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Effect of sample pretreatment on alfalfa silage dry matter and protein degradability in sacco
Animal
Feed Science
and Technology,
38 ( I992
) 69-74
69
Elsevier Science Publishers B.V., Amsterdam
Effect of sample pretreatment on alfalfa silage dry matter and protein degradability in sacco AleksandarHristov
Institute ofAnimal
Science, Feed Tecitnokxy
Department.
Kostinbrod.
Z-32,
Bulgaria
(Received 30 November 1988;accepted23 November 1991)
ABSFRACT Hristov, A., 1992.Effect oisample pretreatmenton alfalfa silagedry maw -mudprotein degradability in sexo. Anim. Feed&i.
Techno/.,
38: 69-74.
Alfalfa silagewas dried and ground (DS) or choppedand frozen (FS) and incubated mminally in saccofor I, 2,4,6.8, I2,24,30.36,48, S4,60,72 and 82 h. The watersolubilityofdty matter (DM) and protein (P) was 27.2 and 50.8%and 25.4 and 66.5%.for DS and FS, respectively.The undegradable fraction and the calculatedslopeofdegradation of DM and P were 27.4% and il.0134 and 16.9% and 0.0181for DS, and 28.6%and0.0131 and 9.6Uand 0.0145 forFS, respectively.Thedata indicate that the methodsof samplepretreatmentstudied havedifferent effectson protein degradationparametersin sacco.Oven drying of alfalfa silagesamplescausesa decreasein protein volubility and extent of rumen degradation.This effect has to be consideredwhen comparing results from experiments where theseprocessingmethodsare used.
INTRODUCTION
The nylon bag method (Van Kenren and Heinemann, 1962; Mehrez and 0rskov, 1977) is frequently used to determine dry matter and protein degradability of silage samples. Different methods of sample processing prior to incubation, such as drying and grinding (Stern and Satter, 1984, De Boer et al., 1987; Nocek, 1987) or chopping (to 0.5-l .5 cm particle length) of fresh samples (Settilit, 1983; Kwaldcel et al., 1986), have been adopted. However, few investigations study (Vik-Mo, 1989) the effect of the silage processing method on the rate and extent of degradation in sacco. The aim of this study was to determine the effect of sample pretreatment (oven drying and freezing) on dry matter and protein degradability of alfalfa sifage in sacco. Correspondence to: Aleksandar Hristov, Institute of Animal Science, Feed Technology Department, Kostinbrod, 22-32, Bulgaria.
D 1992 Elsevier Science Publishers B.V. All rights reserved 0377-8401/92/$05.00
70
A.“Rlsm”
MATERIALS AND METHODS
For the experiment, first-cut alfalfa was silaged (AS) without the addition of preservatives. After 3 months of storage, the silage contained 24.4% dry matter (DM) and (as a percentage of DM) 20.87% protein (P. nitrogenx6.25), 1.1% ammonia (Conway) and 2.11% nitrogen soluble in boratephosphate buffer (Krishnamoorthy et al., 1982) with a pH of 5.88. A representative sample ofthe silage was taken and divided into two equal parts. One part was oven dried at 60°C to constant weight and ground by a rotor-speed mill through a 3 mm screen (dried silage, DS). The other part of the silage sample was chopped with scissors to l-l.5 cm particle length and frozen at - 18°C after weighing in nylon bags (63 pm porosity, I7/10 cm) (frozen silage, FS). Each bag contained about 5 g DM. The experiment was conducted on two steers (340 kg live weight) fed on a diet composed of 14 kg corn silage (24% DM), 5.5 kg fodder concentrate ( 12% crude protein content) and 0.4 kg dehydrated alfalfa meal. Ruminal residence times were 1, 2, 4, 6, 8, 12, 24, 30, 36, 48, 54, 60, 72 and 82 h (duplicated for each time interval, i.e. four replicates per time point). Bags were introduced into the rumen after soaking in water in reverse sequence, removed all at once, washed thoroughly under running tap water, dried (at 60°C) and emptied for further analysis.
Quadruplicate sets of bags for each treatment were incubated in a water bath shaker (20 ml tap water per 1 g of sample DM) at 39°C and 120 c.p.m. for I 5 min (Nocek, 1985 ) in order to find the water-soluble fraction of DM and P. The data were fitted to a non-linear regression model based on use of the Gause-Newton method to iteratively find so!c:ions to the non-linear regression normal equations. The undegraded fraction at time t was found from the equation y=aXexp(bxt) where y is the undegraded fraction at any time point (I) (o/o), a is the rumeninsoluble fraction predicted by the model iintercept) (%), b is the regression constant (slope) (l/h) and t is time (h). Statistically, the data were evaluated using MINITAB (release 6.2) for Macintosh, Dry matter was determined by oven drying at 105°C and nitrogen by the Kjeldahl method. RESULTS AND DISCUSSION
Table 1 shows the results of the experiment and Figs. 1 and 2 illustrate them. For the DM, there is no significant treatment effect for any of the degrada-
EFFECTOFSAMPLE
71
PRETRE’iTMENTONALFALFA
TABLE I In SXCDdegradation paramelers of thealfalfasilagesamples
nem Water-soluble
fraction
(%)
Intercept(9) Slope(I/h) Undegradable
in 82 h fraction’
Prorein Waler-soluble fraclion (%) ln1ercept (%) Slope (I/h) Unhegradable in 82 h fraction
(%)
(%)
‘Al 95% confidence interval. ‘Remaining in the bags after 82 h runten
Dried
MSE’
FWZlZIl
MSE
P*
21.2 64.8 0.0134 27.4
0.6 14.9
25.4 68.3 0.0131 28.6
0.8 15.5
0.140 0.640
0.8
0.320
50.8 49.0 0.0181 16.9
0.8 12.7
66.5 24.5 0.0145 9.6
0.6 4.7
0.000 0.002
0.5
0.001
0.8
0.8
inc!rbakm.
- 2!:1.:: s-1,+yl~~g: ss.mplcr. They axis is DM (k) Fig. I. In saccoDM disappearancrwrsui txxc II! remainingand thexaxis is time (b). 0. d,y +gc-. 3, ciiysliap predicied;n . fiwun silage;0, frozensilagepredicted.
00 0
10
20
30
40
50
60
m
au
so
lo
Fig. 2. In sacco protein disappearance verstts time in alfalfa silage samples. They axis is protein (%) remainingand thexaxisistime (h). Qdtysilag; *,dty silagepredicted; &frozen silage; 0. frozen silage predicted.
The solubility of the protein fraction of DM was higher for FS compared to DS. A significant difference between the treatments was observed for the rumen-soluble fraction of the protein. For FS, as for the DM (both DS and FS), a difference of about lo-12% was observed between the in vitro water-soluble and the in sacco rumen-soluble fraction, predicted by the non-linear regression model. This probably suggests that the last estimate also includes a very rapidly (up to the first hour) degradable fraction of the DM (P). No such effect was observed for the protein in DS. The slopes of the disappearance of the protein and the undegraded fraction at 82 h were higher for DS. The reason for the observed differences seems to be the heating effect. Several workers have clearly shown the adverse effect of high temperature on DM and protein degradability. According to Goering et al. (1973), heat damage of feeds takes place at 60°C in a 24 h heating period and the mechanisms involved in this process are of the Maillard type. Weiss et al. (1986) found DM digestibility of heat-damaged alfalfa silage to be 86.7% of that of unheated silage and for total nitrogen the percentage was 74.7. Vik-Mo ( 1989)
tion parameters.
EF~CTOFSAMPLEPRETREATMENTON
At.FAt.FA
73
found similar results when comparing oven-drying to freeze-drying of silage samples. Heating (at 70°C for 72 h) resulted in a lowering of the rate constant of the disappearance in sacco of DM and nitrogen compared to freezedrying. The author did not find significant differences in digestibility at the end point of incubation (48 h) between the treatments. The results presented here indicate that heating significantly affects in sacco degradation parameters of the protein in alfalfa silage. The lack of difference between the in vitro soluble and in sacco soluble (and rapidly degradable) fraction shows that this could be the fraction of the protein most affected. Thus, surprisingly, the rate of degradation of the remaining protein is slower for FS compared to DS. Heating also affects the extent of protein degradability in 82 h rumen incubation. Obviously, oven drying of such samples (alfalfa silages with a low DM (high ammonia) and high soluble nitrogen content) greatly affects the solubility and in sacco degradability of the protein. Vik-Mo ( 1989) also found that oven drying decreased the rate constant for the initial disappearance of nitrogen. In conclusion the results of the present study show that the parameters of in sacco degradation of alfalfa silage protein are different for oven-dried and frozen samples. Oven drying decreases the solubility and extent of rumen degradation of the protein. Experimental data from trials where these processing methods are used must be compared cautiously. ACKNOWLEDGEMENTS The author thanks Iv5 s. Tzvetkova and Mrs. Ivanova for their technical assistance.
REFERENCES De Boer, G., Murphy, J.J. and Kennely, J.J., 1987. A modified method for determination of in situ rumen degradation of feedstuffs. Can. J. Anim. Sci., 67: 93-102. Goering, H.K., van Soest, P.J. and Hemken, R.W., 1973. Relative susceptibility of forages to heat damage as affected by moisture, temperature and pH. J. Dairy Sci., 56: 137-143. Krishnamc-onhy, U., Miscato, T.V., Sniffen, C.J. and van Soest, P.J., 1982. Nitrogen fractions in selected feedstuffs. J. Dairy Sci., 65: 217-225. Kwakkel, R.P., van Brochew, J., Hof, G. and Boer, H., 1986. The in sacco degradation ofcrude protein and cell wall constituents in grass, alfalfa and maize silages. Neth. J. Agric. Sci., 34: 116-119. Mehrez, AZ and Orskov, E.R., 1977. A study of the artificial tibre bag technique for determining the digestibility of feeds in the mmen. J. Agric. Sci., 88: 645-650. Nocek, J.E., 1985. Evaluation of specific variables affecting in situ estimates of nrminal dry matter and protein digestion. J. Anim. Sci., 60: 1347-1357. Nocek, J.E., 1987. Effect of harvest dry matter concentration and grind on in situ ruminal nu-
74
A.Hatsrcl”
trient fractionation and digestion rate of corn silage stored in bunker silos. J. Dairy Sci.. 70 (Suppl. I ): 174. Seliil$,J., 1983. The nylon bag technique in the determination of ruminal feed protein degradation. 1. Sci. Agric. Sot. Finl., 55: l-78. Stern, M.D. and Satter, L.D., 1984. Evaluation of nitrogen solubility and the dacron bag technique as methods for estimating protein degradation in the rumen. J. Anim. Sci., 58: 714724. Van Keuren, R.W. and Heinemann, W.W., 1962. Study of a nylon bag technique for in viva estimation of forage digestibility. J. Anim. Sci., 21: 340-345. Vik-Mo, L., 1989. Degradability of forages in sacco. 1. Grass crops and silages after oven and freeze drying. Acta Agric. Stand., 39: 43-52. Weis, W.P., Conrad, H.R. and Shokey, W.L., 1986. Digestibility of nitrogen in heat damaged alfalfa. .I. Dairy Sci.,69; 2658-2670.
Feed Science
and Technology,
38 ( I992
) 69-74
69
Elsevier Science Publishers B.V., Amsterdam
Effect of sample pretreatment on alfalfa silage dry matter and protein degradability in sacco AleksandarHristov
Institute ofAnimal
Science, Feed Tecitnokxy
Department.
Kostinbrod.
Z-32,
Bulgaria
(Received 30 November 1988;accepted23 November 1991)
ABSFRACT Hristov, A., 1992.Effect oisample pretreatmenton alfalfa silagedry maw -mudprotein degradability in sexo. Anim. Feed&i.
Techno/.,
38: 69-74.
Alfalfa silagewas dried and ground (DS) or choppedand frozen (FS) and incubated mminally in saccofor I, 2,4,6.8, I2,24,30.36,48, S4,60,72 and 82 h. The watersolubilityofdty matter (DM) and protein (P) was 27.2 and 50.8%and 25.4 and 66.5%.for DS and FS, respectively.The undegradable fraction and the calculatedslopeofdegradation of DM and P were 27.4% and il.0134 and 16.9% and 0.0181for DS, and 28.6%and0.0131 and 9.6Uand 0.0145 forFS, respectively.Thedata indicate that the methodsof samplepretreatmentstudied havedifferent effectson protein degradationparametersin sacco.Oven drying of alfalfa silagesamplescausesa decreasein protein volubility and extent of rumen degradation.This effect has to be consideredwhen comparing results from experiments where theseprocessingmethodsare used.
INTRODUCTION
The nylon bag method (Van Kenren and Heinemann, 1962; Mehrez and 0rskov, 1977) is frequently used to determine dry matter and protein degradability of silage samples. Different methods of sample processing prior to incubation, such as drying and grinding (Stern and Satter, 1984, De Boer et al., 1987; Nocek, 1987) or chopping (to 0.5-l .5 cm particle length) of fresh samples (Settilit, 1983; Kwaldcel et al., 1986), have been adopted. However, few investigations study (Vik-Mo, 1989) the effect of the silage processing method on the rate and extent of degradation in sacco. The aim of this study was to determine the effect of sample pretreatment (oven drying and freezing) on dry matter and protein degradability of alfalfa sifage in sacco. Correspondence to: Aleksandar Hristov, Institute of Animal Science, Feed Technology Department, Kostinbrod, 22-32, Bulgaria.
D 1992 Elsevier Science Publishers B.V. All rights reserved 0377-8401/92/$05.00
70
A.“Rlsm”
MATERIALS AND METHODS
For the experiment, first-cut alfalfa was silaged (AS) without the addition of preservatives. After 3 months of storage, the silage contained 24.4% dry matter (DM) and (as a percentage of DM) 20.87% protein (P. nitrogenx6.25), 1.1% ammonia (Conway) and 2.11% nitrogen soluble in boratephosphate buffer (Krishnamoorthy et al., 1982) with a pH of 5.88. A representative sample ofthe silage was taken and divided into two equal parts. One part was oven dried at 60°C to constant weight and ground by a rotor-speed mill through a 3 mm screen (dried silage, DS). The other part of the silage sample was chopped with scissors to l-l.5 cm particle length and frozen at - 18°C after weighing in nylon bags (63 pm porosity, I7/10 cm) (frozen silage, FS). Each bag contained about 5 g DM. The experiment was conducted on two steers (340 kg live weight) fed on a diet composed of 14 kg corn silage (24% DM), 5.5 kg fodder concentrate ( 12% crude protein content) and 0.4 kg dehydrated alfalfa meal. Ruminal residence times were 1, 2, 4, 6, 8, 12, 24, 30, 36, 48, 54, 60, 72 and 82 h (duplicated for each time interval, i.e. four replicates per time point). Bags were introduced into the rumen after soaking in water in reverse sequence, removed all at once, washed thoroughly under running tap water, dried (at 60°C) and emptied for further analysis.
Quadruplicate sets of bags for each treatment were incubated in a water bath shaker (20 ml tap water per 1 g of sample DM) at 39°C and 120 c.p.m. for I 5 min (Nocek, 1985 ) in order to find the water-soluble fraction of DM and P. The data were fitted to a non-linear regression model based on use of the Gause-Newton method to iteratively find so!c:ions to the non-linear regression normal equations. The undegraded fraction at time t was found from the equation y=aXexp(bxt) where y is the undegraded fraction at any time point (I) (o/o), a is the rumeninsoluble fraction predicted by the model iintercept) (%), b is the regression constant (slope) (l/h) and t is time (h). Statistically, the data were evaluated using MINITAB (release 6.2) for Macintosh, Dry matter was determined by oven drying at 105°C and nitrogen by the Kjeldahl method. RESULTS AND DISCUSSION
Table 1 shows the results of the experiment and Figs. 1 and 2 illustrate them. For the DM, there is no significant treatment effect for any of the degrada-
EFFECTOFSAMPLE
71
PRETRE’iTMENTONALFALFA
TABLE I In SXCDdegradation paramelers of thealfalfasilagesamples
nem Water-soluble
fraction
(%)
Intercept(9) Slope(I/h) Undegradable
in 82 h fraction’
Prorein Waler-soluble fraclion (%) ln1ercept (%) Slope (I/h) Unhegradable in 82 h fraction
(%)
(%)
‘Al 95% confidence interval. ‘Remaining in the bags after 82 h runten
Dried
MSE’
FWZlZIl
MSE
P*
21.2 64.8 0.0134 27.4
0.6 14.9
25.4 68.3 0.0131 28.6
0.8 15.5
0.140 0.640
0.8
0.320
50.8 49.0 0.0181 16.9
0.8 12.7
66.5 24.5 0.0145 9.6
0.6 4.7
0.000 0.002
0.5
0.001
0.8
0.8
inc!rbakm.
- 2!:1.:: s-1,+yl~~g: ss.mplcr. They axis is DM (k) Fig. I. In saccoDM disappearancrwrsui txxc II! remainingand thexaxis is time (b). 0. d,y +gc-. 3, ciiysliap predicied;n . fiwun silage;0, frozensilagepredicted.
00 0
10
20
30
40
50
60
m
au
so
lo
Fig. 2. In sacco protein disappearance verstts time in alfalfa silage samples. They axis is protein (%) remainingand thexaxisistime (h). Qdtysilag; *,dty silagepredicted; &frozen silage; 0. frozen silage predicted.
The solubility of the protein fraction of DM was higher for FS compared to DS. A significant difference between the treatments was observed for the rumen-soluble fraction of the protein. For FS, as for the DM (both DS and FS), a difference of about lo-12% was observed between the in vitro water-soluble and the in sacco rumen-soluble fraction, predicted by the non-linear regression model. This probably suggests that the last estimate also includes a very rapidly (up to the first hour) degradable fraction of the DM (P). No such effect was observed for the protein in DS. The slopes of the disappearance of the protein and the undegraded fraction at 82 h were higher for DS. The reason for the observed differences seems to be the heating effect. Several workers have clearly shown the adverse effect of high temperature on DM and protein degradability. According to Goering et al. (1973), heat damage of feeds takes place at 60°C in a 24 h heating period and the mechanisms involved in this process are of the Maillard type. Weiss et al. (1986) found DM digestibility of heat-damaged alfalfa silage to be 86.7% of that of unheated silage and for total nitrogen the percentage was 74.7. Vik-Mo ( 1989)
tion parameters.
EF~CTOFSAMPLEPRETREATMENTON
At.FAt.FA
73
found similar results when comparing oven-drying to freeze-drying of silage samples. Heating (at 70°C for 72 h) resulted in a lowering of the rate constant of the disappearance in sacco of DM and nitrogen compared to freezedrying. The author did not find significant differences in digestibility at the end point of incubation (48 h) between the treatments. The results presented here indicate that heating significantly affects in sacco degradation parameters of the protein in alfalfa silage. The lack of difference between the in vitro soluble and in sacco soluble (and rapidly degradable) fraction shows that this could be the fraction of the protein most affected. Thus, surprisingly, the rate of degradation of the remaining protein is slower for FS compared to DS. Heating also affects the extent of protein degradability in 82 h rumen incubation. Obviously, oven drying of such samples (alfalfa silages with a low DM (high ammonia) and high soluble nitrogen content) greatly affects the solubility and in sacco degradability of the protein. Vik-Mo ( 1989) also found that oven drying decreased the rate constant for the initial disappearance of nitrogen. In conclusion the results of the present study show that the parameters of in sacco degradation of alfalfa silage protein are different for oven-dried and frozen samples. Oven drying decreases the solubility and extent of rumen degradation of the protein. Experimental data from trials where these processing methods are used must be compared cautiously. ACKNOWLEDGEMENTS The author thanks Iv5 s. Tzvetkova and Mrs. Ivanova for their technical assistance.
REFERENCES De Boer, G., Murphy, J.J. and Kennely, J.J., 1987. A modified method for determination of in situ rumen degradation of feedstuffs. Can. J. Anim. Sci., 67: 93-102. Goering, H.K., van Soest, P.J. and Hemken, R.W., 1973. Relative susceptibility of forages to heat damage as affected by moisture, temperature and pH. J. Dairy Sci., 56: 137-143. Krishnamc-onhy, U., Miscato, T.V., Sniffen, C.J. and van Soest, P.J., 1982. Nitrogen fractions in selected feedstuffs. J. Dairy Sci., 65: 217-225. Kwakkel, R.P., van Brochew, J., Hof, G. and Boer, H., 1986. The in sacco degradation ofcrude protein and cell wall constituents in grass, alfalfa and maize silages. Neth. J. Agric. Sci., 34: 116-119. Mehrez, AZ and Orskov, E.R., 1977. A study of the artificial tibre bag technique for determining the digestibility of feeds in the mmen. J. Agric. Sci., 88: 645-650. Nocek, J.E., 1985. Evaluation of specific variables affecting in situ estimates of nrminal dry matter and protein digestion. J. Anim. Sci., 60: 1347-1357. Nocek, J.E., 1987. Effect of harvest dry matter concentration and grind on in situ ruminal nu-
74
A.Hatsrcl”
trient fractionation and digestion rate of corn silage stored in bunker silos. J. Dairy Sci.. 70 (Suppl. I ): 174. Seliil$,J., 1983. The nylon bag technique in the determination of ruminal feed protein degradation. 1. Sci. Agric. Sot. Finl., 55: l-78. Stern, M.D. and Satter, L.D., 1984. Evaluation of nitrogen solubility and the dacron bag technique as methods for estimating protein degradation in the rumen. J. Anim. Sci., 58: 714724. Van Keuren, R.W. and Heinemann, W.W., 1962. Study of a nylon bag technique for in viva estimation of forage digestibility. J. Anim. Sci., 21: 340-345. Vik-Mo, L., 1989. Degradability of forages in sacco. 1. Grass crops and silages after oven and freeze drying. Acta Agric. Stand., 39: 43-52. Weis, W.P., Conrad, H.R. and Shokey, W.L., 1986. Digestibility of nitrogen in heat damaged alfalfa. .I. Dairy Sci.,69; 2658-2670.