- Email: [email protected]
A note on the fermentation characteristics and rumen degradation of low moisture alfalfa silage treated with sodium hydroxide or ammonia
ANIMAL FEED SCIENCE AND TECHNOLOGY
E LS EVIER
Animal Feed Science and Technology 47 (1994) 297-304
A note on the fermentation characteristics and rumen degradation of low moisture alfalfa silage treated with sodium hydroxide or ammonia N. Nishino *'a, M. Ohshima b, S.
Uchida a
aFaculty of Agriculture, Okayama University, Okayama 700, Japan bThe Farm, School of Agriculture, Nagoya University, Aichi 4 70-01, Japan (Received 30 March 1993; accepted 16 November 1993)
Abstract
The effects of alkali treatment on the fermentation characteristics and rumen degradation of low moisture alfalfa silage were investigated. First crop alfalfa was wilted and ensiled, either directly or after being sprayed with NaOH or NH3 solution ( 1.72% dry matter (DM)). The herbages were stored in laboratory silos and opened at 1, 2, 7, 14, 28 and 56 days after storage. Each NaOH and NH3 addition increased the pH value and delayed lactic acid production of the silage. Both alkali treatments decreased proteolysis during ensilage, and NaOHtreated silage showed the highest protein (hot water insoluble nitrogen) content. Nitrogen solubility in a mineral buffer solution was decreased by the alkali treatments, and NaOHtreated silage showed the lowest solubility. Degradation characteristics of DM and N in the rumen were determined by nylon bag incubation of 56th day silage samples with two rumen fistulated goats. The NaOH treatment significantly decreased the soluble N fraction (63.7%, 54.7% and 60.6% for untreated, NaOH-treated and NHa-treated silage, respectively) but did not influence the extent of degradation. The treatment tended to decrease the rate of N degradation. In contrast, the NH3 treatment significantly increased the rate of DM degradation (0.158 h - ~, 0.151 h-1 and 0.182 h - ' for untreated, NaOH-treated and NHa-treated silage, respectively) without any great changes in the proportions of soluble and degradable fraction.
"Corresponding author. 0377-8401/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSDI0377-8401 (93)00582-G
298
N. Nishinoet al. / Animal FeedScienceand Technology47 (1994) 297-304
1. Introduction
There is evidence that NaOH and NH3 treatment could improve the digestibility of low quality forage having potentially available fibrous components (Klopfenstein et al., 1972; Braman and Abe, 1977; Ben-Ghedalia et al., 1988; Glenn, 1990). Beneficial effects as preservative for hay (Knapp et al., 1975 ) and barley grain (Orskov et al., 1979) were also reported. In addition to the benefits, recent research has found that those treatments could modify the rumen degradation of concentrate feed (Mir et al., 1984; Waltz and Loerch, 1986; Robinson and Kennelly, 1988a). Legume crops generally contain high protein and low fibrous components compared with grass, but the fiber digestibility of legumes is not high so that animals may receive an unbalanced supply of energy and protein. Legume protein is degraded in the rumen at a faster rate than grass protein (Osibe et al., 1987 ); therefore, when alkali treatment increased fiber digestibility and reduced the rate of ruminal protein degradation of alfalfa, animals could receive a better supply of nutrients. This study was undertaken to investigate the effects of NaOH and NH3 treatment on the fermentation characteristics and rumen degradation of low moisture alfalfa silage.
2. Materials and methods 2.1. Silage preparation
First crop alfalfa (Medicago sativa L.) was harvested at the early blooming stage on 26 April 1990. The cutting length of the material was about 5 era. The herbage was wilted for about 24 h in a glasshouse. For each treatment, 500 g of the wilted herbage was put in a grass bottle laboratory silo (800 ml volume ) after spraying with or without NaOH or NH3 solution at a rate of 1.72% of herbage dry matter (DM). The concentrations (w/v) of NaOH and NH3 solution were 50% and 28%, respectively. Twelve silos were prepared for each treatment and duplicate silos were opened after storage for 1, 2, 7, 14, 28 and 56 days at 25°C. 2.2. Nylon bag incubation
Two mature castrated goats equipped with rumen fistulae were used. They were given commercially purchased alfalfa haycube at 2% of their body weight daily in two equal meals at 08:00 and 20:00 h. Nylon bags, each of 42 ~tm pore size and containing 3 g of freeze dried 56th day sample, were inserted in the tureen and removed at 3, 6, 12, 24 and 48 h after incubation. Each sample from individual silos was incubated in the two different goats so that four observations were made for each treatment. Bags were washed in a domestic washing machine and then dried at 60°C for 48 h.
N. Nishino et al. / Animal Feed Science and Technology 47 (1994) 297-304
299
2.3. Analyses The DM content of silage was measured by freeze drying. Total nitrogen was determined by the Kjeldahl procedure. Analyses o f p H value, lactic acid and NH3N of the silages were made using cold water extracts (Nishino et al., 1991 ). Volatile fatty acids were determined by gas chromatography. Hot water insoluble nitrogen was recognized as protein nitrogen. Nitrogen solubility of the silage in bicarbonate-phosphate buffer (pH 6.80) was determined according to K_rishnamoorthy et al. ( 1982 ). In vitro DM digestibility was determined by the method of Tilley and Terry (1963) using rumen fluid taken from goats given alfalfa haycube. Each set of degradability values was fitted to the exponential equation P = a + b ( 1 - e - e l ) recommended by Orskov and McDonald ( 1979 ). The parameters a, b and c were estimated using the non-linear regression analysis procedure of the Statistical Analysis Systems Institute Inc. ( 1985 ), and their treatment means were compared using Duncan's multiple range test. The pH, lactic acid, acetic acid, NH3-N, hot water insoluble nitrogen and buffer insoluble nitrogen values of the silages were subjected to two-way analysis of variance to detect the effects of treatment, ensiling period and the interaction of treatment and ensiling period, using the general linear model (GLM) procedure.
3. Results
Total N of wilted herbage was 2.98% DM, and was increased to 3.27% by NH3 treatment. The DM content of silages averaged 54.8%, and was not greatly influenced by either alkali treatment or ensiling period. The changes in pH value and lactic acid, acetic acid and NHa-N concentrations over the ensiling period are shown in Fig. 1. The two alkali-treated silages consistently showed higher pH values than untreated silage, and NaOH treatment had a slightly but significantly greater effect than NH3 treatment (Fig. 1 (a)). The final pH values of untreated, NaOH-treated and NHa-treated silages were 4.71, 5.25 and 5.11, respectively, and the differences between the silages were similar to those found on the first day of ensiling. A rapid increase of lactic acid was observed on the second day in untreated silage, but in the alkali-treated silages, lactic acid gradually increased during the whole ensiling period (Fig. 1 (b)). The lactic acid concentrations of 56th day silages were about 6.5% DM. The acetic acid content showed no systematic relationship with the ensiling period, but was significantly decreased by NH3 treatment (Fig. 1 (c)). Trace amounts (about 0.1% DM) of butyric acid were detected in all the silages at every sampling, but the data are not shown in Fig. 1. The NH3-treated silage showed the highest NH3 concentration at every ensiling period. The final content was 18.8% compared with 11.7% recorded on the untreated and NaOH-treated silages, although the amount of increase after the first day was similar in all the silages. Except for acetic acid content, period effect was significant ( P < 0.05 ), but no significant interaction was detected.
300
N. Nishino et aL / Animal Feed Science and Technology 47 (1994) 297-304
\\ 6
a
8
---'<>--- Untreated ~ NaOILtreated ~ Nll3-treated
~- 6
~s
b
'2,
t
012
7
i
i
14 28 Days after ensiltng
2.0
H
56
i
i
012 7
i
)
14 28 Days after enslling
56
30
c
~ 1.5
Z
I.O
=
'~ < 0.5 L,
i
012
7
t
,
i
14 28 Days after ensiling
i
SG
012
7
i
i
i
14
28
56
Days after ensiltng
Fig. 1. Changes in pH value (a), and lactic acid (b), acetic acid (c) and NH3-N (d) concentration of low moisture untreated, NaOH-treated and NH3-treated alfalfa silages. Each point represents the mean + SE of duplicate silos. b 2'01 1.8
a
~-~
~ Untreated -----O--- NaOH-treated + Nli3-treated
1.6 ~
1"6i1"8
~# X 1.4
t.2
~ j21
1.0
~
J
012
7
)
i8
14 2 Days after enslling
1.0
,
56
i
012
7
i
,
14 28 Days after ensiling
i
56
Fig. 2. Changes in hot water insoluble nitrogen (a) and buffer insoluble nitrogen (b) content of untreated, NaOH-treated and NH3-treated low moisture alfalfa silages. Each point represents the mean + SE of duplicate silos.
Changes in hot water insoluble nitrogen (HWIN) and buffer insoluble nitrogen (BIN) contents are illustrated in Fig. 2. Both NaOH- and NH3-treated silages consistently showed higher HWIN than untreated silage (Fig. 2 (a)). The HWlN contents of the two alkali-treated silages were similar for the first 2 days; however, it became less in NH3-treated silage than in NaOH-treated silage in the later
N. Nishino et aL /Animal Feed Science and Technology 47 (1994) 297-304
301
Table 1 Parameters from the exponential equation P = a + b ( 1 - e - c t ) l used to describe in situ rumen degradation of dry matter and nitrogen of untreated, NaOH-treated and NH3-treated low moisture alfalfa silages 2 Untreated
NaOH-treated
NH3-treated
SE
Dry matter Soluble fraction (%) Degradable fraction (%) Rate of degradation ( h - 1 )
36.4 42.1 0.158 b
36.6 43.0 0. 151 b
33.4 44.4 0.182 a
1.42 1.01 0.0067
Nitrogen Soluble fraction (%) Degradable fraction (%) Rate of degradation ( h - 1)
63.7 a 27.7 b 0.211
54.7 b 36.2 a 0.190
60.6 a 30.7 b 0.221
1.74 1.51 0.0135
JP, disappearance of dry matter and nitrogen at time t; a, soluble fraction; b, degradable fraction; c, rate of degradation for fraction b. 2Means of four incubation measurements. Values in the same row with different superscripts are significantly different (P< 0.05 ).
ensiling period. Buffer insoluble nitrogen decreased similarly to HWlN during ensilage. The period effect was significant for both HWIN and BIN, and significant interaction was observed with HWIN content. In vitro DM digestibilities for untreated, NaOH-treated and NH3-treated silages stored for 56 days were 68.40/0, 70.7% and 70.5°/0, respectively. The digestibility of untreated silage was significantly lower than those of NaOH-treated and NH3-treated silages. Parameters describing the degradation characteristics of DM and N in the rumen are shown in Table 1. At 48 h incubation, both DM and N of the silages were degraded to almost the maximum amount, and each set of degradability values fitted close to the exponential equation, as judged from the coefficient of determination which was above 0.99 in all cases. The two alkali treatments did not affect the proportions of soluble and degradable fractions of DM. The rates of DM degradation for untreated, NaOH-treated and NH3-treated silage were 0.158 h-J, 0.151 h-~ and 0.182 h-1, respectively. A significant increase was observed in the case of the NH3 treatment. The soluble N fraction of untreated silage was 63.7% but the fraction was significantly reduced to 54.7% by NaOH treatment. The NH3 treatment caused a non-significant decrease of the fraction in spite of the addition of soluble nitrogen as NH3 at ensiling. The NaOH-treated silage contained a larger degradable N fraction than the other silages. The rate of N degradation tended to decrease with NaOH treatment but the difference was not significant. 4. Discussion
The delay in lactic acid production with NH3 treatment was in agreement with the result of corn silage treated with 1% NH3 (Johnson et al., 1982). However,
302
N. Nishino et al. / Anirnal Feed Science and Technology 47 (1994) 297-304
Kung et al. (1984) reported that treatment with 0.7-1.2% NH3 stimulated lactic acid production of alfalfa silage, even though the extent was affected by the DM content of the silage. Flipot et al. ( 1976 ) found that when alfalfa was treated with 3% NaOH, lactate fermentation was enhanced or restricted according to its DM content. The gradual increase of the lactic acid content in both the alkali-treated silages observed in this study might suggest a possibility of further fermentation after 56 days, when all the silages showed almost the same lactic acid concentration. The sustained difference in HWlN content between treated and untreated silages, after the first day of ensiling, indicates that both alkali treatments have greater effects on the reduction of protein breakdown in the initial ensiling period than in the later period. An initial high pH caused by alkali treatment would decrease the plant proteases activity, because its optimum pH ranged around 5.5 (Finley et al., 1980). The difference in response to HWlN reduction between NaOH and NH3-treated silage may suggest that the two alkalis have different effects (pH, osmotic pressure, water activity, etc. ) on microorganisms which also have the proteolytic activity. A significant difference in acetic acid content between the two alkali-treated silages could support the above hypothesis. A slight increase of in vitro DM digestibility by alkali treatment may be due to an improvement of fiber digestibility, although this increase contrasts with the lack of NaOH treatment effect on DM degradation kinetics. As the Tilley and Terry in vitro technique could overestimate in vivo DM digestibility of NaOHtreated forage (Berger et al., 1979) and in situ degradation kinetics did not involve the outflow rate of tureen digesta and the digestion in the lower gut, the different response would be obtained. With late cut alfalfa, the degradation kinetics of DM might be changed, as was found with low quality roughage, while the effect of alkali treatment on in vivo fiber digestibility of alfalfa has not been consistently evaluated (Kung et al., 1989; Glenn, 1990; Nishino et al., 1993 ). The possible role of NH3 treatment for a modification of rumen degradation was described using barley grain as the material (Robinson and Kennelly, 1988a,b). The authors indicated that NH3 treatment increased the degradable fraction of DM, but decreased solubility and rate of DM degradation in the rumen. Ammonia treatment inversely affected the rate of degradation in this experiment. Nitrogen solubility has been considered a factor affecting ruminal protein degradation (Chalupa et al., 1963; Aitchison et al., 1976), while it does not necessarily relate to susceptibility to enzymatic degradation (Mahadevan et al., 1980). The HWlN content of each silage clearly paralleled the BIN content, because the non-protein nitrogen of silage exists in the soluble form. Mir et al. (1984) found that when soya-bean meal was subjected to NaOH treatment, the solubility of protein in the rumen was greatly decreased. In contrast, Waltz and Loerch (1986) reported the NaOH treatment reduced the rate of protein degradation. Our previous study using alfalfa silage treated with 2.44% NaOH (Nishino et al., 1993 ) showed a reduction in the rate of protein degradation in the rumen. In the present experiment, the rate of degradation also seems to decrease by NaOH treatment.
N. Nishino et al. / Animal Feed Science and Technology 47 (1994) 297-304
303
A well known change following alkali treatment of protein is the formation of crosslink amino acids such as lysinoalanine (Finot, 1983). Although amino acid analysis was not performed in this study, our recent experiment suggested that lysinoalanine could be generated only when alfalfa was treated with 4.6% NaOH or more. However, the solubility of nitrogen decreased at a lower treatment level (2.3% NaOH). Other mechanisms might also be responsible for the reduction of nitrogen solubility of alfalfa silage by NaOH treatment. In general, a high proportion of non-protein and soluble nitrogen in naturally fermented alfalfa silage is extensively and rapidly degraded in the rumen and much of the nitrogen is lost as ammonia unless readily available carbohydrate is supplied. The addition of a small amount of NaOH is effective in reducing the formation of soluble nitrogen during ensilage and in enhancing protein utilization by ruminants.
5. Acknowledgment The authors are grateful to Miss Wakita for her care of the goats and helpful assistance in the nylon bag study.
6. References Aitchison, T.E., Mertens, D.R., McGiUiard, A.D. and Jacobson, N.L., 1976. Effect of nitrogen solubility on nitrogen utilization in lactating dairy cattle. J. Dairy Sci., 59:2056-2062. Ben-Ghedalia, D., Yosef, E. and Solomon, R., 1988. Effects of ammonia treatment and stage of maturity of coastal bermuda grass on monosaccharide residue composition and digestibility by steers. J. Sci. Food Agric., 45:1-8. Berger, L., Klopfenstein, T. and Britton, R., 1979. Effect of sodium hydroxide on efficiency of rumen digestion. J. Anita. Sci., 49:1317-1323. Braman, W.L. and Abe, R.K., 1977. Laboratory and in vivo evaluation of the nutritive value of NaOHtreated wheat straw. J. Anim. Sci., 46:496-505. Chalupa, W., Evans, J.L. and Stillions, M.C., 1963. Nitrogen source availability and activity of rumen microorganisms. J. Dairy Sci., 46:1431-1432. Finley, J.W., Pallavicini, C. and Kohler, G.O., 1980. Partial isolation and characterisation of Medicago sativa leaf proteases. J. Sci. Food Agric., 31:156-161. Finot, P.A., 1983. Influence of processing on the nutritional value of proteins. In: C.E. Bodwell and L. Petit (Editors), Plant Proteins for Human Food. Nijhoff/W. Junk, The Hague, Netherlands, pp. 235-249. Flipot, P., Mowat, D.N., Parkins, J.J. and Buchanan-Smith, J.G., 1976. Ensiling characteristics of silages treated with sodium hydroxide. Can. J. Plant Sci., 56:935-940. Glenn, B.P., 1990. Effects of dry matter concentration and ammonia treatment of alfalfa silage on digestion and metabolism by heifers. J. Dairy Sci., 73:1081-1090. Johnson, C.O.L.E., Huber, J.T. and Bergen, W.G., 1982. Influence of ammonia treatment and time of ensiling on proteolysis in corn silage. J. Dairy Sci., 65:1740-1747. Klopfenstein, T.J., Krause, V.E., Jones, M.J. and Woods, W., 1972. Chemical treatment of low quality roughages. J. Anim. Sci., 35:418-422. Knapp, W.R., Holt, D.A. and Lechtenberg, V.L., 1975. Hay preservation and quality improvement by anhydrous ammonia treatment. Agron J., 67:766-769.
304
N. Nishino et al. / Animal Feed Science and Technology 47 (1994) 297- 304
Krishnamoorthy, U., Muscato, T.V., Sniffen, C.J. and van Soest, P.J., 1982. Nitrogen fractions in selected feedstuffs, J. Dairy Sci., 65:217-225. Kung, L., Jr., Grieve, D.B., Thomas, J.W. and Huber, J.T., 1984. Added ammonia and microbial inocula for fermentation and nitrogenous compounds of alfalfa ensiled at various percents of dry matter. J. Dairy Sci., 67:299-306. Kung, L., Jr., Craig, W.M. and Satter, L.D., 1989. Ammonia-treated alfalfa silage for lactating dairy cows. J. Dairy Sci., 72:2565-2572. Mahadevan, S., Erfle, J.D. and Sauer, F.D., 1980. Degradation of soluble and insoluble proteins by Bacteroides amylophilus protease and by rumen microorganisms. J. Anita. Sci., 50:723-728. Mir, Z., Mad-cod, G.IC, Buchanan-Smith, J.G., Grieve, D.G. and Grovum, W.L., 1984. Methods for protecting soybean and canola proteins from degradation in the rumen. Can. J. Anita. Sci., 64:853865. Nishino, N., Ohshima, M. and Yokota, H., 1991. Nutritive value of rice straw ensiled with intact or alkalized Italian ryegrass (Lolium multiflorum, LAM) green juice. J. Jpn. Grassl. Sci., 37:203212. Nishino, N., Ohshima, M. and Yokota, H., 1993. Digestion of alkali-treated alfalfa silage by goats. Asian-AustralasianJ. Anim. Sci., 6:5-11. ~rskov, E.R. and McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci., 92:499-503. Orskov, E.R., Stewart, C.S. and Greenhalgh, LED., 1979. The effect of sodium hydroxide and urea on some storage properties of moist grain. J. Agric. Sci., 92:185-188. Osibe, A., Ogawa, M. and Masubuchi, T., 1987. The degradability of crude forage protein in the rumen of sheep. I. A comparison of leguminous and gramineous crude protein degradability. Bull. Natl. Grassl. Res. Inst., 37:58-63. Robinson, P.H. and Kennelly, J.J., 1988a. Ammonia and sulphur dioxide treatment of high-moisture barley on in situ rumen degradabilityand in situ whole-tract digestibility.Can. J. Anim. Sci., 68:779786. Robinson, P.H. and Kenneily, J.J., 1988b. Influence of ammoniation of high moisture barley on its in situ rumen degradation and influence on rumen fermentation in dairy cows. Can. J. Anim. Sci., 68:839-851. Statistical Analysis Systems Institute Inc., 1985. SAS User's Guide: Statistics. SAS Institute Inc., Cary, NC. Tilley, J.M.A. and Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Sot., 18:104-111. Waltz, D.M. and Loerch, S.C., 1986. Effect of acid and alkali treatment of soybean meal on nitrogen utilization by ruminants. J. Anim. Sci., 63:879-887.
E LS EVIER
Animal Feed Science and Technology 47 (1994) 297-304
A note on the fermentation characteristics and rumen degradation of low moisture alfalfa silage treated with sodium hydroxide or ammonia N. Nishino *'a, M. Ohshima b, S.
Uchida a
aFaculty of Agriculture, Okayama University, Okayama 700, Japan bThe Farm, School of Agriculture, Nagoya University, Aichi 4 70-01, Japan (Received 30 March 1993; accepted 16 November 1993)
Abstract
The effects of alkali treatment on the fermentation characteristics and rumen degradation of low moisture alfalfa silage were investigated. First crop alfalfa was wilted and ensiled, either directly or after being sprayed with NaOH or NH3 solution ( 1.72% dry matter (DM)). The herbages were stored in laboratory silos and opened at 1, 2, 7, 14, 28 and 56 days after storage. Each NaOH and NH3 addition increased the pH value and delayed lactic acid production of the silage. Both alkali treatments decreased proteolysis during ensilage, and NaOHtreated silage showed the highest protein (hot water insoluble nitrogen) content. Nitrogen solubility in a mineral buffer solution was decreased by the alkali treatments, and NaOHtreated silage showed the lowest solubility. Degradation characteristics of DM and N in the rumen were determined by nylon bag incubation of 56th day silage samples with two rumen fistulated goats. The NaOH treatment significantly decreased the soluble N fraction (63.7%, 54.7% and 60.6% for untreated, NaOH-treated and NHa-treated silage, respectively) but did not influence the extent of degradation. The treatment tended to decrease the rate of N degradation. In contrast, the NH3 treatment significantly increased the rate of DM degradation (0.158 h - ~, 0.151 h-1 and 0.182 h - ' for untreated, NaOH-treated and NHa-treated silage, respectively) without any great changes in the proportions of soluble and degradable fraction.
"Corresponding author. 0377-8401/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSDI0377-8401 (93)00582-G
298
N. Nishinoet al. / Animal FeedScienceand Technology47 (1994) 297-304
1. Introduction
There is evidence that NaOH and NH3 treatment could improve the digestibility of low quality forage having potentially available fibrous components (Klopfenstein et al., 1972; Braman and Abe, 1977; Ben-Ghedalia et al., 1988; Glenn, 1990). Beneficial effects as preservative for hay (Knapp et al., 1975 ) and barley grain (Orskov et al., 1979) were also reported. In addition to the benefits, recent research has found that those treatments could modify the rumen degradation of concentrate feed (Mir et al., 1984; Waltz and Loerch, 1986; Robinson and Kennelly, 1988a). Legume crops generally contain high protein and low fibrous components compared with grass, but the fiber digestibility of legumes is not high so that animals may receive an unbalanced supply of energy and protein. Legume protein is degraded in the rumen at a faster rate than grass protein (Osibe et al., 1987 ); therefore, when alkali treatment increased fiber digestibility and reduced the rate of ruminal protein degradation of alfalfa, animals could receive a better supply of nutrients. This study was undertaken to investigate the effects of NaOH and NH3 treatment on the fermentation characteristics and rumen degradation of low moisture alfalfa silage.
2. Materials and methods 2.1. Silage preparation
First crop alfalfa (Medicago sativa L.) was harvested at the early blooming stage on 26 April 1990. The cutting length of the material was about 5 era. The herbage was wilted for about 24 h in a glasshouse. For each treatment, 500 g of the wilted herbage was put in a grass bottle laboratory silo (800 ml volume ) after spraying with or without NaOH or NH3 solution at a rate of 1.72% of herbage dry matter (DM). The concentrations (w/v) of NaOH and NH3 solution were 50% and 28%, respectively. Twelve silos were prepared for each treatment and duplicate silos were opened after storage for 1, 2, 7, 14, 28 and 56 days at 25°C. 2.2. Nylon bag incubation
Two mature castrated goats equipped with rumen fistulae were used. They were given commercially purchased alfalfa haycube at 2% of their body weight daily in two equal meals at 08:00 and 20:00 h. Nylon bags, each of 42 ~tm pore size and containing 3 g of freeze dried 56th day sample, were inserted in the tureen and removed at 3, 6, 12, 24 and 48 h after incubation. Each sample from individual silos was incubated in the two different goats so that four observations were made for each treatment. Bags were washed in a domestic washing machine and then dried at 60°C for 48 h.
N. Nishino et al. / Animal Feed Science and Technology 47 (1994) 297-304
299
2.3. Analyses The DM content of silage was measured by freeze drying. Total nitrogen was determined by the Kjeldahl procedure. Analyses o f p H value, lactic acid and NH3N of the silages were made using cold water extracts (Nishino et al., 1991 ). Volatile fatty acids were determined by gas chromatography. Hot water insoluble nitrogen was recognized as protein nitrogen. Nitrogen solubility of the silage in bicarbonate-phosphate buffer (pH 6.80) was determined according to K_rishnamoorthy et al. ( 1982 ). In vitro DM digestibility was determined by the method of Tilley and Terry (1963) using rumen fluid taken from goats given alfalfa haycube. Each set of degradability values was fitted to the exponential equation P = a + b ( 1 - e - e l ) recommended by Orskov and McDonald ( 1979 ). The parameters a, b and c were estimated using the non-linear regression analysis procedure of the Statistical Analysis Systems Institute Inc. ( 1985 ), and their treatment means were compared using Duncan's multiple range test. The pH, lactic acid, acetic acid, NH3-N, hot water insoluble nitrogen and buffer insoluble nitrogen values of the silages were subjected to two-way analysis of variance to detect the effects of treatment, ensiling period and the interaction of treatment and ensiling period, using the general linear model (GLM) procedure.
3. Results
Total N of wilted herbage was 2.98% DM, and was increased to 3.27% by NH3 treatment. The DM content of silages averaged 54.8%, and was not greatly influenced by either alkali treatment or ensiling period. The changes in pH value and lactic acid, acetic acid and NHa-N concentrations over the ensiling period are shown in Fig. 1. The two alkali-treated silages consistently showed higher pH values than untreated silage, and NaOH treatment had a slightly but significantly greater effect than NH3 treatment (Fig. 1 (a)). The final pH values of untreated, NaOH-treated and NHa-treated silages were 4.71, 5.25 and 5.11, respectively, and the differences between the silages were similar to those found on the first day of ensiling. A rapid increase of lactic acid was observed on the second day in untreated silage, but in the alkali-treated silages, lactic acid gradually increased during the whole ensiling period (Fig. 1 (b)). The lactic acid concentrations of 56th day silages were about 6.5% DM. The acetic acid content showed no systematic relationship with the ensiling period, but was significantly decreased by NH3 treatment (Fig. 1 (c)). Trace amounts (about 0.1% DM) of butyric acid were detected in all the silages at every sampling, but the data are not shown in Fig. 1. The NH3-treated silage showed the highest NH3 concentration at every ensiling period. The final content was 18.8% compared with 11.7% recorded on the untreated and NaOH-treated silages, although the amount of increase after the first day was similar in all the silages. Except for acetic acid content, period effect was significant ( P < 0.05 ), but no significant interaction was detected.
300
N. Nishino et aL / Animal Feed Science and Technology 47 (1994) 297-304
\\ 6
a
8
---'<>--- Untreated ~ NaOILtreated ~ Nll3-treated
~- 6
~s
b
'2,
t
012
7
i
i
14 28 Days after ensiltng
2.0
H
56
i
i
012 7
i
)
14 28 Days after enslling
56
30
c
~ 1.5
Z
I.O
=
'~ < 0.5 L,
i
012
7
t
,
i
14 28 Days after ensiling
i
SG
012
7
i
i
i
14
28
56
Days after ensiltng
Fig. 1. Changes in pH value (a), and lactic acid (b), acetic acid (c) and NH3-N (d) concentration of low moisture untreated, NaOH-treated and NH3-treated alfalfa silages. Each point represents the mean + SE of duplicate silos. b 2'01 1.8
a
~-~
~ Untreated -----O--- NaOH-treated + Nli3-treated
1.6 ~
1"6i1"8
~# X 1.4
t.2
~ j21
1.0
~
J
012
7
)
i8
14 2 Days after enslling
1.0
,
56
i
012
7
i
,
14 28 Days after ensiling
i
56
Fig. 2. Changes in hot water insoluble nitrogen (a) and buffer insoluble nitrogen (b) content of untreated, NaOH-treated and NH3-treated low moisture alfalfa silages. Each point represents the mean + SE of duplicate silos.
Changes in hot water insoluble nitrogen (HWIN) and buffer insoluble nitrogen (BIN) contents are illustrated in Fig. 2. Both NaOH- and NH3-treated silages consistently showed higher HWIN than untreated silage (Fig. 2 (a)). The HWlN contents of the two alkali-treated silages were similar for the first 2 days; however, it became less in NH3-treated silage than in NaOH-treated silage in the later
N. Nishino et aL /Animal Feed Science and Technology 47 (1994) 297-304
301
Table 1 Parameters from the exponential equation P = a + b ( 1 - e - c t ) l used to describe in situ rumen degradation of dry matter and nitrogen of untreated, NaOH-treated and NH3-treated low moisture alfalfa silages 2 Untreated
NaOH-treated
NH3-treated
SE
Dry matter Soluble fraction (%) Degradable fraction (%) Rate of degradation ( h - 1 )
36.4 42.1 0.158 b
36.6 43.0 0. 151 b
33.4 44.4 0.182 a
1.42 1.01 0.0067
Nitrogen Soluble fraction (%) Degradable fraction (%) Rate of degradation ( h - 1)
63.7 a 27.7 b 0.211
54.7 b 36.2 a 0.190
60.6 a 30.7 b 0.221
1.74 1.51 0.0135
JP, disappearance of dry matter and nitrogen at time t; a, soluble fraction; b, degradable fraction; c, rate of degradation for fraction b. 2Means of four incubation measurements. Values in the same row with different superscripts are significantly different (P< 0.05 ).
ensiling period. Buffer insoluble nitrogen decreased similarly to HWlN during ensilage. The period effect was significant for both HWIN and BIN, and significant interaction was observed with HWIN content. In vitro DM digestibilities for untreated, NaOH-treated and NH3-treated silages stored for 56 days were 68.40/0, 70.7% and 70.5°/0, respectively. The digestibility of untreated silage was significantly lower than those of NaOH-treated and NH3-treated silages. Parameters describing the degradation characteristics of DM and N in the rumen are shown in Table 1. At 48 h incubation, both DM and N of the silages were degraded to almost the maximum amount, and each set of degradability values fitted close to the exponential equation, as judged from the coefficient of determination which was above 0.99 in all cases. The two alkali treatments did not affect the proportions of soluble and degradable fractions of DM. The rates of DM degradation for untreated, NaOH-treated and NH3-treated silage were 0.158 h-J, 0.151 h-~ and 0.182 h-1, respectively. A significant increase was observed in the case of the NH3 treatment. The soluble N fraction of untreated silage was 63.7% but the fraction was significantly reduced to 54.7% by NaOH treatment. The NH3 treatment caused a non-significant decrease of the fraction in spite of the addition of soluble nitrogen as NH3 at ensiling. The NaOH-treated silage contained a larger degradable N fraction than the other silages. The rate of N degradation tended to decrease with NaOH treatment but the difference was not significant. 4. Discussion
The delay in lactic acid production with NH3 treatment was in agreement with the result of corn silage treated with 1% NH3 (Johnson et al., 1982). However,
302
N. Nishino et al. / Anirnal Feed Science and Technology 47 (1994) 297-304
Kung et al. (1984) reported that treatment with 0.7-1.2% NH3 stimulated lactic acid production of alfalfa silage, even though the extent was affected by the DM content of the silage. Flipot et al. ( 1976 ) found that when alfalfa was treated with 3% NaOH, lactate fermentation was enhanced or restricted according to its DM content. The gradual increase of the lactic acid content in both the alkali-treated silages observed in this study might suggest a possibility of further fermentation after 56 days, when all the silages showed almost the same lactic acid concentration. The sustained difference in HWlN content between treated and untreated silages, after the first day of ensiling, indicates that both alkali treatments have greater effects on the reduction of protein breakdown in the initial ensiling period than in the later period. An initial high pH caused by alkali treatment would decrease the plant proteases activity, because its optimum pH ranged around 5.5 (Finley et al., 1980). The difference in response to HWlN reduction between NaOH and NH3-treated silage may suggest that the two alkalis have different effects (pH, osmotic pressure, water activity, etc. ) on microorganisms which also have the proteolytic activity. A significant difference in acetic acid content between the two alkali-treated silages could support the above hypothesis. A slight increase of in vitro DM digestibility by alkali treatment may be due to an improvement of fiber digestibility, although this increase contrasts with the lack of NaOH treatment effect on DM degradation kinetics. As the Tilley and Terry in vitro technique could overestimate in vivo DM digestibility of NaOHtreated forage (Berger et al., 1979) and in situ degradation kinetics did not involve the outflow rate of tureen digesta and the digestion in the lower gut, the different response would be obtained. With late cut alfalfa, the degradation kinetics of DM might be changed, as was found with low quality roughage, while the effect of alkali treatment on in vivo fiber digestibility of alfalfa has not been consistently evaluated (Kung et al., 1989; Glenn, 1990; Nishino et al., 1993 ). The possible role of NH3 treatment for a modification of rumen degradation was described using barley grain as the material (Robinson and Kennelly, 1988a,b). The authors indicated that NH3 treatment increased the degradable fraction of DM, but decreased solubility and rate of DM degradation in the rumen. Ammonia treatment inversely affected the rate of degradation in this experiment. Nitrogen solubility has been considered a factor affecting ruminal protein degradation (Chalupa et al., 1963; Aitchison et al., 1976), while it does not necessarily relate to susceptibility to enzymatic degradation (Mahadevan et al., 1980). The HWlN content of each silage clearly paralleled the BIN content, because the non-protein nitrogen of silage exists in the soluble form. Mir et al. (1984) found that when soya-bean meal was subjected to NaOH treatment, the solubility of protein in the rumen was greatly decreased. In contrast, Waltz and Loerch (1986) reported the NaOH treatment reduced the rate of protein degradation. Our previous study using alfalfa silage treated with 2.44% NaOH (Nishino et al., 1993 ) showed a reduction in the rate of protein degradation in the rumen. In the present experiment, the rate of degradation also seems to decrease by NaOH treatment.
N. Nishino et al. / Animal Feed Science and Technology 47 (1994) 297-304
303
A well known change following alkali treatment of protein is the formation of crosslink amino acids such as lysinoalanine (Finot, 1983). Although amino acid analysis was not performed in this study, our recent experiment suggested that lysinoalanine could be generated only when alfalfa was treated with 4.6% NaOH or more. However, the solubility of nitrogen decreased at a lower treatment level (2.3% NaOH). Other mechanisms might also be responsible for the reduction of nitrogen solubility of alfalfa silage by NaOH treatment. In general, a high proportion of non-protein and soluble nitrogen in naturally fermented alfalfa silage is extensively and rapidly degraded in the rumen and much of the nitrogen is lost as ammonia unless readily available carbohydrate is supplied. The addition of a small amount of NaOH is effective in reducing the formation of soluble nitrogen during ensilage and in enhancing protein utilization by ruminants.
5. Acknowledgment The authors are grateful to Miss Wakita for her care of the goats and helpful assistance in the nylon bag study.
6. References Aitchison, T.E., Mertens, D.R., McGiUiard, A.D. and Jacobson, N.L., 1976. Effect of nitrogen solubility on nitrogen utilization in lactating dairy cattle. J. Dairy Sci., 59:2056-2062. Ben-Ghedalia, D., Yosef, E. and Solomon, R., 1988. Effects of ammonia treatment and stage of maturity of coastal bermuda grass on monosaccharide residue composition and digestibility by steers. J. Sci. Food Agric., 45:1-8. Berger, L., Klopfenstein, T. and Britton, R., 1979. Effect of sodium hydroxide on efficiency of rumen digestion. J. Anita. Sci., 49:1317-1323. Braman, W.L. and Abe, R.K., 1977. Laboratory and in vivo evaluation of the nutritive value of NaOHtreated wheat straw. J. Anim. Sci., 46:496-505. Chalupa, W., Evans, J.L. and Stillions, M.C., 1963. Nitrogen source availability and activity of rumen microorganisms. J. Dairy Sci., 46:1431-1432. Finley, J.W., Pallavicini, C. and Kohler, G.O., 1980. Partial isolation and characterisation of Medicago sativa leaf proteases. J. Sci. Food Agric., 31:156-161. Finot, P.A., 1983. Influence of processing on the nutritional value of proteins. In: C.E. Bodwell and L. Petit (Editors), Plant Proteins for Human Food. Nijhoff/W. Junk, The Hague, Netherlands, pp. 235-249. Flipot, P., Mowat, D.N., Parkins, J.J. and Buchanan-Smith, J.G., 1976. Ensiling characteristics of silages treated with sodium hydroxide. Can. J. Plant Sci., 56:935-940. Glenn, B.P., 1990. Effects of dry matter concentration and ammonia treatment of alfalfa silage on digestion and metabolism by heifers. J. Dairy Sci., 73:1081-1090. Johnson, C.O.L.E., Huber, J.T. and Bergen, W.G., 1982. Influence of ammonia treatment and time of ensiling on proteolysis in corn silage. J. Dairy Sci., 65:1740-1747. Klopfenstein, T.J., Krause, V.E., Jones, M.J. and Woods, W., 1972. Chemical treatment of low quality roughages. J. Anim. Sci., 35:418-422. Knapp, W.R., Holt, D.A. and Lechtenberg, V.L., 1975. Hay preservation and quality improvement by anhydrous ammonia treatment. Agron J., 67:766-769.
304
N. Nishino et al. / Animal Feed Science and Technology 47 (1994) 297- 304
Krishnamoorthy, U., Muscato, T.V., Sniffen, C.J. and van Soest, P.J., 1982. Nitrogen fractions in selected feedstuffs, J. Dairy Sci., 65:217-225. Kung, L., Jr., Grieve, D.B., Thomas, J.W. and Huber, J.T., 1984. Added ammonia and microbial inocula for fermentation and nitrogenous compounds of alfalfa ensiled at various percents of dry matter. J. Dairy Sci., 67:299-306. Kung, L., Jr., Craig, W.M. and Satter, L.D., 1989. Ammonia-treated alfalfa silage for lactating dairy cows. J. Dairy Sci., 72:2565-2572. Mahadevan, S., Erfle, J.D. and Sauer, F.D., 1980. Degradation of soluble and insoluble proteins by Bacteroides amylophilus protease and by rumen microorganisms. J. Anita. Sci., 50:723-728. Mir, Z., Mad-cod, G.IC, Buchanan-Smith, J.G., Grieve, D.G. and Grovum, W.L., 1984. Methods for protecting soybean and canola proteins from degradation in the rumen. Can. J. Anita. Sci., 64:853865. Nishino, N., Ohshima, M. and Yokota, H., 1991. Nutritive value of rice straw ensiled with intact or alkalized Italian ryegrass (Lolium multiflorum, LAM) green juice. J. Jpn. Grassl. Sci., 37:203212. Nishino, N., Ohshima, M. and Yokota, H., 1993. Digestion of alkali-treated alfalfa silage by goats. Asian-AustralasianJ. Anim. Sci., 6:5-11. ~rskov, E.R. and McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci., 92:499-503. Orskov, E.R., Stewart, C.S. and Greenhalgh, LED., 1979. The effect of sodium hydroxide and urea on some storage properties of moist grain. J. Agric. Sci., 92:185-188. Osibe, A., Ogawa, M. and Masubuchi, T., 1987. The degradability of crude forage protein in the rumen of sheep. I. A comparison of leguminous and gramineous crude protein degradability. Bull. Natl. Grassl. Res. Inst., 37:58-63. Robinson, P.H. and Kennelly, J.J., 1988a. Ammonia and sulphur dioxide treatment of high-moisture barley on in situ rumen degradabilityand in situ whole-tract digestibility.Can. J. Anim. Sci., 68:779786. Robinson, P.H. and Kenneily, J.J., 1988b. Influence of ammoniation of high moisture barley on its in situ rumen degradation and influence on rumen fermentation in dairy cows. Can. J. Anim. Sci., 68:839-851. Statistical Analysis Systems Institute Inc., 1985. SAS User's Guide: Statistics. SAS Institute Inc., Cary, NC. Tilley, J.M.A. and Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Sot., 18:104-111. Waltz, D.M. and Loerch, S.C., 1986. Effect of acid and alkali treatment of soybean meal on nitrogen utilization by ruminants. J. Anim. Sci., 63:879-887.