Impact of drought stress on vitamin C and anthocyanin content in cultivated lettuces (Lactuca sativa L.) and wild relatives (Lactuca spp.).

Introduction: Lettuce production and quality could be seriously affected by the increasingly limited water resources. Methods: The effect of drought on the content of two antioxidant compounds, vitamin C and anthocyanins, in five cultivated lettuces and two wild relatives was assessed for 2 years. Results and discusion: In leaf samples, Lactuca wild species generally had a higher content of total vitamin C than the cultivated lettuces. In contrast, the commercial varieties usually contained more total anthocyanins than the wild species. Total vitamin C decreased with the drought stress in all accessions, commercial varieties, and lettuce wild relatives, with this tendency being consistent and reproducible across the 2 years. These differences were significant in the case of the green commercial varieties 'Winter Crop' (in 2020/2021) and 'Dolomiti G12' (in 2021/2022) and very significant in the red commercial variety 'Red Sails' (in 2020/2021). However, the only group in which the effect of drought was either significant or very significant in both years was the wild species, Lactuca homblei and Lactuca dregeana, and in the latter also in both tissues (leaf and stem) analyzed. Water stress resulted in an increase of the total anthocyanin content in the leaves from all the accessions, both red commercial varieties and wild relatives, in both years. The most significant enrichment and the only one being either significant or very significant in both years was observed in one of the wild relatives assayed (L. homblei). Stems (L. dregeana) contained more anthocyanins than leaves under control conditions, and it was exactly the opposite under drought. Changes in anthocyanins in the two tissues in response to drought stress were in opposite directions, increasing in leaves and decreasing in stems. This could suggest a translocation of anthocyanins as a first quick mechanism to cope with a severe lack of water. In conclusion, anthocyanins (unlike vitamin C) could play a role in the mechanisms deployed by the plant to tolerate drought stress. The wild species with a robust significant enrichment in anthocyanins as a response to drought (L. homblei) is a promising plant material to breed more resilient lettuces.

Inclusion of Sainfoin in the Concentrate of Finishing Lambs: Fatty Acid Profiles of Rumen, Plasma, and Muscle.

The effects of sainfoin inclusion (Onobrychis viciifolia) in the finishing concentrate for light lambs on the fatty acid (FA) composition of the ruminal digesta, plasma, and meat were evaluated. Twenty-six weaned male lambs were divided into three groups and fed individually ad libitum for 40 days with one of three concentrates differing in the level of sainfoin inclusion: 0% (0SF), 20% (20SF), and 40% (40SF). The rumen digesta showed an increase in C18:3 n-3 concentration and a decrease in C18:1 t10 concentration when sainfoin was included in the concentrate regardless of the level of inclusion. However, the highest C18:1 t11 and the lowest C18:2 n-6 proportions were obtained only in the 40SF rumen, showing a stronger t11 biohydrogenation pathway. In plasma, most effects were associated with changes in the levels of polyunsaturated FA (PUFA) n-3. The meat FA profile of 40SF lambs presented higher percentages of PUFA n-3 and CLA c9,t11 and a lower PUFA n-6/PUFA n-3 ratio compared with those from 0SF and 20SF diets because of the potentiation of the ruminal t11 pathway. Inclusions of 20 and 40% sainfoin both showed beneficial effects on meat quality; furthermore, these effects were most marked in the 40% sainfoin diet.

Nutraceutical Potential of Leafy Vegetables Landraces at Microgreen, Baby, and Adult Stages of Development.

Nutraceutical compounds present in leafy vegetables have gained substantial attention due to the health benefits they offer beyond their nutritional value. The biosynthesis, composition, and concentration of these compounds vary widely among leafy vegetables and carry the influence of genetic, agronomic, and environmental factors. Recently, micro-vegetables are gaining importance among consumers worldwide and are used in gastronomy at different development stages. Another tendency is the utilization of local genetic resources as an integral component of agricultural biodiversity crucial for sustainable production. The present study identifies the nutraceutical potential of 10 leafy vegetables at the microgreen, baby, and adult development stages using local genetic resources from the Spanish Vegetable Genebank (CITA, Aragón). Specifically, two landraces for each of the following crops were used: chard (Beta vulgaris), spinach (Spinacia oleracea), lettuce (Lactuca sativa), borage (Borago officinalis), and chicory (Cichorium intybus). The results reinforce the value of traditional local genetics and demonstrate the potential of these leafy vegetables as a source of functional compounds (fatty acids, vitamin C, carotenoids, polyphenols, antioxidant activity, and tocopherols). The observed variability depending on the crop and the developmental stage recommends the necessity of having a varied diet, since each leafy vegetable product offers a unique nutritional profile.

Colour Stability, Fatty Acid Profile, and Lipid Oxidation in Meat Stored in Modified Atmosphere Packaging from Light Lambs Fed with Concentrate with Carob Pulp (Ceratonia siliqua L.).

There is a growing interest in using by-products rich in polyphenols, such as carob pulp (Cp, Ceratonia siliqua L.), as a dietary source of antioxidants for animals. This study assesses the effects of including Cp in lambs' diet and meat display time (0, 7, 9, and 11 days) in modified atmosphere packaging on meat colour, fatty acid (FA) composition, tocopherol levels, and lipid oxidation values in the Semimembranosus muscle of 40 light lambs. The lambs were fed with concentrates supplemented with increasing Cp levels (0, 150, and 300 g/kg) for 45 days before slaughter. Metmyoglobin (MMb) and malondialdehyde (MDA) contents increased linearly with display time (p < 0.05), regardless of diet (p > 0.05). At 11 days of display, MMb (28 ± 0.8%) and MDA (0.6 ± 0.1 mg MDA/kg of meat) contents remained within the acceptable limits. The α-tocopherol content was lower in the 30% Cp group and meat (p < 0.05). Total saturated and monounsaturated FA contents (934 ± 64 and 823 ± 65 mg/100 g of meat, respectively) did not differ significantly among the groups. However, the meat from lambs fed with 30% Cp showed reduced levels of branched-chain FAs, while polyunsaturated FAs increased (p < 0.05) compared to the control lambs. The inclusion of Cp in the lamb's diet, up to 30%, did not lead to meat deterioration and improved certain quality parameters, including a healthier FA profile. These findings highlight Cp's potential as an alternative antioxidant source in animal diets.

Insights into the role of major bioactive dietary nutrients in lamb meat quality: a review.

Feed supplementation with α-linolenic acid (ALA) and linoleic acid (LA) increases their content in muscle, ALA increases n-3 polyunsaturated fatty acids and decrease n-6/n-3 ratio in muscle, and LA increases rumenic acid. However, high LA supplementation may have negative effects on lambs' lipid oxidative stability of meat. When the sources of ALA and LA are fed as fresh forage, the negative effects are counterbalanced by the presence of other bioactive compounds, as vitamin E (mainly α-tocopherol) and polyphenols, which delay the lipid oxidation in meat. There is a wide consensus on the capability of vitamin E delaying lipid oxidation on lamb meat, and its feed content should be adjusted to the length of supplementation. A high dietary inclusion of proanthocyanidins, phenolic compounds and terpenes reduce the lipid oxidation in muscle and may improve the shelf life of meat, probably as a result of a combined effect with dietary vitamin E. However, the recommended dietary inclusion levels depend on the polyphenol type and concentration and antioxidant capacity of the feedstuffs, which cannot be compared easily because no routine analytical grading methods are yet available. Unless phenolic compounds content in dietary ingredients/supplements for lambs are reported, no specific association with animal physiology responses may be established.

Fatty acid profile, secondary compounds and antioxidant activities in the fresh forage, hay and silage of sainfoin (Onobrychis viciifolia) and sulla (Hedysarum coronarium).

BACKGROUND: Sainfoin (Onobrychis viciifolia) and sulla (Hedysarum coronarium) are forage legumes usually preserved to optimize their utilization as feedstuffs. However, the method of preservation modifies the chemical composition differently in both legume species. Secondary compounds (such as proanthocyanidins, fatty acids, carotenoids and tocopherols) present in forages affect the quality of animal products. Therefore, the effect of preservation on the contents of secondary compounds should be investigated. Accoridngly, samples of sainfoin and sulla were directly freeze-dried (fresh), dried at ambient temperature (hay) and vacuum-packaged for 82 days (silage). RESULTS: In both legumes, the total fatty acid and C18:3 n-3 contents decreased and C16:0 increased with preservation (P < 0.001), with a greater effect for the hays than for the silages. For both legumes, the lutein, neoxanthin and violaxanthin contents decreased to a greater extent in the silages than in the hays (P < 0.001). Both hays exhibited the lowest ß-carotene concentrations (P < 0.001). The α-tocopherol contents decreased in hays, but not in silages, compared to the fresh forages of both legumes (P < 0.001). The antioxidant activities were lower in the silages than in the hays and fresh forages (P < 0.001) and were very strongly related to the contents of polyphenols and proanthocyanidins (P < 0.001). CONCLUSION: Haymaking affected the fatty acid, carotenoid and α-tocopherol contents to a greater extent but had a lesser effect on the antioxidant activities than silage-making, which were very strongly related to their contents of polyphenols and proanthocyanidins. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of Using Rosemary Residues as a Cereal Substitute in Concentrate on Vitamin E, Antioxidant Activity, Color, Lipid Oxidation, and Fatty Acid Profile of Barbarine Lamb Meat.

The shortage of some ingredients and, consequently, the continuous increase in the price of feed encourage the search for other alternatives to maintain animal production and enhance its products. In this line, the use of aromatic plant by-products in animal diet has been recently and widely considered, given their richness in bioactive compounds. Therefore, the aim of this study was to investigate the effects of the inclusion of rosemary residues (RR) and protein sources on lamb meat quality. The experiment was carried out on 24 male Barbarine lambs (3 months old) with an average body weight (BW) of 17.8 ± 2.6 kg, which were divided into three homogeneous groups according to BW. The diet comprised 600 g of oat hay and 600 g of concentrate. Three types of concentrate were evaluated: commercial concentrate as the control group (C); rosemary residues (RR) plus soybean meal as the RRS group, and RR plus faba bean as the RRF group. After an experimental period of 65 days, lambs were slaughtered. The inclusion of RR in both concentrates increased the α-tocopherol and total polyphenol content in meat and protected meat against discoloration (high red index and chroma after 9 days of storage) but did not affect meat lipid oxidation, which was similar for all groups. The FA profile was affected by the inclusion of RR, with no effect from the source of protein (faba bean or soybean). The inclusion of RR in the concentrate increased the C18:2 n-6, C18:3 n-3, C20:4 n-6, C20:5 n-3, and C22:5 n-3 content (p < 0.05). Consequently, the inclusion of RR also increased the total polyunsaturated FA (p < 0.05) and the ratio of polyunsaturated FA to saturated FA (p < 0.05). The results of this study demonstrate that concentrate based on RR could be useful for lamb meat production by improving the nutritional quality of meat, especially the fatty acid profile. In addition, soybean meal can be replaced by faba bean in lamb concentrate without affecting meat quality.

The Inclusion of Pea in Concentrates Had Minor Effects on the Meat Quality of Light Lambs.

The use of pea (Pisum sativum) has been recommended to replace soybean meal in the diet of ruminants, but it may affect meat quality. The aim of this study was to evaluate the effect of the proportion of pea (0%, 10%, 20% and 30%) in fattening concentrates fed to light lambs for 41 days on carcass colour and on the meat quality. Pea inclusion affected neither the colour of the lamb carcasses nor affected most of the parameters of the meat quality. However, the inclusion of pea affected the cholesterol content, and the 20%pea concentrate yielded meat with greater cholesterol contents than the 30%pea concentrate did (p < 0.05). The inclusion of pea had minor effects on individual FAs but affected the total saturated fatty acids (p < 0.01) and the thrombogenicity index (p < 0.05). A greater total saturated fatty acid content was recorded for the 20%pea concentrate than for the rest of the concentrates, and a greater thrombogenicity index was recorded for the 20% concentrate than for the 10%pea concentrate. The results indicated the viability of the inclusion of pea in the fattening concentrate of light lambs without impairing meat quality, with the 30%pea concentrate being the most suitable to reduce the soya-dependency.

Nutritional value of commercial and traditional lettuce (Lactuca sativa L.) and wild relatives: Vitamin C and anthocyanin content.

Lettuce is the most consumed leafy vegetable though the most popular varieties have a low nutritional value. Our objective was to accurately quantify vitamin C and anthocyanins in wild relatives, and commercial and traditional varieties. Wild species and traditional varieties contained more total ascorbic acid (TAA) than commercial varieties (21% and 8%, respectively). In contrast, commercial varieties had significantly higher content of anthocyanins than traditional varieties and wild species (6 and 8 times more, respectively). TAA was significantly higher in green than in red lettuces (18%). TAA was also significantly higher in the leaves of two wild species than in stems. Cyanidin 3-O-(6'-O-malonylglucoside) was the most abundant anthocyanin (97%), present in most samples. The rankings of accessions by vitamin C and anthocyanin contents can be useful for consumers worried about the impacts of food on their wellbeing and for breeders aiming to improve lettuce by biofortification with health-promoting compounds.

Effects of myrtle (Myrtus communis L.) essential oils as dietary antioxidant supplementation on carcass and meat quality of goat meat.

Despite the fact that the use of rosemary and thyme residues and essential oils in animal feeding was widely documented, that of myrtle is scarce. To test the hypothesis that myrtle essential oils (MEOs) could improve goats' carcass characteristics and meat quality traits, twenty-one male goats received a ration consisted of 40% oat hay and 60% concentrate. Experimental goat kids received the control diet supplemented with 0, 0.3 and 0.6% of myrtle essential oils (MEOs) for C, Myrt1 and Myrt2 groups respectively. The administration of MEO did not improve the daily DM intake (p > 0.05). Kids of C and Myrt2 groups had higher average daily gain than Myrt1 group (75 versus 55 g). The goats slaughtered at 19.9 kg of weight did not differ (p > 0.05) in carcass weights and carcass yield in terms of commercial dressing percentage (CDP = 41%) and real dressing percentage (RDP = 52%). The administration of MEO increased the meat polyphenol content, being higher in both Myrtle groups (87 versus. 56 µg gallic acid equivalents g-1 fresh matter, p < 0.05). Myrtle EO administration protected kids' meat against oxidation (0.48 versus. 0.91 mg MDA/kg of meat for Myrtle and C groups, respectively, at the 9th day of storage; p < 0.05). It could be useful to include MEO as a dietary supplement in goats' rations since it improves meat's oxidative status without negative effects on FA profile.

Carotenoids and Liposoluble Vitamins in the Plasma and Tissues of Light Lambs Given Different Maternal Feedings and Fattening Concentrates.

The carotenoids and liposoluble vitamins in the plasma and tissues of the lambs under different maternal feedings and fattening concentrates was studied. During lactation, 21 lambs were housed with their dams, that received a total mixed ration (intensive); 21 ewe-lamb pairs grazed on alfalfa; and 21 pairs grazed on sainfoin. After weaning, half of the lambs in each maternal feeding group received a commercial concentrate (control) and the other half a concentrate with quebracho (Schinopsis balansae), as a source of condensed tannins, until they were slaughtered (23 kg). The analyte concentrations in the plasma of lambs at weaning reflected the content in the feedstuffs. Grazing during suckling more than doubled the contents of lutein in the liver and retinol in the tissues compared to the intensive feeding. The content of α-tocopherol in the tissues was greatest in sainfoin lambs, intermediate in alfalfa lambs, and lowest in the intensive lambs. The quebracho concentrate decreased α-tocopherol (by 41-81%) and γ-tocopherol (by 65-89%) contents in the lamb tissues. The use of the analytes in the plasma at weaning correctly classified 100% of the lambs into the maternal feeding (intensive vs. grazing (alfalfa + sainfoin)) but has to be improved in the carcass and tissues separately.

Physicochemical Characteristics, Fatty Acid Profile, Alpha-Tocopherol Content, and Lipid Oxidation of Meat from Ewes Fed Different Levels of Distilled Myrtle Residues.

The aim of this work was to study the sheep meat physicochemical traits as affected by distilled myrtle residues (MR) supplementation. For this, 27 culled ewes were divided into three groups receiving a ration composed by concentrate and hay for the Control group, concentrate and MR as a total substitute to hay for the Myrt-H group, or hay, less concentrate, and MR as a partial substitute to concentrate for the Myrt-C group. The meat chemical composition, pH, and color parameters were not affected by the MR intake. However, this animal's dietary treatment resulted in higher meat polyphenol and α-tocopherol content for both MR groups (9.38 and 8.05 vs. 3.04 µg g-1 DM for Myrt-H, Myrt-C, and Control, respectively). In addition, since day 3 of meat storage, the lipid oxidation was improved by MR intake being lower for both MR groups than the Control (0.51 vs. 1.11 mg MDA/kg of meat). The total polyunsaturated fatty acid (PUFA) and saturated fatty acid (SFA) were similar among groups. However, the meat of Myrt-H had the highest C18:2n-6 and total PUFAn-6. In conclusion, the MR intake could be useful given it increases the meat content of vitamin E and improves its oxidative status without negative effects on the FA profile.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.).

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized. Samples were collected to represent the entire plant, frozen at -80 °C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.

Effect of Dietary Crude Protein on Productive Efficiency, Nutrient Digestibility, Blood Metabolites and Gastrointestinal Immune Markers in Light Lambs.

This study hypothesized that reducing the level of crude protein (CP) in lambs' feed may improve nutrient utilization and did not negatively affect their productive efficiency, blood metabolites, oxidative status (OS) or intestinal immune barrier function. A total of 120 weaned male Ripollesa lambs (45-60 days old and 15.0 ± 1.5 kg of body weight) were used. Four feed concentrates were formulated for two different phases (growing and finishing): CP20/19 group (20% and 19% of CP on dry matter basis, for each phase, respectively) and CP18/17 group (18% and 17% of CP on dry matter basis, for each phase, respectively). Lambs were randomly assigned to feeding treatments by balancing initial body weight between groups. The reduction of dietary CP level did not impair their growth performance parameters, while it did improve the apparent digestibility of organic matter. Furthermore, the lambs of the CP18/17 group showed lower plasma urea levels with no effect on OS (malondialdehyde levels) or gastrointestinal immunity markers (gene expression of interleukin 10 (IL10), tumor necrosis factor-α (TNFA) and transforming growth factor-ß1 (TGFB)).

Fatty acid profile of muscles and adipose tissues of fat-tail Barbarine lambs as affected by rosemary residue intake.

The rosemary distillation industry produces a considerable amount of rosemary distillation residues (RRs), which can be an alternative for feeding animals in harsh conditions and could enhance animal product quality. Given the meat quality is largely influenced by its fat content and fatty acid composition, the fatty acid (FA) profiles of longissimus thoracis et lumborum (LTL), semi-membranous (SM) muscles, and caudal (CFs) and omental fats (OFs) were determined using 21 Barbarine lambs fed with or without RRs. Diets contained 600 g of concentrate plus 600 g of forage. Forage represented oat hay, RR87 and RR60 pellets containing 87 % or 60 % of RR, respectively. At the end of the study, all lambs were slaughtered, and the fatty acid profile was studied. The inclusion of RR increased the polyunsaturated fatty acid (PUFA) contents and reduced saturated fatty acids (SFAs), and the thrombogenic and saturation indexes in all tissues. The SM muscle was the richest tissue in PUFAs, n -3 and n -6; however, both adipose tissues contained the highest proportions of SFAs. Especially the OF was the richest tissue in oleic acid and SFAs. Feeding RR to lambs enhanced meat quality.

Effect of the maternal feeding on the carotenoid and tocopherol content of suckling lamb tissues.

The objectives of the present study were to quantify the deposition of carotenoids and tocopherols in the tissues of suckling lamb and to use the levels of those compounds to trace the maternal feeding. Twenty suckling lambs were raised with their dams in vegetative-stage pastures, and 19 suckling lambs were stalled indoors with dams that received hay ad libitum, until the lambs reached 10-12 kg. The lambs' weekly intake of carotenoids and tocopherols was estimated from the milk production of the ewes and the carotenoid and tocopherol content of the milk. Samples of the subcutaneous and perirenal fat, longissimus thoracis muscle, and liver of the suckling lambs were collected at 24 h after slaughter. The pasture-raised lambs had greater intake of lutein than their indoor counterparts throughout the suckling period (p < 0.05), more retinol during the second and third weeks of the suckling period (p < 0.05), and more α-tocopherol during the first three weeks of the suckling period (p < 0.05), being similar thereafter. The pasture-raised lambs, when compared to the lambs raised indoors, had greater lutein content in the muscle and liver (p < 0.001), retinol and α-tocopherol content in all tissues (p < 0.001) and muscle and liver γ-tocopherol content (p < 0.05). The maternal feeding could be accurately predicted from the carotenoid and tocopherol content of whole lamb carcasses and muscle tissue but not from those of ewes' milk, lamb liver tissue or lamb fat deposits.

The Inclusion of Concentrate with Quebracho Is Advisable in Two Forage-Based Diets of Ewes According to the In Vitro Fermentation Parameters.

Ewes receive hay or graze on fresh pastures supplemented with concentrates to fulfil their lactation requirements. Quebracho (Schinopsis balansae) can be added to change the ruminal fermentation. Fermentation parameters of forages alone and 70:30 forage:concentrate diets with control and quebracho concentrate were compared after 24 h of in vitro incubation. Fresh forage diets produced less gas (p < 0.05) and had greater IVOMD (p < 0.001), ammonia (NH3-N) content, valeric acid, branched-chain volatile fatty acid proportions, and lower propionic acid proportion than the hay diets (p < 0.01). In the hay diets, methane production increased with control concentrate (p < 0.01) and tended to decrease with quebacho concentrate (p < 0.10). The inclusion of both concentrates increased the acetic:propionic ratio (p < 0.01), and only the inclusion of quebracho concentrate increased the IVOMD (p < 0.01). In the fresh forage diets, gas and methane production increased with the inclusion of the control concentrate (p < 0.05), but methane production decreased with quebracho concentrate (p < 0.01). The inclusion of quebracho concentrate reduced the NH3-N content and valeric acid proportion (p < 0.05). In conclusion, the inclusion of quebracho concentrate would be advisable to reduce the CH4 production and NH3-N content in fresh forage diets and to increase the IVOMD in hay diets in comparison with the forages alone.

Erratum: Rufino-Moya, P.J., et al. Methane Production of Fresh Sainfoin, with or without PEG, and Fresh Alfalfa at Different Stages of Maturity is Similar, but the Fermentation End Products Vary. Animals 2019, 9, 197.

The authors wish to make the following correction to their paper [...].

Methane Production of Fresh Sainfoin, with or without PEG, and Fresh Alfalfa at Different Stages of Maturity is Similar but the Fermentation End Products Vary.

Alfalfa and sainfoin are high-quality forages with different condensed tannins (CT) content, which can be affected by the stage of maturity. To study the effects of CT on fermentation parameters, three substrates (alfalfa, sainfoin, and sainfoin+PEG) at three stages of maturity were in vitro incubated for 72 h. Sainfoin had greater total polyphenol and CT contents than alfalfa. As maturity advanced, CT contents in sainfoin decreased (p < 0.05), except for the protein-bound CT fraction (p > 0.05). The total gas and methane production was affected neither by the substrate nor by the stage of maturity (p > 0.05). Overall, sainfoin and sainfoin+PEG had greater in vitro organic matter degradability (IVOMD) than alfalfa (p < 0.05). Alfalfa and sainfoin+PEG presented higher ammonia content than sainfoin (p < 0.001). Total volatile fatty acid (VFA) production was only affected by the stage of maturity (p < 0.05), and the individual VFA proportions were affected by the substrate and the stage of maturity (p < 0.001). In conclusion, alfalfa and sainfoin only differed in the IVOMD and the fermentation end products. Moreover, CT reduced ammonia production and the ratio methane: VFA, but the IVOMD was reduced only in the vegetative stage.