Effect Of Heat Treatment Of Alfalfa Prior To Ensiling On Nitrogen. Journal Of Dairy Scienc, 1989

Effect of Heat Treatment of Alfalfa Prior to Ensiling on Nitrogen Solubility and In Vitro Ammonia Production I. B. MANDELL, 1 D. N. MOWAT, 1 W. K. BILANSKI, 2 and S. N. RAI 3

University of Guelph Guelph, Ontario N1G 2Wl, Canada ABSTRACT

INTRODUCTION

Two experiments were conducted to assess heat ~eatment of alfalfa prior to ensiling. In Experiment 1, direct-cut alfalfa (26% DM) was subjected to dry heat (air temperature 400"C) for either 0, 7, 14, 21, or 29 s prior to ensiling. Wilted alfalfa (50% DM) was treated similarly for either 0, 5, or 6 s. Heat treatment decreased the soluble N content of silage without inducing heat damage. Across all hours of incubation, heating prior to ensiling decreased in vitro NH 3 N production in comparison with untreated silage (direct-cut or wilted). Duration of treatment affected NH3 production. Treatment of direct-cut material for at least 14 s was necessary to decrease NH3 production in comparison with unheated alfalfa or alfalfa treated for 7 s prior to ensiling. Treatment (6 s duration) following wilting decreased NH3 production in comparison with heat-treated, direct-cut alfalfa (7, 14, 29 s treatments). In Experiment 2, direct-cut alfalfa (36% DM) was subjected to microwave heating for either 0, 30, 60, 120, or 180 s prior to ensiling. Heat treatment decreased the soluble N content of silage. Treatment for at least 60 s resulted in a further decrease in N solubility. In addition, treatment for at least 60 s was necessary to decrease NH 3 production. Thus, short-term heat treatment of alfalfa prior to ensiling may be an effective alternative for improving the utilization of silage N in ruminants.

The CP content of high quality hay crop silages exceeds the recommended requirements for young growing calves (18). However, several recent studies have shown a marked improvement in the performance of calves fed either early cut, wilted alfalfa-grass silages (16) or direct-cut grass silages (5, 25) when supplemented with a protein source of low rumen degradability. This paradox is due to the fact that forage protein undergoes extensive degradation during wilting and the ensilage process (6, 23). Various chemicals (formic acid, formaldehyde, NH3) have been used as additives during ensiling to improve silage stability and N utilization (7, 13). However, these additives do not always prevent extensive proteolysis during ensiling (15) nor improve animal performance (7). Artificial drying of forages improves the utilization of forage protein by the ruminant. Drying decreases N solubility and rumen degradation of forage protein, and increases microbial protein synthesis in the rumen (9, 15). Overall, this increases the amount of amino acids entering the small intestine (9, 23). Unfortunately, due to costs, artificial drying has not been developed for on-farm processing, and few producers are able to take advantage of this technology. Recently, Charmley and Veira (4) demonstrated that steam treatment (60 s duration) of alfalfa prior to ensiling could inhibit proteolysis and markedly improve the utilization of silage N by the ovine. The objective of this study was to determine the effects of heat treatment (HT) of alfalfa prior to ensiling on N solubility and availability to rumen microbes.

Received September 30. t988. Accepted February 22, 1989. 1Department of Animal and Poultry Science. ~School of Engineering. 3present address: National Dairy Research Institute, Kamal 132001, 1-Iaryana,India. 1989 J Dairy Sci 72:2046-2054

MATERIALS AND METHODS Experiment 1

First-cut alfalfa (early bloom) was cut and then chopped by a forage harvester when the

2046

HEAT TREATMENT OF FORAGES PRIOR TO ENSILING DM content was 26%. Random portions of the chopped forage were allowed to wilt 24 h until the DM attained 50%. Direct-cut and wilted alfalfa were heat treated at 400°C (air ambient temperature), using a continuous flow unit (28) in which heat was supplied by 26, 750-W calrod heaters. Forage transit time through the continuous flow unit was either 0, 7, 14, 21, or 29 s for direct-cut alfalfa and either 0, 5, or 6 s for wilted alfalfa. Wilted alfalfa could not be heat treated for more than 6 s due to the development of charring and combustion. Each treatment was processed in quadruplicate. Immediately posttreatment, duplicate samples of alfalfa were either frozen at -20°C or ensiled in polyethylene containers (3200 ml capacity) for 35 d. Nonensiled and ensiled alfalfa were freeze-dried and then ground to pass through a 2-mm screen (Christy-Norris Hammermill Christy and Norris Ltd., Chelmsford, England). Analyses of DM, organic matter (OM), and total N (TN) were conducted according to AOAC (1) procedures. Neutral detergent fiber, ADF, and ADIN were determined according to the sequential methods described by Van Soest and Robertson (24). Hot water insoluble N was determined according to Goering and Van Soest (8) and soluble N calculated. Experimental design was a factorial arrangement within a randomized complete block with the two silos (replicates) serving as blocks. Analysis of variance was conducted on the chemical composition data with storage form of alfalfa, HT, and blocks as factors in the model. Differences among treatment means were determined by nonorthogonal contrasts (22). Samples were selected from wilted and direct-cut alfalfa (nonensiled) and silage and then incubated at 39"C with 10 ml of rumen buffer (8) and 5 ml of strained rumen fluid for 0, 1, 3, 6, 12, and 24 h. Dextrose (.92 g/L) was added to the rumen buffer mixture to provide additional energy for rurnen microorganisms (8). Blank incubations without substrate were conducted at all hours of incubation to determine NH 3 production from rumen fluid alone. Prior to the a.m. feeding, rumen fluid was obtained from a nonlactating, rumen-fistulated dairy cow fed an alfalfa/grass hay. Rumen fluid was strained through four layers of cheesecloth and then kept at 39°C prior to use. After dispensing

2047

5 ml of rumen buffer into each incubation tube, 10 ml of the complete incubation mixture (5 ml rumen fluid, 5 ml rumen buffer) were added prior to the tubes being gassed with CO2, stoppered with bunsen valves, and then incubated in a water bath at 39"C. Incubations were stopped at the appropriate intervals by the addition of 2 ml .4 N H2SO4. Samples of tube contents were collected after vortexing and then centrifuged at 13,000 x g for 3 rain. Supernatant was kept frozen at -20"C until analyzed for NH 3. Ammonia N concentrations were determined using a phenolhypochlorite colorimetric procedure (2). Accumulation of NH3 N at each interval was calculated as the difference between NH 3 N concentrations of tubes containing substrate and NH 3 N concentrations in blank incubations. Experimental design for the NH3 N data was a randomized complete block with the two runs serving as blocks. For each hour of incubation, analysis of variance was conducted with HT and blocks as factors in the model. Differences among treatment means were determined by nonorthogonal contrasts (22). Experiment 2

Third-cut alfalfa (early bloom) was cut and then chopped by a forage harvester when the DM was 36%. Fiberboard trays containing 100 g fresh weight of alfalfa were placed in a conventional kitchen microwave (MW) oven (700 W, 2450 MHz) and then heat treated for either 0, 30, 60, 120, or 180 s. Each HT was replicated 20 times. After treatment, duplicate samples of alfalfa were ensiled in polyethylene containers (550 ml capacity) for 35 d. A sample of nonensiled, nonheat-treated alfalfa was frozen at-20"C. After ensiling, sample processing, chemical analyses, and in vitro NH3 production were conducted according to the methods described in Experiment 1. A randomized complete block design was used in which the two silos (replicates) served as blocks. Analysis of variance was conducted on the chemical composition and in vitro NH 3 data with HT and blocks serving as factors in the model. Differences among treatment means were determined by nonorthogonal contrasts.

Journal of Dairy Science Vol. 72, No. 8, 1989

2048

MANDELL ET AL.

RESULTS AND DISCUSSION

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Journal of Dairy Science Vol. 72, No, 8, 1989

Effect of HT with the continuous flow system on the chemical composition of alfalfa is in Table 1. Heat treatment increased (P<.01) the DM content of alfalfa (direct-cut or wilted). The loss in moisture may be due in part to syneresis associated with thermal denaturation of soluble proteins from the disruption of hydrogen bonding (20). Increasing the duration of HT further increased (P<.01) DM content of direct-cut alfalfa. Heat treating direct-cut alfalfa could be advantageous to producers by increasing forage DM prior to ensiling. This could prevent nulrient losses during wilting without the potential problems of effluent run-off from ensiling a low DM forage (26). Heat treating wilted alfalfa may present a storage problem due to the resultant high DM and the potential for heat damage (10). Heat treatment increased (P<.01) the NDF content of wilted alfalfa but did not affect the NDF content of direct-cut alfalfa. Although artificial heating has been shown to increase the NDF and ADF content of alfalfa haylage (10), the ADF content of alfalfa was unaffected by HT in our study. The TN content of alfalfa was unaffected (P>.10) by HT and is in agreement with Yu and Veira (29). Heat treatment decreased (P<.01) N solubility (or increased the HWIN content) in alfalfa. McDonald (15) noted that artificial drying of feedstuffs can decrease N solubility due to the formation of new linkages within and between peptide chains. These newly formed linkages may be resistant to protease activity or prevent enzymatic access to adjacent peptide bonds. In alfalfa, 32 to 39% of the total leaf protein is composed of a single soluble protein called Fraction-I or ribulose 1,5biphosphate carboxylase (14). Although this protein is totally denatured at a treatment temperature of 80°C for 5 min, partial denaturation will occur at temperatures as low as 50°C (20). Wilting alone did not affect (P>.10) N solubility. This is in agreement with McDonald (15), who stated that wilting prior to ensiling does not necessarily inhibit proteolysis nor provide any beneficial effect in preventing protein breakdown during ensiling. In contrast, Janicki and Stallings (10) reported that as the DM

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content of forage to be ensiled decreased, N solubility after ensiling increased. Increasing the duration of HT decreased (P<.05) N solubility. In the present study, HT of direct-cut alfalfa for 21 and 29 s prevented a decrease in N solubility and may provide an alternative to wilting alfalfa prior to ensiling. Heat treatment for short times (7 and 14 s, direct-cut alfalfa; 5 s, wilted alfalfa) may not inactivate plant enzymes totally (14, 21), enabling moderate proteolysis. Excessive heating of forages increases risk of heat damage, thus limiting N availability in the forage (3, 12). Although HT increased (P<.05) the ADIN content of alfalfa, all values in our study were lower than the 10% figure suggested by Janicki and Stallings (10) to represent limited heat damage. Total N and soluble N content of alfalfa differed (P<.05) between storage forms (nonensiled vs. ensiled). Although the difference in TN content (nonensiled, 3%; ensiled 3.05%) was small, the solubility of ensiled alfalfa exceeded (P<.01) that of nonensiled alfalfa (nonensiled, 36.4% of TN; ensiled, 45.3% of TN). The increase in N solubility upon ensiling is attributed to plant proteolysis during the ensiling process (15). A storage form by treatment interaction (P<.01) occurred for N solubility (Table 2). The interaction was due to a decrease in N solubility from ensiling of control (direct-cut and wilted) alfalfa and alfalfa heat treated for either 5, 7, or 14 s. In contrast, ensiling did not affect N solubility in alfalfa heat treated for 21 or 29 s (direct-cut alfalfa) or 6 s (wilted alfalfa). According to Charmley and Veira (4), HT reduces proteolysis during the ensilage process, resulting in higher concentrations of insoluble N and lower concentrations of NH 3 N. The difference in N solubility between control and heat-processed silages in the present study are quite similar to those previously reported (4). In the latter work, HT prior to ensiling increased the flow of non-NH 3 N to the duodenum in sheep. Effect of HT on NH 3 N production in vitro is in Table 3. Although rumen fluid was obtained from the same fistulated cow on both days of incubation, run-to-run variation (P<.01) occurred throughout all hours of incubation. This resulted in large SE. Broderick (2) also Journal of Dairy Science Vol. 72, No. 8, 1989

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HEAT TREATMENTOF FORAGES PRIOR TO ENSILING noted run-to-run variation in NH 3 N production in vitro using casein as a substrate. In the present study, run-to-run variation was expected, as NH3 N concentrations produced from rumen fluid incubated without substrate differed between days of incubation. Although all NH3 N values were corrected for NH 3 N production from rumen fluid alone, day-to-day variation may be present for the degradative activity of rumen fluid (2). The large run-to-run variation in NH3 N production at 0 h cannot be explained as the fermentation is stopped immediately with H2SO4 after addition of the bufferrumen fluid inoculum. With the exception of 6- and 12-h incubations, Nil 3 N production from the nonensiled alfalfa was less (P<.05) than that from unheated silage (direct-cut or wilted). In vivo studies (6, 9) showed that feeding fresh forages increased the amount of forage amIno acids entering the small intestine in comparison to feeding silages. The inefficient utilization of silage N by the ruminant is due to extensive proteolysis during the ensilage process and to rapid and complete degmd_ation of NPN in the rumen (23). Extensive proteolysis occurred in the control silages as noted in Table 2 by their high soluble N content. Thus, across all hours of incubation, HT prior to ensiling decreased (P<.10) NH 3 N production in both direct-cut and wilted silages. Heat treatment probably decreased N availability to plant and microbial enzymes due to enzyme inactivation (11) and to thermal denaturation of protein, which prevented proteolysis during ensiling and in vitro incubation. This is supported by the similar concentrations of soluble N in heat-treated silages relative to nonensiled control alfalfa (Table 2). Similarly, Krause and Klopfenstein (12) demonstrated that artificial drying of alfalfa decreased NH 3 N production in vitro. Duration of HT affected (P<.05) NH3 N production in vitro. Across all hours of incubation, NH3 N production from alfalfa heattreated for only 7 s was similar to that from the control direct-cut silage. However, HT for 14 s decreased (P<.01) NH 3 N production in comparison with HT for 7 s duration. Wilting prior to HT decreased (P<.05) NH 3 N production in comparison with direct-cut forage. Wilting de-

2051

creased the time required for HT to reduce N solubility of silage and to decrease N availability to rumen microorganisms.

Experiment 2 Effect of MW processing on the DM content and N fractions of alfalfa is in Table 4. Microwave heating increased (P<.01) the DM content of alfalfa. This is consistent with use of the MW for rapid drying of herbage samples (27). Increasing duration of MW treatment resulted in a further increase (P<.01) in DM content. In addition to increasing forage DM, N solubility was markedly decreased (P<.01) over that of control silage. Although MW processing for 30 s decreased N solubility, further decreases in N solubility occurred by increasing the duration of MW treatment. Decreases (P<.01) in N solubility by MW treatment is either due to enzyme inactivation (21) or to thermal denaturation of protein (11). Lower response in N solubility by MW treatmem for 30 s may be related to the sample size (100 g fresh weight) processed in this study or to the power of the MW system employed (19). Wolf and Carson (27) reported that respiration was completely inactivated after 25 g fresh alfalfa was MW processed for 30 s. In addition, Smith (21) noted that rapid drying cannot occur if a thick or massive layer of forage is subjected to heat or freeze drying. Increasing the duration of MW processing beyond 60 s did not result in further decreases (P>.10) in N solubility. This is probably attributed to the fact that 70 to 80% of the TN in fresh alfalfa is true protein (17). Despite increases (P<.05) in the ADIN content of alfalfa with certain MW treatments, values do not indicate even moderate heat damage. Effect of MW treatment on in vitro production of NH 3 N is in Table 5. Run-to-run variation (P<.01) resulted in large SE. Ensiling increased (P<.01) NH 3 N production at 1 h of incubation and decreased (P<.05) NH 3 N production overall at 12 and 24 h of incubation compared with nonensiled control forage. This effect was due to HT prior to ensiling as MW treatment decreased (P<.05) NH3 N production at 1, 3, 12, and 24 h of incubation in comparison with the control silage. With the exception of the 12-h incubations, HT for 30 s prior to ensiling did not affect (P>.10) NH 3 N producJournal of Dairy Science Vol. 72, No. 8, 1989

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1Contrast 1, nonensiled, unheated (0 s) vs. ensiled (0, 30, 60, 120, 180 s heat treated); Contrast 2, ensiled, unheated (0 s) vs. ensiled, heat treated (30, 60, 120, 180 s); Contrast 3, ensiled, unheated (0 s) vs. 30 s heat treated; Contrast 4, 120 s heat treated vs. 180 s heat treated; Contrast 5, 60, 120 s heat treated vs. 180 s heat treated.

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HEAT TREATMENT OF FORAGES PRIOR TO ENSILING

tion in comparison with the control silage. Longer durations of MW treatment appear to be required to inactivate plant enzymes or denature plant protein. Microwave treatment at or exceeding 60 s decreases in vitro NH 3 N production and N solubility from that of the 30 s MW treatment. Unfortunately, operating costs associated with long treatment durations would probably be impractical for on-farm processing (19). Increasing MW treatment beyond 60 s did not reduce further (P>.10) NH 3 N production. Nitrogen solubility was also similar among the 60, 120, and 180 s HT. CONCLUSIONS

These results suggest that short-term HT of alfalfa prior to ensiling may be an effective alternative for improving the utilization of silage N in ruminants. Heat treatment prior to ensiling decreased N solubility in silage and the availability of silage N for rumen degradation. This may increase the amount of rumen microbial protein synthesis as well as forage protein entering the small intestine. Similar findings with dehydrated alfalfa have been demonstrated in vivo in sheep (9). Because the ADIN content of the silage was only marginally increased by HT, postruminal digestion of any bypass protein should not be reduced. This study was restricted to the effects of different I-IT on N quality of alfalfa silage. Other parameters of silage quality (e.g., pH, lactic acid), while important, were not conducted. Viability of HT prior to ensiling will be dependent on installation and operating costs for the treatment technology, and the resultant animal performance relative to that using conventional protein supplements. ACKNOWLEDGMENTS

Financial assistance was provided by the Ontario Ministry of Agriculture and Food. REFERENCES 1 Association of Official Analytical Chemists. 1980. Official methods of analysis. 12th ed. Assoc. Anal. Chem., Washington, DC. 2 Broderick, G. A. 1978. In vitro procedures for estimating rates of ruminal protein degradation and proportions of protein escaping the rumen undegraded. J. Nutr. 108:181.

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3 Bums, J. C. 1981. Drying of fresh herbage samples for laboratory estimates of quality. Page 131 in Forage evaluation: concepts and techniques. J. L. Wheeler and R. D. Moehrie, ed. CSIRO/Am. Forage and Grassl. Counc., Netley, Anst. 4 Charmley, E., and D. M. Veira. 1987. The effect of inl'tibiting plant proteolysis on protein digestion in sheep given lucerne silages. Page 155 in Proc. 8th Silage Conf., Hurley, UK. 5 England, P., and M. Gill. 1985. The effect of fishmeal and sucrose supplementation on the voluntary intake of grass silage and liveweight gain of young cattle. Anim. Prod. 40:259. 6 Flores, D. A., L. E. Phillip, D. M. Veira, and M. Ivan. 1986. Digestion in the lumen and amino acid supply to the duodenum of sheep fed ensiled and fresh alfalfa. Can. J. Anita. Sci. 66:1019. 7Glerm, B. P., and D. R. Waldo. 1986. Alfalfa and orchardgrass silages treated with formaldehyde and formic acid or anhydrous ammonia for heifers. J. Dairy Sci. 69:1317. 8 Goering, H. K., and P. J. Van Soest. 1970. Forage fiber analyses. Agric. Handbook No. 379. Agric. Res. Serv., US Dep. Agric. Washington, DC. 9 Goering, H. K., and D. R. Waldo. 1978. The effects of dehydration on protein utilization in ruminants. Page 277 in Proc. of the 2rid Int. Green Crop Drying Congr., Saskatoon, Can. 10 Janicki, F. J., and C. C. Stallings. 1987. Nitrogen fractions of alfalfa silage from oxygen-limiting and conventional upright silos. J. Dairy Sci. 70:116. 11 Jones, P.I.H., and G. Griffith. 1968. Microwave drying of herbage. J. Br. Grassl. Soc. 23:202. 12 Krause, V., and T. Klopfenstein. 1978. In vitro studies of dried alfalfa and complementary effects of dehydrated alfalfa and urea in ruminant rations. J. Anim. Sci. 46:499. 13 Kung, Jr., L., W. M. Craig, L. D. Satter, and G. A. Broderick. 1986. Effect of adding formaldehyde, glutaraldehyde, or dimethylolurea to alfalfa before ensiling. J. Dairy Sci. 69:2846. 14 Mangan, J. L. 1982. The nitrogenous constituents of fresh forages. Page 25 in Forage protein in ruminant animal production. D. J. Thomas, D. E. Beever, and R. G. Durra, ed. Br. So(:. Anim. Prod. Occas. Publ. No. 6. 15 McDonald, P. 1981. The biochemistry of silage. John Wiley & Sons, Inc., New York, NY. 16 Mowat, D. N., and J. G. Buchanan-Smith. 1988. Protein supplementation of alfalfa/grass silages for cattle. Page 181 in Proc. Am. Forage Grassl. Counc. 17 Muck, R. E.0 and J. T. Dickerson. 1987. Storage temperature effects on proteolysis in alfalfa silage. ASAE Paper No. 87-1078, Am. Soc. Agric. Eng., St. Joseph, MI. 18 National Research Council. 1984. Nutrient requirements of beef cattle. 6th ed. Natl. Acad. Sci., Washington, DC. 19 Nelson, S. O. 1987. Potential agricultural applications for RF and microwave energy. Trans. Am. Soc. Agric. Eng. 30:818. 20 Pathak, J. P., R. H. MacMillan, D. G. Evans, and D. R. Murray. 1978. The effects of temperature on the retention and denaturation of protein in lucerne leaves subjected to mechanical dewatering. J. Sci. Food Agric. 29:835. 21 Smith, D. 1973. Influence of drying and storage conditions on nonstructural carbohydrate analysis of herbage Journal of Dairy Science Vol. 72, No. 8, 1989

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tissue-a review. J. Br. Grassl. Soc. 28:129. 22 Steel. R.G.D., and J. H. Torrie. 1980. Principles and procedures of statistics. McGraw-Hill, New York, NY. 23 Thomas, P. C. 1982. Utilization of conserved forages. Page 55 in Forage protein in ruminant animal production. D. J. Thomson, D. E. Beever, and R. G. Gunn, ed. Occas. Publ. No. 6. Br. Soc. Anim. Prod. 24 Van Soest, P. J., and J. B. Robertson. 1980. Systems of analysis for evaluating fibrous feeds. Page 4 9 / n Standardization of analytical methodology for feeds. W. J. Pigden, C. C. Balch, and M. Graham, ed. IDRC, Ottawa, Can. 25 Veira, D. M., G. Butler, M. Ivan, and J. G. Proulx. 1985.

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Utilization of grass silage by cattle: effect of barley and fishmeal supplements. Can. J. Anim. Sci. 65:897. 26 Wilkinson, J. M. 1981. Losses in the conservation and utilization of grass and forage crops. Ann. Appl. Biol. 98: 365. 27 Wolf, D. D., and E. W. Carson. 1973. Respiration during drying of alfalfa herbage. Crop Sci. 13:660. 28 Woodly, A., J. D. Summers, and W. K. Bilanski. 1972. Effects of heat treatment on the nutritive value of whole rapeseed for poultry. Can. J. Anim. Sci. 52:189. 29 Yu, Y., and D. M. Veira. 1977. Effect of artificial heating of alfalfa haylage on chemical composition and sheep performance. J. Anita. Sci. 44:1112.