Dairy Cow Feed Supplementation Alternatives for Diminishing Methane and Carbon Dioxide Concentration In Vitro

The objective of this study was to evaluate the effect of five different supplementations [YeastSaccharomyces cerevisiae (YS), tea leaves-Camellia sinensis (TL), Red Sorghum (RS), soybean oil (SBO) and Chinese soapberry Sapindus mukorossi (CS)] on the reduction of methane (CH4) in vitro conditions. Tests were done by adding each of the 5 supplementations (0.005 g X1010 for YS; 5 g for TL, RS and CS; and 5 mL for SBO) to the ruminal liquor (1.5 L/digester) of a cow fed Total Mixed Ration and incubated for 24 h. Results show that all the supplementations significantly decreased CH4 concentration compared to the control treatment (p<0.05). In vitro CH4 concentration was reduced from 56.75 to 11.14, 13.32, 19.88, 20.07%, respectively and 30.52% for SBO, TL, RS, CS and YS, respectively. CH4 concentration tended to be higher during the first hours of incubation in the control treatment compared to all supplementations. However, the supplementations reduced CH4 concentration during the first part of the incubation. This demonstrates that the supplementations used in this experiment are very effective to reduce CH4 concentration as early as 2 h after supplemented. Nevertheless, no significant interactions between treatments × time were found in CH4 concentration (p>0.001). CH2 concentration was significantly higher for the control treatment (1769.07 ppm) and YS (1683.41 ppm) compared to TL (1342.98 ppm), CS (1173.61 ppm), RS (1193.39 ppm) and SBO (991.25 ppm) supplementation (p<0.05). All the supplementations, but especially SBO, have the potential to diminish greenhouse gas production in vivo and may be alternatives for the mitigation of global warming.


INTRODUCTION
Global warming, caused by the increasing concentration of greenhouse gases (GHG), is one of the most significant threats facing our world today.GHG is caused by the buildup of gases that trap heat in the atmosphere.According to Milich (1999), these gases are largely anthropogenic in origin and are now at greater concentrations than at any time in the past 160,000 Yrs.
Carbon dioxide (CO 2 ) is the principal GHG, followed by methane (CH 4 ) and nitrous oxide (N 2 O).However, the global warming potential of CH 4 is 23-25 times that of CO 2 (Christophersen et al., 2008).Most of the CH 4 produced within agricultural systems comes from ruminants.CH 4 is a natural by-product of animal digestion through a process known as enteric fermentation.This fermentation leads to an inefficient use of energy from feed and causes ecological problems.The amount of CH 4 produced depends on the type of animal and the amount and kind of feed it consumes, among other variables (Kinsman et al., 1995).
The CH 4 atmospheric lifetime is relatively short (≈10yrs) compared to that of CO 2 (≈100+yrs) (Boucher et al., 2009).Thus, reducing CH 4 emissions would have a more immediate and significant impact on mitigating climate change than just reducing CO 2 emissions.For this reason, there is a worldwide renewed interest in finding sustainable feeding strategies for reducing emissions of CH 4 from ruminants (Jarvis et al., 1995).Several methods have been proposed for CH 4 mitigation, i.e., utilization of condensed tannins (Puchala et al., 2005;Ramírez-Restrepo and Barry, 2005), saponins (Lila et al., 2003 andWina et al., 2005) and oil extracted from various plants (Johnson et al., 2002;Jordan et al., 2006b).However, most of the studies are for only one supplementation at a time, which affects the possibility of comparing effectiveness among treatments.The supplementation of yeast, on the other hand, has been used to enhance nutrition and health in dairy cows, but little research has been done about its effects on reducing CH 4 .This research intends to compare different feed supplements such as oil, yeast and others containing secondary compounds (i.e., tannins, saponins) that might be used in ruminants' diets to diminish GHG.

Treatments:
Five different supplementations [LEVUCELL SC20® yeast-Saccharomyces cerevisiae (YS), Tea leaves-Camellia sinensis (TL), Red Sorghum (RS), soybean oil (SBO) and a byproduct of Chinese soapberry-Sapindus mukorossi (CS)] were added to compare their effect on the concentration of CH 4 in vitro.The amount of each supplementation was 0.005 g×10 10 for YS, 5 g for TL, RS and CS; and 5 mL for SBO.All these supplementations were compared to a control, where no supplementation was added.The amount of supplement included in every treatment was determined after a pre-trial.Food was provided to a cow with different amounts of the supplements every day.Since adding the supplements affected the palatability, the ideal dose of supplementation should be the maximum amount that does not severely compromise the cow's feed intake.

Ruminal liquor collection and preparation:
The protocol for this research was approved by the Innovation and Practical Training Center, National Pingtung University of Science and Technology.Ruminal liquor was collected from 1 cannulated Holstein cow.The cow was fed at libitum with Total Mixed Ration (TMR) (Table 1 and 2) and had access to fresh water at all times.Fresh feed was provided   twice a day at 06:30 and 18:30.Ruminal liquor was collected 3 h after feeding (09:30).The ruminal fluid was then strained through 2 layers of gauze before placing it in the digester (Fig. 1).
Equipment: A BioFlo® 110 Fermentor/Bioreactor was used in the experiment to simulate the rumen conditions.Four digesters (glass vessels) were filled with 1.5 L of ruminal liquor.The pH was set to 6.4 and was automatically controlled with sulfuric acid (H 2 SO 4 ) and sodium hydroxide (NaOH).The temperature was kept stable at 39°C with a heater blanket.The movement conditions of the rumen were simulated with a pump at 150 rpm.CO 2 was flushed in each digester for 2 sec to establish anaerobic conditions (Fig. 2).
Analysis: Thirty two replications were done, with a total of 2304 observations.Every incubation was done during a period of 24 h.CH 4 concentration (%) was measured every 2 h during 24 h of observations.CH 4 measurements were done using a VRAE multigas monitor (PGM-7840) and CO 2 concentration measurements (ppm) were done with an ALNOR Compu Flow CO 2 Meter at the 1 st , 2 nd and 5 th second of opening the valve.

Statistical analysis:
The data from the experiment was subjected to a General Lineal Model (GLM) and Analysis of Variance (ANOVA).The means were later compared for significance using Duncan's test at p<0.05 (SAS, 2000).

RESULTS
In vitro CH 4 concentration was significantly lower for all 5 supplementations when compared to the control treatment (Table 3).CH 4 concentrations were reduced by 80% for SBO, 77% for TL, 65% for RS, 64% for CS and 46% for YS (p<0.05).CH 4 concentration tended to be higher during the first hours of incubation in the control treatment.However, the supplementations reduced CH 4 concentration during the first part of the incubation.This demonstrates that the treatments used in this experiment were effective in reducing CH 4 concentration as early as within 2 h after supplementation.However, no significant interactions between treatments× time were found in CH 4 concentration (p>0.001) (Fig. 1).CO 2 production was significantly higher in the control treatment and YS supplementation compared to TL, CS, RS and SBO supplementations (p<0.05).However, SBO supplementation represented the most important decrement in CO 2 concentration (43%) compared to the control treatment (Table 3).No significant interactions between treatments×time were found in CO 2 concentration (p>0.001) (Fig. 2).

DISCUSSION
Adding oil to ruminants' diets has been shown to decrease CH 4 not only by lowering ruminal substrate fermentability, but also by providing an alternative H sink in the rumen (Johnson and Johnson, 1995).It may also be a consequence of defaunation of protozoa in the Fig. 2: Interaction between treatment and time on CO 2 concentration at 1, 2 and 5 s after opening the valve (p>0.001)rumen (Hegarty, 1999) due to the toxic effect on methanogens associated with ciliate protozoa.Another reason might be hydrogenation, since ruminant bacteria use H to saturate the unsaturated fatty acids.This leads to the use of H for propionate formation instead of acetic acid.Our results are congruent with CH 4 reductions found in previous studies.For example, McGinn et al. (2004) reported a reduction of 21% with sunflower oil supplementation, while Machmüller and Kreuzer (1999) reported a reduction of 28% with coconut oil supplementation.Similarly, two studies by Jordan et al. (2006a, b) found that refined soybean oil supplementation was able to reduce CH 4 by 40 and 18%, respectively.However, those experiments were done in vivo and with higher supplementation levels.
It is known that Camellia sinensis is a saponintannin-rich plant.Saponins are secondary compounds found in many plants.It is believed that saponins kill or damage protozoa by forming complexes with sterols in the protozoal membrane surface (Sliwinski et al., 2002 andWina et al., 2005) and change rumen fermentation patterns (Wang et al., 2000), so this may be an explanation for the reduced CH 4 concentration.Our results for tea leaf supplementation are in line with those of Hu et al. (2005), who reported a reduction of 26% in CH 4 production for in vitro conditions after supplementation of tea saponin at high doses (8 mg).
RS contains condensed tannins, which are secondary plant phenolic compounds that decrease degradation of nutrients in the rumen, which may then be degraded in the hindgut.Hindgut fermentation differs from ruminal fermentation in that there is lower CH 4 production per unit of fermented nutrients (Sliwinski et al., 2002;Min et al., 2003).Our results are in line with those of Hess et al. (2006), who reported a decrease in methanogenesis in vitro by using different plants rich in condensed tannins.
The decrease of CH 4 due to CS supplementation is related to its high saponin content.Results are similar to those of Argawal et al. (2006) who tested Sapindus mukorossi extracted with ethanol, water and methanol, reducing methanogenesis by 96, 20 and 22.7%, respectively.Our results also agree with those of Kamra et al. (2006) who reported complete inhibition of CH 4 production in vitro by supplementing ethanol extract from seed pulp.It is worth mentioning that the lower decrease of CH 4 in our experiment is due to the use of a byproduct from the soap industry instead of an extract.Thus, it could represent an important and feasible alternative for the diet of ruminants in countries where there is a huge amount of this byproduct, e.g., Taiwan.
Results for YS supplementation agree with Lynch and Martin (2002) and Mwenya et al. (2004), who found a significant decrease in CH 4 production when supplementing both live cells of Saccharomyces cerevisiae and yeast culture.Conversely, our results disagree with those of McGinn et al. (2004), where no CH 4 reduction was found when supplementing either Levucell SC or Procreatin-7 yeast to beef cattle.Therefore, there is no conclusive evidence of methanogenesis reduction when supplementing yeast, so it is important to find the variables that affect its effectiveness.Moreover, most of the research has been done in vitro, so in vivo experiments are also needed.
CO 2 is a product of anaerobic fermentation.Thus, at higher fermentation rates, higher CO 2 production is expected.In our research, both CH 4 and CO 2 concentration were higher in the control treatments.This leads to the assumption that the different supplementations used in this experiment change the pattern of fermentation.CO 2 may have captured H 2 to form propionate instead of producing CH 4, as explained by Takahashi (2006).

CONCLUSION
All feed supplementations tested in this experiment significantly decreased CH 4 and CO 2 concentration in vitro conditions.However, SBO was found to be the most efficient in reducing CH 4 and CO 2 , followed by TL (Camellia sinensis).Thus, they may represent new alternatives to lessen GHG (CH 4 and CO 2 ) emissions in vivo and could be a course of action for the dairy cattle industry to mitigate global warming.The present in vitro results indicate that plant secondary compounds seem to have the potential to be used as feed additives for rumen manipulation to reduce CH 4 and CO 2 emissions.However, longer incubation time is advised for future studies to determine if the reduction of CH 4 and CO 2 is transient.Further research may extend the studied supplements to in vivo conditions, which will further help to validate the results.

Fig. 1 :
Fig. 1: Interaction between treatment and time on CH 4 concentration at 1, 2 and 5 s after opening the valve (p>0.001) 60 c Means followed by different letters are significantly different at Duncan's multiple range test (p<0.05);YS: Yeast supplementation; TL: Tea leaf supplementation; RS: Red sorghum supplementation; SBO: Soybean oil supplementation; CS: Chinese soapberry; MSE: Mean squared error

Table 1 :
Composition of TMR fed to milking cows

Table 3 :
CH4 and CO2 concentrations for five different supplementations