1. Introduction
Foods containing a high concentration of n-3 fatty acids in the human diet can reduce the risk of cardiovascular disease and prostate, colon and breast cancer [
1,
2]. Hence, interest in the consumption of polyunsaturated fatty acids (PUFA)-rich dairy products has increased [
3,
4]. Cows receiving typical diets produce milk that contains approximately 70% saturated fatty acids (SFA), whereas mono- and polyunsaturated fatty acids represent approximately 25% and 5% of milk fat, respectively [
5].
Linseed oil, which contains approximately 70% PUFA, of which 50% is α-linolenic acid (ALA), is a rich vegetable source of fatty acid (FA) n-3, and supplementation with linseed oil in cow diets leads to an increase in n-3 FA in their milk. [
6]. The increase in the PUFA content of milk fat is desirable for human health; however, this makes the milk more susceptible to oxidation [
7]. Because PUFA has double bonds, it is susceptible to the loss of electrons because of the action of free radicals, luminosity and other agents, leading to lipid peroxidation [
8]. Lipid peroxidation promotes a rancid flavor and reduces the shelf life of dairy products [
8] and can predispose humans to metabolic diseases [
9,
10]. In the animal body, there is a balance between the formation of free radicals and endogenous antioxidant capacity. Endogenous antioxidant capacity is regulated by the enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX), which can delay lipid peroxidation [
11]. The body can also receive antioxidant compounds, such as vitamin E, selenium, phenolic compounds, from diet (exogenous antioxidants) [
12,
13].
The supply of these sources of antioxidants in dairy cow diets increases the antioxidant activity in milk [
13,
14]. The addition of carotenoids and vitamin E in the feeding of ruminants, can improve the organoleptic quality of the final product [
15], both in meat and milk. This protects the product from the lipoperoxidation effects by improving the oxidative status of meat and milk [
16]. Annatto (
Bixa orellana L.) is a Bixaceae family plant, and its seeds contain carotenoids with antioxidants properties (Bixin and Norbixin). Bixin is a carotenoid that belongs to the apocarotenoid family [
17], and whose antioxidant power is conferred by an extensive chain of double bonds, which allows it to combat singlet oxygens [
18]. Annatto seeds have tocotrienols that, combined with bixin, could synergistically protect PUFAs from oxidation [
19]. The objectives of this study were to evaluate the effects of dietary of annatto seeds, linseed oil, and their combination on dry matter intake (DMI) and nutrient total tract digestibility, as well as changes in the antioxidant capacity and milk FA composition of lactating cows. We hypothesized that linseed oil supplementation would increase n-3 concentration, whereas annatto seeds would increase antioxidant capacity and, consequently, decrease milk oxidation, and the association between the two treatments would increase n-3 concentration in the milk in conjunction with lower oxidation.
3. Results
There was no interaction between annatto seeds and linseed oil on DM and nutrient intake except on EE intake (
p = 0.03) (
Table 2). Indeed, DM, OM, CP, NDF and NFC intake was reduced by annatto seeds addition (
p < 0.001), and linseed oil reduced the NFC intake by 8% (
p = 0.03). There was no interaction between evaluated factors on DM and nutrients digestibility. In addition, annatto seeds did not affect digestibility. However, supplementation of linseed oil increased EE digestibility (
p = 0.005), tended to decrease NDF digestibility (
p = 0.07) and to increase CP digestibility (
p = 0.09).
There was no interaction between annatto seeds and linseed oil on milk yield, composition and antioxidant activity (
Table 3). Diets containing annatto seeds reduced the production of milk (
p = 0.01), protein (
p = 0.01), and lactose (
p = 0.01). Annatto seed addition increased the fat content and total solids content (
p < 0.01) and supplementation with linseed oil decreased milk fat content (
p = 0.03).
An interaction effect between annatto seeds and linseed oil was observed in milk C6:0 (
p = 0.007), C8:0 (
p = 0.01), and C18:2 n6t (
p = 0.04) (
Table 4). The addition of annatto seeds increased the concentrations of C6:0 and C8:0 fatty acids in the absence of linseed oil (
p ≤ 0.05), while in the presence of oil the concentrations of these fatty acids was not changed. Supplementation of linseed oil increased the concentrations of C18:2 n6t FA (
p ≤ 0.05), while in combination with annatto seeds, the concentration of this FA was not affected. It was observed that linseed oil supplementation reduced the concentration of C10:0, C12:0, C13:0, C14:1, C15:0, and C16:0 in milk fat (
p < 0.05), and increased the C18:0, C18:1 n9t, C18:2 n6t, C18:3 n3, C18:2 c9 t11 -CLA, C20:3 n6, C20:4 n6, and C21:0 (
p < 0.05).
An interaction effect between annatto seeds and oil was observed on short chain fatty acids (SCFA) (
p < 0.01), addition of annatto seeds alone increased milk SCFA in the absence of linseed oil (
p ≤ 0.05), while in the presence of oil it did not change the concentrations of these fatty acids (
Table 5). Linseed oil decreased medium chain fatty acids (MCFA) (
p = 0.01) and increased long chain fatty acids (LCFA) (
p = 0.02) in milk fat. Additionally, linseed oil supplementation tended to reduce mono-unsaturated fatty acids (MUFA) (
p = 0.07) and increase PUFA (
P= 0.08) and saturated fatty acids (SFA) (
p = 0.06). There was a tendency for interaction between annatto seeds and linseed oil on
fatty acids n-6 (
p < 0.10), wherein linseed oil supplementation increased the
fatty acids n-6 in the absence of annatto but in the presence of annatto seeds this fatty acid was reduced (
Table 5). There was also an approximately 176% increase of n-3 concentration in milk fat (
p < 0.01) and the n-6/n-3 ratio was improved by linseed oil supplementation once the linseed oil was reduced by 58% (
p < 0.01).
The different treatments, as well as the addition of annatto seeds did not have any effect on the antioxidant concentrations in milk (
Table 3) or blood (
Table 6). The concentration of CD in milk has a tendency to increase with addition of linseed oil (
p = 0.07) to the diet. There was no effect of annatto seeds on blood parameters, linseed oil supplementation has a tendency to increase HDL-cholesterol (
p = 0.08).
4. Discussion
The addition of annatto seeds to the cow diet reduced feed intake. It was observed that cows rejected diets containing annatto seeds. It was associated with undesirable palatability, texture, and odor. Additionally, annatto seeds contain carotenoids, terpenoids, and terpenes [
34,
35,
36], which affect the DMI [
37,
38]. Terpenoids and terpenes can be stored in the form of essential oils and released through structures, such as secretory glands and trichomes; consequently, these compounds are concentrated in the taste buds [
39]. The main essential oil components of annatto seeds are two monoterpenes called α-pinene and β-pinene [
40]. According to Estell et al. [
41], α-pinene in the alfalfa pellets rendered them unpalatable for consumption by sheep. It is possible that the monoterpene α-pinene may be associated with animal rejection of annatto seeds.
The linseed oil supplementation, regardless of annatto seed supply, increased EE intake and reduced the NFC intake. According to the NRC [
20], increased dietary energy density by fat supplementation leads to a replacement of carbohydrates by lipids, which might result in a higher EE content in the diet and a lower proportion of carbohydrates. Lipid supplementation increased the digestibility of EE and CP. Similar results were found by Santos et al. [
42] when dairy cow diets were supplemented with 2.5% linseed oil. Linseed oil had a higher proportion of PUFA and a low melting point facilitating micelle formation and increasing absorption rate in the intestine [
20]. Thus, the addition of linseed oil to diets increased the digestibility of EE.
Exceeding 5% of the dietary lipid DM may cause some adverse effects, such as inhibiting the growth of microorganisms that degrade the fibrous fraction in the roughage and affecting fiber and OM digestibility [
43]. The percentage of EE in diets with oil was 5.6%, which may explain the tendency to reduce NDF digestibility. However, exceeding the 7% lipid limit can have more evident effects on reducing DMI, and digestibility of DM, OM, and NDF [
44].
In the present study, 15 g/kg of annatto seeds in DM did not alter nutrient digestibility. These results were consistent with the findings of Barbosa et al. [
45] who used higher amounts of annatto seeds (100 g/kg, 230 g/kg, and 350 g/kg of DM) in diets for sheep and found that annatto seeds did not influence nutrient digestibility. De Lima Júnior et al. [
46] reported increasing levels (0, 100, 200, and 300 g/kg total DM) of the annatto by-product in the sheep feed did not affect the digestibility of the layers.
In this study, the treatments did not affect the lipid profile (cholesterol, triglycerides, and HDL-cholesterol). These parameters followed the standard values according to Kaneko et al. [
47]. Although some studies have highlighted the potential effects of antioxidants on these parameters, this was not observed in the present study [
48,
49]. Similar results were reported by Lima et al. [
50], where supplementation of the diet with annatto colorum (paprika) in 0.08, 0.12, and 0.16 g/kg of DM for lactating cows did not affect plasma cholesterol concentration but altered the plasma fatty acid profile. In the present study, linseed oil supplementation increased the HDL-cholesterol, which could be attributed to lipid intake.
Milk and lactose production were reduced with diets containing annatto seeds, which also decreased DM and nutrient intake. According to Dado and Allen [
51], milk yield is positively correlated with DMI, resulting in lower net energy intake available for milk synthesis in comparison with the control diet, which had higher production because of higher DMI. Additionally, the lower DMI decreased glucose precursors. As synthesis of lactose is dependent on glucose, lactose is comprised in annatto seed-fed animals [
52]. Milk fat and total solid content increased after consumption of annatto seeds diets. These effects occurred probably because of to a concentration effect because these diets showed a 296 reduction in milk yield [
53].
The addition of annatto seeds did not affect the proportions and ratios of fatty acids in milk, especially the MCFA that are responsible for increasing the fat content, suggesting that these amounts of annatto seeds were not the main cause of the increased content of fat. Additionally, fat-corrected milk production and milk fat production did not show significant effects related to annatto seeds and lipid supplementation.
The addition of annatto seeds did not affect the oxidation products and antioxidant activity, as the seeds were included with the objective of transferring its polyphenolic compounds to milk, to decrease oxidation of milk fat that was enriched with PUFA. Treatments with annatto seeds had approximately 125.25 mg GAE/g DM of total polyphenols, which was not satisfactory to affect concentrations, increase antioxidant activity, or decrease the oxidative profile. Cows supplemented with 24.35 mg GAE/kg DM of total polyphenols from propolis extract did not improve the oxidative stability of milk [
20]. Conversely, Santos et al. [
54] found no significant difference for the oxidative profile but found increased antioxidant activity with the supplementation of 1950 mg GAE/g DM of total polyphenols from grape residue in the presence of soybean oil.
Regarding the antioxidant activity in blood and milk, lipid supplementation increased CD concentration because of increased PUFA intake, which has double bonds that predispose it to lipoperoxidation, thus causing electron loss [
55] and increased CD levels in milk. Supplementation with annatto seeds did not affect the animal or milk oxidative status, which may have been caused by the low absorption range of carotenoids. Bixin is a carotenoid that belongs to the group of carotenes, whose absorptive process is very similar to that of lipids, because carotenes are fat-soluble pigments [
56]. Carotenes are less polar molecules found in the center of emulsions, whose absorptive efficiency depends on the transfer of the emulsion to the micelles, which is not as effective for the carotene group compared to the xanthophyll group [
56].
Supplementation with linseed oil reduced the fat content in milk. The PUFA content in the linseed oil is responsible for this reduction [
57,
58] by altering ruminal biohydrogenation resulting in intermediate products, such as fatty acids trans-9, cis-11-CLA, and trans-10, cis-12-CLA, which also inhibit the synthesis of milk fat in the mammary glands [
59,
60]. However, consumption of material and EE was lower in diets supplemented with annatto seeds, according to Woolpert et al. [
61], and diets with lower EE content led to an increase in de novo fatty acid synthesis. This is the main source of the formation of SCFA in the mammary glands.
Linseed oil diet supplementation reduced the MCFA, owing to the higher concentration of PUFAs in flax oil [
62]. Linseed oil PUFAs can be affected by incomplete biohydrogenation generating the trans-9, cis-11-CLA, and trans-10, cis-12-CLA [
59,
60]. These fatty acids are inhibitors of genes that are involved in the de novo synthesis [
59,
60], such that these CLAs reduced MCFA (C10:0, C12:0, C13:0, C14:1, C15:0, C16:0) and led to a tendency to reduce C14:0. It is noteworthy that these fatty acids are of mixed origin, originating from the de novo synthesis that occurs in the mammary gland and from food.
In general, supplementation with linseed oil increased the LCFA (C18:0, C18:1 n9t, C18:2 n6t, C18:3 n3, C20:3 n6, C20:4 n6, and C21:0). This can be explained by the composition of linseed oil, which is a source of LCFA and PUFA, mainly C18:3 n-3. When the PUFA reaches the rumen, it can be affected by biohydrogenation to protect ruminal bacteria generating intermediates and saturated fatty acids as a final product. The fatty acids that leave the rumen are absorbed in the intestine and incorporated into milk fat [
63]. The C18:2 n6t is produced during the biohydrogenation process, and its reduction may be associated with the addition of carotenoids. Hino et al. [
64] performed an in vitro assay, in which they added 5 or 10 mg of β-carotene, 5 mg α-tocopherol, and 1 g of glucose for each liter of rumen liquid, and observed the growth of ruminal microorganisms. The association of β-carotene and α-tocopherol with sunflower oil decreased the inhibition of the growth of these microorganisms caused by oil; thus, increasing the use of LFCA by ruminal bacteria. Possibly the annatto seed carotenoids promoted the use of the C18:2 n6t fatty acid in the presence of oil, resulting in a lower concentration of n-6 compared to the diet with flaxseed oil.
Supplementation with linseed oil decreased SFA and increased MUFA and PUFA. The increase in PUFA results in a better fatty acid profile for human consumption. The results of the present study are consistent with the findings of Santos et al. [
42] and Suksombat et al. [
65], who reported similar changes in milk fatty acids when linseed oil was used, and those of Caroprese et al. [
66] and Petit and Côrtes [
67], who used linseed to feed dairy cows.
Linseed oil is a rich source of C18:3 n-3, which is partially hydrogenated, and a large part of it becomes overpassed, which promotes an increase in the concentration of n-3 in milk fat. There was an increase of approximately 176% in the concentration of n-3 in milk obtained from cows fed linseed oil supplemented diets as compared to those fed normal diets. Previous studies demonstrated an increase of n-3 concentration in milk from dairy cows receiving linseed oil supplementation [
42,
58,
68]. Fatty acids n-3 has a beneficial effect on human health owing to its potential to reduce cardiovascular disease [
1], prostate, colon, and breast cancer [
2]. Oil supplementation in the cow diet improves the n6:n3 ratio by 58%, which makes their milk healthier, according to the World Health Organization [
69].