Fractionation of dry matter losses of sugarcane silage treated with alkalis or urea

Fraccionamiento de las pérdidas de la materia seca del ensilaje de caña de azúcar tratada con alcalinizantes o urea

Laura M. Oliveira-Borgatti¹, José Pavan-Neto¹, Carolina Tobias-Marino^1*, Paula Marques-Meyer², P. Henrique Mazza-Rodrigues^1,3

¹ Department of Animal Nutrition and Production, Faculty of Veterinary Medicine and Animal Science, University of São Paulo. Duque de Caxias Norte Avenue, 225, Zipcode: 13630-900, Pirassunun.ga, São Paulo, Brazil. *Author for correspondance. (caroltobias@hotmail.com)

³ CNPq Productivity Research Scholarship.

Received: August, 2014.
Approved: April, 2015.

Abstract

Different additives evaluation can contribute fof improving quality and yield of silage production. Therefore, the objective of this study was to evaluate the effects of adding alkalis or urea on fractionation losses due to fermentation and on aerobic stability of sugarcane silage. The experimental design was completely randomized with 13 treatments (6x2) +1: 6 additives (1 or 2 %) plus a control group, and four repetitions per treatment. Data were analyzed by polynomial regression analysis using GLM procedure. The tested additives were sodium hydroxide (NaOH), limestone (CaCO₃), urea (CO(NH₂)₂), sodium bicarbonate (NaHCO₃), quicklime (CaO) and hydrated lime (Ca(OH)₂). The material was ensiled in 52 laboratory silos (plastic buckets; 12 L capacity) and silos were opened 60 d after ensilage. Losses were calculated as the difference among weights at the filling and at the opening of the silos, multiplied by their dry matter (DM) content. Fraction losses were obtained by multiplying initial and final DM amounts by their contents of components. The inclusion of all additives in both concentrations diminished DM loss between 27 to 40 %, as compared to control group. Soluble carbohydrates loss was decreased between 7 to 62 % by NaOH, NaHCO3 and CaCO3 addition, as compared to control group. Sodium bicarbonate, CaO and Ca(OH)₂ inclusion reduced neutral detergent fiber (NDF) loss between 20.5 to 51 % and diminished acid detergent fiber (ADF) between 39 to 58 %, as compared to control group. The use of NaOH, CaO and Ca(OH)₂ improved aerobic stability of silages.

Key words: additive, aerobic stability, conserved forage, ensilage, Saccharum officinarum.

La evaluación de distintos aditivos puede contribuir a mejorar la calidad y el rendimiento de la producción de ensilado. Por lo tanto, el objetivo de este estudio fue evaluar los efectos al añadir alcalinizantes o urea en las pérdidas de fraccionamiento por fermentación y en la estabilidad aeróbica del ensilado de caña de azúcar. El diseño experimental fue completamente aleatorio con 13 tratamientos (6x2) +1: 6 aditivos (1 o 2 %) y un grupo testigo, con cuatro repeticiones por tratamiento. Los datos se analizaron con un análisis de regresión no lineal utilizando el procedimiento GLM. Los aditivos a prueba fueron hidróxido de sodio (NaOH), caliza (CaCO₃), urea (CO(NH₂)₂), bicarbonato de sodio (NaHCO₃), cal viva (óxido de calcio, CaO) y cal hidratada (Ca(OH)₂). El material se ensiló en 52 silos de laboratorio (cubetas de plástico; capacidad de 12 L) y los silos se abrieron 60 d después del ensilado. Las pérdidas de fracciones se calcularon como la diferencia en el peso al llenado y al abrir los silos, multiplicada por su contenido de materia seca (MS). La pérdida de fracciones se obtuvo al multiplicar las cantidades inicial y final de MS por los contenidos de componentes. La adición de todos los aditivos en ambas concentraciones disminuyó la pérdida de MS entre 27 a 40 %, en comparación con el grupo testigo. La pérdida de carbohidratos solubles fue reducida entre 7 a 62 % al agregar NaOH, NaHCO₃ y CaCO₃ respecto al grupo testigo. La incorporación de NaHCO₃, CaCO y Ca(OH)₂ redujo la pérdida de fibra detergente neutra (FND) entre 20.5 a 51 % y disminuyó la pérdida de fibra detergente ácido (FDA) entre 39 a 58 % en comparación con el grupo testigo.

Palabras clave: aditivo, estabilidad aeròbica, forraje conservado, ensilado, Saccharum officinarum.

INTRODUCTION

Sugarcane (Saccharum officinarum L.) is used to replace roughage as a feed source for livestock during the dry season and it is increasing in Brazil due to the high yield and energy per area. Although it is an excellent alternative to decrease operational work, the process of sugarcane ensiling produces alcoholic fermentation, leading to large losses of DM and nutritive value (Cavalli et al., 2010; Pedroso et al., 2011). Ensiled sugarcane has high concentrations of ethanol, due to its soluble carbohydrates content and yeast populations, which convert sugars to ethanol, CO₂ and water, decreasing the content of soluble carbohydrates and increasing the components of cell wall and DM losses, which impair silage quality (Pedroso et al., 2005).

Untreated sugarcane silage has 7.8 to 17.5 % ethanol, which causes up to 29 % DM silage losses (Siqueira et al., 2007). Bacterial inoculants, sodium hydroxide or soybean crop residue were added to sugarcane silage; soybean crop residue improved nutritional quality and reduced DM losses and ethanol production of silages, whereas sodium hydroxide decreased ethanol production, but did not affect nutritional quality neither DM losses (Freitas et al, 2006).

According to Siqueira et al. (2007), the association of Lactobacillus buchneri with sodium hydroxide reduced losses by gases and effluents and increased the DM recovery. Balieiro Neto et al. (2007) applied 0.5, 1 and 2 % of quicklime during ensiling and observed positive correlation with in vitro DM digestibility (IVDMD) and improved non-fiber carbohydrate preservation after silo opening by using 1 and 2 % of quicklime.

The use of alkalizing agents in the preservation process has positive effects (Balieiro Neto et al., 2007; Amaral et al., 2009; Borgatti et al., 2012). In untreated silages, the fraction lost is primarily soluble carbohydrates (Mc Donald et al., 1991), but in alkalis-treated silages, solubilization of fiber components produce sugars, which can be converted to volatile compounds (Pedroso et al., 2007, Borgatti et al., 2012). However, this information is not well documented in the literature reviewed, especially for sugarcane silage. Therefore, the aim of this study was to fractionate DM losses during the fermentation process of sugarcane ensiled with different alkalis or urea, as well as to evaluate the aerobic stability of these silages.

Experimental protocol

The trial was carried out at the Department of Animal Nutrition and Production, Veterinary Medicine and Animal Science Faculty, University of São Paulo (USP; Pirassununga Campus), in September 2007. Sugarcane (34.97 % DM) was chopped (0.95 cm average) when ensiled (Chopper Nogueira, model EM-9F3B). The evaluation of mean theoretical particle size was performed according to the sieves methodology Penn State Particle Size Separator (Lammers et al., 1996).

The experimental design was completely randomized with a factorial arrangement of treatments (6x2) +1: sodium hydroxide (NaOH), limestone (CaCO₃), urea (CO(NH₂)₂), sodium bicarbonate (NaHCO₃), quicklime (CaO) and hydrated lime (Ca(OH)₂), in two concentrations (1 or 2 % of fresh matter), plus a control group (0 % additive). The additives were applied and mixed with chopped sugarcane and homogenized.

There were 52 experimental silos in plastic buckets (252 mm high 245 mm, 12 L capacity) (four replicates per treatment). Then, silages (sugarcanes plus additives) were placed inside each silo and compacted to a density of 500 kg sugarcane m^-3. Silos were sealed with lids, weighed and vertically stored in a covered area at room temperature and opened after 60 d of storage. At opening, silos were weighed to determine DM losses during fermentation as the difference between weights at filling and at opening of silos, multiplied by the DM contents. Losses were transformed in percentage of initial silage; losses of different fractions were obtained by multiplying initial and final DM by the contents of each component.

Nutrient content analysis

Laboratory analyzes were carried out at the Laboratory of Animal Nutrition and Production, Veterinary Medicine and Animal Science Faculty, USP. Silos were opened, homogenized and a sample was taken to analyze: DM (at 55 and 105 °C in a forced-air circulation oven) and crude protein (CP) (AOAC, 1990); neutral (NDF) and acid detergent fiber (ADF) and lignin, according to Van Soest et al. (1991); soluble carbohydrates (SC) according to Johnson et al. (1996); insoluble N in acid detergent (INAD), according to Van Soest and Robertson (1985). Other sample was frozen for counter-proof and another was placed in hydraulic press for silage juice extraction.

Aerobic stability

To determine aerobic stability of silage, 2.0 kg samples of fresh mass were taken from each bucket, placed in Styrofoam boxes (12 L capacity) and stored in a controlled temperature (25 °C) room. Silage temperatures were monitored each hour for 7 d, using the Monitoring and Acquiring Data System (SIMAD), with 12 temperature sensors, 2 data acquiring modules, 1 net converter and 1 software for monitoring, acquiring and controlling environmental variables (MACVA version 1.2 from AUTSENS-Industry and Trade Electronic Devices). Maximum temperatures (°C), time to reach the maximum temperature (h) and time to raise the temperature in 2 °C (h) were recorded. Aerobic stability was calculated as the temperature rise rate (ºC/h), dividing the maximum temperature by the time to reach it (Ruppel et al., 1995).

Statistical analysis

The Relative Biological Efficiency (EBR) of each additive in decreasing silage losses calculated by slope ratio (Ammerman et al., 1995), in which the regression curve slope of the response variable for additive levels (0, 1.0 or 2.0 %) was divided by the regression coefficient (slope) of the standard additive (NaOH) with a 100 % EBR. The slope ratio would be used to compare the angular coefficients two by two as to generate the comparison between the EBR of the additives; however, most responses were non-linear and this method was not used. Therefore, polynomial regression analysis was performed (p≤0.05).

RESULTS AND DISCUSSION

There was a non-linear reduction of DM losses, although urea caused a linear reduction (Table 1). Freitas et al. (2006) report that NaOH did not reduce DM losses of sugarcane silage; however, in our study, the lowest DM losses were due to this additive. The addition of 1% NaOH to sugarcane silage decreased gas losses (6.2 %) compared to no additive (13.2 %) (Siqueira et al., 2007) and, according to Amaral et al. (2009), gas losses were lower for silages with 1 % of quicklime or limestone (13.5 % lower for limestone, as compared to 0 % additive). Santos et al. (2008) report lower gas losses in silages with 1 % of quicklime, as compared to values in our experiment; however, results were similar for control silage (32.1 %) and silage with 1 % limestone (17.2 %). The main reason for DM losses in sugarcane silage is the biochemical reaction of ethanol production, when DM is catalyzed during yeast fermentation since each glucose molecule fermented releases two molecules of ethanol, two of carbon dioxide and two of water (Rodrigues et al., 2005).

Treatments did not change CP losses (Table 1), which may be due to the fact that sugarcane contains only 3 % CP.

Water soluble carbohydrates (WSC) losses were linearly reduced by NaOH and NaHCO₃, but this reduction was non linear when using CaCO₃ and Ca(OH)₂ (Table 1). Probably, these additives inhibit bacteria growth that uses the sugar present in sugarcane in order to grow. The addition of CaO linearly increased the losses of WSC, but the addition of urea did not change these losses (Table 1).

Urea addition to Tanzania grass silage reduced gas losses at 30 d after ensiling, but there was no effect for 60 d (Oliveira et al., 2009) and, in our experiment, with the 60 day-storage period because urea did not change losses of the DM fractions when compared with control. Schmidt et al. (2007) added urea to sugarcane silage and did not observe any effect on soluble carbohydrates content, which may be due to the fact that the carbohydrates in silages with urea were metabolized into more stable products than ethanol, such as other alcohols and aldehydes, not considered in DM losses. The reduction in WSC content is unavoidable during fermentation of silages because they are the substrates for organic acids production, which preserves the silage. Treatment with alkalis agents suggest an inhibitor effect on yeast growth, as treated silages had higher soluble carbohydrates concentration, indicating lower losses of this component, with the exception of quicklime.

In our experiment, sodium hydroxide linearly increased hemicellulose losses, whereas sodium bicarbonate reduced these losses in a curvilinear way (Table 1).

Linearity deviation was observed when quicklime was evaluated and the lowest hemicellulose losses were observed with 1% addition. There was no effect of limestone, urea or hydrated lime on this fraction (Table 1). Hemicellulose losses were calculated as the difference between the initial percentage and that found when the silo was opened; this result could be explained by the partial solubilization and utilization of this fraction by the microorganisms. McDonald et al. (1991) point out that the amount of organic acids produced during silage fermentation is higher than the possible amount to be produced from soluble carbohydrates available in ensiled material; therefore, an extra source of carbohydrates might exist. Probably, hemicellulose is the main carbohydrate hydrolyzed by hemicellulases from bacterial origin of the plant or by the action of alkalis agents. Hemicellulose seems to be the main additional source of substrate for fermentation, which may occur up to 40 % of this fraction utilization (Henderson, 1993), whereas cellulose and lignin remain practically unchanged during ensilage (Morrison, 1993).

Treatments did not change lignin losses (Table 2), lignin losses were numerically negative and a T test was performed for means equal to zero. This test indicated that the value of lignin losses was not statistically different from zero. Therefore, the negative value observed refers to a technique error and should be considered null.

Regarding NDF losses, linearity deviation was observed with the inclusion of sodium bicarbonate or hydrated lime, and both additives reduced the losses of this fraction when compared with control silage. For ADF losses, linearity deviation was also observed when using sodium hydroxide, limestone or quicklime and there was a decrease, as compared to control silage; besides, hydrated lime linearly decreased ADF losses (Table 2). The ADF losses seem to be mainly related to cellulose losses results, since there was no effect of additives on lignin losses in silages.

According to McDonald et al. (1991), NDF losses during ensilage can be relatively high and mainly correspond to the solubilization of hemicellulose fraction. Furthermore, using calcium oxide will change cell wall components and reduce organic soluble fractions disappearance in sugarcane silages (Santos et al. (2008).

Aerobic stability results are presented in Table 3. Sodium hydroxide caused a non-linear decrease of maximum temperature, as well as a linear temperature raise rate in 2 °C, indicating that this alkali improved aerobic stability of sugarcane silage. Pedroso (2003) used 0, 1, 2 and 3 % of NaOH, and found that 1 % increased environment temperature (to evaluate aerobic stability) from 65 (control) to 120 h to reach 2 °C.

In our study, stability was improved by increasing NaOH from 0 to 2 %. In contrast, Siqueira et al. (2005) observed a reduction in stability, probably due to solubilization of cell wall components and an increment in carbohydrates availability, which would improve development of microorganisms. But we did not observe this response as the stability linearly increased with sodium hydroxide dose, suggesting a better growth of fungus and yeasts.

Both doses of limestone addition changed the maximum temperature of silage but did not affect the time to reach maximum temperature, whereas with 1 % the temperature rise rate was lower and the time to raise the temperature in 2 °C was higher (Table 3).

There was linearity deviation for maximum temperature, time to reach maximum temperature and for temperature rise rate, when using sodium bicarbonate; however, the time to raise the temperature in 2 °C did not change (Table 3). There was a non-linear increase of the temperature rise rate for both doses of NaHCO₃, indicating that the aerobic stability of the silages was impaired.

The addition of quicklime caused a linear deviation for maximum temperature and temperature rise rate, but a linear decrease for the time to reach the maximum temperature (Table 3); therefore, aerobic stability of silages was improved. Balieiro Neto et al. (2009) observed that 0.5 % of CaO increased aerobic stability of sugarcane silage through an increase in the number of days needed for rising the silage temperature in 2 °C. According to Rezende et al. (2011), sugarcane silage with 0.5 % of CaO reached first 2 °C above the environmental temperature after 4 d of aerobic exposure, as compared to 0, 1 and 1.5 %; these authors point out that such silage might have more residual carbohydrates, thus promoting a favorable environment for development of deteriorating microorganisms.

There was linear deviation for maximum temperature and time to reach the maximum temperature, when the additive tested was hydrated lime (Table 3). The temperature rise rate linearly decreased, indicating that this additive improved the aerobic stability of the silages.

CONCLUSION

Sodium hydroxide, limestone, sodium bicarbonate and quicklime reduced dry matter losses by the decrease in soluble carbohydrates losses, which can further reduce hemicellulose or cellulose losses or both. Therefore, these additives improved silage aerobic stability and the better effect was brought about by sodium hydroxide.

ACKNOWLEDGMENTS

The authors thank Everson J. Lázaro and Gilmar E. Botteon for the care with the culture and also the technicians Ari Luiz de Castro, Gilson L. A. Godoy and Simi L. D. Aflalo for the assistance with laboratory analysis. Also, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for research funding.

LITERATURE CITED

Amaral, R. C., A. V. Pires, I. Susin, L. G. Nussio, C. Q. Mendes, e A. L. Gastaldello Júnior. 2009. Cana-de-açúcar ensilada com ou sem aditivos químicos: fermentação e composição química. Rev. Bras. Zootec. 38:1413-1421.         [ Links ]

Ammerman, C. B., D. H. Baker, A. J. Lewis. 1995. Bioavailability of Nutrients for Animal: Amino Acids, Mineral and Vitamins. San Diego: Academic Press. 441 p.         [ Links ]

A.O.A.C. (Association of Official Analytical Chemists). 1990. Official Methods of the Association of the Agricultural Chemists. (15 Ed.) Arlington. 1298 p.         [ Links ]

Balieiro Neto, G., G. R. Siqueira, R. A. Reis, J. R. Nogueira, M. T. P. Roth, e A. P. T. P. Roth. 2007. Óxido de cálcio como aditivo na ensilagem de cana-de-açúcar. Res. Bras. Zootec. 36:1231-1239.         [ Links ]

Balieiro Neto, G., G. R. Siqueira, J. R. Nogueira, R. A. Reis, A. P. T. P. Roth, e M. T. P. Roth. 2009. Perdas fermentativas e estabilidade aeróbia de silagens de cana-de-açúcar aditivadas com cal virgem. Rev. Bras. Saúde Prod. Ani.10:24-33.         [ Links ]

Borgatti, L. M. O., J. P. Neto, A. L. V. Conrado, C. T. Marino, P. M. Meyer, and P. H. M. Rodrigues. 2012. Evaluation of relative biological efficiency of additives in sugarcane ensiling. Rev. Bras. Zootec. 41:835-845.         [ Links ]

Cavali, J., O. G. Pereira, S. de C. Valadares Filho, M. O. Porto, F. E. Paiva, and G. Rasmo. 2010. Mixed sugarcane and elephant grass silages with or without bacterial inoculant. Rev. Bras. Zootec. 39:462-470.         [ Links ]

Freitas, A. W. P., J. C. Pereira, F. C. Rocha, E. Detmann, M. D. Ribeiro, M. G. Costa, e F. P. Leonel. 2006. Características da silagem de cana-de-açúcar tratada com inoculante bacteriano e hidróxido de sódio e acrescida de resíduo da colheita de soja. Rev. Bras. Zootec. 35:48-59.         [ Links ]

Henderson, N. 1993. Silage additives. Anim. Feed Sci. Technol. 45:35-56.         [ Links ]

Johnson, R. R., T. L. Balwani, L. J. Johnson, K. E. McClure, and B. A. Dehority. 1996. Corn plant maturity. II Effect on in vitro cellulose digestibility and soluble carbohydrate content. J. Ani. Sci. 25: 617-623.         [ Links ]

Lammers, B. P., D. R. Buckmaster, and A. J. Heinrichs. 1996. A simplified method for the analysis of particle sizes of forage and total mixed rations. J. Dairy Sci. 79: 922-928.         [ Links ]

McDonald, P., A. R. Henderson, and S. J. E. Heron. 1991. The Biochemistry of Silage. 2.ed. Marlow: Chalcomb Publ. 340 p.         [ Links ]

Morrison, W. H., D. E. Akin, D. S. Himmelsbach, and G. R. Gamble. 1993. Investigation of the ester and ether linked phenolic constituents of cell wall types of normal and Brown midrib pearl millet using chemical isolation, microspectrophotometry and 13C NMR spectroscopy. J. Sci. Food Agric. 63: 329-337.         [ Links ]

Oliveira, H. C., A. J. V. Pires, A. C. Oliveira, A. L. Rocha Neto, U. Matos Neto, G. G. P. Carvalho, C. M. Veloso, e U. L. C. Oliveira. 2009. Perdas e valor nutritivo da silagem de capim-tanzânia amonizado com uréia. Arq. Zootec. 58: 195-202.         [ Links ]

Pedroso, A. F. 2003. Aditivos químicos e microbiano no controle de perdas e na qualidade de silagem de cana-de-açúcar (Saccharum officinarum L.). Tese (Doutorado em Agronomia) — Escola Superior de Agricultura Luiz de Queiroz, Piracicaba, SP. 120 p.         [ Links ]

Pedroso, A. F., L. G. Nussio, S. F. Paziani, D. R. S. Loures, M. S. Igarasi, R. M. Coelho, I. H. Packer, J. Horii, L. H. Gomes. 2005. Fermentation and epiphytic microflora dynamics in sugar cane silage. Sci. Agric. 62: 427-432.         [ Links ]

Pedroso, A. F., L. G. Nussio, D. R. S. Loures, T. S. F. Paziani, M. S. Igarasi, R. M. Coelho, J. Horii, e A. A. Rodrigues, 2007. Efeito do tratamento com aditivos químicos e inoculantes bacterianos nas perdas e na qualidade de silagens de cana-de-açúcar. Rev. Bras. Zootec. 36: 556-564.         [ Links ]

Pedroso, A. F., A. A. Rodrigues, W. Barioni Junior, P. F. Barbosa, F. A. P. Santos, L. G. Nussio. 2011. Aditivos químicos e inoculante bacteriano na ensilagem de cana-de-açúcar: efeitos sobre a fermentação das silagens e o desempenho de garrotes. Rev. Bras. Zootec. 40: 1181-1187.         [ Links ]

Rezende, A. V., C. H. S. Rabelo, F. H. S. Rabelo, D. A. Nogueira, D. C. N. A. Faria Junior, e L. A. Barbosa, 2011. Perdas fermentativas e estabilidade aeróbia de silagem de cana-de-açúcar tratadas com cal virgem e cloreto de sódio. Rev. Bras. Zootec. 4: 739-746.         [ Links ]

Rodrigues, P. H. M., L. M. O. Borgatti, R. W. Gomes, R. Passini, e P. M. Meyer, 2005. Efeito da adição de níveis crescentes de polpa cítrica sobre a qualidade fermentativa e o valor nutritivo da silagem de capim-elefante. Rev. Bras. Zootec. 34: 1138-1145.         [ Links ]

Ruppel, K. A., R. E. Pitt, L. E. Chase, and D. M. Galton. 1995. Bunker silo management and its relationship to forage preservation on dairy farms. J. Dairy Sci. 78: 141-153.         [ Links ]

Santos, M. C., L. G. Nussio, G. B. Mourão, P. Schmidt, L. J. Mari, e J. L. Ribeiro. 2008. Influência da utilização de aditivos químicos no perfil da fermentação, no valor nutritivo e nas perdas de silagens de cana-de-açúcar. Rev. Bras. Zootec. 37: 1555-1563.         [ Links ]

Schmidt, P., L. J. Mari, L. G. Nussio, A. F. Pedroso, S. F. Paziani, e F. S. Wechsler. 2007. Aditivos químicos e biológicos na ensilagem de cana-de-açúcar. 1- Composição química das silagens, ingestão, digestibilidade e comportamento ingestivo. Rev. Bras. Zootec. 36: 1666-1675.         [ Links ]

Siqueira, G. R., T. B. Fernandes, e R. A. Reis. 2005. Instabilidade aeróbia de silagens: efeitos e possibilidades de prevenção. In: Volumosos na Produção de Ruminantes. Jaboticabal: Funep. p: 25-60.         [ Links ]

Siqueira, G. R., R. R. Reis, R. P. Schocken-Iturrino, T. F. Bernardes, A. J. V. Pires, M. T. P. Roth, and A. P. T. P. Roth. 2007. Associação entre aditivos químicos e bacterianos na ensilagem de cana-de-açúcar. Rev. Bras. Zootec. 36: 789-798.         [ Links ]

SAS. 2001. SAS User's Guide. 8 ed. Cary, NC: SAS Inst., Inc.         [ Links ]

Van Soest, P. J., and J. B. Robertson. 1985. Analysis of Forages and Fibrous Foods. Ithaca: Cornell University. 202 p.         [ Links ]

Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Carbohydrate methodology, metabolism, and nutritional implications in dairy cattle. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74: 3583-3597.         [ Links ]