Sarsaponin Effects on Ruminal Fermentation and Microbes , Methane Production , Digestibility and Blood Metabolites in Steers

The objective of this study was to evaluate the effects of sarsaponin on methane production, ruminal fermentation, nutrient digestion and blood metabolites using three Holstein steers in a 3×3 Latin Square design. The steers were fed Sudangrass hay plus concentrate mixture at a ratio 1.5:1 twice daily, and sarsaponin (0, 0.5 and 1% of DM), which was given at 09:00 and 17:00 h daily by mixing with concentrate. Rumen samples were collected 0, 2, and 5 h after morning dosing. Ruminal pH was numerically decreased and numbers of protozoa were decreased linearly (p<0.01) by treatment. Ruminal ammonia-N was reduced (linear; p<0.05) and total VFA was increased (quadratic; p<0.05) at 2 and 5 h after sarsaponin dosing. The molar proportion of acetate was decreased (quadratic; p<0.05) and propionate was increased (linear; p<0.01) at all sampling times. Blood plasma glucose was increased and urea-N was decreased (linear; p<0.05) at 2 and 5 h after dosing. Methane was decreased by approximately 12.7% (linear; p<0.05). The apparent digestibility of DM and NDF were decreased (quadratic; p<0.05) and that of CP remained unchanged due to the sarsaponin. The numbers of cellulolytic bacteria were decreased (quadratic; p<0.05), while numbers of total viable bacteria remained unchanged due to the sarsaponin. These results show that sarsaponin can partially inhibit rumen methanogenesis in vivo and improve ruminal fermentation, which supports our previous in vitro results. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 12 : 1746-1751)


INTRODUCTION
Methane production in the rumen represents a substantial loss of feed energy and has also received attention as a potential contributor to global warming (Moss, 1993).Methane production is affected by a variety of nutritional factors including composition of feed ingredients (Lee et al., 2003), and level of intake, type of carbohydrate, forage processing and changes in the ruminal microflora (Johnson and Johnson, 1995).Many different chemical agents such as bromoethanesulfonic acid, pyromellitic diimide and unsaturated fatty acids (Choi et al., 2004), and bromochloromethane (Chalupa, 1984;McCrabb et al., 1997) are also known to reduce methane production.Methanogenesis may be reduced by the addition of electron acceptors such as nitrate and sulfate (Mathison et al., 1998;Sar et al., 2004), malate (Martin and Streeter, 1995) and fumarate (Asanuma et al., 1999;Bayaru et al., 2001;McGinn et al., 2004).A decrease of methanogenesis in the rumen may be useful due to its increasing the efficiency of energy use in the rumen.
Extracts of the Yucca schidigera plant contain sarsaponin, a group of steroidal glycosides that can influence ruminant productivity.Several authors have reported that sarsaponin improves ruminal organic matter digestion (Goetsch and Owens, 1985;Valdez et al., 1986), increases ruminal propionate concentration (Hristov et al., 1999), stimulates the growth of steers (Goodall et al., 1979) and does not affect animal performance (Wu et al., 1994).
Saponins also have antimicrobial properties, particularly in suppressing ciliate protozoa (Wallace et al., 1994;Hristov et al., 1999), peptidase producing bacteria (Wallace et al., 1994;Wang et al., 2000) and cellulolytic bacteria (Wang et al., 2000).Furthermore, methanogenic bacteria were metabolically co-related with ciliate protozoa (Stumm et al., 1982;Finlay et al., 1994;Newbold et al., 1995) and protozoa actively produce hydrogen, which is utilized by methanogenic bacteria (Stumm and Zwart, 1986;Ushida et al., 1997).In recent studies, we have shown that sarsaponin can partially decrease the methane and hydrogen in vitro (Lila et al., 2003).It also decreased the molar proportion of acetate, and increased the propionate and butyrate with a corresponding decrease in the acetate:propionate ratio.The total volatile fatty acid (VFA) was increased and ammonia was decreased by sarsaponin.These changes in ruminal fermentation suggest that sarsaponin behaves like ionophores, and the addition of sarsaponin may improve the efficiency of ruminant production.
Keeping the above facts in view, the present paper investigated the effects of sarsaponin on ruminal fermentation patterns and microbes, nutrient digestion, methane production and blood metabolites in steers.

ABSTRACT :
The objective of this study was to evaluate the effects of sarsaponin on methane production, ruminal fermentation, nutrient digestion and blood metabolites using three Holstein steers in a 3×3 Latin Square design.The steers were fed Sudangrass hay plus concentrate mixture at a ratio 1.5:1 twice daily, and sarsaponin (0, 0.5 and 1% of DM), which was given at 09:00 and 17:00 h daily by mixing with concentrate.Rumen samples were collected 0, 2, and 5 h after morning dosing.

Animals, diet and experimental protocols
Three castrated 10-to 11-mo-old Holstein steers (halfsiblings) (mean body weight, 248±27 kg) were housed in digestion stalls.Equal portions of the daily ration (chopped Sudangrass hay:concentrate mixture, 1.5:1) were fed at 09:00 and 17:00 h at the maintenance level.Water and mineral blocks were continuously available.The ingredients composition of and a chemical analysis of the diet are shown in Table 1.Steers were randomly allotted to treatment sequences in 3×3 Latin squares.Each period consisted of 14 d of adaptation and 10 d of measurement.The sarsaponin doses (0, 11.2, or 22.4 g) were given twice daily at 09:00 and 17:00 h daily by mixing with concentrate.Sarsaponin (DK Sarsaponin 35) was supplied by Desert King International, (Chula Vista, CA, USA) and it contained 6% moisture, 2.4% crude protein, 0.8% crude fat, 24.7% crude fiber, 4.9% ash, and 61.1% carbohydrate.The steers were cared for in accordance with the Guide for the Care and Use of Laboratory Animals prepared by Tokyo University of Agriculture and Technology.

Sampling and measurement
From day 15 to 19 of each period, the 4 d digestion trial was carried out by total collection method.During the digestion trial, feces were collected daily, weighed and composited for each steer, dried in a forced air oven at 60°C for 48 h, ground through a 1 mm screen, and then assayed for DM, NDF and CP.Urine was collected in a 20 liter container, containing 300 ml of 3 N H 2 SO 4 and a 50 to 100 ml sample was removed and frozen until analyzed for nitrogen.From day 20 to 23 of each period, methane and carbon dioxide production were determined.A head hood collection chamber was used for the measurement of methane and carbon dioxide.The concentration of gases was determined at 1-minute intervals throughout the measurement period by an infrared gas analyzer detector (Fuji Electric, Tokyo, Japan).During the measurement of gases, the chamber was maintained at approximately 20°C.On the final day of each period, samples of rumen fluid and jugular venous blood were collected before feeding, and at 2 and 5 h after morning feeding for ruminal protozoa, ammonia and VFA, and plasma glucose and urea-N, respectively.
Approximately 300 ml of ruminal digesta was collected using a flexible polyvinyl chloride stomach-tube and a subsample (100 ml) was taken in a sterilized container for microbiology assay 2 h after feeding.Hungate's method (1969) was used to prepare the media and cultivate the microorganisms.Total viable counts were determined in roll-tubes with the complete medium described by Leedle and Hespell (1980).Cellulolytic medium (Halliwell and Bryant, 1953) was used for the cellulolytic bacterial count.The exact method used for the determination of bacteria has been described previously (Lila et al., 2004b).
The remaining rumen fluid was separated from particulate matter by straining the digesta through four layers of surgical gauze.The pH of the fluid was immediately measured using a portable pH meter.For analysis of VFA and ammonia-N, samples were acidified with 25% meta-phosphoric acid and stored at -30°C until analyzed.After thawing, the rumen fluid was centrifuged at 10,000×g for 10 min at 4°C, and the VFA was analyzed with a gas chromatograph (Model GC-14B, Shimadzu Co. Ltd., Kyoto) using a Thermon-3000 5% Shincarbon A column (1.6 m×3.2 mm I.D., 60-80 mesh, Shinwakako, Kyoto).Ammonia-N was determined with the microdiffusion method.One mL of the rumen fluid was diluted with 4 ml of methylgreen-formalin-saline to count ciliate protozoa (Bayaru et al., 2001).Blood samples were collected into heparinized tubes, immediately placed on ice and centrifuged at 11,000×g for 15 min at 4°C.Plasma was removed and frozen until analyzed for glucose and urea-N.The details of the analyses have been described previously (Mohammed et al., 2004).
Statistical analyses were carried out by a three-way ANOVA using the cow, period, and treatment as the variables, and computation was performed using SAS (SAS, 1994).Significance was declared at p<0.05.The effect of cow and period were not significant; therefore, the main effects of saponin are presented in Table 2, 3 and 4.

RESULTS AND DISCUSSION
The steers were judged to be in excellent health throughout the study.In this study, the steers consumed 2.25 kg DM plus 11.2 g and 22.4 g of sarsaponin twice daily.The effects of sarsaponin on pH, ammonia, protozoa and the molar proportions of VFA in the ruminal fluid are shown in Table 2.The pH value was numerically decreased by sarsaponin supplementation.This result was consistent with the previous in vitro (Lila et al., 2003) and in vivo (Wallace et al., 1994;Hristov et al., 1999) studies.The ciliate protozoa were composed of Entodinium spp., Dasytricha sp. and Isotricha sp. in this study.Headon et al. (1991) explained that the saponin fraction of Y. schidigera inhibited the growth of ruminal ciliate protozoa.The total numbers of protozoa decreased linearly (p<0.01)due to the treatment.These results were consistent with the previous report (Wallace et al., 1994;Hristov et al., 1999;Lila et al., 2003).Supplementation with sarsaponin linearly decreased the ruminal ammonia concentration.Reductions in the ruminal ammonia with saponin have been observed in vitro (Grobner et al., 1982;Wallace et al., 1994;Lila et al., 2003) and in vivo (Hussain and Cheeke, 1995).The in vivo responses have been varied, and are apparently related to the dosing levels and the time of sampling.The effects of sarsaponin on ruminal ammonia likely result from decreased bacterial lysis (as a consequence of inhibited protozoal growth) and, to a lesser extent, from an inhibited deaminative activity and direct binding of ammonia in the rumen (supported by the significantly lower concentration of ammonia 2 h after sarsaponin treatment).
The total VFA was increased (quadratic effect; p<0.05) at 2 and 5 h after dosing.The molar proportion of acetate was decreased (p<0.05) at all sampling times.The molar proportion of propionate (p<0.01) and butyrate (p<0.05) were increased at 2 and 5 h after dosing.Consequently, the acetate:propionate ratio was decreased (p<0.05).The test compound had no effect on iso-butyrate, valerate and iso-  (Grobner et al., 1982;Lila et al., 2003) and in vivo (Valdez et al., 1986).The decrease in the molar proportion of acetate and the increase in butyrate were consistent with the results of the in vivo study (Christopher and Neal, 1987).The blood biochemical measurements after sarsaponin dosing are shown in Table 3.The post-feeding glucose concentration increased (p<0.05)following the treatment.This may be due to the increased gluconeogenesis from propionate, since the ruminal propionate was increased in this study.In contrast, urea-N was decreased (p<0.05),reflecting the decreased ruminal ammonia concentration.These results were consistent with fumaric acid (Bayaru et al., 2001) and cyclodextrin-diallyl maleate (Lila et al., 2004a) reported previously from our laboratory, but dissimilar to the results of Hristov et al. (1999).
Feed intake, gas production, digestibility coefficients, N-metabolism and bacterial counts are shown in Table 4. Feeding of sarsaponin to the steer had no effects on DM intake; however, the digestibility of DM and NDF were decreased (quadratic effect; p<0.05) and CP was unchanged.The effects of sarsaponin on fiber digestibility have been varied.Goetsch and Owens (1985) observed that cattle fed 44 ppm of yucca saponin had an apparent increased digestibility of OM, but a depressed ruminal degradability of ADF.Hristov et al. (1999) reported that the extent of ruminal degradability of DM was not affected by saponin treatment, although the rate of degradation of insoluble DM was increased.Current data also showed that sarsaponin decreased the numbers of cellulolytic bacteria (p<0.05).Similar results were observed with monensin, which also inhibited the cellulolytic bacteria (Chen and Wolin, 1979;Helaszek and White, 1991).
The daily methane production (L/d) and rate of production (L/d.kg-DMI)were 148-182 and 26-32, respectively, throughout the experiment.This is in good agreement with the findings of Shibata et al. (1992), who fed animals at various proportions with hay plus concentrate.Approximately 12.7% of methane was inhibited (p<0.05) and carbon dioxide production was unchanged as a result of the treatment.In the in vitro experiment (Lila et al., 2003), we observed that sarsaponin  reduced the methane and hydrogen concentration in the ruminal gases, which supported the present results, although we did not measure hydrogen concentration in this experiment.Takahashi et al. (2000) reported that Yucca shidigera extract appears to be a promising agent to control rumen methanogenesis in vitro.In the present study, the inhibition of methane production by sarsaponin remained for the 23 d of treatment; therefore, further research is necessary as to whether ruminal adaptation to sarsaponin has occurred or not.The main reason for the methane suppressing effects of sarsaponin may be due to the inhibition of H 2 -producing bacteria such as cellulolytic bacteria (Wang et al., 2000), as well as other bacteria that use pyruvate-ferredoxin oxidoreductase to metabolize pyruvate to acetyl-SCoA.Rumen protozoa also provide hydrogen as a substrate for methanogens (Stumm and Zwart, 1986;Ushida et al., 1997) and a decreased number of rumen protozoa caused by saponin partially contributed to reducing the methane production in this study.Nitrogen retention was increased (p<0.05) by the treatment due to the decrease in urinary N loss.
In conclusion, the present in vivo studies suggest that sarsaponin is a potent inhibitor of rumen methanogenesis because it was effective at a low concentration and had no influence on feed intake.However, further research is necessary to investigate the effect on fiber digestion as well as the ruminal adaptation to sarsaponin.

IMPLICATIONS
Lately, sarsaponin is commercially used to remove the ammonia odor from animal excreta and to improve ruminal fermentation.Furthermore, sarsaponin inhibited methane in the present study.Therefore, it may be possible to inhibit methane production with a combination of direct fed microbials (probiotics) or an exogenous fibrolytic enzyme plus sarsaponin to stimulate digestibility without a detrimental effect on the ruminants.

Table 1 .
Composition of the experimental diet

Table 2 .
Effect of sarsaponin on pH, protozoal number, ammonia-N and volatile fatty acid (VFA) in the rumen fluid of steers

Table 3 .
Effect of sarsaponin on concentration of glucose and urea-N in blood plasma of steers

Table 4 .
Effect of sarsaponin on methane, feed digestibility, nitrogen metabolism, and ruminal total viable counts and cellulolytic a Dry matter intake. 1 NS: Not significant; * p<0.05.L = linear effect due to dose of sarsaponin, Q = quadratic effect due to dose of sarsaponin.