Glycerol supplementation increased growth rates, decreased the acetate: propionate ratio in rumen VFA, and reduced enteric methane emissions, in cattle fattened on cassava pulp-urea, brewers’ grains and rice straw

Phanthavong Vongsamphanh, T R Preston¹ and R A Leng²

Department of Livestock and Fisheries, Ministry of Agriculture and Forestry. PO Box 6644 Vientiane, Lao PDR
vongsamphanh2015@gmail.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
² University of New England, Armidale NSW, Australia

Abstract

Thirty-two “Yellow” cattle, equal numbers of uncastrated males and females, average age 2 years and initial weight 112-170 kg, were housed in eight pens each with 4 animals. They were allocated four (planned) levels of crude glycerol (0, 4, 8 and 12 % of the diet DM) in a random block design with one group of intact males and one of females allocated to each treatment. The basal diet was ad libitum ensiled cassava pulp (with 3% urea DM basis), fresh brewers’ grain (1% of live weight as DM) and rice straw (1% of live weight as DM). The experiment lasted 120 days.

Actual levels of glycerol consumed were 0, 3.56, 7.26 and 10.8% of diet DM. Growth rate increased with a curvilinear trend as the level of glycerol in the diets was increased from 0 to 10.8% in the diet DM. At the highest level of glycerol the increase in live weight gain over the control (frpm 449 to 578g/d) was 29%. Feed conversion rate was not affected by added glycerol. Feeding glycerol resulted in a linear reduction in methane concentration in eructed gas, such that at the highest level of glycerol supplementation the methane concentration was only 30% of that recorded on the control diet without glycerol. The reduction in methane was commensurate with the ratio of acetate: propionate in rumen VFA which decreased linearly from 3.04 to 2.69 as glycerol level was increased from 0 to 10.8% in diet DM.

Keywords: feed conversion, eructed gas, fermentation, in vitro, propionate, “Yellow” cattle

Introduction

World-wide interest in the production of renewable energy has focused attention on the production of biodiesel by trans-esterification of vegetable oils, using methanol as the reacting agent (Kiss and Ignat 2012). A byproduct from the refining process is crude glycerol, which is contaminated in varying degrees with methanol and water.

Purification of the crude glycerol to the pure chemical (which is used in cosmetics, soaps and food additives) is an expensive process. However, crude glycerol can be fed to ruminants as a major energy source replacing grain in the diet (Schröder et al 1999; Donkin 2008)) without the need for purification (Omazic 2013; Omazic et al 2014). The rumen fermentation of glycerol-enriched diets shows increased molar proportion of propionate relative to acetate (Boyd et al 2013), There is also a report that a high proportion of dietary glycerol is absorbed largely from the rumen (Omazic 2013; Omazic et al 2014).

Figure 1: Pathways of glycerol transfer/metabolism in cattle (from Omazic 2013)

Propionate /glycerol are major precursors of glucose. In many ruminant production systems, low in dietary protein, glucose availability from the nutrients absorbed can limit feed efficiency particularly in late pregnancy and early lactation. In feeds based on agricultural byproducts glucose availability is often the factor that limits the efficiency of feed conversion (Kempton et al 1978; Preston and Leng 1987). As an increase in propionate relative to acetate in the rumen fermentation results in less hydrogen being produced, this in turn can be expected to lead to lower methane losses in eructed gases of ruminants. Therefore, theoretically, the feeding of glycerol to cattle should improve both growth rates and feed conversion as well as reducing enteric methane production.

Recent research in Lao PDR has shown that growth rates of local “Yellow” cattle can be increased to levels commensurate with their genetic potential by feeding them with cassava pulp (the by-product of starch extraction from cassava roots) supplemented with urea, cassava foliage and small quantities of brewers’ grains (Phanthavong et al 2014, 2016).

The cultivation of oil palms in Lao PDR is a recent activity, strongly supported by the Government, and has given rise to local production of biodiesel and with it the availability of crude “glycerol”. The aim of the present experiment was therefore to evaluate the effect of crude glycerol on production parameters and enteric methane production in local cattle fed cassava pulp, urea, brewers’ grains and rice straw, in the intensive fattening system developed by Phanthavong et al (2016).

Materials and methods

Location and duration

The experiment was carried out in Natthana Chok Farm, Xaythany District, situated some 30 km from Vientiane Capital, from 26 March to 24 July 2016.

Experimental design

Thirty-two “Yellow” cattle, equal numbers of uncastrated males and females, average age 2 years and initial weight 112-170 kg, were housed in eight pens each with 4 animals. They were injected intramuscularly with Ivomex-F to control internal and external parasites and vaccinated against Foot and Mouth Disease. The planned treatments in a random block design were levels of crude glycerol of 0, 4, 8 and 12 % of the diet DM with one group of intact males and one of females allocated to each treatment.

Feeding system

The feeding system was changed gradually from Napier grass (Pennisetum purpureum) to the experimental diets (Table 1) during a 14 day adaptation period. The fresh cassava pulp, fresh brewers’ grains and glycerol were transported to the farm every 1-2 weeks from local factories in the Vientiane area. The glycerol was the crude byproduct from the processing of oil palm and refining of the oil to biodiesel. Rice straw was collected from nearby rice fields immediately after rice harvest.

Measurements

The cattle were weighed before morning feeding at the beginning of the trial and every 14 days. Feeds offered and refused were recorded daily. After 56 days, the expired breath of the cattle was analyzed for methane and carbon dioxide using an infra-red analyzer Gasmet DX4000, Helsinki, Finland), following the procedure developed by Madsen et al (2010). The cattle were held in a bamboo pen covered with polyethylene sheet (Photo 1) for 10 minutes prior to making 10 consecutive recordings of methane and carbon dioxide concentrations at one minute intervals.

Photo 1: The plastic-covered bamboo cage used for measurement of enteric methane emissions

After 120 days, at the end of the experiment, rumen fluid samples were taken by stomach tube for measurement of pH prior to acidification with sulphuric acid for subsequent analysis of volatile fatty acids using high pressure liquid chromatography (Water model 484 UV detector; column novapak C18; column size 3.9 mm x 300 mm; mobile phase 10 mM H₂PO₄ [pH 2.5]) (Samuel et al 1997) and ammonia by Kjeldahl digestion (AOAC 1990). DM and crude protein in feeds were analysed using the methods of AOAC (1990).

Statistical analysis

The data were analysed by the general linear model option of the ANOVA program in the Minitab (2000) software (version 16.0). In the model the sources of variation were: treatments and error. Live weight gain was estimated from the linear regression of live weight (Y) against days in the experiment (X). Trends in responses (Y) of live weight gain, feed intake, feed conversion, acetate: propionate ratios and methane: carbon dioxide ratios in mixed air-eructed gas, were estimated by quadratic equations relating response (Y) to % glycerol in the diet DM (X).

Results

Feed intake, live weight gain and feed conversion

Cassava pulp accounted for 60% of the DM intake on the control diet, falling to 50% as the level of glycerol was increased to 11% of the diet DM (Figure 1); the remainder of the diet was divided almost equally between brewers’ grains and rice straw.

There was a tendency (p=0.10) for DM intake to be higher for the treatments with added glycerol (Table 2). Growth rate increased with a curvilinear trend (Table 3; Figure 2) as the level of glycerol in the diets was increased. At the highest level of glycerol (10.8% in diet DM), the increase in live weight gain over the control was 29%.

Figure 2: Adding glycerol to the diet increased the rate of live weight gain in both male and female cattle	Figure 3: Trends in DM feed conversion from adding glycerol to the diet

Glycerol supplementation had no effect on rumen pH (Table 4). Molar proportions of acetate decreased and those of propionate increased at the highest level of lycerol supplementation (Table 4; Figure 4). with a resulting linear decrease in the acetate: propionate ratio.  We have no explanation for the decrease in protozoa numbers with the two highest levels of glycerol. The levels of urea in blood were not affected by glycerol supplementation as all diets had similar proportions of protein-N (brewers’ grains) and NPN (urea).

There was a linear reduction in methane concentration in eructed gases, which at the highest level of glycerol supplementation was only 30% of that recorded on the control diet without glycerol (Figure 5).

Figure 4: Adding glycerol to the diet reduced the acetate:propionate ratio in rumen fluid	Figure 5: The ratio of methane:carbon dioxide in mixed expired breath and air was reduced threefold by feeding glycerol at 10.8% of the diet DM

Discussion

The most conclusive study on utilization of crude glycerol as a supplement in fattening diets of cattle was reported by Del Bianco et al (2016) in a large scale feedlot experiment, in which 3,640 Nellore bulls (initial live weight 367 kg) were fed for 100 days on diets with crude glycerol inclusion at up to 15% of the diet replacing maize grain. There was no effect of glycerol level on intake of DM, average daily gain (1.41, 1.37, 1.35 and 1.34 kg/d for 0, 5, 10 and 15% glycerol), feed conversion or carcass quality in terms of Longissimus thoracis muscle area, and back and rump fat thicknesses. The authors concluded that crude glycerol may be included in finishing beef cattle diets at levels up to 15% without impairing performance and carcass characteristics.

In our experiment, the positive curvilinear response in live weight gain, with a 29% improvement at the highest glycerol level is different from the above report, and from others in the literature. Parsons et al (2009) reported a quadratic effect of glycerol level with increases in live weight gain from 1.19 to 1.34 kg/d with 4% glycerol in diet DM, thereafter decreasing gradually to 1.03 kg/d with 16% glycerol in diet DM. The diet was steam-flaked maiza and alfalfa. A similar quadratic response was observed by Hales et al (2013) with an increase in growth rate for 5% glycerol in the diet then a decrease when glycerol level was increased to 7.5%. Most studies show no effect of glycerol level on feed conversion (eg: Hales et al 2013; Del Bianco et al 2016).

The use of cassava root pulp as the source of fermentable carbohydrate in studies with glycerol is a unique feature of the present experiment. In almost all other reported studies the rapidly fermentable carbohydrate source has been maize grain. Cassava pulp, the byproduct of cassava root processing, is similar to maize, in that the basal rapidly fermentable carbohydrate is starch, but the crude protein content is much lower (<2.5% in DM) and the particle size is much smaller. The latter could facilitate the rate of attachment of rumen micro-organisms and hence the rate of fermentation, but it is not clear how this might affect the response to increasing levels of dietary glycerol. The VFA pattern diet on the control diet (22% molar propionate) is similar to what is observed on maize based diets. The change in the fermentation pattern due to added glycerol with reducing proportions of acetate relative to propionate has been observed in other studies (Rémond et al 1993; Kijora et al 1998; Trabue et al 2007; Wang et al 2009; Boyd et al 2013; Del Bianco et al 2013; San Vito et al 2016). It is the logical response in view of the chemical composition of glycerol and its known glucogenic benefits in ruminants suffering from ketosis (Johnson 1954; De Frain et al 2004). These changes in the rumen fermentation pattern would be expected to lead to faster growth rates and better feed conversion (Preston and Leng (1987).

There have been few studies on the effects of glycerol on rumen methane production, and mainly with in vitro systems, where the results have been conflicting. Avila-Stagno et al (2014) studied additions of up to 20% glycerol in a semi-continuous culture system with brome hay and maize silage as the substrate. The acetate: propionate ratio decreased with increasing glycerol level but the production of methane per unit DM digested increased. The authors concluded that their hypothesis: “that supplementing a roughage diet with glycerol would lead to reduction in methane emissions” had not been proven. In a similar experiment but with a substrate composed principally of barley grain (Avila et al 2013), addition of glycerol (20% of the substrate) decreased the acetate: propionate ration in the VFA but the production of methane was unchanged. Lee et al (2011) reported decreases in methane in a feeding trial with goats supplemented with glycerol. The acetate: propionate ratio in the rumen VFA was decreased and it was reported that methane was also decreased. However, methane per se was not measured directly but was predicted from the molar ratios of the VFA . In view of the conflicting results of Avila-Stagno et al (2014) when the reduced ratio of acetate: propionate in VFA did not result in reduced methane, the method of estimating methane from stoichiometric balances derived from VFA proportions appears to be unreliable.

 A more conclusive study was reported by Syahniara et al (2016).These authors applied meta-analysis to 13 experiments and 42 treatments dealing with glycerol supplementation in ruminants. They reported that increasing levels of glycerol decreased molar proportion of acetate and increased molar proportion of propionate and butyrate, thus the ratio of acetate to propionate declined linearly. Methane production decreased linearly, accompanied by an increase of total gas production with increasing levels of glycerol supplementation.

Results of a recent experiment (Inthapanya et al 2017), in which rice straw was treated with glycerol and incubated anaerobically for 14d, provide further evidence of the depressing effect of glycerol on methane production.

Conclusions

• Growth rate increased with a curvilinear trend as the level of glycerol in the diets was increased from 0 to 10.8% in the diet DM. At the highest level of glycerol the increase in live weight gain over the control was 28%.
  
  
• Feed conversion rate was not affected by added glycerol.
  
  
• The ratio of acetate: propionate in rumen VFA decreased linearly from 3.04 on the control treatment to 2.69 with the highest level of glycerol. This was associated with a linear reduction in methane concentration in eructed gases, which at the highest level of glycerol supplementation was only 30% of that recorded on the control diet without glycerol.

Acknowledgements

This research was done by the senior author as part of the requirements for the PhD degree in Animal Production "Improving Livelihood and Food Security of the people in Lower Mekong Basin through Climate Change Mitigation" of Nong Lam University. The authors acknowledge support for this research from the MEKARN II project financed by Sida/MEKARN II. The authors are indebted to Mr. Vakili Vongxay, owner of the Natthanachouk farm where the experiment was carried out, for providing access to cattle, feed resources and infrastructure. The Tropical Feed Resources Research and Development Center (TROFEC) of  Khon Kaen University is acknowledged for assisting with collection of the rumen fluid samples and facilitating the laboratory analyses. The Department of Livestock and Fisheries, (DLF) and the National Agriculture and Forestry Research Institute (NAFRI) pprovided support for many of the activities in this research.

References

AOAC 1990 Official methods of analysis. Association of official Analysis (15th edition). Washington, DC, USA.

Avila-Stagno J, Chaves A V, He M L, Harstad O M and Beauchemin K A 2013 Effects of increasing concentrations of glycerol in concentrate diets on nutrient digestibility, methane emissions, growth, fatty acid profiles, and carcass traits of lambs. Journal of Animal Science 91 (2), 829-837

Avila-Stagno J , Chaves AV , Ribeiro GO Jr , Ungerfeld E M and McAllister T A 2014 Inclusion of glycerol in forage diets increases methane production in a rumen simulation technique system. British Journal of Nutrition 201111(5):829-35.

Boyd J, Bernard J K and West J W 2013 Effects of feeding different amounts of supplemental glycerol on ruminal environment and digestibility of lactating dairy cows. Journal of Dairy Science, 96:470-476 http://dx.doi.org/10.3168/jds.2012-5760

Del Bianco Benedeti P, Galoro da Silva L, Marostegan de Paula E, Shenkoru T, Marcondes M I, Monteiro H F et al 2015 Effects of Partial Replacement of Corn with Glycerin on Ruminal Fermentation in a Dual-Flow Continuous Culture System. PLoS One 10: e0143201 doi: 10.1371/journal.pone.0143201 [ PMC free article ] [ PubMed ]

Del Bianco Benedeti P, Paulino PVR, Marcondes MI, Maciel IFS, da Silva MC, Faciola AP 2016 Partial Replacement of Ground Corn with Glycerol in Beef Cattle Diets: Intake, Digestibility, Performance, and Carcass Characteristics. PLoS ONE 11(1): e0148224. http://dx.doi.org/10.1371/journal.pone.0148224

De Frain J M, Hippen A R, Kalscheur K F and Jardon PW 2004 Feeding glycerol to transition dairy cows: effects on blood metabolites and lactation performance. Journal of Dairy Science 87: 4195–4206. [ PubMed ]

Donkin S S 2008 Glycerol from biodiesel production: the new corn for dairy cattle. Revista Brasileira de Zootecnia Volume 37. http://dx.doi.org/10.1590/S1516-35982008001300032

Inthapanya Sangkhom, Preston T R and Leng R A 2017 Methane production in an in vitro rumen incubation was depressed when rice straw was treated anaerobically with glycerol. Livestock Research for Rural Development. (submitted)

Johnson R B 1954 The treatment of ketosis with glycerol and propylene glycol. Cornell Veterinarian 44(1), 6 -21.

Hales K E, Bondurant R G, Luebbe M K, Cole N A and MacDonald J C 2013 Effects of crude glycerin in steam-flaked corn-based diets fed to growing feedlot cattle. Journal Animal Science 91: 3875–3880. doi: 10.2527/jas.2012-5944 [ PubMed ]

Kempton T J, Hill M K and Leng R A 1978 The effects of varying bypass amino acid and glucose availability on lamb growth an d wool growth. In: Proceedings Australian Society of Animal Production 12:143.

Kijora C , Bergner H , Götz K P , Bartelt J , Szakács J and Sommer A 1998 Research note: investigation on the metabolism of glycerol in the rumen of bulls. Arch Tierernahr. 51(4):341-8.

Kiss A A and Ignat R M 2012 Enhanced methanol recovery and glycerol separation in biodiesel production – DWC makes it happen. Applied Energy. Volume 99: 146-153

Lee S Y, Lee S M, Cho Y B, Kam D K, Lee S C. Kim C H and Seo S 2011 Glycerol as a feed supplement for ruminants: In vitro fermentation characteristics and methane production. Animal Feed Science and Technology 166–167:269–274.

Madsen J, Bjerg B S, Hvelplund T M, Weisbjerg R and Lund P 2010 Methane and carbon dioxide ratio in excreted air for quantification of the methane production from ruminants, Livestock Science 129, 223–227

Minitab 2000 Minitab Software Release 13.2,Minitab Inc., State College Pennsylvania, USA.

Omazic W A 2013 Glycerol Supplementation in Dairy Cows and Calves. Doctoral Thesis. http://pub.epsilon.slu.se/10898/1/werneromazic_a_131115.pdf

Omazic W, Kronqvist C, Zhongyan L, Martens H and Holtenius K 2014 The fate of glycerol entering the rumen of dairy cows and sheep. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag Gm

Parsons G L, Shelor M K and Drouillard J S 2009 Performance and carcass traits of finishing heifers fed crude glycerin. Journal Animal Science 87(2):653-7.

Phanthavong V, Viengsakoun N, Sangkhom I and Preston T R 2014 Cassava pulp as livestock feed; effects of storage in an open pit. Livestock Research for Rural Development. Volume 26, Article #169. http://www.lrrd.org/lrrd26/9/phan26169.htm

Phanthavong V, Khamla S and Preston T R 2016 Fattening cattle in Lao PDR with cassava pulp. Livestock Research for Rural Development. Volume 28, Article #10. http://www.lrrd.org/lrrd28/1/phan 28010.htm l

​Preston T R and Leng R A 1987 Matching Ruminant Production Systems with Available Resources in the Tropics and Sub-Tropics. Penambuk Books, Armidale NSW. http://www.cipav.org.co/PandL/Preston_Leng.htm

Rémond B, Souday E and Jouany J P 1993 In vitro and in vivo fermentation of glycerol by rumen microbes. Animal Feed Science and Technology 41: 121–132.

Samuel M, Sagathewan S, Thomus J and Mathen G 1997 An HPLC method for estimation of volatile fatty acids of rumenfluid. Indian Journal of Animal Science  67:805–807.

San Vito E, Messana J D, Castagnino P S, Granja-Salcedo Y T and Berchielli TT 2016 Effect of crude glycerine in supplement on the intake, rumen fermentation, and microbial profile of Nellore steers grazing tropical grass. Livestock Production Science Volume 192 pp17-24

Schröder A and Südekum K H 1999 Glycerol as a by-product of biodiesel production in diets for ruminants. In: New horizons for an old crop. Proceedings 10th International Rapeseed Congress, Canberra. http:// www.regional.org.au/au/gcirc/1/241.htm

Syahniara T M, Ridlac M, Samsudind A A A B and Jayanegarac A 2016 Glycerol as an energy source for ruminants: a meta-analysis of in vitro experiments. Media Peternakan, 39(3):189-194 DOI: https://doi.org/10.5398/medpet.2016.39.3.189 Available online at http://medpet.journal.ipb.ac.id/

Trabue S, Scoggin K, Tjandrakusuma S, Rasmussen M A and Reilly P J 2007 Ruminal fermentation of propylene glycol and glycerol. J Agric Food Chem 55: 7043–7051. [ PubMed ]

Wang C, Liu Q, Huo W J, Yang W Z, Dong K H, Huang Y X, et al 2009 Effects of glycerol on rumen fermentation, urinary excretion of purine derivatives and feed digestibility in steers. Livestock Science 121: 15–20.

Received 17 November 2016; Accepted 14 January 2017; Published 1 February 2017

Go to top