Effect of different fat supplements on the antioxidant capacity of cow ’ s milk

This study examined the effect of different fat supplements on the antioxidants of cow’s milk. Thirty five Polish Holstein Friesian cows at 180±20 day of lactation and averaging 17.71±2.26 kg/d of milk were divided into five equal groups. There were five treatments groups: 300 g/d of fish oil, 500 g/d of Opal linseed, 500 g/d of Szafir linseed, 150 g/d of fish oil + 250 g/d of Opal linseed, 150 g/d of fish oil + 250 g/d Szafir linseed. Milk samples were collected five times during the 21 day of supplementation period: at the 1st, 14th and 21th day of the experimental period, and also at the 14th and 21st day after the supplement withdrawal. Diet supplementation with the Szafir linseed improved the chemical composition and milk yield at the 21st day of experiment. The highest level of malondialdehyde was recorded in fish oil, Opal linseed, fish oil + Opal linseed, and fish oil + Szafir linseed groups, and the lowest in the Szafir linseed group. After the 21st day of supplementation the concentration of α-retinol was significantly increased in all groups by 23 to 183 %. The highest level of α-tocopherol was found in fish oil + Opal linseed group at the 21st day of supplementation. Total antioxidative status increased in all experimental groups; however, the highest peak was recorded in fish oil + Szafir linseed and Szafir linseed group. The experiment confirmed that manipulation in cows’ diet may improve antioxidative capacity of milk.


Introduction
Recent studies suggest that free radicals have been implicated in the development of over 100 diseases affecting all major organs.Reactive oxygen species are chemically reactive molecules containing oxygen.Antioxidants help to defend the body against free radicals.
One of the most important indicators of oxidation process in milk is malondialdehyde (MDA), which is formed during peroxidation of polyunsaturated fatty acids by the action of reactive oxygen species.Increasing level of MDA modifies the physical properties of the cell membrane, resulting in depletion of antioxidant systems.Mutagenic and carcinogenic properties of reactive oxygen species have also been confirmed by Chen et al. (2000).
Nevertheless, cow's milk contains a range of biologically active substances with antioxidant properties, which reduce the risk of arteriosclerosis and protect against heart disease, reduce the risk of cancer, slow down the progress of Alzheimer's disease, protect the body against environmental pollutants.The typical exogenous antioxidants supplied with food or as supplements are selected vitamins (A, C, E), carotenoids (α-and β-carotene) and also exogenous coenzyme Q10.Beta-carotene (provitamin of vitamin A) reduces the risk of breast, lung, stomach and uterus cancer and provides protection against the onset of myocardial infarction and stroke (O'Grady et al. 2001).Rice-Evans et al. (1997) showed that carotenoids exhibit antioxidant properties, including free radicals as a result of transfer of free electrons, or by creating adducts with them.In addition, α-retinol traps peroxyl radicals, and β-carotene singlet oxygen.Vitamin E is one of the most important antioxidants in milk.It prevents lipid peroxidation through the capture of singlet oxygen and hydroxyl radicals (Kamal-Eldin & Appelquist 1996).Palozza et al. (2000), examining the effect of C20:5 n-3 (EPA)and β-carotene on the growth of tumour cells, also confirmed the role of peroxidation products in this process.According to Gonzalez et al. (1993), in mice implanted with human breast cancer, fed with corn oil or its mixtures with different amounts of fish oil, tumour growth was inhibited when fed with addition of fish oil.Naziroglu et al. (2004) states that vitamin E supplementation decreases the concentration of MDA in the liver of rats.The purpose of this study was to improve the quality of milk fat affecting the health of consumers by elevating antioxidants level in cow's milk.Increasing of the antioxidant capacity of milk by the use of food supplements is a simple method of its modification, which can be advantageous both for the consumers and for the technological quality of milk.There are many studies, in which various forms (whole, rolled, crushed or ground) and quantities of linseeds were analysed as supplements affecting quality of milk (Allred et al. 2006, Nałęcz-Tarwacka et al. 2008, Chilard et al. 2009).Nevertheless, no studies have been conducted to investigate the impact of linseeds variety on the level of fat soluble vitamins, β-carotene and the overall antioxidant effect of milk in one experiment.Also, combined supplementation of fish oil and linseeds is a new solution.Comparison of five different fatty additives in a nutritional experiment allows for selection of the type of supplement, preferably affecting the changes in the content of vitamins soluble in fat and in the overall antioxidant capacity of cow's milk.The purpose of this study was to improve the quality of milk fat affecting the health of consumers by elevating antioxidants level in cow's milk.Obtaining a greater antioxidant capacity of milk by the use of feed supplements is a simple method of its modification, which can be advantageous both for consumers and for the technological quality of milk.

Material and methods
The experiment was conducted at experimental dairy farm belonged to Warsaw University of Life Sciences in Poland.All cows were handled in accordance with the Polish Council on Animal Care regulations.The Warsaw University of Life Sciences Care Committee reviewed and approved the experiment and all procedures carried out in the study.

Animals, treatment, and sampling
Thirty five Polish Holstein Friesian cows, 15 primiparous and 20 multiparous (at second and third lactation) at 180±20 day of lactation and averaging 17.71±2.26kg/d of milk were divided into five equal groups.The treatments were: 1) FO = control with 300 g/d fish oil, 2) LO = control with 500 g/d linseed Opal variety, 3) LS = control with 500 g/d linseed Szafir variety, 4) FOO = control with 150 g/d fish oil + 250 g/d linseed Opal varieties, 5) FOS = control with 150 g/d fish oil + 250 g/d linseed Szafir varieties.Milk collected before the supplementation period (collecting 1) was the reference to further supplementation changes.Crude linseed was given as unprocessed whole seeds.During the first seven days of experiment cows were customized to new supplements in diet.The supplements were administered individually into each animal's feeder every day at the same time (in the morning) for 21 days.Fish oil used in the experiment was manufactured during fish meal production process of the following fish: herring, sprat, silver carp, mackerel and cod, and was purchased in the Agro-Fish company.During the storage the oil was stabilized with antioxidant Randox (Randox, Crumlin, UK), enriched in a-tocopherol (20.04 mg/100 g of oil).The two varieties of linseed, Opal and Szafir, were used in the experiment.
During the experiment the five feeding places were separated in frees stall barn, so that each cow had free access to the trough with supplements.Throughout the experiment, no cases of failure to eat the entire amount of the administered supplements were observed.
The cows were milked daily at 05.30 and 17.30 and milk yield was recorded at each milking.Milk samples were collected five times: at the 1st day of supplementing period (collecting 1) after the 14th (collecting 2) and 21st day of the supplementation period (collecting 3) as well as 14th (collecting 4) and 21th (collecting 5) day after experimental period.Representative milk samples were collected from each cow individually during milking by means of a milk meter in the milking parlour.Combined milk from morning and evening milking were placed in sterile bottles, preserved with Mlekostat CC (Zekar, Brwinów, Poland).The samples were immediately submitted to the Cattle Breeding Division (Milk Testing Laboratory of Warsaw University of Life Sciences) for compositional analysis immediately after milking.
The cows were kept in a free-stall dairy shed and fed on total mixed ration (TMR) diet.All animals had continuous access to water.TMR diet was provided for ad libitum intake, dry matter intake (DMI) was monitored daily throughout the experiment.Cows were fed TMR two times a day at 8.00 and 18.00.The TMR diets were formulated using the French National Institute for Agricultural Research (INRA) system to provide adequate milk energy and milk production for a 650-kg cow producing 20 kg of milk/d containing 3.5 % fat and 3.2 % protein.Representative TMR samples were pooled three times per three week and stored at −20 °C until analysis.Samples were analysed in Cattle Breeding Division (Poland) for dry matter (DM), crude protein, ash, ether extract, acid detergent fibre, neutral detergent fibre.The chemical composition of the TMR was calculated from the chemical composition of individual ingredients of the diet.Daily DMI for individual cows was calculated by subtracting the weekly mean of orts from the weekly mean of feed offered.According to INRA diet offered to cows faced their requirements (±5 %).Chemical composition of the treatment diets is presented in the Table 1.Contained (on 1 000 g): Ca 150 g, P 100 g, Na 50 g, Mg 40 g, Zn 9 000 mg, Mn 7 000 mg, Cu 1 000 mg, J 100 mg, Se 50 mg, vitamin A 1 200 000 j.m., vitamin D3 120 000 j.m., vitamin E 5 000 mg, vitamin K 93 mg, vitamin B1 80 mg, vitamin B6 160 mg, vitamin B2 110 mg, vitamin B12 1 000 mcg, 2 Contained (on 1 000 g): Mg 24 %, P 13.5 %, Ca 1 %

Chemical analyses
Basic parameters of the milk, i.e. fat, protein, casein, lactose and free fatty acid content were determined by automated infrared analysis with a MilkoScan FT120 instrument (Foss, Hillerød, Denmark).Evaluation of hygienic status of the milk was based on somatic cell count on Somacount 150 (Bentley Instruments, Inc., Chaska, MI, USA)).
For examination of the fatty acid composition the fat from the treatment and supplements, were extracted according to Rose-Gottlieb procedure (AOAC 1990) at room temperature.Fatty acid methylation was performed according to the trans esterification method by Kramer et al. (1998).Identification of the individual fatty acids was carried out by gas chromatography using Hewlett Packard 6890 GC with HP Chem software (Hewlett Packard, Palo Alto, CA, USA), a flame-ionization detector, and a Varian Select FAME column (Agilent Technologies, Santa Clara, CA, USA); 100 m length, 0.25 mm in diameter and 0.25 μm thick.The separation was performed at pre-programmed temperature: 130 °C for 1 min; 130-170 °C at 6.5 min; 170-215 °C at 2.75 min; 215 °C for 12 min, 215-230 °C at 20 min and 230 °C for 3 min.Helium at a flow rate of 25 cm/s and constant pressure was used as the carrier gas, the injector temperature was 240 °C, and the detector temperature was 300 °C.
The determination of the antioxidant components was performed using a Randox-TAS (total antioxidant status) test, according to the application described by Randox and Puppel (2011).

Statistical analysis
The obtained data were analysed statistically using a multi-factor analysis of variance (least squares) by means of the SPSS 12.0 packet software (SPSS Inc., Chicago, IL, USA).Only statistically significant (P≤0.01 or P≤0.05) interactions between factors were considered in the study.The level of significance was determined after performing preliminary statistical analyses.
The model used for analysed milk samples was: where Y ijk is the dependent variable, A i is the treatment effect (i=1-6; 1: control; 2: FO; 3: LO; 4: LS; 5: FOO; 6: FOS), B j is the collecting effect (j=1-5), A i ×B j is the fixed interaction effect between treatment and collecting and e ijkl is the random error.

Results
Chemical composition of the TMR is presented in the Table 1, and the content of fatty acids in the supplements in Table 2. Linseeds were an excellent source of C 18:2 cis-9, C 18:2 n-6 and C 18:3 n-3.Opal variety was characterized by higher content of C 18:2 cis-9 and C 18:2 n-6 , but Szafir variety was the richest source of C 18:3 n-3 and C 16:1 acid (Table 2).
The effect of lipid supplementation on the level of fat in milk is well understood, but it should be noted that the obtained results were very diverse.A statistically significant reduction in milk fat content in four groups: FO, LO, FOO, FOS was recorded (Table 3).
As for the total protein content, was a statistically significant increase in its level in milk was recorded after using the following supplements: FO, LO, LS and FOS (Table 3).The fat supplements applied affected slightly the concentration of lactose and SCC levels during the experimental period (Table 3).The addition of LS improved chemical composition and milk yield after the 21st day.Supplementation of LS influenced most preferably the chemical composition of cow's milk; the study has shown increase in the percentage of protein, fat and lactose.The TBA test (thiobarbituric acid) provides information about the level of MDA, a secondary compound formed in lipid oxidation.In all groups, a significant increase of MDA concentration in milk was noted (Table 4) after the 21st day of supplementation.The highest level of MDA was recorded in four groups: FO, LO, FOO and FOS.The lowest MDA content was noted in the LS group (0.378 mg/l of milk), which resulted in an increased concentration of this component by 34 % compared to the first collection (Table 4).One of the main enzymatic reactions causing quality deterioration in raw milk is lipolysis.Lipolysis is the breakdown of triglycerides to free fatty acids (FFA).During the 21st day supplementation period, a very significant increase in FFA content in all dietary groups was found (Table 4).The highest level of FFA was observed in the group with the combined supplementation of fish oil and linseed: FOO and FOS.On the other hand, the lowest level was recorded in LS group -0.520 g/100 g of fat.The effect of supplementation on the content of selected antioxidants and TAS was presented in the  Also, the concentration of α-retinol increased significantly in all experimental groups by: 34.34-183.10%, at 21st day of supplementation.Noteworthy is also a significant increase in vitamin A content in milk of cows receiving the LS supplement (from 0.728 to 2.061 mg/l) compared to other groups.Moreover, there was a higher level of α-retinol compared to control collecting in all supplemented groups (P≤0.05) at the fifth collecting.Concentration of α-retinol in milk after 21 days of supplementation progressed in accordance with the following scheme: LS>FO>FOS>FOO>LO.The use of fatty additives also positively affected the α-tocopherol content.After the 21st day of supplementation, the highest level of α-tocopherol was in FOO group (an increase by 56.58 %).The lowest level was recorded in FOS group (an increase by 36.75 %).In the FO, LO and LS groups an increase of tocopherol concentration was similar, fluctuating between 44.91 % and 49.25 %.A high content of vitamin E was also found after 21 days of administration of supplements (collecting 5) in all of the experimental groups.Amount of α-tocopherol in milk after 21 days of supplementation was changing as follows: FOO>LS>LO>FO>FOS.
The experiments confirmed a significant increase of TAS in milk in all analysed groups (Table 5).The highest level of TAS was recorded in two groups: FOS, increased from 1.282 to 3.331 mmol/l (by 159.82 %) and LS, increased from 1.282 to 3.208 mmol/ (by 150.23 %).In the case of FO, LO and LS, the content of TAS in milk was increased by 84.71 %, 80.73 % and 144.23 %, respectively.During the fifth collecting (after the 21st day of supplementation), the antioxidant potential of milk was still at a higher level compared to the control collection.The capacity of TAS in milk after 21 days of supplementation was shaped as follows: FOS>FOO>LS>FO>LO.

Discussion
Studies conducted over several years show that the introduction of fat supplements (with high concentrations of UFA) in the diet of cows may affect the sensory qualities of milk, through changes in the contents of individual fatty acids (Charmley & Nicholson 1994).However, already in 1967, King (1967) stated that the addition of 1 g of tocopherol per day/cow provides excellent protection against these changes.In the experiments conducted, fish oil was stabilized with Randox antioxidant, and it was enriched with tocopherol by 20.04 mg/100 g of oil.Therefore, the dose for cows receiving 300 g FO was 60.12 mg tocopherol, whereas in the case of cows receiving 150 g FO -it was lesser by half.Organoleptic evaluation of cow's milk in experiments did not show sensory changes in analysed milk samples, this relationship is also confirmed by results obtained by authors, such as Nelson & Martini (2009) and Sympoura et al. (2009), in which the authors showed that the addition of fish oil as a supplement does not affect the sensory quality of milk.
Increased content of C 18:2 n-6 and C 18:3 n-3 fatty acids in milk fat, which is desirable due to its hypocholesterolemic effect, was recognized as an important factor for depressing the oxidative stability of milk fat (Reklewska et al. 2002).Research conducted by Puppel et al. (2012) has shown that combined supplementation of fish oil and linseed has a positive effect on the formation of the levels of these fatty acids in cow's milk; however, the authors do not take into account such factors as the variety of seeds.As it is shown by data on the fatty acid composition of treatment diets (Table 2), the varieties of linseeds were differing in terms of the concentration of individual fatty acids.
According to Doreau et al. (1999), supplementation with fish oil in the amount of 50-100 g/ day/cow may influence the reduced fat concentration in cow's milk.Similar results were also obtained by Bharathan et al. (2008).DePeters & Cant (1992) indicate that addition of fat reduces the synthesis of protein in the mammary gland (including casein and whey proteins), and therefore may be explained by lower protein content during the supplementing period.The authors explained the reduction of this component by increased stimulation of synthesis of fat and lactose.High fat content reduces blood flow through the mammary gland, thereby reducing the concentration of amino acids.The results of this study did not confirm this phenomenon.The increased total protein content in milk in the experimental groups (FO, LO, LS and FOS) was probably the result of additional energy provided in the supplements.The protein content in milk is also associated with the stage of lactation; cows in late lactation are characterized by higher levels of protein, including casein and whey proteins in milk (DePeters & Taylor 1985).Experimental cows were in the late lactation stage, therefore, characterized by higher concentration of milk protein.Positive influence of fatty supplements on protein content was also confirmed by Bobe et al. (2009) experiment. However, Flowers et al. (2008) found that supplementation of cow diet with flax seeds (0.17, 0.34 and 0.51 kg/day) does not influence the performance and content of basic components in the milk protein.
Higher levels of MDA in milk, compared to the results obtained in the experiment carried out by the authors, were showed by Reklewska et al. (2002).Gladine et al. (2007) have demonstrated the protective role of vitamin E in relation to the introduction of PUFA supplementation, and thus demonstrated that tocopherol protects against runaway growth of MDA in milk.Sawosz (2000) observed a reduction in vitamin E content in muscle tissue as a result of feeding pigs with the oil-rich in PUFA, which is evidence of involvement in the synthesis of tocopherol radical.According to Gobert et al. (2009) introduction of supplements in the form of linseed and plant polyphenols (PERP) does not affect the intensification of peroxidation processes in milk, which is also reflected by our study.In the fifth collecting, after 21 days of administration of supplements, the study showed a reduction in the level of MDA in the: FO, LS, FOS and FOO group relative to control collection.The effect of vitamin E on MDA differs between the blood, liver, and mammary gland.Vitamin E supplementation could not prevent the increase in blood MDA at calving, but the significantly lower MDA blood concentrations of supplemented cows in the second week post partum suggests that vitamin E plays a role in recovery from parturition-related oxidative stress (Bouwstra et al. 2008).
Oxidation products are unstable and they tend to react with food constituents, so their detection may not be a valid tool for quality assessment in all cases.After homogenization without pasteurization, natural milk lipase will cause lipolysis of the raw milk, resulting in formation of FFA.Chilliard (1993) confirms that the addition of feed fat may influence the increase in FFA content.Subsequently, FFA will be broken down to aldehydes and ketones (Deeth & Fitz-Gerald 2006).Free fatty acids also cause damage and deformation of the milk fat globule membrane, which affect the deterioration of the technological properties of cow's milk (Kuczyńska 2001).
Similar results in terms of increasing vitamin A content in cow's milk after using linseeds were obtained in the studies of Reklewska et al. (2000) and Nałęcz-Tarwacka et al. (2008).Additionally, Reklewska et al. (2000) also noted the increase in concentration of β-carotene.The increase in milk yield in the following groups: LS, FOS and FOO can also influence the increase of β-carotene content in milk.This dependence is confirmed by research conducted by Nozière et al. (2006).The highest level of β-carotene was demonstrated in two groups that received linseed.However, the higher level was found in the LO group, and highest concentration of vitamin A in LS.According to Yang & Tume (1993) β-carotene to turn into retinol must undergo certain chemical processes.LS supplementation significantly accelerates this process resulting in increasing of both vitamins in milk.
Content of α-tocopherol, one of the most important antioxidants of cow's milk, varies between 13-30 μg/g fat (Jensen & Nielsen 1996).In order to increase its level, it can be injected intramuscularly (Charmley & Nicholson 1994), or included directly in cow feed (Charmley et al. 1993).Adding of oilseeds has a similar effect (Focant et al. 1998, Morales et al. 2000, Bell et al. 2006, Nałęcz-Tarwacka et al. 2008).Increase in the concentration of α-tocopherol in the analysed milk samples may be related to the transfer of supplemented tocopherol (fish oil used in the experiment was nutritionally enriched in α-tocopherol) into milk (Charmley & Nicholson 1994), which could also affect the oxidative stability of milk (Focant et al. 1998).Addition of α-tocopherol to cow feed affects also improves the digestibility and utilization of β-carotene, probably due to the antioxidant properties of vitamin E (Morales et al. 2000).
It should be note that increase of α-tocopherol, α-retinol, and β-carotene content in milk due to use of fat supplements can also be explained by better use of the diet components.TAS enables the determination of total levels of antioxidant compounds (enzymes, antioxidants endo-and exogenous) in the milk in a very short time, and it is also used to monitor individual antioxidant components, as well as the general state of the system to prevent the oxidation process (Puppel et al. 2011). Research Puppel et al. (2012) has shown that levels of TAS were associated with both supplementation and the age of cows.
Summing up, the most favourable changes in the b-carotene content were due to use of linseed of Opal varieties.The content of vitamin A was on the highest level in the milk of cows treated with linseed of Szafir varieties.Formation of β-carotene in milk after 21 days of supplementation takes place in accordance with the following scheme: LO>LS>FO>FOO>FOS, and forα-retinol: LS>FO>FOS>FOO>LO.The concentration of vitamin E was highest in the milk of cows that received a mixture of fish oil and linseed of Opal variety (FOO>LS>LO>FO>FOS).
Despite the fact that the values determined in the fat-soluble vitamins were at a lower level in FOS compared with other groups, the highest level of TAS was obtained in this particular group.This indicates that the determination of antioxidants only on the basis of the level of fat soluble vitamins is inadequate, and therefore all antioxidant components should be taken into account to correctly determine the level of total TAS.Research has confirmed that in nutritional experiments, not only the form, quantity or physical form of linseed, but also the variety should be taken into consideration.

Table 2
Fatty acid composition of the treatment diets: TMR, fish oil and linseed (g/100 g of fatty acid) A-G P≤0.01, a-g P≤0.05, FO: control with 300 g/dfish oil, LO: control with 500 g/d linseed of Opal variety, LS: control with 500 g/d linseed of Szafir variety, FOO: control with 150 g/d fish oil and 250 g/d linseed of Opal varieties, FOS: control with 150 g/d fish oil and 250 g/d linseed of Szafir varieties, 1: at the first day of supplementing period, 2: at the 14th day of supplementing period, 3: at the 21st day of the supplementation period, 4: at the 14th after supplementing period, 5: at the 21th after supplementing period

Table 5 .
Applied fatty additives significantly influenced both the content of soluble vitamins fat and antioxidant properties of milk.β-carotene content at 21st day of supplementing increased significantly in the following groups: FO (by 64.88 %), LO (by 70.92 %), LS (by 69.80 %), FOO (by 46.31 %) and FOS (by 7.16 %).The highest content was noted in the LO group (0.764 mg/l).Formation of β-carotene in milk after 21 days of supplementation was as follows: LO>LS>FO>FOO>FOS.