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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 105oC 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.

Table 1. In vitro dry matter (DMD) and greenhouse gas production at 72 h affected by catfish oil (CFO) supplemented in Exp 1

Item

Treatment

±SEM

p

CFO0

CFO1

CFO2

CFO3

CFO4

CFO5

DMD, %

68.2d

70.7c

71.7b

72.4b

73.5a

74.1a

1.45

0.001

CH4, ml

8.99a

8.74ab

8.69ab

8.67ab

8.44bc

8.27c

0.192

0.001

CH4, ml/g DDM

57.0a

52.3ab

49.4ab

48.8a

46.4b

44.1b

2.23

0.019

CO2, ml/g DDM

287a

265ab

230ab

248ab

230ab

212b

13.9

0.028

Avg. CH4, % (0-72h)

19.1a

19.1ab

18.8ab

18.6bc

18.3c

17.7d

0.119

0.001

CFO0, CFO1, CFO2, CFO3, CFO4 and CFO5: the supplementation levels of Catfish oil of 0, 1, 2, 3, 4 and 5 % to the main substrate (DM basis). DDM: digested dry matter. DMD, DOM: dry mater and organic matter digestibility. a,b,c: Means with different letters within the same rows are different at P<0.05



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.

Table 2. The chemical composition of rice straw and ingredients of concentrate used in Exp 2

Feed

DM

OM

CP

EE

CF

NDF

ADF

Ash

Rice straw

90.8

86.6

4.60

3.23

31.8

68.6

41.2

13.4

Coconut cake meal

88.6

95.4

17.0

10.8

21.9

59.9

42.7

4.60

Soybean cake meal

90.0

94.1

31.6

2.61

5.33

31.3

14.4

5.92

Bone meal

95.6

33.0

21.5

5.51

0.31

4.73

3.93

67.0

Broken rice

86.5

98.7

7.51

1.50

0.58

4.50

1.23

1.35

Rice bran

89.7

89.6

12.8

12.8

8.45

25.9

9.44

10.4

DM: dry matter, OM: organic matter, CP: crude protein, EE: Ether extraction, CF: crude fiber, NDF: neutral detergent fiber, ADF: acid detergent fiber

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.

Table 3. Nutrient intakes and digestible nutrients of cattle supplemented catfish oil levels in Exp 2

Item

Treatment

±SEM

p

CFO0

CFO1

CFO2

CFO3

Nutrient intake, kg

DM

4.89

4.90

4.89

4.83

0.077

0.918

OM

4.44

4.46

4.44

4.41

0.072

0.972

CP

0.427

0.424

0.424

0.417

0.006

0.657

NDF

2.75

2.70

2.63

2.63

0.047

0.261

ADF

2.03

1.99

1.95

1.93

0.029

0.156

Digestible nutrients, kg

DM

2.97

2.96

2.92

2.82

0.089

0.792

OM

2.79

2.77

2.71

2.66

0.068

0.517

CP

0.28

0.30

0.29

0.28

0.007

0.410

NDF

1.83

1.75

1.69

1.76

0.05

0.360

ADF

1.24

1.18

1.14

1.17

0.043

0.525

DM: dry matter, OM: organic matter, CP: crude protein, EE: Ether extraction, CF: crude fiber, NDF: neutral detergent fiber, ADF: acid detergent fiber. CFO0, CFO1, CFO2 and CFO3: Catfish oil supplemented at 0, 1, 2 and 3 % (DM basis), respectively

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.

Table 4. Rumen pH, N-NH3 and total VFAs concentration of cattle supplemented different levels of Catfish oil

Item

Treatment

±SEM

p

CFO0

CFO1

CFO2

CFO3

- pH before feeding

6.92

6.93

6.86

6.89

0.095

0.478

- pH 3 h after feeding

6.86

6.90

6.80

6.79

0.092

0.190

- N-NH3before feeding, mg/100ml

14.2

12.3

12.6

13.65

1.66

0.399

- N-NH3 3 h after feeding, mg/100ml

14.7

15.8

15.2

15.4

2.12

0.913

- TVFAs at 0 h, mM

76.4

77.28

87.2

86.1

4.71

0.144

- TVFA at 3 h after feeding, mM

103

106

107

118

21.3

0.832

Acetic acid, mM

69.3

70.9

69.7

66.0

9.45

0.625

Propionic acid, mM

16.6

18.2

19.7

20.8

4.75

0.453

Butyric acid, mM

10.5

12.8

12.0

11.9

2.75

0.414

CFO0, CFO1, CFO2 and CFO3: Catfish oil supplemented at 0, 1, 2 and 3 % (DM basis), respectively.
TVFAs: total volatile fatty acids, LW: live weight, DMI: dry matter intake and DDM: digested fry matter.
a,b: Means with different letters within the same rows are different at P<0.05

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)


Table 5. Nitrogen intake and retention and greenhouse gas emissions of cattle supplemented different dietary levels of Catfish oil

Item

Treatment

±SEM

p

CFO0

CFO1

CFO2

CFO3

Nitrogen intake & retention

N intake, kg/ani./d

0.068

0.068

0.068

0.067

0.001

0.660

N retention, kg/ani./d

0.032

0.030

0.029

0.032

0.002

0.782

N retention kg/kgLW/d

0.036

0.038

0.039

0.034

0.003

0.727

GHG emissions

CO2, L/kgLW

7.84

7.84

7.81

7.68

0.095

0.618

CH4, L/kgLW

0.777

0.762

0.750

0.698

0.019

0.101

CO2, L/kgDMI

315

311

312

309

3.75

0.748

CH4, L/kgDMI

31.1

30.3

30

28.1

0.65

0.073

CO2, L/kgDDM

521

517

519

512

13.6

0.874

CH4, L/kgDDM

51.3a

50.7ab

49.8ab

47.1b

0.854

0.033

CFO0, CFO1, CFO2 and CFO3: Catfish oil supplemented at 0, 1, 2 and 3 % (DM basis), respectively. TVFAs: total volatile fatty acids, LW: live weight, DMI: dry matter intake and DDM: digested dry matter.
a, b Means with different letters within the same rows are different at P<0.05

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


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.


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