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2 October 2026

15 Pages

Effects of Lipid Supply Form (Whole Seed vs. Free Oil) on In Vitro Ruminal Fermentation and Methane Production of Diets for Dairy and Fattening Sheep

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Departamento de Producción Agraria, Escuela Técnica y Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Ciudad Universitaria, 28040 Madrid, Spain
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Methane2026, 5(4), 33;https://doi.org/10.3390/methane5040033
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Abstract

Enteric methane (CH4) emissions from ruminants contribute substantially to greenhouse gas production. Dietary lipid supplementation is a mitigation strategy, but its effects are variable. We evaluated the effects of lipid source (soybean, sunflower, and cardoon) and lipid supply form (whole oilseed vs. free oil) on in vitro ruminal fermentation and CH4 production of diets representative of those fed to dairy ewes and fattening lambs. For each oilseed, treatments included a control (no added lipids) and either the whole seeds or the extracted free oils at final lipid concentrations of 5.0–5.5 and 6.0–6.5% dry matter for dairy and fattening diets, respectively. Diets were incubated for 17 h with ruminal fluid from sheep fed either dairy or fattening diets. No interactions between diet type and lipid supply form were detected for any oilseed, and lipid supply did not influence ruminal fermentation. Lipid supply form had no effects on methane-related parameters in soybean diets. Compared with the whole seed, the free oil reduced the CH4/total short-chain fatty acids ratio in both sunflower and cardoon diets, and decreased CH4 production and CH4 proportion in total gas in cardoon diets. The effects of lipid supply form depended on the oilseed, with cardoon-free oil showing the greatest potential for CH4 mitigation. These findings highlight the potential of selected free oils as candidates for methane mitigation and the usefulness of in vitro methane production as a screening tool to identify lipid sources with potential for reducing methane emissions, which should subsequently be evaluated in vivo.

1. Introduction

Enteric methane (CH4) emissions from ruminants represent a major environmental concern because CH4 is a potent greenhouse gas with a global warming potential from 27.2 to 29.8 times greater than that of carbon dioxide over a 100-year time horizon [1]. In addition to its environmental impact, methanogenesis constitutes an energy loss for the animal, accounting for up to 12% of gross energy intake depending on diet composition and feeding conditions [2,3]. Consequently, considerable research efforts have focused on developing nutritional strategies capable of reducing enteric CH4 emissions without impairing ruminal function and animal productivity. Among the dietary approaches proposed to reduce CH4 production, lipid supplementation has received considerable attention. Meta-analyses and literature surveys have consistently shown that lipid supplementation can reduce enteric CH4 emissions, although the magnitude of the response is highly variable and depends on factors such as lipid source, fatty acid (FA) profile, inclusion level, and basal diet composition [4,5,6,7,8]. In studies included in these meta-analyses, dietary lipid concentrations commonly reached approximately 5.5–6% of dry matter in supplemented diets, compared with about 2–2.5% in control diets. Under these conditions, reductions in enteric CH4 emissions ranging from 9 to 15% have been reported [5,6,7]. In a recent meta-analysis, Akinropo et al. [8] estimated that each increase of 1 g/kg dry matter in dietary lipids was associated with a reduction of 2.03 g/d in CH4 production in dairy cows. Individual studies have also reported greater reductions with specific lipid sources. For example, supplementation with 5% sunflower oil reduced daily ruminal CH4 emissions by 22.2% in lactating cows [9], whereas supplementation with 6% soybean oil reduced CH4 production by approximately 40% in young bulls [10]. These findings illustrate that the magnitude of the antimethanogenic response to dietary lipids can vary considerably according to lipid source, inclusion level, and dietary characteristics.
Besides the source and inclusion level of dietary lipids, the physical form in which lipids are supplied may also influence their effects on ruminal fermentation and CH4 production [11,12]. Lipids can be supplied either as intact oilseeds or as free oils extracted from the oilseeds, which affects their availability in the rumen and, consequently, their effects on CH4 production and ruminal fermentation. Free oils are immediately accessible to ruminal microorganisms, whereas lipids contained within the oilseeds are released more gradually as the seed matrix is degraded [13]. Several studies have compared the physical form of lipid supply (LS), but the results are inconsistent [11,12,14]. In some studies, whole oilseeds appeared to exert a greater CH4-mitigating effect than their corresponding free oil, whereas other studies reported similar or greater reductions with free oils [5,6,11]. Several factors may explain these contradictory findings, including the lipid inclusion level, the basal diet and the composition of the oilseed [6,12,15]. Soybean (Glycine max (L.) Merr.), sunflower (Helianthus annuus L.) and flax seeds (Linum usitatissimum L.) and their corresponding free oils are among the most extensively studied [10,12,16,17] because of their widespread use in ruminant feeding, whereas information on cardoon (Cynara cardunculus L. var. altilis) seed is scarce. There is a growing interest in this drought-tolerant oilseed crop because of its excellent adaptation to Mediterranean environments and its potential importance under adverse future climate scenarios [18]. In a recent study by our group comparing the effects of soybean, sunflower and cardoon seeds on in vitro CH4 production and ruminal fermentation, cardoon seed showed the greatest potential to reduce CH4 without negative effects on ruminal fermentation parameters [19]. These findings suggest that cardoon may represent a particularly promising alternative lipid source for CH4 mitigation and justify evaluating whether its effects are influenced by the form in which the lipid is supplied. Therefore, the aim of this study is to evaluate the effects of including three lipid sources (soybean, sunflower, and cardoon), either as whole oilseeds or as the corresponding extracted free oils on in vitro ruminal CH4 production and fermentation characteristics of diets representative of those used in dairy-ewes and lamb-fattening systems under intensive production conditions in Spain and other countries. We hypothesized that supplying lipids as free oils would increase the antimethanogenic response because of the greater ruminal availability of unsaturated FA, although the magnitude of this effect would depend on the oilseed and the basal diet. To ensure the same FA profile across both LS treatments, free oils were extracted from the oilseeds used for the study.

2. Results

2.1. Composition of Seeds, Extracted Meals and Oils and Experimental Diets

The three oilseeds differed in chemical composition (Table 1), with soybean seeds having 2.3 and 2.6 times more crude protein concentration than sunflower and cardoon, respectively, whereas sunflower and cardoon seeds contained 1.9 and 2.3 times more neutral detergent fiber than soybean seeds, respectively. Sunflower seeds have the greatest lipid (ether extract) content (2.2 and 1.8 times more than soybean and cardoon seeds). No lipids were detected in any of the extracted meals, which exhibited higher concentrations of crude protein and fiber than the corresponding whole seeds.
Table 1. Chemical composition (g/100 g dry matter) of the whole seeds and the corresponding extracted meals.
The FA profile of each seed and the corresponding extracted oil was similar for all seeds (Table 2). The C18:2n-6 was most abundant FA in all seeds and their extracted free oils followed by C18:1n-9 and C16:0. Although there were differences between the different seeds and free oils in the individual FA profile, those from soybean and cardoon showed similar proportions of total saturated (from 14.5 to 16.3%), monounsaturated (from 26.6 to 28.5%) and polyunsaturated FA (from 55.5 to 58.3%), whereas those from sunflower had lower proportions of saturated FA (10.3%) and monounsaturated FA (21.4 and 21.8% for seed and free oil, respectively) and greater of polyunsaturated FA (68.3 and 67.9%).
Table 2. Fatty acid (FA) profile (g/100 g of total analyzed FA) of the seeds and the corresponding extracted free oil.
The feed ingredients and chemical composition of the experimental diets are shown in Table 3. The diets were designed to be representative of those fed to dairy ewes and fattening lambs in the practice. As expected, within each type of diet (dairy or fattening), all diets had similar levels of crude protein (17.0–17.5 and 15.5–16.0% for dairy and fattening diets, respectively) and of lipids (5.0–5.5 and 6.0–6.5%). The basal diets without additional lipid inclusion from the experimental oilseeds (control diets; SBC, SFC and CAC for soybean, sunflower and cardoon, respectively) had lower lipids content (about 2.0 and 1.8% for dairy and fattening diets, respectively) than those including either the whole seeds (SBS, SFS and CAS for soybean, sunflower and cardoon, respectively) or the corresponding free oils (SBO, SFO and CAO for soybean, sunflower and cardoon, respectively), which showed a similar content of lipids. Thus, the control diets represented basal diets without additional lipid supplementation, whereas the supplemented diets were formulated to achieve a higher lipid concentration. Thus, comparisons between control and supplemented diets reflect the effect of increasing dietary lipid concentration, whereas comparisons between whole seeds and their corresponding free oils allow the effect of lipid supply form to be evaluated at similar total dietary lipid concentrations.
Table 3. Feed ingredients and chemical composition of the dairy and fattening experimental diets including no lipid supplementation (SBC, SFC and CAC) or lipids from soybean, sunflower and cardoon added either as the whole seed (SBS, SFS and CAS) or as free oil (SBO, SFO and CAO).

2.2. In Vitro Fermentation and Methane Production of Diets Containing Soybean

No significant Diet × LS interactions were detected in any fermentation variable for soybean diets (Table 4). Overall, LS had no effects on ruminal fermentation, and only a trend towards a lower NH3-N concentration after 6 h of incubation (p = 0.072) was detected for the diets including free oils compared with those including the whole soybean seeds.
Table 4. Influence of including no extra lipids (SBC) or lipids added as whole soybean seed (SBS) or free soybean oil (SBO) on in vitro ruminal fermentation and CH4 production of dairy and fattening diets after 6 and 17 h of incubation with ruminal fluid from sheep.
In contrast, diet type influenced several fermentation parameters. At both 6 and 17 h of incubation, fattening diets showed lower acetate and valerate proportions and acetate/propionate ratios, together with greater propionate proportions, than dairy diets (p ≤ 0.009). Fattening diets also exhibited greater butyrate proportions after 6 h (p = 0.037) and lower isobutyrate proportions after 17 h (p = 0.001). Moreover, NH3-N concentrations were greater in fattening than in dairy diets at 6 h (p = 0.005), but the opposite was observed after 17 h of incubation (p = 0.006). However, there were no differences between diets in total short-chain FA (SCFA) production, the fermented organic matter, gas and CH4 production or CH4/total SCFA ratios.

2.3. In Vitro Fermentation and Methane Production of Diets Containing Sunflower

There were no significant Diet × LS interactions in any fermentation variable for the sunflower diets (Table 5). The LS of sunflower had no influence on ruminal fermentation, but the form of LS affected several fermentation parameters. Compared with the whole seed, including the free oil tended to increase total SCFA production and the fermented organic matter after 6 h of incubation (p = 0.089 and 0.091, respectively) and the amount of gas after 17 h (p = 0.062). Moreover, adding the free oil tended to reduce the proportion of CH4 in the gas produced (p = 0.054) and resulted in lower CH4/total SCFA ratios (p = 0.042).
Table 5. Influence of including no extra lipids (SFC) or lipids added as whole sunflower seed (SFS) or free sunflower oil (SFO) on in vitro ruminal fermentation and CH4 production of dairy and fattening diets after 6 and 17 h of incubation with ruminal fluid from sheep.
Compared with dairy diets, fattening diets showed lower acetate and valerate proportions and acetate/propionate ratios, but greater butyrate proportions after both 6 and 17 h of incubation (p ≤ 0.039). Fattening diets also showed greater propionate and lower isobutyrate proportions after 17 h (p = 0.005 and 0.002, respectively). Moreover, propionate proportions at 6 h and gas production at 17 h tended to be greater (p = 0.093 and 0.085, respectively) and NH3-N concentrations at 17 h tended to be lower (p = 0.090) for fattening than for dairy diets. No effects of diet type were observed in the remaining fermentation parameters.

2.4. In Vitro Fermentation and Methane Production of Diets Containing Cardoon

No significant diet × LS interactions were detected for any fermentation variable in cardoon diets (Table 6). No global effects of LS were detected at any sampling time, and no differences between the diets containing the whole seed and those with free oil were detected in any parameter at 6 h of incubation (p ≥ 0.201). After 17 h, supplying the lipids as free oil reduced CH4 production (p = 0.012), the proportion of CH4 in the gas produced (p = 0.002), and the CH4/total SCFA ratio (p = 0.003) compared with the supply of the whole seed. No other fermentation parameter was affected by the form of LS.
Table 6. Influence of including no extra lipids (CAC) or lipids added as whole cardon seed (CAS) or free cardoon oil (CAO) on in vitro ruminal fermentation and CH4 production of dairy and fattening diets after 6 and 17 h of incubation with ruminal fluid from sheep.
At both incubation times, fattening diets showed lower acetate and valerate proportions and acetate/propionate ratios together with greater propionate proportions than dairy diets (p ≤ 0.002). Fattening diets also exhibited greater butyrate proportions after 6 h (p = 0.007) and lower isobutyrate proportions after 17 h (p = 0.004) of fermentation. Furthermore, both NH3-N concentrations and the proportion of CH4 in the gas produced after 17 h were lower (p = 0.015 and 0.011, respectively) and CH4 production tended to be lower (p = 0.065) for fattening than for dairy diets. No differences between diet types were detected in the remaining fermentation parameters.

3. Discussion

3.1. Composition of Seeds, Extracted Meals and Oils and Experimental Diets

The chemical composition of soybean and sunflower seeds and their corresponding extraction meals was within the range of values reported in feed tables [20,21]. Similarly, the concentration of the different chemical fractions in cardoon seeds and its corresponding extracted meal was consistent with previous reports [22,23]. The undetectable lipid content of all extracted meals indicates the high efficiency of the oil extraction process used to obtain the free oils.
The experiment was specifically designed to ensure that each free oil had the same FA profile as its corresponding seed, thereby minimizing differences in FA profile as a potential confounding factor influencing the observed responses. Analysis of the FA composition indicated that the whole seeds and their corresponding extracted free oil shared highly similar FA profiles. It should be noted that the free oils were obtained using a laboratory solvent extraction, but this procedure does not fully reproduce conventional oilseed processing, and this difference should be considered when comparing or extrapolating the results to conventionally processed oils. Consistent with previous reports [24,25,26], all three seeds exhibited a predominance of unsaturated FA, with sunflower seed showing the highest polyunsaturated FA concentration [26,27].
The diets were designed to be representative of those typically used in dairy ewes and fattening lambs under intensive production systems in Spain and other countries. Accordingly, the dairy diets were formulated to contain about 17.0–17.5% of crude protein and 5.0–5.5% of lipids (dry matter (DM) basis), whereas the fattening diets contained lower crude protein concentrations (15.5–16.0%) and slightly higher lipid concentration (6.0–6.5%). Within each diet type, all diets including lipids, either as whole oilseeds or free oils, had similar lipid concentrations, within the ranges commonly used in practice [7,28]. The use of a fixed dietary lipid concentration, together with the large differences in lipid content among the oilseeds, resulted in different inclusion rates for each oilseed. In contrast, the inclusion level of free oils was the same across oil sources within each diet type (3.30 and 4.70% of DM for dairy and fattening diets, respectively).

3.2. In Vitro Fermentation and Methane Production of Experimental Diets

The absence of effects of LS on any ruminal fermentation parameter across the three oilseeds indicates that the final dietary lipid levels did not adversely affect ruminal fermentation. In fact, the values of fermentation parameters were within the ranges previously reported for in vitro ruminal fermentation of diets with similar composition [29,30]. However, studies in dairy and beef cattle reported negative effects on DM intake and production performance and modified ruminal fermentation when dietary lipid content was above 5.0% [10,31]. As discussed by several authors [4,5,6,7,8], the effects of lipids on rumen fermentation are highly variable because they depend not only on the nature and concentration of dietary lipids, but also on diet composition among other factors. It should also be noted that the present results were obtained using ruminal fluid from sheep, and therefore differences in rumen physiology and microbial populations between sheep and cattle may influence the magnitude of the responses. Consequently, extrapolation of these in vitro findings to dairy cattle should be made with caution.
Contrary to our initial hypothesis, the response to LS was not affected by diet type, as no diet × LS interactions were detected for any of the parameters evaluated across the tested oilseeds. Similarly, Benchaar et al. [12] found no significant interaction between the dietary forage:concentrate ratio and the form of flax supplementation (whole seed vs. free oil) on ruminal concentrations of SCFA and NH3-N in dairy cows. To the best of our knowledge, no other study has investigated the potential interactions between supplying lipids as whole seeds or as free oils and diet composition on ruminal fermentation parameters. However, the results of the studies comparing the effects of supplying whole oilseeds and free oils using only a single diet are inconsistent.
Consistent with the lack of effects of LS form on rumen fermentation of soybean diets, Freitas et al. [32] detected no changes in fermentation parameters in the rumen of dairy cows when whole soybean seeds were replaced with soybean-free oil in diets containing final lipid concentrations of 4.73 and 5.39% DM, respectively. Similarly, Fiorentini et al. [16] reported no differences in rumen fermentation parameters of heifers by feeding either whole soybean seeds or soybean-free oil in diets containing 4.8% DM of lipids. In contrast, Jordan et al. [10] observed greater reductions in enteric CH4 emissions and shifts in rumen SCFA pattern in beef cattle by replacing whole soybeans with soybean-free oil in diets containing final lipid levels of 11.0 and 8.7% DM, respectively. Although soybean-free oil is more readily available to rumen microorganisms than lipids contained within whole soybean seeds, whose release is constrained by the seed matrix, these results might indicate that differences in the response to LS form are only observed at high dietary levels of inclusion.
Studies comparing the effects of sunflower whole seeds and free oil on ruminal fermentation have also produced contradictory results. Beauchemin et al. [11] reported that feeding sunflower seeds to heifers resulted in similar rumen fermentation parameters but a greater reduction in CH4 production than feeding free oil when both were included in diets containing 5.9% lipids (DM basis). However, the CH4 reduction observed with sunflower seeds was partly due to decreased diet digestibility. Morsy et al. [17] observed no differences in concentrations of SCFA and NH3-N in the rumen of goats fed diets with 4.8% DM of lipids and containing either sunflower seeds or sunflower-free oil. In our study, a reduction in CH4/total SCFA ratio for the free oil diets was the only significant difference observed when comparing sunflower seeds with free oil, although free oil diets also tended to have greater SCFA production and fermented organic matter at short incubation times.
The limited effects of LS form observed in soybean and sunflower might be related to the moderate lipid concentrations used in this study (5.0–5.5 and 6.0–6.5% DM in dairy and fattening diets). Moreover, the diets with whole seeds and free oils were designed to include lipids with a comparable FA profile and to have comparable levels of the corresponding extracted meal to avoid potential confounding effects. Finally, it should be noted that the in vitro system used in this study does not allow long-term adaptation of ruminal microorganisms to the incubated diet [33]; nevertheless, this limitation was partly overcome by inoculating the cultures with ruminal fluid obtained from sheep previously adapted to the same diet.
The most pronounced effects of LS form were observed in cardoon diets. The lower CH4 production and CH4/total SCFA ratio observed with free oils compared with the whole seeds, together with the similar total SCFA production and fermented organic matter in both treatments, suggest a potentially favorable effect of free oils on ruminal fermentation. The results would indicate that cardoon-free oil increased the energetic efficiency of ruminal fermentation, as the CH4/total SCFA ratio is considered an indicator of fermentation efficiency that reflects the balance between the energy lost as CH4 and the energy recovered as SCFA [34]. Although reductions in ruminal NH3-N concentrations following the inclusion of different free oils in the diet have been frequently reported in the literature both in vivo and in vitro [11,17,35], this effect was not observed for any seed in our study.
Interestingly, the greater effects observed with cardoon compared with soybean and sunflower cannot be explained solely by differences in FA composition. Although soybean and cardoon showed relatively similar FA profiles, their effects on ruminal parameters differed, with cardoon showing a more pronounced response, particularly in terms of CH4 production when lipids were supplied as free oils. This suggests that other characteristics of cardoon rather than its FA composition may contribute to the observed differences.

3.3. Influence of Diet Type on In Vitro Fermentation and Methane Production

Differences between dairy and fattening diets were similar across the tested oilseeds. According to our results, shifts in the ruminal SCFA profile towards a greater proportion of propionate, lower proportion of acetate, and lower acetate/propionate ratios have been frequently observed when comparing the fermentation of high-concentrate diets to that of diets with higher forage proportions both in vitro [29,32,35] and in vivo [12,32,35]. The lower NH3-N concentrations observed in fattening diets at 17 h of incubation might be related to their lower crude protein content compared with dairy diets (15.3 vs. 17.6% DM as average). These results are consistent with the reduced proportions of valerate and isobutyrate observed in the fattening diets, as these minor SCFA are produced by the microbial degradation of branched-chain amino acids [36].
In contrast, the effect of diet type on gas and CH4 production varied among oilseeds, with differences between diet types observed only in diets containing cardoon seeds. The trend to lower CH4 production and the lower proportion of CH4 in the gas observed in fattening diets compared with dairy diets is consistent with previous observations showing that ruminal CH4 production is generally reduced by increasing the proportion of concentrate in the diet due to shifts in ruminal fermentation patterns [2,8]. The similar CH4/total SCFA ratios observed across oilseeds in both fattening and dairy diets indicates that the energetic efficiency of ruminal fermentation was unaffected by the diet type.

4. Materials and Methods

4.1. Ethical Statement

All animal procedures complied with the European legislation for the protection of animals used for scientific purposes and were approved by the Institutional Animal Care and Use Committee of the Comunidad Autónoma de Madrid (Approval No. PROEX 035/17).

4.2. Oilseeds and Free Oil Extraction

Soybean, sunflower and cardoon seeds were obtained from a local feed mill, and their chemical composition has been already reported [19]. To obtain the free oils, a representative sample of each oilseed was subjected to extraction with ethyl ether for 2 h using an ANKOM XT10 Extraction System (ANKOM Technology Corporation, Fairport, NY, USA). After extraction, the free oils were collected and kept at 4 °C until incorporation into the experimental diets. The defatted meals obtained were immediately dried at 40 °C for 24 h and stored at 4 °C until analyzed and used as ingredients for experimental diets.

4.3. Experimental Diets

Six mixed diets were formulated for each oilseed (18 diets in total), three representatives of those typically fed to dairy ewes and three of those fed to fattening lambs. Within each type of diet (dairy vs. fattening) and for each oilseed, there were three experimental treatments: (i) a basal diet without additional lipid from the experimental oilseed (control), (ii) including the whole oilseed, and (iii) including the corresponding free oil together with its defatted meal. Both the whole oilseeds and the free oils were incorporated to achieve final dietary lipid concentrations of approximately 5.0–5.5% and 6.0–6.5% of DM for dairy and fattening diets, respectively. Diets including the free oils also contained an amount of the corresponding defatted meal equivalent to that provided by the whole seed. Within each type of diet, all diets were formulated to have similar levels of crude protein. The ingredients and chemical composition of the experimental diets are presented in Table 3.
The dairy diets were composed of 40% forage (32% triticale hay and 8% alfalfa hay) and 60% concentrate, whereas the fattening diets contained 15% of wheat straw and 85% of concentrate. Before preparing the experimental diets, all feed ingredients were individually ground to pass a 1 mm sieve using a Culatti Micro Impact mill (Culatti AG, Steinen, Switzerland). The whole oilseeds had a high lipid content and therefore they were kept at 4 °C overnight before grinding to prevent clogging of the sieve.

4.4. In Vitro Fermentation and Methane Production of Experimental Diets

The rumen inoculum used for the in vitro incubations was obtained from four rumen-cannulated Lacaune ewes (65.2 ± 2.15 kg body weight; 4 years old). The animals were individually housed on straw bedding and had unrestricted access to fresh water throughout the study. To obtain inoculum containing microbial populations representative of ruminants fed either dairy or fattening diets, the ewes were fed two different diets (dairy or fattening) in a crossover design comprising two 21 d experimental periods. During each period, two ewes received each diet. The dairy diet was composed of 40% forage and 60% commercial concentrate, whereas the fattening diet contained 15% wheat straw and 85% commercial concentrate. The diets were fed twice daily (09:00 and 16:00 h) at a daily level of 40 g DM/(kg body weight)0.75. On the last day of each experimental period, rumen content from each ewe was collected immediately before the afternoon feeding and filtered through four layers of cheesecloth. The four ruminal fluids were maintained separately and mixed 1:4 with the culture medium of Goering and Van Soest [37] under continuous CO2 flushing at 39 °C.
Samples of each experimental diet (350 mg DM) were weighed into 120 mL serum bottles. The amount of diet DM in the bottles containing the free oils diets was reduced to account for the weight of the added free oil (3.30 and 4.70% of total diet DM for dairy and fattening diets, respectively). The supply of the free oils was done immediately before the in vitro incubations. The free oils were dissolved in ethanol and 70 µL of the corresponding solution were pipetted directly into each bottle. The rest of the bottles received 70 µL of ethanol. Thirty-five mL of the rumen fluid-culture medium mixture were dispensed into each bottle using a peristaltic pump (Watson-Marlow 520U, Watson-Marlow, Falmouth, UK). Bottles were immediately sealed with rubber stoppers and incubated at 39 °C for 17 h. Within each experimental period, the bottles containing the dairy diets were inoculated with ruminal fluid from sheep receiving the dairy diet, whereas those with fattening diets were incubated with inoculum from sheep fed the fattening diet.
In each experimental period, 2 bottles were incubated for each experimental diet and inoculum (144 bottles in total; 72 bottles in each incubation run). One bottle was used for determining SCFA and NH3-N after 6 and 17 h of incubation. At each sampling time, bottles were hand-shaken and 1.5 mL of their content was taken with a syringe, mixed with 40 μL of 10% H2SO4 and immediately frozen at −20 °C until analysis. The other bottle was used to determine total gas and CH4 production after 17 h. Total gas production was measured using a pressure transducer (HD2304.0; Delta Ohm, Padua, Italy) connected to a calibrated syringe. About 10 mL of gas was collected into evacuated tubes (Terumo Europe N.V., Leuven, Belgium) for CH4 analysis. The production of CH4 was measured only at the end of the incubation because methanogenesis is low at earlier incubation times [38]. In addition, blank bottles containing inoculum but no substrate (2 per rumen inoculum) were included to account for the fermentation of the substrates added with the ruminal fluid and were used for the corresponding corrections of the measured parameters.

4.5. Chemical Analyses

The chemical composition of the whole oilseeds, defatted meals, and experimental diets was determined according to AOAC [39] procedures (methods 934.01, 942.05, and 984.13 for DM, ash and nitrogen, respectively), and crude protein was calculated as nitrogen × 6.25. Fiber fractions (neutral detergent fiber, acid detergent fiber, and acid detergent lignin) were determined sequentially following the procedures of Van Soest et al. [40] using an ANKOM 220 Fiber Analyzer (ANKOM Technology Corporation, Fairport, NY, USA). Sodium sulfite and heat-stable α-amylase were included in neutral detergent fiber analysis, and all values are expressed exclusive of residual ash.
For the analysis of FA profile in the whole seeds, lipids were extracted as described by Sukhija and Palmquist [41], and FA methyl esters were prepared by transesterification with sodium methoxide in methanol followed by acid methylation [42]. The analysis of FA content in both the whole seeds and the extracted free oils was carried out using a gas chromatograph (HP 6890 Series GC System, Hewlett Packard Co., Avondale, PA, USA) fitted with a flame ionization detector and an HP-Innowax capillary column (30 m × 0.316 mm × 0.25 μm; J&W Scientific/Agilent Technologies, Santa Clara, CA, USA) and nitrogen as the carrier gas. Values for individual FA were expressed as a percentage of total identified FA.
Frozen samples of bottles’ content were thawed at 4 °C, centrifuged (13,000× g for 15 min at 4 °C), and the supernatant was used to determine SCFA and NH3-N concentrations [29]. Short-chain FAs (acetate, propionate, butyrate, isobutyrate, valerate and isovalerate) were quantified by gas chromatography [29] using a Shimadzu GC-2010 gas chromatograph (Shimadzu Europa GmbH, Duisburg, Germany) fitted with a flame ionization detector and a TR-FFAP column (30 m × 0.53 mm × 1 μm; Supelco, Madrid, Spain), and NH3-N concentration was determined colorimetrically by the phenol-hypochlorite method [43] using an Epoch microplate spectrophotometer (BioTek Instruments, Swindon, UK). Gas samples were analyzed for CH4 concentration [38] using a Shimadzu GC-14B gas chromatograph (Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto, Japan) equipped with a flame ionization detector and a Carboxen 1000 packed column (Supelco, Madrid, Spain). Helium was used as the carrier gas for both SCFA and CH4 analyses.

4.6. Calculations and Statistical Analyses

The amount of fermented organic matter in each bottle was estimated from individual SCFA (acetate, propionate and butyrate) production as proposed by Demeyer [44]. Statistical analyses were performed separately for each oilseed (soybean, sunflower, and cardoon) using the MIXED procedure of the SAS software version 9.3 (SAS Inst. Inc.: Cary, NC, USA) [45]. Within each oilseed, the statistical model included the fixed effects of diet type (dairy vs. fattening), LS treatment (including no extra lipid, whole oilseeds or free oils) and their interaction, and the random effects of the inoculum and the experimental period. The effect of LS treatment was partitioned into two orthogonal contrasts: C1: no extra lipid inclusion vs. including whole oilseeds and free oils, and C2: whole oilseeds vs. free oils. Statistical significance was declared at p < 0.05, whereas values of 0.05 ≤ p < 0.10 were considered trends.

5. Conclusions

Regardless of the basal diet (dairy vs. fattening), there were no effects of LS from soybean, sunflower or cardoon on in vitro ruminal fermentation indicating that dietary lipid levels up to 6.0–6.5% of DM did not have detrimental effects on fermentation. The influence of the form of LS (whole oilseeds vs. free oils) depended on the oilseed, as replacing the whole oilseed with the corresponding free oil did not alter rumen fermentation characteristics for soybean, whereas some fermentation parameters were affected for sunflower and cardoon seeds, indicating variable responses depending on the oilseed. However, no interactions between LS and the diet type were observed for any of the oilseeds tested.
Regarding methane production, supplying the lipids as free oils rather than as whole seeds reduced CH4/total SCFA ratios for sunflower and cardoon, while free cardoon oil also reduced CH4 production and the proportion of CH4 in the gas produced. These results indicate that free oils from certain oilseeds, particularly cardoon, have potential for methane mitigation. However, these effects were observed under in vitro conditions and should not be interpreted as direct measurements of animal methane emissions. In vitro methane production can be used as a screening tool to compare different lipid sources and dietary conditions and to identify strategies with potential for reducing methane emissions, which should subsequently be evaluated in vivo.

Author Contributions

Conceptualization, J.G. and M.D.C.; methodology, J.G. and M.D.C.; formal analysis, M.D.C. and J.M.A.; investigation, J.G., M.R. and M.D.C.; resources, J.G. and M.D.C.; data curation, M.D.C. and J.M.A.; writing—original draft preparation, J.M.A. and M.R.; writing—review and editing, J.G., M.D.C.; supervision, J.G. and M.D.C.; project administration, J.G.; funding acquisition, J.G. and M.D.C. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Spanish Ministry of Economy and Competitiveness (Project AGL2012-31064).

Institutional Review Board Statement

The care of the ewes and the rumen content extraction followed the European regulations for experimental animal protection and all protocols were approved by the Institutional Animal Care and Use Committee of the Comunidad Autónoma de Madrid (PROEX 035/17). Approval date in: 4 April 2017.

Data Availability Statement

All data can be requested from the authors.

Acknowledgments

We are thankful to the laboratory of Clemente López-Bote for conducting the analysis of fatty acids of seeds and free oils.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CACCardoon diet with no lipids added (control)
CASCardoon diet including cardoon seeds
CAOCardoon diet including cardoon-free oil
DMDry matter
FAFatty acids
LSLipid supply
SBCSoybean diet with no lipids added (control)
SBOSoybean diet including soybean-free oil
SBSSoybean diet including soybean seeds
SCFAShort chain fatty acids
SEMStandard error of the mean
SFCSunflower diet with no lipids added (control)
SFOSunflower diet including sunflower-free oil
SFSSunflower diet including sunflower seeds

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