JOURNAL OF THE SCIENTIFIC AGRICULTURAL SOCIETY OF FINLAND Maataloustieteellinen Aikakauskirja 15 Voi. S4: IS-24, 1982 The degradation and utilization of formaldehyde-treated urea by rumen microbes in vitro JOUKO SETÄLÄ and LIISA SYRJÄLÄ-QVIST Department of Animal Husbandry, University of Helsinki, 00710 Helsinki 71, Finland Abstract. Urea was treated with different levels of formaldehyde (HCHO). The HCHO percentages, on a weight basis, were 0 (F 0), 0.25 (F 0.25) 0.50 (F 0,50), 0.75 (F 0,„), 1.0 (F10), 1.5 (F l s), 2.0 (F 2. 0), 3.0 (F 3. 0) and 5.0 (F 5O). Twenty milligrams of urea was incubated for 5 hours in 40 ml of sheep rumen fluid-buffer solution (1:1) together with 1.5 grams of substrate. The substrate consisted of vacuum-dried and milled feeds: barley (25 %), molassed beet pulp (25 %) and NaOH-treated straw (50 %). The feeds and urea were used in the sameproportions as in the diet of the sheep which yielded the rumen fluid for incubation. Treatment with HCHO decreased hydrolysis of urea to ammonia. The ammonia concentration in contents of fermentors 2 hours after the start of incubation had a highly significant (P < 0.001) negative correlation (r = -0.976, n = 72) with the HCHO treatment level. Microbial protein synthesis was calculated from tungstic acid - sulphuric acid precipitation. Synthesis of protein, expressed as grams of nitrogen per 100 grams fermented organic matter was highest when F, 5-Fj.o urea was used. Treatment with more than 3 % of HCHO decreased the number of protozoa and the general activity of the microbes, thus decreasing fermentation of organic matter and lowering the yield of microbial protein. When F, s urea was used, the total yield (mg protein/hr) was significantly higher than with untreated urea, but the results obtained with F,, s urea did not differ significantly from those with Fc 75 or F 2C urea. Introduction Since the studies of HART et al. (1939), urea has been used in the diets of ruminants. Problems have been caused by the very rapid degradation of urea to ammonia in the rumen. Hydrolysis is often too rapid compared with the capacity of the rumen microbes to utilize ammonia (BLOOMFIELD et al. 1960). This means that ammonia is absorbed through the rumen wall into the blood stream (LEWIS 1957), and levels higher than one percent of NH3-N in the blood cause a risk of ammonia toxification (CHALUPA 1968). The energy source is a very important factor, when ammonia utilization is considered (MOLLER 1973, JOHNSON 1976). Besides using an appropriate energy source, it is necessary to maintain coupled fermentation, a balance between ammonia and energy release (McMENIMAN et al. 1976). This balance could be achieved by lowering the rate of urea hydrolysis. Both urease inhibitors (MAHADEVAN et al. 1976) and slow-release urea products such as https://www.c-info.fi/en/info/?token=xsAiXaSLjfGVZF4h.YccNYINzzDv8uC20eiCPSg.PpKkZDdiua_Bj3A47HOG8ky0h5q9fBTGdXNid9fv95XkYsqvkP1IPhjmqcN5pYRgEC4G1P4GGZTsbDsBlho0MqpF6bS75LYFeqmH7YKxQJJ3u0Rr3HeFMF4IyQyAyLtMrCUyTUZCsy81TjHPDR6fjMwQM0muBUyV7I_xpGjRglMS1N4rf1RZ6z0lM2t9B8I6dLwOSNmIwUSM7iR2q9kCJXuF87pvqA 16 urea-formaldehyde complexes (MILLIGAN et al. 1969, HUSTON et al. 1974, KULASEK et al. 1975) have been used. In practical feedings, however, it seems easier to use slow-release urea than inhibitors. In this experiment the effect of formaldehyde treatments on urea utiliza- tion was studied. A formaldehyde-urea complex has already been used as a fertilizer, but a new processing technique made it seem desirable to test this complex and its possibilities in ruminant feeding as well. Materials and methods Preparation of formaldehyde-urea Formaldehyde (HCHO) was first mixed with urea and water. The liquid mixture contained 26.1 % of urea, 59.9 % of HCHO and 14.0 % of water on a weight basis. Urea slurry was then reacted with this solution. The reaction time was about three minutes and the reaction temperature 4-130°C. After cooling the product was made into prills. The complex was prepared by Kemira Ltd. In vitro method The method was based on the technique used by TILLEY and TERRY (1963), but restricted to incubation in strained rumen fluid and buffer solution (McDOUGALL 1948). Rumen fluid was collected from rumen-fistulated sheep. Their diet consisted of a mixture of barley and molassed beet pulp (1:1), and NaOH- treated wheat straw, both given at the rate of 0.5 kg/animal/day. Rumen contents were taken from different parts of the rumen in a warmed (+39°C) insulated flask before the morning feeding. The contents were squeezed through four layers of cheese-cloth and the fluid was collected directly in a flask held in a water bath (+39°C), and gassed with C0 2 . Warmed (+39°C) buffer solution was then added to the flask and the mixture of rumen fluid and buffer solution (1:1) was gassed with C02 until the pH of the mixture was 6.9. The experimental substrate (not urea) was weighed into fermentors 16-20 hours before the start of incubation and kept in +39°C. The fermentors were glass tubes (100 ml) fitted with rubber stoppers with gas release valves. The incubation time was 5 hours and it started after the rumen fluid- buffer had been added the fermentors. After its addition the tubes were gassed with C02 for about 5 sec. and transferred to an incubator (+39°C). During incubation the pH of the fermentors was followed carefully and kept within the range 6.6-6.9 with warmed 2N Na 2C0 3 . The incubation substrate was 0.75 grams of NaOH-treated wheat straw, 0.75 grams of the concentrate mixture given to the donor animals and 0.020 grams of urea. Before incubation the straw and the mixture were vacuum- dried (+6O°C) and milled with a 1-mm screen. The urea was treated with the following percentages of HCHO, on a weight basis: 0(F 0), 0.25 (F o 2s), 0.50 (Fo.so), 0.75 (F0.75), 1.0 (F,.0 ), 1.5 (F l5), 2.0 (F 2 .0), 3.0 (F 3. 0) and 5.0 (F s, o ).The substrate was incubated in 40 ml of buffer-rumen fluid solution, and there were 10-12 incubations per treatment level, except for levels F 3 0 and F 5 0, for which only four incubations per treatment were performed. The dry matter contents of components in the substrate were determined by oven heating at 100°C or in the analysis for urea, by Fisher titration. The nitrogen content of urea was calculated by the Kjeldahl method. The vacuum-dried samples of straw, barley and molassed beet pulp were analyzed by the methods of PALOHEIMO (1969). True protein content was determined according to BARNSTEIN (1935). The release of ammonia from the urea was followed at intervals of an hour from the start of incubation. For this purpose, there were two fermen- tors per incubation hour. The fermentors were removed and their contents were centrifuged at 2000 rpm for 10 min. The sediment was discarded, and the pH and ammonia content (McCUL- LOUGH 1967) of the supernatant were determined. The release of ammonia from urea was calculated according to principles of DINIUS et. al. (1974). After 5 hours’ incubation microbial protein was determined on the supernatant by sodium tungstate - sulphuric acid precipitation. Protein was determined as nitrogen by the Kjeldahl method, using K 2S04 and HgO as catalysts. The method of WINTER et al. (1964) modified by SETÄLÄ (1981 a) was used. When the synthesis of microbial protein was calculated, account was taken of the protein content of both the substrate and rumen fluid before incubation. In the F O, Fi.q, F 3 0 and F 5 0 treatments the concentration of VFA was determined on the supernatants obtained from the rumen fluid before incubation and from the fermentor contents after incubation. The determina- tion was made by gas-liquid chromatography (HUIDA 1973). Statistical analyses The results were processed with a MONROE 1860 computer using its statistical programs. The differences between treatments means were tested by the Tukey test (STEEL and TORRIE 1960). Results Formaldehyde treatment decreased the dry matter content of urea (Table 1), but had no effect on its nitrogen content. The crude protein (N X 6.25) content of the substrate dry matter was calculated as about 13.2 % with urea and about 8.9 % without. The ammonia concentration in the fermentor contents was highest 2 hours after the start of incubation (Fig. 1). When less than 3 % HCHO was 2 17 18 Table 1. Composition of feeds and urea used in incubations. DM, % Ash Crude True Ether Crude N-free protein 1) protein extract fiber extracts %in DM Barley 87.6 2.7 13.5 12.4 2.1 5.3 76.3 Molassed beet pulp 86.7 9.1 13.2 7.8 0.3 15.4 61.8 NaOH-treated straw 81.6 9.7 4.1 3.7 0.7 45.8 39.7 Urea Untreated (F 0) 99.8 46.5 0.25 % of HCHO (F025) 99.5 46.6 0.50 % of HCHO (F 0,50) 99.7 46.5 0.75 % of HCHO (F 0.„) 99.4 46.7 1.0% ofHCHO(F10 ) 99.4 46.5 1.5 % of HCHO (F,.5 ) 99.3 46.5 2.0 % of HCHO (F 2,0) 99.1 46.5 3.0 % of HCHO (F 3,0) 98.9 46.5 5.0 % of HCHO (F 5.0) 98.2 46.5 *) Values for urea given as N % used, the concentration decreased towards the end of incubation. The ammonia levels in the contents of fermentors after 2 hours’ incuba- tion showed a significant negative correlation (r = -0.976***) with the HCHO treatment levels (Fig. 2). The proportion of untreated urea hydrol- Figure 1. Changes in ammonia concentration in fermentor contents, when feed substrate was incubated alone (FS) or together with ureas (o F O, • F0.75, A F,.0 , AF,S,*F2 0 , □ F3O , ■FS 0 . Fo-F5 0) see Table 1). 19 ysed to ammonia during the first 2 hours was calculated as 97 %. In the HCHO treatments, the F lO urea (1 % HCHO) was the most strongly degraded. In preliminary tests the hydrolysis of F 0 25-F0 50 urea varied widely and these treatments were excluded from further experiments. When the HCHO treatment level was plotted directly against the ammonia concentration in the contents of fermentors, four different levels of urea degradation were found. With treatments of F 0 Fj 5- 2 0, Fj.o and F5 .o, the percentages of urea hydrolysed to ammonia were after 2 hours’ incubation, respectively: 91-94, 82-84, 62 and 44. Microbial protein synthesis calculated as mg/hr was significantly higher (P < 0.01) with F0.75, F) 5 and F 2 .o urea than with the substrate (Fig. 3, Table 2). Addition of FO, F lO and F 3 0 urea to the substrate did not increase protein synthesis significantly. There were no significant differences between the HCHO treatments within these two groups (Table 2), but the difference between these groups was significant (P < 0.01). Treatment with 5 % HCHO had a clearly inhibitory effect on protein synthesis under these in vitro conditions. The results of the F 5 incubations were significantly lower (P < 0.01) than the values obtained with the feed substrate alone and with substrate complemented with untreated urea. When the microbial protein synthesis was calculated as g N/100 g fermented organic matter (OMF), the optimum treatment level was (Table 2). HCHO affected the fermentation and hence the final VFA concentration in the fermentor contents. However, VFA differed significant- ly (P < 0.05) only between the treatment levels F0-i.o and F 5 0. Within the limited pH range used here HCHO caused only minor changes in the nature of fermentation during the incubation period. Figure 2. Effect of HCHO treat- ment on peak values of ammonia concentration in fermentor con- tents after 2-hr incubation (n = 72). 20 Table 2. Volatile fatty acids (VFA), fermentation of organic matter (OM) and microbial protein synthesis in incubations containing urea treated with different levels of HCHO (Fq-Fs o, see Table 1). Incubation FEED SUBSTRATE + UREA Fo F 0.75 Fi.o Fi.s F 2.0 F3O F 5.0 Total VFA after incubation, mmol/1 1) 209’ - 220’ - - 184ab 178b Mole-% of VFA Acetic 70 71 - 67 72 Propionic 20 - 17 - - 19 19 Isobutyric, Butyric 9.7 - 13.0 - - 12.0 7.6 Isovaleric, Valeric 0.3 - + - - 2.0 1.4 OM fermented (OMF), %2 ) 83.5’ - 87.8’ - - 73.7’ b 71.lb Microbial protein synthesis g N/100 g OMF3 ) 1.45 1.81 1.90 2.18 2.11 2.08 1.30 mg protein/hr/40 ml 7.2' 11.5d 8.4' 12.9d 11.7d 8.5 C 4.4' g HCHO/100 g crude protein in whole substrate 0.11 0.15 0.23 0.30 0.45 0.75 Differences between means with different letters are statistically significant: a - b (P < 0.05), c - e (P < 0.01) *) Adjusted for the same initial concentration before incubation. 2 ) Calculated on basis of VFA yield (CZERKAWSKI 1978). 3) Calculated with the formula y = 1.45+0.57X-0.12X2 (SETÄLÄ 1981a), in which X = HCHO treatment, %. Discussion The mechanism of the influence of the HCHO treatment on the rate of urea hydrolysis is very likely the chemical bonds between urea and formal- Figure 3. Effect of HCHO treatment on microbial protein synthesis, when feed substrate (FS) was incubated alone or together with untreated and HCHO-treated ureas. 21 dehyde. At least this is the case when protein is treated with formaldehyde (VAN DOOREN 1972) and it has not been shown that formaldehyde can for instance, inhibit the action of urease. In the experiments of MILLIGAN et al. (1969) and HUSTON et al. (1974) HCHO-treated urea was degraded more slowly to ammonia than untreated urea. When the degradation values for the different HCHO treatments are considered, two points should be noted. Firstly, the values were calculated after 2 hours’ incubation, when the ammonia concentration was at its peak. After this ammonia decreased, due to its utilization in protein synthesis, and it would have been misleading to calculate the degradation at the end of incubation. However, it is possible that considerable degradation of F 3O and F 5O urea occurred during the incubation period from 3 to 5 hours. Secondly we assumed in our calculations that the ’’endogenous ammonia” originating from the rumen fluid, was not changed during incubation. This assumption was probably true, because due to lysis of rumen microbes, changes in the ’’endogenous ammonia concentration” have been observed only when no substrate was included in the incubation (GÖRSCH and BERGNER 1978, SETÄLÄ 1981b). The ammonia level in the fermentor contents cannot have been a factor limiting microbial protein synthesis. The levels were higher than the sug- gested requirements for maximal protein synthesis (SATTER and SETTER 1974, NIKOLIC et al. 1975, SETTER et al. 1979). It, therefore appears that the energy available for microbes (JOHNSONI976, McMENIMAN et al. 1976) was the most important factor in the present conditions. The rate of energy release achieved when urea was treated with 1.5-3.0 % HCHO evidently gave the optimum ammonia/energy ratio for protein synthesis. After treatment levels lower than 1.5 % HCHO, the ammonia/energy ratio was too high. At higher treatment levels there was less ammonia, but, judging from the lower fermentation of organic matter (SETÄLÄ 1981a), also less available energy and the yield of microbial protein was poor. The relatively high yield of protein obtained with F 3 0 urea was also probably partly due to a higher proportion of bacteria in the microbiota of the fermentor contents (MERCER et al. 1980). Changes in the yield of total VFA and in the fermentation pattern at the treatment level of 5 % HCHO may partly be explained by the death of protozoa. In qualitative studies with a microscope it was noted that there were fewer protozoa in the F 3 0 incubations and that they were totally absent after incubation with F5O urea (see also BIRD and LENG 1978). HEMPEL- ZAWITKOWSKA and KULASEK (1974) suggested that formaldehyde could affect the protozoa population at as low a level as 3 % HCHO. THORNTON et al. (1977) suggested that although formaldehyde might not affect the number of microbes, it could decrease their activity at levels of 0.2-0.5 % in the diet. The low protein synthesis with F lO urea was rather unexpected, because the amount of fermented organic matter was highest when this urea was used. 22 Since the ammonia release from F lO urea was also high, it is possible that the reaction between urea and HCHO was not properly balanced. This may also apply to the F0 .25 and F SO urea. According to KRALOVEC and MORGAN (1954), the ratio of urea to HCHO has an important influence on the character of the complex. The values obtained for microbial protein synthesis agreed with those cited in the review by STERN and HOOVER (1979). It should be pointed out that our values represent mainly bacterial protein, because the protozoa were separated with feed particles by centrifugation (WARNER 1966). Nor can the possibility be excluded that feed particles and soluble feed protein were left in the supernatant from which the microbial protein was extracted by precipita- tion. The separation of microbial protein from feed protein is difficult and the other methods also have their shortcomings (see SETÄLÄ 1981 a). According to microscopic studies, contamination by feed particles in the supernatant must have been small. HILLER and Van SLYKE (1922) showed that tungstate precipitated peptides besides protein. CZERKAWSKI (1978) there- fore suggested that when protein is determined as the nitrogen precipitated, the results should be multiplied by 0.7 to obtain microbial protein synthesis on normal diets. This correction was made in the calculation of our results. Acknowledgements. The authors wish to express their acknowledgements to Kemira Ltd. for preparing the treated ureas and to Mr. Risto Kauppinen and Miss Marja Palm for the technical assistance during the experiment. References BARNSTEIN, F. 1935. Bestimmung der Reinproteins. Handbuch der Lebensmittelchemie 11. p. 607 Berlin. BIRD, S. H. & LENG, R. A. 1978. The effects of defaunation of the rumen on the growth of cattle on low-protein high-energy diets. Br. J. Nutr. 40; 163-167. BLOOMFIELD, R. A., GARNER, G. B. & MOHRER, M. E. 1960. Kinetics of urea metabolism in sheep. J. Anim. Sci. 19: 1248. CHALUPA, W. 1968. Problems in feeding urea to ruminant. J. Anim. Sci. 27: 207-219. CZERKAWSKI J. W. 1978. Reassesment of efficiency of synthesis of microbial matter in the rumen. J. Dairy Sci. 61; 1261-1273. DINIUS, D. A., LYON, C. K. & WALKER, H. G., Jr. 1974. In vitro evaluations of protein and protein- safflower oil complexes treated with formaldehyde. J. Anim. Sci. 38: 467-474. GÖRSCH, R. & BERGNER, H. 1978. Priifung einiger Harnstoffderivate unter in vitro-Bedingungen. Arch. Tierern. 30: 221-228. HART, E. 8., BOHSTEDT, G., DEOBALD, H. J. & WEGNER, M. I. 1939. The utilization of sample nitrogenous compounds such as urea and ammonium bicarbonate by growing calves. J. Dairy Sci. 22; 785-798. HEMPEL-ZAWITKOWSKA, J. & KULASEK, G. 1974. Influence of nitrogen components in the sheep’s diet on the protozoa counts and protozoa genera in the rumen. Prace i Materialy Zootechniczne 6: 13-22. HILLER, A. & SLYKE, Van D. D. 1922. A study of certain protein precipitants. J. Biol. Chem. 53: 253-267. HUIDA, L. 1973. Quantitative determination ofvolatile fatty acids from rumen sample and silage by gas- liquid chromatography. J. Scient. Agric. Soc. Finl. 45: 483—488. 23 HUSTON, J. E., SHELTON, B. & BREUER; L. H. 1974. Effect of rate of release of urea on its utilization by sheep. J. Anim. Sci. 39: 618-628. JOHNSON, R. R. 1976. Influence of carbohydrate solubility on nonprotein nitrogen utilization in the ruminant. J. Anim. Sci. 43; 184-191. KRALOVEC, R. D. & MORGAN, W. A. 1954. Condensation products of urea and formaldehyde as fertilizer with controlled nitrogen availability. J. Agric. Food Chem. 2; 92-95. KULASEK, G., LEONTONOWICZ, H. & KRASICKA, B. 1975. Urea-furfural polymer (UFP) as a protein replacer for sheep. Roczniki Nauk Rolniczych, ser. B. 96; 67-76. LEWIS, D. 1957.Blood-urea concentration on relation to protein utilization in the ruminant. J. agric. Sci., Camb. 48: 438-446. MAHADEVAN, S., SAUER, F. & ERFLE, J. D. 1976. Studies on bovine rumen bacterial urease. J, Anim. Sci. 42; 745-753. McCULLOUGH, H. 1967. The determination of ammonia in whole blood by a direct colorimetric method. Clin. Chem. Acta 17: 297-304. McDOUGALL, E. I. 1948. Studies on ruminant saliva 1. The composition and output of sheep’s saliva. Anal. Biochem. 43; 99-109. Mc MENIMAN, N. P., BEN-GHEDALIA, D. & ARMSTRONG, D. G. 1976. Nitrogen-energy interactions in rumen fermentation. Protein metabolism and nutrition. EAAP pubi. 16: 217-227. MEGGISON, P. A., McMENIMAN, N. P. & ARMSTRONG, D. G. 1979. Efficiency of microbial protein synthesis in cattle. Proc. Nutr. Soc. 38; 146A. MERCER, J. R., ALLEN SARAH, A. & MILLER E. L. 1980.Rumen bacterial protein synthesis and the proportion of dietary protein escaping degradation in the rumen of sheep. Br. J. Nutr. 43; 421-433. MILLIGAN. L. P„ ROBBLEE.A. R., WOOD,]. C„ KAY, W. C. & CHAKRABARTTY, S. K. 1969.A new non-protein nitrogen source for ruminants. Can. J. Anim. Sci. 49; 135-141. MÖLLER, P. D. 1973. The influence of different carbohydrate sources on the utilization of urea nitrogen by lactating cows. 412. Beretn. Landokon. Forsogslab. 204 p. NIKOLIC, J. A., JOVANOVIC, M. & FILIPOVIC, R. 1975. Microbial protein synthesis by bovine rumen content in relation to ammonia concentration. Tracer studies on non-protein nitrogen for ruminants 11. p. 43-54. lAEA, Vienna. PALOHEIMO, L. 1969. Weender Analyse. Handb. der Tierern. I. p. 164-171. Hamburg. SATTER, L. D. & SLYTER, L. L. 1974. Effect of ammonia concentration on rumen microbial protein production in vitro. Br. J. Nutr. 32: 199—208. SETÄLÄ, J. 1981 a. Formaldehydi-urean hyväksikäyttö märehtijällä. Lie. thesis, Univ. Helsinki, Dept, of Anim. Husb. 95 p. 1981 b. Unpublished observation. SLYTER, L. L., SATTER, L. D. & DINIUS, D. A. 1979. Effect of ruminal ammonia concentration on nitrogen utilization by steers. J. Anim. Sci. 48: 906-912. STEEL, R, G. & TORRIE, J. H. 1960. Principles and procedures of statistics. New York. 481 p. STERN, M. D. & HOOVER, W. H. 1979. Methods for determining and factors affecting rumen microbial protein synthesis: a review. J. Anim. Sci. 49: 1590-1603. THORNTON, J. H., OWENS, F. N., WILLIAMS, D. E. & PRIGGE, E, C. 1977. Fermentation and digestion of formaldehyde treated ensiled high moisture corn grain. Anim, Sci. Res. Rep., Oklahoma Agric. Exp. Stat. 101: 62—67. TILLEY, J. M. A. & TERRY, R. A. 1963. A two-stage technique for in vitro digestion of forage crops. J. Br. Grass!. Soc. 18: 104-111. VAN DOOREN, P. 1972. The mode of action of formaldehyde on proteins. Repr. 129 p. WARNER, A. C. I. 1966. Diurnal changes in the concentrations of micro-organisms in the rumens of sheep fed limited diets once daily. J. Gen. Microbiol. 45: 213-235. WINTER, K. A., JOHNSON, R. R. & DEHORITY, B. A. 1974. Metabolism of urea nitrogen by mixed cultures of rumen bacteria grown on cellulose. J. Dairy Sci. 47: 793—797. Ms received March 3, 1982. 24 SELOSTUS Formaldehydillä käsitellyn urean hajoaminen ja hyväksikäyttö in vitro -olosuhteissa Jouko Setälä ja Liisa Syrjälä-Qvist Helsingin yliopiston kotieläintieteen laitos. 00710 Helsinki 71 Tutkimuksessa selvitettiin formaldehydi (FICHO)-käsittelyjen vaikutusta urean hajoamiseen ja ureatypen hyväksikäyttöön. Formaldehydi-tasot olivat 0 % (F 0), 0.25 % (F 0 25), 0.5 % (Fo .s), 0.75 % (Fo.rs), 1.0 % (F| 0), 1.5 % (F, j), 2.0 % (F2O ), 3.0 % (F3O ) and 5.0 % (F so) formaldehydiä painoprosent- teina. Urea (20 mg) inkuboitiin yhdessä rehusubstraatin (1.5 g) kanssa. Rehusubstraatti muodostui vakuumi-kuivatusta ja analyysimyllyllä jauhetusta ohrasta (25 %), melassileikkeestä (25 %) ja kuivalipeöidystä oljesta (50 %). Inkubointi suoritettiin pötsineste-puskuriliuos (1:1) -seoksessa (40 ml) ja inkubaatioajan pituus oli viisi tuntia. Käytetyt rehut ja niiden keskinäiset suhteet vastasivat tarkasti ruokintaa, jota käytettiin lampaille, joilta pötsineste otettiin inkubaatiota varten. Formaldehydi-käsittely vähensi urean hajoamista ammoniakiksi. Fermentorin sisällöstä kahden tunnin kuluttua inkubaation alusta mitatun ammoniakin määrän ja HCHO-käsittelytason välillä oli merkitsevä (P < 0.001), negatiivinen (r = -0.976, n = 72) korrelaatio. Mikrobiproteiinisynteesi analysoitiin wolframaatti-rikkihappo-saostuksen avulla. Fermentoitunutta orgaanista ainetta kohti laskettuna synteesi oli suurin, kun käytettiin Fl5 -, F2O- ja F 3O-ureaa. Kolmen prosentin formaldehydi-käsittelystä lähtien formaldehydi alensi alkueläinten lukumäärää, käymisen voi- makkuutta ja orgaanisen aineen sulavuutta inkubaation aikana. Kokonaisproteiinisynteesi (mg proteiinia/ hr) oli merkitsevästi (P < 0.01) suurempi käsittelemättömällä urealla saatuun synteesiin verrattuna, kun käytettiin F, 5 -ureaa. Tällä urealla saadut tulokset eivät poikenneet merkitsevästi F0.75- ja F2O -urealla saaduista proteiinisynteesin arvoista.