Abstract: Background and Objectives: Polyunsaturated fatty acids (PUFA) have human health benefits and essential oils (EOs) possess antimicrobial potential to maintain higher their concentrations in ruminant food products. The main objective of the study was to examine the dose-response effects of anise EO on the fermentation and bio-hydrogenation of n-3 PUFA using batch culture system. Materials and Methods: Six West African Dwarf goats (mean weight 45.0±1.9 kg) were used as rumen fluid donors. A mixture of good quality grass hay, goat pellets, whole ground wheat grain and fish oil were used as basal feedstock. Four treatments replicated into 6 groups were used, after incubation and fermentation 3 replicates were taken from each treatment to determine concentrations of free FA. The remaining replicates were used for concentrations of total volatile fatty acids (TVFA), NH3-N and determination of pH. Results: Gas production, TVFA and ammonia-N concentrations present in culture reduced (p<0.001) in a dose-dependent manner, except for the 150 mg L–1 that did not affect TVFA. The concentrations of n-3 PUFAs (C22: 6n-3, C20: 5n-3 and C18:3 n-3) were maintained at higher (p<0.001) levels in all other treatments than the control. Conclusion: This study concludes that the concentrations of the health beneficial n-3 PUFAs were maintained higher at all doses and the 150 mg L–1 did not affect the levels of total VFA, hence, 150 mg L–1 of anise is observed to be the minimum dose required to maintain a higher concentrations of PUFA and beneficially modify rumen fermentation if effects are repeated in vivo.
INTRODUCTION
Omega-3 long chain polyunsaturated fatty acids (Lc-n-3-PUFA) are essential components of cell membranes, existing as either free molecules or phospholips1,2. The most important members of these class of PUFA are alpha linolenic acid (C18:3 n-3), eicosapentaenoic acid (C20:5 n-3) and docosahexaenoic acid (C22:6 n-3). There is sufficient data on the importance of fat consumption on human health, longevity and general development3 and the volume of available evidence on the fundamental roles of PUFA on human health is increasing. Recent updates indicate that the Lc-n3-PUFA are key regulators in the reduction and prevention of chronic diseases in humans4,5. Therefore, various recommendations regarding daily intake of these fatty acids have been proposed. Available reports suggest that the diets of Australians and Western countries are largely lacking in Lc-n3 PUFA6. This is because seafood and other marine products, which are natural sources of Lc-n3-PUFA are traditionally not made components of most Western countries’ diets7. Hence, in order to meet the recommended daily intakes of Lc-n3-PUFA, there is a need to increase their supply in other human foods (such as milk and meat). Adipose tissues obtained from ruminants are considerably low in Lc-n3-PUFA and high in saturated fatty acids (SFA), suggesting that they are unlikely to be healthy7. This fatty acid composition of ruminant fats is a direct result of the ruminal bio-hydrogenation process of dietary non-esterified unsaturated fatty acids by rumen microbes8. Ruminal outflow of fatty acids are absorbed and incorporated into tissue lipids unchanged4. Therefore, BH generally is perceived as a metabolic reaction to the toxic effect of unsaturated fatty acids by rumen microbes9.
Recently, considerable volume of studies have indicated that the major factor regulating the FA composition of ruminant food products is nutrition10,11. Hence, emphasis has been on the utilization of novel nutritional strategies to modulate rumen fermentation and enhance the Lc-n3-PUFA concentration of ruminant food products7. The richest sources of omega-3 PUFA available to ruminants are forage, oilseeds, fish oil and algae12,13.
In the past few decades, a number of chemical feed additives such as methane inhibitors, ionophores, defaunating agents and antibiotics have been used in ruminant nutrition. However, most of these supplements are not used routinely because of toxicity problems to the host animals and microbial adaptation. Most importantly, a great awareness from public health aspects such as residues of these chemicals in milk and meat and bacterial resistance to antibiotics as a result of increased use in the food chains prohibits their use as feed additives14. In addition, these supplements have been criticized by the consumers' organizations on the ground of product safety and quality. The consumers' demands have stimulated a search for natural alternatives to chemical feed additives. As plants are part of herbivore diets, plants that contain bioactive compounds such as essential oils (EO), saponins and tannins with antimicrobial properties could be explored in animal nutrition to improve feed utilization and health15. Studies have indicated that EOs and their compounds have potential to modify rumen fermentation16.
The possibility of plant essential oils (EOs) and their constituent compounds (EOCs), as feed additive in ruminant nutrition have been evaluated and reviewed14,16,17. Essential oils are complex natural extracts from different parts of plants14. Essential oil compounds are made up of 2 main chemical groups: terpenes and phenylpropanoids, which are responsible for the unique aroma of different plants14,18. In the study by Eburu and Chikunya19, it was observed that out of 10 EOs investigated, anise oil (at 300 mg L–1) expressed the greatest potential to maintain PUFA concentrations (229.5%) but also expressed the second most inhibitory effect on ruminal VFA concentrations (over 80% reduction). Hence, the aim of this study was to identify the minimum concentration and dose response effects of graded doses (150, 300 and 450 mg L–1) of Anise oil as potential feed additive on the extent of rumen bio-hydrogenation of n-3 PUFA and fermentation characteristics of rumen microbe’s in vitro.
MATERIALS AND METHODS
Study area: This study was conducted in the small ruminant units of the Teaching and Research farm of the University of Calabar, Calabar, between the month of June and July, 2018. Calabar is located on latitude 40 571 N and longitude 80 191 E of the equator, an average annual rainfall of between 1260-1280 mm, average temperature of between 25-30°C, a relative humidity of 70 and 90%. Calabar is located at about 98 m.a.s.l above sea levels20. This study was assessed by the University’s ethics committee to comply with the code of ethics for animal experiment.
Animal management: Six West African Dwarf goats (mean weight 45.0±1.9 kg) were used as rumen fluid donors in the research. The goats were offered free access to water and grass (Panicum maximum) as required. The diet was supplemented with 400 g/goat/day of concentrate (divided into two equal halves and fed at 08.00 and 16.00 h). The rumen fluid donor goats were housed in groups of 3/pen with straw bedding. The concentrate was obtained from a reputable dealer in Calabar, Cross River State, Nigeria, whilst Grass (Panicum maximum) was processed from the University of Calabar Teaching and Research farm. Ruminal microbes were adapted to the experimental diet for a period of 2 weeks before slaughter.
On the day preceding the day of slaughter, feed was withdrawn at about 18.00 h. After slaughter, whole rumens were collected and packed in tough plastic bags into insulated boxes to prevent oxygen entry to Animal Science Laboratory, University of Calabar, Calabar. Fluid was obtained as described previous studies by Eburu and Anya21. After straining, the remaining solids were mixed with a volume of buffer (constituted according to the method of Theodorou et al.22, equal to the rumen liquor removed and homogenized using a kitchen blender to detach rumen microbes attached to solids. The mixture was re-strained with 2 layers of cheese cloth and the filtrate added to the rumen fluid to constitute the buffer rumen fluid mixture as the final inoculum. The mixed fluid was held in a water bath maintained at 39°C and flushed with CO to expel oxygen before being dispensed into the in vitro incubation flasks.
Basal feedstock, treatments and in vitro incubation: A mixture of good quality grass hay (Panicum maximum), goat pellets, whole ground wheat grain and fish oil (SPAR SHOP, Calabar, CRS) were used as basal feedstock in the present study. The feed was made from the mixture of a 30:70 goat pellet concentrate and grass hay (Panicum maximum), respectively. Table 1 for the chemical composition and ingredients of the basal feedstock used in incubation. After formulation, the feed mixture was milled through 1 mm screen (Glen Creston Ltd., Stanmore, England). This diet was supplemented with 40% of ground whole wheat grain and 60% of fish oil. The supplementation with both ground whole wheat grain and fish oil was to make provision for extra sources of n-3 PUFAs in the diet.
There were 4 treatments replicated into 6 as follows: Control (An 0, An 150, An 300 and An 450). Into each 125 mL clear glass type 1 serum bottles, 80 mL anaerobic buffer, 1 g of feed substrate and 20 mL inoculums were added before graded doses (0, 150, 300 and 450 mg L–1) of anise oil was supplemented. The bottles were then sealed with rubber cork and incubated at 39°C using incubator (Genlab Ltd., Cheshire, UK) for 24 h.
Fermentation was stopped at 24 h and 3 replicates were taken from each treatment to determine concentration of free fatty acids. The remaining replicates were used for VFA concentration and pH determination using a pH meter.
| Table 1: Ingredients, chemical composition and fatty acid composition of the basal feedstock used in incubations | |
| Components | Composition |
| Feed ingredient (g kg–1 fresh) | |
| Hay | 700.0 |
| Lamb concentrate | 250.0 |
| Soybean oil | 16.0 |
| Wheat grain | 14.0 |
| Fish oil | 20.0 |
| Chemical composition of basal feedstock (g kg–1 DM) | |
| Dry matter | 936.2 |
| Crude protein | 125.0 |
| Neutral detergent fibre | 400.3 |
| Acid detergent fibre | 218.1 |
| Ether extract | 55.9 |
| Fatty acid concentration (g/100 g TFA) | |
| 22:6 n-3 | 2.5 |
| 20:5 n-3 | 3.8 |
| 18:3 n-3 | 11.5 |
| Stearic (18:0) | 2.0 |
Lamb concentrate formulated using maize 19.58, wheat n grain 13.6, Pkc 11.25 kg, salt 25 kg, premix 32 kg, Concentrate: Goat pellet |
|
The dose response effects of anise oil was examined using the in vitro gas production batch culture method. Anise oil was obtained from SPAR SHOP, Calabar, CRS, Nigeria. The percentage composition of major constituent compounds of anise oil (P. anisum) are: trans-Anethole (82.7%), caryophyllene (3.8%) and 2.3% for limonene23.
Sample collection and preservation: Gas pressure in the bottles during incubation was determined at various times (3, 6, 9, 12 and 24 h) using a pressure transducer (Bailey and Mackey Ltd., Birmingham, UK) that was connected to a digital read-out voltmeter. However, only the 24 h results are presented in this paper. Fermentation was stopped at 24 h by freezing the contents of incubation bottles at -20°C for 5 min, then 3 replicates of each treatment (entire vessel content) was emptied for free fatty acid analysis. From the remaining 3 replicates, 5 mL was sampled for ammonia and 4 mL volatile fatty acids (VFA) determinations. The aliquots for ammonia were preserved by mixing 5 mL of 1 M HCL with 5 mL of sample. Samples (4 mL) for VFAs were mixed with 1 mL of a deproteinizing solution and frozen (-20°C) until required for chemical analysis.
Chemical analysis: Phenol-hypochlorite method24,25, was adopted for use on the plate reader for the determination of the concentration of NH3-N in digesta. Gas chromatography (GC) was used for the analysis of VFA as described in previous research by Ottenstein and Bartley26. The concentration of free fatty acids in feed and freeze-dried samples were extracted27, then subjected to GC analysis. The GC (HP 6890+, Agilent Technologies, UK Ltd.) with a flame ionization detector and fitted with a 100 m fused silica capillary column (Varian CP-7489) of 0.2 μm film thickness and 250 μm diameter was used. Approximately 1 μL of fatty acid samples in hexane were injected at 160°C (initial temperature of the column) and held at that temperature for 15 min, before increasing at 1.5 min to 240°C, until the run was completed. Carrier gas was Helium at a flow rate of 1.2 mL min–1. Methyl heneicosanoic (C21:0, Sigma-Aldrich Co. Ltd., UK) was added prior to saponification as an internal standard and fatty acid methyl ester standard mixture (Thames Restek UK) of CLA was used as the standard for identification of peaks.
Experimental design and statistical analysis: The study was a Completely Randomized Design (CRD) and data were analyzed by one-way analysis of variance (ANOVA) using GenStat 16th edition. Differences between treatments were declared by Least Significance Difference (LSD) and significance was declared at p<0.05.
RESULTS
As indicated in Table 1, the dietary concentrations of all n-3 PUFAs are higher than stearic acid (C18:0).
The pH of the rumen during fermentation was not affected by dietary supplementation of anise at all levels (Table 2). The amount of gas produced during fermentation was lowest (p<0.001) in the vessel that contained the highest dose of anise (450 mg L–1) and highest (p<0.001) in the control. This effect was expressed in a dose dependent manner. Relative to the control, all treatments reduced (p<0.001) the concentrations of total VFA in culture and, the level of reductions was proportional to the dose level of supplementation of anise. Except for 150 mg L–1, all treatments decreased the concentrations of NH3-N relative to the control. The level of reduction progressed as the dose level of anise progresses in the vessel.
Relative to the control, the vessel contents of C22:6 n-3 were maintained higher (p<0.001) in all treatments except for 150 mg L–1 (Table 3). Similar observation was made in the level of C20:5 n-3, where all treatments maintained higher (p<0.001, in a linear manner) the level of this fatty acid than the control. Although there was no difference between the vessel contents of C18:2 tr11 CLA in cultures that received 300 and 450 mg L–1, all treatments increased (p<0.001) this concentration than the level observed in the control. The level of C18:0 was higher (p<0.001) in 300 and 450 mg L–1 than in the control and 150 mg L–1.
| Table 2: Effects of graded levels of anise oil on rumen fermentation parameters at 24 h | ||||||
| Levels of anise oil (mg L–1) | ||||||
| Parameters | 0 | 150 | 300 | 450 | SED | p-values |
| pH | 6.65 | 6.55 | 6.65 | 6.65 | 0.55 | NS |
| Gas (mL g–1 OM) | 110.3a | 105.9b | 98.5c | 94.2d | 1.09 | 0.001 |
| TVFA (mM) | 67.9a | 64.2b | 59.9c | 57.5d | 1.17 | 0.001 |
| NH3-N (mM) | 4.9a | 4.8a | 3.9b | 3.0c | 0.62 | 0.001 |
NS: Not significant, TVFA: Total volatile fatty acids, SED: Standard error of the difference of the mean, p-values: Probability values, means within row with different superscripts letters are different (p<0.05) |
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| Table 3: Effects of graded levels of anise oil on concentration of selected fatty acids (g/100 TFA) at 24 h | ||||||
| Levels of anise oil (mg L–1) | ||||||
| Parameters | 0 | 150 | 300 | 450 | SED | p-values |
| C22:6 n-3 | 0.6a | 0.7ab | 0.8b | 0.8b | 0.05 | 0.045 |
| C20:5 n-3 | 0.2a | 0.3b | 0.4c | 0.5d | 0.03 | 0.001 |
| C18:3 n-3 | 0.8a | 1.1a | 1.5b | 1.8c | 0.11 | 0.001 |
| 18:2 c9 t11 CLA | 0.05a | 0.07b | 0.08c | 0.08c | 0.007 | 0.004 |
| Stearic (18:0) | 5.0a | 5.0a | 6.0b | 6.0b | 0.3 | 0.001 |
TFA: Total fatty acids, C-Linoleic acid: Conjugated linoleic acid, SED: Standard error of the difference of the mean, p-values: Probability values, means within row with different superscripts letters are different (p<0.05) |
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| Table 4: Effects of graded levels of anise oil on the bio-hydrogenation of selected polyunsaturated fatty acids at 24 h | ||||||
| Levels of anise (mg L–1) | ||||||
| Parameters | 0 | 150 | 300 | 450 | SED | p-values |
| C18:3 n-3 | 83.2a | 78.6b | 70.5c | 62.8d | 1.11 | <0.001 |
| C20:5 n-3 | 60.1a | 55.2b | 50.4c | 45.3d | 1.24 | <0.001 |
| C22:6 n-3 | 34.9a | 28.5ab | 25.5b | 25.1b | 3.31 | 0.042 |
SED: Standard error of the difference of the mean, p-values: Probability values, means within row with different superscripts letters are different (p<0.05) |
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The bio-hydrogenation of Lc-n3-PUFA (Table 4) was observed to be similar to the reported effects of all treatments on the concentrations of fatty acids (Table 3). Briefly, all treatments reduced the bio-hydrogenation of all PUFAs in a dose related response, relative to the control. Rumen bio-hydrogenation was calculated as the change in the proportion of individual FA such as 18:3 n-3 and 18:3 n-2, in the feed relative to the amount left in incubation vessels at a given time as follows:
DISCUSSION
This study observed that, although the rumen pH was not affected by supplementing with anise oil, other fermentation parameters (total VFA, NH3-N) were substantially and linearly (dose dependent) inhibited. These observations imply that the potency of EO increases with the level of inclusion and the linear effect of anise on gas production, VFA and NH3-N concentrations agrees with previous studies on ruminal fermentation indicators3, where the supplementation of different doses (150, 300, 450 μg mL–1) of oil of Zataria multiflora and Siberian fir needle oil (125, 250 and 500 mg L–1), respectively, were included in culture. It has been reported that anise oil (P. anisum) contained a phenolic compound called Anethole as the main active constituent compound23,28. The antimicrobial effects and mechanism of action of EO is determined by the chemical structure of its constituent compounds29, hence it is speculated that the observation in this study on the effects of graded doses of anise could be a reflection of the activity of the main constituent compound, anethole.
The levels of NH3-N reduced during fermentation and results are consistent with earlier reports by Busquet et al.30, where cinnamon oil and its main constituent compound (cinnamaldehyde), reduced ammonia concentration. These observation leads to the speculation that anise oil could possibly inhibit the activities of proteolytic and hyper ammonia producing bacteria, the 2 groups of microbes responsible for extensive deamination of amino acid31,32. Over 80% of dietary N consumed by dairy cows has been observed to be excreted in faeces and urine as waste33. Therefore, in terms of practical implications, it can be speculated that supplementing ruminant diets with anise oil (at 300 or 450 mg L–1 in this study) could potentially increase ruminal escape of dietary protein and consequently, decrease the production of NH3-N. However, it should be noted that the beneficial effects, in terms of maintaining ruminal concentration of total VFA was only observed in the level of 150 mg L–1 (termed the minimum beneficial effect), where the inhibition was <10%. The rate of VFA inhibition in the present study also corresponds with the changes in the levels of anise in culture and supports previous reports where varying concentrations of different essential oils were used3,34,35. Furthermore, outcome of these results are consistent with those previous observations3,36, where citronella oil (125, 250 and 500 mg L–1) and thyme oil (125, 250 and 500 mg L–1) exhibited a dose related suppression of feed digestibility and total VFA concentrations. Overall, these observations agree that addition of essential oils or their constituent components may have no effect (at low doses) or decrease (at high doses) ruminal concentration of VFA37.
The concentrations of USFA substantially reduced relative to the dietary (initial) levels in the vessel and higher levels of n-3 PUFA were observed in vessels containing all doses of anise than the control. In addition, the bio-hydrogenation of C18:3 n-3 in the present study was above 80% in the control and the. This observations agree with the reports of previous in vitro and in vivo studies38,39, where bio-hydrogenation of PUFA exceeded 80%. The supplementation of anise oil recorded a dose dependent inhibition of n-3 PUFA disappearance, with the highest dose (450 mg L–1) expressing the most inhibitory effect on bio-hydrogenation and support the findings of other researchers40. The accumulation of n-3 PUFAs in this study could suggest that anise oil suppressed the action of Anaerovibrio lipolytica and Butyrivibrio fibrisolvens, the 2 microbes observed to be responsible for the hydrolysis of the ester bond in fatty acids40. In the light of the health benefits of Lc-n3-PUFA (18:3 n-3, 20:5 n-3 and 22:6 n-3) in humans (both infants and adults), a higher concentration in diet is desirable. Updates on the health benefits of Lc-n3-PUFA in adult health (such as delayed decline in cognitive function and reduction in chances of dementia) are available2,41.
In the current study, the concentration of 18:0 in culture doubled the level in the diet. This agrees with previous studies which observed that the bio-hydrogenation of 18:3 n-3 and 18:2 n-6 ends up with 18:0 as the last product8. This is irrespective of the fact that the supplementation of anise (at all doses) maintained the concentration of unsaturated fatty acids higher than the control. A number of theories have been proposed to speculate why the reduction of bio-hydrogenation of PUFA does not always translate to a reduced concentration of 18:0. However, this observation agrees with the previous studies by Jenkins et al.42, where the use of essential oils maintained higher the concentrations of both PUFA and their end-product (C18:0). These speculations are that: 18:0 may be emanating from the metabolism of oleic acid (C18:1 n-9, not determined in this study) in cultures, because about 70% of oleic acid in culture is converted to42 C18:0. Reduced level of lipolysis40 has also been speculated to induce increased concentration of C18:0.
CONCLUSION
This study examined the dose-response effects of anise EO on the fermentation and bio-hydrogenation of n-3 PUFA using batch culture system. The study observed that the concentrations of the health beneficial fatty acids (Lc-n3-PUFA) were maintained higher at all doses and, the 150 mg L–1 did not affect the levels of total VFA. Hence, 150 mg L–1 of anise is observed to be the minimum dose required to maintain a higher concentrations of n-3 PUFA and to beneficially modify rumen fermentation if effects are repeated in vivo.
SIGNIFICANCE STATEMENT
This study discovered that anise oil has potential to positively modify rumen fermentation and improve the health benefits of ruminant food products that can be beneficial for consumers. This study will help the researchers to uncover the critical areas on the potential of plant extracts as additives in modulating rumen fermentation that many researchers were not able to explore. Thus a new theory on the use of essential oil in ruminant nutrition may be arrived at.