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Article

Effects of Different Essential Oil Blends and Fumaric Acid on In Vitro Fermentation, Greenhouse Gases, Nutrient Degradability, and Total and Molar Proportions of Volatile Fatty Acid Production in a Total Mixed Ration for Dairy Cattle

by
Kelechi A. Ike
1,
Oludotun O. Adelusi
1,
Joel O. Alabi
1,
Lydia K. Olagunju
1,
Michael Wuaku
1,
Chika C. Anotaenwere
1,
Deborah O. Okedoyin
1,
DeAndrea Gray
1,
Peter A. Dele
1,
Kiran Subedi
2,
Ahmed E. Kholif
1,3 and
Uchenna Y. Anele
1,*
1
Department of Animal Sciences, North Carolina Agricultural and Technical State University, Greensboro, NC 24711, USA
2
Analytical Services Laboratory, College of Agriculture and Environmental Sciences, North Carolina Agricultural and Technical State University, Greensboro, NC 27411, USA
3
Dairy Science Department, National Research Centre, 33 Bohouth St. Dokki, Giza 12622, Egypt
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(6), 876; https://doi.org/10.3390/agriculture14060876
Submission received: 18 April 2024 / Revised: 21 May 2024 / Accepted: 29 May 2024 / Published: 31 May 2024
(This article belongs to the Section Farm Animal Production)
Versions Notes

Abstract

:
The present study evaluated the inclusion of fumaric acid and essential oil blends (EOBs) containing anise, cedarwood, clove, cumin, eucalyptus, garlic, ginger, lavender, lemongrass, nutmeg, oregano, and peppermint at different proportions on in vitro dry matter (DM) disappearance (DMD), fiber fraction disappearance, the efficiency of microbial production, and the total volatile fatty acids (VFAs). Ten treatments without (control treatment) or with different EOB/fumaric combinations were used in the study with eight replicates. The EOB inclusion level was 200 μL/g of feed (total mixed ration, (TMR)) while fumaric acid was administered at 3% of the TMR (DM basis). The highest DMD, in vitro true degradable DM, partitioning factor (PF24), and in vitro apparent degradable DM were recorded for the fumaric only treatment and the control. Neutral detergent fiber disappearance was reduced with the inclusion of EOB/fumaric combinations. The production of microbial mass and undegraded DM were higher (p < 0.001) for all EOBs and EOB and fumaric treatments. The inclusion of EOB and fumaric combinations reduced (p < 0.001) the total gas production, methane, and ammonia, with a higher PF24 value noted for EOB3 treatment. The inclusion of individual EOB1 containing garlic, lemongrass, cumin, lavender, and nutmeg in a ratio of 4:2:2:1:1 or combined with fumaric acid yielded the highest propionate concentration across all treatments. We concluded that EOBs decreased methane production and nutrient degradability with better results with the individual EOB1 or EOB1/fumaric combination, which showed a potential enhancement in energy production.

1. Introduction

Ruminants produce a significant amount of methane (CH4) due to enteric fermentation [1,2]. Methane production is associated with a loss of about 2–15% of dietary gross energy, resulting in reduced animal productivity [3]. Several strategies have enacted to reduce CH4, with promising results with the administration of antibiotic-based feed additives [2]. The ban on using antibiotics as growth promoters in livestock production due to their potential residual effects in dairy and meat products, along with increased consumer awareness and concerns, has initiated a quest for safe and natural feed additives [4,5]. Alternative products to conventional antibiotics, which serve as growth promoters in animal husbandry, have been investigated over the last two decades [6,7,8]. Studies have shown that various plant extracts, individual or essential oil blends (EOBs), probiotics, prebiotics, and algae rich in biologically active components can modify rumen fermentation, leading to decreases in the production of CH4 and ammonia nitrogen in the rumen with mixed results [6,9,10]. The U.S. FDA has categorized some of these extracts as generally recognized as safe for human consumption [5,11].
Due to the antibacterial properties of the bioactive components in essential oils, they are currently utilized as natural additives in ruminant diets. Essential oils and their blends have been reported to have different molecular structures and biologically active constituents, as they are extracted from different plant sources, making them good alternatives to antibiotics [5,11]. Based on these properties, essential oils have been termed “antibiotic rivals” [12]. The main problem with using a single essential oil is its diverse effect on ruminal fermentation and nutrient utilization, which is dependent on the type and dose of the essential oil. Therefore, the use of several or blends of essential oils over a single essential oil often gives an improved modification of rumen fermentation due to more diverse bioactive compounds [13,14].
Fumarate has been studied for its ability to manipulate rumen microbes and improve the fermentation process in vitro and in vivo [15]. Fumarate functions as a hydrogen acceptor and may be utilized by rumen microorganisms as a propionate precursor [16]. Fumaric plays a key role as one of the metabolic intermediates of the propionate–succinate pathway, making it an option to increase ruminal propionate production by competing with methanogens for available hydrogen [17], resulting in lower CH4 production [15]. Li et al. [18] reported that fumaric acid reduces CH4 emissions, total volatile fatty acids (VFAs), and the acetate-to-propionate ratio but increases the propionate concentration. The effects of fumarate on in vivo studies have been equivocal, presumably because the ideal dosage for supplementation is still unknown. Li et al. [19] included fumaric acid at 32 g/day in the diet of dairy goats and reported a decrease in the CH4 yield by 18.8% and an increase in the molar portion of propionate by 10.2% without affecting the feed intake or nutrient digestibility. Additionally, they observed differences in the microbiota structure, particularly the structure of phylum Firmicutes. In a study by Zhou et al. [20], the daily inclusion of 20 g of di-sodium fumarate in the diets of Hu sheep had no effect on the rumen fermentation parameters; however, Guo et al. [21] observed that the addition of sodium nitrate and disodium fumarate reduced CH4 production and optimized the ruminal volatile fatty acid composition. Furthermore, there is limited information on the synergy of EOBs containing a mixture of four or more individual EOs and FA on in vitro rumen fermentation using inoculum from dairy cattle.
There is limited information on the synergy between EOBs and fumaric administration in the diets of dairy cows. Alabi et al. [15] observed that EOBs at 100 µL/g and fumaric acid at 3% of the diet reduced DM degradability and CH4 production but increased the microbial mass, total VFAs, and propionate in an in vitro study with inoculum from beef cattle. Similarly, Lin et al. [22] observed that mixing fumarate with an EOB containing mainly clove, oregano, cinnamon, and lemon decreased the concentrations of VFAs and ammonia-N (NH3-N), populations of methanogens, and protozoa but increased the propionate proportion. Therefore, the present study evaluated the effects of EOBs and fumaric inclusion in a total mixed ration on in vitro gas production, dry matter (DM) disappearance (DMD), undegraded DM, greenhouse gas emissions (CH4, carbon dioxide (CO2), NH3, hydrogen sulfide (H2S) and NH3-N), total VFAs, and their molar proportions using inoculum from dairy cattle. The hypothesis was that while the EOBs inhibit unfavorable microbial community activities and reduce CH4 production, the accumulated hydrogen ions would be captured by fumaric acid and channeled back to favorable propionate pathway, leading to overall improved rumen fermentation.

2. Materials and Methods

2.1. Experimental Site

The current experiment was executed in the Ruminant Nutrition Laboratory in the Department of Animal Sciences at North Carolina Agricultural Technical State University, Greensboro, NC, USA.

2.2. Experimental Animals and Management

Three cannulated Holstein Friesian dairy cows from the Dairy Research Farm of North Carolina Agricultural and Technical State University were used for this experiment. The cows were healthy and free from any diseases, and allowed to graze freely on a mixed grass pasture and were supplemented with grass hay and a mineral mixture. The cows were managed under an approved protocol: LA22-0019.

2.3. Ingredients and Treatments and Chemical Analysis

Ten treatments were evaluated in the present study. Four EOBs were formulated as follows: EOB1, EOB2, EOB3, and EOB4. EOB1 comprised garlic, lemongrass, cumin, lavender, and nutmeg in a ratio of 4:2:2:1:1. EOB2 comprised anise, clove, oregano, cedarwood, and ginger in a ratio of 4:2:2:1:1. EOB3 comprised cinnamon, chamomile, eucalyptus, and tea in a ratio of 3:2:1:1, and EOB4 comprised oregano, clove, anise, and peppermint in a ratio of 4:3:2:1. Based on earlier studies carried out in our laboratory, we established the ratios for the blends. Fumaric acid (Thermo Fisher Scientific, Branchburg, NJ, USA) was used.
The individual feed ingredients used were procured from the Research Farm of North Carolina A&T State University. A total mixed ration (TMR) containing 60% corn silage, 20% alfalfa hay, and 20% concentrate (DM basis) was formulated without any additives and was considered as the control treatment. The control treatment was administered with fumaric acid alone (FA), essential oil blend 1 alone (EOB1), essential oil blend 2 alone (EOB2), essential oil blend 3 alone (EOB3), essential oil blend 4 alone (EOB4), EOB1 + fumaric acid (EOB1FA), EOB2 + fumaric (EOB2FA), EOB3 + fumaric (EOB3FA), or EOB4 + fumaric (EOB4FA). The dose for the EOBs was 200 μL/g of feed, while fumaric acid was administered at 3% of the TMR (DM basis). The nutrient contents of the TMR are shown in Table 1.
A detailed procedure on the chemical composition has been previously described by Alabi et al. [15]. The dry matter (#930.15) was determined by using the AOAC procedures [23]. Nitrogen (N) quantification (#954.01), ether extract (EE; #920.39), and ash content (#942.05) were determined as described previously [15]. The neutral detergent fiber (NDF) was determined according to the procedure of Van Soest et al. [24], with alpha amylase and sodium sulfite, while the acid detergent fiber (ADF; #973.18) content was analyzed according to the AOAC method [23]. Acid detergent lignin (ADL) was obtained by eliminating cellulose from ADF through soaking with concentrated H2SO4, following the analytical methods recommended by ANKOM Technologies.

2.4. In Vitro Batch Culture, Degradability, and Gas Measurements

Prior to the start of the experiment, 100 mL sample bottles were washed thoroughly and allowed to dry before being used. Ankom F57 fiber filter bags (Ankom Technology Corp., Macedon, NY, USA) were labeled and soaked in acetone in a fume hood for about 10 min and allowed to air dry before being transferred into the oven at 55 °C and dried overnight. Upon retrieving the bags from the oven, they were placed in a desiccator for about 10 min and then weighed with a sensitive scale and recorded accordingly. Samples of the TMR (0.50 ± 0.05 g) were weighed into the bags, and then the bags were sealed with an impulse sealer machine to prevent loss of the samples and immediately inserted into previously labeled 100 mL serum bottles.
The in vitro batch culture technique, as previously described by Anele et al. [25], was used for this study. Ruminal inoculum was collected from three Holstein Friesian cannulated cows. Artificial saliva was prepared according to McDougall’s buffer recipe containing the following (per L): 9.83 g NaHCO3, 3.69 g Na2HPO4, 0.60 g KCl, 0.47 g NaCl, 0.30 g (NH4)2SO4, 0.061 g MgCl2.6H2O, and 0.0293 g CaCl2.2H2O. The buffer was maintained in a water bath at 39 °C. The buffer and ruminal fluid were mixed at 3:1 (v/v), and the pH was measured using a benchtop pH meter (model B10P, VWR International, Randor, PA, USA). Thereafter, 60 mL of the artificial saliva and ruminal liquor were dispensed into the serum bottles containing the substrate [25]. The serum bottles were capped with butyl rubbers, crimped with aluminum seals, and placed inside an incubator equipped with orbital shakers and set to 39 °C and 125 rpm for 24 h.
The sampling and gas analysis were conducted at 24 h post-incubation period. The headspace gas pressure was measured by inserting a 22 G × 1 ½ (0.7 mm × 40 mm) needle to determine the gas pressure using a digital pressure manometer (VWR International, Randor, PA, USA). The concentrations of CH4, CO2, NH3, NH3-N, and H2S were determined using a portable gas analyzer (Biogas 5000, Landtec, Dexter, MI, USA). Thereafter, the liquid content of each bottle was transferred into centrifuge tubes and centrifuged for 15 min at 10,000 rpm. The filter bags in the sample bottles were removed and thoroughly rinsed under cold water until the water was clear. The bags were oven-dried for 48 h at 55 °C. After drying, the bags were placed inside a desiccator for 10 min and weighed for the determination of DMD. The in vitro apparent degradable DM (IVADDM) and in vitro true degradable DM (IVTDDM) were calculated following the methodology outlined by Anele et al. [25]. All treatments were evaluated in two separate runs, with 4 replicates in each run. In each incubation run, 4 bottles with only the buffered inoculum (blanks) were also included to establish the baseline fermentation gas production.

2.5. Microbial Mass Analysis

The determination of microbial mass followed the procedure outlined by Olagunju et al. [26]. The same number of bottles with the same treatments but without using filter bags were incubated for 24 h and used to estimate the pellet weights for both the treatments and the blanks. The microbial mass was calculated as described by Blümmel and Lebzien [27]. The partitioning factor (PF24) at 24 h of incubation was calculated [28] as mg DMD: mL gas.

2.6. Measurement of Volatile Fatty Acids

The total and molar proportions of VFAs were profiled in the ruminal liquid as previously outlined by Olagunju et al. [26]. An internal standard mixture was prepared as described previously [29].

2.7. Statistical Analysis

All data were analyzed using the GLM procedure of SAS (SAS 9.4 version; SAS Institute Inc., Cary, NC, USA) in a complete randomized design using the model Yij = μ + Ti + εij, where Yij is the observation, μ is the mean, Ti is the treatment effect, and εij is the residual error. The PDIFF statement of SAS was used to separate the means, and significant differences were declared at p < 0.05.

3. Results

3.1. In Vitro Nutrient Disappearance and Fermentation Parameters

All treatments with the EOBs had a lower (p < 0.001) DMD, IVADDM, IVTDDM, PF24, and a higher microbial mass and undegraded DM (p < 0.001) compared to the control and FA treatments (Table 2). There were no differences between the FA and control treatments. The highest microbial mass (p < 0.001) was observed in EOB2, EOB3, or EOB4 alone or with fumaric acid.

3.2. Biogas Production

The EOBs alone or with fumaric acid decreased (p < 0.001) the gas production, CH4, CO2, NH3, and H2S compared to the control and FA treatments; however, both EOB1 and EOB1FA treatments were not statistically different from the control (Table 3). The treatments had no effect on NH3-N gas production.

3.3. Volatile Fatty Acid Production

Treatments that had EOBs decreased the total VFAs (p < 0.001), valerate (p = 0.003), and isovalerate (p < 0.001) compared to the control and FA treatments; however, EOB4FA treatment did not affect the valerate proportion compared to the control (Table 4). The lowest total VFA production was observed with EOB2FA treatment. The EOB2, EOB3, EOB4, EOB3FA, and EOB4FA treatments had the highest (p < 0.001) acetate proportions. Also, EOB3FA and EOB4FA treatments had the highest (p < 0.001) acetate/propionate ratio. The lowest proportions of propionate were observed with EOB2, EOB3FA, and EOB4FA, while EOB3, EOB4, EOB3FA, and EOB4FA treatments had the lowest butyrate proportions. The lowest acetate and highest propionate and butyrate proportions were observed with treatments containing EOB1FA (p < 0.001).

3.4. Fiber Degradability

Treatments did not affect the ADFD (Table 5). The EOB1FA and EOB3FA treatments decreased (p = 0.026) the NDFD, with no differences between the control and other treatments. The highest (p = 0.002) ADLD and HEMD and lowest CELD (p = 0.01) were observed with FA and control treatments.

4. Discussion

4.1. In Vitro Nutrient Disappearance and Fermentation Parameters

Different EOBs had variable responses on the parameters, which is consistent with previous studies [5,15] that reported different responses with different EOBs. The lower DMD in EOB-treated diets compared to the control and sole fumaric treatments was probably due to the antimicrobial effects of EOBs on the ruminal microbes responsible for degrading feeds due to the presence of phenolic and non-phenolic compounds in the EOBs [5]. These compounds are known to attach, permeate, and invade the cells of rumen fibrolytic bacteria and protozoa and inhibit their metabolic activities [5]. The decrease in DMD showed that the diets containing the additives would be less digestible, and hence, less nutrients from the diets may be available to animals. Some previous studies [15,30,31] have also reported the negative effects of EOBs and fumaric acids on DM digestibility. The values of DMD recorded in the present study were consistent with those (55.2–58.3%) reported by Susanto et al. [32]. The decrease in DMD also resulted in increases in the undegraded DM in the EOB treatments. Compared with the control and FA diets, treatments containing essential oils and fumaric acid had higher undegraded DM. The undegraded DM values recorded in the current study were within the range (0.106–0.190%) reported by Brice et al. [33]. A lower undegraded value implies that more feed was broken down, thereby releasing more nutrients.
The IVADDM values recorded in this study were higher than the range of 20–46% reported by Brice et al. [33]. As previously noted, essential oils have antiprotozoal and antimicrobial prosperities and markedly inhibit ruminal microbes, resulting in a decrease in DM disappearance [13,34]. Notably, the EOB1 and EOB1FA treatments had the highest values for IVADDM within the EOB–fumaric treatments, indicating that they had a less depressing effect on feed digestibility. This could mean that the constituents of EOB1 had a more synergistic effect individually and with fumaric acid compared with the other EOB-containing treatments. Benetel et al. [35] observed that the inclusion of varying doses of oregano, ginger, clove, and anise did not affect the IVTDDM. Their results are consistent with the other EOB treatments in the present study.
The PF24 in the current study followed a similar trend as the DMD, undegraded residual, and IVTDDM. The partitioning factor is an indication of nutrient sequestration for use by the ruminal microflora [28]. Higher values indicate more nutrient availability for proliferation of rumen microbes, since the PF24 estimation is based on DM degradability, gas production, and the efficiency of microbial biomass synthesis [33,35]. The decrease in PF24 with EOB treatments indicates less nutrients for the microbes, which could invariably reduce their population, and hence, their capacity for feed degradation [36].
Microbial mass production in the present study was higher in the treatments containing EOBs compared to control and FA treatments, which may be related to lower gas production and the negative effects of the EOBs on methanogens [37]. Microbial mass in the rumen is an indication of the capacity of rumen microbes to convert feed sources [38], including proteins and carbohydrates, into microbial biomass and fermentation byproducts, such as VFA.

4.2. Biogases Production

Greenhouse gases, especially CH4, are linked to dietary energy loss and a decrease in productivity. The inclusion of the additives significantly reduced the total gas, CH4, CO2, and NH3 productions. Essential oils are known to impact rumen fermentation by modifying microbial communities [5,33,39]. Kouazounde [40] reported that the addition of essential oils can modify rumen microbial communities, positively impacting rumen fermentation and reducing greenhouse gas emissions. Alabi et al. [15] reported that the administration of 100 µL of EOBs containing garlic, lemongrass, cumin, lavender, and nutmeg at a ratio of 4:2:2:1:1, respectively, alone or combined with fumaric acid at 3% of the incubated substrate (DM basis) reduced greenhouse gas emissions (CH4 by 91% and 86%, CO2 by 66% and 57.9%, respectively) in beef cattle. In the present study, EOB3 alone or in combination with fumaric acid had higher reduction percentages compared to the other EOBs, indicating that the dosage (100 µL in that experiment vs. 200 µL EOB in the present experiment) and rumen liquor donors could have been responsible for the different responses between EOB3 and EOB1, as noted in Alabi et al. [15]. Therefore, not only the type and proportion of essential oils in the blends but also the dosage and source of inoculum (beef vs. dairy) all played a crucial role in the abatement of greenhouse gas emissions.
The observed differences between the EOBs could be as a result of substrate degradation inhibition by the active compounds in each blend. Each individual essential oil targets specific microbial populations or metabolic pathways in the rumen, contributing to a more wholistic approach to mitigating CH4 emissions. In the present study, not only the synergy between the individual essential oils in EOB3 but also higher levels of clove and oregano in the blend could be responsible. Oregano and clove essential oils have shown a higher tendency to reduce enteric CH4 production [41].
The observed reductions in CH4 production with all the EOBs may be due to their ability to directly lyse or reduce the activities of methanogens and reduce their population, thereby reducing methanogenesis. Lin et al. [22] observed significant decreases in the microbial populations of protozoa, methanogens, Fibrobacter succinogenes, and Butyrivibrio fibrisolvens in sheep fed a combination of fumarate and an EOB containing eugenol, carvacrol, citral, and cinnamaldehyde. The importance of fumaric acid as a propionic acid precursor in competing with methanogens for available H2 cannot be ignored [17,22]. The results of CH4 and CO2 productions in this study showed that EOBs and fumaric inclusion in livestock diets could reduce energy loss and greenhouse gas emissions from ruminants, which contribute to the depletion of the ozone layer and the resultant global warming [42].
Lower ruminal NH3 production with the EOBs, especially EOB3 and EOB4 individually or in combination with fumaric acid, indicates less dietary protein degradation and amino acid deamination. Essential oils can terminate the activities and growth of Gram-positive proteolytic bacteria [5,30]. Consistent with the current results, previous studies [15,22] reported significant decreases in the ruminal NH3 production with EOBs and fumaric acid.

4.3. Volatile Fatty Acid Production

The treatments reduced the total VFAs, with the lowest production noted for EOB2FA, EOB2, EOB3, and EOB4 diets, which could reflect lower digestibility. The total VFA content is also impacted by the efficiency of the microbial mass. There is often an inverse relationship between the concentration of total VFAs and microbial mass production, and this trend was observed in the present study, with the control and FA treatments having higher total VFA and lower microbial mass values relative to the EOB treatments. Access to fermentable substrates for microbial growth and metabolism, like proteins and carbohydrates, is necessary for optimum ruminal microbial mass.
Increasing acetate production in EOB2, EOB3, EOB4, EOB3FA, and EOB4FA treatments was not expected, as these additives negatively affected fiber digestion; however, all of these treatments greatly increased the cellulose degradability, indicating its role in increasing acetate production [43].
The acetate/propionate ratio of EOB1FA was 59.8% lower compared to EOB3FA treatment, which had the highest acetate/propionate ratio, and had a 28% reduction in the acetate/propionate ratio compared to control and FA treatments. Different proportions of essential oils in the different blends would explain the different responses. Different chemical compositions, manufacturing processes, and many other factors can change their mode of action on ruminal fermentation and ruminal microbes [44,45]. Possible synergistic/antagonistic interactions among different EOBs and rumen microbial populations cannot be ignored other possible reasons for the different acetate/propionate ratios [5,44,45]. Lower acetate/propionate ratios suggest higher available energy for growth, meat/milk production, fetal development, and other uses [46]. This is because propionate is a glucogenic nutrient that can be converted into glucose, which is a crucial energy source for various tissues and organs in the body [47]. A reduced acetate/propionate ratio increases the effectiveness of ruminal fermentation by providing energy for the activities of rumen bacteria. On the other hand, there are certain benefits of increased acetate/propionate in animal performance, such as higher milk fat in dairy cows.
The inclusion of EOB2, EOB3FA, and EOB4FA reduced the molar portion of propionate in the diets except those containing EOB1 (i.e., EOB1 and EOB1FA treatments), which had the highest propionate values, with 23.52 and 16.67% increases compared to the control and sole FA treatments, respectively. This is probably because essential oils are generally more effective against Gram-positive bacteria than Gram-negative bacteria, and this favors the growth of Gram-negative bacteria [48]. Gram negative bacteria in the rumen are more associated with the propionate pathway [49]. Propionate is unique in its role, being the main precursor for gluconeogenesis in animals [50]. This attribute of propionate as a precursor for glucose generates a steady supply of glucose, even when the diet comprises high-fibrous materials, thereby contributing to the energy and metabolic needs of the animal [50]. Glucose is essential for energy metabolism and various physiological processes in the animal, ultimately leading to improved feed efficiency [18]. A decrease in propionate could increase the CH4 production, thereby leading to more potential for greenhouse gas emissions [16].

4.4. Fiber Degradability

The EOB1FA treatment reduced NDFD by 6% when compared to the control treatment and by 6.12% when compared to EOB3, which had the highest NDFD among the EOBs and fumaric-containing treatments. As previously mentioned, EOBs had antimicrobial effects on ruminal microbes, which could decrease the ruminal DM and fiber degradability [15,30,31]. The NDFD values in the current study were higher than the ranges of 36.2–64.4% and 27.8–46.3% reported for high concentrate and high forage diets treated with essential oil blends in the study by Brice et al. [33]. The inclusion of EOB3FA reduced the ADLD by 25% when compared to the control and FA treatments. EOB1 had the highest ADLD among the EOB–fumaric combinations, which is consistent with our observation that this treatment had a less depressing effect on digestibility compared to the other treatments.

5. Conclusions

Overall, the inclusion of essential oil blends and fumaric acid impacted rumen fermentation and nutrient degradability by reducing DM disappearance and greenhouse gas emissions. However, EOB1 with or without fumaric acid increased the molar proportion of propionate, which is an indication of more energy output for the animals. The treatments used in the present study showed great potential for the abatement of greenhouse gas emissions in dairy cattle. Analyses of the rumen microbiome and in vivo experiments on dairy animals are recommended to determine how the microbial communities and lactational performance will be affected by the essential oil blends. Also, further studies are recommended to investigate the potential of EOB1 and attempt different inclusion levels for this essential oil blend.

Author Contributions

Conceptualization, U.Y.A.; methodology, K.A.I., P.A.D., K.S. and U.Y.A.; formal analysis, K.A.I.; investigation, K.A.I., O.O.A., J.O.A., L.K.O., D.O.O., M.W., C.C.A. and D.G.; resources, K.A.I., P.A.D. and U.Y.A.; data curation, K.A.I.; writing—original draft preparation, K.A.I.; writing—review and editing, A.E.K. and U.Y.A.; supervision, U.Y.A.; project administration, U.Y.A.; funding acquisition, U.Y.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the USDA National Institute of Food and Agriculture, Evans-Allen project 1023327, project number NC.X338-5-21-120-1.

Institutional Review Board Statement

The animal study was approved by the Institutional Animal Care and Use Committee, North Carolina A&T State University, Greensboro (protocol #: LA22-0019; approved 31 July 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

Special thanks to Corey Burgess for taking care of the cannulated dairy cows.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Proximate composition and fiber analysis (%) of total mixed ration.
Table 1. Proximate composition and fiber analysis (%) of total mixed ration.
NutrientTotal Mixed Ration 1
Dry matter66.7
Organic matter93.0
Crude protein13.4
Ether extract4.88
Nonstructural carbohydrates12.7
Neutral detergent fiber62.0
Acid detergent fiber11.9
Acid detergent lignin13.8
1 Contained (DM basis): 60% corn silage, 20% alfalfa hay, and 20% concentrates.
Table 2. Effects of sole essential oil blends and/or fumaric on in vitro DM disappearance and fermentation parameters.
Table 2. Effects of sole essential oil blends and/or fumaric on in vitro DM disappearance and fermentation parameters.
Treatments 1,2DMD (%)Undegraded Residual (g/g DM)Microbial Mass (g/kg DM)IVADDM (g/g DM)IVTDDM (g/g DM)PF24
Control58.5 a0.16 b0.08 c0.54 a0.69 a3.43 a
FA58.6 a0.15 b0.08 c0.53 a0.70 a3.47 a
EOB148.2 b0.18 a0.12 b0.39 b0.63 b3.15 b
EOB247.7 b0.19 a0.18 a0.26 c0.62 b3.11 b
EOB349.0 b0.19 a0.21 a0.20 d0.62 b3.10 b
EOB449.2 b0.19 a0.19 a0.25 c0.62 b3.11 b
EOB1FA48.6 b0.18 a0.13 b0.39 b0.64 b3.19 b
EOB2FA48.4 b0.18 a0.17 a0.29 c0.63 b3.15 b
EOB3FA46.4 b0.19 a0.18 a0.27 c0.63 b3.12 b
EOB4FA48.0 b0.18 a0.19 a0.26 c0.63 b3.15 b
SEM0.690.0200.0030.0200.0400.200
p-value<0.001<0.001<0.001<0.001<0.001<0.001
Means with different superscripts letters along the same column are significantly (p < 0.05) different. DMD = DM disappearance, EOB = essential oil blend, IVADDM = in vitro apparent degradable DM, IVTDDM = in vitro true degradable DM; PF24 = in vitro true degradable DM for partitioning factor, SEM = standard error of means. 1 Treatments: a TMR without any additives (control treatment), supplemented with fumaric acid alone at 3% of diet (FA treatment), or 200 μL/g feed essential oil blend 1 alone (EOB1 treatment), essential oil blend 2 alone (EOB2 treatment), essential oil blend 3 alone (EOB3 treatment), essential oil blend 4 alone (EOB4 treatment), EOB1 + fumaric acid (EOB1FA treatment), EOB2 + fumaric (EOB2FA treatment), EOB3 + fumaric (EOB3FA treatment), or EOB4 + fumaric (EOB4FA treatment). 2 EOB1 contained garlic, lemongrass, cumin, lavender, and nutmeg at 4:2:2:1:1, respectively. EOB2 contained anise, clove, oregano, cedarwood, and ginger at 4:2:2:1:1, respectively. EOB3 contained cinnamon, chamomile, eucalyptus, and tea in the ratio 3:2:1:1, respectively, and EOB4 comprised oregano, clove, anise, and peppermint in the ratio 4:3:2:1, respectively.
Table 3. Effects of sole essential oil blends and/or fumaric on in vitro gas and greenhouse gas production after 24 h.
Table 3. Effects of sole essential oil blends and/or fumaric on in vitro gas and greenhouse gas production after 24 h.
Treatments 1,2Gas Production (mL/g DM)CH4 (mg/g DM)CO2 (mg/g DM)NH3 (mmol/g DM)H2S (mmol/g DM)NH3N (mg/dL)
Control189.0 a8.75 a43.7 a198.5 b880.5 b11.1
FA197.1 a7.10 b38.4 a288.1 a1268.1 a10.8
EOB1104.3 b0.53 c24.1 b102.9 cd890.4 b12.4
EOB246.5 cd0.23 c6.3 c38.2 de153.1 c12.3
EOB330.2e0.08 c3.7 c9.3 e16.3 c12.4
EOB442.9 cd0.22 c8.1 c11.4 e29.9 c11.4
EOB1FA108.9 b0.50 c18.6 b143.7 bc741.0 b11.5
EOB2FA50.3 c0.16 c6.1 c39.8 de86.5 c11.1
EOB3FA37.5 de0.05 c2.4 c12.7 e13.5 c10.7
EOB4FA41.9 cd0.11 c5.5 c6.6 e11.5 c11.2
SEM7.870.411.9713.6968.480.18
p-value<0.001<0.001<0.001<0.001<0.0010.185
Means with different superscripts letters along the same column are significantly (p < 0.05) different. EOB = essential oil blend, NH3 = ammonia, CH4 = methane, CO2 = carbon dioxide, H2S = hydrogen sulfide, SEM = standard error of means. 1 Treatments: a TMR without any additives (control treatment), supplemented with fumaric acid alone at 3% of diet (FA treatment), or 200 μL/g feed essential oil blend 1 alone (EOB1 treatment), essential oil blend 2 alone (EOB2 treatment), essential oil blend 3 alone (EOB3 treatment), essential oil blend 4 alone (EOB4 treatment), EOB1 + fumaric acid (EOB1FA treatment), EOB2 + fumaric (EOB2FA treatment), EOB3 + fumaric (EOB3FA treatment), or EOB4 + fumaric (EOB4FA treatment). 2 EOB1 contained garlic, lemongrass, cumin, lavender, and nutmeg at 4:2:2:1:1, respectively. EOB2 contained anise, clove, oregano, cedarwood, and ginger at 4:2:2:1:1, respectively. EOB3 contained cinnamon, chamomile, eucalyptus, and tea in the ratio 3:2:1:1, respectively, and EOB4 comprised oregano, clove, anise, and peppermint in the ratio 4:3:2:1, respectively.
Table 4. Effects of sole essential oil blends and/or fumaric combinations on total and individual volatile fatty acids.
Table 4. Effects of sole essential oil blends and/or fumaric combinations on total and individual volatile fatty acids.
Treatments 1,2Total (mmol/g DM)Acetate (%)Propionate (%)A:P RatioButyrate (%)Isobutyrate (%)Valerate (%)Isovalerate (%)
Control214.2 a73 d17 b4.2 cd9 b0.1 bc0.5 c0.1 e
FA212.5 a73 d18 b4.1 cd9 b0.2 abc0.5 c0.1 e
EOB1139.7 cb63 e21 a3.1 d15 a0.3 abc1.1 a0.1 de
EOB295.4 de77 bc14 cd5.9 abc8 bc0.3 abc0.8 b0.2 ab
EOB395.4 de.77 bc16 bc5.1 bc6 cd0.4 abc0.8 b0.1 bc
EOB493.2 de78 b15 bc5.1 bc6 cd0.0 c0.8 ab0.1 bc
EOB1FA159.6 b64 e21 a3.0 d14 a0.3 abc0.8 b0.1 e
EOB2FA78.1 e74 cd16 bc4.8 c9 b0.6 a0.9 ab0.2 a
EOB3FA117.9 cde82 a12 d7.4 a5 d0.5 ab0.8 ab0.1 cd
EOB4FA120.6 bcd80 ab13 cd6.7 ab6 cd0.2 bc0.7 bc0.1 cd
SEM7.137.00.40.240.50.0010.030.001
p value<0.001<0.001<0.001<0.001<0.0010.0080.003<0.001
Means with different superscripts letters along the same column are significantly (p < 0.05) different. EOB = essential oil blend, VFAs = total volatile fatty acids, A:P = acetate/propionate ratio, SEM = standard error of means. 1 Treatments: a TMR without any additives (control treatment), supplemented with fumaric acid alone at 3% of diet (FA treatment), or 200 μL/g feed essential oil blend 1 alone (EOB1 treatment), essential oil blend 2 alone (EOB2 treatment), essential oil blend 3 alone (EOB3 treatment), essential oil blend 4 alone (EOB4 treatment), EOB1 + fumaric acid (EOB1FA treatment), EOB2 + fumaric (EOB2FA treatment), EOB3 + fumaric (EOB3FA treatment), or EOB4 + fumaric (EOB4FA treatment). 2 EOB1 contained garlic, lemongrass, cumin, lavender, and nutmeg at 4:2:2:1:1, respectively. EOB2 contained anise, clove, oregano, cedarwood, and ginger at 4:2:2:1:1, respectively. EOB3 contained cinnamon, chamomile, eucalyptus, and tea in the ratio 3:2:1:1, respectively, and EOB4 comprised oregano, clove, anise, and peppermint in the ratio 4:3:2:1, respectively.
Table 5. Effects of sole essential oil blends and/or fumaric combinations on fiber disappearance.
Table 5. Effects of sole essential oil blends and/or fumaric combinations on fiber disappearance.
Treatments 1,2NDFD (%)ADFD (%)ADLD (%)CELD (%)HEMD (%)
Control74.6 a64.719.4 ab37.7 b20.7 a
FA72.0 abc61.819.9 a37.2 b19.5 ab
EOB170.6 bc68.417.1 abc39.5 ab12.9 cd
EOB271.9 abc62.215.6 c40.5 a13.0 cd
EOB374.1 ab59.115.2 c40.3 a17.1 abc
EOB473.8 ab61.215.1 c41.0 a15.8 abcd
EOB1FA69.6 c63.216.6 bc39.4 ab14.8 bcd
EOB2FA71.2 abc63.215.5 c41.2 a15.2 bcd
EOB3FA69.7 c59.814.8 c40.8 a10.8 d
EOB4FA70.5 bc63.315.3 c40.4 a13.0 cd
SEM0.420.970.400.310.66
p value0.0260.7030.0020.0100.002
Means with different superscripts letters along the same column are significantly (p < 0.05) different. EOB = essential oil blend, NDFD = neutral detergent fiber disappearance, ADFD = acid detergent fiber disappearance, ADLD = acid detergent lignin disappearance, HEMD = hemicellulose disappearance, CELD = cellulose disappearance, SEM = standard error of means. 1 Treatments: a TMR without any additives (control treatment), supplemented with fumaric acid alone at 3% of diet (FA treatment), or 200 μL/g feed essential oil blend 1 alone (EOB1 treatment), essential oil blend 2 alone (EOB2 treatment), essential oil blend 3 alone (EOB3 treatment), essential oil blend 4 alone (EOB4 treatment), EOB1 + fumaric acid (EOB1FA treatment), EOB2 + fumaric (EOB2FA treatment), EOB3 + fumaric (EOB3FA treatment), or EOB4 + fumaric (EOB4FA treatment). 2 EOB1 contained garlic, lemongrass, cumin, lavender, and nutmeg at 4:2:2:1:1, respectively. EOB2 contained anise, clove, oregano, cedarwood, and ginger at 4:2:2:1:1, respectively. EOB3 contained cinnamon, chamomile, eucalyptus, and tea in the ratio 3:2:1:1, respectively, and EOB4 comprised oregano, clove, anise, and peppermint in the ratio 4:3:2:1, respectively.
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Ike, K.A.; Adelusi, O.O.; Alabi, J.O.; Olagunju, L.K.; Wuaku, M.; Anotaenwere, C.C.; Okedoyin, D.O.; Gray, D.; Dele, P.A.; Subedi, K.; et al. Effects of Different Essential Oil Blends and Fumaric Acid on In Vitro Fermentation, Greenhouse Gases, Nutrient Degradability, and Total and Molar Proportions of Volatile Fatty Acid Production in a Total Mixed Ration for Dairy Cattle. Agriculture 2024, 14, 876. https://doi.org/10.3390/agriculture14060876

AMA Style

Ike KA, Adelusi OO, Alabi JO, Olagunju LK, Wuaku M, Anotaenwere CC, Okedoyin DO, Gray D, Dele PA, Subedi K, et al. Effects of Different Essential Oil Blends and Fumaric Acid on In Vitro Fermentation, Greenhouse Gases, Nutrient Degradability, and Total and Molar Proportions of Volatile Fatty Acid Production in a Total Mixed Ration for Dairy Cattle. Agriculture. 2024; 14(6):876. https://doi.org/10.3390/agriculture14060876

Chicago/Turabian Style

Ike, Kelechi A., Oludotun O. Adelusi, Joel O. Alabi, Lydia K. Olagunju, Michael Wuaku, Chika C. Anotaenwere, Deborah O. Okedoyin, DeAndrea Gray, Peter A. Dele, Kiran Subedi, and et al. 2024. "Effects of Different Essential Oil Blends and Fumaric Acid on In Vitro Fermentation, Greenhouse Gases, Nutrient Degradability, and Total and Molar Proportions of Volatile Fatty Acid Production in a Total Mixed Ration for Dairy Cattle" Agriculture 14, no. 6: 876. https://doi.org/10.3390/agriculture14060876

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