Evidence That Forage-Fed Cows Can Enhance Milk Quality

: Researching the distinguishing factors of nutritional milk quality is key to sustainable production and addresses increasing media and scientiﬁc scrutiny regarding human health e ﬀ ects and ecological impacts of dairy products. Modern Western diets have high omega-6 relative to omega-3 fatty acid (FA) consumption. This ratio in milk can be manipulated by management practices; increasing forage in dairy diets raises omega-3 in milk. Whilst studies identify higher concentrations of nutritionally beneﬁcial FAs in organic dairy, milk from 100% forage-fed cows in the UK has not been investigated. This study explores di ﬀ erences in FA composition between supermarket conventional and organic and Pasture for Life Association (PFLA) milk, collected in April, July and October, 2017. PFLA milk had higher concentrations of conjugated linoleic acid ( + 94%) and omega-3 ( + 92%) than conventional milk. Additionally, concentrations of palmitic acid ( + 11%), omega-6 ( + 69%) and the ratio of omega-6 / omega-3 ( + 201%) were higher in conventional than PFLA milk. PFLA milk had higher concentrations of alpha-linolenic acid ( + 39%), conjugated linoleic acid ( + 30%) and omega-3 ( + 21%) and lower concentrations of omega-6 ( − 36%) and a lower ratio of omega-6 / omega-3 ( − 44%) than organic milk. This supports previous studies and demonstrates the scope to improve milk FA proﬁles further for potential health beneﬁts through pasture-based management. n-6/n-3 by month and management from conventional and organic supermarket and PFLA milk (mean values ± SD).


Introduction
Fats from dairy products have undergone major scrutiny by both the media and scientific community. Though ruminant agriculture has been criticised for its land use and emissions, a recent study indicates that lower-yielding, pasture-based production can have lower emissions per litre of milk compared to more-intensive, higher-yielding systems [1]. Additionally, despite claims that dairy products are unnecessary sources of saturated fat and calories, dairy fats are a complex of important fatty acids (FAs) [2], many beneficial to human health. The more forage that cows consume, the higher the concentration of beneficial fatty acids [3,4], linking environmental benefits with nutritional gains.
Long-chain omega-3 FAs (n-3)-eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA)-have been shown to lower the risk of coronary heart disease (CHD) and are anti-inflammatory, anti-thrombotic and anti-atherogenic [5,6]. Additionally, long-chain FAs are essential in brain development and function and have protective effects that potentially support healthy aging [7,8]. Conjugated linoleic acid (C18:2, c9t11 isomer-CLA9) was also linked to a lower CHD risk and enhances immune response, improves body composition and reduces

Experimental Design
Milk was collected from eight PFLA dairy farms (across southern UK) and five supermarkets (located centrally to the PFLA farms-within 10 miles (16 km) West of Bristol) in April (4 th -6 th ), July (4 th -6 th ) and October (10 th -12 th ), 2017. There is no winter sampling because PFLA farmers predominantly spring calve, and therefore cows do not produce milk from November to January/February. The locations of the PFLA farms and the supermarkets are in Appendix A Figure A1. A representative 500 mL milk sample was taken from the bulk tank on each farm (after stirring to disperse the cream) on the morning of collection. This coincided with the purchase of half-litre (500 mL), own-brand organic and conventional whole, pasteurised and homogenised milk from five supermarkets: Asda, Marks and Spencer, Sainsbury's, Tesco and Waitrose. None of the dairy farms supplied milk to the supermarkets.

Milk Analysis
All samples were kept below 4 • C during transportation (maximum 3 days) and then frozen at −20 • C until analysis. Milk from supermarkets was purchased on the same day as each farm sampling date. All were in date and their shelf life went beyond the date they were frozen. The samples were thawed overnight at 4 • C, freeze dried and then 130 µg of lyophilized milk was methylated and esterified to prepare for gas chromatography (GC), using the method described by Chilliard et al. [25] and Stergiadis et al. [26]. The chemicals used for extraction of FAs, correction factors for short-chain FAs (C4:C10:0), analytical standards and identification of peaks followed the methodology of and are described by Stergiadis et al. [27].
Fatty acid methyl esters (FAMEs) were separated and quantified using GC (Shimadzu, GC-2014, Kyoto, Japan). The GC had a flame ionisation detector and a 100 m × 0.25 mm ID, 0.2 µm film thickness, Varian CP-SIL 88 fused silica capillary column. To optimize peak separation, modifications to the chromatographic conditions from the original method by Chilliard et al. [25] were updated as described by Stergiadis et al. [27].
Although purchased milk was pasteurised and homogenised and farm milk was in a raw state at sampling, milk FA profiles are not changed by these processes [28], making this an appropriate comparison. There is some evidence that time after pasteurisation could reduce CLA concentration [29]. However, further research is required to confirm this. Fatty acid results are expressed as a percentage of the whole FA profile. Thus, there is no need to adjust for differences in fat content between the whole supermarket milk (3.5% fat) and the raw farm milk (~3.5%-4.5% fat).

Data Analysis
Microsoft Excel was used for data handling and statistical analysis was completed using the statistical software 'R' [30]. Tests included a linear mixed-effects model (R package 'nlme') to allow for nested random effects and unbalanced design, analysis of variance and Tukey's honestly significant difference. The factors were month (April, July, October (n = 18 each month)) and management (conventional (n = 15), organic (n = 15), PFLA (n = 24)), with source (supermarket (own-brand) name and farm name) treated as a random factor. The concentrations of FAs identified as having either a positive or negative impact on human health are explored in the results.

Results
Milk fat composition varied by management system but rarely by month and interactions between management and month were not significant. The concentrations of the main nutritionally relevant FAs are presented in Table 1, although full details of the FA profiles are presented in Appendix A, Table A1. Unless stated otherwise, all differences mentioned here and in the discussion were significant (p < 0.05).

Effect of Sampling Month on Composition
The only FA that showed seasonal variation was C12:0, which was~8% higher in April compared with milk collected in July and October.

Effect of Management and Month
There was no significant interaction between management and sampling month for the fatty acid profiles. However, Figure 1 demonstrates that for conventional and organic milk, the ratio of n-6/n-3 was significantly higher in April compared with other months. PFLA milk had a more consistent ratio across the three sampling dates and also had greater consistency between the individual farms compared with variation between samples bought from the various supermarkets (as indicated by the standard deviation).

Milk Fat Composition
This study explored differences in milk fat composition between conventional and organic UK supermarket own-brand and PFLA products and found significant differences between the three

Milk Fat Composition
This study explored differences in milk fat composition between conventional and organic UK supermarket own-brand and PFLA products and found significant differences between the three types of milk. Overall, these findings support previous studies (summarised in Table 2), showing benefits in organic compared with conventional milk but also, for the first time, demonstrate the scope for further improvement beyond that of typical organic milk in the UK through forage-only feeding.

Omega-6 and Omega-3
The main n-6 (LA) and n-3 (ALA) PUFAs must both come from our diet, as they cannot be synthesised by humans. However, the balance of these two essential FAs is important. Both are metabolised into long-chain FAs-LA to arachidonic acid and ALA to EPA, DPA and DHA. However, ALA and LA compete for the same enzymes for elongation/desaturation, resulting in lower EPA, DPA and DHA generation from ALA at high dietary LA intake levels [16]. In the past 150 years, the n-6/n-3 ratio in our diet has increased dramatically from approximately 1/1 to 10-25/1. Historically, n-3 came from meat (reared on pasture), fish, leafy green vegetables, nuts and berries. However, this has decreased, whilst n-6 consumption has increased with the inclusion of vegetable oils (including sunflower, soya bean, and corn/maize oil), cereal grains and animal products produced from grain-based, rather than forage-based diets over the last 50 years [31]. There is increasing evidence that this excess n-6 consumption and increase in dietary n-6/n-3 has contributed to a sharp rise in obesity, atherosclerosis and diabetes [15,16,32]. Therefore, working towards a n-6/n-3 ratio closer to 1-4/1 is thought to be important for human health [31]. While, even the n-6/n-3 ratio of conventional milk (2.2) was within the recommended range, it was three times lower in PFLA grass-fed milk (0.73)-more n-3 than n-6, with the potential to counterbalance high n-6 intake levels in many Western diets.
Results from other studies presented in Table 2 show a lower n-6/n-3 ratio in organic milk compared with conventional milk, and an even lower ratio for milk from low-input/forage-based systems. Details of the diet and management system used on farms producing the conventional and organic supermarket milk assessed in this study were not available. However, results from previous farm surveys including feeding details (Table 2) also showed clear positive associations between the ratio of n-6/n-3 and forage consumption in dairy diets. For example, PFLA-type farms producing Grassmilk ® in the US [3] showed that the highest proportion of forage in the diet had the lowest n-6/n-3 ratio (0.95) and low-input, organic farms that used moderate levels of concentrate had an intermediate ratio (2.3), while milk from conventional feedlot dairy systems had the highest n-6/n-3 ratio (5.8). It is well known that the inclusion of grain in livestock diets increases n-6 and suppresses n-3 in both dairy and meat produce [3,21,33]. Additionally, modelling by Benbrook et al. [3] suggests that a dietary switch from moderate consumption of conventional dairy products to high consumption of Grassmilk ® products reduces the LA/ALA ratio (LA and ALA are the major PUFAs, and this ratio tends to mirror that of n-6/n-3) in the total diet by 47%, hypothesised to have a direct benefit to human health. The milk collected from PFLA farms here (n-6/n-3: 0.73) show similar results to those reported for Grassmilk ® in the US (n-6/n-3: 0.954) by Benbrook et al. [3] and other studies ( Table 2), suggesting that these systems maximising grazing and other forages in dairy diets significantly alter the FA composition of milk, providing potential health benefits to consumers. Current study † Conventional low-input farms are not certified organic but generally follow some organic principles, often are spring calving and feed very little or no concentrate during lactation (the outdoor season), but, unlike organic farms, may use nitrogen fertiliser.

Conjugated Linoleic Acid
The main CLA in cow's milk is the isomer CLA9, which is almost exclusively from ruminant animal fats including dairy products [36]. This study found higher concentrations of CLA9 in milk from cows on pasture, and this is supported by previous research [37,38]. Milk from conserved forage diets has lower concentrations of CLA than milk from cows grazing fresh forage, although many studies have supplemented silage-based dairy diets with oilseeds, fish and/or various vegetable oils to improve the milk FA profile by elevating CLA concentrations [25,36,39]. In this study, the 100% grass-fed milk (1.2%) had double the concentration of CLA9 compared to the conventional supermarket milk (0.62%), suggesting that it may not be necessary to feed expensive oils or seeds, when high-quality, fresh forage could be used.

Effect of Management and Season
Seasonal variation in milk composition is expected due to the difference in feeding throughout the year on most systems (often cows are outdoors grazing during the summer and indoors on silage or hay diets during winter) [19]. Different patterns in the ratio of n-6/n-3-conventional and organic milk follows the expected trend of a high ratio of n-6/n-3 in April (when high-yielding cows might still be fed conserved forages with grains or cereal by-products) and a decrease in the ratio across July and October, when diets are likely to have increasing proportions of fresh forage from grazing ( Figure 1). In both the conventional and organic milk, n-3 concentrations gradually increased across the three sampling dates, whilst n-6 either remained the same (conventional) or decreased (organic), which is a similar pattern reported by Butler et al. [28] and Kliem et al. [40] (only conventional) in seasonal retail milk comparison studies. However, the PFLA milk did not follow this pattern; both n-3 (higher in PFLA than the other milks) and n-6 (lower than the other milks) increase incrementally from July to October, resulting in the n-6/n-3 ratio remaining relatively constant. PFLA farms were spread across the southern UK, with a wide range of herd sizes, breeds, sward types and management, but the main constant between farmers was their feeding policy. PFLA does not feed any grains, only forage, suggesting the continuity in forage-based feeding creates a more consistent composition, especially in the ratio of n-6/n-3, which has a stronger effect than location or other aspects of management (e.g., breed). Although the supermarket milk samples may have come from a smaller geographic area, they had a wide variation, partially attributed to the unknown of how many farms contributed to each sample. Despite this inherent variability, likelihood is high (especially in conventional systems) that cows come from a similar genetic lineage, suggesting that management, including feeding, is diverse in farms supplying the different supermarkets.

Forage in the Diet
A common theme of this paper is that concentrations of nutritionally beneficial FAs were significantly higher with pasture-based feeding. Fatty acid manipulation/improvement has been extensively researched with oils [39] and/or conserved forage [41]. However, evidence from this paper and work by Benbrook et al. [3] and others (Table 2) demonstrate that fatty acid profiles can improve by increasing pasture within dairy diets. This could be a cheaper and more effective way of modifying FA profiles whilst supporting the natural function of cow digestion and increasing beneficial FAs in the human diet. Increasing forage (and decreasing concentrate feed) could lower yields but the cost saved on inputs could be worth the compromise. Thus, a full economic analysis is required. The PFLA farm business case for feeding ruminants solely on pasture and conserved forages [42] finds that, with the right management, raising ruminants on forage can be profitable. Additionally, in the USA, Grassmilk ® farmers receive a 15% premium (over and above that for organic milk) if concentrations of beneficial FAs reach a threshold [3]. Whilst this is not yet available in the UK, interest in the nutritional composition of foods and encouraging high welfare and sustainable farming standards is high.

Conclusions
In conclusion, this study has found that milk from 100% forage-based dairy systems has a nutritionally more desirable FA profile compared to conventional and organic supermarket milk. Of particular interest is the ratio of n-6/n-3, which decreases dramatically from conventional to organic to PFLA milk. Additionally, the ratio of n-6/n-3 remained constantly low over the seasons in PFLA milk yet peaked (end of winter) and troughed (end of summer) in conventional and organic milk. Whilst these findings are consistent with previous studies, the novel finding in this study shows further improvement in the FA profile beyond organic milk. This provides scope for further and larger-scale research into producing 'more nutritious' milk and how dairy consumption can alter the ratio of n-6/n-3 in the total diet.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Figure A1. Map of the PFLA farms on the left and the supermarkets on the right [43].  Figure A1. Map of the PFLA farms on the left and the supermarkets on the right [43].