cd de c e
D I SCUSSION (Experiments 1 - 3 )
Experiments 1 -3 were des i gned t o inve s t igate whether arginine o r methion ine o r both were required to any s ignificant extent for the detoxificat i on of compounds in LPC . Ini t i ally (Experiment 1 ) L - met yl ( h 1 4c) met 1 on1ne was fe Wlth an PC d1et t o an adult h · · d · 1 ·
cockerel . The excreta of this cockerel was coll ected, extracted and p ar t i tioned into a " l i p id " and a "water fraction" ; radioac t ivi ty was present in these two frac t i ons in a rat i o of 1 : 2 . 3 ( "wat er <+ : " l ipid "
fraction ) .
Approximately 70% of the radioac t iv i ty in t he "water frac t i on" app eared to be present in phenolic compounds . Thes e compounds had presumably been methylated by e i t her the chicken or the gut microflora . These phenolic compounds were no t complexed with
proteins in the excret a . Therefore the frequent ly made sugges t i on (e . g . Pirie ,1 9 7 8) that such an as soc i at i on may occur during the produc t i on of LPC and so reduce the diges t i bi l i ty of LPC is not suppor t ed by these results . S imilarly , the resul ts do not indicate that phenolics and proteins as sociate during diges t i on . Methylated phenolic compounds s imi l ar to those found in the excre t a o f LPC fed chickens may have been present i n the " water fract ion" of excreta from chickens fed the SBM diet but this was not
inve s t i gated . However, when 1-(methyl 1 4c) methionine was given to chickens fed e i ther LPC or SBM diets (Exper iment 2 ) , there
were no s i gnificant differences in the percentage total radioac t ivity whi ch was recovered in the "water fraction" obtained from the excre ta of these two groups of chickens . Therefore the requirement for methyl groups for the detoxificat i on of aromat ic compounds was apparen t ly s imi l ar for chickens fed e i t her the LPC or SBM diets . In contr as t , the percentage radioac t ivi ty recovered in the " l i p i d frac tion" was s i gnificantly increased in the excre t a o f the
chickens fed the· LPC d i e t compared to those fed the SBM diet . As mos t of the r adioac t ivi ty in this fract i on was found to be in a
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cyclopropane fatty aci d ( 95% in t he " l i p id frac t i on" of the chickens fed the LPC diet and 70% in the " l ipid fract ion" of chickens fed the SBM diet), thi s sugges ted that t he ut i l is at ion of methionine in the LPC di e t was probably being influenced by t he gut micro-organisms . This was also indicated by the resul ts obtained when antibiotic was added to the diets . The add i t i on of antibiotic to LPC die t fed to chickens t ended to decreas e the level of radioac t ivi ty in the " l i p id fraction"
whereas t he rad i oac t ivity level increased in the " l ipid frac t i on " of chickens fed the SBM + ant ibiotic diet . Such resul t s ,
together with the s ignificant difference between radioac tive levels in t he LPC and SBM " lipid frac t i ons " , indicate that differences in t he diets can modify t he act ivity and/or the population structure of the microflor a ;
by Adams ( 1 98 0 ) .
. as was also suggested
Another possible indicator of detoxificat ion was measurement of the amount of orn i thine excreted by LPC and SBM fed cockerels . Compared to the result s of Nes he im and Garl ich ( 1 96 3 ) who found a substantial effect due to feeding 2% (w/w) benzoic acid , the increase in orni thine excret ion due to feeding a 1 5% LPC diet compared to a 1 5% SBM diet was smal l . Neshe im and Gar lich ( 1 96 3 ) calcul ated the arginine intake over a test period . They found that approximate ly 2% of the arg inine e at en was excreted in t he form of orni thine conjugates when chickens were fed a basal di e t , but when 2% benzoic aci d was introduced into the bas al diet approximate ly 40% of t he inges ted arginine was excreted as orn i t hine conjugated products .
The l ow amount of orn i th ine excre t ed due to feeding LPC compare d to SBM t herefore , indicated that l arge amounts o f arginine were
not being required for ornithine synthes i s and that t he detox i fi c at i on of aromatic compounds via orn i thine was not a maj or contribut ing
In conclus i on , therefore , the resul t s of this s tudy ind icated t hat the detoxification of aromat ic compounds by ei ther met hyl ation or conjugati on with orni thine p l ayed an ins ignificant role in the poor growth response of chickens fed LPC di ets . This conclus i on
reflected t he resul ts of Eggum and Chr i s tensen ( 1 97 5 ) who found no s ignificant effect on growth due to detoxificat i on mechani sms operati ng in rats fed e ither bar l ey and/or tannins . The
feeding of LPC compared to SBM , however , increas ed t he l evel o f cyc lopropane fatty acid in the excret a . This indicated that the gut microflora may be influencing the nut r i t i onal value of LPC and/or that LPC may be influencing the met abol i sm and/or structure of the gut microflora .
4 :
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Exhaust ive Enzyme D i ge s t ion of "Pure" LPC and SBM
The in vi tro exhaust ive enzyme diges t ion was des igned to
inve s t i gate whe ther LPC protein could be hydrolysed by enzymes to a s imilar extent as SBM protein . The in vitro di ges t i on was carri ed out over 4 days as the result s of a shorter di gest i on period and/or l ow enzyme l evel may be masked by an inhibi t ory property inherent in LPC .
Method
(i) The enzyme d i ge s t mixture includes p ancreatin (BDH L td . , England) and pronase (S i gma Chemical s , U . S . A .).
Pancreatin was included to provide lipase and amylase act ivity in order t o remove any pos sible " shield ing " effect s due t o the pres ence of fats and carbohydrates . The 1 0% (w/v) pancreat in solut i on was centrifuged (5 , 000 x g for 5 minutes) before use to remove insolub l e matter .
Pronase i s a mixture of several proteolyt ic enzymes including endopept i dases and exopeptidases whi ch are produced by a s train
of The enzymes present are neutral and
alkal ine pro t e ases , amino pept idase and carboxypep t i dase (Narahashi , 1 970). The enzyme mixture provid� the required enzyme act ivi t ies wi thout excess ively increas ing the amount of protein material .
(i i) Buffer : The pH 7 . 5 buffer solut ion used was 0 . 1 M sodium borate in 0 . 05 M hydrochloric aci d .
0 . 0 1 M calcium chlori de .
I t also contained
(i i i) Exhaus t i ve Exhaust ive enzyme digests were carried out on 200 mg of LPC or SBM in 1 0 mls of buffer solut ion of pH 7 . 5 . Two al i quots of each of the enzymes , pancreat in and pronas e , were added at 24-3 6 hour intervals to give for each enzyme a final enzyme/substrate rat io of 1 %(w/v). Pancreat in was added fir s t foll owed by p ronase wi th the pH of 7 . 5 being maintained
t hroughout by the addit i on of 0 . 1 M sodium hydroxide . The t emperature of t he diges t was maintained at 37°C and the
soluti on was cont inuously agi t at ed . Toluene was added to all diges t s . Dup l icate SBM and triplicat e LPC samples wer e di ges ted . After centrifugation ( 1 0 , 000 x g for 5 minutes) the supernatants wer e analysed for amino aci ds . Enzyme blanks were also analysed so t hat the amino aci d diges t i bi l i ty values could be corrected for the add i t i onal amino acids due to enzyme addi t ion . The percentage of each amino acid digested was calculated from the fol lowing formula : -
% Amino Aci d Di ges t ed Resul ts
Amino Aci d Total Amino Aci d (g)
1 00
X
The resul ts given in Table XXI demonstrated t hat t he in vitro mean diges t ion l evel of protein in LPC was l ower by approximat ely 6% compared to SBM protein. There was some var i abil i ty in LPC resul t s , which may have resulted from t he fine powdery nature of t he produc t . Thi s caused i t to s t ick t o t he glassware whil e bei ng agi t at ed over t he digest i on period .
The result s do s how t hat none of the individual amino aci d di ges t i bi l i ty values f o r L P C were markedly lower t han the mean amino aci d diges tibil i ty value for LPC . These resul ts , therefore , indicate that e it her the LPC protein can be almost hydrolysed as effect ively as SBM by enzyme act i on , or that the method used here was unsui t ab l e to detect any d ifferences . However , because t he
in vitro data often differs from the in vivo' results (Akeson and S tahman , 1 96 5 ) �hese results mus t be viewed with caution .
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Table XXI : Results of in vi tro Exhaus t ive D i gest ion
Cyst ine Threonine Serine Valine t1ethionine Isoleuc ine Leuc ine Tyros ine Phenyl alanine Hist i dine Lysine Arginine Overall Mean
Overall Mean Range
% Amino Acid diges t ed
* Mean % Amino * "Pure "LPC 6 9 79 78 79 66 76 7 2 8 0 69 6 8 79 74 74 66 - 79 Aci d D " 1 "Pure "SBM+ 86 80 79 8 6 69 8 6 76 90 76 73 75 80 80 78 - 82
Supernatant amino aci d(g) 1 00
X
Total amino aci d (g) Tripl icate - LPC batch number 5
5 : Compar i son of the " Correct ed ( CAAA ) and Appar ent ( ApAAA) Amino Ac id Availab i l i ty
of LPC and SBM Ingredients and of diets containing LPC and SBM''
In this experiment " apparent " and " corrected " ( corrected for endogenous secretion ) amino aci d avai l ab i l i t ies were measured by analys i s of the excreta from cockerels which had been fed e i ther LPC or SBM or diets cont aining these protein concentrates . The met hod was based on t he TME met hod ( p 1 7 ) .
Likuski and Dorrell ( 1 97 8 ) were the first to pub l i sh amino ac i d avai l ab i l i t y resul t s derived b y t h e TME method . The t erms " apparent " and " corrected" amino acid avail ab i l i ty are defined by the equations given below in the Methods sect i on .
The avai l ab i l i ty values of amino acids as determined by t he
excre t a method can be over-est imated because of the act ivi t ie s o f the microflora i n the large intes t ine . The extent of the influence by the microflora on t he avai l ab i l i ty est imate is unclear ( McNab , 1 980 ) . HowevPr , microbi al fermentation is considered to have l ess of an
effect on protein degradat i on in poultry than in other monogas t ri cs , e . g . p igs ) because of the fas ter rate of diges t a p ass age through t he
l ower gut ( Zebrowska , 1 97 8 ) . Other factors , such as var i at i ons in dietary protein l evel are bel i eved to influence the amount of endogenous s ecret i on ( McNab , 1 98 0 ) and so influence the amino acid avai l ab i l i ty e s t imates . As the prote in l evels of the d i e t s used in this experiment were s imilar , i t was assumed that any variation in endogenous output was a character i s t i c of the " t e s t " ingredi ent as both prote in sources were s tudi ed under comparab l e cond i t ions .
O ther factors , e . g . rates of di ges t i on and/or absorp t i on , which may effect the availab i li t y of dietary amino acids to growing ch ickens cannot be measured by· this type of as say . The purpo s e o f t h i s s tudy was therefore t o compare two s im i l ar protein source s under comparable cond i t i ons .
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Method
The ingredient and d i etary amino aci d ava i l ab i l i t i es were measured us ing the True Met abol i s ab l e Met hod (TME) described on p 1 7 • The amino ac i d avai l ab i l i ty values were e s t imated in terms of the "apparent" amino aci d avail ab i l i ty (ApAAA) and the " corrected " amino acid avai l ab i l i ty (CAAA). These were calculated using the formulae out l ined by Likuski and Dorrell ( 1 97 8) :-
g: amino acid fed - g a.-:1:no acid excreted l OC % Appa::--ent Ar.lir.o Acid Ava�lab i l i ty ( A?AAA.)
q , amino acid i'ed X
% "'2rue" or 11Correcte<!" Amino Acid Availabi l i ty (CAM)
g am:.no acic:! fed - ( G: a:r:ino acid excreted
= - g. e�ldcger..o�s ar.jino ac td excreted)
gi ami no acid fed
From t he equati ons , three sets of amino acid analys es were required for the calculat i on of CAAA and ApAAA .
(i) The amino acid compos i t ion of the feed which . i s fed to the cockerel .
·. oo X -
(i i) The amount of amino aci d excreted by a fed cocker e l . ( i i i) The amount of amino aci d excreted under s t arvat i on
cond i t ions to give a value for the endogenous excretion l eve l .
Thirty-two adult Sykes cockerels were housed 1n s ingle cockerel cages in an environment al ly controlled room . All cockerel s were maintained o n a commerci al 1 8% protein ( N x 6 . 25 ) chick grower diet s o t hat any pos s ible carry-over effect of t he maintenance d i e t to the subsequent avail ab i l i ty e s t imates was
conside red to be equally distr ibuted across all treatments . Two separate " feeding " p eriods of 24 hours each were carri ed out a week apart . Before e ach experimental " feeding" period each cockere l was s t arved for 24 hour s .
In order to have an endogenous amino acid est imate for e&ch individual cockerel a ::cross-over" des i gn was adopted . For each exper iment al feeding period , four groups of 4 cockerels were force-fed 1 6 g " pure " SBM or 1 4 g " pure " LPC or
2 5 g SBM diet or 2 5 g LPC d i e t . S imilarly four groups of 4 cockerels were s tarved for a further 24 hours for the est imat i on of endogenous output . The excre t a collect i on period was 24 hours after whi ch the excret a were individual ly p l aced in pots and fro z en .
The following week , during the experiment al " feeding " per iod the order of t he cockerels was reversed , i . e . the previously fed cockerels were now s t arved and the previous ly s t arved cockerels were force-fed wi t h t he relevant ingredi ent or diet . The excreta collec t i on period was again , 24 hours . The excr e t a were frozen , freeze-dried , ground and wei ghed . The amino acid analyses were carri ed out as described on p 1 6
Resul ts
The mean values for ApAAA and CAAA for the ingredients LPC and SBM are given in Table XXI I for each of the essent i al amino acids . Comparab l e f i gures for t he diets cont aining LPC or SBM are given in Table XXI I I . The s t at i s t i cal analyses for Exper iment 5 are summarised in Appendix B , Tab les XI I I and XIV . The mean overal l amino acid avai l ab i l i ty was 81 . 6% for "pure " LPC and 92 . 4% for " pure " SBM on a CAAA bas is ( a di fference of 1 2% ) , or 70 . 4% and 85 . 3% for "pure " LPC and " pure " SBM respect ively on an ApAAA bas i s ( i . e . a difference of 1 5% ) . Cys t i ne had t he lowes t avai l ab i l i ty value in "pur e " LPC ( 51 . 2% CAAA and
1 1 . 9% ApAAA compared t o 80 . 8% CAAA and 7 5 . 7% ApAAA in " pure " SBM ) . The effect of the above differences in amino acid avail ab i l i ty were brought into perspective when the diets containing LPC or SBM were compared us ing t he s ame ass ay procedure (Tab l e XXI I I).
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Table XXII : Summary of the Resul t s of Experiment 5 - LPC an� SBM Ingredient Amino Aci d Avai lability us ing the TME Method
ApAAA � ''?ure" LPCCAAA � ApAAA'lure" SBM CAAA %