Digestibility, methane production and nitrogen balance in sheep fed ensiled or fresh mixtures of sorghum–soybean forage

Livestock Science 141 (2011) 36–46

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Livestock Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i

Digestibility, methane production and nitrogen balance in sheep fed ensiled or fresh mixtures of sorghum–soybean forage R. Lima a, c, R.F. Díaz a, b, A. Castro a, b, V. Fievez c,⁎ a b c

Universidad Central “Marta Abreu” de Las Villas, Department of Veterinary Medicine and Zootechny, Carretera a Camajuaní km 5 ½, 54830 Santa Clara, Cuba Universidad Central “Marta Abreu” de Las Villas, Agriculture Science Institute (CIAP), Carretera a Camajuaní km 5 ½, 54830 Santa Clara, Cuba Ghent University, LANUPRO, Proefhoevestraat 10, 9090 Melle, Belgium

a r t i c l e

i n f o

Article history: Received 28 December 2010 Received in revised form 27 April 2011 Accepted 29 April 2011 Keywords: Sorghum Soybean Ensiling Bag and fecal digestibility

a b s t r a c t The feeding value of a mixture of sorghum and soybeans plants, either fresh or ensiled, was evaluated with sheep. Sorghum and soybeans were harvested during the Cuban rainy season and ensiled in a ratio of 0.6/0.4 (w/w, as feed) with molasses and a bacterial inoculant. Silos were opened between 162 and 182 d post ensiling during the Cuban dry season and silages were fed to six pelibuey sheep (including two fistulated). Six other sheep (also including two fistulated) were fed sorghum and soybean in the same proportion, but freshly harvested during the dry season. The experiment lasted 21 d (14 d adaptation and 7 d data collection period). Silage quality parameters included pH, ammonia, lactate, and short chain fatty acids (SCFA). Further, both fresh and ensiled diets were offered to study the rumen fermentation characteristics (pH, ammonia, lactate, SCFA, in situ degradability and methane), duodenal flow of microbial protein (assessed through urinary purine derivatives secretion), fecal degradability of nutrients and urinary N excretion. From these measurements ME value and degradable CP supply at the small intestine (DCPSI) were estimated. Silage was of excellent quality. Compared to fresh forage feeding silage increased molar propionate proportion and rumen microbial protein synthesis and reduced methane emission. Fresh forage showed lower rumen degradability and total digestibility. Further, the higher ME concentration (11.2 vs. 10.3 MJ/kg DM) and DCPSI (84.7 vs. 56.1 g/kg DM) of the silage would allow a higher milk production or daily gain as compared with fresh forage available during the Cuban dry season. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Own (Lima et al., 2010) and former (e.g., Marrero et al., 2000; Rezende et al., 2001; Silva et al., 2004) research illustrated intercropped cultivation of sorghum (Sorghum bicolor

Abbreviations: ADF, acid detergent fiber; CP, crude protein; DCPSI, digestible crude protein in small intestine; DE, digestible energy; DM, dry matter; DMI, dry matter intake; ERD, effectively rumen degraded; FM, fresh material; GE, gross energy; GEI, gross energy intake; ME, metabolizable energy; MNPD, daily duodenal flux of microbial N; NDF, neutral detergent fiber; OM, organic matter; PDa, microbial purines absorbed from the small intestine; PDe, urinary excretion of purine derivatives; SCFA, short chain fatty acid. ⁎ Corresponding author at: Ghent University, Department of Animal Production, Proefhoevestraat 10, 9090 Melle, Belgium. Tel.: + 32 9 264 90 02; fax: + 32 9 264 90 99. E-mail address: [email protected] (V. Fievez). 1871-1413/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2011.04.014

(L.) Moench) and soybean (Glycine max (L.) Merr.) during the Cuban rainy season showed promising dry matter and crude protein yields, with soybean proportions reaching between 350 and 400 g/kg DM. Further, ensilibility of such a mixture (0.6/0.4 sorghum/soybean, w/w) as well as the response to molasses and lactobacilli inoculation was assessed through a Rostock fermentation test and from lab scale silages which showed excellent silage quality was reached [according to Ojeda et al. (1991): pH ≤4, lactate N14 mg/g FM, NH3–N b2.5 g/100 g N, 0.10b acetate in total fermentation acids b0.30 and negligible concentrations of butyrate and alcohol] when 35 g molasses/kg FM and bacterial inoculant were added (Lima et al., 2011). Furthermore, in vitro rumen simulations suggested this ensiled mixture to be highly fermentable (Lima et al., 2010). Accordingly, combined sorghum–soybean silages could offer a possibility to compensate the lack of feed during long

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periods with shortness of rainfall (e.g., on average yearly 210 d in Cuba), which is one of the major challenges for ruminant production in the tropics (Lima et al., 2009). Indeed, an efficient ruminant production system requires a stable feed quality throughout the year, e.g. through conservation of feed (Ojeda et al., 1991). However, in addition to in vitro assessments (Lima et al., 2010), in vivo trials are required to fully assess the nutritional value and environmental impact of this mixture. Such in vivo trials should generate information on the extent of rumen degradability, metabolizable energy and protein value to incorporate in feed evaluation systems (Madsen et al., 1997). Additionally, emissions towards the environment could be evaluated from N balances and rumen gas collections. Hence, the first objective of this in vivo digestion trial was to assess rumen degradable and bypass fractions, methane production, metabolizable energy content and N balances of sorghum–soybean silage produced during the Cuban rainy season. A second objective of this experiment was to compare these feed values as well as emissions (methane and nitrogen) with those of the same combination of fresh sorghum–soybean forage produced during the dry season to illustrate the nutritional superiority of the ensiled material. The latter is mainly of practical importance as ensiling is not yet common practice in Cuba and many tropical regions.

2. Materials and methods 2.1. Plant material Sorghum (S. bicolor (L.) Moench) (CIAP 2E-95) and soybean (G. max (L.) Merr.) (INCASOY-35) were sown on a research farm (22°43′N, 79°90′W) from the Agriculture Research Institute (CIAP) of the Central University of Las Villas, Santa Clara, in the center of Cuba. Soybean and sorghum were sown as reported by Lima et al. (2010) at two different dates (22/06/2009 and 05/11/2009 for ensiling and to feed as fresh forage, respectively) on two different fields but with similar fertility and same soil type (Calcisol soil, Nerey et al., 2010). Monthly average precipitation, temperature and humidity during the growing season to produce forage for ensiling and fresh feed were 214 ± 21 mm and 61.5 ± 18 mm, 27 ± 0.8 °C and 21 ± 0.8 °C and 79 ± 2% and 77 ± 4%, respectively. Crops were not fertilized and the crop for ensiling was not irrigated. For the production of fresh forage, irrigation was needed as the experiment took place during the dry season: crops were irrigated five times, i.e. from the first month after sowing every 3 weeks, during two hours/day for three days/week. The pasty grain state (the best state to ensiling according to Marrero et al. (2000); Romero, (2004)) of sorghum determined the harvesting date (25/09/2009 and 12/03-01/04/2010 for ensiling and to feed as fresh forage, respectively). Bean formation was completed at the time of harvest for the soybean crop. For experimental purposes, soybean and sorghum plants were harvested separately around noon in both periods. Sorghum harvests included the whole plant with all green leaves as well as the highest leaf with yellow coloration. Soybean was harvested about 20 cm above the soil. The sorghum and soybean were chopped separately to a particle size of 2 cm.

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2.2. Small scale silages Plants (170 kg of fresh material (FM) (102 kg of sorghum + 68 kg of soybean)) were packed into experimental silos (tanks with volume of 208 L) in triplicate. The additive used in this study consisted of 20 g of WSC per kg of FM by means of molasses (35 g/kg FM) and of a bacterial inoculant of Lactobacillus plantarum (DSMZ 8862 and DSMZ 8866, BIOSIL®, Dr. Pieper Technologie- und Produktentwicklung GmbH, Wuthenow, Germany; applied according to Lima et al. (2010)). The compaction density was 817 kg FM/m 3. The samples were homogenized manually (by hand with aid of the hay fork) before packing the experimental silos and compacted by foot (walking over material) assuring air expulsion and until the desired compaction density. The experimental silos were covered with polyethylene and left at farm temperature (20 ± 5 °C) until their opening between 168 and 182 d later. 2.3. Sampling and chemical analysis of fresh and ensiled forage 2.3.1. Fresh forage After chopping and homogenizing, 350 g of fresh forage was dried at 65 °C for 72 h in triplicate before ensiling and before feeding. Afterwards, the dried material was ground to pass a 1 mm screen and stored in glass bottles at room temperature (28 ± 3 °C) until chemical analysis. 2.3.2. Ensiled forage After 168 to 182 d of ensiling and immediately at the opening of the silos, subsamples were taken on alternate days for determination of pH, ammonia nitrogen to total nitrogen ratio (NH3–N/N), lactic acid, short chain fatty acids (SCFA) and alcohol in sample extracts. Another subsample was dried (65 °C during 72 h) and stored, after which it was used for proximate analysis. Extracts were prepared and stored as described by Lima et al. (2010). In all extracts, pH was measured before acidification (780 pH/ion, Metrohm, Herisau, Switzerland), lactic acid was oxidized to acetaldehyde using Conway microdiffusion chambers and measured spectrophotometrically (224 nm) according to Conway (1957a). Ammonia and alcohol were determined before acidification according to Conway (1957b) and Voigt and Steger (1976), respectively. For SCFA analysis, acidified extracts were centrifuged (10 min at 22,000 × g, Beckman J2-HS, CA, USA), before determination by gas chromatography on a Shimadzu GC-14A (Shimadzu Corporation, ´s-Hertogenbosch, The Netherlands), according to Van Ranst et al. (2010). 2.3.3. Proximate chemical analysis Samples were assayed (in duplicate) for proximate Weende fractions: dry matter (DM; ID 930.15), ether extract (920.39), crude fiber (978.10) and CP (ID 954.01) (AOAC, 1995), organic matter (OM) content (EEC, 1971), neutral detergent fiber analyzed without sodium sulfite and heat stable amylase and expressed exclusive of residual ash (NDF) (Van Soest et al., 1991), acid detergent fiber determined by sequential analysis of the residual NDF and expressed exclusive of residual ash (ADF) (Van Soest et al., 1991) and hemicellulose was calculated as difference between NDF and ADF. Cellulose and lignin were determined according to Van

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Soest et al. (1991), where lignin was oxidized with permanganate. Dry matter was corrected for silages considering the pH and assuming SCFA, lactate, alcohol and ammonia losses according to Beyer et al. (2003). Detailed formulas were presented by Lima et al. (2011). 2.4. In vivo trial 2.4.1. Animals and management The experiment, which utilized fresh forage and silage of combined sorghum–soybean, took place between March and April 2010 in Santa Clara, Cuba. The work described was carried out in accordance with the recommendations of the Ethical Commission of Ghent University and was supervised by a qualified veterinarian. Eight non rumen-fistulated and four rumen-fistulated pelibuey lambs (at the beginning 27 ± 5 kg and at the end of experiment 29 ± 5 kg), were housed in metabolic cages (1.25 m × 0.65 m) under continuous lighting during the experiment. During the 14 d of adaptation preceding the collection period of 7 d, the lambs were removed from the cages twice per week to clean the metabolic cages and allow same movement to the animals. The animals were fed three times a day at 08:00, 14:00 and 20:00 h. 2.4.2. Experimental diets Fresh drinking water was available ad libitum. Fresh forage and silage of combined sorghum–soybean diets were offered in three equal portions a day. Six sheep (2 rumen-fistulated and 4 non rumen-fistulated) were offered either fresh forage or silage (1.1 kg DM/sheep/day) during 21 d. 2.4.3. Feed intake Feed intake was registered during 7 d of the experimental period by weighing the feed offered and refused. Feed refusals were collected just prior to the distribution of the 08:00 h feeding. A representative sample of both the distributed and refused feed was dried (65 °C, 72 h) and stored for proximate chemical analysis. Average DM intake (±standard deviation, n = 6) of the fresh forage and silage was 1.04 ± 0.05 and 0.98 ± 0.08, respectively. 2.4.4. Urine and feces collection Total urine and feces were collected during the 7 d of the collection period. Prior to the urine and feces collection, sheep were removed from the metabolic cages, which were cleaned completely. Urine and feces were separated by using a urine device collector (Cao et al., 2010) connected to two recipients, one for storage at pH b3 (urea analysis) and the other at 3 b pH b 4 (purine derivatives analysis). The pH was adjusted gradually as described by Fievez et al. (2001). Fecal material was collected daily and a subsample (10–20%) was dried (65 °C, 72 h). After measuring total urine excretion, each day a subsample (150 mL) was diluted to 700 mL (to prevent precipitation of uric acid (Chen et al., 1992) and a new subsample of 250 mL was stored until chemical analysis. Prior to analysis, two urine samples per animal were composed, one from day 1–4 and the other from day 4–7, and this was according to daily urine production. Feces samples were pooled similarly.

2.4.5. Collection of rumen gasses Gas was collected over 6 h (from 08:00 until 14:00 h) for 3 d (first, third and fifth) of the collection period. Total gas expelled through the rumen fistula of each sheep was collected through one tube (1.5 cm diameter) connected to an airtight polyethylene bag (100 L) using a one way valve as described by Berra et al. (2008). After collection, 100 mL of gas was stored at room temperature into a 120 mL gastight incubation flask for further analysis. The polyethylene bag was connected to a glass column for measurement of the total gas volume by water displacement. 2.4.6. Sampling of rumen liquid On the first five days of the collection period, rumen liquid was sampled, as described, just before the morning (08:00 h) and the afternoon (14:00 h) feeding, as well as at 11:00 (on the days of gas collection) or at 10:00 and 12:00 h (on the second and fourth day of the collection period). The pH was measured and the fermentation stopped (phosphoric/formic acid, 10/1 v/v, final concentration 2%). The rumen liquid was centrifuged (22000 × g for 15 min) and, after filtering, the supernatant was kept refrigerated at 4 °C for chemical analysis. 2.4.7. In sacco incubations During the second week of the adaptation period, samples were taken from the fresh forage and silage, distributed to the animals. Samples were immediately carried to the laboratory, mixed thoroughly, cut and dried (65 °C, 72 h). The dried samples were ground in a hammer mill (1-mm sieve) and stored in glass bottles at room temperature until utilization. In sacco degradability was measured twice during the collection period as described by Mbanzamihigo et al. (2002). One bag was not incubated and immediately washed, whereas the incubated bags were removed from the rumen after 3, 6, 9, 12, 24, 48 and 72 h. After removal from the rumen, the bags were washed till the water was clear, dried (65 °C, 72 h) and weighed for determination of degraded dry matter (DM). 2.4.8. Analysis 2.4.8.1. Chemical composition of ingested material and feces. Both offered and refused feeds, as well as feces were analyzed for DM, OM, CP, NDF, ADF, hemicellulose, cellulose and lignin as described above (section 2.3.3). In addition, gross energy was determined by bomb calorimetry (Parr 1261 ISOPERIBOL, Moline, IL, USA). The fecal digestibility of DM, OM, CP, NDF, hemicellulose, cellulose and energy were calculated with data collected from total feces collection. 2.4.8.2. Urinary excretion of urea and purine derivatives. Total N in urine samples was determined by Kjeldahl (EC, 1993). In addition, urea was determined by conversion to ammonia through urease as described by Fievez et al. (2001). Ammonia was determined according to Conway (1957b). Purine derivatives (allantoin, uric acid and xanthine) were determined in the urine samples by HPLC using an Äkta Purifier 10 HPLC (Amershm Pharmacia Biotech AB, Uppsala, Sweden), equipped with an autosampler injecting 100 μl of sample by means of a loop. A precolumn (Supelguard™ LC-18, 2 cm, 4.6 mm Ø, Bellefonte, PA, USA) was used to protect the

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column (SUPELCOSILTM LC-18, 25 cm RP column, 4.6 mm internal diameter and 5 μm particle size, Bellefonte, PA, USA). Buffers were prepared as described by Fievez et al. (2001) and adjusted to pH 4.0 with NaOH (10 M) for buffer A and with phosphoric acid concentrated (85%) for buffer B. The two buffers were run according to following chromatographic conditions at 1 mL/min with the effluent monitored at 205 nm. The sequential gradient (CV = column volume) steps for buffer B was as follows: 1.5 CV equilibration with 0% solvent B (2.5 min), 7.82 CV to 20% solvent B (13 min), 9.02 CV to 100% solvent B (15 min), 12.03 CV to 0% solvent B (20 min) and 1 CV with 0% B for re-equilibration (1.7 min). The sample was injected after the first equilibration phase of 2.5 min. Standards and samples were prepared as described by Fievez et al. (2001). Allantoin, uric acid and xanthine were identified from retention times (3.5, 7.5 and 9.2 min, respectively) (Fig. 1). 2.4.8.3. Composition of rumen gasses. Rumen gasses were analyzed for CH4 by gas chromatography (3000 micro-GC, Agilent, USA) according to Hassim et al. (2010). 2.4.8.4. Rumen SCFA and ammonia concentration. Total and individual (acetate, propionate, butyrate, isobutyrate, valerate and isovalerate) SCFA concentrations, as well as ammonia were determined in pre-treated rumen liquid as described by Van Ranst et al. (2010) and Lima et al. (2010), respectively. Daily averages (n = 5/sheep) were calculated from individual determinations.

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2002) to correct for those losses. As ethane was not available under Cuban experimental conditions, a preliminary total gas collection trial was performed in Belgium (July 2009) (approved by the Ethical commission of Ghent University, CaseNumber 2009_070) to assess the proportion of infused ethane recovered in the total gas collected. Gas collections in Cuba and Belgium were identical, with gasses collected from the rumen by means of the fistula as explained before. During eight days (6 h per day) split into two weeks, gasses were collected from two mature sheep, with methane and ethane analysis by gas chromatography as described by Hassim et al. (2010). The recovery of ethane in this preliminary experiment was 0.4977 ± 0.0119 in sheep A and 0.4979 ± 0.0086 in sheep B with an average of 0.4978 ± 0.0102. From the low variation between collection days and the very similar results between both sheep, we assumed that almost half of the gasses are recovered through the collection via this type of rumen fistula. Hence, in the Cuban experiment, total methane emissions (CH4, L/d) were calculated as: CH4 = %CH4 ⁎ðTGC = 0:498Þ where % CH4 is the proportion of methane (%, v/v) in the collected gas, TGC (L/d) is the total gas collected assuming that the total gas collection in 6 h was a quarter of the daily gas production and 0.498 is the proportion of the gas collected from the rumen fistula to the total gas produced in the rumen. Methane expressed as percentage of gross energy intake (GEI, MJ) was calculated as:

2.4.9. Calculations

%GEI = CH4 ⁎0:7167⁎0:05583 = GEI⁎100;

2.4.9.1. Rumen CH4 production. As a portion of the gas produced in the rumen is expired or eructed or might be lost during sampling of rumen contents, the use of ethane infusion as a tracer gas was proposed (Mbanzamihigo et al.,

where CH4 is the daily methane emission (L), 0.7167 represents the weight (g) of 1 L of CH4, 0.05583 is the combustion energy of methane (MJ/g) (Holter and Young, 1992) and GEI is the gross energy intake (MJ/d).

Fig. 1. Example of a separation chromatogram of allantoin in pelibuey sheep urine.

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2.4.9.2. Effective rumen degradability. Data of in sacco degradability (D, as proportion) of DM, OM and CP were fitted to the equation:   −k t D = a + b 1−expð f Þ ;

2.4.9.5. Digestible CP supply at small intestine. The CP digestible in the small intestine (DCPSI) was calculated as follows:

where t is time (h), “a” is the soluble fraction (as proportion), “b” is the potentially rumen degradable fraction (as proportion), and kf is the degradation rate (/h) (Ørskov and McDonald, 1979). From in sacco degradability of DM and CP, effective rumen degradability (ERD, as proportion) was then calculated as:   ERD = a + b  kf = kf + kp ;

with NI, N intake (g/kg DM), NF, fecal N excreted (g/kg DM) and NL, rumen N losses (g/kg DM) calculated as:

where kp (0.03 per h) is the rumen passage rate (Ørskov and McDonald, 1979). 2.4.9.3. Duodenal flow of microbial N. The duodenal flow of microbial N was assessed from the excretion of the purine derivatives (allantoin, uric acid and xanthine). Conversion of total daily renal excretion of purine derivatives (PDe) to daily duodenal flux of microbial N (MNPD, g N/d) was based on the equation of Chen and Gomes (1992): MNPD = ðPDa ⁎70 Þ = ð0:116⁎0:83⁎1000Þ;

DCPSIðg = kg DMÞ = ðNI −NF −NL Þ⁎6:25 + CPendogenous

NL = ðNI ⁎ERDNÞ−MNPD with ERDN, the effectively rumen degraded N (g/g NI) and MNPD, duodenal flow microbial N (g/kg DM). The first part of the DCPSI-equation [(NI − NF − NL) ⁎ 6.25 excludes endogenous protein lost during digestive processes as they are recovered in the feces. Similarly, in the Belgian/Dutch protein evaluation system these losses are subtracted from the rumen bypass and microbial protein digested in and amino acids absorbed from the small intestine as they were considered to depend more on feed than animal characteristics. However, as endogenous losses are accounted for at the level of animal maintenance requirements in the Cuban system (Roche et al., 1999), DCPSI values needed to be corrected for these losses. These losses were estimated from the difference in maintenance requirements for sheep of 28 kg (BW) at 1 kg/d of DMI according to French (Theriez et al., 1987) and the Belgian/Dutch system (CVB et al., 2001) as:

where 70 is the N content of purines (70 mg N/mmol), 0.116 is the ratio of purine–N to total N in mixed rumen microbes, 0.83 is the digestibility of microbial purines, and PDa is the microbial purines absorbed from the small intestine (mmol/ d) calculated as:   0:75 ð−0:25⁎PDe Þ PDa = 0:84⁎PDe + 0:15⁎BW ; ⁎e

CPendogenous

where, BW 0.75 is the metabolic body weight of the animal (kg), the slope 0.84 represents the recovery of absorbed purines in the urine, the component within brackets represents the endogenous contribution of purine derivatives to total excretion after correction for the utilization of microbial purines by the animal, and PDe is the total purines excreted in the urine (mmol/d) calculated as the sum of daily urinary excretion of allantoin, uric acid and xanthine.

To compare feed characteristics of ensiled and fresh feed, general linear model (GLM) analyses were performed with SPSS 15.0 (SPSS software for Windows, release 15.0., Inc., Chicago, IL, USA). The GLM was used to assess the effect of diet type on digestion coefficients, energy content, urinary urea and purine derivatives following the model:

2.4.9.4. Energy content. The digestible (DE) and metabolizable energy (ME) were calculated as: DE = GEI⁎DGE=100 ME = DE−CH4 E−UE where, GEI, the gross energy intake; DGE, the digestibility of gross energy (%), CH4E is the energy losses from methane assuming the combustion energy of methane of 55.83 kJ/g and UE the energy losses from urine estimated according to Paladines et al. (1964):   UEkJ = d = 17:257kcal = g⁎urinary Ng = 100 ml + 1:09kcal ⁎4:184kJ = kcal⁎daily urine excretionml = 100 Additionally, ME (MJ/kg DM) was estimated from Cáceres and González (2000) and compared with our assessment: MEestimated = ð37:20 kcal = kg⁎DOM−148:9 kcal = kgÞ ⁎4:184 kJ = 1000 where, DOM is the digestibility of organic matter (%).

ð g = kg DMÞ

= 2:5⁎BW 0:75 −1:5⁎BW 0:75 :

2.5. Statistical analysis

  Y = μ + FSi = 1−2 + FS Aj = 1−6 + ε with FSi = 1–2, the diet (fresh vs. silage) and FS(Aj = 1–6), animal, as a random factor within diet. In addition, GLM was used to assess the effect of diet type on rumen fermentation parameters (pH, total SCFA and individual SCFA, lactate and ammonia), rumen degradabilities and methane production, following the model:   Y = μ + FSi = 1−2 + FS Aj = 1−2 + ε with FSi = 1–2, the diet (fresh vs. silage) and FS(Aj = 1–2), animal, as a random factor within diet. 3. Results 3.1. Chemical characterization of the diets The chemical composition of the fresh forage and silage offered to animals is shown in Table 1. The contents of DM, OM, CP, NDF, ADF, hemicellulose and cellulose in the fresh forage were higher in the silage. The silage fermentation

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Table 1 Chemical composition of a fresh and ensiled mixture of sorghum–soybeans and silage fermentation characteristics. Parameters

Fresh forage-dry season SG

a

SB

a

Fresh forage-rainy season Combined

Average

SG SD b

Average

7.23 0.40 0.96 6.05 10.4 11.6 5.88 9.96 1.44 0.76 – –

328 932 81.0 56.0 155 423 249 174 181 49.3 633 600

SB

Silage Combined SD

Average

SD

11.6 2.48 1.67 2.70 2.99 4.09 7.08 5.56 8.12 1.79 – –

309 915 116 85.8 192 419 256 164 193 54.3 – –

6.68 1.63 1.29 3.18 3.78 12.0 8.24 17.1 1.12 0.27 – –

3.98 2.24 18.3 2.95 nd d nd 0.09 0.86

0.04 0.13 0.22 0.88 – – 0.01 0.03

c

Chemical composition (g/kg DM) DM (g/kg FM) 388 OM 954 CP 84.0 EE 36.4 CF 223 NDF 551 ADF 280 Hemicellulose 271 Cellulose 206 Lignin 61.8 ratio 615 ratio (g/kg FM) 600

365 894 185 83.4 392 465 391 74.0 315 59.4 385 400

379 931 123 54.5 288 518 323 195 248 60.9 – –

285 878 187 142 275 402 333 69.0 303 51.3 367 400

311 912 120 87.5 199 415 280 135 226 50.0 – –

Parameters of silage fermentation (mg/g FM) pH NH3–N/N (g/100 g) Lactate Acetate Butyrate Propionate Alcohol L/TFa e a

SG: sorghum; SB: soybean. SD: standard deviation results from triplicate sampling. c DM: dry matter; FM: fresh material; OM: organic matter; CP: crude protein; EE: ether extract; CF: crude fiber; NDF: neutral detergent fiber; ADF: acid detergent fiber. d nd: not detected. e L/TFa: lactic acid in total fermentation acids. b

parameters (pH, NH3–N/N, lactate, acetate, butyrate, propionate, alcohol and proportion of lactic acid in total fermentation acids (L/TFa)) suggested an excellent silage quality (Table 1).

3.2. Methane production and chemical composition of rumen fluid Table 2 shows the effect of diet on daily methane production, and the production expressed per kg of effectively rumen degraded OM (ERDOM) as well as percentage of gross energy intake (%GEI). Those parameters were lower (P b 0.001) for the animals fed silage. In addition, some parameters of rumen fluid are presented in Table 2. Acetate proportion was higher (P b 0.01) when fed fresh forage compared with silage while the opposite was observed for propionate and valerate proportions. However, the pH, lactate, total SCFA, butyrate, isobutyrate and isovalerate were similar (P N 0.05). Finally, urinary purine derivative excretion as well as individual urinary excretion of allantoin, uric acid and xanthine were higher (P b 0.01) in silage fed animals than animals fed fresh forage. Moreover, the proportion of allantoin (0.46 vs. 0.51, fresh forage vs. ensiled, respectively), uric acid (0.47 vs. 0.44) and xanthine (0.09 vs. 0.05) in total urinary purine derivatives was dependent of the feeding system.

3.3. Degradability and energy content The effective rumen degradability of DM and CP (ERDCP), fecal apparent digestibility and energy content of combined forage and silage of sorghum–soybean are presented in Table 3. Generally, degradability both in the rumen as well as total digestibility was higher for the ensiled as compared with the fresh forage (Table 3). Higher rumen degradability was related to an increased rate of fermentation (kf) as well as an increase in the soluble material (a value). Consequently, the energy content of the ensiled diet was higher (P b 0.001) as compared with the fresh material; with the latter containing only 92% of the ME of the ensiled forage. 3.4. Nitrogen intake and output The nitrogen balance is presented in Table 4. The N intake and N in feces were higher (P b 0.001) in animals fed fresh forage than in silage fed animals. However, urinary N excretion when feeding silage was about 9% higher as compared with fresh forage feeding. Urinary purine derivatives and as a consequence duodenal flow of microbial N, calculated thereof, were higher (P b 0.01) when feeding silage as compared with fresh forage, despite the similar ammonia concentration in the rumen fluid (P N 0.05). Finally, silage showed a greater (P b 0.001) DCPSI as compared with fresh forage. In addition, the positive N balance in both diets is

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Table 2 Methane production (n = 2 sheep × 3 d), production and proportions of SCFA in rumen fluid (n = 2 sheep × 5 d) and urinary excretion of purine derivatives (n = 6 sheep × 2 periods) by sheep fed either a fresh or ensiled mixture of sorghum–soybean(60/40, w/w). Parameters

Fresh forage Average

CH4 expressed as L/d % GEI b L/kg ERDDM c

32.8 7.17 81.6

Average

1.95 0.43 4.84

19.8 4.75 42.3

SD

Total SCFA (mmol/L) and proportion of individual SCFA (mmol/mol total SCFA) Total SCFA 153 17.4 Acetate 670 20.2 Propionate 204 17.7 Butyrate 106 7.33 Isobutyrate 3.45 1.93 Isovalerate 9.83 1.24 Valerate 6.98 0.73 Others pH Lactate, mmol/L Allantoin (mmol/d) Uric acid (mmol/d) Xanthine (mmol/d) PDe (mmol/kg ERDOM) a b c d

6.61 3.76 5.22 4.46 0.48 25.5

P value d

Silage a

0.07 0.27 1.53 0.33 0.22 4.60

SD

FS

FS(A)

1.25 0.45 2.70

0.001 0.018 b 0.001

0.872 0.206 0.886

162 599 265 99.8 2.31 19.5 13.8

13.4 12.1 22.9 12.9 1.57 7.70 1.64

0.049 0.002 0.012 0.167 0.177 0.219 0.001

0.931 0.854 0.621 0.708 0.633 0.002 0.923

6.66 3.98 7.85 8.18 1.20 37.3

0.10 0.37 2.48 1.61 0.43 8.76

0.218 0.465 0.006 b 0.001 b 0.001 0.007

0.535 0.039 0.693 0.082 0.789 0.065

SD: standard deviation. GEI: gross energy intake. ERDDM: effectively rumen degraded dry matter. Statistics significance of FS: fresh forage vs. silage; FS(A): animal random factor within diet.

related with animal weight gain during the experiment (on average 2 kg during the experimental period of 21 d). 4. Discussion 4.1. Chemical characterization of the diets Fresh forage available during the Cuban dry season showed higher fiber content than fresh forage available during the Cuban rainy season (Lima et al., 2010). The higher ADF and lignin contents of fresh forage as compared with silage suggest a lower quality of the former. In addition, all parameters of ensiling fermentation were in the range of an excellent silage (Ojeda et al., 1991), which is in line with reports of Lima et al. (2010) studying the same proportion of combined silage of sorghum–soybean in laboratory silages. 4.2. Nutritive values of soybean–sorghum 4.2.1. Metabolizable energy content of ensiled and fresh soybean–sorghum Metabolizable energy values obtained through correction of digestible energy by energy losses from methane and urinary N are in line with those estimated from digestible OM (Cáceres and González, 2000) (ME estimated[fresh forage]/ ME calculated[fresh forage] = 0.95; ME estimated[silage]/ME calculated[silage] = 0.98). This is an indication of the reliability of the methane and urine collection systems we established for this experiment. ME content of ensiled and fresh sorghum–soybean are in line with other studies reporting ME of fresh and ensiled soybean between 10.5 and 11.8 MJ/kg DM (Tobía et al., 2007; 2008) and ME of fresh and ensiled sorghum between 8.97 and

10.1 MJ/kg DM (Gallardo and Gagiotti, 2004; Marrero et al., 2000; NRC and Nutrition, 1985). Moreover, ME contents are comparable with values reported for corn silages (10.0– 12.0 MJ/kg MD) (Barahona and Sánchez, 2005). Hence, we assessed the validity of equations developed for corn silages (De Boever et al., 1997) to predict ME based on crude nutrients (Table 5). Particularly, equations based on fat, ADF, ash and lignin (De Boever et al., 1997) seemed to precisely predict ME content of sorghum–soybean mixtures, both fresh as well as ensiled. Moreover equations of grass silages based on crude fiber, crude protein and crude ash (Schenkel, 1998) are valuable predictors. This merits verification with more data of combined sorghum–soybean mixtures to assess prediction accuracy and precision of these equations. 4.2.2. Rumen protein degradability and microbial protein production Although average rumen ammonia concentrations are beyond those considered limiting for microbial growth (50 mg NH3-N/L; Satter and Slyter, 1974), the proportion of rumen degradable N to rumen degradable OM (20.6 g N/kg ERDOM) is below the ratio considered ideal (24 g N/kg ERDOM) e.g. in the Belgian–Dutch protein evaluation system (Tamminga et al., 1994). Hence, some additional rumen degradable protein might be of interest to supplement. Alternatively, a higher soybean proportion in the silage could be considered (e.g., 0.50 or 0.60) as good quality silage was obtained with those soybean proportions (Lima et al., 2010). A slight lack of rumen degradable protein eventually also might be at the origin of microbial N flow efficiencies (22.8 ± 5.32 g microbial N/kg ERDOM, Table 4) at the lower end of those reported when feeding (ensiled) grasses (22–30 g microbial N/kg ERDOM reported) (Givens and Rulquin, 2004; Owens et al., 2009;

R. Lima et al. / Livestock Science 141 (2011) 36–46

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Table 3 Rumen degradability (n = 2 sheep × 2 periods), energy content and total digestibility (n = 6 sheep × 2 periods) by sheep fed either a fresh or ensiled mixture of sorghum–soybean (60/40, w/w). Parameters

Fresh forage Average

P value e

Silage Average

SD

0.01 0.03 0.01 0.02 0.993

0.42 0.36 0.07 0.67 –

b 0.01 0.01 b 0.01 0.01

0.013 0.082 0.027 0.007

0.003 0.017 0.732 0.664

0.36 0.42 0.06 0.63 0.985

0.01 0.01 b 0.01 0.01 0.989

0.48 0.32 0.07 0.71 –

b 0.01 0.01 b 0.01 0.01

0.003 0.003 0.027 0.003

0.037 0.282 0.692 0.804

Rumen degradability CP a b kf (/h) ERD R2

0.34 0.36 0.05 0.56 0.961

0.01 0.01 0.01 0.01 0.980

0.52 0.22 0.08 0.68 –

b 0.01 0.01 0.01 0.01

0.001 0.002 0.001 0.001

0.815 0.611 0.960 0.667

Fecal apparent degradability c DM OM GE CP NDF Hemicellulose Cellulose

0.64 0.66 0.66 0.61 0.57 0.69 0.56

0.03 0.03 0.04 0.05 0.05 0.04 0.06

0.01 0.01 0.02 0.02 0.03 0.07 0.05

b0.001 b0.001 b0.001 b0.001 0.012 0.005 0.002

0.349 0.340 0.193 0.139 0.202 0.902 0.707

Energy (MJ/kg DM) expressed as d DE 11.3 ME 10.3 MEestimated 9.73

0.71 0.16 0.49

0.44 0.16 0.19

0.001 b0.001 b0.001

0.055 0.111 0.353

Rumen degradability DM b a b kf (/h) ERD R2

0.31 0.44 0.06 0.60 0.992

Rumen degradability OM a b kf (/h) ERD R2

SD

a

0.71 0.74 0.73 0.72 0.62 0.75 0.64

12.7 11.2 11.0

FS

FS(A)

a

SD: standard deviation. a, b, kf: parameters of rumen degradability; “a”: soluble fraction, “b”: potential rumen degradable fraction and kf: degradation rate; ERD: effectively rumen degraded. c DM: dry matter, OM: organic matter, GE: gross energy, CP: crude protein, NDF: neutral detergent fiber. d DE: digestible energy, ME: metabolizable energy, MEestimated: metabolizable energy estimated from digestible OM according to Cáceres and González (2000). e Statistics significance of FS: fresh forage vs. silage; FS(A): animal random factor within diet. b

Verbic et al., 1999). Nevertheless, the considerably higher (Pb 0.01) urinary excretion of purine derivatives by silage fed animals reflects an increased efficiency of microbial protein synthesis in the rumen as compared with animals fed fresh forage, which showed a low efficiency of microbial N supply to the intestine (15.9 ± 2.74 g microbial N/kg ERDOM, Table 4).

production rates of 10.4 kg milk and growth of 715 g were estimated from DCPSI and ME content of silage when fed as sole diet (12.0 or 6.00 kg DMI/d, respectively) (Table 6); while daily production rates based on fresh forage available during the Cuban dry season are limited to 6.22 kg of milk or 480 g growth, with DCPSI as limiting factor.

4.2.3. Comparison of nutritive values of ensiled and fresh soybean–sorghum material Metabolizable energy of fresh forage only represented 92% of the ME of the ensiled material. This result is related to the higher rumen degradability and fecal digestibility as well as reduced methane excretion associated with the ensiled diet. However, the energy lost through urinary N excretion was increased. The increase in apparent organic matter digestibility might be related to the release of degradable fiber (e.g., Lima et al., 2010) and/or reduction of tannins (e.g., de Oliveira et al., 2009; Hartmann et al., 2005) in ensiled as compared with fresh forage. In addition, the DCPSI of fresh forage only represented 66% of that of silage. Consequently, daily

4.3. Environmental impact of livestock in feeding systems based on fresh or ensiled forages 4.3.1. Rumen methane production Rumen methane production from ensiled sorghum– soybean only represented 61% of those excreted when fresh sorghum–soybean was fed. A similar difference in methane production (14.7 L CH4/d) between both diets was calculated from the equation of Kirchgessner et al. (1995) based on chemical composition of the diet. The lower methane production when feeding silage as compared with fresh forage is in line with the lower NDF and ADF content. Further, methane excretion from ensiled material might be reduced

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Table 4 Nitrogen balance (n = 6 sheep × 2 periods) of sheep fed either a fresh or ensiled mixture of sorghum–soybean (60/40, w/w). Parameters

Fresh forage

P value d

Silage Average

SD

FS

0.56 0.90 1.29 0.57

18.5 5.16 8.53 7.41

1.38 0.62 0.64 0.57

b0.001 b0.001 0.106 0.002

0.001 0.071 0.342 0.979

N intake and output (g N/kg BW 0.75/d) Intake 1.92 Feces 0.76 Urinary N 0.70 Urinary N (g N/L) 5.64 Urinary urea 0.61 Urinary urea (g N/L) 4.95 b g DCPSI/kg DM 56.1

0.12 0.11 0.15 0.94 0.06 0.52 5.93

1.59 0.44 0.74 6.14 0.64 5.34 84.7

0.19 0.06 0.11 0.48 0.11 0.54 2.74

0.007 b0.001 0.631 0.084 0.524 0.045 b0.001

b 0.001 0.002 0.021 0.781 0.027 0.883 0.127

Rumen N retention c Ammonia, mmol/L PDe (mmol/d) PDa (mmol/d) MNPD (g N/d) MNPD (g N/kg ERDOM)

0.96 1.59 2.57 0.94 2.74

5.54 17.3 14.5 10.6 22.8

1.82 3.40 2.82 2.05 5.32

0.857 b0.001 b0.001 b0.001 0.008

0.048 0.321 0.308 0.308 0.058

N intake and output (g N/d) Intake Feces Urinary N Urinary urea

Average

SD

21.3 8.37 7.75 6.79

a

5.76 10.2 9.23 6.31 15.9

FS(A)

a

SD: standard deviation. DCPSI: digestible crude protein at small intestine. c PDe: total purines excreted in urine, PDa: microbial purines absorbed from the small intestine, MNPD: daily duodenal flux of microbial N; ERDOM: effectively rumen degraded organic matter. d Statistics significance of FS: fresh forage vs. silage; FS(A): animal random factor within diet. b

through increased propionate formation (Demeyer and Fievez, 2000), provoked by lactate in the silage (Lima et al., 2010). Nevertheless, lactate concentrations in rumen fluid were similar for both fresh and ensiled forage, which can be due to the rapid metabolism of lactate in the rumen (half-life of 25 min, Chamberlain et al., 1983), particularly in animals adapted to silage feeding (Jaakkola and Huhtanen, 1992). Although, lactate is mainly converted to propionate (52%) (Jaakkola and Huhtanen, 1992)), it also is partially transformed to valerate (Nagaraja and Titgemeyer, 2007), which could be related to the higher (P b 0.001) proportion of the latter fatty acid (Table 2) in the rumen of silage fed animals. 4.3.2. Nitrogen utilization efficiency The proportion of protein digestible at the small intestine (Table 4) represents 46% of the total crude protein in fresh

sorghum–soybean whereas it represents 73% of the crude protein of ensiled material. Consequently, environmental nitrogen emissions are assumed to be considerably lower when feeding silages as compared to fresh material. 5. Conclusions Combined ensiling of sorghum–soybean with molasses and bacterial inoculant, produced with minimal technical resources and at small scale, showed excellent ensiling fermentation characteristics and lower methane emissions, resulting in 8% greater ME than fresh forage available during the Cuban dry season. Moreover, both total apparent as well as rumen degradability were greater as compared with those of fresh forages. Further, the higher ME content and crude protein digestible at the small intestine in the sorghum–soybean silages

Table 5 Metabolizable energy estimated from crude nutrients. Originally equations were developed for corn silage (1–4), fresh and ensiled grasses (5–6) with R2 reflecting the determination coefficient of the original equation. Reported equations now were used to predict ME of mixtures of sorghum and soybean (60/40, w/w) fed either fresh as available during the Cuban dry season or ensiled with forage harvested during the Cuban rainy season. Equation a

1 2 3 4 5 6

ME b (MJ/kg DM)

13.31 − 0.0098 ADF 12.7 1 − 0.0108 ADF + 0.0262 EE 12.76 + 0.0285 fat − 0.0075 ADF − 0.0166 ash 12.86 + 0.0265 fat − 0.0056 ADF − 0.0153 ash − 0.0253 lignin 14.06-0.0137 CF + 0.00483 CP − 0.0098 ash 13.99 − 0.01193 CF + 0.00393 CP − 0.01177 ash

R2

Fresh forage

Silage

0.54 0.72 0.78 0.80 NR c NR

9.93 11.4 11.4 10.5 10.1 10.3

10.6 12.2 11.9 11.0 11.2 11.2

a Equations 1–4 according to De Boever et al. (1997); equations 5–6 according to Schenkel (1998). ADF: acid detergent fiber; EE: ether extract; CP: crude protein; CF: crude fiber; all chemical composition in g/kg DM. b ME: metabolizable energy. c NR: not reported by the author.

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Table 6 Estimation of milk production and average of daily gain from digestible protein at small intestine (DCPSI) and metabolizable energy (ME) obtained from a mixture of sorghum and soybean (60/40, w/w) fed either fresh as available during the Cuban dry season or ensiled with forage harvested during the Cuban rainy season. Parameters

Dairy cattle a

Beef cattle b

ME

DCPSI

ME

DCPSI

Requirements Maintenance (/d) Locomotion (/d) Growth of cows in 2nd lactation (/d) Milk production or daily gain (/kg)

MJ 55.2 14.2 7.40 5.52

g 317 – 31.0 52.2

MJ 41.1 – – 36.5

g 204 – – 276

Production d Fresh forage Silage

Milk (kg/d) 8.48 10.4

6.22 c 12.8 c

Growth (g/d) 567 715 c

480 c 1102

a Requirements were calculated according to Roche et al. (1999) for dairy cows (Cuban breed) with 450 kg of body weight, second lactation, high energy expenditure for locomotion and referred milk production. b Requirements were calculated according to Roche et al. (1999) for growing cattle (Cuban breed) with 250 kg of body weight and referred average of daily gain. c Limiting factor. d Production rates according to feed values of fresh and ensiling roughages (Tables 3 and 4) and dry matter intake of 12.0 and 6.00 kg/d for dairy and beef cattle, respectively.

harvested during the Cuban rainy season should allow for a higher milk production or daily gain as compared with fresh forage available during the Cuban dry season. Acknowledgements This research was supported by Ghent University, Belgium and Central University of Las Villas, Cuba, through Cuba– Flanders VLIR project. Special acknowledgements to the staff of the Laboratory for Animal Nutrition and Animal Product Quality of Ghent University and to the staff from the Experimental Stations in CIAP of the Central University of Las Villas for the technical assistance during this research. References AOAC, 1995. Official Methods of Analysis, 16th edition. Association of Official Analytical Chemists, Arlington, VA, USA. Barahona, R.R., Sánchez, S.P., 2005. Limitaciones físicas y químicas de la digestibilidad de pastos tropicales y estrategias para aumentarla. Rev. CORPOICA 6, 22. Berra, G., Finster, L., Valtorta, S.E., 2008. Use of tannins to mitigate methane emission in grazing dairy cows, Livestock Environment VIII. Proceedings of the 8th International Symposium. ASABAE, St. Joseph, Michigan, USA, pp. 299–302. Beyer, M., Chudy, A., Hoffmann, L., Jentsch, W., Laube, W., Nehring, K., Schiemann, R., 2003. Reference numbers for characterisation of feed value and their calculation. In: Jentsch, W., Chudy, A., Beyer, M. (Eds.), Rostock Feed Evaluation System. Gottlob Volkhardtsche Druckerei, Miltenberg-Frankfurt, Germany, p. 13. Cáceres, O., González, E., 2000. Metodología para la determinación del valor nutritivo de los forrajes tropicales. Rev. Pastos y Forrajes 23, 87–103. Cao, Z.J., Mo, F., Wang, C.Q., Xian, Y.L., Ma, M., Li, S.L., Xu, C.C., Zhang, X.M., 2010. A simple urine collecting apparatus and method for steers. J. Anim. Vet. Adv. 9, 99–101. Chamberlain, D.G., Thomas, P.C., Anderson, F.J., 1983. Volatile fatty acid proportions and lactic acid metabolism in the rumen in sheep and cattle receiving silage diets. J. Agr. Sci. 701, 47–58. Chen, X.B., Chen, Y.K., Franklin, M.F., Ørskov, E.R., Shand, W.J., 1992. The effect of feed intake and body weight on purine derivative excretion and microbial protein supply in sheep. J. Anim. Sci. 70, 1534–1542. Chen, X.B., Gomes, M.J., 1992. Estimation of microbial protein supply to sheep and cattle based on urinary excretion of purine derivatives — an overview of the technical details. International Feed Resources Unit, Occasional Publication, Rowett Research Institute, Aberdeen, UK. Conway, E.J., 1957a. Acetaldehyde from lactic acid and threonine with bisulphite absorption. In: Conway, E.J. (Ed.), Microdiffusion Analysis and Volumetric Error. Crosby Lockwood & sons Ltd., London, UK, pp. 276–280.

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