In situ degradability and selected ruminal constituents of sheep fed with peanut forage hay Gisele Machado Fernandesa*, Rosana Aparecida Possentib, Waldssimiler Teixeira de Mattosc, Eliana Aparecida Schammassd and Evaldo Ferrari Juniore
a
Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade
de São Paulo, Ribeirão Preto, SP, Brazil;
b
Laboratório de Referência em Nutrição
Animal, Instituto de Zootecnia, APTA/SAA, Nova Odessa, SP, Brazil; c Laboratório de Referência em Forragicultura, Instituto de Zootecnia, APTA/SAA, Nova Odessa, SP, Brazil; d Laboratório de Metodologias Quantitativas em Pesquisa Zootécnica, Instituto de Zootecnia, APTA/SAA, Nova Odessa, SP, Brazil;
e
Centro de Pesquisa e
Desenvolvimento de Nutrição Animal e Pastagens, Instituto de Zootecnia, APTA/SAA, Nova Odessa, SP, Brazil
*Corresponding author: Gisele Machado Fernandes. E-mail:
[email protected]
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Abstract Inclusion levels of hay Arachis pintoi cv. Belmonte in the diet of sheep were studied through the dry matter intake, concentration of volatile fatty acids, ammonia nitrogen concentration, ruminal pH, in situ degradability of dry matter and crude protein. Significant interaction was observed between treatments and sampling times for ammonia nitrogen and acetate, propionate and butyrate concentration and the acetate:propionate ratio. The ruminal pH and total volatile fatty acids concentration were not affected by interaction between treatments and sampling time. The dry matter and crude protein degradation were similar, rising with the increasing content of Arachis, showing linear effect. The treatment containing 60% of Arachis showed best results, with good levels of daily weight gain and higher ruminal concentrations of volatile fatty acids. This legume showed high levels of crude protein, high digestibility and appropriate levels of fiber, with excellent standards of degradation and ruminal characteristics. The use of legume Arachis for ruminants is as promising option of nutrient supply to meet production demands of these animals. Keywords: volatile fatty acids, Arachis pintoi, crude protein, ammonia nitrogen.
1. Introduction Studies on animal nutrition have shown factors that affect livestock yield and their interaction with the environment, through assessments on food nutritional values, nutrient metabolism in livestock and biochemical parameters related to nutrition and animal breeding. Legumes are a very important food source for ruminants and can be used as hay, silage or grazing, therefore, chemical characterization of these plants can help choose the best use in animal feed. The forage potential of legumes and ruminant production such as sheep, justify studies on digestive traits of these plants, benefiting the animal production and contributing to an economically viable use of livestock (Argel & Pizarro, 1992). 2
The legume Arachis pintoi is native to the Brazilian Cerrado and shows great potential due to its characteristics of adaptation to acid soils of low fertility, high forage production of good-quality and high capacity for nitrogen fixation and shade tolerance confer to this legume species a growing importance as an alternative to improve the quality of cultivated pastures in tropical climates (Lascano, 1994; Ladeira et al., 2002). This legume present results for digestibility of dry matter between 60% and 70%, with protein rates between 30% to 25%, been highly accepted by the grazing animals which select the A. pintoi throughout the year, providing weight gain at the rate of 500 to 1000 grams per day (Silva, 2004). Ladeira et al (2002a), in a trial of digestibility in vivo, found rates of crude protein, for Arachis pintoi, around 14.3% as being a great source of protein when compared to tropical graminea. Fernandes et al (2000) tested the quality of the forage of A. pintoi in floodplain area and found average values for crude protein and in vitro digestibility of 21.88% and 66.48%, respectively - being the cv. Belmonte one of the forage which presented a better quality. The aim of this study was to evaluate the ideal level of inclusion of hay of Arachis pintoi cv. Belmonte in the sheep diet by measuring the dry matter intake (DMI), concentration of volatile fatty acids (VFA), ammonia nitrogen concentration (NH3-N), ruminal pH, in situ degradability of dry matter (DM) and crude protein (CP).
2. Materials and methods 2.1. Animals, diets and experimental design Four males Santa Inês sheep with average body weight (BW) of 40 kg with cannulas in the rumen were dewormed and kept in individual pens (2.20 m long, 1.70 m wide) equipped with a feeder and a water fountain. The animals were weighed every beginning and end of experimental period, fed ad libitum twice daily at 8:00 a.m. and
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4:00 p.m., the amount of diet offered was adjusted according to the feed intake during the adaptation period of the animals, and no orts greater than 100 g/kg were allowed and received mineral supplementation also ad libitum with the following composition: Ca 220 g/kg; P - 55 g/kg; Mg – 35 g/kg; S - 22 g/kg; Cl – 105 g/kg; Na - 70 g/kg; Mn 1500 mg/kg; Fe - 500 mg/kg; Zn - 1550 mg/kg; Cu - 440 mg/kg; Co - 50 mg/kg; I - 40 mg/kg; Se - 20 mg/kg. The chemical composition of the diets is shown in Table 1. Table 1. Chemical composition (g/kg DM) of diets containing Arachis pintoi cv. Belmonte and Cynodon dactylon cv. Coastcross. Diets 100% GH#
Dry matter
906
30% LH +
60% LH +
70% GH
40% GH
899
100% LH
891
887
DM* basis Crude protein
107
142
173
209
Ether extract
17
17
17
15
Ash
53
60
70
82
Acid detergent fiber
377
369
360
349
Neutral detergent fiber
771
687
574
447
Lignin
46
55
66
80
Non-fiber carbohydrates
52
95
166
247
In vitro digestibility
542
635
591
710
Gross energy (J/kg)
18841
18530
18693
18492
Notes: *DM, Dry matter; #GH, Grass hay; LH, Legume hay.
An experimental design was used in 4 x 4 Latin Square with four periods and four treatments that corresponded to the levels of Arachis pintoi cv. Belmonte included to replace grass hay Cynodon dactylon cv. Coastcross in the diet. The experimental protocols were approved by the ethics committee of the Animal Science Institute. The diets were composed of legume hay (LH) and grass hay (GH) at the following rates:
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Treatment 1= 100% GH; Treatment 2 = 30% LH + 70% GH; Treatment 3 = 60% LH + 40% GH; Treatment 4 = 100% LH. The four experimental periods lasted 21 days each, and the first ten days were for adaptation, management and consumption adjustment of diets. On the 11 th day, we placed the nylon bags containing diets for the in situ study and from the 16th to the 20th day, we evaluated DMI by weighing the feed offered and the feed remaining in the feeders, obtained on the following morning. The sampling of rumen fluid was performed on 21th day.
2.2. Hay production The biomass of Arachis pintoi cv. Belmonte for hay production was obtained in two different cutting times. The soil remediation was done in a uniformity cut performed, in the planted area of 270 m2, applying: 54 kg of dolomitic lime, 6.75 kg simple superphosphate and 810 g of micronutrients, according to soil composition. The material was cut with a mower coupled to a tractor, spread out to the sunlight in a concrete area, and after hacked, it was stored in a warehouse. At the end of the cuts, all the material was mixed to reduce the differences in periods of cut, then, it was chopped and stored in raffia bags in a warehouse. The hay of grass Cynodon dactylon cv. Coastcross was obtained from a privateowned property, milled, bagged and stored in the warehouse.
2.3. Ruminal degradability For DM and CP degradability, we used the technique of in situ nylon bag, according to the method proposed by Huntington and Givens (1995). The bags used for incubation
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of the ruminal food were size 9 x 5 cm, with porosity of 50 x 70 m (ANKOM®), and samples weighing 2 g in each bag. Incubation times were 0, 3, 6, 24, 48, 72 and 96 hours, carried out in reverse so that all were removed simultaneously. The bags containing the samples for the time zero were not incubated, however the wash was carried out with the others. After removed from the rumen, the bags were washed with water to remove excess of ruminal content and, subsequently washed in a washing machine until the water was clean. Later, the bags were placed in a forced-circulation oven at 55°C for 72 hours as recommended in Cummins et al. (1983). After removed from the oven, the bags were weighed to determine the amount of residue. Next, the samples were milled in a micro-mill with a 2 mm-mesh sieve and kept in the laboratory for the analysis of nutritional values.
2.4. Laboratory analysis The procedures of the official methods of analysis (Anonymos ,1995) were used to determine the DM (105º C in a forced air oven for 24 h), ash (Method #942.05), CP (Method #976.06) e Ether extract (Method #920.29). Sequential detergent fiber analysis using heat-stable α-amilase and sodium sulfite. The neutral detergent fiber (NDF) content was determined by the method proposed by Van Soest (1994), acid detergent fiber (ADF) and lignin according to Goering and Van Soest (1970). The NDF and ADF concentrations were ash-corrected. The non-fiber carbohydrates (NFC) was determined according to the equation of Sniffen et al. (1992). The evaluation of in vitro digestibility (IVD) in the hay was performed according to the methods of Tilley & Terry (1963). A calorimetric pump C5000
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Calorimeter System, IKA Labortechnik was used to evaluate the gross energy value of the biomass. For the determination of VFA and NH3-N, ruminal fluid was collected in a plastic beaker from the ventral region, covered with ice and filtered through four layers of gauze-like fabric. The samplings were performed at: 0 (before the first feeding), 2, 4 and 8 hours after the first feeding. The ruminal fluid was centrifuged immediately after sampling and 5 ml of supernatant was removed. For the samples used in the determination of VFA and NH3-N, supernatant was added to 1 ml of formic acid and 0.5 ml of sulfuric acid, respectively. After preparation, the samples were frozen for further analysis. To determine VFA, 1.6 ml of buffered ruminal fluid was centrifuged (Sorvall Superspeed RC2-B, Newton, CT, USA) for 15 minutes at 4°C with 0.4 ml of 3:1 solution of 25% metaphosphoric acid with 98 – 100% of formic acid and 0.2 ml of 2ethyl-butyric solution 100 mM (internal standard). After centrifugation, 1.2 ml of each sample was transferred to chromatographic vials. Of the extract obtained, 1µL was injected in gaseous chromatographic (GC HP 7890ª; Injector HP 7683B, Agilent Technologies) equipped with capillary column HP-FFAP (19091F-112; 25 m; 0.320 mm; 0.50 µm; J & W Agilent Technologies). The injection was performed automatically, the carrier gas is H2, kept at 31.35 ml/min flow. The temperature in the injector and detector was 260°C. The total time of the chromatographic run was 16.5 minutes, divided into three heating stages as follows: 80°C (1 min), 120°C (20°C/min, 3 min) 205°C (10°C/min, 2 min). The VFA concentration (mM) was determined based on an external calibration curve. The concentration of NH3-N was determined according to the method of Fenner (1965). After freezing, we used 1 ml of each sample, to which 15 ml of sodium
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hydroxide (18N) was added and the tube was immediately coupled to the apparatus of micro distillation Kjedhal (Method #976.06, AOAC, 1995). Boric acid (2%) was used as receptor solution and sulfuric acid (0.05 N) for titration. The pH was measured immediately after sampling completion of 100 ml of ruminal fluid for each time 0 (before the first feeding), 2, 4 and 8 hours after the first feeding. The values were obtained using a digital potentiometer (Analyzer ® - model pH300) calibrated with buffer solutions of pH 4.0 and 7.0.
2.5. Statistical analysis The analysis were performed by SAS software (2009). Initially, data were tested for variance homogeneity and residue normality through the GUIDED DATA ANALYSIS to perform the assumptions of the variance analysis. It was necessary to convert the variable of dry matter effective degradation at 5%/h (ED5) into log (x). Degradation of DM and CP, DMI and BW were analyzed using PROC GLM, according to the mathematical model: Yijk = μ + Ai + Pj + Nk + eijk where μ= constant; Ai = random effect of animal ( i-1,...,4); Pj = random effect of the period ( j=1,..,4); Nk= fixed effect of level of Arachis (K=1,..4); eijk = residual error, in a normal distribution with mean zero and variance δ2. Linear, quadratic and cubic effects were estimated for the inclusion levels of Arachis. Because cubic effects were not significant, they are not shown on tables. Effects were declared significant at P
