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Effects of species and maturity stage on nutritional and fermentation characteristics of Sarcobatus species
Animal Feed Science and Technology 211 (2016) 241–245
Contents lists available at ScienceDirect
Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
Short communication
Effects of species and maturity stage on nutritional and fermentation characteristics of Sarcobatus species Teshome Shenkoru, Barry L. Perryman ∗ The College of Agriculture, Biotechnology, and Natural Resources, University of Nevada-Reno, Mail Stop 202, Reno, NV 89557, USA
a r t i c l e
i n f o
Article history: Received 20 July 2015 Received in revised form 16 November 2015 Accepted 19 November 2015 Keywords: Sarcobatus spp. Ammonia nitrogen Volatile fatty acids
a b s t r a c t The aim of this paper was to investigate the effect of maturity stage and species on nutritional and rumen fermentation characteristics of two species of greasewood, Black [Sarcobatus vermiculatus (Hook) Torr], and Bailey [Sarcobatus baileyi (Coville) Jepson]. Crude protein (CP) content of both species for all stages ranged from 114.1 to 204.8 g/kg, which is greater than the maintenance requirement of cattle. Highest CP content (204.8 g/kg, P = 0.045) was observed for Black greasewood pre-bloom. Highest acid detergent fiber (ADF) and lignin (sa) fractions (311.3 and 103.9 g/kg, P < 0.05, respectively) were recorded for Bailey post-bloom, while the lowest ADF and Lignin (sa) fractions (163.4 g/kg and 36.3 g/kg, P < 0.05, respectively) were obtained for Black greasewood pre-bloom stage. In vitro dry matter (IVDMD) and organic matter (IVOMD) disappearance, ammonia nitrogen (NH3 N), and volatile fatty acid (VFA) content were determined at the end of a 48 h incubation period. Black greasewood was higher (409.0 vs. 366.1 g/kg; P = 0.006) in IVOMD than Bailey greasewood. The pre-bloom stage yielded more (P = 0.015) IVOMD than the bloom and postbloom stages. No IVOMD difference was observed between bloom and post-bloom stages. Rumen ammonia nitrogen was highest (32.13 mg/100 ml; P = 0.005) for Black greasewood pre-bloom. Total VFA was significantly affected by species and maturity. The highest total VFA content was observed for both species pre-bloom. Total VFA remained the same for Black greasewood at bloom and post-bloom stages, while it declined significantly with increased maturity for Bailey. Based on this study Black greasewood has greater potential than Bailey greasewood as a late summer and early fall forage source in salt desert shrub plant communities of the Great Basin. © 2015 Published by Elsevier B.V.
1. Introduction Native forages with inherently high protein content can be important forages for rangeland animal agriculture operations (wild ungulates as well) in the Great Basin and other areas of western Northern America. Two species of greasewood, Black [Sarcobatus vermiculatus (Hook) Torr.], and Bailey [Sarcobatus baileyi (Cov.) Jeps.], occur in the Great Basin. The former is distributed throughout the region on alkaline or saline floodplains with relatively high water tables, often functioning as
Abbreviations: ADF, acid detergent fiber (inclusive of residual ash); Lignin (sa), lignin determined by solubilization of cellulose with sulphuric acid; CP, crude protein; DM, dry matter; IVDMD, in vitro dry matter digestibility; IVOMD, in vitro organic matter digestibility; aNDF, neutral detergent fiber assayed with heat stable amylase and expressed with residual ash; OM, organic matter; NH3 N, ammonia nitrogen; VFA, volatile fatty acid. ∗ Corresponding author. E-mail address: [email protected] (B.L. Perryman). http://dx.doi.org/10.1016/j.anifeedsci.2015.11.010 0377-8401/© 2015 Published by Elsevier B.V.
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T. Shenkoru, B.L. Perryman / Animal Feed Science and Technology 211 (2016) 241–245
a phreatophyte (Perryman, 2014). Black greasewood covers over 4.8 million ha from Mexico to Canada primarily in cold deserts (Robertson, 1983). Bailey greasewood, a non-phreatic, upland form, occurs on fine to course textured soils derived from saline lake sediments along the east face of the Sierra Mountains, covering approximately 0.2 of western Nevada and eastern California (Robertson, 1983). Black greasewood has been recognized as a palatable rangeland forage known for its oxalate toxicity potential for decades (Fleming et al., 1928). It was first analyzed by Forbes and Skinner (1903) to determine nutritional content by proximate analysis: crude protein (198.1 g/kg), fat (24.5 g/kg); and soluble carbohydrates (342.8 g/kg). Nutritional content suggests that Black greasewood is excellent native forage. However, Forbes and Skinner (1903) also state, “The figures quoted give no grounds for rejection of the plant for grazing purposes; yet, under actual feeding conditions, the greasewood fails to fulfill the promise given by its analysis.” Although Black greasewood is recognized as excellent rangeland forage, it has been understood that under field conditions, animal performance has not matched the nutritional analysis. Ensminger et al. (1990) reported nutritional content of greasewood including crude fiber, ash, ether extract, N-free extract, crude protein, total digestible nutrient, and digestible metabolizable and net energy for ruminants. However, these values were taken from bulk samples at one point in time, or combined across several points in time. They also list the values as Sarcobatus spp., indicating the mixing of greasewood species for their analysis. Nutritional characteristics often change with maturity stage. Smith et al. (1992) demonstrated that mean crude protein levels reached 160 g/kg ranging between 210 g/kg (spring) and 140 g/kg (fall) across the growing season for Black greasewood in ephemeral stream channels of Wyoming. In order to optimize grazing animal efficiency on native rangeland forages, it is necessary to understand how nutritional characteristics change as plants mature. Additionally, nutritional characteristics of Bailey greasewood as a separate species have not been investigated since only recently the genus was formally recognized as having two distinct species (Perryman, 2014). Rumen fermentation characteristics of both species have not been investigated. Our aim was to study the nutritional and fermentation characteristics of two greasewood species at three maturity stages: pre-bloom, bloom, and post-bloom. 2. Materials and methods 2.1. Experimental design The chemical composition experiment was organized as a completely randomized design (CRD), arranged in a 2 × 3 factorial treatment structure with 30 experimental replications (individual plants), and two laboratory replicates run simultaneously. The in vitro experiment was organized identically but with laboratory replication that consisted of three incubation tubes run on each of two consecutive days. Treatments included: the two greasewood species (Black and Bailey) and three maturity stages (pre-bloom, bloom, post-bloom). Chemical composition variables included: dry matter (DM), organic matter (OM), neutral detergent fiber assayed with heat stable amylase (aNDF), acid detergent fiber inclusive of residual ash (ADF), lignin determined by solubilization of cellulose with sulfuric acid [lignin (sa)], and crude protein (CP). In vitro analysis included in vitro dry matter digestibility (IVDMD), in vitro organic matter digestibility (IVOMD), total volatile fatty acids (VFA) and individual fatty acids all measured at 48 h incubation time. 2.2. Field methods Vegetation samples for both species were taken simultaneously in 2003. Pre-bloom samples were obtained in mid-May; bloom stage samples in early-July; and post-bloom in late-September. Samples were taken from ecological sites located approximately 25 miles northeast of Reno, Nevada. Black greasewood was sampled from a gravelly loam 4–8 precipitation zone (027XY018NV) ecological site and Bailey greasewood from a sandy 5–8 precipitation zone (027XY009NV) ecological site (NRCS, 2003) less than 100 m from the former site. Approximately, 20 g (dry weight) of non-woody biomass were collected from 30 random plants of both greasewood species at four random locations in the upper plant canopy and combined into a single, pooled sample for each plant (80 g total per plant). The purpose of multiple canopy locations was to collect material that had a high potential for being consumed by grazing animals. The non-woody biomass included leaves, and current year vegetative and reproductive stems. Sampling also included any reproductive organs and tissues that might be present. For both species, and for each stage of maturity, samples from all 30 plants were oven dried at 55 ◦ C for 48 h and ground to a 2 mm sieved particle size. 2.3. Laboratory methods Chemical composition samples were analyzed for DM and ash according to methods 930.15 and 942.05 of AOAC (2000), respectively; and ADF, Lignin (sa), aNDF, according to Van Soest et al. (1991); neutral detergent fiber was assayed with heat stable alpha amylase, without sodium sulphite and expressed with residual ash. Kjeldahl nitrogen was determined using a Kjelteck 2300 analyzer unit (Foss North America, Eden Prairie, MN). For the in vitro analysis, 0.5 g of substrate from each plant-subspecies-maturity stage was loaded into three separate 50ml centrifuge tubes, and three corresponding blank tubes, for two separate runs (two runs × three tubes). Rumen fluid from two mature, rumen fistulated Angus steers was combined and used to inoculate fermentation tubes (Care and handling of
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fistulated steers, including ruminal cannulation, were conducted under the protocols approved by the University of Nevada Reno Institutional Animal Care and Use Committee). Steers were fed an alfalfa-grass ration for four weeks prior to rumen fluid collection. Fluid was collected 2 h post-feeding and strained through four layers of cheese cloth into a pre-warmed, insulated thermos and transported to the University of Nevada-Reno Animal Nutrition Laboratory. Rumen fluid was then mixed with McDougall’s buffer (McDougall, 1948) on an equal volume basis (with carbon dioxide bubbling), and 30 ml of the mixed inoculum were added to each centrifuge tube (containing a substrate or a blank). Tubes were flushed with carbon dioxide, capped with stoppers equipped with one-way gas release valves, and placed in an incubator/shaker (Innova 4400, New Brunswick Scientific, Edison, NJ) at 39 ◦ C (Tilley and Terry, 1963). At the end of the 48 h incubation period, tubes were placed in storage at 5 ◦ C for 2 h to stop the fermentation process. Supernatant digesta samples were filtered through a pre-weighed Whatman 541 filter paper and washed with 0.78 ethanol and acetone then dried at 105 ◦ C in a drying oven, weighed and ashed in a muffle furnace at 500 ◦ C overnight and weighed again to determine OM residue. In vitro organic matter digestibility was calculated as follows: (1 − ((OM residue − OM blank)/original OM)) × 100. A 4-mL aliquot of supernatant solution was drawn from each substrate tube and prepared according to Erwin et al. (1961) for short chain fatty acid analysis. Volatile fatty acid concentrations in the supernatant were determined with a gas chromatograph (Varian Model 3800, Varian Inc. Walnut Creek, CA, equipped with a glass column (180 cm × 4 mm i.d.) packed with GP 0.1 SP-1200/0.01 H3 PO4 on 80/100 Chromosorb WAW [Supelco, Bellefonte, PA], with nitrogen used as the carrier gas at a flow rate of 85 ml/min. The oven, injection port, and detector (FID) port temperatures were 125, 175, and 180 ◦ C, respectively. The concentration of VFA were expressed on a mM basis and summed. The concentration of each VFA was divided by the sum and multiplied by 100 to get mol/100 mol of total VFA. Ammonia nitrogen content was determined by the Chaney and Marbach (1962) procedure. Post hoc comparisons were performed using least significant difference (LSD). Data were analyzed with JMP Statistical Analysis Software version 7.0.2 (SAS, 2008) and differences determined at (P < 0.05) for all analyses. 3. Results 3.1. Chemical composition Interactions between species and maturity stage were observed for OM, CP, aNDF, ADF, and lignin (sa) contents (Table 1). Organic matter of Bailey greasewood at post-bloom was the highest compared to other stages in both species. Crude protein content of Black greasewood was highest during the pre-bloom stage (204.8 g/kg) and lowest post-bloom (114.1). Lowest CP values were observed at bloom and post-bloom stages for Bailey greasewood (114.5 and 125.3 g/kg). Neutral detergent fiber content was highest (468 g/kg) for Bailey post-bloom, while Black was lowest (313 g/kg) pre-bloom. The highest ADF (249 g/kg) was observed for Bailey post-bloom, while the lowest (164.3 g/kg) was recorded for Black at the pre-bloom stage. Bailey had higher ADF at all stages than Black greasewood. Post-bloom Bailey was higher in lignin (sa) (104 g/kg) than all other stages in both species, and Bailey had higher lignin (sa) at all stages of growth than Black greasewood. 3.2. Fermentation characteristics No interactions were observed for IVOMD and IVDMD (Table 2), so only main effect results are presented. Black greasewood had greater IVOMD and IVDMD (456.4 vs 402.6 g/kg, 555.6 vs 478.1 g/kg) than Bailey greasewood, respectively. Both parameters were also significantly affected by maturity stage. Pre-bloom stage yielded greater IVOMD than bloom and postbloom stages. No difference was noted in IVOMD values for bloom and post-bloom stages. In vitro dry matter digestibility was higher at pre-bloom and bloom stages than post-bloom. Two-way interactions (species × maturity stage) were observed for NH3 N, total VFA, acetate, propionate, isobutyrate, isovalerate, and actate:propionate concentrations (Table 2). Rumen ammonia N concentration was significantly higher for black greasewood at pre-bloom stage compared to all other stages in both species. No difference was observed for Bailey among stages. The lowest value of NH3 N (8.41 mg/100 ml) was observed for Black greasewood, post-bloom. Total VFA Table 1 Effect of species × maturity stages on chemical composition of greasewood species, 2003. Item
OM (g/kg) CP (g/kg) aNDF (g/kg) ADF (g/kg) Lignin (sa) (g/kg)
Baileya
Blacka
SEM
V
B
PB
V
B
PB
795.9b 125.3c 396.3b 242.3b 87.5b
773.2c 114.5d 387.6c 248.5b 74.4c
825.8a 114.0d 467.8a 311.3a 103.9a
772.8c 204.8a 313.3e 164.3d 36.3e
753.6d 129.7b 335.5d 196.2c 49.7d
753.8d 114.1d 336.6d 199.2c 53.9d
16 13 18 47 22
Row means with different letters differ at P < 0.05. OM, organic matter; CP, crude protein; aNDF, neutral detergent fiber assayed with heat stable amylase, and expressed inclusive of residual ash; ADF, acid detergent fiber expressed inclusive of residual ash; Lignin (sa), sulfuric acid lignin. a V, pre-bloom; B, Bloom; PB, Post-bloom.
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Table 2 Main effect of means on in vitro organic matter digestibility (IVOMD), dry matter digestibility (IVDMD) and simple effects of means on ammonia N, total volatile fatty acids (VFA), individual VFA as affected by species and maturity stages interaction, 2003. Stage of maturitya
IVOMD (g/kg)
IVDMD (g/kg)
Ammonia N (mg/ 100 ml)
Total VFA (mM)
Individual VFA (mol/100 mol of total VFA)
Acetate
Propionate Butyrate Isobutyrate Isovalerate Valerate
Ac:Prc
Plant species V Bailey B PB
396.2 370.5 331.5
473.4 472.5 395.8
14.76b 11.65bc 14.47b
44.30a 31.97b 23.68c
66.58a 69.46a 55.88b
22.54b 22.22b 34.48a
7.91 6.06 5.03
0.48b 0.65ab 0.54b
0.94a 0.96a 0.97a
1.55 1.69 2.08
3.11ab 3.52a 2.68b
V B PB
446.2 383.9 397.0
539.5 499.8 508.4
32.13a 11.93bc 8.41c
45.47a 32.72b 33.15b
66.61a 68.45a 69.83a
25.35b 24.68b 23.72b
5.20 4.84 4.61
1.01a 0.46b 0.42b
0.98a 0.66b 0.63b
0.85 0.91 0.79
2.75b 2.87b 3.20ab
*
*
*
*
nsb
ns
ns
ns
ns
ns
ns
*
*
*
*
ns
ns
*
ns
ns
ns
ns
ns
ns
*
*
*
*
ns
*
*
ns
*
10.5
8.10
1.91
1.25
0.95
0.14
0.07
0.13
0.18
Black
Significance of effects Plant species Stage of maturity Species × maturity SEM * a b c
1.36
1.78
P < 0.05. V, pre-bloom; B, Bloom; PB, post-bloom; SEM, standard error of means. ns, not significant P > 0.05. Ac:Pr, acetate to propionate ratio.
concentration was higher for pre-bloom than all other maturity stages for both species. The lowest value (23.68 mM) was recorded for Bailey at the post-bloom stage. Acetate concentration of Bailey at post-bloom was significantly lower than all other stages in both species. Propionate concentration was higher for Bailey at post-bloom; otherwise its concentration was equal for all maturity stages for both species. 4. Discussion Chemical composition and in vitro fermentation characteristics changed as the two species matured. Ash content characteristically high in desert shrubs (Haddi et al., 2003) was also relatively high in both species. Crude protein content of both species at all maturity stages ranged between 114 and 205 g/kg was similar to that of Smith et al. (1992) that reported CP content of Black greasewood harvested in spring, summer and fall to be 213, 126, 139 g/kg, respectively. Bailey greasewood CP content was much lower than Black in the early to mid-growing seasons. Although secondary plant compounds such as the oxalates known to be present in greasewood can make protein less available, CP levels in both species were well above cattle maintenance requirements (75 g/kg, NRC, 1996). The IVOMD of the two species indicated that both are low in digestible energy, and IVOMD declined precipitously as maturity increased. The IVDMD values, initially similar, also decreased with maturity. Generally, Black greasewood had lower ADF levels than Bailey greasewood. The ADF and lignin (sa) (often referred to as ADL), estimate the quantity of cellulose and lignin fractions. Lower ADF and lignin in Black greasewood indicated more digestible dry matter than Bailey. Bailey had higher lignin at all stages of growth compared to Black greasewood indicating the lignification process was faster than for Black greasewood. Volatile fatty acids are the major sources of carbohydrates for ruminant animals. According to Fahey and Berger (1988) VFAs supply about 0.7–0.8 of the host animal’s total caloric requirement. The molar proportion of propionate in post-bloom Bailey was substantially higher than in the other stages. This is due to a reduction in acetate and butyrate as fermentation time increases. The actual propionate fraction remains constant, while its molar proportion increases. This is common for forages with low concentrations of fermentable carbohydrates (Uden, 2011). Total VFA mM was highest at the pre-bloom stage for both species. A major problem when feeding browse to cattle is a lack of synchronization between nitrogen and energy release in the rumen (Broderick et al., 1991). According to Owens and Zinn (1988) for maximal rumen activity and bacterial growth there should be an adequate supply of both carbon and nitrogen, and the degradation of carbon has to match the degradation of nitrogen for efficient microbial fermentation. Our findings indicate that ammonia production was higher for Black greasewood at the pre-bloom stage than Bailey greasewood. However, despite its high CP content and high rumen NH3-N concentration, Black greasewood total VFA production was the same as Bailey greasewood at the pre-bloom stage. This indicates a lack of degradation synchronization of protein and carbohydrate sources in Black greasewood. Although we did not measure bacterial protein synthesis, we hypothesized that there was more CP than available energy for synthesis. As a result, rumen bacterial protein synthesis decreased and the excess ammonia nitrogen was not captured as microbial protein. According to Cook and Harris (1968), during winter periods or dry
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seasons, grasses are generally low in protein, but high in energy content, while shrubs like greasewood are high in protein and relatively low in energy. This makes greasewood which is high in CP, a good supplement for early fall grazing animals when CP levels in senescent grasses fall below required maintenance levels, but are still relatively high in energy. In the Great Basin, early fall corresponds with the time when cattle in particular, are typically at a low cyclical nutrient demand stage. Thus, a mix of grasses and greasewood during these grazing periods should provide forage of adequate nutritional quality for grazing cattle. 5. Conclusions The nutritional value of both species of greasewood native to North America, have been analyzed as separate species for the first time. Bailey had less nutritional value than Black greasewood in terms of DMD and CP content. Crude protein and in vitro DMD decreased with increasing age of greasewood species. Total VFA concentrations of Bailey and Black greasewood were the same at pre-bloom and bloom stages, but VFA dropped significantly for Bailey at the post-bloom stage. The lignification process was faster for Bailey than Black greasewood. Both species showed adequate CP concentration to supply cattle during late-summer and early-fall grazing periods when the CP levels in senescent grasses fall below required maintenance levels. Conflict of interest None declared. Acknowledgment This study was funded by the Nevada Agricultural Experiment Station. References AOAC, 2000. Official Methods of Analysis, seventeenth ed. Association of Official Analytical Chemists, Gaithersburg, MD. Broderick, G.A., Wallace, R.J., Ørskov, E.R., 1991. Control of digestion and extent of protein degradation. In: Tsuda, T., Sasaki, V., Kawashima, R. (Eds.), Physiological Aspects of Digestion and Metabolism in Ruminants. Academic Press, New York, pp. 541–592. Chaney, A.L., Marbach, E.P., 1962. Modified reagents for determination of urea and ammonia. Clin. Chem. 8, 130–132. Cook, C.W., Harris, L.E., 1968. Nutritive Values of Seasonal Ranges. Bull 472. Utah Agricultural Experiment Station, Logan, UT, pp. 55. Ensminger, M.E., Oldfield, J.E., Heinemann, W.W., 1990. Feeds and Nutrition. The Ensminger Publishing Company, Clovis, CA, pp. 1479–1481. Erwin, E.S., Macro, G.J., Emery, E.M., 1961. Volatile fatty acids analysis of blood and rumen fluid by gas chromatography. J. Dairy Sci. 44, 1768–1771. Fahey Jr., G.C., Berger, L.L., 1988. Carbohydrate nutrition of ruminants. In: Church, D.C. (Ed.), The Ruminant Animal, Digestive Physiology, Nutrition. Prentice Hall, Englewood Cliffs, NJ, pp. 269–297. Fleming, C.E., Miller, M.R., Vawter, L.R., 1928. The greasewood (Sarcobatus vermiculatus), a range plant poisonous to sheep. Nevada Agric. Exp. Sta. Bull. 115, 2–22. Forbes, R.H., Skinner, W.W., 1903. Greasewood analysis. In: Annual Arizona Agric Exp. Sta. Rep., fourteenth ed., pp. 349–350. Haddi, M.L., Filacorda, S., Meniai, K., Rollin, F., Susmel, P., 2003. In vitro fermentation kinetics of some halophyte shrubs sampled at three stages of maturity. Anim. Feed Sci. Technol. 104, 215–225. McDougall, R.I., 1948. Study on ruminant saliva. 1. The composition and output of sheep’s saliva. Biochem. J. 43, 99–109. National Research Council (NRC), 1996. Nutrients Requirement of Beef Cattle, seventh ed. National Academy Press, Washington, DC. Natural Resource Conservation Service (NRCS), 2003, April. Nevada Ecological Site Descriptions: Major Land Resource Area 27. Natural Resource Conservation Service, USDA. Owens, F.N., Zinn, R.A., 1988. Protein metabolism of ruminants. In: Church, D.C. (Ed.), The Ruminant Animal, Digestive Physiology and Nutrition. Prentice-Hall, Englewood Cliffs, NJ, pp. 227–249. Perryman, B.L., 2014. A Field Guide to Nevada Shrubs. IRM Press, Lander, WY. Robertson, J.H., 1983. Greasewood [Sarcobatus vermiculatus (Hook.). Torr.]. Phytologia 54, 309–324. SAS, 2008. Statistical Analysis Software JMP Version 7.0.2. SAS Institute Inc., SAS Campus Drive, Cary, NC, USA. Smith, M.A., Rodgers, J.D., Dodd, J.L., Skinner, L., Quentin, D., 1992. Habitat selection by cattle along an ephemeral channel. J. Range Manag. 45 (4), 385–390. Tilley, J.M.A., Terry, R.A., 1963. A two stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Soc. 18, 104–111. Uden, P., 2011. Using a novel macro in vitro technique to estimate differences in absorption rates of volatile fatty acids in the rumen. J. Anim. Phys. Anim. Nutr. 95, 27–33. Van Soest, P.V., Robertson, J., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597.
Contents lists available at ScienceDirect
Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
Short communication
Effects of species and maturity stage on nutritional and fermentation characteristics of Sarcobatus species Teshome Shenkoru, Barry L. Perryman ∗ The College of Agriculture, Biotechnology, and Natural Resources, University of Nevada-Reno, Mail Stop 202, Reno, NV 89557, USA
a r t i c l e
i n f o
Article history: Received 20 July 2015 Received in revised form 16 November 2015 Accepted 19 November 2015 Keywords: Sarcobatus spp. Ammonia nitrogen Volatile fatty acids
a b s t r a c t The aim of this paper was to investigate the effect of maturity stage and species on nutritional and rumen fermentation characteristics of two species of greasewood, Black [Sarcobatus vermiculatus (Hook) Torr], and Bailey [Sarcobatus baileyi (Coville) Jepson]. Crude protein (CP) content of both species for all stages ranged from 114.1 to 204.8 g/kg, which is greater than the maintenance requirement of cattle. Highest CP content (204.8 g/kg, P = 0.045) was observed for Black greasewood pre-bloom. Highest acid detergent fiber (ADF) and lignin (sa) fractions (311.3 and 103.9 g/kg, P < 0.05, respectively) were recorded for Bailey post-bloom, while the lowest ADF and Lignin (sa) fractions (163.4 g/kg and 36.3 g/kg, P < 0.05, respectively) were obtained for Black greasewood pre-bloom stage. In vitro dry matter (IVDMD) and organic matter (IVOMD) disappearance, ammonia nitrogen (NH3 N), and volatile fatty acid (VFA) content were determined at the end of a 48 h incubation period. Black greasewood was higher (409.0 vs. 366.1 g/kg; P = 0.006) in IVOMD than Bailey greasewood. The pre-bloom stage yielded more (P = 0.015) IVOMD than the bloom and postbloom stages. No IVOMD difference was observed between bloom and post-bloom stages. Rumen ammonia nitrogen was highest (32.13 mg/100 ml; P = 0.005) for Black greasewood pre-bloom. Total VFA was significantly affected by species and maturity. The highest total VFA content was observed for both species pre-bloom. Total VFA remained the same for Black greasewood at bloom and post-bloom stages, while it declined significantly with increased maturity for Bailey. Based on this study Black greasewood has greater potential than Bailey greasewood as a late summer and early fall forage source in salt desert shrub plant communities of the Great Basin. © 2015 Published by Elsevier B.V.
1. Introduction Native forages with inherently high protein content can be important forages for rangeland animal agriculture operations (wild ungulates as well) in the Great Basin and other areas of western Northern America. Two species of greasewood, Black [Sarcobatus vermiculatus (Hook) Torr.], and Bailey [Sarcobatus baileyi (Cov.) Jeps.], occur in the Great Basin. The former is distributed throughout the region on alkaline or saline floodplains with relatively high water tables, often functioning as
Abbreviations: ADF, acid detergent fiber (inclusive of residual ash); Lignin (sa), lignin determined by solubilization of cellulose with sulphuric acid; CP, crude protein; DM, dry matter; IVDMD, in vitro dry matter digestibility; IVOMD, in vitro organic matter digestibility; aNDF, neutral detergent fiber assayed with heat stable amylase and expressed with residual ash; OM, organic matter; NH3 N, ammonia nitrogen; VFA, volatile fatty acid. ∗ Corresponding author. E-mail address: [email protected] (B.L. Perryman). http://dx.doi.org/10.1016/j.anifeedsci.2015.11.010 0377-8401/© 2015 Published by Elsevier B.V.
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a phreatophyte (Perryman, 2014). Black greasewood covers over 4.8 million ha from Mexico to Canada primarily in cold deserts (Robertson, 1983). Bailey greasewood, a non-phreatic, upland form, occurs on fine to course textured soils derived from saline lake sediments along the east face of the Sierra Mountains, covering approximately 0.2 of western Nevada and eastern California (Robertson, 1983). Black greasewood has been recognized as a palatable rangeland forage known for its oxalate toxicity potential for decades (Fleming et al., 1928). It was first analyzed by Forbes and Skinner (1903) to determine nutritional content by proximate analysis: crude protein (198.1 g/kg), fat (24.5 g/kg); and soluble carbohydrates (342.8 g/kg). Nutritional content suggests that Black greasewood is excellent native forage. However, Forbes and Skinner (1903) also state, “The figures quoted give no grounds for rejection of the plant for grazing purposes; yet, under actual feeding conditions, the greasewood fails to fulfill the promise given by its analysis.” Although Black greasewood is recognized as excellent rangeland forage, it has been understood that under field conditions, animal performance has not matched the nutritional analysis. Ensminger et al. (1990) reported nutritional content of greasewood including crude fiber, ash, ether extract, N-free extract, crude protein, total digestible nutrient, and digestible metabolizable and net energy for ruminants. However, these values were taken from bulk samples at one point in time, or combined across several points in time. They also list the values as Sarcobatus spp., indicating the mixing of greasewood species for their analysis. Nutritional characteristics often change with maturity stage. Smith et al. (1992) demonstrated that mean crude protein levels reached 160 g/kg ranging between 210 g/kg (spring) and 140 g/kg (fall) across the growing season for Black greasewood in ephemeral stream channels of Wyoming. In order to optimize grazing animal efficiency on native rangeland forages, it is necessary to understand how nutritional characteristics change as plants mature. Additionally, nutritional characteristics of Bailey greasewood as a separate species have not been investigated since only recently the genus was formally recognized as having two distinct species (Perryman, 2014). Rumen fermentation characteristics of both species have not been investigated. Our aim was to study the nutritional and fermentation characteristics of two greasewood species at three maturity stages: pre-bloom, bloom, and post-bloom. 2. Materials and methods 2.1. Experimental design The chemical composition experiment was organized as a completely randomized design (CRD), arranged in a 2 × 3 factorial treatment structure with 30 experimental replications (individual plants), and two laboratory replicates run simultaneously. The in vitro experiment was organized identically but with laboratory replication that consisted of three incubation tubes run on each of two consecutive days. Treatments included: the two greasewood species (Black and Bailey) and three maturity stages (pre-bloom, bloom, post-bloom). Chemical composition variables included: dry matter (DM), organic matter (OM), neutral detergent fiber assayed with heat stable amylase (aNDF), acid detergent fiber inclusive of residual ash (ADF), lignin determined by solubilization of cellulose with sulfuric acid [lignin (sa)], and crude protein (CP). In vitro analysis included in vitro dry matter digestibility (IVDMD), in vitro organic matter digestibility (IVOMD), total volatile fatty acids (VFA) and individual fatty acids all measured at 48 h incubation time. 2.2. Field methods Vegetation samples for both species were taken simultaneously in 2003. Pre-bloom samples were obtained in mid-May; bloom stage samples in early-July; and post-bloom in late-September. Samples were taken from ecological sites located approximately 25 miles northeast of Reno, Nevada. Black greasewood was sampled from a gravelly loam 4–8 precipitation zone (027XY018NV) ecological site and Bailey greasewood from a sandy 5–8 precipitation zone (027XY009NV) ecological site (NRCS, 2003) less than 100 m from the former site. Approximately, 20 g (dry weight) of non-woody biomass were collected from 30 random plants of both greasewood species at four random locations in the upper plant canopy and combined into a single, pooled sample for each plant (80 g total per plant). The purpose of multiple canopy locations was to collect material that had a high potential for being consumed by grazing animals. The non-woody biomass included leaves, and current year vegetative and reproductive stems. Sampling also included any reproductive organs and tissues that might be present. For both species, and for each stage of maturity, samples from all 30 plants were oven dried at 55 ◦ C for 48 h and ground to a 2 mm sieved particle size. 2.3. Laboratory methods Chemical composition samples were analyzed for DM and ash according to methods 930.15 and 942.05 of AOAC (2000), respectively; and ADF, Lignin (sa), aNDF, according to Van Soest et al. (1991); neutral detergent fiber was assayed with heat stable alpha amylase, without sodium sulphite and expressed with residual ash. Kjeldahl nitrogen was determined using a Kjelteck 2300 analyzer unit (Foss North America, Eden Prairie, MN). For the in vitro analysis, 0.5 g of substrate from each plant-subspecies-maturity stage was loaded into three separate 50ml centrifuge tubes, and three corresponding blank tubes, for two separate runs (two runs × three tubes). Rumen fluid from two mature, rumen fistulated Angus steers was combined and used to inoculate fermentation tubes (Care and handling of
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fistulated steers, including ruminal cannulation, were conducted under the protocols approved by the University of Nevada Reno Institutional Animal Care and Use Committee). Steers were fed an alfalfa-grass ration for four weeks prior to rumen fluid collection. Fluid was collected 2 h post-feeding and strained through four layers of cheese cloth into a pre-warmed, insulated thermos and transported to the University of Nevada-Reno Animal Nutrition Laboratory. Rumen fluid was then mixed with McDougall’s buffer (McDougall, 1948) on an equal volume basis (with carbon dioxide bubbling), and 30 ml of the mixed inoculum were added to each centrifuge tube (containing a substrate or a blank). Tubes were flushed with carbon dioxide, capped with stoppers equipped with one-way gas release valves, and placed in an incubator/shaker (Innova 4400, New Brunswick Scientific, Edison, NJ) at 39 ◦ C (Tilley and Terry, 1963). At the end of the 48 h incubation period, tubes were placed in storage at 5 ◦ C for 2 h to stop the fermentation process. Supernatant digesta samples were filtered through a pre-weighed Whatman 541 filter paper and washed with 0.78 ethanol and acetone then dried at 105 ◦ C in a drying oven, weighed and ashed in a muffle furnace at 500 ◦ C overnight and weighed again to determine OM residue. In vitro organic matter digestibility was calculated as follows: (1 − ((OM residue − OM blank)/original OM)) × 100. A 4-mL aliquot of supernatant solution was drawn from each substrate tube and prepared according to Erwin et al. (1961) for short chain fatty acid analysis. Volatile fatty acid concentrations in the supernatant were determined with a gas chromatograph (Varian Model 3800, Varian Inc. Walnut Creek, CA, equipped with a glass column (180 cm × 4 mm i.d.) packed with GP 0.1 SP-1200/0.01 H3 PO4 on 80/100 Chromosorb WAW [Supelco, Bellefonte, PA], with nitrogen used as the carrier gas at a flow rate of 85 ml/min. The oven, injection port, and detector (FID) port temperatures were 125, 175, and 180 ◦ C, respectively. The concentration of VFA were expressed on a mM basis and summed. The concentration of each VFA was divided by the sum and multiplied by 100 to get mol/100 mol of total VFA. Ammonia nitrogen content was determined by the Chaney and Marbach (1962) procedure. Post hoc comparisons were performed using least significant difference (LSD). Data were analyzed with JMP Statistical Analysis Software version 7.0.2 (SAS, 2008) and differences determined at (P < 0.05) for all analyses. 3. Results 3.1. Chemical composition Interactions between species and maturity stage were observed for OM, CP, aNDF, ADF, and lignin (sa) contents (Table 1). Organic matter of Bailey greasewood at post-bloom was the highest compared to other stages in both species. Crude protein content of Black greasewood was highest during the pre-bloom stage (204.8 g/kg) and lowest post-bloom (114.1). Lowest CP values were observed at bloom and post-bloom stages for Bailey greasewood (114.5 and 125.3 g/kg). Neutral detergent fiber content was highest (468 g/kg) for Bailey post-bloom, while Black was lowest (313 g/kg) pre-bloom. The highest ADF (249 g/kg) was observed for Bailey post-bloom, while the lowest (164.3 g/kg) was recorded for Black at the pre-bloom stage. Bailey had higher ADF at all stages than Black greasewood. Post-bloom Bailey was higher in lignin (sa) (104 g/kg) than all other stages in both species, and Bailey had higher lignin (sa) at all stages of growth than Black greasewood. 3.2. Fermentation characteristics No interactions were observed for IVOMD and IVDMD (Table 2), so only main effect results are presented. Black greasewood had greater IVOMD and IVDMD (456.4 vs 402.6 g/kg, 555.6 vs 478.1 g/kg) than Bailey greasewood, respectively. Both parameters were also significantly affected by maturity stage. Pre-bloom stage yielded greater IVOMD than bloom and postbloom stages. No difference was noted in IVOMD values for bloom and post-bloom stages. In vitro dry matter digestibility was higher at pre-bloom and bloom stages than post-bloom. Two-way interactions (species × maturity stage) were observed for NH3 N, total VFA, acetate, propionate, isobutyrate, isovalerate, and actate:propionate concentrations (Table 2). Rumen ammonia N concentration was significantly higher for black greasewood at pre-bloom stage compared to all other stages in both species. No difference was observed for Bailey among stages. The lowest value of NH3 N (8.41 mg/100 ml) was observed for Black greasewood, post-bloom. Total VFA Table 1 Effect of species × maturity stages on chemical composition of greasewood species, 2003. Item
OM (g/kg) CP (g/kg) aNDF (g/kg) ADF (g/kg) Lignin (sa) (g/kg)
Baileya
Blacka
SEM
V
B
PB
V
B
PB
795.9b 125.3c 396.3b 242.3b 87.5b
773.2c 114.5d 387.6c 248.5b 74.4c
825.8a 114.0d 467.8a 311.3a 103.9a
772.8c 204.8a 313.3e 164.3d 36.3e
753.6d 129.7b 335.5d 196.2c 49.7d
753.8d 114.1d 336.6d 199.2c 53.9d
16 13 18 47 22
Row means with different letters differ at P < 0.05. OM, organic matter; CP, crude protein; aNDF, neutral detergent fiber assayed with heat stable amylase, and expressed inclusive of residual ash; ADF, acid detergent fiber expressed inclusive of residual ash; Lignin (sa), sulfuric acid lignin. a V, pre-bloom; B, Bloom; PB, Post-bloom.
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Table 2 Main effect of means on in vitro organic matter digestibility (IVOMD), dry matter digestibility (IVDMD) and simple effects of means on ammonia N, total volatile fatty acids (VFA), individual VFA as affected by species and maturity stages interaction, 2003. Stage of maturitya
IVOMD (g/kg)
IVDMD (g/kg)
Ammonia N (mg/ 100 ml)
Total VFA (mM)
Individual VFA (mol/100 mol of total VFA)
Acetate
Propionate Butyrate Isobutyrate Isovalerate Valerate
Ac:Prc
Plant species V Bailey B PB
396.2 370.5 331.5
473.4 472.5 395.8
14.76b 11.65bc 14.47b
44.30a 31.97b 23.68c
66.58a 69.46a 55.88b
22.54b 22.22b 34.48a
7.91 6.06 5.03
0.48b 0.65ab 0.54b
0.94a 0.96a 0.97a
1.55 1.69 2.08
3.11ab 3.52a 2.68b
V B PB
446.2 383.9 397.0
539.5 499.8 508.4
32.13a 11.93bc 8.41c
45.47a 32.72b 33.15b
66.61a 68.45a 69.83a
25.35b 24.68b 23.72b
5.20 4.84 4.61
1.01a 0.46b 0.42b
0.98a 0.66b 0.63b
0.85 0.91 0.79
2.75b 2.87b 3.20ab
*
*
*
*
nsb
ns
ns
ns
ns
ns
ns
*
*
*
*
ns
ns
*
ns
ns
ns
ns
ns
ns
*
*
*
*
ns
*
*
ns
*
10.5
8.10
1.91
1.25
0.95
0.14
0.07
0.13
0.18
Black
Significance of effects Plant species Stage of maturity Species × maturity SEM * a b c
1.36
1.78
P < 0.05. V, pre-bloom; B, Bloom; PB, post-bloom; SEM, standard error of means. ns, not significant P > 0.05. Ac:Pr, acetate to propionate ratio.
concentration was higher for pre-bloom than all other maturity stages for both species. The lowest value (23.68 mM) was recorded for Bailey at the post-bloom stage. Acetate concentration of Bailey at post-bloom was significantly lower than all other stages in both species. Propionate concentration was higher for Bailey at post-bloom; otherwise its concentration was equal for all maturity stages for both species. 4. Discussion Chemical composition and in vitro fermentation characteristics changed as the two species matured. Ash content characteristically high in desert shrubs (Haddi et al., 2003) was also relatively high in both species. Crude protein content of both species at all maturity stages ranged between 114 and 205 g/kg was similar to that of Smith et al. (1992) that reported CP content of Black greasewood harvested in spring, summer and fall to be 213, 126, 139 g/kg, respectively. Bailey greasewood CP content was much lower than Black in the early to mid-growing seasons. Although secondary plant compounds such as the oxalates known to be present in greasewood can make protein less available, CP levels in both species were well above cattle maintenance requirements (75 g/kg, NRC, 1996). The IVOMD of the two species indicated that both are low in digestible energy, and IVOMD declined precipitously as maturity increased. The IVDMD values, initially similar, also decreased with maturity. Generally, Black greasewood had lower ADF levels than Bailey greasewood. The ADF and lignin (sa) (often referred to as ADL), estimate the quantity of cellulose and lignin fractions. Lower ADF and lignin in Black greasewood indicated more digestible dry matter than Bailey. Bailey had higher lignin at all stages of growth compared to Black greasewood indicating the lignification process was faster than for Black greasewood. Volatile fatty acids are the major sources of carbohydrates for ruminant animals. According to Fahey and Berger (1988) VFAs supply about 0.7–0.8 of the host animal’s total caloric requirement. The molar proportion of propionate in post-bloom Bailey was substantially higher than in the other stages. This is due to a reduction in acetate and butyrate as fermentation time increases. The actual propionate fraction remains constant, while its molar proportion increases. This is common for forages with low concentrations of fermentable carbohydrates (Uden, 2011). Total VFA mM was highest at the pre-bloom stage for both species. A major problem when feeding browse to cattle is a lack of synchronization between nitrogen and energy release in the rumen (Broderick et al., 1991). According to Owens and Zinn (1988) for maximal rumen activity and bacterial growth there should be an adequate supply of both carbon and nitrogen, and the degradation of carbon has to match the degradation of nitrogen for efficient microbial fermentation. Our findings indicate that ammonia production was higher for Black greasewood at the pre-bloom stage than Bailey greasewood. However, despite its high CP content and high rumen NH3-N concentration, Black greasewood total VFA production was the same as Bailey greasewood at the pre-bloom stage. This indicates a lack of degradation synchronization of protein and carbohydrate sources in Black greasewood. Although we did not measure bacterial protein synthesis, we hypothesized that there was more CP than available energy for synthesis. As a result, rumen bacterial protein synthesis decreased and the excess ammonia nitrogen was not captured as microbial protein. According to Cook and Harris (1968), during winter periods or dry
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seasons, grasses are generally low in protein, but high in energy content, while shrubs like greasewood are high in protein and relatively low in energy. This makes greasewood which is high in CP, a good supplement for early fall grazing animals when CP levels in senescent grasses fall below required maintenance levels, but are still relatively high in energy. In the Great Basin, early fall corresponds with the time when cattle in particular, are typically at a low cyclical nutrient demand stage. Thus, a mix of grasses and greasewood during these grazing periods should provide forage of adequate nutritional quality for grazing cattle. 5. Conclusions The nutritional value of both species of greasewood native to North America, have been analyzed as separate species for the first time. Bailey had less nutritional value than Black greasewood in terms of DMD and CP content. Crude protein and in vitro DMD decreased with increasing age of greasewood species. Total VFA concentrations of Bailey and Black greasewood were the same at pre-bloom and bloom stages, but VFA dropped significantly for Bailey at the post-bloom stage. The lignification process was faster for Bailey than Black greasewood. Both species showed adequate CP concentration to supply cattle during late-summer and early-fall grazing periods when the CP levels in senescent grasses fall below required maintenance levels. Conflict of interest None declared. Acknowledgment This study was funded by the Nevada Agricultural Experiment Station. References AOAC, 2000. Official Methods of Analysis, seventeenth ed. Association of Official Analytical Chemists, Gaithersburg, MD. Broderick, G.A., Wallace, R.J., Ørskov, E.R., 1991. Control of digestion and extent of protein degradation. In: Tsuda, T., Sasaki, V., Kawashima, R. (Eds.), Physiological Aspects of Digestion and Metabolism in Ruminants. Academic Press, New York, pp. 541–592. Chaney, A.L., Marbach, E.P., 1962. Modified reagents for determination of urea and ammonia. Clin. Chem. 8, 130–132. Cook, C.W., Harris, L.E., 1968. Nutritive Values of Seasonal Ranges. Bull 472. Utah Agricultural Experiment Station, Logan, UT, pp. 55. Ensminger, M.E., Oldfield, J.E., Heinemann, W.W., 1990. Feeds and Nutrition. The Ensminger Publishing Company, Clovis, CA, pp. 1479–1481. Erwin, E.S., Macro, G.J., Emery, E.M., 1961. Volatile fatty acids analysis of blood and rumen fluid by gas chromatography. J. Dairy Sci. 44, 1768–1771. Fahey Jr., G.C., Berger, L.L., 1988. Carbohydrate nutrition of ruminants. In: Church, D.C. (Ed.), The Ruminant Animal, Digestive Physiology, Nutrition. Prentice Hall, Englewood Cliffs, NJ, pp. 269–297. Fleming, C.E., Miller, M.R., Vawter, L.R., 1928. The greasewood (Sarcobatus vermiculatus), a range plant poisonous to sheep. Nevada Agric. Exp. Sta. Bull. 115, 2–22. Forbes, R.H., Skinner, W.W., 1903. Greasewood analysis. In: Annual Arizona Agric Exp. Sta. Rep., fourteenth ed., pp. 349–350. Haddi, M.L., Filacorda, S., Meniai, K., Rollin, F., Susmel, P., 2003. In vitro fermentation kinetics of some halophyte shrubs sampled at three stages of maturity. Anim. Feed Sci. Technol. 104, 215–225. McDougall, R.I., 1948. Study on ruminant saliva. 1. The composition and output of sheep’s saliva. Biochem. J. 43, 99–109. National Research Council (NRC), 1996. Nutrients Requirement of Beef Cattle, seventh ed. National Academy Press, Washington, DC. Natural Resource Conservation Service (NRCS), 2003, April. Nevada Ecological Site Descriptions: Major Land Resource Area 27. Natural Resource Conservation Service, USDA. Owens, F.N., Zinn, R.A., 1988. Protein metabolism of ruminants. In: Church, D.C. (Ed.), The Ruminant Animal, Digestive Physiology and Nutrition. Prentice-Hall, Englewood Cliffs, NJ, pp. 227–249. Perryman, B.L., 2014. A Field Guide to Nevada Shrubs. IRM Press, Lander, WY. Robertson, J.H., 1983. Greasewood [Sarcobatus vermiculatus (Hook.). Torr.]. Phytologia 54, 309–324. SAS, 2008. Statistical Analysis Software JMP Version 7.0.2. SAS Institute Inc., SAS Campus Drive, Cary, NC, USA. Smith, M.A., Rodgers, J.D., Dodd, J.L., Skinner, L., Quentin, D., 1992. Habitat selection by cattle along an ephemeral channel. J. Range Manag. 45 (4), 385–390. Tilley, J.M.A., Terry, R.A., 1963. A two stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Soc. 18, 104–111. Uden, P., 2011. Using a novel macro in vitro technique to estimate differences in absorption rates of volatile fatty acids in the rumen. J. Anim. Phys. Anim. Nutr. 95, 27–33. Van Soest, P.V., Robertson, J., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597.