Effect of cow’s milk with different PUFA n-6: n-3 ratios on performance, serum lipid profile, and blood parameters of grower gilts

The concern with human health has increased the interest in producing foods enriched with polyunsaturated fatty acids (PUFA), directly or naturally, by inclusion in the animals’ diet. The positive effects such as antithrombotic, anti-inflammatory, and hypolipidemic have been observed in pigs and rats, used as human models for study. The present study evaluated the effect of cow’s milk with different lipid profiles on performance, serum fatty acid profile, biochemical analysis, and a complete blood count of gilts used as a human model. At 34 days, thirty gilts were equally distributed in three treatments. Experimental treatments were milk from cows without the oil supplementation (C), milk from cows fed an enriched diet with linseed oil (n-3), and milk from cows fed an enriched diet with soybean oil (n-6). Milk supplementation was performed until 190 days old, provided once in the morning. The n-3 and n-6 milk reduced the concentration of myristic acid in the blood and increased the leukocytes. Milk enriched with n-3 compared to n-6 reduced the stearic acid. In conclusion, milk with a better PUFA profile can reduce saturated fatty acids in the blood and alter the concentration of cells in the defense system.


Introduction
The consumption of cow's milk and its derivatives in human nutrition has been reduced or even replaced by other beverages of vegetable origin due to the lipid profile [1] and lactose intolerance and some proteins [2].
It is known that the composition of fatty acids in cow's milk can be changed and is highly influenced by the nature of the forage consumed, supplementation with vegetable or oilseed oils, and even vitamin E, and this change can be verified in a few hours [3,4]. Researchers  requirements of the females according to [18]. The diets were based on corn and soybean feedstuffs and did not contain any oil as an ingredient (Table 1). All animals received ad libitum water. Daily milk supplementation was provided for pigs at 08:00 am, after individual feeding. Each gilt was supplemented with 200 mL (from 34 to 76 days), 300 mL (from 77 to 128 days), 400 mL (from 129 to 174 days), and 500 mL (175 to 190 days) of cow's milk. The enriched milk provided to the gilts in this current study was obtained from the study carried out by [13] using Holstein cows. Briefly, the cows were supplemented or not with 2,5% (dry matter basis) of linseed or soybean oil, sources of n-3 and n-6, respectively. The effects of these vegetable oils' inclusion on the lipid fraction profile of dairy cows' milk are presented in Table 2.

Growth
Body weight (BW) was assessed using a digital scale (Toledo 1 , model MGR-3000, São Bernardo do Campo, SP), consisting of initial (from 34 to 76), grower (from 77 to 112), and finisher (from 113 to 190) phases. Additionally, average daily feed intake (ADFI), and from these measurements, the average daily gain (ADG) and the feed conversion ratio (FCR) were calculated.

Blood sampling and analysis
Whole blood, serum, and plasma were collected by jugular venipuncture using a 1.2 × 40 mm collection needle (Becton Dickinson & Company, Juiz de Fora, MG, Brazil). Whole blood and  The complete blood count was evaluated by Sysmex 1 equipment (model XS-800i, Kobe, Japan). The serum FA profile was quantified by gas chromatography-mass spectrometry as follows [19]. The plasma sample was analyzed for total cholesterol, HDL, total protein, and serum urea by enzymatic kits (VIDA Biotecnologia 1 , Minas Gerais, Brazil) using a spectrophotometer. Triacylglycerol and LDL were analyzed by enzymatic kits (LABTEST 1 , Minas Gerais, Brazil) using a spectrophotometer. The very-low-density lipoprotein (VLDL) concentration was determined indirectly by the following equation [20]: VLDL cholestrol mg dl À � ¼ triglyceride concentration � 5. All analyses were according to the manufacturer's instructions.

Statistical analyses
All statistical analyses were performed using SAS 9.4 (SAS Inst. Inc., Cary, NC). The data were analyzed in a completely randomized design, and the animal was considered an experimental unit. The animals were randomly distributed in 1 of 3 treatments. The normality of the residues was verified by the Shapiro-Wilk test (UNIVARIATE procedure of SAS), and information with student residues greater than +3 or less than -3 was excluded from the analysis. The Levene test compared the homogeneity of the variances. Variables with a continuous distribution such as body weight, average daily gain, average daily feed intake, feed conversion rate, complete blood counts, biochemical parameters, and fatty acids profile of blood were analyzed using the MIXED procedure of SAS. When the time factor was present, repeated measures models were fitted by multiple mixed linear models using the MIXED procedure of SAS. The model included: the fixed effects of treatment, time and their interaction, and the random effect of the animal. The experimental unit was the gilt. The SLICE option using the LSMEANS/PDIFF command was used to explore the interactions between treatment and the day of data collection. The Kenward-Roger method was used to correct the degree of freedom  of the denominator for the F test. The covariance structure was determined based on the lowest Akaike information criteria value. The treatments were analyzed using two orthogonal contrasts: contrast 1 (C1) control group vs. n-3 + n-6 groups, and contrast 2 (C2) n-3 group vs. n-6 group. Significance level was set at P < 0.05 and tendency towards significance at 0.05 < P < 0.10.

Results
All gilts consumed their supplementation successfully so that the milk remains were not significant.

Gilts' performance
There was no difference in the treatment x time interaction on performance. Females supplemented with n-3 and n-6 had similar growth performance between themselves, and in comparison, to those of the control groups (see S1 Fig). Only time effect was observed for all these variables (P < 0.001, Table 3).

Fatty acids profile in gilts serum
At 77 days old, the control group had greater myristic acid concentration than those that received enriched cow's milk (n-3 and n-6 P = 0.062, Figs 1 and S2). At 132 days old, the n-3 gilts had an increased γ-linolenic acid concentration than other groups (P = 0.079, Fig 2). The control gilts group had raised myristic acid concentration compared to the females of different groups (P = 0.031, Table 4). Besides, the n-3 gilts group had a small SFA concentration (P = 0.090) and ratio SFA: USFA (P = 0.097) and greater USFA than those of the control and n-6 groups (P = 0.090, Table 4). Also, the n-3 female groups had lower stearic acid concentration (P = 0.002, Table 4 Table 4). The other fatty acids were similar among groups (P > 0.05, Table 4).

Biochemical parameters
At 77 days old, the n-6 gilts group had greater LDL than those of other groups (P = 0.083, Fig  3). Besides, these females increased HDL compared to the n-3 group (P = 0.063, Table 5). The other biochemical parameters were similar among females of the control group and those supplemented with enriched cow's milk (P > 0.05).

Hemogram and leukogram
Control gilts had a tendency to reduced red blood cells (RBC), platelets, and lymphocytes compared to those that received enriched cow's milk (P < 0.10, Table 6). Besides, these females had lower leukocytes (P = 0.049, Table 6) than the gilts of the omega group. These females also had increased mean corpuscular hemoglobin (MCH) compared to those of the omega group (P = 0.070, Table 6). Milk supplementation did not affect the other counts (P > 0.05, Table 6).

Discussion
Polyunsaturated fatty acids are essential for normal physiological function and the health of humans and domestic animals. Considering the opposing effects of n−3 and n−6 PUFA, the proportion of these PUFA, especially dietary linoleic acid and α-linolenic acid, may regulate diverse sets of homeostatic processes by themselves or by locally acting bioactive signaling lipids called eicosanoids derived from arachidonic acid, eicosapentaenoic acid, and docosanoids from docosahexaenoic acid [21]. Through the consumption of PUFA by diet, it is possible to reduce plasma levels of LDL and triacylglycerols. Therefore, SFA has a high potential to increase LDL and long-chain polyunsaturated fatty acids n-6 and n-3 to reduce it [22]. The supplementation of gilts with cow's Total n-6: Sum of n-6 fatty acids; n-6/n-3, ratio of total n-6 to total n-3 fatty acids; ARA/EPA: Ratio of arachidonic acid to eicosapentaenoic acid. b Gilts fed with non-enriched milk (Control); or supplemented with cow's milk enriched with n-3 or n-6. c C1, contrast between Control vs n-3+n-6; C2, contrast between n-3 vs n-6.
https://doi.org/10.1371/journal.pone.0258629.t004 milk enriched naturally with PUFA from 34 to 190 days old led to changes in fatty acid profile and leukogram, as well as a trend was observed for increased HDL and reduced LDL, and hemogram; however, the growth performance had been similar among the females' groups. Liu and Kim [23] did not report any change in the growth performance of crossbred pigs fed diets in different n-6: n-3 ratios (15.17, 9.68, and 4.87, high, medium, and lower, respectively). Nevertheless, in blood lipids, a reduction in total cholesterol, LDL, and triglycerides were observed for the lower ratio n-6:n-3 PUFA compared to animals in the other groups.  Similarly, Li et al. [24] reported based on a meta-analysis in humans that low-ratio n-6/n-3 PUFA reduced triglyceride concentration and increased HDL concentration (P < 0.05), and the beneficial effects of low-ratio n-6/n-3 PUFA on blood lipids were enhanced with time.
Besides these, researchers also verified that n-3 PUFA derived from plants significantly reduced total cholesterol and LDL concentrations, and n-3 PUFA as eicosapentaenoic acid (EPA), and DHA significantly reduced triglyceride and increased HDL concentration. In the present study, different n-6:n-3 PUFA ratios were observed in cow's milk enriched with n-3 and n-6 sources (2.72, 7.92 and 8.26, n-3, group control, and n-6, respectively), but in female serum, the ratio was similar (16.35, 27.27 and 25.53, n-3, control group, and n-6, respectively) after 22 weeks of supplementation. Growth performance results were similar to those reported by Liu and Kim [23]. On the other hand, HDL trended an increase in the n-6 group compared with n-3, and total cholesterol was similar among the group. Considering the overall period, the LDL was numerically lower in animals from the n-3 and n-6 groups. Still, a trend towards the difference between the PUFA groups was observed at 77 and 190 days old. An increase in LDL concentration from 34 to 77 days was verified in three groups, but from that moment on, the animals in the n-6 group had a reduction while the other groups remained elevated. Several factors may influence these results, such as the presence or absence of lipoprotein disorders, the relation between VLDL and total cholesterol, the dose of oil administered, varying effects of EPA vs. DHA, the source of n-3, the type of individual the study is being conducted on (normolipidemic or hypercholesterolemic), and the period of supplementation [25,26].
The importance of lipids consumed in the diet on the effects on human health has been widely discussed, as they influence the lipid composition of blood, colostrum, milk, and tissues, and it is even possible to observe the manipulation of gene expression and immunity [27][28][29]. Myristic and stearic acids are the most abundant saturated fatty acids in mammals [30]. Myristic acid has for 8 to 12% of total fat in milk, and a more substantial intake of this lipid raises LDL concentrations [31]. Cow's milk from the study by [13] was supplemented for gilts in this current study, and this milk had a lower concentration of myristic acid than those without the oil supplementation (7.59, 7.98, and 10.36, soybean, linseed, and control, respectively, P < 0.05), as well as reduction of SFA (56.52, 56.69 and 66.89, soybean, linseed, and control, respectively, P < 0.05). This fact may have directly influenced the serum fatty acid profile of gilts of n-3 and n6 groups. Besides, myristic acid may be produced through de novo lipogenesis, but it represents a small fraction [32]. Thus, we believe that the alteration found in the blood myristic acid may have come directly from the lipids present in milk through the transfer from the small intestine by diffusion or facilitated transport.
A higher concentration of stearic acid was observed in milk from cows that received soybean oil and linseed compared to control (14.50, 14.94, and 11.28, soybean, linseed, and control, respectively, P < 0.05), and no differences between the PUFA-supplemented groups were reported by [13]. The higher concentration of stearic acid found in the serum of gilts from the n-6 group may have been originated from de novo lipogenesis due to the higher elongation activity of palmitic acid by fatty acid elongase. Furthermore, PUFA is known to repress the stearoyl-CoA desaturase-1 (SCD-1) gene expression [33] that it is a lipogenic enzyme responsible for the biosynthesis of oleic acid (18:1) by desaturating stearic acid (18:0) [34][35][36]. The length, structure, and number of double bonds in the fatty acid chains also seem to influence the activity of SCD, according to [37]. The linoleic acid and EPA can directly regulate SCD activity at the level of transcription via sterol regulatory element-binding protein 1c (SREBP-1c), but the same does not occur with α-linolenic acid (ALA) [38]. Therefore, the difference in stearic acid found between n-3 and n-6 groups may have been influenced by all these factors since cow's milk enriched with soy oil had a higher concentration of linoleic acid (2.13, 2.62, and 2.03, control, soybean, and linseed, respectively [13].
In the present study, control gilts tended to have a reduced RBC, platelets, and lymphocytes and enhanced MCH compared to those that received enriched cow's milk. These females also had lower leukocytes and higher monocytes than the gilts of the PUFA group.
Inflammation is a positive state for the body, as it denotes an immediate response to injury or infection. Thus, several cells are moved towards the affected site, including leukocytes, which can be modulated by fatty acids. This is because leukocytes have an essential role in inflammation and infection through the synthesis of important cytokines, some with more inflammatory functions, arising from PUFA n-6, and others with more anti-inflammatory functions, mainly from PUFA n-3 [39,40]. In this case, the n-6 PUFA is a substrate for the synthesis of eicosanoids, such as prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs). The series 4 LTs are B 4 , C 4 , D 4, and E 4 , and LTB 4 is a potent chemotactic agent for leukocytes [39].
Thus, it is possible that the PUFA-enriched milk, treatments n-3 and n-6 in this study, have stimulated this higher concentration of leukocytes due to the performance they can perform in the body in terms of cytokine production, cell proliferation, adhesion molecules, and cell death.
The consumption of PUFA can cause different effects on the cells that make up the blood plasma. According to Xia et al. [41], dietary n-3 polyunsaturated fatty acids (fish oil) in mice alters the hematopoietic microenvironment and induces the expression of matrix metalloproteinase 12 (MMP12) in stromal cells, providing new insights into diet-mediated regulation of hematopoiesis. Similar results were found by [42], in which oral administration of PUFA in mice increases hematopoiesis in them by generating active metabolites, which activate the Wnt and CXC Chemokine Receptor Type 4 (CXCR4) pathways and promote thrombopoiesis through the Notch proteins (Notch1) pathway. Although, how the dietary fatty acids can influence the hematopoiesis remains yet largely unclear. Unlike the previous results, except for the segmented ones, the other variables related to hematopoiesis had similar results between groups n-3 and n-6, without the effect of n-3 PUFA in our study.
The increased intake of EPA and DHA replaces the arachidonic acid in the cell membranes of erythrocytes, platelets, neutrophils, monocytes, and liver cells [43] and according to [44] and [45], as a consequence, may occur: 1) reduced prostaglandin (PG) synthesis of series 2; 2) reduction of thromboxane A2 (TXA2), which acts by aggregating platelets and causes vasoconstriction; 3) reduced production of leukotrienes B4, which induces inflammation; 4) increased of thromboxane A3 (TXA3), which are weak as platelet aggregators and as vasoconstrictors; 5) higher synthesis of prostaglandin I3 (PGI3) without reducing PGI2, which act as active vasodilators and inhibitors of platelet aggregation; 6) higher concentrations of LT of type B5, which is a chemotactic agent and a weak inducer of inflammation.
Lymphocytes become active and initiate cytokine secretion stimulating the immune response, although their proliferation may be impaired due to γ-linolenic acid or EPA [46,47]. Caughey et al. [48] suggest a reduction in the function of immune cells after supplementing humans with a high concentration of n-3 PUFA (linseed oil) due to reduced synthesis of interleukin-1 and tumor necrosis factor (TNF) by blood monocytes.
Ingestion of PUFA leads to their distribution to virtually every cell in the body, resulting in effects on membrane composition and function, cellular signaling, and regulation of gene expression through various transcription factors. Although the results obtained for gilt in growing were not expressive in the current study, more studies are necessary to clarify how the consumption of cow's milk enriched naturally with PUFA can influence the development of animals e humans.
In conclusion, gilt that received the cow's milk enriched naturally with PUFA n-3 or n-6 had similar growth performance, but the blood fatty acid profile and hematological parameters were altered without impairing the health of gilts. This study provided insight on how food enriched can modulate the serum lipid profile and cells of the defense system, promoting health benefits.