Influence of different levels of dried citrus pulp on in vitro ruminal fermentation kinetics of total mixed ration in goat rumen inocula

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increased linearly (P<0.01) with the inclusion of DCP in rations.The ration at 30% DCP (DCP30) had the greatest value of ME and SCFA, but no differences were observed in the PF 96 among treatments.The data suggest that the DCP30 ration had the best impact on rumen gas production and IVDMD, IVOMD, ME, and SCFA.KEY WORDS: dried citrus pulp, gas production, in vitro fermentation, goat INTRODUCTION Conventional alternative feeds have been developed to meet the nutritional requirements of livestock during dry seasons.Food processing produces large amounts of by-products, but only a few of them have been successfully integrated as alternatives in livestock feeding because most of them are low in the major nutrients, and/or not balanced for them (Gado et al., 2011).
In Mexico, a large quantity of by-products from the citrus juice industry is obtained in the form of dehydrated citrus pulp (Ku et al., 1993).Dried citrus pulp (DCP) is a widespread by-product used mainly in ruminant diets (Volanis et al., 2006).Ensiled and dried citrus pulp have been extensively studied on dairy cows (Belibasakis and Tsirgoyianni, 1996), fattening calves (Hadjipanayiotou and Louca, 1976), and lambs (Gado et al., 2011).Citrus pulp (DCP) modifies ruminal fermentation (Pinzón and Wing, 1976), improves fibre digestion (Gado et al., 2011), and increases microbial protein synthesis in dairy cows (Belibasakis and Tsirgoyianni, 1996;Gado et al., 2009), and sheep (Aregheore, 2000).Citrus pulp could be an alternative energy source replacing some grain in concentrate diets (Gado et al., 2011).It is noteworthy that the effects of including DCP in diets for goats on fermentation and nutrient metabolism have not yet been fully examined.Therefore, the objective of this study was to investigate the effect of different levels of DCP in diets on in vitro fermentation of gas production kinetics and energy utilization in goat rumen inocula.

Experimental diets
Four total mixed rations (TMR) were formulated to include 4 levels of dried citrus pulp (0%, 10%, 20%, and 30% of the TMR).The rations met the nutritional requirements for non-lactating mature does in early gestation (40 kg body weight, feed consumption of 1.05 kg DM; NRC, 2007; Table 1).

Preparation of diet extract to determine secondary metabolites
Secondary metabolites were determined as outlined previously in Salem et al. (2011).Briefly, the experimental diets were ground in a Willey-mill to pass a 1 mm screen and immediately extracted at 1 g/8 ml of solvent mixture.The mixture of solvents contained 10 ml methanol (99.8/100, analytical grade, Fermont ® , Monterrey, Mexico), 10 ethanol ml (99/100, analytical grade, Fermont ® , Monterrey, Mexico), and 80 ml distilled water.Rations were individually soaked and incubated with the solvent mixture in the laboratory at 25-30°C for 48-72 h in closed flasks.After incubation, all flasks were incubated in a water bath at 39°C for one h and then immediately filtered; the filtrates were collected and stored at 4°C for further use.

Proximate analysis and secondary metabolites
Samples of each diet were analysed for DM, ash, CP (N×6.25), and ADF according to AOAC (1990).The NDF analyses were according to the Van Soest et al. (1991) procedure, using an ANKOM 200 Fibre Analyzer Unit (ANKOM Technology Corporation, Fairport, NY, USA).Total mixed rations were extracted using a mixture of ethanol, methanol and water (40:40:20) and secondary metabolites were determined using 10 ml of different TMR.The extract was fractionated using ethyl acetate (99.7/100, analytical grade, Fermont ® , Monterrey, Mexico) to determine total phenolics by drying and quantifying the layer in the funnel.After total phenolics separation, n-butanol (99.9/100, analytical grade, Fermont ® , Monterrey, Mexico) was added to fractionate the saponins (Makkar et al., 1998).The remaining solution was considered to be the aqueous fraction (lectins, polypeptides, starch;Cowan, 1999).

Donor animal's inocula
Rumen fluid was collected by a stomach tube before the morning meal from 8 gestating goats (Boer×Saanen, body weight 39±2.3 kg).The animals had access to clean water and were fed twice daily at 07.00 and 16.00 h for 15 days before sampling.Ruminal fluid was obtained using a stomach tube from multiple sites in the rumen, strained through two layers of muslin and then kept for 30 min at 39°C under a continuous CO 2 stream.

Gas production assay
The gas production assay was carried out according to Theodorou et al. (1994).A sample of 1 g DM (each one of the TMR; Table 1) was weighed in triplicate into 160 ml serum bottles.Anaerobic buffer solution (90 ml, containing microand macroelements, a reducing agent, and a reduction indicator of resazurin) was added to the bottles containing 10 ml of ruminal fluid.Negative controls (blank) containing buffered rumen fluid but no substrate were also included in triplicate for correction of gas produced from small particles present in the ruminal fluid.Cumulative gas production (ml/g DM) was recorded at 2, 4, 6, 8, 10, 12, 15, 19, 24, 30, 36, 48, 72, and 96 h after incubation at 39°C.The volume of gas produced after 24 h of incubation (GP 24 ) was used as an index of energy feed value of tree fodder samples (Menke and Steingass, 1988).

In vitro degradability
At the end of incubation (96 h), the contents of each serum bottle were filtered through sintered glass crucibles (coarse porosity no. 1, 100-to 160 µm pore size, Pyrex, Stone, UK) under a vacuum.Fermentation residues were dried at 105ºC overnight and then incinerated in a muffle furnace at 550ºC for 12 h.Loss in weight after incineration was used as a measure of ash.The in vitro organic matter degradability (IVOMD) at 96 h of incubation was calculated as the difference between the OM content of the substrate and its undegradable OM.

Calculations
The pressure generated by the gas accumulated in the upper part of the incubation bottles was measured through a pressure transducer connected to a digital reader.The equation previously obtained using regression analysis was as follows: Y = -0.807+ 6.86X + 0.083X 2 where: Y -volume (ml), X -pressure (psi); R 2 = 0.99.
The gas production data (ml/g DM) were then fitted using the NLIN option of SAS ( 2002) to the model of France et al. (2000) as follows: where: A -volume of gas production at time t; b -asymptotic gas production (ml/g DM); c -rate of gas production (h), and L (h) -lag time.
Metabolizable energy (ME, MJ/kg DM) was estimated according to the procedure by Menke and Steingass (1988), by the following equation: ME (MJ / kg DM) = 2.20 + 0.136 GP + 0.057 CP where: GP 24 -24 h gas volume and CP (% DM) of the experimental diets.
Partitioning factors (PF, used as a measure of fermentation efficiency) were calculated as the ratio of OM degradation to total gas production at 96 h (i.e., OM disappearance (IVOMD)/total gas production (GP 96 ); Blümmel et al., 1997).

Statistical analysis
Data for in vitro gas production, gas production parameters (b, c and L), in vitro degradability (IVDMD and IVOMD), ME, SCFA, and PF 96 were analysed using the general linear model procedure (SAS, 2002) for a completely randomized design with four treatments of three repetitions; means were tested using the Tukey test (Steel and Torrie, 1980).Linear and quadratic contrasts of DCP level were performed.

RESULTS
The effect of including different levels of DCP in TMR on in vitro ruminal fermentation parameters (b, c and L), IVDMD, IVOMD, ME, SCFA, and PF 96 are shown in Table 2 and Figure 1.Inclusion of DCP linearly increased (P<0.01)cumulative gas production at 24, 48, and 96 h after inoculation.  .GP -gas production (ml/g DM at 24, 48 and 96 h); b -asymptotic gas production (ml/g DM); c -fractional rate of gas production (/h); L -lag time (h); IVDMD -in vitro dry matter degradability (g/kg DM); IVOMD -in vitro organic matter degradability (g/kg DM); SCFAshort-chain fatty acid concentration (mmol); ME -metabolizable energy content (MJ/kg DM); PF -partitioning factor; OM -disappearance/total gas production (GP 96 ) Figure 1.In vitro gas production profiles total mixed rations with the different levels of dried citrus pulp (DCP) in goat rumen inocula Dried citrus pulp increased (P<0.01)asymptotic gas production (b) of TMR, while the fractional rate of gas production (c) was different among treatments.Rations at 30% and 10% DCP had the highest values, whereas control and 20% DCP presented the same values (quadratic effect P<0.01).The lag phase (L) decreased linearly (P<0.001) with DCP addition.The IVDMD and IVOMD increased linearly (P<0.01) with the inclusion of DCP in the diet.The ration at 30% DCP had the greatest ME and SCFA values, but no differences were observed among treatments in the PF 96 .

DISCUSSION
Increase the DCP level in TMR (i.e., to DCP30) linearly increased gas production (Table 2; Figure 1), maybe because DCP contains a high amount of the neutral detergent soluble fibre fraction that includes pectin (250 g pectin/kg DM; Arthington et al., 2002)), soluble sugars (120 to 400 g/kg DM), and less than 10 g starch/kg DM (Volanis et al., 2006).Pectin is the main carbohydrate in dried citrus pulp, and is quickly and extensively degraded by ruminal bacteria (Sunvold et al., 1995).Moreover, higher concentrations of fermentable carbohydrates in DCP increase gas production, degradable N compounds, and decrease gas production because of the binding of CO 2 to ammonia (Krishnamoorthy et al., 1995).Substitution of feeds having high starch concentrations in TMR by others rich in rapidly fermentable carbohydrates, such as citrus pulp, avoids, at least in part, the negative effect on forage digestibility caused by high dietary starch levels (Barrios-Urdaneta et al., 2003).
The lower concentration of secondary compounds in DCP (Table 1) also improved gas production of the different TMR used, possibly due to the ability of rumen microorganisms to degrade them.These microorganisms can degrade alkaloids (Wachenheim et al., 1992), saponins (Hart et al., 2008) and phenolics (Varel et al., 1991) in plant extracts, however, and utilize them as an energy source.Administration of lower doses of leaf extracts rich in secondary compounds to ruminants as feed additives modified in vitro ruminal fermentation of high concentrate diets in lambs fed a daily dose of S. babylonica and L. leucocephala extract (Jiménez-Peralta et al., 2011), and improved in vivo digestibility and average daily gain of lambs (Salem et al., 2011).
Gas production reflects more on the digestible energy content of protein and fat (Aregheore, 2000) and it is generally a good indicator of digestibility, fermentability, and microbial protein production (Sommart et al., 2000).Macías-Cruz et al. (2010) indicated that the DM, OM, and CP apparent digestibility and intake increased with the inclusion of citrus pulp at 75% of the diet DM.The improvements observed in the apparent digestibility of the diet may be due to the better nutrient profile and higher solubility of carbohydrates in the rumen, however, in the present study, IVDMD and IVOMD were improved with the increasing levels of DCP (linear effect P<0.01; Table 2).

CONCLUSIONS
Inclusion of dried citrus pulp (DCP) at the level of 30% in goat rations positively influenced ruminal microbial fermentation and digestion of the carbohydrates of rapid soluble DCP, suggesting the possibility of using this citrus by-product as a potential alternative feed in goat nutrition.

Table 1 .
Ingredients, chemical composition and secondary compounds (g/kg DM) of the total mixed rations (TMR) with the different levels of dried citrus pulp (DCP)

Table 2 .
In vitro fermentation characteristics of total mixed rations (TMR) with the different levels of dried citrus pulp (DCP) in goat rumen inocula