Effect of Multispecies Swards on the Fat Composition and Oxidative Stability of Milk from Dairy Cows in Mid-Late Lactation

Multispecies swards are considered a sustainable alternative to traditional ryegrass systems, offering diverse phytochemicals that may improve milk quality. This study compared the fat composition and oxidative stability of milk from cows fed multispecies swards (MULTI), ryegrass monoculture (PRG), and ryegrass–white clover mixtures (PRG + WC).

Milk from MULTI-fed cows contained the highest levels of polyunsaturated and bioactive fatty acids, while PRG milk had more saturated and monounsaturated fatty acids. Antioxidants such as β-carotene and α-tocopherol were higher in PRG milk, leading to greater oxidative stability. These findings show that multispecies swards can enhance the nutritional profile of milk by boosting bioactive fatty acids, making them a promising alternative to conventional feeding systems.

The original article

Effect of Multispecies Swards on the Fat Composition and Oxidative Stability of Milk from Dairy Cows in Mid-Late Lactation

Samuel Rapisarda, Kate M. McCarthy, Tommy M. Boland, Helen Sheridan, Frank J. Monahan, Finbar J. Mulligan, Graham O’Neill, Nissreen Abu-Ghannam*

ACS Agric. Sci. Technol. 2025, 5, 4, 499–512

https://doi.org/10.1021/acsagscitech.4c00584

licensed under CC-BY 4.0

Selected sections from the article follow. Formats and hyperlinks were adapted from the original.

Milk and dairy products are consumed by more than 6 billion people worldwide (1) and are regarded as staples in many diets due to their complete nutritional profile and potential health benefits. (2) According to the FAO, (3) there has been a 59% increase in global milk production over the last three decades, from 530 million tons in 1988 to 843 million tons in 2018. A further 35% increase is projected by 2030, resulting in an annual production of 1.1 billion tons of product. However, the growth and intensification of milk production have led to a rise in agricultural emissions, primarily because of the fertilization of grasslands and enteric fermentation in ruminants. Indeed, the use of fertilizer is responsible for nitrogen (N) loss via nitrate leaching, causing groundwater pollution, detrimental effects on biodiversity, and emissions in the form of ammonia, nitric oxide, and nitrous oxide. (4) Simultaneously, ruminant digestion of the grass fibers produces acetate and butyrate, enhancing emissions of methane. (5)

In Ireland, agriculture is responsible for 38.4% of the total national greenhouse gas emissions, with the main contributors being nitrous oxide and methane. (6) Considering that Ireland has committed to have all its waters in good status by 2027 (European Water Framework Directive), (7) enhance its recovery of biodiversity by 2030, (8) and reduce 30% of its 2005 emissions levels by 2030 (European Green Deal), (9) there is a strong need to identify scientific strategies that can meet the growing demand for dairy production while also mitigating the negative environmental implications associated with current ruminant production systems.

Perennial ryegrass (Lolium perenne) monoculture systems provide high dry matter (DM) yields and enhanced sensory properties in milk in comparison to total mixed ratios. (10) However, they require intense management and fertilizer N inputs, representing a significant cost for farmers and having a negative impact on the environment. Multispecies swards, consisting of grasses, legumes, and forage herbs, have recently been recognized as a more sustainable alternative as they can increase soil properties and enhance biodiversity. (11) Legumes, such as white clover and red clover, have rhizobia bacteria on the nodules of their roots which can convert atmospheric N into plant available N, reducing N fertilizer inputs in comparison to perennial ryegrass monoculture systems. (12) Furthermore, legume inclusion improves earthworm activity and associated water infiltration rates, (13) while herbs such as ribwort plantain and chicory contain phytochemicals that have been shown to interact with the rumen microbiota and potentially reduce methane emissions by up to 20%. (14,15)

Multispecies swards also have other beneficial properties beyond environmental factors. For instance, feeding ruminants with multispecies swards has been associated with anthelmintic effects, animal weight gain, and increased milk production. (16,17) Plant species diversity has been shown to increase milk production potential and yields per hectare, which in turn can increase revenues and reduce production risks. (18) Including legumes improves weed suppression, increases herbage DM production, and enhances forage feed value in comparison to perennial ryegrass monoculture. (16,19) Also, legumes and herbs contain a variety of phytochemicals which are precursors to compounds linked to animal performance and improved milk quality. (20) In this regard, there is evidence suggesting that polyphenols might reduce plant-mediated lipolysis, lower the ruminal hydrogenation of PUFAs, and increase the levels of health-promoting fats in milk. (21) Consumption of dairy products containing high levels of PUFAs has been linked to various health benefits in humans, including reduced incidence of cardiovascular diseases and arteriosclerosis, (22) while consumption of omega-3 PUFA α-linolenic acid and its intermediate products from rumen biohydrogenation (e.g., conjugated linoleic acid (C18:2 c9t11) and vaccenic acid) has been associated with anti-inflammatory, antidiabetic, antiobesity, and anticancerogenic properties. (23,24) However, it is worth noting that high levels of PUFAs might also influence the oxidative stability of milk, potentially leading to the formation of off-flavor compounds (e.g., aldehydes) and diminishing consumer acceptability of the product. Although multispecies swards have a lower environmental impact than monoculture systems, information on the impact of the multispecies swards on the fat composition and oxidative stability of milk still needs to be assessed.

The objectives of this study were to compare the fat composition (i.e., fatty acids, carotenoids, fat-soluble vitamins) and fat oxidative stability of milk produced by cows fed on multispecies swards [consisting of perennial ryegrass, timothy (Phleum pratense), white clover (Trifolium repens), red clover (Trifolium pratense), chicory (Chicorium intybus), and ribwort plantain (Plantago lanceolata) and receiving 95 kg N/ha] (MULTI) with two other types of milk produced from cows fed on conventional swards [a perennial ryegrass monoculture receiving 250 kg N/ha (PRG) and a binary mixture of perennial ryegrass and white clover receiving 115 kg N/ha (PRG + WC)] over the Irish summer grazing season (July–September).

This paper is the first to assess the impact of multispecies swards grown in Ireland on the fatty acid and fat-soluble vitamin profile of bovine milk. Although previous Irish-based research has focused on the link between multispecies swards and animal productivity, there are no studies that have demonstrated the effect on the chemical properties of milk over late lactation. Information on the impact of multispecies swards on the quality of milk might help farmers in choosing sustainable feeding strategies that can enhance the nutritional quality of dairy products, increase farm potential revenues, and benefit the health of the consumer.

2. Materials and Methods

2.4. Identification and Quantification of Fatty Acids Using GC–QqQ–MS

Mass spectrometry was used to identify and quantify the FAs present in the milk samples using a method previously established by Rapisarda et al. (26) Chromatographic separation was achieved using a CP-Sil 88 column [(100 m × 0.25 mm 0.20 mm) Agilent Technologies, Cork, Ireland] using an Agilent Technologies GC 7890B coupled with an Agilent Technologies Triple-Quadruple-Mass-Spectrometry detector. The carrier gas was helium, and the split ratio was set at 15:1. The injection volume was 1 μL with an inlet temperature of 280 °C. The oven was programmed to start at 80 °C, then increased to 220 °C at a rate of 3.5 °C/min, and held for 5 min. Subsequently, the mixture was heated to 225 °C at a rate of 2 °C/min and held for 22.5 min, resulting in a total run time of 70 min. The mass spectrometer operated in positive scan mode, scanning m/z 29 to 550 every 0.3 s. The ion source was set at 230 °C and electron impact at 70 eV was used to obtain the spectra. Commercial standards and the NIST08 mass spectral library were used to identify the compounds. Calibration curves were constructed using different standard concentrations (ranging from 20 to 200 μg/mL), and the detector response was used to calculate linear regression equations (R² = 0.99).

2.5. Identification and Quantification of Carotenoids and Fat-Soluble Vitamins Using LC–QqQ–MS

Carotenoids and fat-soluble vitamins were extracted from milk following the work of Plozza et al. (27) and using a hot saponification method which involved incubating the samples at 60 °C for 20 min at 200 rpm. Briefly, 0.2 g of sample and 50 μL of β-tocopherol internal standard were added to a 15 mL propylene tube containing 2.5 mL of 50% aqueous potassium hydroxide and 2 mL of 0.2% of ethanolic BHT. After incubation, the sample was cooled on ice for 5 min and the analytes of interest were extracted twice with 2 mL of hexane with 1% BHT. The hexane aliquots were pooled together and subsequently evaporated to dryness under nitrogen flow. The dried extract was reconstituted with 1 mL of methanol, filtered through a 0.2 μm PTFE filter (Captiva, Agilent Technologies, Cork, Ireland), and stored at −18 °C before LC-QqQ-MS analysis.

Carotenoids and fat-soluble vitamins were analyzed using an Agilent Technologies 1290 Infinity series HPLC coupled with an Agilent Technologies 6470 series electrospray ionization Triple-Quadruple-Mass-Spectrometry detector. A 5 μL sample was injected, and separation was carried out using an Agilent Technologies Poroshell 120 column [(3.0 × 100 mm × 2.7 μm), Cork, Ireland] maintained at 40 °C. The mobile phase consisted of two components: (A) 0.1% formic acid in water and (B) 0.1% formic acid in methanol. Elution was carried out at a flow rate of 0.7 mL/min, with gradient B starting at 85%, increasing to 90% within 10 min, then to 92% in 4 min, and finally to 100% in 6 min. This composition was maintained for 7 min with a post-time of 3 min. Multiple reaction monitoring (MRM) was utilized to detect and quantify the eluted vitamins, using a positive ion source, a gas flow of 5 L/min, a nebulizer pressure of 45 psi, a sheath gas temperature of 250 °C, and a sheath gas flow of 11 L/min. The parameters of the MRM mode are detailed in Table S2. Calibration curves were constructed from peak areas of different standard concentrations (ranging from 0.1 to 1 μg/mL), and sample concentration was calculated using the equation for linear regression obtained from the calibration curve (R² = 0.99).

2.7. Statistical Analysis

All the data were reported as means ± standard deviations of triplicate determinations. Statistical analysis was performed with SPSS Statistic Software (vers 28.0.0), using one-way analysis of variance (ANOVA) and multivariate analysis of variance (MANOVA) to assess the significant differences across feeding treatments (PRG, PRG + WC, and MULTI) and seasonality (July, August, September). Posthoc analysis included the Tukey and Kruskal–Wallis test. Pearson correlation analysis was also performed to assess the relationship between fatty acids, fat-soluble vitamins, and oxidative stability of the milk samples. Differences at p < 0.05 were considered statistically significant. GC–MS and LC–MS data were analyzed using Agilent Mass Hunter Workstation Software-Qualitative Analysis (vers 10.0) and Mass Hunter Workstation Software-Quantitative Analysis (vers 9.0). Graphs were plotted using GraphPad Prism Software (vers 8.0.1).

3. Results and Discussion

3.1. Identification and Quantification of Milk Fatty Acids

The FA profile of the milk produced from cows fed on three types of swards (PRG, PRG + WC, and MULTI) was determined during the Irish late summer (July–September) (Tables 1 and 2). To enhance chromatographic separation, free FAs were converted into FAME; this was done to reduce the polarity of the FAs, which would otherwise interact through hydrogen bonding with other molecules in the sample and interfere with the separation. (29) Individual FAME were identified and quantified using highly selective GC–QqQ–MS. A representative chromatogram of the predominant FAME identified in milk is shown in Figure S1.

Milk from cows fed PRG contained higher total saturated fatty acid (SFAs) mean concentrations (783.45 mg/g) in comparison to those of PRG + WC (704.17 mg/g) and MULTI (676.09 mg/g) (p = 0.321). High concentrations of total monounsaturated fatty acids (MUFAs) were found in milk from cows fed on PRG (284.68 mg/g), while lower concentrations were observed in PRG + WC (255.06 mg/g) and MULTI (261.37 mg/g) (p = 0.580). Analysis of individual MUFAs in milk from cows fed on PRG found oleic acid (222.42 mg/g) being somewhat higher than in PRG + WC (196.16 mg/g) and MULTI (207.24 mg/g) (p = 0.638), whereas the levels of myristoleic acid, palmitoleic acid, and elaidic acid (10.60, 17.11, 31.06 mg/g, respectively) were similar to PRG + WC (9.57, 17.56, 27.65 mg/g, respectively) and MULTI (8.23, 14.50, 24.24 mg/g, respectively) (p > 0.05).

Mean concentrations of PUFAs were significantly higher in milk from cows fed on MULTI (36.83 mg/g) and PRG + WC (36.72 mg/g) in comparison to PRG (28.00 mg/g) (p = 0.017); in particular, linoleic acid, α-linolenic acid, and vaccenic acid concentrations in milk from MULTI (16.47, 10.98, and 7.17 mg/g, respectively) were almost twice those in PRG (9.74, 6.27, and 3.50 mg/g, respectively). There was a lower omega-6:3 ratio in milk from cows fed on MULTI (1.58) in comparison to PRG + WC (1.65) and PRG (1.70) (p = 0.716).

Mean concentrations of SFAs (particularly palmitic acid, stearic acid, and myristic acid) increased in milk from cows fed on PRG (from 715.35 mg/g in July to 893.21 mg/g in September) (p = 0.370), PRG + WC (from 667.03 mg/g in July to 774.58 mg/g in September) (p = 0.369) and MULTI (from 485.08 mg/g in July to 680.48 in September) (p = 0.066). Between one-and-a-half and 2-fold increases in concentrations of MUFAs were observed in milk from MULTI (from 146.57 mg/g in July to 291.99 mg/g in September) and PRG + WC (from 236.21 mg/g in July to 331.20 mg/g in September) (p = 0.011), while the increase was lower in PRG (from 237.36 mg/g in July to 299.88 mg/g in September) (p = 0.361). There were more than 2-fold increases in concentrations of PUFAs in milk from MULTI (from 16.74 mg/g in July to 49.78 mg/g in September) (p < 0.001), PRG + WC (from 23.94 mg/g in July to 54.08 mg/g in September) (p < 0.001), and PRG (from 17.49 mg/g in July to 37.92 mg/g in September) (p = 0.002). A decreasing omega-6:3 ratio was observed from July to September in milk from cows fed on PRG (from 1.82 to 1.52) (p = 0.591), PRG + WC (from 1.93 to 1.46) (p = 0.112), and MULTI (from 1.81 to 1.49) (p = 0.145).

This study found that mean levels of α-linolenic acid and linoleic acid were higher in milk from cows fed on MULTI (10.97 and 16.46 mg/g) and PRG + WC (9.50 and 15.11 mg/g), with over 2-fold increases in concentration from July to September (p < 0.05). The lower levels of α-linolenic acid and linoleic acid in milk from PRG remained constant throughout the weeks of the summer season (p > 0.05) (Figure 2 1,2). The presence of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in milk was also investigated, but these FAs were not detected.

ACS Agric. Sci. Technol. 2025, 5, 4, 499–512: Figure 2. Variations in the α-linolenic acid (1), linoleic acid (2), conjugated linoleic acid (C18:2 c9t11) (3), and vaccenic acid (4) levels in milk from PRG (perennial ryegrass), PRG + WC (perennial ryegrass and white clover), and MULTI (perennial ryegrass, white clover, timothy, red clover, chicory, and ribwort plantain) systems estimated weekly (W1, W2, and W3) throughout the summer months (July, August, and September). Lower-case and upper-case superscript letters indicate significant differences (p < 0.05) within each sward system and between sward systems, respectively.

3.2. Identification and Quantification of Milk Carotenoids and Fat-Soluble Vitamins

The carotenoid (β-carotene and lutein) and fat-soluble vitamin (D₂, D₃, K₁, α-tocopherol, γ-tocopherol, and β-tocopherol) composition of milk produced from cows fed on three types of swards (PRG, PRG + WC, and MULTI) was determined during the Irish late summer (July–September) (Tables 4 and 5) using highly selective LC–QqQ–MS in positive MRM acquisition mode. MRM mode enables precise monitoring of specific ions corresponding to target compounds, as well as leading to heightened quantification accuracy at lower detection levels.

ACS Agric. Sci. Technol. 2025, 5, 4, 499–512: a Values are presented as mean ± standard error of the mean (SEM). Significant differences (p < 0.05) between treatments are indicated with letter superscript. Ergocalciferol, cholecalciferol, phytomenadione, and menaquinone were not detected, while lutein and γ-tocopherol were not quantified.

ACS Agric. Sci. Technol. 2025, 5, 4, 499–512: a Values are presented as mean ± standard error of the mean (SEM). Significant differences (p < 0.05) between months are indicated with letter superscript. Ergocalciferol, cholecalciferol, phytomenadione, and menaquinone were not detected, while lutein and γ-tocopherol were not quantified.

Mean concentrations of β-carotene were higher in milk produced from cows fed on PRG (15.29 mg/kg) (p = 0.007), with levels being 38% higher than those on PRG + WC (11.09 mg/kg) and 49% higher than those on MULTI (10.28 mg/kg). Concentrations of β-carotene decreased from July to September in milk from PRG (from 20.20 to 15.34 mg/kg) (p = 0.049) and PRG + WC (from 13.83 to 10.19) (p = 0.021) (Figure 3,1), while they increased in MULTI (from 7.97 to 9.27) (p = 0.667). Concentrations of lutein were below the quantification limit of the equipment.

ACS Agric. Sci. Technol. 2025, 5, 4, 499–512: Figure 3. Variations in the β-carotene (1) and α-tocopherol (2) levels of milk from PRG (perennial ryegrass), PRG + WC (perennial ryegrass and white clover), and MULTI (perennial ryegrass, white clover, timothy, red clover, chicory, and ribwort plantain) systems estimated weekly (W1, W2, and W3) throughout the summer months (July, August, and September). Lower-case and upper-case superscript letters indicate significant differences (p < 0.05) within each sward system and between sward systems, respectively.

4. Implications of Using Multispecies Swards in Dairy Production Systems

As consumers become more aware of the environmental issues associated with the dairy industry, there is a strong need to find economic and ecofriendly strategies to support the expansion of dairy production. In several investigations conducted in New Zealand, (67) UK, (68) France, (69) and most recently in Ireland, (70) MULTI have been suggested to be a more sustainable and viable feeding strategy in comparison to perennial ryegrass-based systems. From an environmental point of view, the clover species can fixate atmospheric N, while herb species contain a higher water content which can effectively dilute N in the urine and reduce N leaching through the soil. (71) Inclusion of clover and herbs can also augment N utilization efficiency within the rumen by reducing the conversion of crude protein into ammonia. (72) Furthermore, addition of herbs could inhibit the activity of methanogenic ruminal microorganisms that are accountable for methane production. (73)

From a nutritional point of view, this study showed that milk derived from cows fed on MULTI had elevated levels of health-promoting omega-3 PUFAs (1.49% of total FAs), as compared to milk from cows fed on other freshly harvested grasslands (PRG + WC = 0.70–1.21%; PRG = 0.60–0.92%) and traditional ensiled forages (legume = 1.20%; grass = 0.51%). (74) The European Commission considers a high source of omega-3 PUFAs any food containing at least 600 mg of α-linolenic acid in 100 g. (75) As the findings of this study indicate that the average concentration of α-linolenic acid in milk MULTI was 1097 mg/100 g, milk from cows fed on MULTI could be considered a good source of omega-3 PUFAs. However, further research is needed to investigate the enhancement and stability of these fatty acids in milk and their potential impact on nutritional quality. Interestingly, Kearns et al. (76) also reported similar findings to this study when investigating the effect of MULTI on the chemical composition of beef. Particularly, beef produced by cows fed on MULTI had higher levels of PUFAs (e.g., α-linolenic acid and linoleic acid) compared to beef from PRG and PRG + WC. The author also observed lower levels of α-tocopherol and reduced lipid oxidative stability in beef from MULTI, suggesting a correlation between the effects of MULTI on milk and beef chemical composition.

MULTI systems contain compounds that have the potential to exert a positive influence on the health and productivity of ruminants, potentially reducing the reliance on antibiotics and medications. (77) For instance, phytochemicals in herbs, such as aucubin and acteosides, can have anthelmintic and anti-inflammatory properties, while the plethora of minerals in chicory and ribwort plantain can enhance ruminant health and performance. MULTI can also generate higher levels of dry matter with 55% less fertilizer, while also preventing excessive weed growth. (12) Consequently, MULTI enhances farm productivity, reduces production risks associated with biomass yield loss, and ultimately increases revenues. (18) Finally, perennial ryegrass, ribwort plantain, and timothy exhibit favorable growth during the spring, whereas white clover, red clover, ribwort plantain, and chicory had optimal growth during the summer. (78) Therefore, implementation of MULTI has the advantage of maintaining diversified pastures all year around.

Multispecies swards have the potential to serve as an alternative sward for conventional pasture systems as they can increase the concentration of bioactive fatty acids in milk, particularly α-linolenic, linoleic, and vaccenic acid. Feeding ruminants with multispecies and conventional swards resulted in milk with similar levels of conjugated linoleic acid (C18:2 c9t11). However, milk from cows fed on multispecies contained lower levels of antioxidant compounds, such as α-tocopherol and β-carotene, which consequentially might have resulted in higher lipid oxidation.