The effect of cattle breed, season and type of diet on the fatty acid profile of raw milk

The aim of the study was to determine the effect of cow breed, season and type of diet on the fatty acid (FA) profile of raw milk. A 2-year study was conducted on bulk milk samples collected from eight herds consisting of Czech Fleckvieh (CF, four herds) and Holstein (H, four herds) breeds. One half of the herds of each breed was grazed (G), while the other half was not (N). Samples were collected twice in winter (W) and twice in summer (S). Milk yield in CF (5385.50 kg) was lower than in H (7015.15 kg, P < 0.05). The effect of breed was found in odd-chain, branch-chain and hypercholesterolemic FAs (P < 0.05). The content of fat was lower in summer (S) than in winter (W), being 3.71 and 3.91 g 100 g−1, respectively (P < 0.05). The proportion of saturated and polyunsaturated FAs was lower in S than in W (P < 0.05). The content of monounsaturated FAs was higher in S (30.69 g 100 g−1) than in W (27.72 g 100 g−1, P < 0.05). Milk yield in grazing herds (G, 5197.50 kg) was lower (P < 0.05) than in non-grazing herds (N, 7203.75 kg). The sum of saturated and hypercholesterolemic FAs was lower and the sum of monounsaturated and odd-chain FAs was higher in G than in N (P < 0.05). Content of conjugated linoleic acid (CLA) and C18:3n3 was higher in G (0.93 and 0.64 g 100 g−1) than in N (0.42 and 0.39 g 100 g−1, respectively, P < 0.001).


Introduction
Bovine milk contains on average 4 % of fat that is from 97 to 98 % composed of triacylglycerols (Jensen, 2002).Milk fat can contain up to 400 different fatty acids (FAs; Jensen, 2002) that are usually grouped according to saturation of their carbon chain into saturated (SFAs), monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids.Bovine milk typically contains 70 % SFAs, 25 % MUFAs and 5 % PUFAs (Grummer, 1991;Shingfield et al., 2008).From the view of human health, attention has recently also been paid to so-called hypercholesterolemic FAs (HCFAs, C12:0, C14:0 and C16:0), which increase deposition of fat in the vascular walls and are related to atherosclerotic diseases (Jensen, 2002), and to odd-and branch-chain FAs (OCFAs and BCFAs, respectively), which are capable of inhibiting cancer cell proliferation and differentiation as well as inducing apoptosis in a number of cancer cell lines (Adamska et al., 2014).
Many factors can affect the FA composition of bovine milk fat, including breed (Adamska et al., 2014;Samková et al., 2014), parity (Stádník et al., 2013) or stage of lactation (Kirchnerová et al., 2013), as well as animal factors and diet composition (reviewed by Kalač and Samková, 2010), season (Frelich et al., 2012;Adler et al., 2013), geographical location (Collomb et al., 2008), access to fresh grazing Published by Copernicus Publications on behalf of the Leibniz Institute for Farm Animal Biology.(Frelich et al., 2012;Shingfield et al., 2013), grazing sward type (reviewed by Roca Fernandez and Gonzalez Rodriguez, 2012), silage type (Kalač and Samková, 2010), feeding of cereal and oil seeds, and oil supplementation in feed (Angulo et al., 2012;Stergiadis et al., 2014;Siurana and Calsamiglia, 2016) as nutritional and management factors.The abovementioned studies showed the possibility of altering FA profile of milk fat.However, as Coppa et al. (2013) pointed out, the majority of studies investigating the effect of diet and animal-related factors on milk FA profile were controlled trials not always reflecting common practice in commercial farms or applying measurements of farming practices not suitable on farms.Thus, the aim of the study was to determine the effect of the two predominant breeds reared in the Czech Republic and the effects of season and feeding management on the fatty acid profile of raw bovine milk.

Herd management and diet
A 2-year study was conducted on bulk milk samples collected from eight commercial dairy herds consisting of Czech Fleckvieh (CF, four herds) and Holstein (H, four herds) dairy cows.The average herd size was 185 ± 149 (from 66 to 439) dairy cows and the average milk yield was 6200.6 ± 1455.1 kg (from 3836 to 8124 kg) (see Table 1 also for details about selected farms' environmental characteristics).Cows were fed diets consisting of maize silages, clover-grass haylages, meadow hay, locally available feedstuffs, concentrate and mineral mixtures according to relevant milk yield and standard requirements.One half of the herds of each breed was grazed (G) during the summer season, while the other half was not (N).Composition of diets for each herd is given in Table 2.

Sampling and analysis
Cows were milked twice a day, and samples were collected regularly two times in the winter (W) and two times in the summer (S) period.In each group a total of 32 bulk milk samples were examined.Data concerning daily milk performance were obtained from animal records kept from milk recording kept by the Czech-Moravian Breeders' Corporation.
Milk fat was extracted with petroleum ether from freezedried milk samples.FAs of isolated fat were re-esterified to their methyl esters by methanolic solution of potassium hydroxide.The identification of FA methyl esters was carried out using analytical standards (SUPELCO, USA) and acetonitrile chemical ionisation mass spectrometry (Varian MS 4000 detector).Calibration was performed using a quantitative analytical standard (SUPELCO, USA).The proportions of individual FAs were calculated from the ratio of their peak area to the total area of all the observed FAs.

Statistical analysis
The GLM procedure of the SAS v. 9 program package (SAS Institute Inc., Cary, NC, USA) was used for the calculation.
Multi-factor analysis of variance with fixed effects as breed, season and feeding was used for statistical evaluation of the data set according to following model: where y ij k is the independent variable, µ is the general mean, b i is the effect of breed (i = 2), s j is the effect of season (j = 2), f k is the effect of feeding (k = 2), and e ij k is the random effect.

Effect of the breed
Milk yield, content of milk fat and its FA composition in dependence on breed, season and type of feeding is presented in Table 3. Milk yield in CF was 5385.50 kg and was lower than the milk yield in H (7015.15 kg, P < 0.001).The concentration of milk fat was higher in CF than in H (P < 0.05).In general, contents of FA groups in the CF and H breeds, respectively, determined in our study were as follows: SFAs, 65.34 and 67.09 g 100 g −1 ; MUFAs, 29.09 and 28.49 g 100 g −1 ; and PUFAs, 3.89 and 3.82 g 100 g −1 .

Effect of the season
The concentration of milk fat was lower during the summer season (S; 3.71 g 100 g −1 ) than in the winter season (W; 3.91 g 100 g −1 ) (P < 0.05).The content of total SFAs was lower in S (64.9 g 100 g −1 ) than in W (67.53 g 100 g −1 , P < 0.05).No effect of season was observed on the content of HCFA in our study (P > 0.05).The content of MUFAs was higher in S than in W, being 30.69 and 27.72 g 100 g −1 , respectively (P < 0.05), mainly due to significantly increased values of C18:1 in S compared to W (P < 0.05).The content of PUFAs in S (3.66 g 100 g −1 ) was lower than in W (4.06 g 100 g −1 , P < 0.05); however, content of C18:2n6 and CLA was not affected by the season.

Effect of the type of feeding
Milk yield in grazing herds (G, 5197.50 kg) was lower (P < 0.05) than in non-grazing herds (N, 7203.75 kg).Content of fat was higher in G than in N (P < 0.05).
The sum of SFAs in G (64.74 g 100 g −1 ) was lower than in N (67.69 g 100 g −1 , P < 0.05) mainly due to significant differences in C8:0, C10:0, C11:0, C13:0, and C14:0 (P < 0.05), even if major SFAs such as C16:0 and C18:0 did not differ between G and N feeding systems.The content of MUFAs was higher in G than in N (P < 0.05), mainly due to higher proportion of C18:1 in G (26.49 g 100 g −1 ) in comparison to N (24.15 g 100 g −1 , P < 0.05).Furthermore, higher contents of minor FAs, such as C19:1 and C20:1 in G (P < 0.05), contributed to differences in total MUFAs as well.Although the content of PUFAs in our study was not affected by the type of feeding (P > 0.05), higher content of CLA and C18:3n3 in G compared to N was observed (P < 0.001).The content of BCFAs was higher in G (1.91 g 100 g −1 ) than in N (1.71 g 100 g −1 , P < 0.05).

Discussion
Our findings concerning milk yield and concentration of milk fat are in agreement with data characterising an ordinary population of dairy cows of these two breeds bred in the Czech Republic (e.g.Wolfová et al., 2007;Ducháček et al., 2014).Contents of SFAs, MUFAs and PUFAs determined in our study are within the range that has been recently reported for these FA groups in H and CF cows (Samková et al., 2014;Stádník et al., 2013;Ducháček et al., 2014).As mentioned earlier, breed belongs to factors affecting FA composition of milk fat, as proved in recent studies (e.g.Palladino et al., 2010;Van Eijndhoven et al., 2011;Samková et al., 2014); however, many authors agree that the effect of breed on FA profile is minor compared with the effects of diet (Garnsworthy et al., 2006;Ferlay et al., 2011;Nantapo et al., 2014).According to Kelsey et al. (2003) and Roca Fernandez and Gonzalez Rodriguez (2012) breed contributes less than 1 % of the variation in milk FAs.This was also confirmed in our study because we found significant breed effects only in OCFAs, BCFAs and HCFAs and some individual mediumand long-chain SFAs and MUFAs and CLA (P < 0.05).Our findings are in agreement with studies performed on the same breeds (Samková et al., 2014).Minor breed effects on medium-or long-chain SFAs and MUFAs or on HCFAs have also been found in other studies (Morales et al., 2000;Drackley et al., 2001;White et al., 2001;Ferlay et al., 2006).
www.arch-anim-breed.net/59/373/2016/Arch.Anim.Breed., 59, 373-380, 2016 Season is considered a substantial source of variation in the FA composition of milk fat (Frelich et al., 2012;Adler et al., 2013).In our study, the content of total SFAs was lower in S than in W (P < 0.05).Similar findings were reported by Adler et al. (2013), who found higher proportions of total SFAs and most short-and medium-chain SFAs in milk from the indoor feeding season compared to the outdoor feeding season.A positive effect of season on SFA proportion has also been proved by Lindmark-Månsson et al. (2003), Collomb et al. (2008) and Ozcan et al. (2015), with the highest differences mainly in C16:0 and C18:0.Frelich et al. ( 2012) described lower content of short-chain SFAs (C4:0 − C10:0) and C12:0 in S compared to W in herds kept indoors (P < 0.05).Seasonal variations among SFAs were also noted by Adamska et al. (2014).
Increased levels of C18:1 in S compared to W (P < 0.05) are in agreement with Adler et al. (2013); however, in their study, significant differences in individual MUFAs were also noted in C14:1 and C16:1 FAs, which were not affected by the season in our study.On the other hand, we observed seasonal variation in few minor FAs such us C12:1 and C20:1.Lock and Garnsworthy (2003) and Wiking et al. (2010) also mentioned higher content of C18:1 in summer milk.Among individual PUFAs, significant differences were observed in C18:3n3 and also in some minor FAs (P < 0.05).Higher values of C18:3n3 in S were also reported in other studies (Lindmark-Månsson et al., 2003;Lock and Garnsworthy, 2003;Collomb et al., 2008;Wiking et al., 2010;Adler et al., 2013).On the other hand, Frelich et al. (2012) and Ozcan et al. (2015) found no effect of season on C18:3n3 content.In our study, content of C18:2n6 and CLA was not affected by the season.Similar findings were reported by Lindmark-Månsson et al. (2003), Frelich et al. (2012) and Ozcan et al. (2015).In contrast, Collomb et al. (2008) and Adler et al. (2013) observed a positive effect of the outdoor/summer season on content of these two FAs (P < 0.05).Discrepancies in the effect of season on FA profile as discussed above can be at least partly explained by different management of herds during the summer season, when pasture or feeding of fresh forage to cows kept indoors can be applied.This is also the case in our study because one half of each of the studied herds was grazed during the summer, while the other half was not.This fact can influence seasonal differences in FA profile; however, it represents the common practice in the Czech Republic and thus characterises the FA profile of milk fat in this part of the year.
The breeding of the two most common dairy breeds, H and CF, in the Czech Republic relies on two feeding strategies: a pasture-based feeding system with a seasonal pasture from May to October followed by silage feeding indoors for the rest of the year, and a silage-based feeding system with the indoor silage feeding without any access to pasture throughout the year (Frelich and Šlachta, 2011).Concerning the total SFAs, our results are in agreement with many studies comparing pasture-based and silage-based feeding systems (Frelich et al., 2009(Frelich et al., , 2012;;Kirchnerová et al., 2013;Shingfield et al., 2013); however, there are some differences in the effect of feeding system on individual SFAs.In the study of Frelich et al. (2009) content of C4:0 was lower in the indoor feeding season; furthermore, they found significant differences in C16:0 and C18:0.Similarly, in their subsequent study Frelich et al. (2012) mentioned lower content of C12:0, C14:0, C15:0 and C16:0 and higher content of C18:0 in grazing herds in comparison to indoor kept herds (P < 0.05).According to Dewhurst et al. (2006) and Coppa et al. (2013), a lower amount of C16:0 in milk from feeding of fresh herbage is a well-known and wellpredicted trend that is attributed to a lower C16:0 proportion in herbage in comparison to maize silage (Elgersma et al., 2006).
The lower content of HCFAs in G is in disagreement with Kirchnerová et al. (2013), who did not find significant differences in total HCFAs.The content of MUFAs was higher in G than in N (P < 0.05) mainly due to higher proportion of C18:1, C19:1 and C20:1.This is in agreement with Chilliard et al. (2009), Frelich et al. (2012) and Shingfield et al. (2013).Also, Kirchnerová et al. (2013) proved a higher content of MUFAs in a pasture-based feeding system.On the other hand, Adamska et al. (2014) did not find differences in total MUFAs but confirmed the positive effect of pasture on C18:1 FAs in Polish indigenous cattle.
In addition to our study, higher content of BCFAs in grazed herds has been reported by Ferley et al. (2008), Frelich et al. (2009), Slots et al. (2009) and Kirchnerová et al. (2013).Furthermore, changes in BCFAs that originate from rumen fermentation can be well predicted by an increase in fresh herbage and hay and a decrease in maize silage and concentrates in the cows' diet (Coppa et al., 2013) because high-neutral detergent fibre diets (such as fresh herbage-based diet) favour ruminal populations of cellulolytic bacteria instead of the amylolytic bacteria favoured by starch-rich diets (Vlaeminck et al., 2006).Furthermore, the FA profile of milk fat can be influenced by the FA composition of pastures, which is dependent upon species, variety, growing conditions and forage maturity, as well as by grazing management strategies implemented at the farm level such as timing of cutting or grazing (Chouinard et al., 1998;Dewhurst et al., 2003;Elgersma et al., 2006;Ferlay et al., 2006).

Conclusions
In conclusion, this study confirmed that breed has only a minor influence on the FA profile of milk fat.Seasonal variability was observed in 14 FAs.In the summer season the content of saturated and polyunsaturated FAs was lower and content of monounsaturated FAs was higher than in the winter season.A positive effect was observed mainly in C18:1 FAs.Cow feeding had a major effect on milk FA composition.The variability in FA proportion was observed in 26 FAs.Pasture, compared to year-round feeding based on silages, decreased the contents of saturated and hypercholesterolemic FAs and increased the proportion of C18:1, 18:3n-3 and conjugated linoleic acid (CLA).

Table 1 .
Basic information about environment of studied dairy cow herds.

Table 2 .
Composition of diets (kg day −1 , as fed basis) of dairy cows used in experimental herds.

Table 3 .
Effect of cattle breed, season and type of feeding on the fatty acid profile (g 100 g −1 of total fatty acids) of milk fat.