Effects of Yucca schidigera and Quillaja saponaria with or without β 1–4 galacto-oligosaccharides on ruminal fermentation, methane production and nitrogen utilization in sheep

Animal Feed Science and Technology 138 (2007) 75–88

Effects of Yucca schidigera and Quillaja saponaria with or without ␤ 1–4 galacto-oligosaccharides on ruminal fermentation, methane production and nitrogen utilization in sheep B. Pen, K. Takaura, S. Yamaguchi, R. Asa, J. Takahashi ∗ Department of Animal Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan Received 17 April 2006; received in revised form 7 November 2006; accepted 21 November 2006

Abstract Effects of Quillaja saponaria extract (QSE) and Yucca schidigera extract (YSE) with or without ␤ 1–4 galacto-oligosaccharides (GOS) on ruminal fermentation, methane production and N utilization in wether sheep were evaluated. Four wethers fitted with permanent ruminal fistulae were assigned in a 4 × 6 Youden square design experiment and fed a basal diet comprised of concentrate and Italian ryegrass hay (2:3, on a DM basis) at 55 g/kg metabolic body weight. Treatments were: (1) control (no addition of supplement); (2) 14 ml of QSE; (3) 14 ml of YSE; (4) 20 g of GOS; (5) 14 ml QSE + 20 g GOS; (6) 14 ml YSE + 20 GOS per day. Digestibility of NDFom increased (P<0.05) in sheep treated with QSE, QSE + GOS, and YSE + GOS, but was not affected by YSE or GOS. Ruminal fluid pH increased (P<0.05) in sheep administered with YSE compared with other treatments. Ammonia N and total VFA concentrations declined (P<0.001) with administration of QSE and YSE compared with the control. Propionate and acetate molar proportions were not affected, while butyrate molar proportion declined (P<0.001) with QSE alone or in combination with GOS. Digestible energy increased (P<0.05)

Abbreviations: CH4 , methane; CO2 , carbon dioxide; VFA, volatile fatty acid; GOS, ␤ 1–4 galactooligosaccharides; QSE, Quillaja saponaria extract; YSE, Yucca schidigera extract ∗ Corresponding author. Tel.: +81 155 49 5421; fax: +81 155 49 5421. E-mail address: [email protected] (J. Takahashi). 0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2006.11.018

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with all treatments, except YSE. Results suggest that administration of QSE and YSE reduced ruminal ammonia N and total VFA concentration, and numerically decreased methane emissions, without having adverse effects on fiber digestion. In addition, administration of YSE, with or without GOS, numerically reduced protozoal numbers. QSE, YSE and YSE + GOS may have potential as beneficial manipulators of ruminal fermentation. © 2006 Elsevier B.V. All rights reserved. Keywords: Natural products; Saponin containing plant extract; Methane production; Microbial N synthesis

1. Introduction Global warming due to an increase in the atmospheric concentration of greenhouse gases such as carbon dioxide and methane is an important issue. Generation of methane from livestock, particularly from ruminants, represents 2–12% of gross energy intake (Johnson and Johnson, 1995) and contributes approximately 16% of global methane emissions (Moss, 1993). A growing concern about global climate change has increased attention on ways to abate ruminal methanogenesis. Antimicrobial compounds are routinely incorporated into ruminant diets to improve efficiency, suppress methanogenesis and reduce excretion of N in urine and feces. In recent years, there has been increased concern regarding use of in feed antibiotics in ruminants due to the progressive increase of antibiotic resistance among pathogenic microorganisms (Carro and Ranilla, 2003). Therefore, current interest is focused on use of safe natural products, versus chemical compounds, to beneficially manipulate ruminal fermentation. Previous studies have indicated that supplementation of saponins, or saponin-rich feeds such as Yucca schidigera and Quillaja saponaria, decreased ciliate protozoa population in vitro (Makkar et al., 1998; Pen et al., 2006a) as well as in vivo (Valdez et al., 1986). A decreased ruminal ciliate protozoa population may enhance the flow of microbial N from the rumen, increase efficiency of N utilization and decrease methane production since 9–25% of methanogens associate with ciliate protozoa in the rumen (Newbold et al., 1995). In addition, Y. schidigera supplementation increased propionate production, and decreased methane production, in vitro (Takahashi et al., 2000; Pen et al., 2006a). Galactooligosaccharides (GOS), a mixture of galactose and glucose, are synthesized enzymatically from lactose by ␤-d-galactosidase derived from Bacilus circulans or Cryptococcus laurentii (Matsumoto et al., 1990). GOS is a prebiotic, which is non-digestible in monogastric animals, but known to stimulate beneficial microbes in the gastrointestinal tract (Mwenya et al., 2005a,b). Increased proportion of propionate (Santoso et al., 2004), and reduced methane production (Mwenya et al., 2004), have been reported in ruminants supplemented with GOS. Supplementation of GOS to sheep administered with QSE and YSE might have synergistic effects on ruminal fermentation versus use of QSE or YSE alone. The objectives of the present study, therefore, were to evaluate effects of QSE and YSE alone, or in combination with GOS, on ruminal fermentation, methane production and N utilization in sheep.

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2. Materials and methods 2.1. Animals, diets and supplements Four Cheviot wethers (initial weight: 60.9 ± 3.7 kg and final weight: 59.9 ± 4.4 kg) fitted with permanent ruminal fistulae were used in a 4 × 6 Youden square design experiment (six experimental periods and six treatments). Wethers were kept in an individual metabolic cage and fed a basal diet at 55 g of DM/kg metabolic body weight (BW0.75 ) daily. The basal diet, a mixture of Italian ryegrass hay and concentrate (3:2, w/w DM basis), was fed to the wethers at 8:30 and 16:30 h, while mineral blocks (Fe = 1232 mg; Cu = 150 mg; Co = 25 mg; Zn = 500 mg; I = 50 mg; Se = 15 mg and Na = 382 mg/kg) and clean drinking water were available at all times. Feed refusals were removed before each morning feeding, weighed and kept for chemical analysis. The treatments were: (1) control (no addition of supplement); (2) 14 ml of QSE; (3) 14 ml of YSE; (4) 20 g of GOS; (5) 14 ml QSE + 20 g GOS; (6) 14 ml YSE + 20 GOS per day. GOS powder which was resistant to gastric enzymes was applied to wethers by mixing with concentrate, but Q. saponaria extract (QSE) or Y. schidigera extract (YSE) were administered directly into the rumen via ruminal fistulae twice daily immediately after morning and afternoon feedings, to prevent neutralization of saponin-induced antiprotozoal activity in saliva (Odenyo et al., 1997). Doses of QSE and YSE at 14 ml/day, corresponding to 0.8–1.13 g of quillaja saponins and 1.31–1.64 g of yucca saponins per wether per day were used. Each wether received the treatments in a different sequence according to a 4 × 6 Youden square arrangement. The experimental protocol was approved by Obihiro University of Agriculture and Veterinary Medicine Committee for Animal Use and Care, Japan. Commercial concentrate and Italian ryegrass hay (Taisei feed, Hokkaido, Japan) and galacto-oligosaccharides (GOS, Yakult Co. Ltd., Tokyo, Japan) were used. The QSE and YSE were dark brown liquids with specific gravities of 1.15 and 1.17, respectively, and were supplied by Mitsuba Trading Co., Ltd. (Tokyo, Japan). According to the manufacturer, QSE contained 50–70 g/kg saponins, whereas YSE contained 80–100 g/kg saponins. 2.2. Experimental procedure The study had six experimental periods, and each lasted for 18 days with 10 days of adaptation to the respective experimental diets, 5 days of complete collection of feces and urine, 2 days of quantitative measurement of gaseous exchange and a final day for ruminal fluid sampling. The 10 day adaptation allowed adaptation of ruminal metabolism to the treatments (Grubb and Dehority, 1975). The BW was measured at the beginning and the end of each experimental period. Feces was collected into fecal bag and urine was collected into a bucket containing 100–120 ml of 100 ml/l sulfuric acid to reduce pH below 3 and prevent bacterial degradation of N compounds including purine derivatives. Feces and urine were collected twice daily and stored at −10 ◦ C. At the end of each collection period, feces urine and feed refusals were composited among days within period for each wether. Additional urinary samples were diluted 1:4 with distilled water for allantoin, uric acid and xanthine + hypoxanthine determination. All samples were stored at −20 ◦ C until analysis. Oxygen consumption and carbon dioxide and methane release were quantitatively measured

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in two 23 h runs by an open circuit respiratory system using a hood over the wether’s head as described by Takahashi et al. (1999). Ruminal fluid samples were collected 0, 1, 2, 4, 6 and 8 h after the morning feeding through the ruminal fistulae equipped with a valve to avoid gas losses during sampling and to avoid disturbing rumen fermentation. The pH and oxidation–reduction potential (ORP) of the ruminal fluid were measured immediately using a pH meter (HM-21P, DKK TOA Electronics Ltd., Tokyo, Japan) equipped with appropriate electrodes. For protozoa enumeration, 1 ml of ruminal fluid samples collected at 0, 2, 4, and 8 h were preserved in 14 ml methyl-green:formalin:saline solution (Ogimoto and Imai, 1981) in tubes with tight caps and stored in the dark. The remaining samples were stored at −20 ◦ C without addition of preservatives for subsequent analyses of volatile fatty acids (VFA) and ammonia N concentrations. 2.3. Laboratory analysis Feed, feed refusals and fecal samples were dried at 60 ◦ C for 72 h in a forced-air oven and moisture loss was determined after air-drying at room temperature for 24 h. The samples were ground to pass through 1 mm sieve and stored in air tight containers. Samples were analyzed for dry matter (DM; method 930.15) and organic matter (OM; method 942.05) according to the AOAC procedures (1995). The N was determined by a Kjeldahl method (method 976.05; AOAC, 1995) using an electrical heating digester (DK 20, Actac, Tokyo, Japan) and an automatic distillation apparatus (UDK, 130D, Actact, Tokyo, Japan), and crude protein (CP) was determined as N × 6.25. Neutral detergent fiber (NDFom, without ␣-amylase and without sodium sulfite) of Italian ryegrass, feed refusal and fecal samples and aNDFom (with ␣-amylase and without sodium sulfite) of concentrate were analyzed according to the methods described by Van Soest et al. (1991) and acid detergent fiber (ADFom) was determined (method 973.18; AOAC, 1995). Values of aNDFom, NDFom and ADFom are expressed exclusive of ash content. Additionally, feed and feed refusal samples were analyzed for sulfuric acid lignin (sa) by the method of Van Soest et al. (1991). Cellulose was calculated as difference between ADFom and lignin (sa). Total N concentration of urinary samples was determined as described in the feed analysis. Gross energy contents of feed, feed refusals, feces, urine and dietary supplements were determined by using a bomb calorimeter (CA-4P, Shimadzu, Tokyo, Japan) as described by Pen et al. (2006b). Diluted urinary samples were analyzed for allantoin concentration using a spectrophotometer (U-2000, Hitachi, Tokyo, Japan) according to the procedure of Chen and Gomes (1995), and uric acid and xanthine + hypoxanthine were measured together as uric acid using a commercial kit (Uric Acid C, Wako Pure Chemical Industries, Tokyo, Japan) after treatment of urine with xanthine oxidase as described by Chen and Gomes (1995). Ciliate protozoa numbers were enumerated via light microscopy using a 0.2 mm deep Fuchs-Rosenthal counting chamber (Kayagaki IRIKA, Kogyo Co. Ltd., EKDS, Tokyo, Japan). Ammonia N concentration of ruminal fluid samples was analyzed according to the procedure of Conway and O’Malley (1942). Ruminal fluid samples for VFA determination were first centrifuged at 12,500 × g for 15 min at 4 ◦ C and deproteinized with 250 g/l metaphosphoric acid (0.2 ml/ml of ruminal fluid), centrifuged at 8000 × g for 15 min at 4 ◦ C, and then analyzed using a gas–liquid chromatography (GC-2014, Shimadzu, Kyoto, Japan)

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equipped with a flame-ionization detector and a capillary column (Ulbon HR-52, 0.53 mm ID × 30 m, 3.0 ␮m). 2-Ethyl-n-butyric acid was used as an internal standard. The operation conditions were: injector temperature, 190 ◦ C; detector temperature, 280 ◦ C; column temperature 50–90 ◦ C (5 ◦ C/min). 2.4. Calculations of energy balance and microbial N supply Total methane gas volume obtained from the open circuit respiratory system was converted to its gross energy value using the conversion factor 39.54 kJ/l. Heat production (kJ/day) was calculated using the equation: 16.18 O2 (l/day) + 5.02 CO2 (l/day) − 2.17 CH4 − 5.99 N (g/day) (Brouwer, 1965). Energy retention was calculated as difference between metabolizable energy (ME) and heat production. The amount of microbial purines absorbed (X, mmol/day) corresponding to the purine derivatives excreted (Y, mmol/day) and microbial N supply to duodenum were calculated based on the following equations (Chen and Gomes, 1995): Purine derivatives (mmol/day) = 0.84X + (0.150 W 0.75 e−0.25X ) Microbial N supply(gN/day) =

X(mmol/day) × 70 = 0.727X; 0.116 × 0.83 × 1000

where W0.75 is the metabolic BW of the wether, 70 the N content of purines (mg/mmol), 0.116 the ratio of purine N:total N in mixed rumen microbes and 0.83 is the digestibility of microbial purines (Chen and Gomes, 1995). The efficiency of microbial N synthesis is expressed as grams of microbial N/kg of digestible OM apparently fermented in the rumen. 2.5. Statistical analysis Data of nutrient digestibility, N and energy balance, and microbial N supply were subjected to analysis of variance for Youden square design using the MIXED procedure of SAS (1996). Data of ruminal fluid characteristics and gaseous exchanges were analyzed using the MIXED procedure of SAS (1996) with time treated as a repeated measure and an unstructured (type = un) as a covariance structure. The model included fixed effects of dietary treatment and experimental period and animal served as random effect. Multiple comparisons among means were performed using the least squares means procedure (PDIFF option) of SAS (1996). Treatment effects were declared significant if P<0.05 and trends were accepted at P<0.10.

3. Results 3.1. Chemical composition and nutrient digestibility The gross energy contents of dietary supplements (Table 1) were: galactooligosaccharides 15.25 MJ/kg, Q. saponaria extract 11.23 MJ/kg and Y. schidigera extract 13.32 MJ/kg. There were no effects of administration of QSE and YSE, with or without

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Table 1 Chemical composition (g/kg DM) of Italian ryegrass hay and concentrate and gross energy of diet components and feed additives Chemical composition

Italian ryegrass hay

Concentratea

Dry matter Organic matter Crude protein Neutral detergent fiber Acid detergent fiber Acid detergent lignin Cellulose

907.6 938.5 61.5 649.8 376.6 47.7 329.0

875.9 929.1 213.8 178.9 76.4 15.1 61.4

Gross energy Italian ryegrass hay (MJ/kg DM) Concentrate (MJ/kg DM) Q. saponaria extract (MJ/kg)b Y. schidigera extract (MJ/kg)b Galacto-oligosaccharides (MJ/kg)b

18.08 18.20 11.23 13.32 15.25

Values are means of six sub-samples of each material (n = 6). a Contained 540 g/kg of grains (heat treated corn, maize, heat treated oat and rye), 250 g/kg of oil seed meals (soybean meal and rapeseed meal), 130 g/kg of corn gluten feed, wheat bran and starch pulp, 80 g/kg others (alfalfa meal, molasses, calcium carbonate powder, salt, corn syrup, malt, yeast, lactic bacteria). b MJ/kg of fresh material.

GOS, on apparent nutrient digestibility of DM, OM, CP and ADFom (Table 2). Moreover, NDFom digestibility increased (P<0.05) in sheep treated with QSE, QSE + GOS and YSE + GOS. 3.2. Ruminal fermentation characteristics Wether that received intraruminal administration of YSE had higher (P<0.05) ruminal fluid pH compared with other treatments (Table 3). Redox potential (ORP) of ruminal fluid was increased (P<0.05) with all treatments except YSE. Ammonia N and total VFA concenTable 2 Effects of Q. saponaria extract (QSE) and Y. schidigera extract (YSE) with or without galacto-oligosaccharides (GOS) on apparent digestibility of nutrients in sheep fed on Italian ryegrass hay and concentrate (3:2, on a DM basis) Treatmenta

Dry matter Organic matter Crude protein Neutral detergent fiber Acid detergent fiber

S.E.M.

Control

QSE

YSE

GOS

QSE + GOS

YSE + GOS

0.669 0.686 0.705 0.547b 0.526

0.683 0.701 0.708 0.584a 0.557

0.675 0.692 0.697 0.568a,b 0.539

0.676 0.692 0.701 0.564a,b 0.540

0.688 0.704 0.699 0.586a 0.557

0.686 0.703 0.711 0.575a 0.558

0.0153 0.0161 0.0127 0.0331 0.0287

All values are means of four animals (i.e., 4 observations). Mean values within a row with different letters (a and b) differ (P<0.05). a Treatments were: Control; QSE 14 ml/day; YSE 14 ml/day; GOS 20 g/day; QSE + GOS; YSE + GOS.

Parameters

Treatmenta

S.E.M.

Control

QSE

YSE

GOS

QSE + GOS

YSE + GOS

pH Redox potential (mV) NH3 -N (mg/l)

6.38a −302.6a 177.90a

6.40a −291.2b,c 158.74b,c

6.51b −298.3a,b 143.01c,d

6.38a −291.0b,c 159.00a,b,c

6.34a −286.8c 168.56a,b

6.38a −289.6b,c 167.99a,b

0.125 9.17 13.965

Volatile fatty acids Total VFA (mmol/l)

104.37a

91.93b

85.81b

98.12a

100.55a

7.807

Molar proportions Acetate Propionate Butyrate Other VFAb Acetate:propionate ratio Ciliate protozoa (×105 /ml)c

0.729 0.150 0.111a 0.009a 4.88 16.03

0.734 0.160 0.101b 0.004b 4.83 15.02

0.732 0.155 0.108a 0.004b 4.79 13.96

101.65a 0.730 0.154 0.109a 0.007a,b 4.77 16.51

0.737 0.154 0.103b,c 0.005b 4.92 15.06

0.732 0.155 0.107a,c 0.006b 4.91 12.65

0.0138 0.0076 0.0078 0.0017 0.293 4.111

All values are means of four animals across 6 sampling times (i.e., 24 observations) unless otherwise stated. Mean values within a row with different letters (a–c) differ (P<0.05). a Treatments were: Control; QSE 14 ml/day; YSE 14 ml/day; GOS 20 g/day; QSE + GOS; YSE + GOS. b Including valerate, isovalerate and isobutyrate. c Values are means of four animals across 4 sampling times (i.e., 16 observations).

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Table 3 Effects of Q. saponaria extract (QSE) and Y. schidigera extract (YSE) with or without galacto-oligosaccharides (GOS) on ruminal fluid properties and ciliate protozoa numbers in sheep fed on Italian ryegrass hay and concentrate (3:2, on a DM basis)

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Table 4 Effects of Q. saponaria extract (QSE) and Y. schidigera extract (YSE) with or without galacto-oligosaccharides (GOS) on nitrogen balance and urinary excretion of purine derivates in sheep fed on Italian ryegrass hay and concentrate (3:2, on a DM basis) Treatmenta

S.E.M.

Control QSE

YSE

GOS

QSE + GOS YSE + GOS

Nitrogen intake (g/day)

22.20

22.45

22.48

22.85

22.08

22.39

0.611

Nitrogen excretion (g/day) Feces Urine

6.55 10.21

6.56 9.71

6.83 10.39

6.82 9.35

6.68 10.10

6.49 10.37

0.415 0.515

Nitrogen retention (g/day)

5.45

6.18

5.26

6.68

5.31

5.52

0.579

0.292 0.433 0.275

0.303 0.463 0.234

0.299 0.409 0.292

0.301 0.458 0.241

0.289 0.462 0.248

0.0127 0.0256 0.0247

Nitrogen balance (proportion of N intake) Feces 0.295 Urine 0.460 Retention 0.245 Urinary purine derivatives (mmol/day) Allantoin 8.92 Uric acid + xanthine + hypoxanthine 2.98 Total purine derivatives 11.90

9.56 2.98 12.53

10.30 3.67 13.98

10.30 2.90 13.20

10.34 3.09 13.43

10.11 2.89 13.00

0.339 0.492 0.668

Microbial N supply (g/day) EMNS (g/kg DOMR)b

10.78 22.46

12.05 25.46

11.32 23.37

11.56 24.63

11.19 23.26

0.589 1.494

10.22 22.10

All values are means of four animals (i.e., 4 observations). a Treatments were: Control; QSE 14 ml/day; YSE 14 ml/day; GOS 20 g/day; QSE + GOS; YSE + GOS. b EMNS, efficiency of microbial N synthesis; DOMR, digestible organic matter apparently fermented in the rumen. DOMR = DOMI (digestible organic matter intake) × 0.65 (ARC, 1984).

trations in the ruminal fluid declined (P<0.001) with administration of QSE and YSE. Molar proportions of acetate and propionate were unaffected by any treatments, while butyrate molar proportion declined (P<0.01) with QSE and QSE + GOS. Molar proportions of other VFAs (i.e., isobutyrate, isovalerate and valerate) declined (P<0.05) with administration of QSE and YSE alone, or in combination with GOS. 3.3. Nitrogen utilization and microbial N synthesis Nitrogen intake, excretion of N in feces and urine, and N retention were unaffected by any treatments (Table 4); and total excretion of urinary purine derivatives (i.e., allantoin, uric acid and xanthine + hypoxanthine) and microbial N supply were also unaffected by any treatment. Efficiency of microbial N synthesis (g of N/kg OM apparently fermented in the rumen) was also not affected by any treatments. 3.4. Energy balance and utilization Although administration of plant extracts (i.e., QSE and YSE) and GOS to the basal diet provided additional GE, no differences in GE intake in sheep among all treatments occurred

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Table 5 Effects of Q. saponaria extract (QSE) and Y. schidigera extract (YSE) with or without galacto-oligosaccharides (GOS) on energy balance and utilization in sheep fed on Italian ryegrass hay and concentrate (3:2, on a DM basis) Treatmenta

S.E.M.

Control QSE

YSE

GOS

QSE + GOS YSE + GOS

GE intake (MJ/day)

20.14

20.70

20.71

21.34

20.65

20.91

0.738

Energy loss (MJ/day) Feces Urine Methane Heat production Total loss

6.94 0.44 1.54 8.37 17.08

6.68 0.52 1.28 8.14 16.73

6.93 0.53 1.36 8.34 17.22

7.05 0.48 1.49 8.90 17.94

6.57 0.46 1.47 8.60 17.10

6.66 0.52 1.33 8.47 17.02

0.454 0.031 0.069 0.372 0.680

13.19b 11.42

14.01a 13.78a,b 14.29a 12.11 11.83 12.31

14.08a 12.15

14.26a 12.37

0.480 0.430

3.05

3.97

3.55

3.89

0.340

Utilization of GE (MJ/MJ) DE/GE ME/GE RE/GE

0.656b 0.568 0.153

0.677a 0.667a,b 0.669a,b 0.684a 0.585 0.572 0.576 0.589 0.191 0.167 0.158 0.171

0.683a 0.592 0.187

0.0154 0.0128 0.0155

Utilization of ME (MJ/MJ) RE/ME

0.268

0.326

0.316

0.0246

Digestible energy (DE, MJ/day)b Metabolizable energy (ME, MJ/day)c Retained energy (RE, MJ/day)d

3.48

0.293

3.40

0.274

0.290

All values are means of four animals (i.e., 4 observations). Mean values within a row with different letters (a and b) differ (P<0.05). a Treatments were: Control; QSE 14 ml/day; YSE 14 ml/day; GOS 20 g/day; QSE + GOS; YSE + GOS. b DE was calculated as difference between GE intake and GE loss in feces. c ME was calculated as difference between DE and energy loss in urine and methane. d RE was calculated as difference between ME and heat production.

(Table 5). Energy loss through feces, urine, methane and heat production were unaffected by any treatments. Digestible energy increased (P<0.05) with all treatments, except YSE. 3.5. Methane production Supplementation of GOS, QSE and YSE had no effect on oxygen consumption and releases of methane and carbon dioxide (Table 6). A combination of GOS with QSE and YSE also had no effect on oxygen consumption as well as methane and carbon dioxide emissions.

4. Discussion 4.1. Effects of GOS supplementation In the present study, GOS had no effect on apparent digestibility of nutrients. A number of studies with GOS in sheep (Santoso et al., 2004) and cattle (Mwenya et al., 2005a,b) also

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Table 6 Effects of Q. saponaria extract (QSE) and Y. schidigera extract (YSE) with or without galacto-oligosaccharides (GOS) on gaseous exchange in sheep fed on Italian ryegrass hay and concentrate (3:2, on a DM basis) Treatmenta

Gaseous exchange (l/day) Methane release Oxygen intake Carbon dioxide release Relative methane release Metabolic body weight (l/kg) Dry matter intake (l/kg) OM digested (l/kg)

S.E.M.

Control

QSE

YSE

GOS

QSE + GOS

YSE + GOS

38.84 427.71 404.81

32.55 376.14 342.68

34.61 403.69 363.20

37.78 438.48 398.81

37.02 399.70 386.29

33.63 385.03 374.40

1.966 14.709 16.640

1.86 34.48 53.43

1.53 29.17 44.77

1.64 30.93 47.87

1.78 32.49 50.27

1.76 33.49 51.04

1.60 30.61 46.48

0.100 2.165 2.361

All values are means of four animals (i.e., 4 observations). a Treatments were: Control; QSE 14 ml/day; YSE 14 ml/day; GOS 20 g/day; QSE + GOS; YSE + GOS.

showed no differences in nutrient digestibility compared with non-supplemented animals. Supplementation of GOS, which is a readily fermentable carbohydrate, would be expected to increase propionate production because of production of lactate, which is an intermediate in propionate production. However, molar proportion of propionate and other VFA were not affected by GOS in the present study and that of Mwenya et al. (2005a), which contrast to the report by Santoso et al. (2004). Protozoa numbers in ruminal fluid were unaffected by GOS in the present study and in a previous study by Mwenya et al. (2005b) with dry cows. GOS supplementation had no effect on ruminal methane production in the present study, and in previous studies (Santoso et al., 2004; Mwenya et al., 2005a,b). In contrast, reduced methane production has been reported in ruminants supplemented with GOS (Mwenya et al., 2004). In the present study, GOS supplemented at 20 g/day did not affect ruminal ammonia N concentration, which is consistent with previous reports (Santoso et al., 2004; Mwenya et al., 2005a,b). The effect of GOS on ruminal ammonia N concentration might be diet dependent, as Mwenya et al. (2004) reported that sheep fed a basal diet comprising 400 g/kg timothy hay, 300 g/kg alfalfa hay cube and 300 g/kg concentrates supplemented with 20 g/day of GOS had lower ammonia N concentration of ruminal fluid compared to unsupplemented animals. In the present study, GOS did not alter N metabolism or microbial N synthesis. Lack of response in N metabolism in cows receiving GOS has also been reported (Mwenya et al., 2005a), and effects of GOS on microbial N supply in ruminants has been variable. Mwenya et al. (2005b) reported a decrease in microbial N supply and efficiency of microbial N synthesis in GOS supplemented ruminants, whereas Santoso et al. (2004) reported an increase. 4.2. Effects of intraruminal administration of saponin containing plant extracts with or without galacto-oligosaccharides A major expected effect of administration of QSE and YSE was a decline in ruminal ciliate protozoa population. The antiprotozoal effect of saponins or saponin containing plant extracts has been reported in vitro (Valdez et al., 1986; Hess et al., 2003) as well as in vivo

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(Diaz et al., 1993). Saponins have membranolytic properties, as they form a complex with cholesterol in protozoa cell membranes causing cell lysis. Pen et al. (2006a) reported that protozoa numbers were strongly inhibited by addition of QSE and YSE in vitro. However, only a numerical decline in ciliate protozoal numbers in sheep administered with YSE (14 ml/day) and YSE + GOS (14 ml + 20 g/day) occurred in the present study. The inefficacy of saponin containing plant extracts on suppressing ruminal protozoa numbers in ruminants has also been reported (Hristov et al., 1999, 2003; Sliwinski et al., 2002). According to Wallace et al. (2002), adaptation of the mixed ruminal microbial population to saponins is one of the factors contributing to variability of antiprotozoal activity of saponins or saponin containing plants. Moreover, effects of saponins, or saponin containing plants, on protozoal population in the rumen have varied markedly with diet, feeding level and dose of saponins (Lu and Jorgensen, 1987). According to Lu and Jorgensen (1987), saponins may cause a shift in nutrient digestion from the rumen to the hindgut, which could cause lower methane production (Fievez et al., 1999). In our previous in vitro study, Pen et al. (2006a) reported that methane production was markedly inhibited by YSE addition, but not by QSE addition. Wang et al. (1998) reported that supplying YSE at 0.5 mg/ml in a buffer did not affect methane production in vitro. Saponins are believed to modify digestion and utilization of dietary nitrogenous compounds in ruminants. According to Wang et al. (1998), proteolysis and molar proportions of branched-chain VFA (i.e., isobutyrate, isovalerate) were lower when saponin containing extracts of Y. schidigera was added in vitro. In the present study, total molar proportion of isobutyrate, isovalerate and valerate decreased with intraruminal administration of QSE and YSE, suggesting that less protein was degraded in the rumen (France and Siddons, 1993). Ammonia N concentration of ruminal fluid decreased in sheep treated with QSE and YSE, which is consistent with previous studies in vitro (Wang et al., 1998; Pen et al., 2006a) and in vivo (Sliwinski et al., 2002). This was likely due to reduced protein degradation and protozoa numbers or, presumably, from ammonia binding of QSE and YSE in the rumen. YSE might have an ability to bind ammonia when concentrations of ammonia are high and release bound ammonia when the concentration is low in the rumen (Makkar et al., 1998). Urinary excretion of allantoin and total purine derivatives, which are indicators for microbial protein arriving at the duodenum (Balcells et al., 1991), were not increased with any treatment in the present study. The effect of saponin containing extracts on urinary excretion of allantoin and total purine derivatives in ruminants probably depends on the ratio of concentrate:forage in the diet. According to Hess et al. (2003), use of a high proportion of concentrate in diets, which generally supports higher microbial synthesis, masks effects of saponin containing extracts. Santoso et al. (2006) reported that supplementing with Y. schidigera powder at 0.24 g/kg diet DM increased microbial N supply in sheep fed a grass silage based diet with a low proportion of concentrate. Intraruminal administration of QSE, QSE + GOS and YSE + GOS increased apparent digestibility of NDFom, but YSE alone had no effect on apparent nutrient digestibility. Lu and Jorgensen (1987) reported increased OM and cellulose digestibility when alfalfa saponins were fed to sheep. In contrast, Hess et al. (2003) reported a decrease in NDF and ADF digestibility with supplementation of the saponin containing plant Sapindus saponaria. These differences among studies seem to be mainly related to the diet dependent nature of the saponin effects (Lu and Jorgensen, 1987; Wang et al., 2000), and comparisons of results

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from studies with different basal diets can be misleading. Furthermore, other constituents of Y. schidigera and Q. saponaria extracts besides saponins may also influence ruminal fermentation (Pen et al., 2006a). In the present experiment, QSE and YSE administration at 14 ml/day decreased total rumen VFA concentration, but did not alter the propionate molar proportion. In contrast, Pen et al. (2006a) reported that QSE and YSE had no effect on total VFA concentration, but increased propionate production in vitro. Valdez et al. (1986) and Wu et al. (1994) found no change in propionate production in vivo with supplementation of yucca saponin at 77 and 396 ppm, respectively. Butyrate molar proportion declined with administration of QSE and QSE + GOS. A slight increase in butyrate molar proportion with yucca saponins has also been reported (Wu et al., 1994; Santoso et al., 2006).

5. Conclusion Administration of Q. saponaria and Y. schidigera extracts at 14 ml/day into the rumen decreased total VFA and ammonia N concentration in the ruminal fluids of wether sheep. Administration of QSE with or without GOS (20 g/day) increased NDFom digestibility, while QSE alone numerically reduced methane production. Administration of YSE alone only numerically decreased protozoa numbers and methane production without modifying NDFom digestibility. But administration of YSE together with GOS increased NDFom digestibility, and numerically decreased protozoa numbers and methane production. Results suggest that saponin containing plants of Q. saponaria and Y. schidigera, with or without galacto-oligosaccharides, may have potential as beneficial manipulators of rumen fermentation.

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