A study of in vitro and in vivo greenhouse gas emissions, digestion, rumen environment and nitrogen retention of growing crossbred cattle supplemented by Catfish oil

N V Thu and N T K Dong¹

College of Agriculture, Can Tho University, Vietnam
nvthu@ctu.edu.vn
¹ College of Applied Biology, Tay Do University, Vietnam

Abstract

Two experiments was implemented to determine nutrient digestibility, rumen environment, nitrogen retention and in vitro and in vivo methane and carbon dioxide production of Lai Sind cattle effected by catfish oil (CFO) supplementation. In the first experiment, it was a complete randomized design with 6 treatments and 3 replications. The treatments were 0, 1, 2, 3, 4, 5 % of CFO in the total substrate (DM basis) by using a glass syringe system and Para grass (Brachiara mutica) used as the main substrate. In the second experiment, it was a 4x4 Latin square design with 4 growing crossbred cattle (Red Sindhi x local cattle) and 4 treatments. The treatments were the supplementation of CFO at a level of 0, 1, 2 and 3% corresponding to the CFO0, CFO1, CFO2 and CFO3 treatments. In all the treatments rice straw was fed ad libitum, while concentrate feed was fed at a level of 1 kg per 150 kg LW. The results showed that the in vitro CH₄ and CO₂ production from 0-48 h were significantly different (P<0.05) among the treatments and they were gradually abated (P<0.05) when increasing CFO levels from 0 to 5%. In Exp 2 the nutrient intakes and digestion of cattle supplemented with different levels of catfish oil were not significantly different (P>0.05) among the treatments and rumen parameters and nitrogen retention of cattle were similar (P>0.05) in different treatments, however, the rumen N-NH₃ and VFAs concentrations were higher at 3 h after feeding as compared to those before feeding. The CH₄ production (L/kg DMI) had a trend of gradual reduction from CFO0, CFO1, CFO2 and CFO3 treatments, however this (L/kg DDM) gradually decreased among the treatments (P<0.05) with the values of 51.3, 50.1, 49.8 and 46.1, respectively, and the linear relationship between the CH₄ emissions and CFO supplementation being y= 51.7-1.59x (R²=0.836). It was concluded that a consistent reduction of CH₄ production (L/kg DDM) and no adverse effect on nutrient digestibility and rumen environment was found when increasing the CFO from 0 to 3% in the in vitro and in vivo experiments of the crossbred cattle.

Key words: climate change, environment, feedstuffs, lipid, nutrition, ruminants

Introduction

In the World ruminant production is increasing rapidly for providing good quality meat, milk, and their products to the large population. Therefore greenhouse gases (GHGs) production is also rising proportionally that contribute negatively to climate change (Islam and Lee, 2019). There are many methods of GHGs mitigation by the rumen microbes using various techniques have been investigated extensively with varying degree of success, however the reduction of GHGs and improvement of ruminant performance and safe products should be considered. Fat could reduce CH₄ emissions from ruminants because the unsaturated fatty acids act as an H₂ sink within the rumen through dehydrogenation and reduction occurs with the highest rate (Boadi et al 2004). It was also reported that supplementation of fat often reduces carbohydrate fermentation due to the toxic effects of fat on cellulolytic bacteria and protozoa, whereas starch fermentation was not affected (Grainger et al 2011). In Vietnam beef cattle population was 5,964,000 (2019) with an increase of 2.40% compared to that in 2018 (DLH, 2020) and the crossbred cattle (Red Sindhi x local cattle) is an important breed for beef production in many provinces. Production of Catfish (Pangasius hypophthalmus) was about 5,400 ha and 1.42 million ton in 2018 (Vasep, 2019) in the Mekong delta. As a result the byproduct as Catfish oil is available with a big amount for use, while it takes possession from 11.0 to 14.0% with high unsaturated fatty acids (80.0%) and metabolizable energy (27.0 MJ/kg DM). Rice production in the Mekong delta is the main product for domestic consumption and exportation. Beef cattle production is traditionally raised by natural grasses and rice straw for income of the producers in the Mekong delta provinces of Vietnam. Thus the rational rice straw utilization in diets for improving beef cattle performance has a significant importance. This study aimed to evaluate in vitro and in vivo CH ₄ and CO₂ production, nutrient intake and digestibility and rumen parameters of growing cattle fed rice straw by fish oil supplementation for the further research and application in term of effective utilization of the fish oil and GHGs mitigation for beef production.

Materials and method

This study included two experiments, the first experiment was an in vitro gas experiment, which was carried out at the Laboratory E205 of College of Agriculture of Can the University (CTU) and the second one was implemented at the experimental farm of the JIRCAS-CTU project, College of Rural Development of the CTU. The Catfish oil (CFO) used in both two experiments was bought at the Catfish oil Product for Animal Feeds Company in Dong Thap Province. It was collected from the extraction from raw Catfish oil to be used as the animal supplement (Photo 1).

Photo 1. Catfish oil used in the experiment

Experiment 1

Experimental design and materials

The experiment 1 was arranged in a complete randomized design with 6 treatments and 3 replications. The treatments were 0, 1, 2, 3, 4 and 5 % of the CFO in the total substrate (DM basis). The substrate used in the experiment was the Para grass (Brachiaria mutica), which has been popularly used for feeding ruminants in the Mekong delta of Vietnam (Doan Huu Luc et al 1996; Nguyen Van Thu and Preston, 1999 and Vo Duy Thanh et al 2012). It was cut into small pieces, about 1 cm of length and then dried at 65^°C during 24 h, then ground through 1mm sieve. Representative samples were put into the incubation 50 ml-syringes, which were added buffer solution and cattle rumen fluid, prior to filling each bottle with carbon dioxide following the method described by Menke et al. (1979). The rumen fluid used in the Exp was collected via an esophagus tube in the morning before feeding from the 2 growing crossbred cattle (Red Sindhi x local cattle) fed by Para grass.

Measurements taken

The incubation was for 72 h with measurement of gas, methane and carbon dioxide being recorded at 6, 12, 24, 36, 48 and 72 h. The CH ₄ and CO₂ percentage of the gas was measured by passing the gas samples through the Biogas analyzer (BM2K2 – E000, UK). Unfermented solids at 24, 48 and 72 h were determined by filtering two layers of cloth and dried at 105^oC for 24 h and ashed at 550 ^oC for 5 hrs to measure DMD and OMD, respectively.

Experiment 2

Experimental design and feeding

The experiment was arranged in a 4x4 Latin square design with the growing crossbred cattle (Red Sindhi x local cattle) of 192 ± 8.24 kg. Four treatments were the rice straw and concentrate as control, rice straw, concentrate + 1% Catfish oil, rice straw, concentrate + 2 % Catfish oil and rice straw, concentrate + 3% Catfish oil (based on DM intake) corresponding to the CFO0, CFO1, CFO2 and CFO3 treatments. In all the treatments the rice straw was fed ad libitum, while concentrate (16% CP and 3000 kcal ME/kg DM) was at a level of 1 kg per 150 kg LW. Feeds were offered twice a day at 7:00 AM and 2:00 PM with the concentrate fed in advance then the rice straw offered. The fish oil was mixed with the concentrate before feeding and the mixture was assured to completely be consumed by the animals.

Measurements taken

The experimental period was 14 days with 7 days for adaptation and then 7 days for sampling. The measurements of GHG emissions, nutrient digestibility, rumen parameters and N balance werere done over the last 7 days. During the 7 days collection period, feeds offered and refused, feces and urine will be collected daily, and the rumen liquid was collected via an esophagus tube before and 3 hours post feeding, weighed and pooled weekly for analysis. Rumen NH₃-N concentration was determined by the method of Kjeldahl and the VFA concentration determination was carried out through GC assay as described by Pirondini (2012). Apparent digestibility coefficients for DM, OM, CP, NDF, ADF and nitrogen retention were determined by the method described of Japan Livestock Technology Association (2000). Nitrogen retention was also calculated by measurements of the N intakes, in feces and urine. CH₄ and CO₂ production was measured over a 24 h period with three consecutive days while the cattle heads were in ventilated hood following the method described by Bhatta et al (2007). Concentrations of CH₄ and CO₂ in chamber were automatically recorded during the measurement period by using Infrared Gas Analyzer, Model IR200, Style S3; YOKOGAWA, Japan (Sakai 2016).

Chemical analysis

In both experiments the samples of feeds, refusals and feces were analyzed for DM (dry matter), OM (organic matter), CP (crude protein), neutral detergent fiber (NDF) and Ash. DM, OM, Ash and nitrogen (N) were analyzed according to the standard methods of AOAC (2000) and NDF was determined by the methods of Van Soest et al (1991).

Statistical analysis

The data of two experiments were subjected to an analysis of variance (ANOVA) using the Linear Model procedure (GLM) following the Complete randomized and 4x4 Latin square design of Minitab Reference Manual Release16.0 (Minitab, 2016). When the F test is significant (p<0.05), Tukey’s test for paired comparisons was used.

Results and discussion

Experiment 1

The main substrate used for this in vitro study was Para grass (Brachiaria mutica), a natural grass, which is popularly used for feeding cattle in the Mekong delta provinces of Vietnam for both cut and carried system or grazing. It contained (% in DM) 16.2, 90.6, 11.1, 3.55, 61.9, 35.4 and 9.38 for DM, OM, CP, EE, NDF, ADF and ash, respectively.

The in vitro gas production of the treatments over time is in Figure 1.

Figure 1. In vitro gas production (ml) of different CFO levels from 6 to 72 h

In general, the gas volume of treatments fast produced from 6 to 36h, then it did slowly from 36 to 72 h and was significantly different (P<0.05) among the treatments. Particularly at 72 h it was significantly lower (P<0.05) for the CFO3, CFO4 and CFO5 treatment.

In Table 1 the In vitro dry matter (DMD) and CH₄ and CO₂ production at 72 h were presented.

Figure 2. The relationship between in vitro CH 4 production (ml/gDDM) and CFO (%) supplementation at 72h in Exp 1

It was found that the CH₄ production at 6, 12 and 24 h was not significantly different (P>0.05) among the treatments, while the CO₂ production was significantly different (P<0.05) with the lowest values for the CFO5 treatment. The CH4 and CO₂ production at 48 was significantly different (P<0.05) among the treatments. At 72 h the in vitro DMD was significantly different (P<0.05) among the treatments with the higher values for the CFO supplemented treatments (Table 1). While there were significantly gradual reductions of accumulated CH₄ (ml, ml/g DDM) and CO₂ (ml/g DDM) when increasing CFO levels from the CFO0 to the CFO5 treatment with the linear relationship according to the function y=-2.34x + 64.5 with R²=0.925 (Fig. 2) for the CH₄ (ml/g DDM). Patra and Yu (2013) in an in vitro study concluded that fish oil decreased methane quadratically and digestibility linearly with increasing doses. Similarly, Sondakh et al (2017) found that the addition of Cakalang fish oil from 0 to 7.5% gradually reduced in vitro methane gas production (P<0.05) and fatty acid profile without any effects on total and partial VFA concentrations. It was also recommended to use the fish oil at 5% as unsaturated fatty acid source in feed composing of Napier grass and concentrate at 60:40 ratio.

Experiment 2

The chemical composition of feeds used in Exp 2 was presented in Table 2.

The chemical composition of rice straw fed cattle in the experiment was similar to that reported by Rahman et al (2009) being 3.20 % CP, 67.4 % NDF and 45.2 %NDF (DM basis). The chemical composition of ingredients for concentrate was also showed in Table 2.

The nutrient intakes and digestible nutrients of cattle in the experiment were showed in Table 3.

In Table 3 the intake and digestion of DM, OM, CP, NDF and ADF of cattle supplemented different levels of catfish oil were not significantly different (P>0.05) among the treatments. The results agreed with those reported by Beauchemin et al (2007), who compared animal fat (tallow) and sunflower oil (about 48 percent higher unsaturated FA concentration in the diet); both supplemented at 3.4 percent of dietary DM, and reported no effect on DM and NDF digestibility, feed intake and average daily weight gain in cattle.

Rumen pH, N-NH₃ and VFAs concentration before and 3 hrs after feeding of cattle in Exp 2 were presented in Table 4.

The rumen pH values were slightly lower for cattle after feeding, while the N-NH₃ and total VFAs concentration were higher for cattle after feeding. The pH values, N-NH₃ and total VFAs concentration were not significantly different (P>0.05) among the treatments. After 3 h feeding the rumen propionate concentrations (mM) of cattle were 16.6, 18.2, 19.7 and 20.8 for the CFO0, CFO1, CFO2 and CFO3, however they were not significantly different (P>0.05). Patra and Yu (2013) in a 3×2 factorial design for the in vitro study of fish oil and coconut oil with three concentrations (0, 3.1, and 6.2 mL/L medium) indicated that there was no significant difference of their pH values and total VFA concentrations compared to those of the control. The results of pH values and total VFA in present study also agreed with those found by Pirondini et al. (2012) in a study to evaluate the effects of diets with different starch concentrations and fish oil supplementation on in vivo total-tract nutrient digestibility, N balance, and methane (CH₄) emissions in lactating dairy cows with a 4×4 Latin square design. Particularly propionic acid (HPr) concentration had a tendency of increase from the CFO0 to CFO3 (Fig 3) and there was a close linear relationship between HPr and CFO supplementation with y=1.41x+16.71 (R²=0.993).

Figure 3. The impact of dietary CFO (%) on rumen propionic acid (HPr, mM)

The results in Table 5 indicated that there was no effect of CFO supplementation of nitrogen intake and retention of cattle among different treatments (Table 5). In this study it was found that CH ₄ production (L/animal/day) of cattle was gradually reduced but not significantly different (P>0.05) and was 153, 148, 147, and 136 for the CFO0, CFO1, CFO3 and CFO4, respectively. The CH₄ production (L/cattle/d) was similar to that stated by Chuntrakort et al . (2013) being about 135.4 of Thai cattle with average live weight of 290 kg. CH₄ and CO₂ production (L/kg LW and L/kg DMI) had a tendency of reduction when increasing the CFO levels in the treatments, however, they were not significantly different (P>0.05). However, there was a linear relationship between methane production (L/kg DMI) reduced when increasing the Catfish oil in the diets of cattle with R² = 0.892.

The CH₄ production (L/kg DMI) in the present study was similar to that reported by Vu Chi Cuong (2010) being 30.2 L/kg DMI and Terada (2001) being 30.0 L/kg DMI. The CH₄ production (L/kg DDM) was significantly different (P<0.05) among the treatments with the lowest value for the CFO3 treatment (46.1). There was also a linear relationship between CH4 emissions (L/kg DDM) and CFO (%) supplemetation with the regression equation being y=51.7-1.59 x (R²=0.836). In a review of fat effects on enteric CH₄ production (Martin et al 2010), compared a total of 67 in vivo diets with beef, sheep and dairy cattle, reporting an average of 3.8% (g/kg DMI) less enteric CH₄ with each 1% addition of fat (Singh, 2010). Similarly, Beauchemin et al (2007) stated that methane production of ruminants was reduced by about 12 percent by both lipid sources of animal fat and sunflower. Roger (2010) also reported that including 2% fish oil in the diet of cattle reduced flatulence, apparently due to the omega 3 fatty acids in the oil, and the technique cut methane output of three cows by 21%. Fat supplementation is considered as a dietary option for mitigating enteric methane emissions (Beauchemin et al 2008). Besides this it also improves energy (Coppock and Wilks 1991) in animal diets and increase concentrations of n-3 PUFA and n-3 conjugated linoleic acids (CLA) in milk and meat, depending on the fat source, which have beneficial effects on human health (Lock and Bauman 2004). But when we feed the animal with more than 10% of fat or lipid, it has a negative effect on digestion and feed intake of animals. Traditionally, fat supplementation to the diet has been used to enhance the dietary energy content to meet the energy demand for high-producing dairy cows. However, recently, fat has been used for CH₄ mitigation. If the energy supplementation in a ruminant’s diet is changed from carbohydrate to fat, then less fermentation and CH ₄ production will occur (Haque 2018).

Figure 4. Relationship between Methane production and HPr in the experiment

In the present study it was also indicated that the increase of HPr (Fig 4) reduced methane production following the function y=-0.934x²+37.8x-884 (R²=0.998). Similarly in in vitro and in vivo experiment, Lana et al. (1998), Christophersen et al (2008) and Hristov et al (2013) reported that increasing the rumen HPr production would reduce methane production.

Conclusion

• There was a consistent reduction of CH₄ production (L/kgDDM) investigated in in vitro and in vivo experiments, when supplementing the CFO in the experimental Para grass substrate and diets, respectively.
  
  
• No adverse effects on nutrient intakes and digestibilities and rumen parameters of cattle were found in the present study.

Acknowledgement

This research is funded in part by the Can Tho University Improvement Project VN14-P6, supported by a Japanese ODA loan. The Authors also thank to the JIRCAS project and Dept. of Animal Sciences of College of Agriculture, Can Tho University for facilitating the equipments use and laboratory works.

References

AOAC 2000 Official methods of Analysis of AOAC International. 17^th edition. AOAC International. Gaithersburg, Maryland, USA. Vol 1, Chapter 5, pp. 5-15.

Beauchemin K A, Kreuzer M, O’Mara F and McAllister T A 2008 Nutritional management for enteric methane abatement: A review. Aust. J. Exp. Agric. 48:21–27.

Beauchemin K A, McGinn S M and Petit H V 2007 Methane abatement strategies for cattle: Lipid supplementation of diets. Can. J. Anim. Sci., 87, pp.431–440.

Bhatta R, Enishi O and Kurihara M 2007 Measurement of methane production from ruminants. Asian-Australasian Journal of Animal Sciences, 20(8), pp. 1305-1318.

Boadi D A, Wittenberg K M, Scott S L, Burton D, Buckley K and Small J A 2004 Effect of low and high forage diet on enteric and manure pack greenhouse gas emissions from a feedlot. Can J Anim Sci. 84:445-53.

Christophersen C T, Wright A D G and Vercoe P E 2008 In vitro methane emission and acetate:propionate ratio are decreased when artificial stimulation of the rumen wall is combined with increasing grain diets in sheep. Journal of Animal Science, Volume 86, Issue 2, 384–389.

Chuntrakort P, Otsuka M, Hayashi K, Takenaka A, Udchachon S and Sommart K 2013 Effect of dietary whole cottonseed, sunflower seed, and coconut kernel on methane production in Thai native cattle and Brahman crossbred beef cattle. JIRCAS Working report, 79, pp. 51-54.

Coppock C E, and Wilks D L 1991 Supplemental fat in high energy rations for lactating cows: Effects on intake, digestion, milk yield, and composition. J. Anim. Sci. 69:3826–3837.

Department of livestock Husbandry 2020 Animal production situation in Vietnam. http://nha channuoi.vn /tinh-hinh-chan-nuoi-nam-2019/

Doan Huu Luc, Nguyen Van Thu and Preston 1997 A study of feed degradability and rumen environment of swamp buffaloes. Livestock Research for Rural Development 9 (3) 1997 . http://www.lrrd.org/lrrd21/7/luc21111.htm.

Grainger C and Beauchemin K A 2011 Can enteric methane emissions from ruminants be lowered without lowering their production? Anim Feed Sci Technol., 166-167:308-20

Haque Md N 2018 Dietary manipulation: a sustainable way to mitigate methane emissions from ruminants. Journal of Animal Science and Technology 60:15. https://doi.org/10.1186/s40781-018-0175-7

Hristov A N, Oh J, Lee C, Meinen R, Montes F, Ott T, Firkins J, Rotz A, Dell C, Adesogan A, Yang W, Tricarico J, Kebreab F, Waghorn G, Dijkstra J and Oosting S 2013 Mitigation of greenhouse gas emissions in livestock production – A review of technical options for non-CO2 emissions. In: Gerber, P.J., Henderson, B. and Makkar, H.P.S. (eds). FAO Animal Production and Health Paper No. 177. FAO, Rome, Italy.

Islam M and Sang-Suk L 2019 Advanced estimation and mitigation strategies: a cumulative approach to enteric methane abatement from ruminants. J. Anim Sci. Technol. 61(3):122-137.

Japan Livestock Technology Association (JLTA) 2000 Technical Manual for Feed Analysis, pp. 2-19.

Lana RP, Russell J B and Van Amburgh M E 1998 The role of pH in regulating ruminal methane andammonia production. J. Anim. Sci., 76, 2190–2196.

Lock A L and Bauman D E 2004 Modifying milk fat composition of dairy cows to enhance fatty acids beneficial to human health. Lipids 39:1197–1206.

Martin C, Morgavi D P and Moreau D 2010 Methane mitigation in ruminants: from microbe to the farm scale. Animal, 4, pp. 351-365.

Menke K H, Raab L, Salewski A, Steingass H, Fritz D and Schneider W 1979 The estimation of the digestibility and metabolizable energy content of ruminant feeding stuffs from the gas production when they are incubated with rumen liquor in vitro. J. agric. Sci., 93: 217-222

Minitab 2016 Minitab reference manual release 16.1.0 Minitab Inc.

Nguyen Van Thu and Preston T R 1999 Rumen environment and feed degradability in swamp buffaloes fed different supplements. Livestock Research for Rural Development 11 (3) 1999. http://www.lrrd.org/lrrd11/3/thu 113.htm

Patra A K and Yu Z 2013 Effects of coconut and fish oils on ruminal methanogenesis, fermentation, and abundance and diversity of microbial populations in vitro. J. Dairy Sci. http://dx.doi.org/ 10.3168/jds.2012-6159. 96: 1782–1792.

Pirondini M, Malagutti L, Colombini S, Amodeo P and Crovetto G M 2012 Methane yield from dry and lactating cows diets in the Po Plain (Italy) using an in vitro gas production technique. Ital. J. Anim. Sci. 11:330–335

Rahman M M, Akbar M A, Islam K M S, Khaleduzzaman A B M and Bostami A B M R 2009 Nutrient digestibility and growth rate of bull calves fed rice straw treated with wood ash extract. Anim. Sci., 38(1&2), pp. 42 – 52.

Rogers S 2010 Fish Oil Could Control Methane-Packed Cow Farts. Earth first. http://earthfirst.com/fish-oil-could-control-methane-packed-cow-farts/

Sakai T, Tran Kim Chi, Nguyen Van Thu, Suzuki T, Hayshi K and Higuchi K 2016 Ventilated Hood System for Measurements of Enteric Methane Emissions in Vietnam. Working Paper · March 2016. DOI: 10.13140/RG.2.2.34692.9408. https://www.researchgate.net/publication/317002110

Singh B 2010 Some nutritional strategies for mitigation of methane emissions. In: International conference on “Physiological capacity building in livestock under changing climate scenario”. Physiology and Climatology division, Indian Veterinary Research Institute, Izatnagar, 243122, Uttar Pradesh, India, November 11-13, 2010. pp. 142- 158.

Sondakh E H B, Waani M R and Kalele J A D 2017 Changes In Vitro Methane Production and Fatty Acid Profiles in Response to Cakalang Fish Oil Supplementation. Media Peternakan. DOI: https://doi.org/10.5398/medpet.2017.40.3.188. 40(3):188-193.

Terada F 2001 Global warming and agriculture animal in Japan. In proc. Of 1^st Internal Conference GGAA2001. Nov. 7-11. Obihiro, Japan, pp. 21-26, 2001.

Van Soest P J, Robertson J B and Lewis B A 1991 Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition J. Dairy Sci. 74, pp. 3583 – 3598.

Vasep 2019 A review of Catfish production in Vietnam (Vietnamese). http://vasep.com.vn/Tin-Tuc/1018_56184/Tong-quan-nganh-ca-tra.htm.

Vo Duy Thanh, Nguyen Van Thu and Preston T R 2012 Effect of potassium nitrate or urea as NPN source and levels of Mangosteen peel on in vitro gas and methane production using molasses, Operculina turpethum and Brachiaria mutica as substrate. Livestock Research for Rural Development 24 (4) 2012. http://www.lrrd.org/lrrd24/4/thanh 24063.htm

VVu Chi Cuong 2013 A direct estimation of CO₂ and CH₄ emissions by dairy heifers 75% HF (Vietnamese). Journal of Animal Science and Technology. ISSN: 1859-0802. National Institute of Animal Sciences (NIAS), 31, 74-79, 2013.