Enzimas fibrolíticas

J. Appl. Anim. Res. 38 (2010) : 191-196

Effects of Fibrolytic Enzymes on Ruminal Fermentation and Digestibility in Steers Fed Diets with Sodium Bicarbonate O.D. Montañez-Valdez1*, J.M. Pinos-Rodríguez2, J.J. Martínez-Tinajero3, R. Rojo-Rubio4, J. Salinas-Chavira5, J. H. Avellaneda-Cevallos6 1

Departamento de Desarrollo Regional Centro Universitario del Sur de la Universidad de Guadalajara Ciudad Guzmán, Jalisco. 49000. México 2

Instituto de Investigación de Zonas Desérticas Universidad Autónoma de San Luis Potosí. S.L.P. 78377. México 3

Facultad de Ciencias Agrícolas Universidad Autónoma de Chiapas, México 4

Centro Universitario UAEM-Temascaltepec Universidad Autónoma del Estado de México 5

Facultad de Medicina Veterinaria y Zootecnia Universidad Autónoma de Tamaulipas

6

Unidad de Investigación Científica y Tecnológica Universidad Técnica Estatal de Quevedo, Ecuador (Received February 4, 2010; accepted August 10, 2010)

Abstract Montañez-Valdez, O.D., Pinos-Rodríguez, J.M., Martínez-Tinajero, J.J., Rojo-Rubio, R., Salinas-Chavira, J. and Avellaneda-Cevallos, J.H. 2010. Effects of fibrolytic enzymes on ruminal fermentation and digestibility in steers fed diets with Sodium bicarbonate. J. Appl. Anim. Res., 38: 191-196.

The objective of this study was to evaluate the effect of exogenous fibrolytic enzymes (ENZ) on ruminal fermentation and disappearance in steers fed high-concentrate diets with sodium bicarbonate (SB, 3% of total diet). Six ruminally cannulated steers (450±15 kg) were randomly assigned to a replicate 3 x 3 Latin square. Experimental high-concentrate diets (70% concentrate DM) were as follow: control; ENZ (0.3% enzymes) and ENZ+BS (0.3% enzymes plus 3% SB). Dry matter intake, ruminal pH, ammonia N, ruminal disappearance of dry matter and neutral detergent fiber of diets were not affected by ENZ or ENZ+SB. Steers fed ENZ and ENZ+SB spent more time ruminating and resting and less time eating than steers on control diet. Ruminal pH and ammonia N concentrations in steers fed ENZ+BS were greater than in steers fed ENZ or not supplemented. There were scarce effects of fibrolytic enzymes on ruminal disappearance and fermentation of high-concentrate diet with sodium bicarbonate for steers.

Key words: Fibrolytic enzymes, sodium bicarbonate, digestibility. *

Corresponding author e-mail: montanez77@hotmail.com

191 J. Appl. Anim. Res. 0971-2119/2010/$10.00 © GSP, India.

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O.D. Montañez-Valdez and coworkers

Introduction Added fibrolytic enzymes (e.g. cellulases, xylanases) to diets for ruminants have been viewed with considerable skepticism for years.

Despite the considerable number of studies conducted recently, the mechanisms by which fibrolytic enzymes could improve fiber digestion in the rumen have not been clearly elucidated and seem to be affected by several factors such as the type of enzyme and the nature of the substrate (Giraldo et al., 2007).

Inconsistent responses are thought to be due in part to differences in physical and chemical conditions under which the enzymes have been evaluated; a study showed that the highest activity of carbohydrate degrading enzymes was at pH from 3 to 7 for β-glucanase and from 6 to 7 for xylanase (Ao et al., 2008). It suggests that these enzymes seem to work better at close to neutral ruminal pH conditions (Colombatto et al., 2007). Thus, sodium bicarbonate, which alleviates the adverse effects of acidic pH induced by high concentrate diets, could help to enhance the effects of fibrolytic enzymes on fiber degradation. Therefore, the objective of this study was to evaluate the effects of fibrolytic enzymes on dry matter intake (DMI), ruminal digestibility and ruminal fermentation of high concentrate diets with sodium bicarbonate (SB) for steers.

were simultaneously conducted. Sorghum grain, soybean meal based high concentrate complete feeds were offered daily at 7:00 and 18:00 h. Experimental diets were as follows: control (without ENZ or SB); ENZ (0.3% enzymes of total diet); and ENZ+BS (0.3% enzymes plus 3% SB of total diet).

The fibrolytic enzyme preparation containing xylanase and cellulase activities (Fibrozyme, Alltech Inc., Nicholasville, KY, USA) was extensively characterized by Ranilla et al. (2008), who showed that 1 g of enzyme preparation liberated 583 μmol of xylose per min from oat spelt xylan and 163 μmol of glucose from carboxymethylcellulose when incubated at pH 6.5 and 39C. Sodium bicarbonate was a feed grade ruminal buffer (BS; Arm & Hammer, Church and Dwight Co., Inc.). Three days before the beginning of the feeding period the enzyme mixture was first mixed with alfalfa hay and then with the rest of the feed ingredients. Experimental periods lasted 11 d for adaptation to diets and 4 d of experimental measurements.

The experiment was conducted under the supervision and approval of the Academic Committee of the Department of Animal Science of Colegio de Postgraduados, Montecillo in México, according to the regulations established by the Animal Protection Law, enacted by the Estado de México.

Feed offered and orts were recorded, sampled daily and composited by period. On d 12, ruminal fluid samples were collected and eating, rest and chewing time were recorded from all five steers every two hours during 10 h, starting after the morning feeding. On d 13, in sacco disappearance was performed according to Vanzant et al. (1998). Feeds offered and refused were analyzed for organic matter (OM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF). Feed samples were ground to pass a 2 mm screen for in situ ruminal disappearance determination. Ash, CP and ADF (AOAC, 1997) and NDF (Van Soest et al., 1991) were also determined in feed samples.

Six steers (450±15 kg) fitted ruminal cannulae (7.5 cm center diameter, Bar Diamond, Parma, ID, USA) were randomly assigned to a replicated 3x3 Latin square with 2 different squares for balancing carryover effects. Two squares with 3 steers each square

The pH values in ruminal fluids were recorded (Orion 210, Orion Research Corp., Boston, Massachusetts; USA) and samples (5 ml) of fluids were immediately acidified with 3 M metaphosphoric acid (1:10 dilution), cooled at 4C and centrifuged (25,000xg; 4C; 20 min).

Materials and Methods

193

Fibrolytic enzyme and ruminal fermentation

Volatile fatty acid (VFA; GC 6890, GC 6890 Hewlett Packard, Hewlett Packard Co., Wilmington, DE, USA) and ammonia N (I-E Cary, Varian, Mulgrave, Victoria, Australia) were determined by the Erwin et al. (1961) and McCullough (1967) procedures, respectively. To evaluate ruminal total bacteria (Cobos et al., 2002) and protozoa (Neubauer chamber) 150 ml of the original ruminal fluid samples were utilized.

Ruminal kinetics of DM and NDF disappearance were estimated by the model of Ă˜rskov and McDonald (1979): P = a + b (1– e-kt); where P = denotes proportion (%) of the material disappearance (lost through the bag) at time t; a = ruminal soluble, readily disappearance fraction (%) at 0 h; b = is the insoluble, potentially disappearance fraction (%); and k = is the disappearance constant for fraction b (%/h). The DM remaining at each incubation time was fitted to the nonlinear regression model using NLIN procedure of SAS (1999).

Data were analyzed using MIXED procedure (SAS, 1999) to account for effects of square, period within square, steer within square and treatment. Diet was considered a fixed effect; square, period within square and steer within square were considered random effects. Data for ruminal pH, ammonia N, VFA and protozoa were summarized by time of sampled and then analyzed using the same mixed model but with time of sample as a repeated measure (Littell et al., 1998). Bacteria concentration data was analyzed by Harrigan

and McCance (1976) methodology. Means were compared by Tukey procedure (SAS, 1999).

Differences of treatments were declared at P<0.05.

Results and Discussion Treatments did not affect DMI of diets (Table 1). Steer fed ENZ and ENZ+SB spent less time eating, but more time ruminating and resting than steer not supplemented. In agreement with our findings, evidences suggest that the same enzyme product used in this experiment do not affect DMI in ruminants (Giraldo et al., 2008). In all cases, ruminal pH dropped shortly after feeding (Fig. 1), but all ruminal pH values found in this experiment were >6.5, which indicated that ruminal digestibility was not limited at any time (Fuentes et al., 2009). For the dietary treatments BS+ENZ, but not ENZ, rumial pH increased compared to control. A significant effect of BS on ruminal pH values was expected because cattle fed ENZ+BS ruminated 0.38 h/d longer than cattle fed control diet, which could cause an increase in salivary secretion (Krause et al., 1998).

The lack of effects of fibrolytic enzymes on ruminal disappearance and ruminal fermentation (Table 2) may be related to the application method of the enzymatic product.

In effect, Krueger et al. (2008) found that fibrolytic enzymes were most effective at improving digestion and intake when applied at cutting as compared to applied at feeding or baling forage, which could result from greater moisture availability for enzyme

Table 1 Effect of fibrolytic enzymes on eating behaviour of steers fed diets with sodium bicarbonate1

Dry matter intake, kg/d Eating, h/d Ruminating, h/d Resting, h/d 1

Control

ENZ

ENZ+SB

SEM

13.0

13.1

13.3

0.44

a

2.5

b

2.3

b

0.30

4.4

a

4.5

a

0.30

2.9

b

4.0

17.0

b

17.3

a

ENZ, enzyme; BS, sodium bicarbonate; ENZ+SB, enzyme plus sodium bicarbonate.

Different superscripts in the same row refer to differences (P<0.05).

a,b

17.2

a

0.17

194

O.D. Monta単ez-Valdez and coworkers

Table 2 Effect of fibrolytic enzymes on ruminal kinetics disappearance in steers fed diets with sodium bicarbonate Control

ENZ

ENZ+SB

SEM

Soluble (a), %

38.0

37.2

37.6

6.32

Potentially disappearing (b)

49.9

51.6

51.1

7.11

a+b

87.9

88.8

88.7

9.41

4.2

4.3

4.4

0.65

56.6

59.8

60.5

7.91

2.2

2.4

2.5

0.28

Dry matter fraction, %

Disappearance rate (c), /h Neutral detergent fiber fraction, % Potentially disappearing (b) Disappearance rate (c), /h a

ENZ, enzyme; BS, sodium bicarbonate; ENZ+SB, enzyme + sodium bicarbonate; SEM, standard error of means.

Control

ENZ

dispersal and activity when the enzyme was applied at cutting, as well as the longer enzyme-substrate interaction time for this treatment. In our experiment the enzyme was mixed with the alfalfa hay and then with the rest of the dietary ingredients 3 d before feeding; the total mixed rations had <20% moisture. Yang et al. (2000) reported that fibrolytic enzyme addition to concentrates one month before feeding increased diet digestion in lactating dairy cows. Contrastingly, PinosRodriguez et al. (2008) and Giraldo et al. (2008) found that the same enzymatic product increased ruminal digestibility and affected ruminal fermentation without a pre-feeding feed-enzyme interaction.

ENZ+SB

7.1 7 6.9

pH

6.8 6.7 6.6 6.5 6.4

Ammonia N, mg/l

200

150

100

50

0 0

2

4

6

8

10

After feeding, h

Fig. 1. Effect of fibrolytic enzyme (ENZ) on pH values and ammonia N concentration in rumen of steers fed diets with sodium bicarbonate (SB).

Few studies have shown that changes in fermentation pattern are due to concomitant changes in rumen bacteria populations. Giraldo et al. (2007, 2008) reported that treating substrates with exogenous fibrolytic enzymes increased the numbers of rumen bacteria that utilize hemicellulose or secondary products of cellulose digestion, as well as, increased numbers of cellulolytic bacteria in Rusitec fermenters. Similarly to our findings (Fig. 1), Beauchemin et al. (2000) found that exogenous enzymes decreased ruminal NH 3 N concentrations possibly as a result of an increment of degradations of slow fermentating

195

Fibrolytic enzyme and ruminal fermentation

Table 3 Effect of fibrolytic enzymes on volatile fatty acids, protozoa and bacteria population of steer fed diets with sodium bicarbonate1 Control

ENZ

ENZ+SB

SEM

Acetate, mol/100 mol

70.2

71.1

70.5

2.26

Propionate, mol /100 mol

16.5

16.0

16.7

0.63

Butyrate, mol/100 mol

13.3

12.9

12.8

0.60

Total VFA, mmol/l

83.6

70.7

76.6

3.39

4.2

4.4

Acetate:propionate ratio 6

Cellulolytic bacteria x10 /ml 6

Protozoa x10 /ml

0.4

b

14.0

6.1

4.9

4.2 a

15.0 6.0

0.10 a

0.06

1

ENZ, enzyme; BS, sodium bicarbonate; ENZ+SB, enzyme + sodium bicarbonate; SEM, standard error of means.

Different superscripts in the same row refer to differences (P<0.05).

a,b

carbohydrates, this was not confirmed in the current study.

Our results suggest that ENZ and ENZ+SB increased cellulolytic bacteria but did not change on VFA concentrations and molar proportion of acetate, propionate and butyrate (Table 3). We failed to show significant effect of EZN+SB on ruminal disappearance. The hypothesis that EZN+SB would have significant effect on ruminal disappearance was not confirmed. Perhaps, the ruminal pH found was not low enough for the ENZ+SB to alleviate the dropped digestibility that occurs in presence of low ruminal pH.

Colombatto, D., Mould, F.L., Bhat, M.K. and Owen, E. 2007. Influence of exogenous fibrolytic enzyme level and incubation pH on the in vitro ruminal fermentation of alfalfa stems, Anim. Feed Sci. Technol., 137: 150-162.

Erwin, E.S., Marco, G.J. and Emery, E. 1961. Volatile fatty acids analysis of blood and rumen fluid by chromatography. J. Dairy Sci., 44: 1768-1771.

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