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Nitrogen fractions and in sacco dry matter and crude protein degradability of fresh and frozen alfalfa
Animal Feed Science Technology 71 Ž1998. 351–355
Short communication
Nitrogen fractions and in sacco dry matter and crude protein degradability of fresh and frozen alfalfa Aleksander N. Hristov
)
Institute of Animal Sciences, Kostinbrod 22-32, Bulgaria Received 21 April 1997; accepted 28 August 1997
Abstract Second cut alfalfa was harvested at 163 g kgy1 DM content and processed fresh or after being frozen for 30 days at y188C. Concentrations of ammonia– and free amino acid–N were clearly increased in frozen samples compared to the fresh crop. Freezing resulted in increased extent of initial dry matter and crude protein disappearance in sacco. Rumen degradability parameters significantly differed from those of the fresh forage with the soluble and potentially degradable fraction being the most affected. The effect of freezing on rumen degradability of alfalfa tended to disappear after 24 h of rumen incubation. q 1998 Elsevier Science B.V. Keywords: Alfalfa; Freezing; Nitrogen fractions; Rumen degradability
1. Introduction Several papers have indicated effect of freezing on chemical composition and protein degradability of forages. Abdalla et al. Ž1988. reported freezing and subsequent freezedrying of various forage species increased the crude protein solubility and digestibility in vitro compared to oven drying. Kohn and Allen Ž1992. found that freezing of fresh alfalfa resulted in a significant decrease in buffer soluble crude protein level and in an increase in neutral detergent insoluble nitrogen. Dry matter degradability in situ was increased in grass samples that were subjected to freezing compared to fresh herbage ŽHuntington and Givens, 1995.. ) Corresponding author. Agriculture and Agri-Food Canada, Lethbridge Research Center, P.O. Box 3000, Main Street, Lethbridge, Alberta T1J 4B1, Canada. Tel: q1-403-327-4561; fax: q1-403-382-3156; e-mail: [email protected].
0377-8401r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 7 - 8 4 0 1 Ž 9 7 . 0 0 1 4 4 - 2
352
A.N. HristoÕr Animal Feed Science Technology 71 (1998) 351–355
The objective of the present study was to test the effect of freezing on N distribution and in sacco degradability parameters of alfalfa dry matter and protein.
2. Materials and methods Second cut alfalfa Ž Medicago satiÕa L.. harvested at about 10% bloom stage with 163 g kgy1 DM content was treated fresh ŽF. or after being stored frozen for 30 days at y188C in sealed nylon bags ŽFR.. Water extracts were analyzed for concentration of NH 3 –N, total free amino acids ŽTFAA. and TCA Žtrichloroacetic acid. –soluble N ŽHristov, 1994.. Oven dried samples were analyzed for Total Kjeldahl N ŽTN.. About 30 g fresh alfalfa sample Žchopped with scissors to 1.0 cm approximate particle length. was incubated in sacco in nylon bags Ž63 m m porosity, 17r10 cm, Hristov, 1992. in the rumen of three steers fed 10 kg dayy1 of an ammoniated strawrfodder concentrate diet Ž60:40. for 1, 3, 8, 24, 48 and 72 h. The F samples were weighted into the bags and incubated in the rumen within 30 min after harvesting. Each bag was replicated so six independent estimates were obtained for each time-point. Dry matter and crude protein were determined after oven drying at 658C to constant weight. Rumen degradability parameters ŽØrskov and McDonald, 1979. and the effective degradability ŽED. of alfalfa DM and crude protein ŽCP. were estimated as follows: P s a q b ) Ž 1 y expyc t . ED s a q Ž b ) cr Ž c q k . . where P Ž%. denotes proportion of the material degraded Žlost through the bag. at time t; a Ž%. is the soluble potentially degradable fraction; b Ž%. is the insoluble potentially degradable fraction; c Žhy1 . is the degradation rate constant for fraction b; k is the particle flow rate constant assumed to be 0.05 hy1 ŽHristov and Broderick, 1996.. Statistically, the means of the in sacco experiment were separated by t-test ŽMinitab, Minitab, St. College, PA..
3. Results and discussion Freezing greatly increased NH 3 –N and TFAA–N Ž6.5 fold. concentrations compared to fresh alfalfa ŽTable 1.. In vivo, Beever et al. Ž1974. found an increased amount of amino-N entering the small intestine after feeding frozen compared to fresh herbage. The level of TCA–soluble N was slightly decreased by the treatment. Similarly, Kohn and Allen Ž1992. reported significantly lower concentration of buffer soluble nitrogen in frozen compared to fresh samples and Deinum and Maassen Ž1994. found a decreased forage protein solubility after freezing Žand consecutive drying at 708C.. Freezing significantly increased the soluble and potentially degradable fraction of DM and the ED of DM compared to F ŽTable 1.. Other studies have shown similar effect of freezing on rumen degradability of forages ŽDanley and Vetter, 1971; Ould-Bah and Michalet-Doreau, 1988; Vik-Mo, 1989; Kamoun and Thewis, 1990; Peyraud, 1990;
A.N. HristoÕr Animal Feed Science Technology 71 (1998) 351–355
353
Table 1 Effect of freezing on N partitioning and in sacco rumen degradability parameters of alfalfa dry matter and crude protein Item
Treatment Fresh
Frozen
218.2
236.3
106.9 25.1 36.4
90.5 43.7 235.7
Rumen degradability parameters (ns 6) Dry matter a4 b c d
17.4"1.51a 62.7"2.06 a 0.076".002 55.3"0.12 a
33.7"0.50 b 44.9"1.24 b 0.079"0.006 61.1"0.71b
Crude protein a b c d
17.6"1.50 a 74.9"1.40 a 0.096"0.008 a 66.5"0.87 a
42.9"0.56 b 49.1"0.55 b 0.108"0.011b 76.0"0.97 b
Crude protein Žg kgy1 DM. N fractions (g kg y 1 TN 1) TCA–soluble N 2 Ammonia–N Amino acid–N 3
1
Total nitrogen; 2 Trichloroacetic acid soluble N Žnon-protein N.; 3 Total free amino acids; 4According to the nonlinear model P s aq b ) Ž1yexp ct ., where: P Ž%. denotes proportion of the material degraded Žlost through the bag. at time t; a Ž%. is the soluble potentially degradable fraction; b Ž%. is the insoluble potentially degradable fraction; c Žhy1 . is the degradation constant for fraction b ŽØrskov and McDonald, 1979.; and d Ž%. is the effective degradability of DM and protein ŽED. found through the following equation: EDs aqŽ bcrŽ cq k .., where k Žhy1 . is the particle flow rate constant assumed to be 0.05 ŽHristov and Broderick, 1996.. ab Means in a row not having a common superscript differ at P - 0.05.
Karn, 1991.. In the present experiment the rate of in sacco DM disappearance did not differ among the treatments. The effect of the freezing tended to disappear beyond 24-h rumen incubation ŽFig. 1.. Freezing of the alfalfa resulted in a significantly higher initial extent and rate of rumen disappearance of CP compared to the fresh sample ŽTable 1.. As for the DM the effect of the treatment tended to diminish at the later stages of rumen incubation ŽFig. 2.. The often use of the in sacco approach in rumen studies necessitates standardization of the sample preparation procedures ŽAFRC, 1987. which reportedly, could be a major factor of variation in determining rumen degradability parameters of feedstuffs ŽVik-Mo, 1989; Hristov, 1992.. In the present experiment the Effective degradabilities of DM and CP of the frozen alfalfa were by 5.8 and 9.5 points, respectively higher than those of the fresh alfalfa. The frozen samples had higher soluble and lower insoluble fractions of DM and CP. The 94% and 144% higher rumen soluble fraction of DM and CP, respectively of the frozen compared to the fresh alfalfa reported here could not be explained without involving disruption of cell membranes due to crystallization of the intracellular content during freezing despite the fact that some authors did not find microscopically any
354
A.N. HristoÕr Animal Feed Science Technology 71 (1998) 351–355
Fig. 1. In sacco disappearance of dry matter in fresh Žopen symbols. and frozen alfalfa Žclosed symbols..
changes in surface morphology of frozen plant material ŽKohn and Allen, 1992.. The higher effective degradabilities of DM and CP in the rumen of FR vs. F indicate more intensive microbial digestion was suggested by Engels Ž1989., impossible without microbial penetration through the outer cell wallsx and also indirectly support the hypothesis of the disruptive effect of freezing on cell structure. The results of the present experiment clearly showed that the rumen degradability parameters of dry matter and crude protein of alfalfa samples as estimated through the in sacco procedure differ from those of the fresh forage with the soluble and potentially degradable fraction being the most affected.
Fig. 2. In sacco disappearance of crude protein in fresh Žopen symbols. and frozen alfalfa Žclosed symbols..
A.N. HristoÕr Animal Feed Science Technology 71 (1998) 351–355
355
References Abdalla, H.O., Fox, D.G., Van Soest, P.J., 1988. An evaluation of methods for preserving fresh forage samples before protein fraction determinations. J. Anim. Sci. 66, 2646–2649. AFRC Technical committee on responses to nutrients. Characterization of feedstuffs: nitrogen. Report no. 2, Nutr. Abstr. Rev. ŽSer. B., 57 Ž1987. 713–736. Beever, D.E., Cammell, S.B., Wallace, A., 1974. The digestion of fresh, frozen and dried perennial rye-grass. Proc. Nutr. Soc. 33, 73A–74A. Danley, M.M., Vetter, R.L., 1971. Changes in carbohydrate and nitrogen fractions and digestibility of forages: method of sample processing. J. Anim. Sci. 33, 1072–1077. Deinum, B., Maassen, A., 1994. Effects of drying temperature on chemical composition and in vitro digestibility of forages. Anim. Feed Sci. Technol. 46, 75–86. Engels, F.M., 1989. Some properties of cell wall layers determining ruminant digestion. In: Chesson, A., Ørskov, E.R. ŽEds.., Physico-chemical Characterization of Plant Residues for Industry and Feed Use. Elsevier, London, pp. 80–87. Hristov, A.N., 1992. Effect of sample pretreatment on alfalfa silage dry matter and protein degradability in sacco. Anim. Feed Sci. Technol. 38, 69–74. Hristov, A.N., 1994. Effect of sodium methabisulphite on proteolysis and rumen degradability of alfalfa silage protein. Anim. Feed Sci. Technol. 48, 143–151. Hristov, A.N., Broderick, G.A., 1996. Microbial protein synthesis in ruminally cannulated cows fed alfalfa silage, alfalfa hay, or corn silage. J. Dairy Sci. 79, 1627–1637. Huntington, J.A., Givens, D.I., 1995. The influence of sample preparation and incubation sequence on in situ dry matter degradability of fresh herbage in non-lactating dairy cows. Ann. Zootech. 44 ŽSuppl. 37.. Kamoun, M., Thewis, A., 1990. Influence du mode de conditionnement d’un fourrage vert sur sa composition chimique, le digestibilite in vitro de la matiere organique et la degradabilite in sacco de l’azote dans le rumen. Reprod. Nutr. Dev. Suppl. 2, 159s–160s. Karn, J.F., 1991. Chemical composition of forage and feces as affected by microwave oven drying. J. Range Manage. 44, 512–515. Kohn, R.A., Allen, M.S., 1992. Storage of fresh and ensiled forages by freezing affects fiber and crude protein fractions. J. Sci. Food Agric. 58, 215–220. Ørskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. ŽCamb.. 92, 449–503. Ould-Bah, M.Y., Michalet-Doreau, B., 1988. Influence du traitement des fourrages verts sur la cinetique de degradation in sacco de l’azote dans le rumen. Reprod. Nutr. Dev. Suppl. 1, 103–104. Peyraud, J.L., 1990. Influece du mode de sechage et de la finesse de broyage des echantillons de fourrages sur l’estimation de la degradabilite de l’azote dans le rumen. Reprod. Nutr. Dev. Suppl. 2, 153s–154s. Vik-Mo, L., 1989. Degradability of forages in sacco: 1. Grass crops and silages after oven and freeze-drying. Acta Agric. Scand. 39, 43–52.
Short communication
Nitrogen fractions and in sacco dry matter and crude protein degradability of fresh and frozen alfalfa Aleksander N. Hristov
)
Institute of Animal Sciences, Kostinbrod 22-32, Bulgaria Received 21 April 1997; accepted 28 August 1997
Abstract Second cut alfalfa was harvested at 163 g kgy1 DM content and processed fresh or after being frozen for 30 days at y188C. Concentrations of ammonia– and free amino acid–N were clearly increased in frozen samples compared to the fresh crop. Freezing resulted in increased extent of initial dry matter and crude protein disappearance in sacco. Rumen degradability parameters significantly differed from those of the fresh forage with the soluble and potentially degradable fraction being the most affected. The effect of freezing on rumen degradability of alfalfa tended to disappear after 24 h of rumen incubation. q 1998 Elsevier Science B.V. Keywords: Alfalfa; Freezing; Nitrogen fractions; Rumen degradability
1. Introduction Several papers have indicated effect of freezing on chemical composition and protein degradability of forages. Abdalla et al. Ž1988. reported freezing and subsequent freezedrying of various forage species increased the crude protein solubility and digestibility in vitro compared to oven drying. Kohn and Allen Ž1992. found that freezing of fresh alfalfa resulted in a significant decrease in buffer soluble crude protein level and in an increase in neutral detergent insoluble nitrogen. Dry matter degradability in situ was increased in grass samples that were subjected to freezing compared to fresh herbage ŽHuntington and Givens, 1995.. ) Corresponding author. Agriculture and Agri-Food Canada, Lethbridge Research Center, P.O. Box 3000, Main Street, Lethbridge, Alberta T1J 4B1, Canada. Tel: q1-403-327-4561; fax: q1-403-382-3156; e-mail: [email protected].
0377-8401r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 7 - 8 4 0 1 Ž 9 7 . 0 0 1 4 4 - 2
352
A.N. HristoÕr Animal Feed Science Technology 71 (1998) 351–355
The objective of the present study was to test the effect of freezing on N distribution and in sacco degradability parameters of alfalfa dry matter and protein.
2. Materials and methods Second cut alfalfa Ž Medicago satiÕa L.. harvested at about 10% bloom stage with 163 g kgy1 DM content was treated fresh ŽF. or after being stored frozen for 30 days at y188C in sealed nylon bags ŽFR.. Water extracts were analyzed for concentration of NH 3 –N, total free amino acids ŽTFAA. and TCA Žtrichloroacetic acid. –soluble N ŽHristov, 1994.. Oven dried samples were analyzed for Total Kjeldahl N ŽTN.. About 30 g fresh alfalfa sample Žchopped with scissors to 1.0 cm approximate particle length. was incubated in sacco in nylon bags Ž63 m m porosity, 17r10 cm, Hristov, 1992. in the rumen of three steers fed 10 kg dayy1 of an ammoniated strawrfodder concentrate diet Ž60:40. for 1, 3, 8, 24, 48 and 72 h. The F samples were weighted into the bags and incubated in the rumen within 30 min after harvesting. Each bag was replicated so six independent estimates were obtained for each time-point. Dry matter and crude protein were determined after oven drying at 658C to constant weight. Rumen degradability parameters ŽØrskov and McDonald, 1979. and the effective degradability ŽED. of alfalfa DM and crude protein ŽCP. were estimated as follows: P s a q b ) Ž 1 y expyc t . ED s a q Ž b ) cr Ž c q k . . where P Ž%. denotes proportion of the material degraded Žlost through the bag. at time t; a Ž%. is the soluble potentially degradable fraction; b Ž%. is the insoluble potentially degradable fraction; c Žhy1 . is the degradation rate constant for fraction b; k is the particle flow rate constant assumed to be 0.05 hy1 ŽHristov and Broderick, 1996.. Statistically, the means of the in sacco experiment were separated by t-test ŽMinitab, Minitab, St. College, PA..
3. Results and discussion Freezing greatly increased NH 3 –N and TFAA–N Ž6.5 fold. concentrations compared to fresh alfalfa ŽTable 1.. In vivo, Beever et al. Ž1974. found an increased amount of amino-N entering the small intestine after feeding frozen compared to fresh herbage. The level of TCA–soluble N was slightly decreased by the treatment. Similarly, Kohn and Allen Ž1992. reported significantly lower concentration of buffer soluble nitrogen in frozen compared to fresh samples and Deinum and Maassen Ž1994. found a decreased forage protein solubility after freezing Žand consecutive drying at 708C.. Freezing significantly increased the soluble and potentially degradable fraction of DM and the ED of DM compared to F ŽTable 1.. Other studies have shown similar effect of freezing on rumen degradability of forages ŽDanley and Vetter, 1971; Ould-Bah and Michalet-Doreau, 1988; Vik-Mo, 1989; Kamoun and Thewis, 1990; Peyraud, 1990;
A.N. HristoÕr Animal Feed Science Technology 71 (1998) 351–355
353
Table 1 Effect of freezing on N partitioning and in sacco rumen degradability parameters of alfalfa dry matter and crude protein Item
Treatment Fresh
Frozen
218.2
236.3
106.9 25.1 36.4
90.5 43.7 235.7
Rumen degradability parameters (ns 6) Dry matter a4 b c d
17.4"1.51a 62.7"2.06 a 0.076".002 55.3"0.12 a
33.7"0.50 b 44.9"1.24 b 0.079"0.006 61.1"0.71b
Crude protein a b c d
17.6"1.50 a 74.9"1.40 a 0.096"0.008 a 66.5"0.87 a
42.9"0.56 b 49.1"0.55 b 0.108"0.011b 76.0"0.97 b
Crude protein Žg kgy1 DM. N fractions (g kg y 1 TN 1) TCA–soluble N 2 Ammonia–N Amino acid–N 3
1
Total nitrogen; 2 Trichloroacetic acid soluble N Žnon-protein N.; 3 Total free amino acids; 4According to the nonlinear model P s aq b ) Ž1yexp ct ., where: P Ž%. denotes proportion of the material degraded Žlost through the bag. at time t; a Ž%. is the soluble potentially degradable fraction; b Ž%. is the insoluble potentially degradable fraction; c Žhy1 . is the degradation constant for fraction b ŽØrskov and McDonald, 1979.; and d Ž%. is the effective degradability of DM and protein ŽED. found through the following equation: EDs aqŽ bcrŽ cq k .., where k Žhy1 . is the particle flow rate constant assumed to be 0.05 ŽHristov and Broderick, 1996.. ab Means in a row not having a common superscript differ at P - 0.05.
Karn, 1991.. In the present experiment the rate of in sacco DM disappearance did not differ among the treatments. The effect of the freezing tended to disappear beyond 24-h rumen incubation ŽFig. 1.. Freezing of the alfalfa resulted in a significantly higher initial extent and rate of rumen disappearance of CP compared to the fresh sample ŽTable 1.. As for the DM the effect of the treatment tended to diminish at the later stages of rumen incubation ŽFig. 2.. The often use of the in sacco approach in rumen studies necessitates standardization of the sample preparation procedures ŽAFRC, 1987. which reportedly, could be a major factor of variation in determining rumen degradability parameters of feedstuffs ŽVik-Mo, 1989; Hristov, 1992.. In the present experiment the Effective degradabilities of DM and CP of the frozen alfalfa were by 5.8 and 9.5 points, respectively higher than those of the fresh alfalfa. The frozen samples had higher soluble and lower insoluble fractions of DM and CP. The 94% and 144% higher rumen soluble fraction of DM and CP, respectively of the frozen compared to the fresh alfalfa reported here could not be explained without involving disruption of cell membranes due to crystallization of the intracellular content during freezing despite the fact that some authors did not find microscopically any
354
A.N. HristoÕr Animal Feed Science Technology 71 (1998) 351–355
Fig. 1. In sacco disappearance of dry matter in fresh Žopen symbols. and frozen alfalfa Žclosed symbols..
changes in surface morphology of frozen plant material ŽKohn and Allen, 1992.. The higher effective degradabilities of DM and CP in the rumen of FR vs. F indicate more intensive microbial digestion was suggested by Engels Ž1989., impossible without microbial penetration through the outer cell wallsx and also indirectly support the hypothesis of the disruptive effect of freezing on cell structure. The results of the present experiment clearly showed that the rumen degradability parameters of dry matter and crude protein of alfalfa samples as estimated through the in sacco procedure differ from those of the fresh forage with the soluble and potentially degradable fraction being the most affected.
Fig. 2. In sacco disappearance of crude protein in fresh Žopen symbols. and frozen alfalfa Žclosed symbols..
A.N. HristoÕr Animal Feed Science Technology 71 (1998) 351–355
355
References Abdalla, H.O., Fox, D.G., Van Soest, P.J., 1988. An evaluation of methods for preserving fresh forage samples before protein fraction determinations. J. Anim. Sci. 66, 2646–2649. AFRC Technical committee on responses to nutrients. Characterization of feedstuffs: nitrogen. Report no. 2, Nutr. Abstr. Rev. ŽSer. B., 57 Ž1987. 713–736. Beever, D.E., Cammell, S.B., Wallace, A., 1974. The digestion of fresh, frozen and dried perennial rye-grass. Proc. Nutr. Soc. 33, 73A–74A. Danley, M.M., Vetter, R.L., 1971. Changes in carbohydrate and nitrogen fractions and digestibility of forages: method of sample processing. J. Anim. Sci. 33, 1072–1077. Deinum, B., Maassen, A., 1994. Effects of drying temperature on chemical composition and in vitro digestibility of forages. Anim. Feed Sci. Technol. 46, 75–86. Engels, F.M., 1989. Some properties of cell wall layers determining ruminant digestion. In: Chesson, A., Ørskov, E.R. ŽEds.., Physico-chemical Characterization of Plant Residues for Industry and Feed Use. Elsevier, London, pp. 80–87. Hristov, A.N., 1992. Effect of sample pretreatment on alfalfa silage dry matter and protein degradability in sacco. Anim. Feed Sci. Technol. 38, 69–74. Hristov, A.N., 1994. Effect of sodium methabisulphite on proteolysis and rumen degradability of alfalfa silage protein. Anim. Feed Sci. Technol. 48, 143–151. Hristov, A.N., Broderick, G.A., 1996. Microbial protein synthesis in ruminally cannulated cows fed alfalfa silage, alfalfa hay, or corn silage. J. Dairy Sci. 79, 1627–1637. Huntington, J.A., Givens, D.I., 1995. The influence of sample preparation and incubation sequence on in situ dry matter degradability of fresh herbage in non-lactating dairy cows. Ann. Zootech. 44 ŽSuppl. 37.. Kamoun, M., Thewis, A., 1990. Influence du mode de conditionnement d’un fourrage vert sur sa composition chimique, le digestibilite in vitro de la matiere organique et la degradabilite in sacco de l’azote dans le rumen. Reprod. Nutr. Dev. Suppl. 2, 159s–160s. Karn, J.F., 1991. Chemical composition of forage and feces as affected by microwave oven drying. J. Range Manage. 44, 512–515. Kohn, R.A., Allen, M.S., 1992. Storage of fresh and ensiled forages by freezing affects fiber and crude protein fractions. J. Sci. Food Agric. 58, 215–220. Ørskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. ŽCamb.. 92, 449–503. Ould-Bah, M.Y., Michalet-Doreau, B., 1988. Influence du traitement des fourrages verts sur la cinetique de degradation in sacco de l’azote dans le rumen. Reprod. Nutr. Dev. Suppl. 1, 103–104. Peyraud, J.L., 1990. Influece du mode de sechage et de la finesse de broyage des echantillons de fourrages sur l’estimation de la degradabilite de l’azote dans le rumen. Reprod. Nutr. Dev. Suppl. 2, 153s–154s. Vik-Mo, L., 1989. Degradability of forages in sacco: 1. Grass crops and silages after oven and freeze-drying. Acta Agric. Scand. 39, 43–52.