The Effect of CLA-Rich Isomerized Poppy Seed Oil on the Fat Level and Fatty Acid Profile of Cow and Sheep Milk

Simple Summary Conjugated linoleic acid (CLA) has attracted significant interest due to its health-related properties. The use of isomerized poppy-seed oil (IPO) enriched with CLA in cow and sheep feed reduced the fat content in milk and favorably modified the fatty acid profile. The content of saturated fatty acids (SFAs) in milk fat, especially medium-chain fatty acids showing adverse atherogenic and thrombogenic effects, decreased, while the content of polyunsaturated fatty acids (PUFAs), including biologically active fatty acids with pro-health properties (i.e., CLA isomers and trans-vaccenic acid (TVA)), increased. In conclusion, IPO with a high concentration of CLA could be used in dairy animal feed to change the nutritional quality and health value of milk, which is beneficial from a human point of view. Abstract The aim of the study was to examine the effect of dietary supplementation of isomerized poppy seed oil (IPO) enriched with conjugated dienes of linoleic acid (CLA) on cow and sheep milk parameters (fat content, fatty acid profile, Δ9-desaturase index, and atherogenic index). The process of poppy seed oil alkaline isomerization caused the formation of CLA isomers with cis-9,trans-11, trans-10,cis-12, and cis-11,trans-13 configurations in the amounts of 31.2%, 27.6%, and 4.1% of total fatty acids (FAs), respectively. Animal experiments were conducted on 16 Polish Holstein Friesian cows (control (CTRL) and experimental (EXP), n = 8/group) and 20 East Friesian Sheep (CTRL and EXP, n = 10/group). For four weeks, animals from EXP groups received the addition of IPO in the amount of 1% of dry matter. Milk was collected three times: on days 7, 14, and 30. Diet supplementation with IPO decrease milk fat content (p < 0.01). Milk fat from EXP groups had higher levels of polyunsaturated fatty acids, including FAs with beneficial biological properties, that is, CLA and TVA (p < 0.01), and lower levels of saturated fatty acids, particularly short- (p < 0.01) and medium-chain FAs (p < 0.05). The addition of IPO led to a decrease in the atherogenic index.


Introduction
Fatty acid (FA) composition plays a crucial role in milk nutritional quality. Based on potential benefits for long-term human health, there is interest in developing sustainable nutritional strategies Michałko (IHAR, Plant Breeding and Acclimatization Institute-National Research Institute, Poznań, Poland) in a screw press. The synthesis also involved ethylene glycol (analytical standard, a.s.), potassium hydroxide (a.s.), concentrated hydrochloric acid (a.s.), anhydrous sodium sulfate (a.s.), and hexane as a solvent (all reagents were obtained from Avantor Performance Materials Poland Inc., Gliwice, Poland).
In order to carry out alkaline isomerization of poppy seed oil, ethylene glycol was placed in a reactor, and after heating to about 50-60 • C, approximately 25% mass potassium hydroxide (KOH) relative to glycol was introduced. After dissolving potassium hydroxide and increasing the temperature to 140 • C, poppy oil was added to the system. The isomerization process was carried out for 3 h at 185 ± 5 • C. When the reaction was completed and the reactor content cooled down to 70-80 • C, water was added (1:1 by volume) in order to dilute the resulting soap solution, and then hydrochloric acid was introduced into the reactor in order to acidify the soaps to free FAs. After the soaps were completely acidified, the reaction mixture was transferred to a separator where the aqueous layer was separated, and the FA layer was rinsed with water (about 90 • C) and then dried over sodium sulfate.
The process of poppy seed oil pressing, its isomerization, and chromatographic analyses of FA profiles were performed at the Ignacy Mościcki Institute of Industrial Chemistry in Warsaw (Poland).

Animals and Treatments
All the cows and sheep were handled in accordance with the regulation of the Polish Council on Animal Care, and all procedures for this trial were approved by the 2nd Local Ethical Committee for Experiments on Animals in Wrocław (No. 6/2009).
The study was carried on sixteen Polish Holstein Friesian cows of black-white variety (582 ± 12.1 kg of BW, at 90 ± 12 d of lactation (multiparous cows), and an average milk yield of 31.4 ± 3.57 kg/d), divided into two groups (n = 8/group). The animals were selected on the basis of stage of lactation, milk yield, and live weight and they were allocated at random to particular groups. The cows were kept in a stall system, individually blocked, and fed total mixed ration (TMR) based mainly on maize and grass silage with free access to fresh water. Cows from the experimental group (EXP) additionally received isomerized poppy seed oil (IPO) in the amount of 1% dry matter (DM), that is, 220 g per head/day on the mineral carrier Humokarbowit, while the animals from the control group (CTRL) received Humokarbowit in an analogous amount ( Table 2). Dry matter intake (DMI) in both groups was 21 kg/cow per day, allowing 5% refusals.  4 Feed unit for lactation. 5 Protein digested in the intestine when rumen fermentable nitrogen is limited. 6 Protein digested in the intestine when rumen fermentable energy is limited.
In the case of sheep, the research material consisted of twenty Friesian breed ewes (62 ± 2.6 kg of BW, at 110 ± 12 d of lactation (multiparous sheep in 3rd-4th lactation), and an average milk yield of 1.96 ± 0.34 kg), divided into two groups (n = 10/group). The sheep were kept individually indoors and fed grass hay and complex mixture (62%:38% of DM) with free access to fresh water. Ewes from the experimental group (EXP) additionally received isomerized poppy seed oil (IPO) in the amount of 1% DM, that is, 20 g per head/day on the mineral carrier Humokarbowit, while sheep from the control group (CTRL) were given Humokarbowit in the same amount (Table 3). In both groups, DMI was 1.9 kg/ewes per day, allowing 7% refusals.  4 Feed unit for lactation. 5 Protein digested in the intestine when rumen fermentable nitrogen is limited. 6 Protein digested in the intestine when rumen fermentable energy is limited.
Animals (cows and ewes) with similar body weight and milk yield (no statistical differences) were selected for the study and the allocation to particular groups (CTRL and EXP) was random.

Experiment Design, Measurements, and Sampling Procedures
Before the start of the experiment, all cows and sheep were fed the same CTRL diet for 10 days (considered the adaptation period; Tables 2 and 3). The study was conducted for a 4-week period. The supplements (Humokarbowit in CTRL and Humokarbowit with isomerized poppy seed oil in EXP) were administered in the morning for 30 d (cows at 05:00 h with TMR and sheep at 07:00 h with complex mixture).
The animals were milked twice a day (cows at 06:00 and 16:00 h, sheep at 08:00 and 18:00 h). Individual milk samples (100 mL each) from the morning milking were collected from cows and sheep after 7, 14, and 30 days for analysis of fat content and FA profiles. Milk samples were immediately cooled to a temperature of 4 • C; transferred to the Laboratory of Milk Assessment and Analysis in the Institute of Animal Breeding, Wrocław University of Environmental and Life Sciences (Poland); and analyzed within 4 h of collection. Milk fat content was determined by automated infrared (Infrared 150 apparatus; Bentley Instruments Inc., Chaska, MN, USA) and is presented in Tables 4a and 5a. Table 4. (a) Means of fat and fatty acid content of total lipids of milk from cows that were fed the two diets (CTRL and EXP) at the three sampling times (7, 14, and 30 d). (b) Means of fatty acid (FA) groups with varying degrees of saturation and carbon chain length, desaturase index, and atherogenic index of milk from cows that were fed the two diets (CTRL and EXP) at the three sampling times (7, 14, and 30 d a-c Means within a row with different superscripts differ (p < 0.05). A-C Means within a row with different superscripts differ (p < 0.01). 1 CTRL, control group fed with total mixed ration (TMR) containing no additional oil; 2 EXP, experimental group fed with basal diet (TMR) containing the addition of 220 g/d (1% of DM) of isomerized poppy seed oil (IPO); 3 SEM, standard error of the mean; 4 probability of significant effects due to diet (D), time (T), and their interaction (D × T); * p < 0.05, ** p < 0.01, NS, not significant; 5 SFAs, saturated fatty acids; 6 SCFAs, short-chain fatty acids (FAs with C4-10); 7 MCFAs, medium-chain fatty acids (FAs with C12-16:0); 8 MUFAs, monounsaturated fatty acids; 9 PUFAs, polyunsaturated fatty acids; 10 Σ, CLA cis-9,trans-11; trans-10,cis-12; trans-9,cis-11; trans-11,cis-13 and trans-11,trans-13; 11 DI, desaturase index represents the ratio of product and substrate for ∆ 9 -desaturase (double bonds are in the cis orientation unless otherwise indicated); 12 AI, atherogenic index calculated from (C12:0 + (4 x C14:0) + C16:0)/(MUFAs + PUFAs).  Milk fat (1 g) for chromatographic analysis was obtained after milk centrifugation at 4000 × g for 15 min at 4 • C (MPW 260RH apparatus; Med. Instruments, Warsaw, Poland). Lipid extraction was performed according to the modified Folch procedure (chloroform and methanol in a volume ratio of 2:1), and methylation was performed using 2 M KOH in methanol [19].
Chromatographic analyses in this part of the study were conducted in the Laboratory of Chromatography and Meat Analysis, Institute of Animal Breeding, Wrocław University of Environmental and Life Sciences (Poland).

Statistical Analysis
All variables were tested for normality using the Shapiro-Wilk test. Experimental data were analyzed using SAS (Statistical Analysis System, version 9.3 for Windows; SAS Institute, Cary, NC, USA). The fat content and composition and fatty acid profile of cow and sheep milk were calculated by two-way ANOVA with dietary treatments (D) and sampling time (T) as fixed effects and their interactions (D × T) according to the model: where Y ijk is the dependent variable, µ is the overall mean, D i is the effect of dietary treatment (i = CTRL from days 7, 14, and 30; EXP from days 7, 14, and 30), T j is the effect of sampling time (j = CTRL and EXP from day 7, CTRL and EXP from day 14, and CTRL and EXP from day 30), D × T ij is the interaction between dietary treatments and sampling time, and ε ijk is the residual error.
Differences in ANOVA were determined using Tukey's multiple range test at p ≤ 0.05 levels of significance.
The data are presented as average values and accompanied by standard error of the mean.

Milk Fat Content and Fatty Acid Profiles
As a result of supplementation of dairy cows' TMR with IPO, a decrease in milk fat content after 7 days by 13.1% (p < 0.01), after 14 days by 16.8% (p < 0.01), and after 30 days by 20.2% (p < 0.01) was observed compared with the CTRL group (Table 4a). A decrease in milk fat content, as a result of IPO additive administration, was also found in sheep milk: 11.5% (7 d, p < 0.05), 20.2% (14 d, p < 0.01), and 24.4% (30 d, p < 0.01). In both groups of animals, in addition to the diet, the fat content of milk was also affected by the duration of IPO supplementation (p < 0.05) and a diet x time interaction was recorded (p < 0.01) ( Table 5a).
The addition of IPO also modified FA profiles of milk fat and affected the value of atherogenic index and the product/substrate ratio for ∆ 9 -desaturase (Tables 4a,b and 5a,b).

CLA Synthesis and Elaboration of Feed Additives
The content of CLA synthesized as a result of the alkaline isomerization process is significantly affected by the FA profile of the substrate. A high content of LA, which is the main substrate in the process of CLA synthesis, and a low content of ALA, which is also subject to isomerization leading to the formation of positional and geometric isomers (CLnA), determine the high degree of usefulness of the oil.
From the point of view of CLA isomer synthesis, poppy seed oil containing 72.8% LA and 0.7% ALA (Table 1) is characterized by a very favorable FA profile. Similar to other unprocessed oils of plant origin, this does not contain CLA. The distribution of double bonds (positional) and the arrangement of radicals in relation to the double bond axis (geometric) in LA change as a result of the alkaline isomerization process. Consequently, three conjugated dienes of linoleic acid with c9,t11, t10,c12, and c11,t13 configurations, having biological properties other than LA, in amounts of 31.2%, 27.6%, and 4.1% of total FAs, respectively, were synthesized. Apart from CLA, the IPO also included SFAs (C16-18:0), OA, LA, ALA, and CLnA (Table 1).
Due to the oily form of IPO, in order to facilitate its use in animal feed, it was nozzle-sprayed on the mineral carrier, Humokarbowit ® , a humus-mineral preparation characterized by high sorption capacity and antioxidant properties. The composition of Humokarbowit®was previously described by Bodkowski and Patkowska-Sokoła [34], and it was demonstrated to be suitable for farm animal feed [4,19].

Fat Content
According to literature data, certain types of diet may cause a significant decrease in milk fat secretion. Low-fat milk syndrome, more commonly referred to as milk fat depression (MFD), is a state that naturally occurs in dairy production when animals are fed highly fermentable diets or dietary plant or fish oil supplements [35,36]. As a result of disturbances in the rumen biohydrogenation process, FAs are produced as intermediate products, which are strong inhibitors of milk fat synthesis [37,38]. The MFD mechanism focuses on the mammary gland and includes a coordinated reduction of mRNA genes of key enzymes associated with all aspects of milk fat synthesis [38,39]. These processes include de novo synthesis of FAs, collection and transport of previously produced FAs, and denaturation and incorporation into triglycerides (TGs). The trans-10,cis-12 CLA isomer has been proven to be a potential nutritional tool to manipulate milk fat synthesis and FA profiles in lactating animals [17,38,40]. The activity of desaturase ∆ 9 , responsible for fat synthesis in the mammary gland, was probably also inhibited by TVA formed from CLA during ruminal transformations [41].
In the present study, the IPO supplement contained two major CLA isomers: trans-10,cis-12 and cis-9,trans-11. In previous studies, Baumgard et al. [53] and Bauman et al. [54] had shown that the c9,t11 CLA isomer had no or little effect on milk fat synthesis in dairy cows; hence, the lowering effect of fat can be attributed to the t10,c12 CLA isomer. In our study, the effective amount of t10,c12 isomer was 60.7 and 5.5 g/head/day in cows and sheep, respectively, and the decrease in milk fat concentration was from 13.1% to 20.1% in cows and from 11.5% to 24.4% in sheep, depending on the duration of IPO application.

Fatty Acid Profiles
Milk fat synthesis depends on two general sources of FAs, namely, de novo synthesis of FAs in the mammary gland and transfer of preformed FAs from blood TGs. The short-and medium-chain FAs (SMCFAs, C4-C14) and half of C16 FAs are synthesized de novo, whereas the rest of the FAs including C16 and other long-chain FAs (LCFAs) are derived from TGs in the blood or from non-esterified fatty acids (NEFAs), mainly during negative energy balance [54]. About half of the fat in milk is synthesized in the udder from acetate and β-hydroxybutyrate and the remaining part is transported from the pool of FAs circulating in the blood (originating from body fat mobilization, absorption from diet, or from fat metabolized in the liver) [55]. During diet-induced MFD, the de novo synthesized SMCFAs are reduced to a much greater extent than the other saturated FAs [6,18,56]. An effect of CLA isomers on lipid metabolism and decrease in the short-and medium-chain FA (SMCFA) level in milk is confirmed in the studies by Bauman et al. [54] and Harvatine et al. [57], and this is probably related to the activity of many lipogenic enzymes, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) [58], and decreases lipogenic rates and expression of genes involved in milk lipid synthesis [56].
In our study, the concentrations of all individual SCFAs, including C4, C6, C8, and C10 (p < 0.01), and MCFAs, including C12, C14, and C16 (p < 0.01, p < 0.05), were significantly reduced as the result of cow and sheep diet supplementation with IPO, while the concentration of C18 (p < 0.01) was increased. In the case of cows, except the diet, the content of C4, C10, C12, and C14 was also affected by the duration of the IPO supplementation (p < 0.05, p < 0.01); and for C4, C10 (p < 0.01) as well as C6, C12, and C16 (p < 0.05), the diet x time interaction was also observed. On the other hand, in the case of sheep, the IPO supplementation time affected the content of C4, C6, C8, C10, C12, and C16 (p < 0.05, p < 0.01), while diet x time interaction was recorded for C4 and C6 (p < 0.01) as well as C8, C10, C14, and C16 (p < 0.05). Similar changes in FA profiles in milk, as the result of infusion or supplementation of CLA isomers and their mixture, have been demonstrated in other studies on cows [43,44,59] and sheep [46,52]. The decrease in the share of MCFAs in milk fat (i.e., laurynic, myristic, and palmitic acids) and the unquestionable role of total cholesterol and its LDL fractions in blood as dietary factors for the cause of many cardiovascular diseases [32,60] are very important observations from previously conducted research.
On the other hand, the content of biologically active FAs with pro-health properties [2,61] (i.e., isomers c9,t11 and t10,c12 CLA and TVA, as well as PUFAs and MUFAs) significantly increased as a result of IPO application in milk fat of cows and sheep. In both groups of animals, in the case of CLA and TVA isomers, in addition to diet, the spectrum of changes was also affected by the duration of the IPO application and diet x time interaction was found (p < 0.05, p < 0.01). The increase in the content of CLA isomers and TVA was probably related to the high supply of CLA isomer mixtures in the feed dose of cows and sheep (1% DM) and their transformations. According to Salsinha et al. [62], Bauman et al. [54], and Shingfield and Wallace [63], most dietary CLA isomers are subject to microbiological biohydrogenation in the rumen: first to trans-11 C18:1 (TVA) (from isomer C18:2 cis-9,trans-11) and trans-10 C18:1 (from isomer C18:2 trans-10,cis-12), and then to stearic acid (C18:0). A part of rumenic acid (c9,t11 CLA, RA) which is not hydrogenated to TVA or C18:0 in the rumen is absorbed from the gastrointestinal tract and, together with blood, is transported into the mammary gland. However, the contribution of this pathway in RA synthesis is negligible [64]. The predominant source of RA in milk fat (about 78%) is endogenous synthesis in the mammary gland from TVA as the rumen origin substrate, with ∆ 9 -desaturase as the key enzyme [65,66]. A similar increase in the concentration of CLA and TVA in milk fat, as a result of infusion or supplementation of CLA isomers or their mixture, has been observed in other studies on cows [19,43,44] and sheep [17,46,52].
Given the negative role of C12:0, C14:0, and C16:0 acids, Ulbricht and Southgate [32] proposed the atherogenic index (AI). Conclusions concerning fat quality from the point of view of human diet may be drawn based on AI values. In our study, the AI value decreased from 10.6% to 20.5%, depending on the species of animal and the duration of the IPO additive administration.

Conclusions
The supplementation of the rations of dairy cows and ewes with isomerized poppy seed oil (IPO) reduced the content of milk fat, which had been confirmed in earlier studies, showing that dietary CLA lowered milk fat content in lactating animals. The decreased milk fat secretion was accompanied by a lower proportion of short-chain FAs (C4-10) as well as medium-chain FAs (C12-16) (i.e., laurynic, myristic, and palmitic acids) exhibiting atherogenic and thrombogenic effects, which is, in turn, consistent with the inhibitory effect of trans-10,cis-12 CLA on de novo FA synthesis. On the other hand, the content of biologically active FAs with pro-health properties (i.e., CLA isomers c9,t11 and t10,c12 and TVA) increased significantly in milk fat in both animal species as a result of IPO supplementation. In addition, it was observed that for sheep, the time of IPO supplementation had a greater effect on the spectrum of changes in individual fatty acids than in cows. This study demonstrated that isomerized poppy seed oil with high concentrations of CLA could be used in dairy animals to change the nutritional quality and associated health value of milk, which is beneficial from a human point of view.