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15 July 2026

14 Pages

In Vitro Evaluation of Cardoon as a Replacement for Soybean and Sunflower Seeds in Ruminant Diets: Effects on Ruminal Fermentation and Methane Production

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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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Fermentation2026, 12(7), 337;https://doi.org/10.3390/fermentation12070337
This article belongs to the Section Animal and Feed Fermentation
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Abstract

Including oilseeds in the diet is a widely studied strategy to reduce CH4 emissions in ruminants, but the effects on ruminal fermentation depend on oilseed composition. This study compared the effects of including three oilseeds (soybean, sunflower and cardoon) differing in fiber content and lignification on in vitro ruminal fermentation and CH4 production of dairy and fattening diets. Oilseeds were included to achieve two dietary lipid levels: low (3.5–4.0%) and high (5.5–6.0%). Diets were incubated in vitro with buffered ruminal fluid from sheep for 17 h. The high lipid level reduced microbial activity only in the fattening diet, particularly after 6 h of incubation, although this effect largely disappeared by 17 h. The most consistent differences among oilseeds were observed in NH3-N concentrations, with soybean yielding lower values than sunflower and cardoon, suggesting reduced protein degradation due to soybean processing. In the fattening diet, cardoon at the low lipid level reduced CH4 production compared with soybean and sunflower (44.9, 50.5 and 52.3 mL/g incubated dry matter) but also reduced total volatile fatty acid production and fermented organic matter, whereas no differences were detected for dairy diets. At equal lipid supply, sunflower and cardoon can replace soybean in dairy diets without negatively affecting ruminal fermentation, whereas cardoon may reduce CH4 production and fermentation in fattening diets at low lipid levels.

1. Introduction

Reducing CH4 emissions in ruminant production has become an important objective both to mitigate its greenhouse effect and to improve dietary energy efficiency. Among the proposed mitigation strategies, the inclusion of dietary lipids, particularly through oilseeds, has been widely explored as a practical and feasible nutritional approach [1]. However, although lipid supplementation may decrease CH4 production, excessive lipid levels can negatively affect ruminal fermentation and impair fiber digestion, limiting its practical application [1,2]. Within this context, oilseeds such as cardoon (Cynara cardunculus) are of particular interest because of their dual role as a source of oil and fibrous hull material. Cardoon is a perennial Mediterranean crop well adapted to drought-prone environments and low-input production systems, characteristics that may increase its agronomic relevance under future climate-change scenarios [3]. In addition to its use as an energy and industrial crop, its seeds contain relatively high concentrations of oil with a fatty acid (FA) profile comparable to that of sunflower oil [4]. Furthermore, cardoon meal has moderate protein content and may represent a locally sourced protein ingredient for ruminant feeding [4]. Despite these potential advantages, information regarding the nutritive value of cardoon seeds and their effects on ruminal fermentation and CH4 production remains scarce.
The antimethanogenic effects of lipid supplementation are mainly related to the action of free FA released during lipid hydrolysis. These compounds interact with the cell membranes of rumen microorganisms, reducing nutrient uptake and exerting toxic effects on protozoa, methanogenic archaea and Gram-positive bacteria [5,6,7]. In addition, unsaturated FA may decrease CH4 production because their biohydrogenation competes with methanogenic archaea for hydrogen utilization [8,9]. However, free FA tend to adhere to feed particles and may also interfere with microbial colonization, particularly affecting primary colonizers such as cellulolytic bacteria. Consequently, reductions in fiber degradation and overall diet digestibility may occur, although these effects can be influenced by the type of diet [1,10]. The magnitude of these effects can also depend on the FA profile of the oilseed and its structural characteristics. For example, sunflower seed hulls are characterized by a waxy epidermis and relatively high lignin concentrations, features associated with reduced microbial colonization [11]. Such characteristics may attenuate the contact between free FA and ruminal microorganisms, thereby reducing their negative effects on bacterial growth and fiber degradation. Likewise, the hull-to-kernel ratio may influence the response to lipid supplementation. In small seeds such as cardoon, this ratio is relatively high, suggesting that adverse effects of FA on ruminal fermentation may be less pronounced than in oilseeds with a lower proportion of hull material. A greater proportion of hulls may reduce the immediate exposure of ruminal microorganisms to free FA released during lipid hydrolysis by limiting the accessibility of the lipid-rich kernel, thereby moderating their inhibitory effects on microbial colonization and fiber degradation. Nevertheless, this should be regarded as a working hypothesis rather than a general rule, as the extent to which hull material modulates lipid availability is also likely to depend on factors such as seed structure, the degree of lignification, seed processing, and the characteristics of the basal diet, all of which may influence microbial access to lipids and the subsequent fermentation response. Consequently, the balance between the potentially protective effect of the hull and the inhibitory effect of free FA is expected to vary with feeding conditions and should not be assumed to be consistent across different diets. To our knowledge, this hypothesis has not been directly evaluated in cardoon seeds. Supporting this possibility, Cajarville et al. [12] reported that including up to 30% whole cardoon seeds in a mixed diet (1:1 alfalfa hay) did not negatively affect fiber digestibility in sheep. Moreover, supplementation with cardoon seeds reduced the acetate-to-propionate ratio, a fermentation shift commonly associated with decreased CH4 production [13]. Although these findings suggest that cardoon seeds may represent a promising lipid source for mitigating methane emissions without compromising ruminal fermentation, the study evaluated cardoon in isolation and did not compare its effects with those of other oilseeds differing in structural characteristics but providing similar amounts of dietary lipid. Therefore, it remains unclear whether the response is specific to cardoon or reflects more general effects of lipid supplementation.
The aim of this study was therefore to compare the effects of three oilseeds rich in unsaturated FA (soybean, sunflower and cardoon), but differing in fiber content and degree of lignification, on in vitro ruminal fermentation and CH4 production of diets representative of those fed to dairy and fattening ruminants. We therefore hypothesized that differences in seed structure would influence the balance between the antimethanogenic effects of lipids and their potential negative effects on ruminal fermentation, with cardoon seeds expected to show a more favorable response because of their higher hull-to-kernel ratio. However, this response was expected to depend on the dietary characteristics.

2. Materials and Methods

2.1. Oilseeds and Experimental Diets

Samples of the three oilseeds (soybean, sunflower and cardoon) were obtained at a local feed mill. Half of the sample of each oilseed was subjected to an extraction of the oil with ethyl ether for 2 h using an ANKOMXT10 Extraction system (ANKOM Technology Corporation, Fairport, NY, USA). The resulting extraction meal was dried at 40 °C for 24 h and stored until being used in the formulation of the diets incubated in vitro. Chemical composition of the whole oilseeds and their extraction meals is shown in Table 1, whereas Table 2 shows the FA profile of the whole seeds.
Table 1. Chemical composition (g/kg dry matter) of the whole oilseeds (soybean, sunflower and cardoon), the extracted meals of each oilseed, and of the forage, cereals and wheat straw used as ingredients in the experimental diets incubated in vitro with ruminal fluid from sheep 1.
Table 2. Fatty acid (FA) profile (g/100 g of analyzed FA) of the whole oilseeds.

2.2. Experimental Diets

A total of 12 mixed diets representative of those fed to dairy (6 diets) and fattening ruminants (6 diets) were formulated. All dietary ingredients were ground to pass a 1 mm screen before preparing the diets. Dairy diets were composed of 40% of forage and 60% of concentrate, and in all of them, the forage was a 1:4 mix of alfalfa hay and triticale hay. Fattening diets were composed of 15% wheat straw and 85% of concentrate. In all diets, the concentrate contained a 1:1 mixture of corn and wheat as cereals, and one of each tested oilseeds. The oilseeds were included in the diets to reach two different lipid levels (LL) in the diet: low (3.5–4.0% fat) and high (5.5–6.0% fat). To achieve isonitrogenous diets, the extraction meal of the corresponding oilseed was included in some diets. Feed ingredients and chemical composition of the 12 experimental diets are given in Table 3.
Table 3. Feed ingredients (g/kg) and chemical composition (g/kg dry matter) of the experimental diets containing the oilseeds at two levels of lipid inclusion.

2.3. Donor Animals and In Vitro Incubations

Animals’ management and rumen content withdrawal were carried out in accordance with the European guidelines for experimental animal protection and all the experimental procedures were approved by the Institutional Animal Care and Use Committee of the Comunidad Autónoma de Madrid (Approval number PROEX 035/17).
Four adult rumen-fistulated Lacaune ewes (65.2 ± 2.15 kg body weight and four years old) were used as donors of rumen fluid (inoculum) for the in vitro incubations. Sheep were housed in individual floor pens with straw bedding and had free access to fresh water throughout the experimental period. To obtain rumen inocula representative of different production systems, sheep were fed either a diet representative of those fed to dairy or to fattening ruminants in a cross-over design with two 21-d periods. In each period, two sheep were fed each diet. The dairy diet consisted of 40% forage and 60% of a commercial concentrate (fresh matter basis), whereas the fattening diet consisted of 15% wheat straw and 85% of a commercial concentrate. Both diets were offered at a fixed rate of 40 g dry matter (DM)/kg body weight0.75 per day and distributed in two equal meals (9:00 and 16:00 h). Rumen content from each donor sheep was obtained immediately before the afternoon feeding and strained through 4 layers of cheesecloth. The rumen fluid from each sheep was mixed independently with the buffer solution of Goering and Van Soest [14] in a 1:4 ratio (v/v) at 39 °C under continuous flushing with CO2.
Samples (350 mg of DM) of each experimental diet were carefully weighed into 120 mL glass vials. Each vial was filled up with 35 mL of the ruminal fluid-buffer mixture using a peristaltic pump (Watson Marlow 520U, Watson Marlow, Falmouth, UK) and was sealed with a rubber stopper. In each period, the vials containing the dairy diets were inoculated with the ruminal fluid from the sheep fed the dairy diet, whereas those containing the fattening diets were inoculated with the ruminal fluid from the sheep fed the fattening diet. Vials were incubated at 39 °C for 17 h, which is considered the average rumen retention time for medium-producing ruminants [15] (6%/h rumen passage rate). Two vials were incubated for each diet and inoculum and each was used for different analytical purposes. One vial was used for volatile fatty acids (VFA) and NH3-N determination and was sampled at 6 and 17 h. The other vial was used for total gas production and CH4 measurement at 17 h of incubation. Gas and CH4 production were not measured at 6 h because CH4 generation at early incubation times is low, whereas 17 h better reflects overall substrate degradation and methanogenic activity. Samples of 1.5 mL of vials’ content were taken with a syringe, mixed with 40 μL of 10% H2SO4 (in volume) to cease fermentation, and immediately frozen at −20 °C until VFA and NH3-N analysis. Total gas production was measured using a pressure transducer (HD2304.0 pressure gauge, Delta Ohm, Padua, Italy) and a calibrated syringe. A gas sample (10 mL) was taken in a vacuum tube (Terumo Europe N.V., Leuven, Belgium) for CH4 analysis. Blank vials (no substrate; two per inoculum) were included to correct the gas production values for gas release from endogenous substrates supplied by the ruminal fluid.

2.4. Chemical Analyses

Dry matter (ID 934.01), ash (ID 942.05) and nitrogen (N; ID 984.13) content of feeds and experimental diets were determined according to the Association of Official Analytical Chemists [16]. Crude protein (CP) content was calculated as N × 6.25. Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) analyses were performed sequentially according to Van Soest et al. [17] using an ANKOM220 Fiber Analyzer unit (ANKOM Technology Corporation, Fairport, NY, USA). Sodium sulfite and heat-stable amylase were used in the analysis of NDF, and all values were expressed exclusive of residual ash.
The extraction of lipids from oilseeds (200 mg) followed the procedure of Sukhija and Palmquist [18]. Fatty acid methyl esters were then prepared by transesterification using a mixture of sodium methylate–methanol and were methylated in the presence of sulfuric acid as described by Segura and López-Bote [19]. The FA methyl esters were separated 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 polyethylene glycol column (30 m × 0.316 mm × 0.25 μm; J&W Scientific/Agilent Technologies, Santa Clara, CA, USA). Nitrogen was used as a carrier gas and results were expressed as a percentage of total FA identified.
Samples for measuring NH3-N and VFA in the content of the vials were defrosted at 4 °C, centrifuged (13,000× g; 15 min; 4 °C) and the supernatant used for the analysis of VFA and NH3-N. Concentrations of VFA were measured by gas chromatography as described by García-Martínez et al. [20] using a Shimadzu GC 2010 chromatograph (Shimadzu Europa GmbH, Duisburg, Germany) equipped with a flame ionization detector and a TR-FFAP column (30 m × 0.53 mm × 1 μm; Supelco, Madrid, Spain). Concentrations of NH3-N were determined by the phenol-hypochlorite method [21] using an Epoch microplate spectrophotometer (Bioteck Instruments, Swindon, UK). Analysis of CH4 was performed following the procedure of Martínez et al. [22], using a gas chromatograph (Shimadzu GC 14B; Shimadzu Europa GmbH, Duisburg, Germany) fitted with a flame ionization detector and a column packed with Carboxen 1000 (Supelco, Madrid, Spain). For both SCFA and CH4 analysis, helium was used as a carrier gas.

2.5. Calculations and Statistical Analyses

The amounts of VFA produced in each vial were calculated by subtracting the amount initially added with the rumen fluid and buffer mixture from those measured at each sampling time (6 and 17 h). Gas production values were corrected for gas released from the corresponding blank incubations. The amount of fermented OM (FOM) in each vial was calculated from acetate, propionate and butyrate production according to the equation proposed by Demeyer [23].
The effects of the inclusion of the oilseeds at the two LL on ruminal fermentation parameters and CH4 production were analyzed independently for dairy and fattening diets. Data were analyzed by analysis of variance, considering the fixed effects of the oilseeds, LL, and the oilseeds × LL interaction, and the random effects of the experimental period and the inoculum (rumen fluid and buffer mixture). When a significant effect of the oilseed was detected, differences among means within the same LL level were assessed by Tukey’s multiple comparison test. Statistical analyses were performed using the PROC MIXED procedure of SAS version 9.3 (SAS Institute Inc., Cary, NC, USA) [24]. The experimental unit was defined as the diet × inoculum combination, and individual incubation vials were not considered independent experimental replicates. Significance was declared at p < 0.05, whereas 0.05 < p < 0.10 values were considered as a trend.

3. Results

3.1. Chemical Composition of Oilseeds and Experimental Diets

As expected, the three oilseeds differed in NDF content and fiber lignification, which was low for soybean, medium-high for sunflower and high for cardoon seeds (2.91, 18.4 and 22.0%, respectively; Table 1). The FA profile of soybean and cardoon seeds was rather similar (Table 2), except for C18:2 n-6, which was lower in soybean than in cardoon (50.1 vs. 58.3%), and C18:3 n-3, which was almost absent in cardoon seed (0.1%) compared with soybean seed (5.70%). However, the proportions of total saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) FA were comparable for both oilseeds. The FA profile of sunflower seeds showed lower proportions of C18:1n-9 (20.7%) and greater of C18:2n-6 (68.2%) compared with the other two oilseeds, resulting in greater proportions of total PUFA (55.8, 58.3 and 68.3% of total FA for soybean, sunflower and cardoon seeds, respectively) and lower of both SFA and MUFA.
Due to the differences in lipid content between the oilseeds, it was necessary to include about 2.2 and 1.3 times more soybean and cardoon seeds (averaged values for dairy and fattening diets), respectively, than sunflower seeds to achieve the two prefixed LL in the diets (Table 3). Variations in NDF and ADF content in the oilseeds resulted in differences in fiber content in the diets, and levels of NDF in sunflower and cardoon diets were, on average, 1.1% greater than in the diets containing soybean seeds for both dairy and fattening diets. Similarly, ADF content was 1.1 and 1.2% greater for sunflower and cardoon diets, respectively, than for soybean diets for dairy diets, and 1.3 and 1.5% greater for fattening diets. As expected, within each type (dairy vs. fattening), diets were isonitrogenous.

3.2. Fermentation of Dairy Diets

No significant LL × oilseed interactions (p > 0.05) were observed for dairy diets at either 6 or 17 h of incubation (Table 4). Likewise, the lipid level did not significantly affect any of the fermentation parameters evaluated. The lowest p-value associated with the effect of LL was 0.210 (total VFA at 6 h of incubation), indicating that none of the measured variables was significantly influenced by increasing the dietary lipid level. Differences among oilseeds were limited. At 6 h of incubation, diets containing sunflower showed lower acetate/propionate ratios (p = 0.022 and p = 0.018 for the low and high LL, respectively) than those including cardoon at both LL. In addition, at the high LL, sunflower diets showed a greater proportion of propionate than soybean diets, whereas cardoon diets showed intermediate values. Furthermore, NH3-N concentrations differed among substrates, with soybean and cardoon diets showing the lowest and highest values, respectively, at both LL, while sunflower diets showed intermediate values. At 17 h, differences among oilseeds in NH3-N concentrations were maintained, and isobutyrate and isovalerate proportions were minimum in soybean diets and maximum in cardoon diets. No differences among oilseeds were detected in gas, CH4, VFA production or the amount of FOM at any incubation time.
Table 4. Effect of including soybean (DSB), sunflower (DSF) or cardoon (DCA) oilseeds at two lipid levels (LL) in dairy diets on ruminal fermentation parameters after 6 and 17 h of in vitro incubation with ruminal fluid from sheep.

3.3. Fermentation of Fattening Diets

No significant LL × oilseed interactions were detected for fattening diets, except for a tendency (p = 0.092) in the CH4/total VFA ratio after 17 h of incubation (Table 5). Increasing the LL in the diet reduced (p < 0.001) total VFA production and the amount of FOM at 6 h of incubation, and tended (p = 0.070) to increase NH3-N concentrations. In contrast, after 17 h of incubation, the effect of LL was much less pronounced and was limited to a small but significant reduction (p = 0.018; about 1.5%) in gas production.
Table 5. Effect of including soybean (FSB), sunflower (FSF) or cardoon (FCA) oilseeds at two lipid levels (LL) in fattening diets on ruminal fermentation parameters after 6 and 17 h of in vitro incubation with ruminal fluid from sheep.
Differences among oilseeds were more evident than those associated with LL. At 6 h of incubation, sunflower diets showed greater VFA production than those with cardoon at the low LL (p = 0.001) and greater than soybean diets at the low LL (p < 0.001), whereas at the high LL, VFA production was greater in soybean than in cardoon diets (p < 0.001). Likewise, the FOM was greater in sunflower than in cardoon (p = 0.023) and soybean diets (p = 0.001) at the low LL, whereas at the high LL, sunflower diets showed greater FOM than soybean diets (p = 0.015). No differences in the molar proportions of individual VFA were detected among oilseeds. After 17 h of incubation, sunflower diets continued to show greater VFA production (p = 0.045 and p = 0.017 for the low and high LL, respectively) and FOM (p = 0.040 and p = 0.019 for the low and high LL, respectively) than cardoon diets. However, no differences between sunflower and soybean diets were detected except for isovalerate proportion, which was greater in sunflower diets (p < 0.001 and p = 0.005 for the low and high LL, respectively).
Gas production was lower in cardoon diets than in both sunflower and soybean diets (p < 0.001 for both diets and at both LL). Likewise, CH4 production was lower in cardoon diets than in sunflower and soybean diets (p = 0.032, respectively) at the low LL, whereas no differences were detected at the high LL. Propionate proportions were also lower in cardoon than in sunflower diets (p = 0.048 and p = 0.044 for the low and high LL, respectively). Cardoon diets also showed greater isovalerate proportions than both sunflower (p = 0.001 and p < 0.001 for the low and high LL, respectively) and soybean diets (p < 0.001 at both LL), whereas sunflower diets also showed greater isovalerate proportions than soybean diets (p < 0.001 and p = 0.005 for the low and high LL, respectively). Finally, at the high LL, NH3-N concentrations were greater in cardoon than in soybean diets (p = 0.018), whereas no differences were detected between cardoon and sunflower diets.

4. Discussion

4.1. Chemical Composition of Oilseeds and Experimental Diets

Despite differences in fiber content and composition, soybean and cardoon seeds showed relatively similar FA profiles, with comparable proportions of SFA, MUFA and PUFA. The main differences were the greater proportion of C18:2n-6 and the almost complete absence of C18:3n-3 in cardoon seeds. The FA profile observed for cardoon seeds agrees with previous reports [25,26], which identified C18:1n-9 and C18:2n-6 as the predominant FA. In accordance with earlier studies [27,28,29], both soybean and sunflower seeds contained a high proportion of unsaturated FA (82.7 and 89.7%, respectively), with C18:2n-6 (50.1 and 68.2%), C18:1n-9 (25.4 and 20.7%) and C16:0 (13.1 and 6.4%) being the most abundant.

4.2. Fermentation of Dairy and Fattening Diets

Results from previous studies [30,31] highlighted the importance of using ruminal fluid from donors fed a diet similar to that being incubated in in vitro systems, as the characteristics of the donor diet can influence the in vitro fermentation results [31]. Accordingly, dairy and fattening diets were incubated with ruminal inocula collected, in each case, from sheep previously adapted to similar diets, and therefore, the results for each type of diet were analyzed independently. Moreover, both LL were slightly different in dairy and fattening diets, which would also preclude a joint analysis of the data from both types of diets.
The absence of LL × oilseed interactions for both diets indicates that the effects of increasing dietary LL on ruminal fermentation were similar for all tested oilseeds. The absence of LL effects on any fermentation parameter of the dairy diet indicates that the high LL had no negative impact on ruminal fermentation, although previous studies in dairy cows reported negative effects on DM intake, ruminal fermentation and milk production when LL in the diet was above 5.0% [32,33]. In contrast, other work [34] showed no negative effects of including 6% of soybean oil in the diet of dairy sheep on feed intake, milk yield and composition, or in the in vitro rumen fermentation of the diet. In contrast, increasing LL in the fattening diet reduced total VFA production and FOM after 6 h of incubation, suggesting a transient inhibition of microbial activity in concentrate-rich diets because this inhibitory effect almost disappeared after 17 h of incubation, when only small reductions in gas production were detected. As discussed by Fiorentini et al. [35], the effects of lipids on rumen fermentation are highly variable and depend not only on the nature and concentration of the lipids in the diet but also on diet composition (forage, concentrate, minerals, etc.).
Differences among seeds in CH4 production were only observed in the fattening diets, with cardoon diets showing the lowest values. This reduction was accompanied by lower total VFA production and FOM, indicating reduced overall fermentation. However, the reduction in CH4 production with cardoon seeds was proportionally greater than the reductions observed for total VFA and FOM, suggesting that mechanisms other than reduced overall fermentation may also contribute to this response. Nevertheless, this interpretation should be considered with caution, as other fermentation indices do not provide consistent evidence for a specific antimethanogenic effect. One possible explanation relates to the greater proportion of concentrate in the fattening diets. The decrease in forage proportion from 40% in the dairy diets to 15% in the fattening diets may have increased the adsorption of free FA onto concentrate particles. As previously discussed [1], the CH4-suppressing effects of fats may be more pronounced with high-concentrate diets because in high-forage diets, the FA can readily be adsorbed onto the fiber particles and this may lower the effective inhibitory concentrations in the rumen fluid or adsorption onto bacteria, including methanogens. Contrary to the expectations based on the different fiber composition of the oilseeds, sunflower and cardoon seeds in the dairy diets did not consistently reduce diet fermentation compared with soybean seeds, as indicated by the similar VFA production and FOM observed at both incubation times. This may have been due to a compensatory effect associated with the greater ruminal DM degradability of sunflower and cardoon seeds [12,36], compared with toasted soybean seeds [37]. For fattening diets, cardoon reduced total VFA production and the amount of FOM compared with sunflower at both LL, but differences with soybean diets were evident only at the high LL. As discussed above, this could be related to the greater negative effect of free FA in diets rich in high-fermentable substrates [1].
Within each type of diet (dairy or fattening), differences in NH3-N and branched-chain VFA concentrations further support variations in ruminal protein degradability among oilseeds. At 17 h of incubation, soybean diets consistently showed lower NH3-N concentrations and proportions of isobutyrate and isovalerate than cardoon diets, whereas sunflower diets had intermediate values. Because these branched-chain VFA are generated from microbial degradation of branched-chain amino acids [38], their concentrations can indicate lower ruminal protein degradation for toasted soybean compared with raw sunflower and cardoon seeds. Previous studies reported protein degradability values around 58–60% for toasted soybean seeds [37], whereas higher values (>80%) have been determined for sunflower and cardoon seeds [12,36].
Protein degradation may also contribute indirectly to CH4 production through hydrogen generation associated with the fermentation of amino acids [39]. Therefore, the greater ruminal degradability of sunflower and cardoon protein could contribute to CH4 generation and may have partially masked the direct inhibitory effects of their FA on methanogenesis.
The CH4 production generally followed the observed fermentation pattern. The absence of differences among substrates in CH4 production in the dairy diets agrees well with the similar VFA profile observed for all diets, as CH4 mitigation is frequently associated with increased propionate formation [20]. However, this was not observed in the fattening diets containing cardoon seeds that showed both reduced CH4 production and lower propionate proportions than the diets with soybean and sunflower. These results, together with the similar CH4/total VFA ratios in all fattening diets, suggest that the lower fermentability of cardoon seeds contributed substantially to the reduction in CH4 production. However, when considered together with the proportionally greater reduction in CH4 than in total VFA production and FOM, the available evidence is not sufficient to distinguish unequivocally between a reduction in fermentability alone and the contribution of additional antimethanogenic mechanisms. An additional limitation of the present study is that gas and CH4 production were measured only at the end of the 17 h incubation period. Consequently, the temporal development of gas production and methanogenesis could not be assessed, and potential differences among oilseeds during the early stages of fermentation may have remained undetected. For all diets, values of CH4 proportion in total gas and CH4/total VFA ratios were within the range previously reported for in vitro ruminal fermentation of diets of different compositions [20].

5. Conclusions

Altogether, the results indicate that sunflower or cardoon seeds can replace soybean seeds in dairy diets without major negative effects on in vitro ruminal fermentation despite their greater fiber content. Nevertheless, the soybean diets consistently resulted in lower NH3-N concentrations and lower branched-chain VFA proportions than the other oilseeds, indicating differences in protein degradation among oilseeds despite the similar overall fermentation pattern.
In fattening diets, sunflower seeds did not affect ruminal fermentation activity compared with soybean seeds. In contrast, cardoon seeds reduced CH4 production only at the low lipid level, but also reduced total VFA production and FOM, indicating lower fermentation. These effects were likely related to the high content of lignified fiber in cardoon seeds, which may have limited substrate fermentability. Further in vivo studies are needed to confirm these findings and to evaluate the practical implications of including cardoon seeds in ruminant diets. In addition, practical recommendations for the inclusion of oilseeds in ruminant diets should consider not only the total dietary lipid concentration but also the fatty acid profile of the oilseed, as this may impose additional nutritional constraints under specific production conditions.

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

Institutional Review Board Statement

The ewes were cared for according to the European regulations for experimental animal protection. Protocols for rumen content extraction were approved by the Institutional Animal Care and Use Committee of the Comunidad Autónoma de Madrid (Approval number PROEX 035/17; Approval date in: 2017-04-04).

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.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ADFAcid detergent fiber
ADLAcid detergent lignin
CPCrude protein
DCADairy diet including cardoon seeds
DCA1Dairy diet including cardoon seeds to reach about 3.5% fat in the diet
DCA2Dairy diet including cardoon seeds to reach about 5.5% fat in the diet
DM Dry matter
DSBDairy diet including soybean seeds
DSB1Dairy diet including soybean seeds to reach about 3.5% fat in the diet
DSB2Dairy diet including soybean seeds to reach about 5.5% fat in the diet
DSFDairy diet including sunflower seeds
DSF1Dairy diet including sunflower seeds to reach about 3.5% fat in the diet
DSF2Dairy diet including sunflower seeds to reach about 5.5% fat in the diet
EEEther extract
FAFatty acids
FCA1Fattening diet including cardoon seeds to reach about 4.0% fat in the diet
FCA2Fattening diet including cardoon seeds to reach about 6.0% fat in the diet
FOMFermentable organic matter
FSB1Fattening diet including soybean seeds to reach about 4.0% fat in the diet
FSB2Fattening diet including soybean seeds to reach about 6.0% fat in the diet
FSF1Fattening diet including sunflower seeds to reach about 4.0% fat in the diet
FSF2Fattening diet including sunflower seeds to reach about 6.0% fat in the diet
LLLipid level
MUFAMonounsaturated fatty acids
NDFNeutral detergent fiber
OMOrganic matter
PUFAPolyunsaturated fatty acids
SFASaturated fatty acids
SEMStandard error of the mean
VFAVolatile fatty acids

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