27, 1989 to April 13, 1990to include 4 feeding cycles. A feeding cycle was defined as 3 weeksof starvation followed by 3 weeks of refeeding. Before theexperiment began the fish were allowed to acclimate for 14 days.The fish to be sampled were then tagged and allowed to acclimate foranother 7 days to eliminate any sick or dying fish from theexperiment. The details of the experimental procedure follow.Preparation of Experimental GroupsDuring the 2 week acclimation period the fish were fed tosatiation once a day and then starved for 2 days prior to the taggingprocedure. The fish that were to be sampled during the experiment(1,050 fish) were randomly chosen from the population. These fishwere individually netted from the holding tanks and placed three ata time in a dark, well-aerated anesthetic tank containing 0.4 ml/liter2-phenoxy-ethanol. Once anesthetized, the fish were removed from8Figure 1: The layout of the experimental facility.Legend^A: Hot water supplyB: Cold water supplyC: Air CompressorD: Carbon mechanical filtration systemE: Thiosulphate injection systemF: Air lineG: Water lineH: Experimental tanksI: Water supply valvesJ: Air supply valvesK: Tank drainsL: Gutter91 0the anesthetic tank and tagged with a 1" long number coded anchortag (Floy Tag Co., Seattle, Washington, U.S.A.). These fish (1,020)would be recognized as part of the terminal sample population thatwould be analyzed for plasma growth hormone levels and carcasscomposition as well as growth. Thirty fish were tagged withindividually numbered fingerling tags that were sutured through thedorsal muscle just posterior to the dorsal fin. These fish were part ofa population that would be monitored for changes in growth only.The standard length (±0.5 cm), weight (Mettler Balance P1200 ± 0.01grams), and tag number were recorded for each fish. The tagged fishwere then divided into the following groups; Group A-experimental,Group B- control 1, and Group C- control 2. The fish tagged withfingerling tags will be referred to as Group A', Group B' and Group C'and are subgroups of the main population.The remainder of the fish were divided equally into ReserveTanks A, B, and C to correspond with the experimental groups. Thesefish were used to replace terminally sampled fish during theexperiment such that the holding density was kept constant.Experimental GroupsEach experimental group was divided between two tanks with170 terminal tagged fish and 5 monitored tagged fish per tank. Theration level was calculated as a percentage of the total body weightof the fish in an experimental group and expressed in percent bodyweight per day (% B.W./day). Group A and Reserve Tank A were fedat 3% B.W./day and maintained on a 3 week cyclic feeding regime.Group B and Reserve Tank B fish were fed daily at 3% B.W./day and11Group C and Reserve Tank C fish were fed daily to satiation. Fig. 2shows the distribution and environmental conditions of each group offish.Sampling ProcedureAll fish were sampled weekly and starved two days prior tosampling to empty the gut. Ten fish (5 per tank) from each groupwere individually netted and placed in an anesthetic tank containinga lethal dose of 2-phenoxy-ethanol. When near death the fish wasremoved, identified by the tag number and its weight and standardlength recorded. Approximately 3 c.c.s of blood was collected fromthe caudal artery using a heparinized syringe.^The fish was thenfrozen (dry ice), placed in a sealed bag and stored in a freezer forcarcass composition analysis. The blood samples were centrifugedand the plasma collected and stored in plastic screw-cap vials at-20 °C.Five fish from groups A', B', and C' were sampled on the sameday as the terminal sampling. The tank that these five fish camefrom alternated weekly (Fig. 2). These fish were identified by thefingerling tag, lightly anesthetized, and the tag number, weight, andlength were recorded before being returned to the appropriate tank.A fish from the corresponding reserve tank was weighed andthen added to the sampling tank to replace each fish that wasremoved for terminal sampling. The weight of the substitute fishwas recorded so that the appropriate ration level was maintained.The water temperature of each tank was recorded on a weekly basisand the mortalities for each group recorded daily.12Figure 2: The experimental layout and conditions for the three experimental Groups A,B, and C. The reserve tanks are maintained on the same feeding regime and at the sameration level as the experimental group they will be placed with during the samplingprocedure.136 tanks^12L/1 2D1 75 fish/tank^photoperiodGROUP ARation- 3% B.W./dayFed on a 3-week feeding cycleRESERVE TANKSR.T.A^R.T.C.R.T.BGROUP BRation- 3% B.W./dayFed dailyGROUP CRation-satiationFed daily46Experimental Design14Data AnalysisThe mean weight and length were calculated for both themonitored and terminal sample population of each group. The meanspecific growth rate (% growth/day) and condition factor (K) werecalculated using the terminal sample data.The condition factor was calculated using Ricker's formula(Ricker, 1975), which provides the best index when lengths varybetween groups being compared:K = 100 W/Lwhere K is condition factor, W is weight (g), and L is length (cm).The specific growth rates (S.G.R.) were calculated betwensample periods as follows:S.G.R. = 1nS2 - 1nS1 x 100 x d-1where In is the natural logarithm, Si and S2 are the weights at time1 and 2, and d-1 is the number of days between time 1 and 2 (Clarkeand Shelbourn, 1986). A Kruskal-Wallis analysis of variance test wasused to test the significance of the results. The null hypothesis wasrejected with a significance level of alpha less then or equal to 0.05(Zar, 1984).RESULTSThe results of this experiment indicate that a C.G.R. did occur inthe fish that were placed on a cyclical feeding regime (Figs. 3, 4, 5, 6,7, Appendix 1, 2). The average mean weight and length of the fishfrom Groups A, B, and C during the 24 week experiment are shown inFigs. 3 and 5 respectively. The average mean weight of Groups A', B'and C' are shown in Fig. 4 and both the mean average weight and15length of these groups are shown in Appendix 2. A Kruskal-Wallisanalysis of variance on the Groups A, B, and C data shows nosignificant difference in the final weight of fish fed cyclically andthose placed on a daily feeding regime for three of the four feedingcycles. Group C fish that were fed to satiation daily weresignificantly larger at the end of the fourth feeding cycle than theGroup A and B fish which were fed restricted rations. The cyclicfeeding regime resulted in a significant increased variability inweight within the Group A population (Appendix 1). The pattern ofweight loss and gain in Group A shows that the greatest weight lossoccurs during the first week of the starvation portion of the feedingcycle. This initial weight loss, however, significantly decreasesduring the subsequent feeding cycles (Figs. 8, 9).There was no significant difference between the final length ofthe fish from all three groups. The condition factor of the threegroups of fish shows no significant difference at the end of all thefeeding cycles except for cycle four. Again, during this cycle theGroup C fish show a higher average weight and thus higher conditionfactor (Fig. 7).The rate of weight loss and gain is shown in Fig. 6. The S.G.R ofthe cyclically fed fish shows that the fish lose weight most quicklyduring the first week of starvation and the response decreases withsubsequent feed cycles. There was a moderate increase in S.G.R.during the first week of refeeding that significantly increased withthe number of feeding cycles (Fig. 10). The highest S.G.R. wasrecorded in the third week of refeeding in the cyclically fed fish.16Figure 3: The mean weight (g) of Groups A, B, and C for weeks 0-24. The letter Ssignifies the begining of the starvation phase of the feeding cycle and letter F therefeeding phase. Vertical lines represent the S.D. from the mean.1 7Group AGroup BGroup C..........■■Ca...........w.....■......■......■..er....12...■■■•3 6SF0S242118S15FF9^12F SWeek st00Figure 4: The mean weight (g) of Groups A', B', and C' for weeks 0-24. The letter Ssignifies the beginning of the starvation phase of the feeding cycle and the letter F therefeeding phase. Vertical lines represent the S.D. from the mean.19—a—Group A'Group B'Group C'1518^21^24Figure 5: The mean length (cm) of Groups A, B, and C for weeks 0-24. Vertical linesrepresent the S.D. from the mean.211 8 2 1 24-s- GROUP A----•-- GROUP B----°-- GROUP CWeek *Figure 6: The mean specific growth rate (%/day) for Groups A, B, and C for weeks 0-24. Vertical lines represent the S.D. from the mean.230t—a--- Group A--•-- Group B—a-- GrOUD C>-:ca:/.3.e......a)7:2.c4io;En067.._C)a)0.C')cCoa)2.1.CAFigure 7: The mean condition factor for Groups A, B, and C for weeks 0-24. There isno significant difference (P s , .05) between Groups A, B, and C at the end of feed cycles1, 2, and 3 (weeks 6, 12, 18, and 24).251.81.62'8 1 4.1 .Cuc073-C00 1.2C03a)21.0--ci--- Group A--*--- Group B--o— Group C0.80 96 12Week #24211815Figure 8: The greatest mean weight loss (g) of Group A for all four feed cycles. A feedcycle includes 3 weeks of starvation followed by 3 weeks of refeeding. The equation ofthe line is included in the figure. There is a significant (P< .05) decrease in the amountof weight lost per cycle.27y = 21.955 - 1.7490x RA2 = 0.98202120193)N(t)2 18.5a)._a)3C^17asa)E_,ccow 16CL1514Figure 9: The greatest mean weight loss (g) for Group A' for all four feed cycles. A feedcycle includes 3 weeks of starvation followed by 3 weeks of refeeding. The equation ofthe line is included in the figure. There is a significant (P< .05) decease in the amount ofweight lost per cycle.291816o)..-•U,(r)04-,.a^14a)a)3ccoa)E..)4coaa)^121013^ y = 19.845 - 2.1230x IR^2 = 0.9711110,0^ 1^ 2 5Feed cycleFigure 10: The S.G.R. of Group A during week 1 of the refeeding phase (weeks 4, 10,16, and 22) for the four feed cycles. The equation of the line is included in the figure.There is a significant (P< .05) increase in the rate of growth during this week per cycle.3112Feed cycleI -I-3>,CZ73^0, 0a- as_c(D CD--'^0CO 0)1.- .s"C 0a) 0o —L._ a)cy) %-.0 0.—^0a) ...Q. 0u) 1:1) 0Cas z5;a) .....2 0There was a tendency for the peak S.G.R. in Group A to decrease withthe number of feeding cycles (Fig. 11). One-way analysis of variance,however, show P. 0.103.The feeding behavior of the fish in all three groups wasobserved. The fish in Group A were lethargic in the first feedingcycle of the experiment. They showed little interest in food when itwas initially re-introduced and often did not consume the entireration available on the first week of refeeding. The feeding responseincreased with the subsequent refeeding week. This behaviorcontinued throughout the second feed cycle but changed during thethird feed cycle. At this time, the fish were less lethargic during thestarvation phase and showed a good feeding response on the firstweek of the refeeding phase.Mortality data from each group during the experiment aregiven in Table 1. During week 19 and 20 there was a significantincrease in the mortalities recorded in all groups, but mostsignificantly in the cyclically fed fish. The majority of the mortalitiesin all groups including the reserve populations occurred on March7th, with 25 mortalities from Group A, 5 from Group B, and 3 fromGroup C. There is a decrease in S.G.R. in the control groups duringthis time. Week 19 corresponds to the first week of starvation in thecyclically fed fish and a temporary malfunction of the sodiumthiosulfate pump on March 6th.Fluctuations in water temperature during the experiment areshown in Fig. 12. Lower water temperatures did not restrict theability of the cyclically fed fish to elicit a C.G.R. The fish fed tosatiation (Group C) consumed between 2-4% B.W./day depending on33Figure 11: The greatest mean S.G.R. of Group A (week 3 of the refeeding phase).for allfour feed cycles. A feed cycle includes 3 weeks of starvation followed by 3 weeks ofrefeeding. The equation of the line is included in the figure (P = .103).34y = 1.5350 - 0.14800x 13^2 = 0.8050rn00.-^1.2a)0cnCuca)E- 1.1caa)CLFeed cycleFigure 12: The fluctuation in water temperature during the 24 week experiment(October 27-April 13).36a)a)4-J4-,986 9 2eel<8Table 1 : The number of mortalities for each group of fish during theexperiment (October 27, 1989 - April 13, 1990).^The numberrecorded is the total number of mortalities for that sample week.The abbreviation R.T. A, B, and C indicates the reserve tank for theappropriate group of fish.38DATE WEEK GROUPAGROUPBGROUPCR.T.A R.T.B. R.T.C.Oct. 27 0 1 0 0 1 0 0Nov.^3 1 0 0 0 0 0 010 2 2 1 0 0 0 017 3 0 0 0 0 2 024 4 0 0 0 1 0 0Dec.^1 5 0 0 0 0 0 18 6 0 0 1 0 015 7 0 0 0 0 0 022 8 0 0 2 0 0 029 9 2 0 0 0 1 0Jan.^5 10 0 1 0 2 0 012 11 0 0 1 0 0 019 12 0 0 0 0 0 026 13 0 0 0 0 0 0Feb.^2 14 0 0 1 0 0 29 15 1 0 0 0 0 016 16 0 0 0 0 0 023 17 0 1 0 1 0 0Mar.^2 1 8 1 0 0 0 1 09 19 28 6 4 19 3 716 20 4 2 3 3 5 223 21 2 0 0 0 0 030 22 0 1 0 0 0 1April 6 2 3 0 0 1 0 0 013 24 0 0 0 0 0 0TOTAL 40 10 13 27 12 1339Table 2: Body and gonad weight of individual fish from each experimentalgroup at week 24. The letter M or F indicates the sex of the fish (M-male;F-female). An * indicates precocious maturation.40GROUP A GROUP B GROUP CFishWe ig ht(g)GonadWeight(g)FishWe ig ht(g)GonadWeight(g)FishWeig ht(g)GonadWeight(g)83.79 0.15 F 99.53 0.02 F 227.36 0.46 M164.13 *2.88 M 144.13 *4.15 M 179.83 0.21 F168.04 0.28 M 125.15 0.19 F 196.81 0.31 M151.35 0.29 M 180.22 0.29 M 238.30 0.44 M174.34 0.21 M 185.34 0.33 M 135.63 0.17 F82.19 0.13 F 183.25 0.32 M 186.01 0.28 M123.04 0.18 F 136.44 0.28 M 178.33 0.24 F187.32 0.30 M 196.81 0.20 F 224.30 0.67 M161.40 *3.01^M 167.18 0.30 M 213.95 0.07 F195.67 0.41 M 208.33 0.36 M 167.44 *2.23 M41the water temperature. At water temperatures of 9-10 °C consuming3-4% B.W./day while consuming 1-2% B.W./day at the lower watertemperatures of 7-8 ° C. Because Group C fish consumed up to 4%B.W/day the feed conversion efficiency was not calculated as fish inGroups A and B were on a set ration level of 3% B.W./day and thuswere unable to vary their food intake. Therefore any changes infeed conversion efficiency between the groups could not beaccurately calculated.The sexual maturity of the fish from week 24 showed thatsome precocious maturation occurred (Table 2 ). Group A had 2%and Groups B and C had 1% precocious maturation. All the fishexhibiting this early maturation were male.DISCUSSIONIn this study, growth rates of fish that had been on a cyclicfeeding regime were significantly higher than those fed daily. This isattributed to the C.G.R. Because the "compensating fish" increased inlength as well as weight and there was no significant difference inthe condition factor between the three groups for three of the fourfeeding cycles, 'real' growth was occurring (Weatherly and Gill,1987). The increase in condition factor approximating that of thecontrol group is consistent with the trend of 'conservatism' in bodyproportions observed by Weatherly and Gill (1982) in rainbow trout.The results from Groups A', B', and C' (Figs. 4, 9, Appendix 2)indicate that fish that were sampled and then returned to thepopulation to continue to grow throughout the experiment showed42similar growth patterns to the fish that were sampled once.Therefore, the sample of the population used to provide theinformation on carcass composition and plasma growth hormonelevels was a valid representative of growth in that populationUnfortunately, only a portion of Groups A', B', and C', was sampledper week, therefore there may be a bias in the measurement of thegrowth pattern of these fish as some of them were sampled moreoften then others.The cyclically fed fish were able to attain weight similar tothose fed daily in all the feeding cycles except the last. Duringfeeding cycle four, the sodium thiosulfate pump failed allowing highlevels of chlorine to enter the water system. Large numbers ofmortalities were recorded in the cyclically fed fish that werebeginning the starvation phase of the feeding cycle. Watertemperature during this cycle was higher than previous weeks whichprobably intensified the problem by increasing the metabolic rate ofthe fish.Quinton and Blake (1990) suggested that chlorine poisoninglead to the inability of the cyclically fed fish to achieve growth ratecomparable to the controls. This could be the reason that thecyclically fed fish in this study were unable to equal the final weightof control Group C. Cyclic feeding therefore significantly increases therisk of mortality due to poor water quality.The pattern of weight loss and gain in the cyclically fed fish issimilar to that recorded by Dobson and Holmes (1984) and Quintonand Blake (1990). The greatest weight loss occurred during theinitial week of starvation. This large initial loss is probably due to43dealimentation (Elliott, 1972; Quinton and Blake, 1990). The greatestincrease in growth occurred during the third week of refeeding.Quinton and Blake (1990) propose that this is due to changes inprotein turnover rates. They suggest that a reduction in proteinmetabolism may be a physiological mechanism underlying the C.G.R.This view is based on the changes in the metabolic rate of muscletissues that occurs shortly after starvation (Loughna and Goldspink,1984). In particular, during starvation the basal metabolic rate andactivity levels drops and both protein degradation and synthesis(protein turnover rate) is decreased (Love, 1970; Smith, 1981;Loughna and Goldspink 1984). This may explain the generallethargy and feeding behavior of the fish during the initiation of thestarvation phase of the feeding cycle.Quinton and Blake (1990) suggest that if the proteindegradation levels remain low but protein synthesis increases duringrefeeding more protein could be laid down and thus could accountfor the high S.G.R. This is consistent with the observation that amoderate period of starvation is required for a C.G.R. to occur.Brown and rainbow trout adapt their basal metabolic rate tomaintenance food rations so that if the amount of food available isreduced the fish first lose weight but then become "adapted" to thenew level of feeding so that they gain weight (Brown, 1946; Dobsonand Holmes, 1984). The results of this experiment also indicate thatthe fish are adapting to the feeding regime. There was a significantdecrease in the percent of weight loss during starvation phases of thefeeding cycles and a significant increase in the feeding response andgrowth rate of the fish during the first week of refeeding. This tends44to indicate that the fish were acclimating to the restricted rations. Nosignificance was found in the tendency for the S.G.R. to decrease withsubsequent refeeding cycles. This was due to a lower S.G.R. for Cycle3 than Cycle 4. There was however, a problem with water qualityduring the beginning of Cycle 4 that may have also effected the finalrefeeding week of Cycle 3. The peak S.G.R. recorded in Cycle 3 maytherefore be artificially low and there may be a real' decrease ingrowth response to subsequent feeding cycles. If this is the case,repetitive cycles may eventually reduce the strength of the C.G.R. asthe strength of the response to restricted rations declines. The properfeeding cycle may therefore have to be adjusted as the fishacclimate.The restricted ration level used in this experiment did notallow for the calculation of a feed conversion efficiency value. Duringthe initial week of refeeding in the cyclically fed group the rationlevel offered to the fish was not fully consumed. The control groupfed to satiation daily also consumed a higher ration level (4%B.W./day) than the set level for the other groups (3% B.W./day). Thepractise of feeding the fish once a day may have had a negativeeffect on the growth rate in all the experimental groups. Feedingonly once a day results in a decrease of assimilation efficiency duringdigestion (Jobling, 1981) while feeding spread throughout the dayincreases feed conversion efficiency (Wurtsbaugh and Davis, 1977).45CHAPTER 3CARCASS COMPOSITIONINTRODUCTIONMost fish experience periods of starvation at some point duringtheir lives. The degree of starvation may vary from moderate(wintering) to severe (spawning). They have therefore become veryadept at using different body constituents as fuel for survival duringperiods of prolonged food shortage (Love,1970).^The four mainconstituents are water, lipid, protein, and ash (mineral).^Insalmonids the live body weight is generally made up of water (70-80%), lipid (2-12%), protein (15-20%) and ash (2-3%) (Love, 1970and Bakshish Dosanjh, personal communication).The total percentage water content of all animals tends toincrease with periods of starvation (Love, 1970). However, there isa strong correlation between water content and lipid levels in fish.Generally water content shows an inverse relationship with lipid inthe muscle of "fatty" fish (Love, 1970; Weatherly and Gill, 1987).Therefore a steady decline in lipid levels characterizes fooddeprivation in most cases. This relationship can be explained by themobilization of lipids as an energy source during the onset ofstarvation. It is likely that visceral adipose and muscle lipids areused for fuel in early starvation (Weatherly and Gi11,1984; Quintonand Blake, 1990) and that the muscle lipid is proportionally replacedwith water (Parker and Vanstone, 1966; Quinton and Blake, 1990).46The transport of these lipids is seen in the free fatty acidcontent of the blood which rises during periods of starvation (Love,1970). Which of the lipid stores available to fish are used first wasexamined by Jezoerska et al (1982). They found that in rainbowtrout the visceral fat deposits are a very important energy sourceduring early starvation and that saturated fatty acids arepreferentially mobilized from there even though they are present inhigher percentages in muscle and liver tissue.Protein tends to vary little in healthy fish and is utilized onlyduring prolonged periods of food deprivation. Long-term starvationcauses a decrease in overall intestinal size (Love, 1970). Whitemuscle tissue is then used as an energy source (Johnson andGoldspink, 1973). Significant protein utilization in rainbow troutdoes not occur until 7 or 8 weeks of starvation (Elliot, 1976;Weatherly and Gill, 1984; Quinton and Blake, 1990). Therefore, withmoderate starvation and lipid loss the body weight of the fish ismaintained through an increase in water content (Love, 1970).Ash or mineral levels tend to vary with water and proteinlevels in fish (Love, 1970). This is logical as muscle depends on aproper balance of water, protein, and ionic components to functionnormally and the proper functioning of muscle tissue is imperative tothe survival of the fish.The effects of starvation on carcass composition are thereforequite well understood. The effects of starving and refeeding, orrecovery growth, of agricultural animals has been studied bystudying compensatory growth. The results of these studies arehowever inconsistent.^Wilson and Osbourne (1960) reviewed some47of these studies and found an increase in fat content in animals thathad shown compensatory growth while other authors studying sheepreport an increase in total protein content (McManus et al., 1972).The way in which a fish resumes growth after subsequentperiods of starvation and refeeding is not well documented. It hasbeen suggested however, that once irreversible gut atrophy occursdue to prolonged starvation, a fish is no longer able to utilize feedproperly and thus is unable to recover the growth lost (Bilton andRobins, 1973).Weatherly and Gill (1982) have remarked on the"conservativeness" of proportional body weights independent of theoverall somatic growth rate of a fish. This means that theproportional tissue weights will not change greatly with a fishincreasing in size. Therefore, when a fish is moderately starved thevarious tissues will proportionally be reduced. In this way the fishcan function normally until prolonged starvation producesirreversible damage. They found that, as stated earlier, visceral fatis the first tissue utilized during starvation but that the gut will alsoshrink more than the other body tissues. If starvation is notprolonged however, the fish will recover not only in somatic growthbut in the relative growth of their tissues as well (Weatherly and Gill,1987).Quinton and Blake (1990) elicited a C.G.R. in rainbow trout andfound after 3 weeks of starvation an increase in water and proteinand a decrease in lipid content. After 3 weeks of refeeding the testanimals were found to be similar in body composition to the controlfish that had not been previously starved.48Farbridge, Flett, and Leatherland (1991) looked at the effect ofrestricted ration and the refeeding on the body composition ofrainbow trout as well. They found a reduction in visceral adiposetissue and carcass and liver lipid content, and an increase in carcasswater content in fish put on the restricted rations. By the end of therefeeding period there were no differences between theexperimental and control groups.It can therefore be assumed that compensatory growth is 'real'growth and not just an increase in water content. How this growth isdivided amongst the body constituents is not well understood. Thissection of the study focuses on how the changes in growth associatedwith a C.G.R corresponds to changes in the body composition of thefish.METHODS AND MATERIALSPreparation of SampleThe following steps were repeated for each fish that was to beanalyzed. The identification number of the fish was recorded. Four1 1/2" sections were cut (electric band-saw) from the frozen fishsample to be used for chemical analysis. Section one was removedfrom directly in front of the orbital, section two from behind theoperculum, section three included the last 1/3 of the dorsal fin andsection four was removed from behind the caudal peduncle andincluded portions of the tail fin (Fig. 13). These sections areconsidered a representation of the whole fish (Analysis of49Figure 13: The four sections chosen to represent the whole fish for carcass compositionanalysis.50dorsal finlateral line^ adipose fin caudal finnostrilsgill cover(operculum) pectoral finpelvic fincaudal peduncleanal fin51Agricultural Materials, 1973).^The four frozen sections were thenchopped into smaller pieces and placed in a blender. The tissue wasblended into a homogeneous mixture which was then placed in alabeled container to be used for chemical analysis to determine thepercent protein, lipid, ash, and moisture of each fish.Percent ProteinThe percent protein of each fish was based on nitrogendetermination using a colorimetric method (Technicon AutoAnalyzerII). The advantages of using this system were that large numbers ofsamples could be analyzed and because the samples and standardswere treated equally there was less possibility of there being errorbetween the samples. The amount of sample used per test was 0.5grams. The procedure for this analysis is described in the TechniconAutoAnalyzer II manual-Industrial Method No. 334-74W/B. Thenitrogen levels produced from this analysis were then multiplied bya factor of 2.3436 to give the percent protein in these tissue samples.Percent tissue protein levels are calculated from :% Protein= Corrected nitrogen reading x 2.3436weight of sample (mg)52Percent LipidTotal lipid content was determined using a modification of theBligh and Dyer (1959) extraction method described in Herbes andAllen, (1983) using 5.0 grams of the homogeneous mixture. Lipidcontent of the sample was calculated as:% Lipid= weight of oil in 5m1 chloroform layer x chloroform volume x100^5^ weight of samplePercent AshThe percent of ash in each sample was determined by the dryash method described by Chapman and Pratt (1961). The results ofigniting a 2 gram sample were then used in the following equation todetermine total ash content of the sample.% Ash= ^weight of ash ^x 100sample dry weightPercent MoistureTotal moisture content of the sample was determined using 5.0grams of the sample and the procedures outlined in the Analysis ofAgricultural Materials (1973). The results of this test were used inthe following equation.% Moisture=100 - (dry dish +dry sample weight) - dry dish weight x100original sample weight53RESULTSThe results of the proximate analysis for Groups A, B, and C areshown in Figs. 14, 15, 16, 17, Appendix 3. The results of Groups A',B', and C' at week 24 are shown in Table 3. The significance of theseresults was analyzed using the Kruskal-Wallis analysis of variance.For each feeding cycle there was a significant increase in moisture(Fig. 14) and a decrease in the fat (Fig. 15) content of the Group Afish after 3 weeks of starvation. By the third week of refeedingthere was no significant difference in the water content of the fish,but the cyclically fed fish did maintain a slightly higher proteincontent then the other groups. The lipid (Fig. 15) content in thecyclically fed fish remained significantly lower than the fish fed dailythrough out the experiment. The fish in Group C, which were fed tosatiation daily, had the highest fat content and lowest protein level ofthe three groups by the end of the experiment. The results of theproximate analysis at the end of the experiment on Groups A', B', andC' revealed no significant difference in carcass composition fromGroups A, B, and C. (Table 3). There was no increase in variabilitywithin the population of Group A as the experiment progressed(Appendix 3) as was observed in the weight, length data.The changes in carcass compositon due to the phases ofstarvation and refeeding were gradual in Group A. It was observedthat in feed cycle 3 and 4, the moisture levels were not as high andone-way analysis of variance showed a significant (P. 0.05) decreasein the amount of lipid lost after the starvation phase of the feed cycle(Fig. 15).54Figure 14: The mean moisture level of fish in Groups A, B, and C for weeks 0-24.The vertical lines represent the S.D. from the mean.55Group AGroup BGroup CWeek Figure 15: The mean lipid level of fish in Groups A, B, and C for weeks 0-24.The vertical lines represent the S. D. from the mean.57242 1181512Week 4t9630Group A—4-- Group B—II-- Group C5Figure 16: The mean protein level of fish in Groups A, B, and C for weeks 0-24.The vertical lines represent the S. D. from the mean.59,-,V.^1 6.5a)>a)c 1 6.3,...a)4-,0Lfa^16.1cpaa)E—e,^Group A--•-- Group B—II-- Group CFigure 17: The mean ash level of fish in Groups A, B, and C for weeks 0-24.The vertical lines represent the S. D. from the mean.6 1Group AGroup BGroup C■•■•■•■14,1■•■12Week6^90^315^18^21^242.8 —a.).cfVCeaCUTable 3. The carcass composition of Groups A', B', and C' at week 24.There is no significant difference (ft .05) between the values fromthis group and the main population at this time.63WEEK 24GROUP (Y0MOISTURE % LIPID % PROTEIN %ASHA' 71.52 9.97 16.51 2.38B' 71.42 10.62 16.14 2.41C' 70.63 10.81 16.13 2.3464DISCUSSIONThe fish maintained on a cyclical feeding regime exhibited aconsistent pattern of tissue weight loss and gain. The initial responseto starvation was an increase in moisture level and a correspondingdecrease in fat content. This pattern reflects the preferential use ofvisceral fat deposits and muscle lipids (Parker and Vanstone, 1966;Smith, 1981; Weatherly and Gill, 1981) with the subsequentreplacement of water.Comparing the changes in moisture and lipid levels during afeeding cycle with the changes in weight and growth reveals someinteresting patterns. Although the greatest percentage weight lossoccurs in the first week of the starvation phase, there is no largedecrease in fat content during that week. There is no large decreasein moisture, protein or ash levels either. This initial weight loss istherefore associated with the emptying of the gut. The fish continuesto lose weight at a much lower rate during the second week as thefat stores are progressively depleted. By the third week ofstarvation the fish appear to stop losing weight and there is a smallincrease in the S.G.R. This increase correlates with low lipid levelsand high moisture levels and is due to an increase in water contentas fat is mobilized as an energy source. Protein levels do not appearto cycle with the feeding cycle and therefore it is not likely used asan energy source during the starvation period.The fat content of the cyclically fed fish was significantly lowerthan the control groups throughout the experiment.^These results65differ from observations by Farbridge, Flett, and Leatherland (1991)where no significant differences were found in carcass compositionbetween fish fed on a restricted ration and those on a cyclic feedingregime after 98 days. The restricted feeding regime used in theFarbridge, Flett, and Leatherland (1991) study included feeding thefish to satiation 1 day per week as compared to this study thatstarved the fish for 3 consecutive weeks. The difference in restrictedfeeding regimes probably explains the different results. Quinton andBlake (1990) used the same protocol as that used in this experimentand found no significant difference in carcass composition betweenthe control group and cyclically fed fish after one feeding cycle, butmention that there was a tendency for the cyclically fed fish to be'leaner'. This experiment confirms this tendency.The cyclically fed fish had a significantly higher protein levelafter refeeding than the control groups and by the fourth feedingcycle maintained consistently higher protein levels. This increase inprotein content in the cyclically fed fish support the view of Quintonand Blake (1990) that the protein degradation rate reduced inresponse to starvation remains depressed during refeeding while theprotein synthesis rate returns to normal. In rainbow trout rapidgrowth is normally achieved through the recruitment of new smallmuscle fibre, as opposed to an increase in the diameter of theexisting ones (Weatherly et al., 1979; Weatherly and Gill, 1984). Thismay account for the increase in protein levels.Once again the data reflected the ability of the fish to acclimateto restricted rations. The amount of fat lost during the starvationphase of the feeding cycle gradually declined as the experiment66progressed so that less of the fat gained during the refeeding phasewas used as a energy source during feed restriction. Subsequently,lower moisture levels were recorded as well. Protein was not usedas a substitute, therefore, the fish must be acclimating to the feedrestriction. The cyclically fed fish appear to adjust their bodychemistry to the effects of starvation. In this way, once feed is re-introduced, it can be directed towards growth.67CHAPTER 4CHANGES IN PLASMA GROWTH HORMONE LEVELSINTRODUCTIONThe mechanism underlying the C.G.R. is not well understood.Several studies on rats by Mosier (Mosier and Jansons, 1985; Moisieret al, 1985) however, indicate that growth hormone may play a role.Growth hormone or somatotropin (an anabolic protein) isproduced in the alpha cells of the pars distalis of the pituitary glandand plays an important role in regulating growth and metabolism(Weatherly and Gill, 1984; Muller, 1974; Donaldson et al; 1979).Injections of mammalian growth hormone preparations (see reviewby Donaldson et al, 1979) and recombinant piscine growth hormone(Down et al, 1988, 1989) can increase the growth rate in salmonids.Danzmann et al (1990) however, found that under optimal conditionsgrowth hormone did not promote growth in trout.The control of growth hormone in teleosts is not fullyunderstood. The possible role of the hypothalamus in influencinggrowth hormone secretion has been suggested for some species offish (Ball, 1981). Hypophysectomy has been found to stunt growth infish with injections of growth hormone preparations reactivating thegrowth (Ball, 1969; Donaldson et al, 1979; Weatherly and Gill, 1987).Growth hormone may not stimulate growth in fish directly butmay involve a secondary hormonal mediator called somatomedinwhich acts at the cellular level (Donaldson et al, 1979; Weatherly and68Gill, 1987).^According to Muller (1974), somatomedin and insulinhave very similar qualitative effects.Komourdjian et al (1976b) observed that Atlantic salmon whichwere exposed to longer day length had higher growth rates andlower condition factors than those on shorter day lengths. They alsohad an increase in the amount and activity of pituitary somatotrophs.Injecting salmonids with purified mammal growth hormone has beenfound to decrease condition factor (review Donaldson, 1979).It therefore appears that increased growth hormone levelsstimulate a greater increase in length than weight. Weatherly andGill (1983a), when comparing the allometric carcass weight againstbody weight, found little difference in the percentage weightsbetween controls and rainbow trout that had received an injection ofbovine growth hormone. They therefore believe that visceral fatdifferences may account for the differences in condition factor.Physiologically there appear to be three main mechanisms inmammals which trigger hormonal secretion from the pituitary; adecrease in nutrient levels, variations in plasma amino acids, andstress associated with emotional excitement, anaesthesia, andexercise (review by Muller, 1974).In certain fish species both stress and starvation will stimulatethe release of growth hormone (McKeown, Leatherland and John,1975). Sustained exercise has also been found to increase plasmagrowth hormone levels (Barrett and McKeown, 1988,1987). Kayes(1977b) suggests that temperature and seasonal differences mayaffect the ability of growth hormone to stimulate growth. This is notsurprising as in the wild most of fish growth occurs in spring and69summer months with increasing day length, high water temperatureand an increased food supply (Baker and Wigham, 1979).It has been suggested that growth hormone enhances growthby stimulating the appetite of fish and by improving food conversionefficiency (Weatherly and Gill, 1987). Market et a_L (1977) foundthat growth hormone affected the appetite of fish through a directeffect on the control centers in the hypothalamus and by metabolicchanges that feedback to those control centers.The mechanisms by which growth hormone improves foodconversion in fish is not fully understood, however, Market et al.(1977) speculate that there are three possible mechanisms. Firstthere is a greater mobilization of lipid so that more of the ingestedamino acids are available for protein growth as lipid is usedpreferentially as an energy source. This theory is supported bySheridan (1986) who found that growth hormone has the specificeffect of causing the release of non-essential fatty acids (NEFA) fromtissues and thus increasing NEFA levels in the blood. He also foundthat fat is used as an energy source preferentially to bothcarbohydrates and protein when under the influence of growthhormones.Secondly growth hormone stimulates protein synthesis bypromoting amino acid accumulation in tissues.^This is apparentlyhow it mediates growth. Isaksson et al. (1980) found that ratsachieve somatic growth by alternating phases of accelerated andreduced protein synthesis in muscle correlated with surges in plasmagrowth hormone levels.70Finally, directly or indirectly growth hormone may stimulateinsulin synthesis and release (Weatherly and Gill, 1987). Increasedinsulin synthesis and release would result in greater proteinsynthesis and therefore improved protein conversion. In mammalsthere is a recognized relationship in growth regulation betweengrowth hormone and insulin. In mammals growth hormone isconsidered to control muscle growth by nuclear replication whileinsulin controls cytoplasm growth. This situation may exist in fish aswell (Donaldson et al, 1979).Because of the important growth promoting action of growthhormone it is reasonable to assume that it may play a role in thecompensatory growth response. The plasma growth hormone levelsof fish showing compensatory growth were therefore recorded to seeif the hormone levels would cycle with the growth of these fish.METHODS AND MATERIALSFive fish from each group (A,B,C,) were randomly chosen fromweek 24 (termination date of experiment) for gonadal somaticanalysis. The weight of each fish was recorded and then the gonadsexcised and weighed. The sex of the fish was recorded at this time aswell.Plasma AnalysisThe determination of plasma growth hormone levels in thesamples collected from this experiment was done by Dr. J.Leatherland from the Department of Zoology at the University ofGuelph in Ontario. The assay technique this laboratory uses for71salmonid growth hormone determination is a "two-site",noncompetitive enzyyme-linked immunosorbent assay (ELIZA)(Farbridge, 1989). This assay is based on generating monoclonalantibodies (MCAs) that will bind to growth hormone in coho salmonand rainbow trout pituitaries. These MCAs are then used to purifyrainbow trout growth hormone (m.w. 22,500). The sensitivity of thismethod is recorded to be < 1.56 ng/ml and plasma growth hormonelevel in rainbow trout was measured as 35 ng/ml (Farbridge, 1989).The results obtained from this analysis were correlated withchanges in growth to see if plasma growth hormone levels werecycling with growth rates.RESULTSThe results of the plasma growth hormone levels for the firstfeeding cycle are presented in Fig.18 and Table 4. Variance withinthe sample groups was large (Table 4) although the majority of thevalues were within the range (20-80 ng/ml) of plasma growthhormone levels identified by Wagner and McKeown (1986) forrainbow trout. Plasma growth hormone levels in Group A showed noevidence of any compensatory increase or cycling of any kind. AKruskal-Wallis analysis of variance test confirms that there is nosignificant difference between the cyclically fed group and thecontrols.All three groups show a decrease in plasma growth hormonelevel at week 4. This result cannot be explained by feeding behavior72Figure 18: The mean plasma growth hormone levels for Groups A, B, and C for weeks0-6 (1 feed cycle). The vertical lines repesent the S.D. from the mean.734000) 30(r)a)a)!:5 2.c0(.4coG)eeTable 4: Rainbow trout plasma growth hormone (G.H.) assay results. Meanplasma G.H. level, standard deviation (S.D.), range and sample size (n) forGroups A, B, and C during feed cycle 1 (October 27-December 12).75Mean G.H.^S.D.^Range^nGroup A^(ng/mI)Week #0 19.36 6.15 9.49-30.22 91 13.57 6.72 5.60-26.15 102 13.59 9.77 2.90-31.93 83 14.56 6.81 3.94-29.07 104 9.42 3.71 3.00-13.06 105 19.56 10.75 8.05-39.08 86 17.56 5.79 9.01-26.95 11Mean G.H. S.D. Range n(ng/ml)Group BWeek #0 22.96 9.44 11.35-37.59 101 16.61 11.84 8.18-47.35 102 9.69 3.55 6.23-18.71 103 12.30 4.53 7.49-20.35 104 10.57 6.00 5.79-26.71 105 12.65 7.08 4.95-22.53 76 33.41 21.90 12.13-70.47 10Mean G.H. S.D. Range n(ng/ml)Group CWeek #0 18.99 8.07 12.30-37.91 101 18.97 7.11 9.73-28.18 102 18.97 9.80 9.30-42.52 103 15.96 5.00 7.50-29.74 104 9.27 3.30 6.79-17.80 95 14.37 8.60 4.67-30.35 86 19.64 6.40 10.57-29.92 1076or a significant drop in water temperature, and is not reflected in thegrowth data.DISCUSSIONWhile a number of hormones have been implicated ingoverning somatic growth and metabolism, the most significant isgrowth hormone (McLean and Donaldson, 1992). As stated earlier,growth hormone is believed to be a component of the C.G.R. in rats(Moiser and Janson, 1985). Rats achieve somatic growth on a dailybasis through alternating phases of accelerated and reduced proteinsynthesis in muscle tissue correlated with surges in plasma growthhormone levels (Isaksson and Abertsson-Wikland et a1,1980).Plasma growth hormone levels in this study, however, did not showany compensating response to the cyclic feeding regime even thoughthe growth rates of fish exposed to this feeding technique did showcompensation in growth. These findings confirm a report byFarbridge (1989) of compensatory growth occurring without anyassociated compensation in plasma growth hormone levels.Farbridge (1989) found the plasma growth hormone levels in theanimals on the most restricted feeding regime (1 day/week) to havelower plasma growth hormone levels throughout the experimenteven though they showed the greatest C.G.R. response. The resultsfrom this experiment show no significant difference between theplasma growth hormone levels in any of the groups. The differencebetween the findings of the research by Farbridge (1989) and this77study may be due to the less severe starvation phase used byFarbridge.It is suspected that there is an interrelated physiology betweenthyroid hormone and growth hormone (Dondaldson et al, 1979;Leatherland, 1982b) as thyroid hormone potentiates the growth-promoting effect of administered growth hormone (Higgs et a1,1970;Donaldson el a1,1979). Farbridge (1989) found the the plasma L-thyrocine (T4) and triiodo-L-thyronine (T3) levels in fish that hadbeen starved were lower than in fish maintained on a daily feedingregime and once these fish were refed, the plasma T4 and T3 levelsincreased to levels observed in the control groups. However, theyfound no compensatory increases in plasma thyroid hormone levelwith the C.G.R. Farbridge, Flett, and Leatherland (1991) suggest thatperhaps tissue sensitivity increases in the fish fed reduced rations sothat when they are refed, lower levels of T3 produce a greater tissueresponse.Rhythms in growth rate occur naturally (Brown, 1946a; Wagnerand McKeown,1985). A schedule of the lunar calender for the sixmonth experimental term was acquired to determine if biweeklypatterns in plasma growth hormone levels were evident as reportedby Farbridge and Leatherland (1991). Unfortunately, due to theweekly sampling schedule of this experiment the resolution was toolow for this type of analysis. Also Farbridge and Leatherland (1991)associated the increase in plasma growth hormone levels on new andfull moons with increases in food consumption. Two of the threegroups of fish in this study were on restricted rations and thereforewould be unable to increase food consumption levels. The effect of78naturally occurring rhythms in growth rate is a feature of fishgrowth that should be taken into consideration when manipulatingfish growth.79CHAPTER 5CONCLUSIONS AND FISH CULTURE IMPLICATIONSThe results of this experiment confirm that the C.G.R. occurs inrainbow trout and that fish placed on a cyclic feeding regime willcatch up in growth to fish fed daily. The C.G.R. has been shown tooccur even in cooler water temperatures of 7-10 ° C with high specificgrowth rates achieved making it applicable for salmonid grow outfacilities.The mechanism of this response is still not fully understood.This research, though, has provided significant insight into thechanges in body composition during cyclical growth. The initialweight loss during the starvation phase is due to the emptying of thegut. The fish then mobilizes lipid stores for energy, increasing thewater content of the fish. This increase in water content is realizedin an increase in S.G.R. during the third week of starvation. Asfeeding cycles progress the fish acclimate to the restricted rationsand less of the lipid stores are used for energy. Protein is notutilized as an energy source during the C.G.R. as revealed in thereduction of total weight loss during the starvation phase.Carcass composition analysis from this study shows increasedprotein levels in cyclically fed fish, supporting the suggestion that anincrease in protein synthesis with reduced protein degradation maybe occurring (Quinton and Blake, 1990). The results of criticalswimming tests performed on cyclically fed fish show a return fromdepressed fitness levels to normal fitness once the fish is refed80(Raymond and Blake, unpublished). Further histological studies intothe muscle dynamics that occur during the C.G.R. is indicated bythese findings.Another advantage to this feeding technique is that itmay assist in reducing the occurrence of precocious maturation insalmonid populations. There is a positive correlation betweenincreased growth rates in the spring and an increase in the numberof precociously maturing salmonids (Rowe and Thorpe, 1990b).Reducing growth in the spring by feed restriction has been found tosuccessfully reduce precocious maturation in atlantic salmon (Roweand Thorpe, 1990b). As well, several authors have speculated thatthe initiation of maturation may be linked to the accumulation of fatreserves during the spring (Thorpe, 1986; Meyers et al, 1986). Asresults from this study show, cyclically fed fish have a significantlylower fat content than fish fed daily and this may as well reduce thepotential for precocious maturation to occur. Because of theincreased S.G.R. that occurs during the C.G.R., however, care must betaken in deciding the extent and duration of the feeding cycle to beused as a prolonged increase in growth rate may increase thepotential for precocious maturation.Brown and rainbow trout acclimate their basal metabolic rateto maintenance food rations, so that if the amount of food is reducedthe fish first lose weight but then become "adapted" to the new levelof feeding so that they gain weight (Brown, 1946; Dobson andHolmes, 1984). Raymond and Blake (unpublished) show that there isno significant difference in the standard metabolic rate between fishon a cyclic or constant feeding regime even though the 'fitness' of the81cyclically fed fish increased after refeeding. This experiment alsoshows acclimation to reduced feed rations in cyclically fed fish. Thiswas indicated in the reduced weight loss during the starvation phaseof subsequent feeding cycles. Dobson and Holmes (1984) foundsimilar results. The ability of fish to acclimate to restricted feedingregimes means that there may be a point where the starvationresponse required to elicit a C.G.R. does not occur. At least threeweeks of starvation were necessary to produce a significant C.G.R.(Quinton and Blake,1990). Therefore, cyclic feeding regimes mayhave to be monitored and the length of the starvation phasemodified to the extent the fish have acclimated to the regime.This experiment confirms findings of Farbridge, Flett, andLeatherland (1991), that increases in plasma growth hormone levelsdo not explain the increases in S.G.R. observed in the C.G.R. Undercertain conditions, exogenous piscine and mammalian growthhormone preparations increase the growth rate of fish (Weatherlyand Gill, 1987) and affect the size of muscle fibres (Weatherly andGill, 1982). However, in rainbow trout reared at optimal conditionsfor growth, recombinant piscine growth hormone had no growth-promoting activity (Danzmann et al, 1990). It has also beensuggested that the periods of reduced growth in rainbow trout maybe analogous to the anabolic refractory period seen in rats whentissues are unresponsive (Wagner and McKeown, 1985). Thus, it isunlikely that a simple relationship between growth hormonesecretion and growth exists.It is more probable that growth hormone works in combinationwith other hormones such as insulin, cortisol, and thyroid hormones82to promote overall growth in fish. For instance, growth hormonedirectly or indirectly stimulates insulin synthesis and release(Weatherly and Gill, 1987) that then stimulates protein synthesis inanimals (Ablett, 1981).The results of this research highlight the potential of the C.G.R.to reduce feed costs and improve product quality. Several of thefindings from this study, however, indicate that caution should beapplied when using this technique in high density productionfacilities. There is a tendency for fish populations on a cyclicalfeeding regime to have an abnormally high number of 'runts' that donot catch up in growth once refed. This was indicated by the highvariability in the weight data of the cyclically fed fish. Frequent'culling' may reduce their occurrence. Another problem with using acyclic feeding regime is that the fish are very sensitive to waterquality and probably more highly susceptible to disease during thestarvation phase. This may be a serious problem in high densityproduction facilities. If however, the fish become acclimated to thisform of feeding as indicated in this research, this increasedsensitivity may decrease with time.as the starvation phase becomesless stressful to the fish.The C.G.R. can be a powerful economic tool for fish production.Although the mechanism of this response is still not fully understood,findings from this study provide insight into the way the body"adjusts" to cyclical feeding to allow for the increased growthassociated with the C.G.R. The fish grown by this technique areleaner with a higher protein content then fish fed daily thusproducing a more desirable market fish. Findings from this study83monitored when applying this feeding technique to fish culture. Thepossible economic benefits to industry make further research intothe C.G.R. desirable. 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The Biology of Fish Growth,Academic Press, London.Wilson, P.N. and Osbourne, D.F. (1960). Compensatory growth afterundernutrition in mammals and birds. Biol. Rev. Camb. Phil.Soc. 37, 324-363.Wurtsbaugh, W.A. and Davis, G.E. (1977). Effects of fish size andration level on the growth and food conversion efficiency ofrainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 11, 99-1 0 4Zar, J.H. (1984). Biostatistical Analysis, Second Edition, Prentice-Hall,New Jersey.91Appendix 1: The mean weight and length of Groups A, B, and C for weeks 0-24. The abbreviation S.D. indicates the standardDATEdeviation from the mean.GROUP AWeek#^Mean^S.D.weight(g)Meanlength(cm)S.D.GROUP BMeanweight(g)S.D. Meanlength(cm)S.D.GROUP CMeanweight(g)S.D. Meanlength(cm)SDOct. 27 0 78.41 6.31 16.20 1.03 79.81 4.96 16.88 1.12 75.51 8.54 16.11 1.32Nov. 3 1 63.33 10.54 16.34 1.11 81.72 9.24 17.03 1.07 74.92 12.89 15.93 1.7410 2 58.26 13.02 15.98 1.05 86.03 6.93 17.23 1.15 78.01 13.85 16.45 1.0317 3 70.32 9.42 16.31 0.97 90.16 10.47 17.42 1.08 76.13 9.74 17.04 1.8224 4 72.03 10.34 16.56 1.09 84.21 16.83 18.04 1.42 83.43 5.49 16.86 1.32Dec. 1 5 74.91 9.43 16.46 1.08 91.07 11.85 18.12 1.15 85.61 12.29 17.53 1.138 6 98.08 8.54 15.91 0.96 95.43 3.49 18.05 1.29 99.04 10.04 17.50 1.7315 7 80.14 11.32 18.17 1.07 88.51 10.06 17.61 1.45 88.11 15.57 18.01 1.27■.c) 22 8 79.76 10.58 17.26 1.16 90.69 14.95 18.03 1.10 93.07 11.66 17.79 1.35t■) 29 9 83.53 15.11 17.54 1.08 94.73 18.32 19.49 0.97 100.88 20.86 18.10 1.31Jan. 5 10 79.11 13.60 17.40 1.04 95.86 15.48 19.85 1.07 112.65 17.94 18.65 1.7212 11 83.01 17.38 17.70 1.13 98.71 16.30 19.34 1.52 118.27 12.58 19.97 1.2519 12 113.89 16.99 18.02 0.98 114.67 9.54 19.75 1.62 117.34 10.48 18.97 2.0626 13 96.61 9.54 18.88 1.32 119.83 13.06 18.90 1.13 125.07 17.59 19.32 1.20Feb. 2 14 96.74 23.08 18.74 1.02 120.09 8.35 19.42 2.01 121.34 14.97 20.25 1.369 15 101.68 19.06 18.95 1.26 124.31 14.85 19.34 1.36 130.21 20.56 21.63 1.2416 16 107.49 21.55 18.71 1.89 120.07 15.93 19.70 1.21 136.93 17.46 20.70 2.4123 17 109.34 20.04 18.44 2.05 128.93 10.45 19.89 1.53 144.17 14.85 21.00 1.28Mar. 2 18 129.95 22.79 17.38 1.89 132.11 11.49 20.21 2.43 143.31 11.83 20.90 1.349 19 115.24 16.43 19.49 2.18 151.03 20.51 20.17 1.85 151.42 16.46 21.16 1.0716 20 118.39 23.51 18.24 1.55 150.88 18.43 21.02 1.18 160.18 14.96 20.93 1.5223 21 129.27 28.31 19.31 2.02 157.31 21.32 20.76 1.20 163.03 20.07 20.84 2.0630 22 141.03 24.79 20.42 2.14 164.33 13.29 21.34 1.42 159.91 17.95 21.07 1.20Apr. 6 23 138.19 23.56 20.13 1.62 172.18 16.93 20.75 1.92 182.63 12.97 21.67 1.1713 24 162.86 22.57 22.01 2.11 199.32 11.84 21.77 1.15 203.62 15.32 22.22 1.29Appendix 2: The mean weight and length of Groups A', B', and C' for weeks 0-24. The abbreviation S.D. indicates the standarddeviation from the mean.GROUP A^ GROUP B^ GROUP CWeek#^Mean^Std^Mean^S.D.^Mean^S.D.^Mean^S.D.^Mean^S.D.^Mean^S.DDATE^weight(g) length(cm)^weight(g) length(cm)^weight(g) length(cm) Oct.^2;^0^78.41^5.89^16.31^0.64^80.62^6.08^16.53^0.41^74.32^5.84^16.03^0.53Nov.^3^1^67.02^6.11^16.51^0.42^82.41^4.98^17.62^0.36^76.11^6.97^16.34^0.32^10^2^61.13^4.34^16.34^0.55^85.62^7.16^17.34^0.27^79.23^4.77^16.21^0.4117^3^67.24^9.03^16.26^0.51^83.41^4.75^18.23^0.42^74.77^8.04^16.94^0.5324^4^70.21^6.11^15.87^0.68^85.41^5.16^18.26^0.31^80.62^6.84^16.32^0.73Dec.^1^5^75.81^8.02^16.34^0.57^89.62^7.42^18.21^0.28^89.32^9.03^16.12^0.518^6^93.16^5.47^17.94^0.49^92.41^5.90^18.25^0.31^87.43^6.88^17.27^0.5815^7^77.89^4.62^17.76^0.68^89.76^7.04^18.26^0.60^96.50^6.04^17.83^0.83(4.)^ 22^8^77.02^3.31^18.23^0.54^93.11^3.98^17.94^0.73^91.42^4.63^18.16^0.6929^9^84.03^6.12^16.94^0.76^96.21^7.03^18.62^0.52^114.06^6.97^18.21^0.79Jan.^5^10^86.11^5.36^17.64^0.60^94.71^4.11^19.32^0.84^116.51^3.55^18.47^1.1512^11^87.62^4.96^17.51^0.43^96.67^9.39^19.42^0.90^112.49^6.97^18.62^0.6219^12^115.02^3.86^17.96^0.64^99.26^5.69^19.51^0.63^122.32^7.94^19.54^0.9526^13^101.32^7.62^18.59^0.69^112.62^6.32^19.82^0.74^127.16^9.04^19.33^0.79Feb.^2^14^103.62^6.62^18.32^0.96^117.89^7.14^19.80^0.41^129.32^6.48^19.96^0.719^15^106.62^9.37^18.44^0.54^126.12^5.64^20.01^0.85^127.99^5.18^21.06^0.9616^16^110.41^8.76^18.62^0.71^123.62^5.11^19.62^0.81^135.76^6.84^20.99^0.7523^17^109.99^6.11^19.73^0.67^125.71^4.96^19.83^0.63^139.89^9.31^22.04^0.89Mar. 2^18^128.42^8.62^19.57^0.96^129.67^8.55^20.34^0.68^147.62^5.20^21.76^0.929^19^117.41^11.55^19.96^1.22^136.82^5.63^20.26^0.39^146.66^7.41^21.84^0.681 6^2 0^120.06^10.55^19.83^1.04^147.31^7.04^22.13^1.05^154.17^5.74^20.63^0.3923^21^121.43^9.62^19.81^0.81^159.98^6.98^20.76^0.89^162.33^6.39^21.55^0.6730^22^140.06 14.31^19.97^0.96^160.07^5.27^21.03^0.49^159.48^5.74^22.61^0.85Apr.^6^23^142.21^12.11^20.43^1.08^175.06^6.74^21.37^0.96^166.30^8.59^22.74^0.6413^24^159.99 12.62^21.21^1.18^181.62^7.04^21.46^0.77^189.46^9.69^22.54^0.79 GROUP AWeek#^MeanDATE^moisture (%)GROUP BS.D.^Meanmoisture (0/0 )GROUP CS.D.^Mean^S.D.moisture (%)Appendix 3: The mean carcass composition values of Groups A, B, and C for weeks 0-24. The abbreviation S.D. indicat(deviation from the mean.MOISTURE0.210.340.350.730.180.870.760.430.370.570.260.730.230.670.540.550.290.891.040.670.490.530.460.350.640.560.780.360.941.030.750.340.470.850.750.360.750.730.930.250.380.640.750.261.60.840.630.720.910.820.730.580.260.190.480.930.630.730.520.490.730.290.470.370.590.670.880.240.740.850.250.840.640.750.53Oct.^2 -/^0^71.14Nov.^7^1 71.35^10^2^72.1817^3^73.0424^4^72.63Dec.^1^5^71.818^6^71.4215^7^71.4422^8^72.8129^9^73.11Jan.^5^10^72.8912^11^71.6419^12^71.2726^13^71.53Feb.^2^14^72.019^15^72.6116^16^72.6423^17^71.36Mar.^2^18^71.019^19^71.3216^20^72.2123^21^72.4130^22^72.39Apr.^6^23^71.4513^24^71.2370.6470.4271.0271.1970.8770.6371.3970.5170.3870.2170.8371.0170.9670.3870.4171.0670.4270.3970.6170.8871.0170.9170.7270.9671.2171.3170.8170.6270.5870.970.7270.9770.6170.5471.0270.7170.7371.1770.3670.6170.7270.4170.4670.8370.9170.0570.2370.9670.5371.19LIPIDDATEWeek#GROUP AMeanlipid^(%)S.D.GROUP BMeanlipid^(%)S.D.GROUP CMeanlipid^(%)S.D.Oct. 2; 0 10.01 0.18 10.31 0.35 10.21 0.63Nov. 7 1 9.01 0.64 10.13 0.63 10.34 0.5910 2 7.25 0.56 10.51 0.42 10.36 0.3717 3 7.14 0.74 9.98 0.36 10.23 0.2124 4 7.87 0.93 10.16 0.73 10.22 0.36Dec. 1 5 8.31 0.64 10.47 0.42 10.38 0.718 6 9.24 0.76 10.38 0.55 10.21 0.8215 7 8.41 0.42 9.81 0.63 10.32 0.52■ID22 8 7.91 0.38 10.33 0.72 10.51 0.43til 2 9 9 7.33 0.64 10.43 0.54 10.32 0.46Jan. 5 10 7.51 0.53 10.02 0.25 10.44 0.7212 11 7.98 0.26 10.17 0.74 10.47 0.3719 12 9.01 0.42 10.54 0.41 10.29 0.7326 13 8.96 0.64 10.37 0.31 10.54 0.91Feb. 2 14 8.31 0.55 10.29 0.26 10.49 0.529 15 7.52 0.94 10.43 0.73 10.62 0.7516 16 7.91 0.63 10.57 0.81 10.87 0.5623 17 8.74 0.52 10.53 0.52 10.91 0.59Mar. 2 18 9.71 0.45 10.49 0.44 10.73 0.619 19 9.36 0.73 10.38 0.72 10.58 0.8316 20 9.01 0.63 10.42 0.41 10.61 0.3323 21 7.91 0.42 10.31 0.65 10.56 0.7430 22 7.84 0.69 10.61 0.28 11.47 0.41Apr. 6 23 8.46 0.42 10.59 0.36 11.22 0.3913 24 9.57 0.37 10.57 0.29 11.36 0.27PROTEINDATEWeek#GROUP AMeanprotein (%)S.D.GROUP BMeanprotein (%)S.D.GROUP CMeanprotein (%)S.D.Oct. 2; 0 16.83 0.49 16.79 0.53 16.88 0.61Nov. 7 1 16.79 0.83 16.72 0.64 16.83 0.4510 2 16.73 0.52 16.64 0.95 16.67 0.8117 3 16.81 0.44 16.66 0.73 16.74 0.5524 4 16.83 0.91 16.55 0.66 16.59 0.81Dec. 1 5 16.76 0.53 16.37 0.75 16.65 0.488 6 16.63 0.93 16.49 0.84 16.71 0.3515 7 16.78 0.42 16.58 0.81 16.62 0.4122 8 16.66 0.61 16.52 0.94 16.57 0.68■0C^ 29 9 16.58 0.73 16.39 0.51 16.39 0.91Jan. 5 10 16.64 0.53 16.59 0.38 16.55 0.5312 11 16.73 0.53 16.74 0.49 16.82 0.7619 12 16.65 0.59 16.54 0.59 16.47 0.4926 13 16.59 0.72 16.49 0.88 16.52 0.78Feb. 2 14 16.67 0.58 16.33 0.71 16.32 0.589 15 16.63 0.83 16.51 0.36 16.29 0.7216 16 16.58 0.51 16.63 1.05 16.54 0.8223 17 16.61 0.47 16.47 0.52 16.33 0.63Mar. 2 18 16.73 0.68 16.73 0.59 15.99 0.499 19 16.88 0.83 16.35 0.62 16.09 1.0416 20 16.81 0.69 16.72 0.93 16.29 0.4723 21 16.82 0.41 16.41 0.51 15.82 0.6230 22 16.78 0.38 16.49 0.77 16.03 0.81Apr. 6 23 16.89 0.86 15.93 0.43 16.22 0.6913 24 16.92 0.59 16.52 0.81 15.97 0.98ASHDATEWeek#GROUP AMeanash (%)S.D.GROUP BMeanash (%)S.D.GROUP CMeanash (%)S.D.Oct. 2; 0 2.44 0.12 2.36 0.09 2.26 0.09Nov. 7 1 2.43 0.09 2.32 0.11 2.15 0.0810 2 2.38 0.12 2.41 0.12 2.37 0.1317 3 2.17 0.09 2.37 0.09 2.33 0.0824 4 2.35 0.08 2.39 0.09 2.27 0.19Dec. 1 5 2.49 0.13 2.28 0.11 2.26 0.098 6 2.36 0.13 2.29 0.08 2.34 0.0915 7 2.35 0.14 2.31 0.09 2.42 0.1422 8 2.41 0.17 2.36 0.15 2.43 0.17,I 29 9 2.04 0.09 2.48 0.07 2.32 0.09Jan. 5 10 2.27 0.09 2.28 0.17 2.36 0.1212 11 2.25 0.07 2.44 0.15 2.31 0.1419 12 2.38 0.19 2.38 0.09 2.38 0.1726 13 2.31 0.13 2.37 0.18 2.39 0.19Feb. 2 14 2.37 0.09 2.43 0.15 2.28 0.179 15 2.22 0.09 2.41 0.09 2.37 0.0916 16 2.31 0.14 2.44 0.09 2.35 0.0923 17 2.37 0.16 2.34 0.09 2.31 0.14Mar. 2 18 2.25 0.11 2.38 0.12 2.39 0.099 19 2.35 0.09 2.27 0.16 2.33 0.1616 20 2.31 0.07 2.43 0.13 2.37 0.1523 21 2.21 0.09 2.24 0.08 2.44 0.0930 22 2.65 0.11 2.21 0.09 2.31 0.08Apr. 6 23 2.31 0.12 2.36 0.13 2.35 0.1813 24 2.46 0.09 2.57 0.14 2.34 0.13

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