Protein Quality Evaluation in Livestock

DEPARTMENT OF ANIMAL NUTRITION BIHAR VETERINARY COLLEGE, PATNA (BIHAR ANIMAL SCIENCES UNIVERSITY, PATNA, BIHAR) Lecture Topic: Methods of Feed Protein Quality Evaluation in Livestock Dr. Kaushalendra Kumar Associate Professor

EVALUATION EVALUATION OF OF PROTEIN PROTEIN QUALITY QUALITY IN IN MONOGASTRIC MONOGASTRIC ANIMALS ANIMALS Protein Protein is is mainly mainly required required for for maintenance maintenance & & building building of of body body tissue tissue. . EAA EAA come come from from diet diet. . D Depend epend on on digestible digestible protein protein quantity quantity of of feed feed but but digestible digestible protein protein figures figures are are not not exclusively exclusively measures measures of of the the value value of of a a protein protein to to livestock livestock. . Because Because the the efficiency efficiency of of absorbed absorbed protein protein differs differs from from sources sources of of protein protein. .

Cont..... So, So, in in order order to to allow allow such such differences, differences, different different methods methods for for evaluating evaluating proteins, proteins, such such as as; ; P Protein rotein efficiency efficiency ratio ratio (PER) (PER) N Net et protein protein retention retention (NPR) (NPR) G Gross ross protein protein value value (GPV) (GPV)

Cont..... PER PER = = gain gain in in body body weight weight (g) (g) / / protein protein consumed consumed (g) (g) NPR NPR = = weight weight gain gain of of TPG TPG weight weight loss loss of of NPG NPG / / weight weight of of protein protein consumed consumed Where Where; ; TPG TPG = = group group given given the the test test protein protein and and NPG NPG = = group group of of protein protein- -free free diet diet. .

Cont..... Gross Gross protein protein value value (GPV) (GPV) GPV GPV = = A/A A/A Where Where; ; A A = = g g increased increased weight weight gain/g gain/g test test protein protein & & A A = = g g increased increased weight weight gain/g gain/g casein casein. . L Live ive weight weight gains gains of of birds birds receiving receiving a a basal basal diet diet containing containing 80 80 g g CP/kg CP/kg are are compared compared with with those those of of birds birds receiving receiving the the basal basal diet diet plus plus 30 30 g/kg g/kg of of a a test test protein, protein, and and of of yet yet others others receiving receiving the the basal basal diet diet plus plus 30 30 g/kg g/kg of of casein casein. . E Extra xtra live live weight weight gain gain per per unit unit of of supplementary supplementary test test protein protein stated stated as as a a proportion proportion of of the the extra extra live live weight weight gain gain per per unit unit of of supplementary supplementary casein casein is is the the GPV GPV of of the the test test protein protein. .

Cont..... Nitrogen Nitrogen balance balance Live Live weight weight gain gain may may not not be be related related to to protein protein stored stored & & more more accurate accurate evaluation evaluation of of a a protein protein may may be be obtained obtained by by using using the the results results of of N N- - balance balance experiments experiments. . N Nitrogen itrogen consumed consumed in in the the food food is is measured, measured, together together with with that that voided voided in in faeces faeces, , urine urine & & any any other other N N- -containing containing products products such such as as milk, milk, wool wool & & eggs eggs. . When When the the N N- - intake intake is is equal equal to to the the output, output, the the animal animal is is said said to to be be in in nitrogen nitrogen equilibrium equilibrium. .

Cont..... Factors Factors affecting affecting Balance Balance trials trials Inadequate Inadequate adaptation adaptation of of trial trial animals animals to to the the diet diet & & environment environment Collection Collection & & weighing weighing of of faeces faeces & & and and urine urine Storage Storage of of faeces faeces & & urine urine S Sampling ampling of of faeces faeces & & urine urine for for chemical chemical analysis analysis. .

Biological Biological value value (BV) (BV) Cont..... D Direct irect measure measure of of proportion proportion of of food food protein protein that that can can be be utilized utilized by by animal animal for for synthesizing synthesizing body body tissues tissues & & may may be be defined defined as as proportion proportion of of absorbed absorbed nitrogen nitrogen i i. .e e. . retained retained by by the the body body. . B Balance alance trial trial is is conducted conducted in in which which N N- -intake intake & & urinary urinary & & faecal faecal excretions excretions of of N N are are measured, measured, along along with with endogenous endogenous fractions fractions in in these these two two attribute attribute. . BV BV = = N N intake intake ( (faecal faecal N N MFN) MFN) (urinary (urinary N N EUN) EUN) / / N N intake intake ( (faecal faecal N N MFN) MFN) Where Where; ; MFN MFN = = metabolic metabolic (endogenous) (endogenous) faecal faecal nitrogen nitrogen & & EUN EUN = = endogenous endogenous urinary urinary nitrogen nitrogen. .

Cont.....

Cont..... BV BV of of a a food food protein protein also also depends depends upon upon the the number number & & kinds kinds of of AA AA present present in in the the diet diet. . C Closer loser the the AA AA composition composition of of food food protein protein approaches approaches that that of of the the body body protein, protein, the the higher higher will will be be its its biological biological value value. . P Product roduct of of BV BV & & digestibility digestibility is is termed termed the the Net Net P Protein rotein U Utilisation tilisation (NPU) (NPU) & & is is the the proportion proportion of of N N- -intake intake retained retained by by the the animal animal. .

Cont.....

Chemical Chemical score score Cont..... Considered Considered that that the the quality quality of of a a protein protein is is decided decided by by the the AA AA i i. .e e. . in in greatest greatest deficit deficit when when compared compared with with a a standard standard. . S Standard tandard generally generally used used been been egg egg protein protein, , but but many many workers workers now now use use a a defined defined AA AA mixture mixture. . C Content ontent of of each each of of the the EAAs EAAs of of a a protein protein is is expressed expressed as as a a proportion proportion of of that that in in the the standard standard (standard (standard pattern pattern ratio) ratio) & & the the lowest lowest proportion proportion taken taken as as the the score score. .

Calculation Calculation of of chemical chemical score score Cont..... In In wheat wheat protein, protein, ex ex- - EAAs EAAs in in greatest greatest deficit deficit is is lysine lysine. . C Contents ontents of of lysine lysine in in egg egg & & wheat wheat proteins proteins are are 72 72 g/kg g/kg & & 27 27 g/kg, g/kg, respectively, respectively, & & the the chemical chemical score score for for wheat wheat protein protein is is therefore, therefore, 27 27/ /72 72 = = 0 0. .37 37. . They They are are useful useful for for grouping grouping proteins proteins but but suffer suffer a a serious serious disadvantage disadvantage in in that that no no account account is is taken taken of of the the deficiencies deficiencies of of AA AA other other than than that that in in greatest greatest deficit deficit. .

Cont..... E Essential ssential amino amino acid acid index index (EAAI) (EAAI) G Geometric eometric mean mean of of the the egg, egg, or or standard standard pattern, pattern, ratios ratios of of EAAs EAAs. . Has Has a advantage dvantage of of predicting predicting the the effect effect of of supplementation supplementation in in combinations combinations of of proteins proteins. . Has Has the the disadvantage disadvantage that that proteins proteins of of very very different different AA AA composition composition may may have have the the same same or or a a very very similar similar index index. . C Chemical hemical score score & & EAAI EAAI are are based based upon upon gross gross AA AA composition composition. .

Cont..... Net Net Protein Protein Utilization Utilization (NPU) (NPU) It It is is the the proportion proportion of of the the N N- - intake intake retained retained by by the the animal animal. . P Product roduct of of BV BV & & digestibility digestibility is is termed termed the the NPU NPU. . Net Net Protein Protein Values Values (NPV) (NPV) M Measure easure of of protein protein actually actually available available for for metabolism metabolism by by animals animals. . P Product roduct of of NPU NPU & & % % CP CP. .

Cont..... Ileal Ileal digestibility digestibility A Availability vailability of of AA AA is is primarily primarily determined determined by by their their digestibility digestibility measured measured at at the the end end of of small small intestine, intestine, i i. .e e. ., , at at the the ileum ileum level level, , generally generally there there is is no no AAs AAs absorption absorption from from large large intestine intestine. . However, However, m micro icro flora flora metabolize metabolize some some of of the the undigested undigested AA AA in in large large intestine intestine thus thus preventing preventing them them from from appearing appearing in in faces faces. . That That is is why why, , the the concept concept of of ileal ilealdigestibility digestibility has has been been established established. .

Cont..... Measurement Measurement of of ileal ileal digestibility digestibility Ileal Ileal digestibility digestibility can can be be measured measured from from either either total total recovery recovery of of ileum ileum flow flow from from pigs pigs fitted fitted with with ileo ileo- -rectal rectal anastomosis anastomosis (IRA), (IRA), or or From From determination determination of of concentration concentration of of indigestible indigestible marker, marker, generally generally chromic chromic oxide oxide, , in in a a sample sample of of ileal ileal digesta digesta from from pigs pigs fitted fitted with with cannula cannula. . Apparent Apparent ileal ileal digestibility digestibility: : DApp DApp = = [(AA [(AA Diet Diet DM DM x x DMI) DMI) - - (Digesta (Digesta x x DME DME x x 100 100)] )] x x 100 100 / / (AA (AA Diet Diet DM DM x x DMI) DMI) DApp DApp = = [(AA [(AA Diet Diet FM FM x x FMI) FMI) - - (Digesta (Digesta x x DME DME x x 100 100)] )] x x 100 100 / / (AA (AA Diet Diet FM FM x x FMI) FMI)

Cont..... Corrected Corrected apparent apparent ileal ileal digestibility digestibility C Corrected orrected apparent apparent ileal ileal digestibility digestibility can can be be calculated calculated either either from from apparent apparent digestibility digestibility or or from from the the standardised standardised digestibility digestibility. . D D App App Corr Corr = = D D App App + + Endo Endo DMI DMI x x 10 10 x x ( (1 1/AA /AA Diet Diet DM DM - - 1 1/ / AARMDM) AARMDM) D D App App Corr Corr = = D D Std Std Endo Endo DMI DMI x x 10 10 / / AARMDM AARMDM D D App App Corr Corr = = D D App App + + Endo Endo FMI FMI x x 10 10 x x ( (1 1/AA /AA Diet Diet FM FM - - 1 1/ / AARMFM) AARMFM) D D App App Corr Corr = = D D Std Std Endo Endo FMI FMI x x 10 10 / / AARMFM AARMFM

Cont..... Standardized Standardized ileal ileal digestibility digestibility is is now now used used for for evaluating evaluating proteins proteins for for pigs pigs. . Advantage Advantage of of using using ileal ileal digestibility digestibility & & so so avoiding avoiding microbial microbial AA AA production production in in the the hind hind gut gut inherent inherent in in using using whole whole- -tract tract digestibility, digestibility, & & also also accounting accounting for for basal basal endogenous endogenous AA AA loss loss. . For For poultry poultry, , evaluation evaluation of of protein protein sources sources is is also also based based on on standardised standardised ileal ileal digestibility digestibility of of three three major major AA AA : : lysine, lysine, methionine methionine & & tryptophan tryptophan. . For For horses horses, , evaluation evaluation of of protein protein sources sources is is based based on on CP CP or or DCP DCP. .

Cont..... Biological Biological assay assay of of available available amino amino acids acids A Available vailable AA AA content content of of a a food food protein protein may may be be assayed assayed by by measuring measuring the the live live weight weight gain, gain, FCE FCE or or N N- -retention retention of of animals animals given given the the intact intact protein protein as as a a supplement supplement to to a a diet diet deficient deficient in in the the particular particular AA AA under under investigation investigation & & the the response response to to the the test test material material is is compared compared with with the the response response to to supplements supplements of of pure pure AAs AAs. .

Cont..... M Method ethod has has been been used used successfully successfully for for lysine, lysine, methionine methionine and and cystine cystine. . But, But, disadvantages disadvantages associated associated with with biological biological methods methods; ; time, time, technical technical expertise expertise and and supply supply of of suitable suitable animals animals major major problem problem of of preparing preparing diets diets deficient deficient in in specific specific AA AA but but adequate adequate in in other other respects respects. .

Cont..... Microbiological Microbiological assay assay of of essential essential amino amino acids acids Certain Certain microorganisms microorganisms have have amino amino acid acid requirements requirements similar similar to to those those of of higher higher animals animals & & have have been been used used for for the the evaluation evaluation of of food food proteins proteins. . M Methods ethods based based on on measuring measuring the the growth growth of of microorganisms microorganisms in in culture culture media media that that include include the the protein protein under under test test. . Best Best results results have have been been obtained obtained with with Streptococcus Streptococcus zymogenes zymogenes and and Tetrahymena Tetrahymena pyriformis pyriformis. .

Cont..... F Former ormer is is used used after after an an AA AA or or enzymic enzymic pre pre- -digestion digestion of of the the food food protein protein; ; estimates estimates of of availability availability of of lysine lysine & & methionine methionine. . T T. . pyriformis pyriformis has has intrinsic intrinsic proteolytic proteolytic activity activity & & used used for for soluble soluble proteins, proteins, without without pre pre- -digestion digestion. . I Improved mproved method, method, where where pre pre- -digestion digestion with with enzyme enzyme pronase pronase (proteases) (proteases) & & measuring measuring response response in in terms terms of of the the tetrahymanol tetrahymanol content content of of the the culture culture medium, medium, gives gives results results for for available available lysine, lysine, methionine methionine & & tryptophan tryptophan that that correlate correlate well well with with those those of of biological biological assays assays. .

Cont..... Chemical Chemical methods methods M Most ost widely widely used used method method is is FDNB FDNB- -reactive reactive lysine lysine ( (proposed proposed by by K K. .J J. . Carpenter) Carpenter). . Practically, Practically, only only source source of of utilizable utilizable lysine lysine in in foods foods is is that that which which has has the the epsilon epsilon- -amino amino group group free free to to react react with with various various chemical chemical reagents reagents. . P Protein rotein under under test test is is allowed allowed to to react react under under alkaline alkaline conditions conditions with with fluoro fluoro- -2 2, ,4 4- -dinitrobenzene dinitrobenzene (FDNB) (FDNB) to to give give DNP DNP- -lysine, lysine, concentration concentration can can be be measured measured colorometrically colorometrically. .

Cont..... In In practice, practice, method method has has been been agree agree well well with with biological biological procedures procedures for for evaluating evaluating proteins proteins as as supplements supplements to to diets, diets, those those containing containing high high proportions proportions of of cereals, cereals, where where lysine lysine is is limiting limiting. . With With vegetable vegetable protein protein & & diets diets containing containing high high levels levels of of carbohydrate, carbohydrate, but but method method is is not not so so satisfactory satisfactory, , results results being being too too low low owing owing to to destruction destruction of of the the coloured coloured lysine lysine derivative derivative during during acid acid hydrolysis hydrolysis. .

Cont..... Dye Dye- -binding binding methods methods U Used sed widely widely for for estimating estimating protein protein in in foods foods like like cereals cereals & & milk milk. . M Methods ethods are are rapid rapid & & give give reproducible reproducible results, results, used used for for measuring measuring total total basic basic AAs AAs & & reactive reactive lysine lysine. . L Latter atter requires requires blocking blocking of of epsilon epsilon- -amino amino group group to to prevent prevent reaction reaction with with dye dye. . Orange Orange- -G G has has been been used, used, along along with with 2 2, ,4 4, ,6 6- -trinitrobenzene trinitrobenzene sulphonic sulphonic acid acid & & propionic propionic anhydride anhydride as as blocking blocking agents agents & & has has proved proved effective effective for for estimating estimating the the lysine lysine content content of of cereals cereals. . It It is is less less effective effective for for fish fish & & meat meat meals meals. .

Cont..... Factors Factors affecting affecting AA AA assays assays Changes Changes in in concentration concentration of of one one or or more more AAs AAs may may increase increase the the amounts amounts of of others others required required to to maintain maintain growth growth rates rates. . Certain Certain AAs AAs like like tryptophan tryptophan & & histidine histidine, , may may be be toxic, toxic, at at concentrations concentrations far far greater greater than than normally normally occur occur in in food food proteins proteins. . Antagonisms Antagonisms may may exist exist between between specific specific acids acids that that affect affect their their utilisation utilisation. . Antinutritional Antinutritional factors factors (ANF) (ANF) E Enzyme nzyme inhibitors, inhibitors, lectins, lectins, polyphenols polyphenols & & certain certain non non- -protein protein AAs AAs. .

MEASURES MEASURES OF OF PROTEIN PROTEIN QUALITY QUALITY FOR FOR RUMINANT RUMINANT ANIMALS ANIMALS Crude Crude Protein Protein Digestibility Digestibility Crude Crude Protein Protein True True Protein Protein Protein Protein Equivalent Equivalent Metabolizable Metabolizable protein protein RDP RDP & & RUP RUP

Cont..... CRUDE CRUDE PROTEIN PROTEIN (CP) (CP) P Protein rotein content content of of a a food food is is calculated calculated from from its its N N- -content content determined determined by by a a modification modification of of the the Kjeldahl Kjeldahl technique technique or or the the Dumas Dumas Method Method. . Two Two assumptions assumptions made made for for calculating calculating protein protein content content from from that that of of nitrogen nitrogen; ; first, first, that that all all the the N N of of the the food food is is present present as as protein protein second, second, that that all all food food protein protein contains contains 160 160 g g N/kg N/kg. . So, So, N N- -content content of of the the food food is is then then expressed expressed in in terms terms of of crude crude protein protein. . CP CP (g/kg) (g/kg) = = g g N/kg N/kg * * 1000 1000/ /160 160 or or CP CP (g/kg) (g/kg) = = g g N/kg N/kg * * 6 6. .25 25

Cont..... DIGESTIBLE DIGESTIBLE CRUDE CRUDE PROTEIN PROTEIN (DCP) (DCP) D Determined etermined by by digestibility digestibility trials trials. . When When CP CP content content of of feed feed stuffs stuffs is is multiplied multiplied by by digestibility digestibility coefficient coefficient of of protein protein gives gives the the DCP DCP. . M Most ost common common way way of of expressing expressing protein protein values values & & requirements requirements of of ruminants ruminants. . DCP DCP taken taken as as measure measure for for the the expressing expressing the the proteins proteins values values of of feeds feeds for for ruminants ruminants in in India India. .

Cont..... True True protein protein (TP) (TP) T Term erm TP TP is is used used to to denote denote the the proteins proteins only only. . C Can an be be separated separated from from NPN NPN compounds compounds by by precipitation precipitation with with cupric cupric hydroxide hydroxide. . Then Then N N estimation estimation performed performed by by kjeldahl kjeldahl method method. . Protein Protein equivalent equivalent (PE) (PE) NPN NPN fraction fraction is is given given half half the the nutritive nutritive value value of of true true protein protein. . C Calculated alculated as as PE PE = = ( (% % DCP DCP + + % % DTP) DTP) / / 2 2

Cont..... Metabolizable Metabolizable protein protein (MP) (MP) Microbial Microbial demand demand for for protein protein expressed expressed in in terms terms of of effective effective rumen rumen- -degradable degradable protein protein (ERDP) (ERDP) & & foods foods have have to to be be evaluated evaluated in in the the same same terms terms. . ERDP ERDP = = CP CP x x ( (0 0. .8 8a a + + bc bc / / (c (c + + r)) r)) Where Where; ; a, a, b b & & c c are are the the fitted fitted parameters parameters derived derived from from the the determination determination In In- -sacco sacco degradability degradability of of the the food, food, 0 0. .8 8 is is the the efficiency efficiency of of capture capture of of the the N N of of the the readily readily degradable degradable fraction, fraction, & & r r is is the the outflow outflow rate rate. .

Cont..... T The he differential differential capture capture of of rapidly rapidly & & slowly slowly degradable degradable proteins proteins & & the the rate rate of of passage passage through through the the rumen rumen. . D Demand emand for for AAs AAs at at tissue tissue level level is is quantified quantified in in terms terms of of truly truly digestible digestible protein protein required required to to be be absorbed absorbed from from SI SI & & designated designated as as MP MP . . Y Yield ield of of MCP MCP is is related related to to energy energy available available to to the the rumen rumen microorganisms microorganisms in in terms terms of of fermentable fermentable metabolizable metabolizable energy energy. . MEferm MEferm is is assumed assumed to to be be 0 0. .1 1 ME ME for for silages silages & & 0 0. .05 05 ME ME for for brewery brewery & & distillery distillery by by- -products, products, & & MEfat MEfat is is 35 35 MJ/kg MJ/kg. .

Cont..... The The assumption assumption made made here here for for silage silage must must be be suspect suspect. . M Major ajor fermentation fermentation product product in in well well- -made made silages silages is is lactate lactate, , & & several several rumen rumen bacteria, bacteria, notably notably Megasphaera Megasphaera elsdenii elsdenii are are able able to to utilise utilise lactate lactate with with the the production production of of propionate propionate. . MCP MCP yield yield (g) (g) is is calculated calculated as as follows follows: : FME FME (MJ)y (MJ)y Where Where; ; y y is is 9 9 at at maintenance, maintenance, 10 10 for for growth growth & & 11 11 for for lactation lactation; ; or or alternatively alternatively: : y y = = 7 7 + + 6 6( (1 1 - - e e- -0 0. .35 35L) L)

Cont..... P Proportion roportion of of MCP MCP present present as as true true protein protein is is assumed assumed to to be be 0 0. .75 75 & & the the true true digestibility digestibility to to be be 0 0. .85 85, , & & Contribution Contribution of of microbial microbial protein protein (DMP) (DMP) to to the the truly truly absorbed absorbed amino amino acids acids is is: : DMP DMP (g/kg (g/kg DM) DM) = = FME FME (y (y * * 0 0. .75 75 * * 0 0. .85 85) ) = = 0 0. .6375 6375( (FMEy FMEy) ) When When this this contribution contribution is is taken taken into into account, account, then then remains remains a a residual residual metabolisable metabolisable protein protein requirement, requirement, that that can can be be calculated calculated as as MP MP - - DMP DMP & & has has to to be be satisfied satisfied by by truly truly digestible digestible undegraded undegraded protein protein of of the the diet diet. .

Cont..... True True digestibility digestibility of of dietary dietary undegraded undegraded protein protein is is calculated calculated on on the the assumption assumption that that ADIN ADIN content content is is indigestible indigestible & & the the remainder remainder has has a a true true digestibility digestibility of of 0 0. .9 9. . So, So, truly truly digestible digestible undegraded undegraded true true protein protein (DUP) (DUP) is is then then: : DUP DUP = = 0 0. .95 95 (CP (CP ( (1 1 - - a a bc bc/(c /(c + + r)) r)) - - 6 6. .25 25 (ADIN) (ADIN) Where Where; ; a, a, b b & & c c are are the the usual usual In In sacco sacco constants constants & & DUP, DUP, CP CP & & ADIN ADIN are are g/kg g/kg DM DM. . The The metabolizable metabolizable protein protein supplied supplied by by the the food food may may be be calculated calculated as as MP MP (g/kg (g/kg DM) DM) = = DMP DMP + + DUP DUP. .

Cont..... M Metabolizable etabolizable protein protein content content of of a a food food is is of of no no use use as as a a guide guide to to satisfy satisfy the the residual residual demand demand for for MP, MP, which which includes includes a a contribution contribution from from ERDP ERDP & & has has already already been been taken taken into into account account in in the the form form of of DMP DMP. . So, So, P Protein rotein content content of of foods foods is is thus thus stated stated in in terms terms of of ERDP ERDP & & DUP DUP. .

Cont..... RDP RDP & & RUP RUP Rumen Rumen Degradable Degradable Protein Protein (RDP) (RDP) is is the the fraction fraction of of Crude Crude Protein Protein consumed, consumed, which which is is broken broken down down by by rumen rumen microbes microbes. . Where, Where, the the r remaining emaining protein protein which which reaches reaches SI SI without without degradation degradation called called as as Rumen Rumen undegradable undegradable protein protein (UDN) (UDN). . NRC NRC ( (2001 2001) ) suggests suggests that that maximum maximum milk milk & & milk milk protein protein yields yields occur, occur, when when RDP RDP is is 12 12. .2 2% % of of diet diet dry dry matter matter. .

Cont..... Rumen Rumen undegradable undegradable protein protein (UDN) (UDN) is is also also called called Bypass Bypass P Protein rotein or or R Rumen umen U Undegradable ndegradable Protein Protein (RUP) (RUP). . P Portion ortion of of intake intake protein protein that that escapes escapes rumen rumen degradation degradation & & is is digested digested directly directly in in the the SI SI. . About About 80 80 to to 85 85 % % of of the the microbial microbial protein protein & & or or true true protein protein that that flows flows out out of of the the rumen rumen is is digested digested in in SI SI & & is is expressed expressed as as a a % % of of crude crude protein protein. .

Discussions... Questions, if any ?? THANKS 19-08-2025 Kaushalendra Kumar, BVC, BASU, Patna 40