Abstract: Background and Objective: Dietary manipulation can decrease rumen CH4 production by competing for hydrogen utilization or inhibiting methanogenesis. The combination of ingredients, such as sunflower oil and nitrate may reduce CH4 production and increasing efficiency of N utilization in meat goats fed rice straw. This study was aimed to study the interaction between nitrate and sunflower oil on feed intake, rumen production and microbial population in goats. Materials and Methods: Eight male rumen-fistulated 50% Anglo-nubian and 50%, Thai native goats with an initial body weight of 28.8±5 kg (Mean±SD) were used in this study. Goats were randomly assigned according to a 2×2 factorial arrangement in a 4×4 Latin square design to investigate the effect of the potassium nitrate (2 and 3% KNO3 concentrate) and sunflower oil (3 and 6% Sunflower oil concentrate) levels on feed intake, rumen production and microbial population. Results: There was no interaction between the levels of potassium nitrate (KNO3) and sunflower oil on any parameters. Voluntary feed intake, nutrient digestibility and nitrogen utilization were not influenced by the SFO and KNO3 levels. An increased SFO level significantly increased (p<0.05) NH3-N, propionate and CH4 production, but an increased SFO level significantly decreased (p<0.05) the copies of R. albus, P. bryantii and P. ruminicola per millilitre of rumen fluid. An increased KNO3 level significantly increased (p<0.05) propionate in the rumen but significantly decreased (p<0.05) the C3: C4 ratio and CH4 production. Copies of Archaea mcrA were significantly increased (p<0.05) with increasing KNO3 levels. Conclusion: This study discovered the feeding the combination between nitrate and sunflower oil can be beneficial to reduce CH4 production by goats fed rice straw. So that this study would be pointed out that ruminant fed low quality roughages can be improved by combining nitrate and sunflower oil.
INTRODUCTION
Methane (CH4) emission from enteric fermentation of ruminants is responsible for global warming and CH4 generated by livestock contributes up to 40% of greenhouse gas (GHG) emissions1. The CH4 is also a key factor that indicates inefficient energy utilization. Thus, there have been many attempts to decrease CH4 production from ruminants, including CH4 vaccination, dietary manipulation and dietary management. Dietary manipulation that can decrease organic matter (OM) fermentation in the rumen may help mitigate CH4 emission from ruminants2. Counteracting methanogenesis or competing for Hydrogen (H) utilization from CH4 production may decrease CH4 emission.
Fats and oils are supplemented in ruminant diets to enhance energy density and to manipulate ruminal fermentation. The use of oils, such as soybean oil (SO), sunflower oil (SFO) and fish oil (FO), modifies the biohydrogenation process, thereby managing rumen fermentation end products. The SFO a high polyunsaturated fat, has been demonstrated to reduce CH4 production3. Non-protein nitrogen is generally used to increase nitrogen (N) in ruminant diets due to it is inexpensive and high contents of urea, nitrate (NO3), nitrite. In addition, supplemental NO3 has been reported to reduce CH4 production. The mode of action of NO3 induced decreases in CH4 production may be associated with nitrate reduction during the fermentation process4-7. NO3 is an alternative electron acceptor that can be modified to ammonia (NH3) via nitrite reduction8,9, which supplies N for microbial growth10-14.
Based on the papers above, the combination of CH4 and SFO could be potentially inhibited CH4 production and then improved rumen fermentation and product. Therefore, the objectives of current study were to study the effect of SFO and NO3 level in concentrate on feed intake, nutrient digestibility and rumen microorganisms of meat goats fed rice straw as the main roughage source.
MATERIALS AND METHODS
Animals and treatments: Eight male rumen-cannulated Anglo-Nubian×Thai native crossbred goats with a mean body weight (BW) of 28.8±5 kg were used. This study had the following two factors: factor A was SFO level (3 and 6% in concentrate diet) and factor B was potassium KNO3 (3 and 6% in concentrate diet). Treatment combinations were as follows: 3% SFO+3% KNO3 (T1); 3% SFO+6% KNO3 (T2); 6% SFO+3% KNO3 (T3) and 6% SFO+6% KNO3 (T4). The composition of dietary treatments and rice straw used was shown in Table 1. The trial had a 2×2 factorial arrangement in a 4×4 Latin square design.
| Table 1: | Ingredient and chemical composition of diet in the experiment (g kg–1 DM) |
AMineral and vitamin mix: Provided per kg of concentrate including Vitamin A: 5000 IU, Vitamin D3: 2,200 IU, Vitamin E: 15 IU, Ca: 8.5 g, P: 6 g, K: 9.5 g, Mg: 2.4 g, Na: 2.1 g, Cl: 3.4 g, S: 3.0 g, Co: 0.16 mg, Cu: 100 mg, I: 1.3 mg, Mn: 64 mg, Zn: 64 mg, Fe: 64 mg, Se: 0.45 mg, B3% SFO: 3% of sunflower oil in diets, C6% SFO: 6% of sunflower oil in diets, D2% KNO3: 2% of potassium nitrate in concentrate diets, E3% KNO3: 3% of potassium nitrate in concentrate diets |
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| Table 2: | PCR primers for real-time PCR assay |
Animals were fed twice daily at 08.00 am and 17.00 pm. All goats were fed ad libitum with rice straw water and mineral salt block. Prior to the experiment, goats were dewormed by means of Ivomectin (IVOMEC F plus, Bangkok, Thailand) and injected with vitamins A (500,000 I.U.), D3 (75,000 I.U.) and E (50 I.U.) (Biotecnocem, Dallas, USA). Goats were individually housed in pens (60×120×90 cm). The study consisted of 4 periods of 28 days each consisting of 21 days of adjustment followed by 7 days for measurements. During the last 7 days, rumen fluid and blood were collected. Body weights were measured at the last day of each period. The experimental protocol was complied with the Thailand Code for the Care and Use of Animals for Scientific Purposes with the Project code SUT 4/2558 Certificate U1-02632-2559. The experiment was conducted at Suranaree University of Technology Farm during August to December, 2016.
Data collection and sampling procedures: Rice straw and concentrate samples were collected daily during the collection period and were combined prior to analyses. Composite samples were dried at 60°C and ground (1 mm screen using Cyclotech Mill, Tecator, Sweden) and then analyzed for contents of dry matter, ether extract, ash, crude protein AOAC15, neutral detergent fiber (NDF) and acid detergent fiber (ADF) Goering and Van Soest16. The rumen fluid samples were collected at 0, 3 and 6 h post-feeding. Rumen fluid was immediately measured for pH (HANNA instrument HI 8424 microcomputer). The filtrates were divided into two portions. The first portion was used for ammonia nitrogen (NH3-N) analyses using the micro Kjeldahl method and Volatile fatty acids (VFA) was measured by gas chromatography (GC) analysis17. The second portion of rumen fluid and digesta were used for DNA extraction for use in real-time PCR using primers provided in Table 2. A blood sample was drawn from the jugular vein at the same time as rumen fluid sampling, separated by centrifugation at 5,000×g for 10 min and stored at -20°C until analysis of blood urea nitrogen (BUN) according to the method of Crocker18. The calculation of ruminal CH4 production was based on VFA proportions according to Moss et al.19 as follows:
CH4 production = 0.45 (acetate)-0.275 (propionate)+0.4 (butyrate) |
Statistical analysis: All data obtained from the experiment were subjected to ANOVA for 4×4 Latin square design with a 2×2 factorial arrangement of treatments consisting of two levels of SFO and two levels of KNO3 of the Statistical Analysis System Institute (SAS)25. Treatment means were compared by Duncan’s New Multiple Range Test (DMRT) Steel and Torrie26. Differences among means with p<0.05 were accepted as statistically significant differences. The variation is given as standard error of the least square means (SEM).
The model was:
| Yijk | = | Dependent variable |
| μ | = | Overall mean |
| ρi | = | Effect of period |
| αj | = | Effect of factor A (SFO and KNO3) |
| βk | = | Effect of factor B (level of SFO and KNO3) |
| αβjk | = | Interaction AB between SFO and KNO3 |
| εijk | = | Experimental error for ijk on the observation |
RESULTS AND DISCUSSION
Voluntary feed intake and apparent digestibility of nutrients: When expressed as g day–1, BW (%) and g kg–1 BW0.75, feed intake was not significantly different according to levels of SFO and KNO3 and there was also no interaction between the level of SFO and level of KNO3 with regard to feed intake. Li et al.12 observed no difference in feed intake in lactating goats when the diets were supplemented with safflower oil or linseed oil. Pal et al.27 reported that NO3 fed to sheep at 2% of the concentrate mixture does not reduce feed intake, which is low for exhibiting any adverse effect on intake. Other studies6 have shown that 2.6% NO3 and 4% KNO3. Nolan et al.28 in sheep diets do not affect Dry matter intake (DMI). The apparent digestibilities of dry matter (DDM), OM, ether extract (EE), NDF and ADF were not significantly different according to levels of SFO and KNO3 and there was also no interaction between the level of SFO and level of KNO3 with regard to the DDM of nutrients (Table 3). Mewara et al.29 also reported that digestibility of DM, Crud protein (CP) and fibre components is unaffected by added oil but that EE is increased by the addition of SFO to the concentrate mixture.
Nitrogen utilization: Data in Table 4 showed the N utilization of goats fed different levels of SFO and KNO3. N intake, N excretion (urine and faeces), N absorption and N retention were not significantly different according to levels of SFO and KNO3 (p>0.05) and there was also no interaction between the level of SFO and level of KNO3 with regard to N utilization (p>0.05). These results contrast those reported by Doranalli and Mutsavangwa30, who found that diets supplemented with SFO improve N retention in sheep compared to non-supplemented diets.
Ruminal fermentation end products: Ruminal pH, total volatile fatty acids (TVFAs), butyrate concentration and bacterial count were not significantly different (p>0.05) according to the level of SFO and KNO3 or their interaction presented in Table 5. Alaboudi and Jones31 observed that nitrate and nitrite quickly clear from rumen fluid within 3 h and that the molar proportion of VFAs quickly returns to the pre-nitrate levels. The increasing level of NO3 had no influence on TVFAs. The proportion of acetate and the acetate (C2)/propionate (C3) ratio tended to increase and the proportion of C3 tended to be less for the NO3 diet7-9-12. NH3-N concentration was decreased (p<0.05) with increasing levels of KNO3. There was no interaction between the level of SFO and KNO3 with regard to C2 concentration. Increasing levels of SFO decreased (p<0.05) the C3 concentration, while increasing levels of KNO3 increased (p<0.05) the C3 concentration. Increasing levels of SFO decreased (p<0.05) the C3 concentration, while increasing levels of KNO3 increased (p<0.05) the C3 concentration.
| Table 3: | Effect of dietary treatment on feed intake and digestibility of nutrients in meat goats |
3% SFO: 3% of sunflower oil in concentrate diets, 6% SFO: 6% of sunflower oil in concentrate diets, 2% KNO3: 2% of potassium nitrate in concentrate diets, 3% KNO3: 3% of potassium nitrate in concentrate diets, SFO: Effect of sunflower oil, KNO3: Effect of KNO3, SFO×KNO3: Interaction between SFO×KNO3, SEM: Standard error of the mean, ANDF: Neutral detergent fiber, BADF: Acid detergent fiber |
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| Table 4: | Effect of dietary treatment on nitrogen balance |
3% SFO: 3% of sunflower oil in concentrate diets, 6% SFO: 6% of Sunflower oil in concentrate diets, 2% KNO3: 2% of Potassium nitrate in concentrate diets, 3% KNO3: 3% of Potassium nitrate in concentrate diets, SFO: Effect of Sunflower oil, KNO3: Effect of KNO3, SFOxKNO3: Interaction between SFO x KNO3, SEM: Standard error of the mean |
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| Table 5: | Effect of dietary treatment on rumen ecology and fermentation characteristics in meat goats |
33% SFO: 3% of sunflower oil in concentrate diets, 6% SFO: 6% of sunflower oil in concentrate diets, 2% KNO3: 2% of potassium nitrate in concentrate diets, 3% KNO3: 3% of potassium nitrate in concentrate diets, SFO: Effect of Sunflower oil, KNO3: Effect of KNO3, SFO×KNO3: Interaction between SFO×KNO3, SEM: Standard error of the mean, ANH3-N: Ammonia nitrogen (mg %), BBUN: Blood urea nitrogen (mg %), CTVFA: Total Volatile fatty acids (mM L–1), DCH4 = (0.45×acetate) (0.275×propionate)+(0.4×butyrate) according to Moss et al.19 |
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Van Zijderveld et al.6 noted that there is no difference in TVFAs concentration and molar proportion of C2 and C3 in the rumen fluid. There was no interaction between the levels of SFO and KNO3 with regard to C3 concentration.
CH4 production was increased (p<0.05) with increasing levels of SFO and increasing the levels of KNO3 increased (p<0.05) CH4 production. There was no interaction between the levels of SFO and KNO3 with regard to CH4 production. McGin et al.32 demonstrated that SFO decreases protozoa populations and that the lowest protozoa population is found in a treatment containing 6% SFO and 2% KNO3 (p<0.05). There was no influence of the levels of SFO and KNO3 on protozoa population. Lipids have also been shown to inhibit methanogenesis, even in the absence of rumen protozoa, probably due to the toxicity of long chain fatty acids (LCFA) to methanogenic bacteria. These LCFA have the capacity to attract more H2 atoms and, thus, may be more able to influence the H2 balance in the rumen when large quantities are included in the diet compared to short fatty acids (SFA) Ellis et al.33, Jouany et al.34 showed that utilization of polyunsaturated fatty acids (PUFA) may decrease rumen methanogens.
Microbial population: There was no interaction between the level of SFO and level of KNO3 with regard to real-time PCR parameters. Total fungi decreased (p<0.05) with increasing levels of KNO3 and SFO did not affect total fungi as shown in Table 6. The level of KNO3 did not influence real-time PCR parameters. Zhou et al.35 reported that supplemental NO3 does not significantly change the population of Ruminococcus albus and Ruminococcus flavefaciens because they adapt to NO3. In contrast, NO3 has been found to reduce cellulolytic bacteria, total bacteria, R. flavefaciens, Butyrivibrio fibrisolvens and Fibrobacter sucinogenes36. In the present study, R. albus, Prevotella bryantii and Prevotella ruminicola decreased (p<0.05) with increasing levels of SFO. This finding correlated with decreasing NDF and ADF digestibilities with 6% SFO as compared to 3% SFO regardless of KNO3 level.
| Table 6: | Effect of dietary treatment on population of rumen microbial population using real-time PCR |
3% SFO: 3% of sunflower oil in concentrate diets, 6% SFO: 6% of sunflower oil in concentrate diets, 2% KNO3: 2% of potassium nitrate in concentrate diets, 3% KNO3: 3% of potassium nitrate in concentrate diets, SFO×KNO3: Interaction between SFO×KNO3, SEM: Standard error of the mean |
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It is well known that R. albus is a predominant fibre-degrading bacteria in the rumen. Moreover, polyunsaturated fatty acids have been reported to be toxic toward rumen microorganisms37. The present experiment was showed that addition of 6.0% SFO reduced total bacteria population when compared with 3.0% SFO.
CONCLUSION
It can be concluded that there was no interaction between the levels of SFO and KNO3 on any parameters. Voluntary feed intake, nutrient digestibility and nitrogen utilization were not influenced by the levels of SFO and KNO3. Increasing SFO levels significantly increased (p<0.05) NH3-N, propionate and CH4 production but significantly decreased (p<0.05) copies of R. albus, P. bryantii and P. ruminicola per milliliter of rumen fluid. Increasing levels of KNO3 significantly increased (p<0.05) propionate in the rumen but significantly decreased (p<0.05) the C3: C4 ratio and CH4 production. Copies of Archae mcrA were significantly increased (p<0.05) with increasing KNO3 levels. Thus, these findings suggested that SFO can be used as an energy source and that KNO3 can be used as a CH4 inhibitor in goat diets.
SIGNIFICANCE STATEMENT
This study discovers the feeding combination between nitrate and sunflower oil can be beneficial to reduce CH4 production by goats fed rice straw. So that the study would be pointed out that ruminant fed low quality roughages can be improved by combining nitrate and sunflower oil.
ACKNOWLEDGMENTS
The authors would like to express their sincerest gratitude and appreciation to the Thailand Research Fund (TRF) via “The Royal Golden Jubilee Ph.D. Program” and Suranaree University of Technology for providing laboratories and facilities and financially supporting this research with grant number (RU3-303-55-01).