Milk fatty acids profile and arterial blood milk fat precursors concentration of dairy goats fed increasing doses of soybean oil

Small Ruminant Research 114 (2013) 152–160

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Milk fatty acids profile and arterial blood milk fat precursors concentration of dairy goats fed increasing doses of soybean oil O.C. Almeida a , A.V. Pires b,∗ , I. Susin b , R.S. Gentil b , C.Q. Mendes d , M.A.A. Queiroz e , E.M. Ferreira c , M.L. Eastridge f a Departamento de Zootecnia, Universidade Federal Rural de Pernambuco, Unidade Acadêmica de Garanhuns, Garanhuns, Pernambuco 55296-901, Brazil b Departamento de Zootecnia, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Piracicaba, PO Box 09, São Paulo 13418-900, Brazil c Departamento de Zootecnia, Universidade Estadual de Ponta Grossa, Ponta Grossa, Paraná 84.030-900, Brazil d Faculdade de Agronomia e Medicina Veterinária, Universidade de Brasília, PO Box 4508, 70.910-970 Brasília, DF, Brazil e Colegiado de Zootecnia, Fundac¸ão Universidade Federal do Vale do São Francisco, Petrolina, Pernambuco 56310-770, Brazil f Department of Animal Sciences, The Ohio State University, Columbus 43210, United States

a r t i c l e

i n f o

Article history: Received 10 August 2012 Received in revised form 29 April 2013 Accepted 30 April 2013 Available online 2 June 2013 Keywords: CLA Fatty acids Mammary metabolism Milk fat precursors

a b s t r a c t The provision of fatty acid sources in ruminant diets allows the manipulation of fatty acid profiles in milk and meat, permitting to increase the fatty acids of interest, such as conjugated linoleic acid. The objectives of this experiment were to evaluate the inclusion of increasing doses of soybean oil (30, 60 or 90 g/d) on intake, total tract digestibility of nutrients, milk production and composition, arterial and milk fatty acid profiles, and arterial concentrations of glucose, acetate, and ␤-hydroxybutyrate in dairy goats. Four multiparous Saanen goats with mean initial DIM 32 ± 6 and mean initial BW 63 ± 7 kg were assigned in a 4 × 4 Latin square. Does were housed in tie stalls and fed a 40% of corn silage and 60% concentrate diet. Experimental treatments consisted of: (1) basal diet without soybean oil infusion (control); (2) basal diet plus an oral infusion of 30 mL soybean oil; (3) basal diet plus an oral infusion of 60 mL soybean oil; and (4) basal diet plus an oral infusion of 90 mL soybean oil. The DM and OM intakes decreased linearly (P < 0.05) with increasing soybean oil supply. Dry matter digestibility was negatively influenced by oil (P < 0.01); however, the OM digestibility was not affected by soybean oil doses. Milk production tended to decrease (P = 0.07) by soybean oil supply, although when this was corrected to 3.5% fat, it decreased linearly (P < 0.01) as a result of the decreasing (P < 0.05) milk fat content. Vaccenic acid concentration in arterial blood increased (P < 0.01) 127% with 90 g/d of soybean oil addition. Appearance of C18:2 t10, c12 in milk caused a reduction (P < 0.01) in milk fat and reached a 31% reduction when 90 g/d of soybean oil was offered compared to the control diet, while the opposite occurred with C18:2 c9, t11, which decreased (P < 0.05) with increasing doses of soybean oil. Increasing soybean oil had no effect on arterial blood concentrations of glucose, acetate and ␤-hydroxybutyrate. Therefore, the decrease in milk fat concentration was due to the inhibition of mammary synthesis of fatty acids, but not by a limitation of its precursors for fatty acid synthesis. © 2013 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author. E-mail address: [email protected] (A.V. Pires). 0921-4488/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.smallrumres.2013.04.014

Lipid sources have been the object of many studies on ruminant nutrition, especially those focused on the

O.C. Almeida et al. / Small Ruminant Research 114 (2013) 152–160

assessment of energy needs, reduction of methane production, and manipulation of fatty acid (FA) profile in milk and meat. When ingested by lactating animals, the FA can be targeted to 3 metabolic purposes: incorporation into the fat tissue, oxidation to energy supply, and direct secretion in milk (Palmquist, 1994). The last one can request up to 75% of absorbed FA (Palmquist and Mattos, 1978). Sources of FA have been shown to be a very promising tool to meet current market demands, once the manipulation of FA profile in milk and in meat occur through the incorporation of compounds of interest, such as conjugated linoleic acid (CLA). This group of isomers is derived from the activity of rumen bacteria on dietary linolenic and linoleic acid (Bauman and Griinari, 2003) and has aroused interest in the scientific community due to its ability to perform various metabolic functions in the human body, particularly the ability to inhibit lipogenesis and some types of carcinomas (Bhattacharya et al., 2006). Dairy products account for about 75% of CLA ingested by humans (Bauman et al., 2006), where the isomer C18:2 c9, t11 (rumenic acid) represents over 75% of CLA present in these products (Sieber et al., 2004). Although this isomer is also produced in the rumen, the main source of rumenic acid present in milk derived from intramammary synthesis is via 9-desaturase enzyme activity on vaccenic acid (C18:1 t11). The C18:1 t11 is one of the intermediates of ruminal biohydrogenation of linoleic and linolenic acids, being transported to the mammary gland via bloodstream. Another CLA isomer that has attracted attention is the C18:2 t10, c12; its relationship with milk fat depression in cows has already been defined (Bauman et al., 2006). However, its effects on lactating goats are controversial (Andrade and Schmidely, 2006; Erasmus et al., 2004; Lock et al., 2008). The present study aimed to evaluate the effects of increasing doses of soybean oil (30, 60 or 90 g/d) on intake, nutrient total tract digestibility, milk production and composition, milk and arterial FA profiles, and concentrations of glucose, acetate, and ␤-hydroxybutyrate in the arterial blood of lactating goats. 2. Materials and methods The research protocol and all animal care followed the guidelines recommended in the Guide for the Care and Use of Agricultural Research and Teaching (FASS, 1998). All procedures were approved by “Luiz de Queiroz” College of Agriculture Animal Ethics Committee. 2.1. Animal, treatments and experimental design Four lactating Saanen goats with mean initial DIM 32 ± 6 and mean initial BW 63 ± 7 kg were allotted in individual tie-stall pens (0.50 m × 1.2 m), provided with a feed bunk and water. Animals were assigned to a 4 × 4 Latin square design. Experimental periods consisted of 28 d; d 1 through 24 served as an adjustment period and d 25 to 28 were for data collection. Two months before start of the experiment, the animals were surgically prepared for subcutaneous exteriorization of both carotid arteries to facilitate the collection of arterial blood. All animals were fed ad libitum a total mixed diet (basal diet, Table 1). The basal diet was formulated to meet the requirements for lactating goats (AFRC, 1998). Experimental treatments consisted of: (1) basal diet without soybean oil infusion (control); (2) basal diet plus an oral infusion of 30 mL soybean oil; (3) basal diet plus an oral infusion of 60 mL soybean oil; and (4) basal diet plus an oral infusion of 90 mL soybean oil. Soybean oil was provided by oral infusion to have an exact control of the amount

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Table 1 Ingredient and chemical composition of experimental basal diet (% of DM). Item Ingredients Corn silage Ground corn Soybean meal, 48% CP Urea Mineral mixturea Chemical analysis Dry matter, as-fed basis Crude protein Ether extract Neutral detergent fiber Ash

Basal diet 40.0 42.5 13.7 0.90 2.90 70.3 17.8 3.2 34.0 7.1

a Composition: Ca, 24.1%; P, 7.5%; Mg, 1.0%; S, 7.0%; Cl, 21.8%; Na, 14.5%; Mn, 1100 mg/kg; Fe, 500 mg/kg; Zn, 4600 mg/kg; Cu, 300 mg/kg; Co, 40 mg/kg; I, 80 mg/kg; and Se, 15 mg/kg.

consumed, and thereby investigate the effects of increasing amounts of soybean oil on dairy goat performance, milk fatty acid composition and some metabolic parameters. 2.2. Feeding management Concentrate ingredients were mixed previously in a horizontal mixer with a 500 kg capacity. Experimental diets were fed at 0800 and 1600 h daily as TMR (except for soybean oil) for ad libitum intake. Animals were allowed free access to fresh water. Silage and concentrate were individually weighed in an electronic scale for each pen and manually mixed in the feed bunks. The amounts of TMR offered and refused for each animal were recorded daily to maintain feed refusals less than 10%. Soybean oil was administrated orally with the aid of a syringe twice a day (50% of daily dose each time) at the same time as feeding the TMR (0800 and 1600 h). The oral administration of the oil was used to ensure the increasing oil doses ingestion independently of the other ingredients of the diet. 2.3. Sample and data collection Digestibility was measured on d 25–28 of each period. A capsule containing 1.5 g of chromic oxide was dosed orally on d 14–28. Fecal grab samples were taken on d 27–28 of each period to represent every 2 h in a 12-h period. Samples were frozen during collection, dried in a forced-air oven at 60 ◦ C for 72 h and composited for each animal by equal sample weight at the end of each period. Goats were milked at 0730 and 1600 h daily. Milk yield was recorded from the d 21 to 27 of each period using a Tru-Test (Tru-Test® , Manukau, New Zealand) milk meter. Two consecutive milk samples were taken at a.m. and p.m. milkings on d 27–28 of each period, and each sample was split into 2 aliquots. The a.m. and p.m. milk samples were composited, and the first aliquot was stored at 4 ◦ C with bronopol Broad Spectrum Microtubs II (2-bromo-2-nitropropane-1-3-diol, D&F Control Systems Inc., Dublin, CA), and sent to Clínica do Leite (ESALQ/USP, Piracicaba, São Paulo, Brazil) to determine milk composition. The second aliquot of milk was stored at −18 ◦ C until it was analyzed for FA profile by GLC analysis. Arterial blood samples were collected from d 21 to 27 of each period at 0, 2, 4 and 6 h after offering the diets at the a.m. feeding, taken by carotid arteries using BD Vacutainer® tubes containing EDTA (BDBrazil, Juiz de Fora, Minas Gerais). Plasma was immediately separated by centrifugation at 3000 × g for 20 min and stored at −18 ◦ C for later laboratorial determination of FA profile, glucose, ␤-hydroxybutyrate, and acetate. 2.4. Chemical analysis and calculations The samples of feed offered, orts, and fecal contents were dried in a forced air oven at 60 ◦ C for 72 h, ground to pass through a 1-mm screen (Marconi, Piracicaba, São Paulo, Brazil), and were analyzed for DM, OM, N and EE (AOAC, 1990). The NDF and ADF contents of feed, orts, and fecal samples (correted for ash and protein) were determined according

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Table 2 Fatty acid profile (g/kg DM) of main dietary ingredients. Fatty acid

C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:1 Other

2.5. Statistical analysis

Ingredient Corn silage

Ground corn

Soybean oil

0.22 4.98 0.78 7.35 9.50 1.81 0.23 1.65

0.40 5.56 1.64 12.26 14.52 0.39 – 0.84

1.30 140.20 30.00 225.30 511.50 74.40 1.80 15.50

Intakes, milk production and composition, milk and arterial FA profile, and total tract digestibility of nutrients were analyzed as a 4 × 4 Latin square design using PROC MIXED (SAS, 2003), according to the following model: Yijk =  + Ti + Pj + ck + TWjk + eijk , where Yijkl is the dependent variable;  is the overall mean; Ti is the fixed effect of treatment i (i = 1–4); Pj is the fixed effect of period j (j = 1–4); ck is the random effect of goat k (k = 1–4); and eijk is the residual error associated with the ijk observation. Arterial glucose, acetate, and ␤-hydroxybutyrate were analyzed using PROC MIXED with repeated measures and Compound Symmetry (CS) as the covariance structure. All means were obtained by using the LSMEANS option and linear and quadratic contrasts were performed. Significant differences were declared at P < 0.05.

3. Results to Van Soest et al. (1991). Chromic oxide and fecal samples were analyzed for Cr by the Energy Dispersive X-ray Fluorescence (EDXRF) technique (Nascimento Filho, 1999). The excitation was done with Mo-Ka X-rays (17.44 keV) from a Mo target X-ray tube with a Zr filter, operated at 25 kV and 10 mA. Samples were excited for 200 s and the Ka characteristic X-rays were detected by a multichannel spectrometer, based on a Si(Li) semiconductor detector and conventional nuclear electronics. The AXIL software (Van Espen et al., 1977) was used to interpret the spectra, obtaining the characteristic Ka X-ray intensities. After determination of the chromium concentration, the values of digestibility were placed at the formula proposed by Ferret et al. (1999). Milk samples were analyzed for true protein, fat, lactose, and total solids by infrared analysis with a spectrophotometer milk analyzer Bentley 2000 (Bentley Instruments, Chaska, MN; AOAC, 1990). Fat-corrected milk (3.5% FCM) was calculated as (0.432 + 0.1625 × (% of milk fat)) × milk yield according to equation proposed by Sklan et al. (1992). In order to determine the feed FA profile, 2 g of each feed sample was mixed with 20 mL of chloroform–methanol (2:1, v/v) (Folch et al., 1957). The total arterial FA was obtained from 2 mL composited samples that were collected 0, 2, 4 and 6 h after a.m. feeding, which were subjected to FA extraction by the protocol of Folch et al. (1957), with the following modifications: 2 mL of plasma, 20 mL of chloroform and methanol solution (2:1), and use of a 1.5% NaCl solution for the separation phases. The milk FA was extracted by centrifugation of the 20 mL at 17,000 × g for 30 min at 8 ◦ C; then, 300–350 mg of fat cake was sampled. The FA of the feed were trans methylated in two steps, first with 5 mL of 10% methanolic HCl (2 h at 90 ◦ C) and after with 1 mL of hexane and 10 mL of 6% K2 CO3 ; the samples were centrifuged for 5 min at 500 × g to separate solvent layers (Kramer et al., 1997). The organic layer was transferred to a 13 mm × 100 mm culture tube containing 1 g each of sodium sulfate and charcoal, capped with a Teflon-lined screw cap, centrifuged and transferred into 1 mL GLC auto sampler vials, capped, and stored at −20 ◦ C until GLC analysis. To avoid the migration of conjugated double bonds, the milk and arterial FA were methylated by the alkaline method in two steps with 2 mL of 0.5 M sodium methoxide (2 min at 50 ◦ C) and with 3 mL of 10% methanolic HCl (10 min at 80 ◦ C) (Kramer et al., 1997). To detect possible loss of FA during the transmethylation, stearic acid (C18:0) was used as an external standard, while nonadecanoic acid (C19:0) was used as an internal standard. The later was used in order to quantify the arterial FA content since the extraction process did not allow its determination by weighing. The fatty acid methyl esters were separated by using a HP 6890 gas chromatograph (Hewlett Packard Co., Avondale, PA) equipped with a fused silica capillary column (CP-SIL 88; 100-m length, 0.25-mm id, and 0.2 ␮m film thickness, Varian Inc., Walnut Creek, CA). Helium was used as the carrier gas. Injector and flame ionization detector temperatures were set at 223 and 260 ◦ C, respectively, and the split ratio was set at 80:1. Oven temperature was set for 140 ◦ C, held for 5 min, then increased by 4 ◦ C/min to 240 ◦ C, held for 15 min, decreased by 20 ◦ C/min to 140 ◦ C, and held for 0.5 min. Retention times and response factors were determined with methyl ester standards purchased from Nu-Check Prep (Elysian, MN). Arterial glucose was determined by direct reading using a biochemical auto-analyzer (YSI 2700 Select, Biochemistry Analyser, Yellow Spring, OH, USA). The ␤-hydroxybutyrate concentrations were determined using commercial kits (Wako Chemicals® , Richmond, VA, USA). Arterial acetate concentration was determined by GLC, according to the methodology proposed by Pethick et al. (1981).

The increasing soybean oil supply caused different responses depending on the dose and the analyzed parameter. 3.1. Intake and digestibility The DM and OM intakes (P < 0.05), as well as the dry matter digestibility (P < 0.01) decreased, but OM digestibility was not affected (Table 3). 3.2. Milk yield and composition Milk production tended to decrease (P = 0.07), although when the production was corrected to 3.5% fat, it decreased linearly (P < 0.01) as a result of the decreasing milk fat content (Table 3). Milk fat content and yield also decreased linearly (P < 0.05 and <0.01, respectively). The concentrations of milk protein and lactose did not change (P > 0.05 for both), however their production tended to decrease (P = 0.07, Table 3). Concentration and yield of total milk solids decreased (P < 0.05), although the non-fat solids concentration did not change statistically (P > 0.05), but tended to decrease (P = 0.07). 3.3. Arterial blood fatty acids profile Concentration of fatty acids in arterial blood with chain less than 14 carbons were not influenced by treatments (P > 0.05, Table 4), neither that of FA with 15 carbons (pentadecanoic acid and 10-pentadecenoic). The heptadecanoic (C17:0) concentration was reduced linearly (P < 0.01), and that of heptadecenoic acid (C17:1) was not influenced (P > 0.05, Table 4). Concentrations of long-chain FA (≥C18) and stearic acid (C18:0) increased linearly (P < 0.05 for both, Table 4, Fig. 1), conversely to what was observed for oleic acid (C18:1; P < 0.05). The vaccenic acid (C18:1 t11; P < 0.01); the linoleic acid (C18:2; P < 0.01) and the total trans FA (P < 0.05) concentrations in arterial blood increased linearly. 3.4. Milk fatty acid profile The treatments resulted in broad changes in milk FA profile (Table 5). It was observed a linear decreasing in the concentrations of the following FA of milk: C10 (P < 0.05),

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Table 3 Performance of dairy goats supplemented with soybean oil. Variable

DMI, kg/d OMIa , kg/d DMDb (%) OMDc (%) Milk, kg/d 3.5 FCMd , kg/d FCM/DMI Fat, % Fat, g/d Protein, % Protein, g/d Lactose, % Lactose, g/d Total solids, % Total solids, g/d SNFe , % SNF, g/d

Soybean oil dose (g/d)

SEM

0

30

60

90

2.49 2.21 68.6 78.1 3.76 3.73 1.50 3.50 126 3.21 117 4.46 169 12.36 448 8.86 322

2.65 2.36 67.9 77.4 3.72 3.51 1.33 3.31 116 3.25 115 4.74 166 12.31 432 9.01 316

2.33 2.06 64.7 76.2 3.64 3.20 1.37 2.84 90 3.26 104 4.67 150 11.78 376 8.94 286

2.22 1.96 63.8 75.5 3.55 2.89 1.30 2.41 69 3.31 96 4.78 138 11.52 332 9.22 267

P-value

0.05 0.05 3.8 6.7 0.08 0.08 0.05 0.12 11 0.04 8 0.06 11 0.21 32 0.10 20

Linear

Quadratic

*

ns

*

*

**

ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns

Tf T **

ns * **

ns T ns T * *

ns T

ns: not significant (P > 0.05). a Organic matter intake. b Dry matter digestibility. c Organic matter digestibility. d 3.5 Fat-corrected milk. e Non-fat solids. f T: tendency (0.05 < P < 0.10). * P < 0.05. ** P < 0.01.

Table 4 Arterial fatty acid profile of dairy goats supplemented with soybean oil. Item

Fatty acids (g/100 g of fatty acids)
Soybean oil dose (g/d)

SEM

0

30

60

90

4.27 1.57 0.61 0.41 23.28 0.77 0.93 0.33 20.83 19.76 2.11 nd nd 14.43 1.76 8.69 58.93 51.93 39.60 22.37 16.03 3.01 0.76

4.98 0.68 0.57 0.33 19.59 0.94 0.81 0.31 22.17 17.92 2.93 nd nd 17.82 1.75 8.95 61.84 49.17 42.05 22.42 16.72 3.33 0.86

4.98 1.01 0.58 0.30 20.4 0.25 0.71 0.40 23.27 15.43 3.37 nd nd 18.25 1.80 9.05 62.12 51.29 42.73 19.78 20.05 4.03 0.78

3.37 0.64 0.48 0.38 19.33 0.69 0.48 0.55 24.45 12.01 4.79 nd nd 22.48 2.51 7.75 66.23 48.92 43.40 18.97 24.98 5.01 0.89

nd: non detected; ns: not significant (P > 0.05). a Monounsaturated fatty acids. b Polyunsaturated fatty acids. c Sum of the t11 C18:1, c9, t11 C18:2 and t10, c12 C18:2. * P < 0.05. ** P < 0.01.

0.64 0.45 0.02 0.10 0.69 0.27 0.05 0.11 0.68 0.54 0.52 – – 1.38 0.69 1.66 1.87 1.51 1.03 2.73 0.58 0.53 0.06

P-value Linear

Quadratic

ns ns ns ns ns ns

ns ns ns ns ns ns ns ns ns ns ns – – ns ns ns ns ns ns ns ns ns ns

**

ns * * **

– – **

ns ns *

ns *

ns * *

ns

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18:1 (P < 0.05), other short-chain acids (C4–C10; P < 0.05), medium-chain acids (C12–C16; P < 0.01) and total saturated (P < 0.01). The concentration of C18:2 and of C18:2 t10, c12 responded positively to the treatments (P < 0.05 and P < 0.01, respectively), conversely to what was observed for the isomer C18:2 c9, t11 (P > 0.05). Soybean oil intake increased linearly (P < 0.05) the total concentration of C18:1 t11 (P < 0.01), long-chain FA (≥C18), total unsaturated (P < 0.05), polyunsaturated acids (P < 0.05), the unsaturated:saturated ratio (P < 0.05) and also of trans fatty acids concentration (P < 0.01) in milk. The concentration of the total monounsaturated fatty acids did not change (P > 0.05), although cis9 monounsaturated fatty acids reduced linearly in milk (P < 0.05). 3.5. Arterial concentrations of metabolites There was no effect (P > 0.05) of soybean oil doses on arterial blood concentrations of glucose (63.91 ± 1.21 mg/dL), acetate (1.33 ± 0.18 mMol/L) and ␤hydroxybutyrate (1.19 ± 0.18 mMol/L), neither in the concentration of these metabolites in periods subsequent to the morning diet supply (0, 2, 4 and 6 h).

Fig. 1. Concentration of fatty acids in milk from goats supplemented with different doses of soybean oil.

Table 5 Milk fatty acid profile of dairy goats supplemented with soybean oil. Item

Fatty acid (g/100 g of fatty acids) C10:0 C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 C18:0 C18:1 C18:1 t11 C18:2 c9,t11 C18:2 C18:2 t10,c12 C18:3 Other Short-chain (C4–C10) Medium-chain (C11–C16) Long-chain (≥C18) Total saturated Total unsaturated MUFAa cis9 MUFAb PUFAc Total transd Vaccenic:rumenic Unsaturated:saturated

Soybean oil dose (g/d)

SEM

0

30

60

90

7.99 4.98 9.63 0.41 1.19 30.25 0.63 9.45 28.34 nd 1.03 2.16 nd 0.06 3.81 8.07 46.63 41.06 62.99 32.51 29.38 28.44 3.25 0.46 nd 0.52

6.95 3.56 7.61 0.34 0.88 29.95 0.12 12.64 27.89 0.79 0.20 2.19 nd 0.98 4.33 7.02 42.11 44.79 61.19 32.60 29.14 26.35 3.37 1.21 nd 0.53

6.94 3.11 7.60 0.25 0.89 28.51 0.13 13.22 26.07 5.78 0.15 2.67 0.10 1.01 3.57 6.98 40.09 49.08 59.79 36.18 32.23 23.23 3.95 4.54 15.79 0.61

3.30 1.61 4.14 0.09 0.78 27.39 0.08 18.27 25.92 7.19 0.12 2.82 0.14 1.84 5.68 4.43 33.67 57.60 55.05 39.20 34.28 20.37 4.92 6.58 38.54 0.71

nd: non detected; ns: not significant (P > 0.05). a Monounsaturated fatty acids. b cis9 monounsaturated fatty acids. c Polyunsaturated fatty acids. d Sum of the t11 C18:1; c9,t11 C18:2; and t10,c12 C18:2. * P < 0.05. ** P < 0.01.

1.77 0.99 1.51 0.13 0.10 1.87 0.21 1.37 2.03 0.13 0.02 0.23 0.02 0.04 1.86 2.07 3.12 2.53 3.03 2.61 3.98 2.16 0.18 0.80 1.40 0.02

P-value Linear

Quadratic

*

ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns

* ** **

ns **

ns * * ** * * ** *

ns * ** * ** *

ns * * ** ** *

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4. Discussion 4.1. Intake and digestibility The decrease of DM and OM intakes with increasing soybean oil supply may be interpreted as a consequence of the higher energy intake. These findings do not agree with the verified by Bouattour et al. (2008), which included 2.5% of soybean oil in the diet (DM basis) to lactating goats and found no effect on DMI, although the supply of 6% (DM basis) of soybean oil to ewes also did not affect DMI (Gómez-Cortés et al., 2008). This discrepancy of results may be a consequence related to the method used for oil supply: in our experiment it was used in oral doses, which probably influenced in rumen environment. The difference between DM and OM digestibility may be related to the increasing lipids amount reaching the lumen of the digestive tract, as well as the saponiphication of some of the mineral fraction; part of this fraction may not have been effectively dissociated in the intestine, affecting absorption (Rahnema et al., 1994). 4.2. Milk yield and composition Milk production was in agreement with the results obtained by Dhiman et al. (2000), when evaluated the effect of providing FA sources to dairy cows and observed that animals supplemented with 4.4% linseed oil and 3.6% soybean oil had similar milk production. However, when corrected for fat content, milk production of both treatments were lower than the others supplemented with raw soybeans, roasted soybeans, and 2.2% linseed oil. In this current study, feed efficiency was not altered by soybean oil doses, since it was recorded a linearly decrease on milk production corrected to 3.5% fat and on DMI (Table 3). Linearly decrease of milk fat content for intake of 30, 60 and 90 g/d of soybean oil led to reductions of 5.4, 18.9 and 31.1% in fat percentage of milk, resulting in daily losses of 2, 36 and 57 g of fat, respectively. Although these results corroborates the literature, the effect of supplementation of goats with soybean oil on milk fat content needs further investigation, since our results are contrary to those obtained by Bouattour et al. (2008), which observed increases of milk fat content and yield in response to the addition of 2.5% (DM basis) of soybean oil in the diet. Probably, the oral supply of soybean oil (2 times/day) in our experiment is the main reason for this divergent result: oral doses containing free fat are more available for rumen microbial activity, and consequently increase of C18:2 t10, c12 synthesis. The progressive reduction in milk fat content coincided with an increase of C18:2 t10, c12 (Table 5 and Fig. 1), and may be explained by the fact that this FA is the most likely responsible for the milk fat inhibition (Baumgard et al., 2000), in a dose-dependent manner. The potential of lipogenic inhibition and the dosedependence was also recorded in goats by Lock et al. (2008), when gave 30 or 60 g/d of an encapsulated supplement of C18:2 t10, c12 and verified a reduction in milk fat content from 5 and 18%, respectively. In the present experiment, raising C18:2 t10, c12 concentration in goat milk was a

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result of increasing linoleic acid in the rumen, since it is the main precursor used by the microbiota to synthesize this isomer (Bauman et al., 1999). According to Bauman and Griinari (2003), the reduction of milk fat imposed by the isomer C18:2 t10, c12 is a result of the inhibition of its synthesis in mammary tissues, with consequent decrease of short and medium-chains FA milk concentration, and also of FA capture in arterial blood. In the present experiment, the inhibitory effect of C18:2 t10, c12 was exclusively of the mammary fatty acid synthase enzyme, since there were only reduction in short and medium-chain FA (Table 5 and Fig. 1). The inhibition of this enzyme is confirmed by the fact that there was no reduction in the supply of the main precursors of these FA (acetate and ␤-hydroxybutyrate) by arterial blood. The ingestion of FA sources generally influence milk fat, although the concentration and production of milk protein, lactose and non-fat solids do not change (Li et al., 2009, 2012; Sutton and Morant, 1989). Chilliard et al. (2003), however, suggested that the protein content of cow’s milk may vary when the animals are supplemented with some lipid sources, but this is not observed in dairy goats. The negative impact of the soybean doses upon the yield of total milk solids was exclusively consequence of the decreasing milk fat yield. This interpretation is confirmed by the fact that the non-fat solids tended to increase with oil doses (Table 3). All changes in milk composition observed here were also recorded by Erasmus et al. (2004), who supplemented lactating goats with increasing levels of protected CLA (1.3, 2.6, or 3.9 g/d) and reported that fat was the only milk component modified.

4.3. Arterial blood fatty acids profile The treatments did not influence the concentration of FA in arterial blood with chain less than 14 carbons probably due to the absence of these compounds in soybean oil (Table 2) and absence of the synthesis of such fatty acids in the rumen, as pointed by Wu and Huber (1994). These authors also observed high activity of the rumen microbiota on them, which may be responsible for the disappearance of more than 90% of the amount ingested. Similarly, the treatments did not alter the FA concentrations with 15 carbons in arterial blood because they also are not present in soybean oil, additionally, their ruminal synthesis was not changed since they are of microbial origin (Jenkins, 1993). The linear reduction of heptadecanoic (C17:0) concentration with higher doses of soybean oil can express the inhibition of its synthesis by the rumen microbiota, since it is also synthesized exclusively by microorganisms (Jenkins, 1993). The increase of arterial blood concentration of longchain FA (≥C18) with increased soybean oil doses is probably due to the higher intake of C18 FA, especially linoleic acid, originating from the rising doses of soybean oil. In this experiment, these long-chain FAs were most likely from the diet, since animals were not in energy deficit. Similarly, the arterial blood concentration of stearic acid (C18:0) increased linearly with increasing doses of soybean oil, a

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result that is justified by the increased supply of substrate for ruminal biohydrogenation, especially of linoleic acid. Contradicting the increase of the intake of oleic acid, the arterial concentration of this FA decreased with increasing of soybean oil dose, probably as a result of the biohydrogenation and of the intestinal 9-desaturase inhibition by the C18:2 t10, c12 raise, as happened in the mammary tissue. The increase of arterial concentration of the vaccenic acid (C18:1 t11), reaching a 127% increase with the greater dose, is due to rise of linoleic acid to the rumen, which is the main substrate for the synthesis of vaccenic acid. The detection of vaccenic acid and not of the C18:2 c9, t11 demonstrates the importance of the mammary gland supply of such isomer via arterial blood, since this is the precursor of more than 70% of CLA present in milk (Griinari et al., 2000). The elevation in vaccenic acid is due to its increased uptake given that the amount reaching the duodenum is about 20 times that of C18:2 c9, t11 (Hristov et al., 2005). Therefore, treatments that promote vaccenic acid supply to the mammary tissue are of great interest, since the vast majority of CLA present in milk is derived from mammary synthesis of this FA. The linoleic acid (C18:2) concentration in arterial blood was linearly increased by the treatments due to the high concentration of this FA in soybean oil, which led to an increase in its absorption. The low concentration of linolenic acid in soybean oil explains the lack of response in the arterial blood with increasing doses of soybean oil and, in addition, some of the C18:3 was biohydrogenated in the rumen. The enhancing of total trans FA concentration in arterial seems to be a response to the higher ruminal synthesis and subsequent intestinal absorption. This result was a consequence of the increasing intake of linoleic acid provided by soybean oil and the biohydrogenation of C18:2 to C18:1 t11 (Griinari et al., 2000). 4.4. Milk fatty acid profile The reduction in the C10:0 concentration after the administration of the maximum oil dose of soybean oil, with 73.1% of reduction, is probably due to intra-mammary synthesis inhibition, which was caused by the increased incorporation of long-chain FA (≥C18) from the arterial blood into milk, and also by increased influx of C18:2 t10, c12 to the mammary gland (Baumgard et al., 2001). Although not detected in arterial blood, it can be deduced that there was an increase in the contribution of this isomer to the mammary gland via bloodstream, since C18:2 t10, c12 found in milk is from the rumen (Bauman et al., 1999). The reduction in the C10:0 and other short-chain acids concentrations in goat’s milk can be seen as an important tool to reduce its characteristic odor, since C10:0, along with caproic and caprylic acids, in free form, are responsible for such features in the product, that lead to some resistance by humans in consuming both milk and its derivatives (Chilliard et al., 2003). As in the case of short-chain FA, the reduction of the concentration of medium-chain FA (C12–C16) in milk

decreased about 38% when the dose was 90 g/d. This result is probably due to the inhibition of intramammary synthesis by C18:0 and C18:2 t10, c12, since the C12, C14 and C14:1, and about half of the C16 are resulted from mammary lipogenesis (Chilliard et al., 2003). The inhibition of C16 intramammary synthesis is confirmed because the arterial supply was not influenced by soybean oil doses. The lowering of these FA (lauric, myristic and palmitic) in milk is of interest because they are part of a group of compounds responsible for development of cardiovascular diseases (Palmquist and Mattos, 2006). The intramammary inhibition hypothesis is reinforced with the similar acetate and ␤-hydroxybutyrate concentrations in arterial blood among treatments. Therefore, it can be concluded that the inhibition of synthesis was not caused by reduction in substrate availability for the mammary gland. This effect was responsible for the dissonance between arterial concentration of short and medium-chain FAs, which were not altered by doses of soybean oil, and milk concentration, that was reduced, clearly demonstrating the inhibition of mammary synthesis of these FAs. As well as in arterial blood (Table 4), the doses of soybean oil increased the concentration of C18:0 in milk due to an increase in C18 FA supply for ruminal biohydrogenation, especially the hydrogenation of oleic and linoleic acids and the mammary 9-desaturase inhibition by the C18:2 t10, c12 raise. Similar results were recorded by Mir et al. (1999) when offered increasing doses of canola oil (0, 2, 4 and 6%) for lactating goats, concluding that this response was a consequence of the ruminal microbiota activity on the higher ruminal intake of C18:2. The linear increase of milk C18:1 t11 concomitantly with the increasing dose of soybean oil was a result of the isomerization and incomplete ruminal biohydrogenation of the great amounts of linoleic and linolenic acids provided by the soybean oil, combined with the possible inhibition of its hydrogenation in the ruminal environment (Harfoot et al., 1973). In addition to the increased amount of vaccenic acid released to the mammary gland by arterial blood (Table 4), the raising concentration of C18:2 t10, c12 caused mammary 9-desaturase inhibition, with a subsequence increase in the direct passage of C18:1 t11 to the milk and concomitant decrease of C18:2 c9, t11 (Fig. 2), as well as decrease of the other fatty acids 9 milk concentration (C18:1) and concomitant rising of the by-products of saturation (18:0). Such findings are in agreement with those recorded by Baumgard et al. (2001) that showed C18:2 t10, c12 as the most potent inhibitor of this enzyme. The decrease of C18:1 in milk with increasing doses of soybean oil can be justified by the reduction of its supply by the arterial blood (Table 4) due to rumen microbiota activity on this FA, which converted it into stearic acid via hydrogenation (Harfoot and Hazlewood, 1988) and intestinal and mammary 9-desaturase inhibition by C18:2 t10, c12. Similarly to what was observed in blood, milk concentration of C18:2 responded positively to the treatments as a consequence of the high concentration of this FA in soybean oil.

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The acetate concentration was not influenced by soybean oil doses and its average was consistent with the values observed by Animut and Chandler (1996). Similarly, it was not observed any effect in arterial ␤-hydroxybutyrate concentration, probably because the animals were receiving diets that met their energy requirements, since energy deficiency is the main reason of changes in blood concentration of this metabolite (Baldwin and Kim, 1993).

5. Conclusions

Fig. 2. Arterial changes in the concentrations of C18:1 t11; C18:2 c9, t11; and C18:2 t10, c12 in milk with increasing dose of soybean oil.

The non-interference of soybean oil doses upon the total concentration of monounsaturated fatty acids in milk is possibly a consequence of losses in components of this fraction, such as oleic acid having been offset by the increase in the C18:1 t11 concentration. The linear increasing of unsaturated and polyunsaturated acids is especially due to the elevation in the linoleic and linolenic acids concentrations from the soybean oil and its by-product of FA isomerization (C18:2 t10, c12). The trans FA milk concentration increased with higher doses of soybean oil, as consequence of the higher amount of FA susceptible to isomerization, especially to C18:1 t11 synthesis. The vaccenic:rumenic ratio in milk increased as a consequence of the elevation of vaccenic, associated with the decrease of rumenic concentration. Similarly, the elevation of unsaturated:saturated ratio in milk resulted from the increase of the unsaturated fatty acids, combined with lower concentration of the saturated fatty acids. The soybean oil doses increased the total concentration of long-chain FAs (≥C18) in milk, which is explained by the fact that soybean oil is rich in this fatty acids group. The similarity between concentration of long-chain FAs (≥C18) in arterial blood and milk reinforce the hypothesis of the non-inhibition of mammary lipoprotein lipase activity by C18:2 t10, c12, since due to this enzyme uptake them of the bloodstream (Annison, 1983). The cis9 monounsaturated FA of the milk was reduced by soybean oil increase, due to the inhibition of 9-desaturase by the C18:2 t10, c12, similarly to what was observed for the oleic acid and the rumenic acid (both cis9 FA). 4.5. Arterial blood concentrations of metabolites The invariability of glucose concentration is consistent with the results observed by Sasaki (2002), who concluded that the body of a ruminant maintains an accurate glycemic control, not being influenced by nutrition as in monogastric animals. The maintenance of optimal physiological glucose concentrations is dictated by hormonal discharges strictly controlled, especially by insulin and glucagon.

Soybean oil supplementation increased the C18:2 t10, c12 in milk, resulting on decrease of milk fat due to the inhibition of the FA mammary synthesis, represented by short and medium-chain FA reduction. Arterial concentration of precursors for FA mammary synthesis (glucose, acetate and ␤-hydroxybutyrate) was not influenced by soybean oil. Therefore, decreased milk fat content was observed as a result of the inhibition of mammary synthesis of FA, but not by the limitation of precursors for its synthesis. The increase of milk concentration of C18:2 t10, c12 reduced the milk concentration of C9 FAs, especially the C18:2 c9, t11.

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