Maataloustieteellinen Aikakauskirja Vol. 56: 155—161, 1984 In vitro evaluation of protein digestibility in the abomasum and small intestine of ruminants JOUKO SETÄLÄ, HANNA VÄÄTÄINEN 1 and TERTTU ETTALA Valio Finnish Co-operative Dairies’ Association, Research and Development Department, P.O. Box 176, SF-00181 Helsinki, Finland Abstract. The investigation included studies with microbial protein, 0,5 % formaldehyde (HCHO) treated protein in soybean meal, and ruminally undegradable feed proteins in cotton seed and sunflower seed cakes. Microbial mass was separeted with centrifugations from the rumen contents of roughage-fed sheep. Undegradable feed proteins were prepared with the nylon bag technique. The proteins were digested in solutions of pepsin-HCI and trypsin-chymotrypsin. The average digestibility for microbial protein was 80.7 %. The corresponding values for the un- degradable proteins in cottonseed cake and sunflower seed cake were 83.7 % and 82.7 %. In the incubation with pepsin-HCI, pH in the solution affected significantly (P < 0.001) the di- gestibility of HCHO-treated protein in soybean meal. The average digestibilities at pH 1.5—2.0, pH 2.5—3.0, and pH 3.1—4.0 were 97.2, 76.0, and 71.0 %, respectively. Introduction In vitro tests of protein digestibility have mainly been used for non-ruminants. In the studies for ruminants Kowalczyk et al. (1977, 1978 a, b) used abomasal fluid from a fistulated animal and they also evaluated protein digestibilities with rats. Multi-enzyme tests were used when the digestibilities of ru- minally undegradable feed proteins (in sac- co) were analyzed. In nylon bag studies in vitro methods would most obviously be rele- vant, because the digestibility of undegrad- able protein has to be estimated from a very small sample. The digestibility of microbial protein separated from the rumen contents could also be determined (e.g. Bergen et al. 1967). The aim of the present study was to inves- tigate possibilities to use in digestibility anal- yses a method which included protein treat- ments with pepsin-HCI and trypsin-chymo- trypsin. This method was chosen, because it Department of Animal Flusbandry, University of Hel- sinki, SF-00710 Helsinki, Finland Present address: Agricultural Research Center, Department of Animal Flusbandry, SF-31600 Jokioinen, Finland Index words: digestibility, protein, in vitro, ruminant 155 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=22mrZtd9iJzaOwhC.-f4jM2YGz92yTKo3ioSzDg.3v_2QDQ--T2osSoBeQuwGqBc0KrHZA6HTTZTcpPqIaNzaZFHBDqoIpcu9wzQpasy27ClpWFNrAMysMahcJ40aIKP_0j2p_laT7moChcewWaXtSC9f1A7k5CoNMHQKBwZb-5KWp-9WVa7JNYXfA4WqkbgIUPem3VXhYdmBbIFmOgJL7h2QVFCMryMpKUPx_erz7jnW4w36Y2VCUQGKEJZv3YlmMNBuebMpUW_k_hBZTAzys78md8 was reported e.g. by Mozersky and Panet- tieri (1983) that the methods based on a pH drop in the incubations (e.g. Hsu et al. 1977), would not be applicable to proteins of which the in vivo digestibility is not known. The present study included investigations mainly with the protein in microbial mass, which was separated from the rumen con- tents of sheep. The digestibility of ruminally undegradable protein in sunflower seed cake, cottonseed cake, and formaldehyde treated protein (0.5 °7o treatment level) in soybean meal was also studied. Materials and methods In vitro technique The technique was modified using pro- cedures described in the literature, mainly those by Akeson and Stahman (1964), and Anon (1970). The procedure was divided into two phases, incubations with pepsin- HCI and with trypsine and chymotrypsine. In the first phase samples of 500 mg were in- cubated at + 39°C in pepsin-HCI solution for 16 hours. Pepsin (Merck 7190, 1 : 10000, 2000 FIP-U/g) concentration in the solution of 0.05 N HCI was 0.1 °/o. The ratio of the solution to the sample protein was 0.7 ml/mg protein. The amount of pepsin-HCI was hence changed according to the amount of protein in the sample, because Van Bruc- hem and Van’t Klooster (1980) suggested that proteins stimulated abomasal secretions of acid in vitro. Incubations were made in glass tubes (volume 100 ml) with rounded bottoms and the tubes were carefully shaken at the beginning of the incubations. The pH in the incubations was always kept under pH 2.0. In the second phase contents of the tubes were centrifuged at the speed of 400 g for 10 min. Supernatant in the tube was carefully discarded and the sediment was neutralized (pH 7.0) using washings with phosphate buf- fer (pH 7.0). The contents of the tubes were centrifuged as described above and the sedi- ment was taken for further analyses. Solutions of bovine chymotrypsine (Merck 2307, 45 U/mg) and bovine trypsine (Merck 24579, 3.5 U/mg) were prepared in phos- phate buffer (pH 7.0) using 50 mg of chy- motrypsine, or 5 mg of trypsine for 50 ml of the buffer. The solutions of trypsine and chymotryp- sine were added into the tubes so that the amounts were 10 ml and 1 ml, respectively. In addition, 9 ml of the buffer was transfer- red into the tubes. The incubations in the second phase were made (at pH 7) at + 39°C for 24 hours. After this the samples were centrifuged as de- scribed earlier. The supernatants in the tubes were carefully discarded and the sediments (undigestible protein of the sample) were transferred for the analysis of protein N. Analysis of protein N in the samples The sample was transferred into the Kjel- dahl bottle and incubated in 80 ml of dis- tilled water, at + 70°C for 1.5 hours. The contents of the bottles were carefully stirred 4—5 times during the incubation. After this incubation 1.5 ml of A1 2(S04)3K 2S04 - 12H,0, 15 ml of the solution of copper sulphate (156.4 g CuS04-5H 20 in 1000 ml of distilled water), and 15 ml of 2.5 % NaOH were added into the bottles. When the contents of the bottles were cool and clear they were squeezed through an N-free filter paper, and the sample on the fil- ter paper was washed with 750 ml of warm, distilled water. The sample and the paper were carried to N determination which was made using the Kjeldahl method but exclud- ing CuS04 as a catalyst. Blank tests includ- ing filter paper and reagents were used in all determinations. The method described is based on the procedure used at the Depart- ment of Animal Husbandry, University of Helsinki. 156 157 Separation of microbial mass from the rumen contents Microbial mass was separated from the ru- men contents of fistulated sheep in the hay or hay and grass silage (65 : 35 on DM basis) diets. Rumen contents were collected from different parts of the rumen, squeezed through a cheese cloth, and the filtrate was centrifuged at 400 g for 5 min. The sediment was discarded and the filtrate was further centrifuged over 30 000 g at + 2°C for 20 min. The sediment was carefully separated and stored frozen for the analyses. Samples of feed proteins The samples of the formaldehyde treated protein in soybean meal, and ruminally un- degradable (in sacco) proteins in sunflower seed cake, and cotton seed cake were pre- pared by Dr. Torben Hvelplund, National Institute of Animal Science, Denmark (see Hvelplund 1983). Results and discussion Studies with microbialprotein Composition of the microbial mass Fresh, separated microbial mass was used in incubations. Samples were not dried be- fore incubation, because Wallace (1983) among others suggested that for instance freeze drying might affect digestibility. Crude protein (Nx6.25) content in the ran- dom-sampled (n = 7) mass was 37.1 ± 1.0 % in fresh weight. The proportion of protein N in total N was 86.7 ± 0.7 % (n = 7). Effect of HCI and the enzymes on digestibility According to our results HCI alone gave a poor digestibility value for microbial pro- tein (Table 1). Moreover, the combination of HCI and pepsin was not sufficient eith- er, and it could be suggested that one of the enzymes in the pancreatic fluid is needed for a proper protein digestion. However, pepsin-HCI might have more importance in the digestion of the protein of plant origin when the sample is less digestible owing to the fibrous structure etc. On the other hand, Rojahn and Wagner (1961) ob- served the difference of 9 %-units only in the digestibility of barley protein when the estimation was made either with pepsin- HCI (lower values) or with pepsin-HCI and pancreatin. Those incubations in which a stronger HCI was used, tended to give higher digest- ibilities for microbial protein. Korte (1979) wondered whether results of this kind could be caused by a better digestibility of nucleic acids in microbial cells. However, our results emphasized the importance of the enzymes of the pancreatin in the di- gestion of microbial protein. Digestibility of microbial protein When the digestibility of microbial pro- tein was determined with the pepsin-HCI and trypsin-chymotrypsin, the mean of the digestibility was 80.7 % (see Table 1). This value is the same average value which could be calculated (e.g. 80.4 %) from the papers of Tas et al. (1977), Hagemeister et Table 1. The effect of different factors on the digestibility-% of microbial protein in vitro. Digestibility-% x SEM N 0.05 N HCI 20.5 0.9 2 0.05 N HCI + pepsin 69.2 1.0 2 0.05 N HCI + pepsin, 86.1 0.3 2 chymotrypsin Trypsin + chymotrypsin 81.2 1.4 4 0.05 N HCI + pepsin, 80.7 0.8 14 chymotrypsin and trypsin 0.14 N HCI + pepsin, 86.5 1.1 4 chymotrypsin and trypsin N = number of incubations 158 al. (1980), Storm & orskov (1982), Hvelp- lund (1983), Storm et al. (1983), and Wal- lace (1983). In some of the incubations di- gestibility of microbial protein differed quite clearly from the mean value. This type of va- riation was also found by Buchmann (1979) between in vitro incubations. In the present study the coefficientof the variation (CV-%) of the results between the incubations was 3.7 %, being higher than reported by Buch- mann (1979). In the experiments reviewed from the liter- ature there was also a great variation in the results between different investigations. This can be partly explained by the different ex- perimental techniques. However, Bergen et al. (1967) and Wallace (1983) showed great differences in protein digestibility between bacterial strains and therefore part of the differences between different studies and in- cubations (present study) might be explained by the different composition of the bacterial mass digested. Ecpecially the increase in the proportions of Gram + (Wallace 1983) or cellulolytic bacteria (Bergen et al. 1967) in microbial mass can decrease digestibility of microbial protein. In our technique, occa- sional difficulties in squeezing the small sample in different phases of the work con- tributed also most evidently to the variation of the results. Studies with microbialprotein and feed protein Effect of pH and incubation period During the incubations the pH in the con- tents of the tubes was carefully followed. The corresponding values in pepsin-HCI in- cubations and in trypsin-chymotrypsin incu- bations were pH 1.6 ± 0.02 and pH 6.9 ± 0.01. It is doubtful, however, whether pH in the contents of the abomasum and small intes- tine remains so stable in vivo. In the experi- ment with goats von Engelhard? and Hauf- fe (1975) found pH 2.3 ±0.3 in the aboma- Table 2. Effect of incubation period in trypsin-chymotrypsin incubations and pH in pepsin-HCI incubations on the digestibility-% of microbial and feed proteins. „ . . Undegradable protein Protein in _ Microbial protein HCHO-soybean meal Sunflower seed cake Cottonseed cake 18 hours 24 hours 18 hours 24 hours 18 hours 24 hours 18 hours 24 hours pH 1.5—2.0 83.0 85.0 84.0. 83.0 98.0 97.0 97.0 97.0 84.0 83.0 85.0 83.0 85.0 85.0 86.0 86.0 x 84.0 x 83.5 x 97.5» x 97.0" x 83.5 x 84.0 x 85.0 x 86.0 pH 2.5—3.0 80.0 82.0 86.0 86.0 75.0 75.0 76.0 78.0 83.0 82.0 82.0 83.0 81.0 82.0 83.0 84.0 x 81.0 x 86.0 x 75.0 b x 77.0" x 82.5 x 82.5 x 81.5 x 83.5 pH 3.1—4.0 81.0 81.0 81.0 82.0 69.0 69.0 73.0 73.0 83.0 82.0 83.0 81.0 83.0 82.0 84.0 84.0 x 81.0 x 81.5 x 69.0 b x 73.0 b x 82.5 x 82.0 x 82.5 x 84.0 P < 0.001, a—b; meanbetween pH treatments with different letters differ significantly sum. McAllan (1981) reported an average value, pH 2.9, for steers and the pH varied form 2.50 to 3.17. Moreover, he also sug- gested that pH in the small intestine can vary being 5.69 at the beginning of the intestine and approaching pH 7.3 at the terminal end of the small intestine. In order to study the pH effect, the pH in pepsin-HCI incubations was adjusted to three different levels (Tables 2 and 3). How- ever, any clear changes in the digestibility of undegraded feed proteins and microbial pro- tein were not observed when the pH varied from 1.5 to 4.0. Ambrose and Snyder (1964) also found only a limited effect on protein digestibility caused by a change in the pH from 1.2 to 2.5. The digestibility of the for- maldehyde treated feed protein was signi- ficantly (P < 0.001) decreased when a pH higher than 1.5—2.0 was used. These results demonstrated the importance of acidic con- ditions for the release of formaldehyde-pro- tein complex as suggested for instance by Ferguson et al. (1967) although on the basis of their results as low a pH as in our study would not be necessary. The effect of the pH on the incubations with trypsin-chymotrypsin was not studied. Johnson et al. (1983) reported that a pH higher than 6.9 could change the activity of some enzymes of the pancreatin. At least this was the case with a-amylase. However, ac- cording to the paper of McAllan (1981) the effect of pH in vivo may not be significant in controlling the activity of trypsin and chy- motrypsin. When protein digestibilities in vitro are applied to in vivo a critical point might be the length of the incubation period, e.g. the retention time of the feed particles in the abomasum and in the small intestine of the ruminant. In pepsin-HCI incubations length of the incubation may not be important if the activity of pepsin is reasonably high (e.g. AO AC-standard). In that case Ambrose and Snyder (1964) did not report clear differ- ences in digestibilities evaluated after the pe- riods of 2,4, 6, or 16 hours. In the literature there is not very much in- formation available about the retention time of feed particles in the abomasum and small intestine. In one of the publications Colucci et al. (1982) reported retention times from 11 to 17 hours for concentrate and roughage in the omasum, abomasum, and small intestine. According to these findings an incubation period of 24 hours in our study was too long. When the incubation period was shortened to 18 hours, which is still reasonable regard- ing the practical work, there were very small and non-significant changes in the digest- ibility values of microbial and feed proteins (Table 2). The digestibilities of undegradable pro- teins in cotton seed cake and sunflower seed cake were, however, generally higher than reported in vivo by Hvelplund (1983). It is Table 3. Statistical parameters of the data in table 2 (analysis of variance, factorial experiment). Source of Sum of Degrees of Mean F-value variation squares freedom square Incubation 0.0083 1 0.0083 S (substrate) 33.4167 3 11.1389 P (period) 18.7500 1 18.7500 pH 588.2917 2 294.1458 36.2168*** SxpH 1005.7083 6 167.6181 20.6380*** PxpH 9.1250 2 4.5625 SxpHxP 19.2083 6 3.2014 Error 186.8017 23 8.1218 *** P < 0.001 159 possible that the in vitro conditions created optimum circumstances for the activity of enzymes and therefore higher digestibilities were obtained. In conclusion, the in vitro technique is a reasonable method in evaluation of protein digestibility in the lower digestive tract of a ruminant when it is used in studies with mi- crobial protein or nylon bags. However, the analysis has to be carefully made if HCHO- treated feeds are studied. It is clear that the described technique is relatively complicated and laborous, but it is more simple than the in vivo techniques generally available. However, direct compar- isons between in vitro and in vivo determina- tions have to be made, before in vitro results can be applied to in vivo conditions. References Akeson, W. R. & Stahman, M. A. 1964. A pepsin pancreatin digest index of protein quality evaluation. J. Nutr. 83: 257—261. Ambrose, M. E. & Snyder, D. G. 1964. Pepsin digest- ibility: As an index of quality in fish meal: Part I. Some studies in the USA. Fishing News International 3 (3): 210, 212—213. Anon 1970. Official methods of analysis of the Asso- ciation of Official Analytical Chemists. 11th ed. p. 127 —128. Washington. Bergen, W. G., Purser, D, B. & Cline, J. H. 1967. En- zymatic determination of the protein quality of indi- vidual rumen bacteria. J. Nutr. 92: 357—364. Buchmann, N. B. 1979. In vitro digestibility of protein from barley and other cereals. J. Sci. Fd. Agric. 30: 583—589. Colucci, P. E., Chase, L. E. & Van Soest, P, J. 1982. Feed intake, apparent diet digestibility, and rate of particulate passage in dairy cattle. J, Dairy Sci. 65: 1445—1456. Engelhardt, W., von & Hauffe, R. 1975. Role of the omasum in absorbtion and secretion of water and electrolytes in sheep and goats. Digestion and me- tabolism in the ruminant, ed. McDonald, I. W. and Warner, A. C. I. p. 222. The University of New En- gland pubi, unit. Ferguson, K. A., Hemslev, J. A. & Reis, P, J. 1967. The effect of protecting dietary protein from micro- bial degradation in the rumen. Aust. J. agric. Sci. 30: 215—217. Hagemeister, H., Lopping, E. & Kaufmann, W. 1980. Microbial protein synthesis and digestion in the high- yielding dairy cow. Resent advances in animal nutri- tion 1980, ed. Haresign, W. p, 67—84. Butter- worths. Hsu, H. W., Vavak, D. L., Satterlee, L. D. & Miller, G. A. 1977. A multienzyme technique for estimating protein digestibility. J. Food Sci. 42: 1269—1273. Hvelplund, T. 1983. Digestibility of rumen microbial protein and undegraded dietary protein in the small intestine of sheep. Protein metabolism and nutrition 11. p. 283—286. INRA. Johnson, D, D., Mitchell, G. E., Jr., Tucker, R. E. & Muntifiering, R. B. 1983. Ovine pancreatic alpha- amylase digestion of purified, feed, or abomasal starch as affected by pH, J. Anim. Sci. 57, suppl. I. Abstract 13, p. 6. Korte, H. 1979. Vergleichende Untersuchungen zur Proteinverdaulichkeit mit Hilfe der Pepsin-HCI- Methods. Diplomarbeit, Kiel, 47 p. Kowalczyk, J,, Pastuzewska, B. & Otwinowska, A. 1977. Biological value of protein for rats and diges- tibility of nutrients of a diet containing formalde- hyde-treated rapeseed oilmeal. Rocz. Nauk Rolnicz. 98 B: 91—97. —, Jaczewska, A. & Drozdz, A. 1978 a. Decomposi- tion of protein in formaldehyde treated or untreated feeds by the contents of the rumen or abomasum in vitro. Rocz. Nauk Rolnicz. 98 B: 17—24. —, Bartik, M. & Jaczewska, A, 1978 b. A note on en- zymic digestion of protein treated with formaldehyde. Rocz. Nauk Rolnicz. 99 B: 123—127. McAllan, A. B. 1981. Changes in the composition of digesta during passage through the small intestines of steers. Br. J. Nutr. 46: 431—440. Mozerskv, S. M. & Panettieri, R. A. 1983. Is pH drop a valid measure of extent of protein hydrolysis. J. Ag- ric. Food Chem. 31: 1313—1316. Rojahn, J. & Wagner, J. 1961. In vitro Verdaulich- keitsversuche des Rohproteins und einiger Aminosäu- ren der Nackt- und Spelzgerste. Arch, fur Tierern. 11: 145—156, Storm, E. & Orskov, E. R. 1982. Biological value and digestibility of rumen microbial protein in lamb small intestine. Proc. Nutr. Soc. 41: 78 A. —, ORSKOV, E. R. & Smart, R. 1983. The nutritive val- ue of rumen micro-organisms in ruminant. 2. The ap- parent digestibility and net utilization of microbial N for growing lambs, Br. J. Nutr, 50: 471 —478. Tas, M, V., Axford, R. F. E. & Evans, R. A. 1977. 160 The digestibility of rumen microbial protein in the small intestines of sheep. Proc, Nutr. Soc. 36: 79 A Wallace, R. J. 1983. Digestion of rumen bacteria in vitro. Br. J. Nutr. 49: 101 —108. Van Bruchem, J. & Vant Klooster, A. Th. 1980. Ef- fect of protein on abomasal secretion of acid in sheep. Br. J. Nutr. 44: 307—312. Ms received 16 May, 1984 SELOSTUS Proteiinin sulavuuden määrittäminen märehtijän juoksutusmahassa ja ohutsuolessa in vitro Jouko Setälä, Hanna Väätäinen 1 ja Terttu Ettala Valion tutkimus- ja tuotekehittelyosasto, PL 176, 00181 Helsinki Tutkimuksessa selvitettiin pötsin sisällöstä sentrifu- goimalla erotetun mikrobiproteiinin sekä nailonpussi- menetelmällä tuotetun pötsissä hajoamattoman rehu- proteiinin sulavuutta in viira -menetelmällä. Mikrobi- proteiini eristettiin lampailta, jotka olivat joko heinä- tai heinä-säilörehu -ruokinnalla. Tutkittavat rehut oli- vat auringonkukkakakku ja puuvillasiemenkakku. Li- säksi tarkasteltiin formaldehydi-käsittelyn (0,5 % -kä- sittely) vaikutusta soijarouheen valkuaisen sulavuuteen. In vitro -menetelmässä tutkittava substraatti inkuboi- tiin pepsiini-HCI- ja trypsiini-kymotrypsiini -liuoksessa. Mikrobiproteiinin keskimääräinen sulavuus oli 80,7 % ja aurinkokukkakakun sekä puuvillasiemenkakun pöt- sissä hajoamattoman proteiinin sulavuus vastaavasti 83,7 % ja 82,7 %. Pepsiini-HCI -inkubaatiossa käytetty pH vaikutti merkitsevästi (P < 0.001) formaldehydi-käsitellyn soi- jarouheen valkuaisen sulavuuteen. Sulavuudet pH-alueil- la11.2.0,5 —2.0,22.3.05—3.0 ja 33. 4.0—4.0 olivat vastaavasti 97,2 %, 76,0 % ja 71,0 °/o. 1 MTTK, kotieläinhoito-osasto, 31600 Jokioinen 161