Oxidative Stability of Flaxseed (Linum usitatissimum L.) Cake and its Inclusion in the Diet of Lactating Cows

Abstract

The objective was to evaluate the oxidative stability of flaxseed cake and its inclusion in the diet of lactating cows, on milk production and composition and blood parameters (total cholesterol, triglycerides, high density lipoprotein -HDL and low density lipoprotein - LDL). Oxidative stability was evaluated by acidity index (AI) and peroxides (PI), determined from day zero to the ninth day of storage. Eighteen crossbreed Holstein x Jersey cows were used, nine animals per treatment (without and with the inclusion of cake). The experimental design was completely randomized, with two treatments and nine replications. There was an increase in PI during the first six days of storage, followed by a decrease on the ninth day. Supplying 4.5% flaxseed cake to lactating cows for 90 days did not promote changes in production, chemical composition and fatty acid profile of milk, somatic cell counts and blood parameters. Thus, flaxseed cake can be used to feed lactating cows.

Keywords:
Animal nutrition; Dairy cattle; Fat; Linolenic acid; Milk composition; Peroxide index

HIGHLIGHTS

Flaxseed cake is susceptible to lipid oxidation, but without significant changes in its quality.

Flaxseed cake does not alter milk production in lactating cows.

Flaxseed cake does not alter the chemical composition and fatty acid profile of milk.

INTRODUCTION

Feed is the factor with the greatest impact on production costs in animal production systems. To meet the nutritional requirements of ruminants, less costly alternative feeds of high nutritional value are being included in the diet, for example, cakes and meals extracted from oilseeds [¹].

There is currently a trend in the population to consume healthy and functional foods, i.e., foods that improve the body’s functions, reducing the risk of diseases [²]. Flaxseed (Linum usitatissimum L.) is an oleaginous plant characterized by a high content of polyunsaturated fatty acids (PUFAs) such as linolenic acid (C18:3 n3), fibers, and lignans [³,⁴]. Linolenic acid improves the PUFA profile in ruminant milk and meat [⁵]. The inclusion of this fatty acid in animal feed can have positive effects, such as reducing the accumulation of fat in the liver and increasing the milk yield during the transition period, as well as lower rates of embryo death, increased postpartum fertility, and shorter calving intervals of dairy cows [⁶,⁷].

Although beneficial, foods rich in PUFAs, especially linolenic acid, are susceptible to oxidation when stored for long periods of time [⁸]. The oxidation process decreases the shelf life of the food, alters its color, flavor and odor, reduces its nutritional quality, and can lead to the formation of potentially toxic substances that can affect consumer health [⁹]. On rural properties, raw materials and feeds destined for use in animals are usually stored for long periods of time, a fact that can compromise the quality of the diet and affect productive and reproductive performance. Furthermore, in the case of oil-rich feeds, secondary compounds formed by lipid oxidation cause sensory alterations in the feed [¹⁰], with a consequent reduction in its consumption by animals.

Lipid oxidation is mediated by various reaction mechanisms that are related to the number and nature of unsaturated bonds, exposure to light and heat, and the presence of pro-oxidants or antioxidants [¹¹]. The lack of control of the storage conditions of raw material, processing conditions, and heat treatment can also result in lipid oxidation [¹²]. In ruminants, PUFAs are partially transformed into conjugated linoleic acid (CLA) and linolenic acid in the rumen, which will be part of the fatty acid composition of milk. Studies have shown that CLA present in milk can reduce the development of malignant tumors, prevent diabetes, and modulate the immune system in humans [¹³], in addition to improving the animal’s immune response [¹⁴].

Within this context, the aim of the present study was to evaluate the oxidative stability of flaxseed cake and the effects of its inclusion in the diet of lactating cows on milk yield milk composition, and blood parameters [cholesterol, triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL)].

MATERIAL AND METHODS

Characterization of flaxseed cake

Flaxseed cake was prepared at Instituto Agronômico do Paraná (IAPAR), Londrina, PR, Brazil, by cold pressing using an experimental press. A 10-kg sample of flaxseed cake was ground in a Cyclone rotor mill (1-mm sieves) for laboratory analyses. The chemical composition of the cake was: dry matter (DM) - 90%; crude protein - 31.80%; neutral detergent fiber - 33.37%; acid detergent fiber - 23.74%; ether extract - 20.56%; mineral matter - 3.90%, according to AOAC [¹⁵]. Analysis of zearalenone and aflatoxins revealed the absence of these compounds.

Assessment of oxidative stability

Oxidative stability was assessed by testing accelerated oxidation in an oven (Schaal method) [¹⁶]. Subsamples were collected from the previously homogenized 10-kg cake sample and distributed on aluminum trays, each containing 50 g of cake. The subsamples were placed in an oven at 65 ^oC for 9 days, simulating nine months of storage. Samples were collected daily for oil extraction as described by Bligh & Dyer [¹⁷] to analyze oxidative stability in triplicate.

The acidity index (AI) and peroxide index (PI) were evaluated from 0 to day 9 of storage. The IA of flaxseed cake oil was determined according to the AOAC method Ca 5a-40 [¹⁵] and is expressed as milligrams of potassium hydroxide per gram of sample (mg KOH g^-1). The peroxide index (PI) was determined according to the AOAC method Cd 8-53 and is expressed as milliequivalents of peroxide per kg of sample (mEq kg^-1).

Fourier-transform infrared spectroscopy was performed from day 0 to day 9 of storage. For this analysis, 5-μL aliquots of the oil were applied to potassium bromide tablets (100 mg) and infrared spectra were obtained with a Shimadzu 8400 spectrophotometer operating in the range of 4,000 to 500 cm^-1.

The ¹H nuclear magnetic resonance (NMR) spectra of the oils were obtained on days 0, 3, 6 and 9 with a Bruker Avance 400 NMR spectrometer operating at 9.4 Tesla, by observing the hydrogen nucleus at 400.13 MHz. An aliquot (10 μL) of each sample was dissolved in 600 μL of CDCl₃ containing 0.1% tetramethylsilane (TMS). After homogenization, 500-μL aliquots were transferred directly to 5-mm NMR tubes. The concentrations of linolenic acid, linoleic acid, oleic acid, and total saturated fatty acids were calculated as described by Barison and coauthors [¹⁸].

Evaluation of flaxseed inclusion in the diet of dairy cows

The experiment was conducted at the Dairy Cattle Sector of Fazenda Escola Capão da Onça (FESCON), which belongs to the State University of Ponta Grossa (UEPG), Ponta Grossa, PR, Brazil. The municipality is located at an altitude of 990 m (25°05'49" south latitude and 50°03'11" west longitude). The climate of the region is characterized as humid subtropical, mesothermal, Cfb type (Köppen classification). The average temperature in the coldest month is below 18^oC, with the occurrence of frost, and the average temperature in the warmest month is below 22^oC. There is no defined dry season. Average annual rainfall ranges from 1,600 to 1,800 mm, with an annual relative humidity of 70-75% (Climatempo).

Eighteen black-and-white Holstein and crossbred cows with a mean live weight of 643 ±109 kg and a mean age of 4 years, including primiparous and multiparous cows of 1^st to 4^th calving order, were used. For evaluation of the inclusion of flaxseed cake in the concentrate, the animals were divided into two treatments: no inclusion of flaxseed cake (control group) and inclusion of 4.5% flaxseed cake in the diet (treated group). The ingredients of the concentrate and the chemical composition of the diets are shown in Table 1. The ration was formulated to meet the requirements of lactating cows according to the NRC [¹⁹]. The cows were allowed to adapt to the diets for 14 days.

Cows were kept in a semi-intensive system where they received bulk feed (corn silage) and concentrate (with or without flaxseed cake) three times a day. The concentrate consisted of corn, soybean meal, wheat bran, mineral supplement, and buffer with or without the addition of flaxseed cake. The animals were milked twice a day, at 8:00 and 15:00 h. After the afternoon milking, the cows were transferred to paddocks where they remained until milking the next morning.

Table 1
Ingredients of the concentrate (g/kg DM) and chemical composition of the diet.

Cows diet
	Concentrate
Ingredient	Without flaxseed cake	With flaxseed cake
Corn	551.0	547.0
Soybean meal	266.0	241.0
Flaxseed cake	-	46.0
Wheat bran	138.0	121.0
Bovigold	35.0	35.0
Mineral supplement1	10.0	10.0
Chemical composition
Dry matter (g/kg natural material)	893.2	90.65
Crude Protein (g/kg DM)	158.3	159.0
Neutral detergent fiber (g/kg DM)	458.	453.9
Total digestible nutrient (g/kg DM)	738.6	734.4
Net energy of lactation (Mcal/kg)2	1.69	1.68

• ¹ Calcium (min) 190.00 g/kg; phosphorus (min) 60.00 g/kg; sulfur (min) 20.00 g/kg; magnesium (min) 20.00 g/kg; potassium (min) 35.00 g/kg; sodium (min) 70.00 g/kg; cobalt (min) 15.00 mg/kg; copper (min) 700.00 mg/kg; chromo (min) 10.00 mg/kg; iron (min) 700.00 mg/kg; iodine (min) 40.00 mg/kg; manganese (min) 1,600.00 mg/kg; selenium (min) 19.00 mg/kg; zinc (min) 2,500.00 mg/kg; vitamin A (min) 200,000.00 IU/kg; vitamin D3 (min) 50,000.00 IU/kg; vitamin E (min) 1,500.00 IU/kg; fluoride (max) 600.00 mg/kg. ²NEL, net energy for lactation: 0.0245 x TDN (%) - 0.12 (NRC, 2001).

Milk yield and composition

Daily milk yield was measured by individual sampling using electronic milk meters. Milk samples from each cow were stored in duly identified sterile bottles containing bronopol as preservative. These samples were collected during the afternoon milking. The samples were sent to the laboratory of Associação Paranaense dos Criadores de Bovinos da Raça Holandesa (APCBRH) for analysis of the percentages of fat, protein, lactose, and total solids by infrared spectroscopy in an automated B2300 Combi analyzer (Bentley) and of somatic cell count (SCC) by spectrometry (Somacount 500). For statistical analysis, the SCC values ​​were log₁₀ transformed to normalize the distribution of the data.

Fatty acid analysis in milk

On day 40 of the experiment, milk samples were collected from each cow during the morning and afternoon milkings for analysis of saturated, unsaturated and monounsaturated fatty acids, linoleic acid (18:2), and linolenic acid (18:3) by NMR using a Bruker Avance 400 NMR spectrometer. An aliquot (10 μL) of each sample was dissolved in 600 μL of CDCl₃ containing 0.1% TMS. After homogenization, 500-μL aliquots were transferred directly to 5-mm NMR tubes. The spectra obtained were calibrated in relation to the TMS signal at 0.00 as an internal reference. These transformations and/or adjustments were performed using Bruker’s TopSpin software, which was also used to acquire the spectra. Butyric acid was calculated according to Brescia and coauthors [²⁰].

Blood parameters

Blood samples were collected from the cows for determination of the following blood parameters: total cholesterol, triglycerides, HDL, and LDL. Blood was collected directly from the mammary vein on day 82 of the experiment, three hours after the morning feeding, and stored in vacutainer tubes containing anticoagulant. The blood parameters were determined according to Friedewald, Levy & Fredrickson [²¹].

Statistical analysis

The results of the oxidative stability tests were expressed as mean ± standard deviation. Data were analyzed by analysis of variance (ANOVA), followed by Fisher’s test for differences between means, using the Action Stat statistical program. A p-value <0.05 was considered significant. A completely randomized design was used for analysis of the inclusion of flaxseed cake in the diet of dairy cows, with two treatments and nine replicates. Repeated measures over time were used to evaluate the interaction between treatment and time. When the interaction was not significant, the simple treatment effect was evaluated by the F test. Statistical analysis was performed using the SAS 8.1 program (2009), with a p-value <0.05 being considered significant.

RESULTS

Oxidative stability of flaxseed cake

There were differences (p<0.05) in the AI and PI of flaxseed cake oil over the storage period (Table 2). An increase in AI (p<0.05) was observed until day 9 of storage. The PI increased over the first 6 days of storage, followed by a decrease due to the degradation of peroxides and the formation of secondary oxidation products.

Table 2
Mean and standard deviation of acidity index (AI) and peroxide index (PI) of the oil of flaxseed cake submitted to accelerated oxidation for 9 days of storage at 65^oC.

Storage period (days)	AI (mg KOH g-1)	PI (mEq kg-1)
0	1.80 ± 0.02e	0.99 ± 0.01c
1	2.06 ± 0.44de	1.96 ± 0.03b
2	2.38 ± 0.01cd	2.01 ± 0.01b
3	2.38 ± 0.01cd	2.97 ± 0.03a
4	2.47 ± 0.21cd	2.97 ± 0.09a
5	2.78 ± 0.16bc	2.97 ± 0.03a
6	3.01 ± 0.19b	3.51 ± 0.70a
7	3.18 ± 0.07b	2.00 ± 0.01b
8	3.69 ± 0.18a	1.48 ± -0.71bc
9	3.77 ± 0.12a	1.47 ± 0.65bc
p-value	<0.001	<0.001

• ¹ Means in the same column followed by different letters differ by the Fisher’s test (p<0.05). Each day corresponds to one month of storage.

The infrared spectrum of flaxseed cake oil submitted to accelerated oxidation (Figure 1) showed unsaturated carbon hydrogen stretching by the double bond (C=C-H) in the 2,955 cm^-1 region. The 2,915 and 2,840 cm^-1 bands refer to the carbon-hydrogen bond of alkanes. The band found in the 1,746 cm^-1 region represents the carbonyl (C=O) stretching frequency characteristic of esters [²²]. The stretching frequency of C=C and out-plane deformation of the same bond appear between 1.654-1.648 cm^-1 and at 723 cm^-1, respectively [²³]. The absorption band at 3,000-3,500 cm^-1, which is more intense in the oil after 9 days of accelerated oxidation, corresponds to carboxyl and hydroxyl groups. This band is a qualitative indicator of the increase in the acidity of the oil and the consequent increase in oxidation.

Figure 1
Infrared spectrum of flaxseed cake oil submitted to accelerated oxidation obtained on day 0 (pink) and day 9 (green).

The 1H NMR method provides information on the structural changes that occur in a molecule during oxidation. Figure 2 shows the 1H NMR spectrum of the flaxseed cake oil studied. Letter A represents the methylene hydrogens between two double bonds (diallylic), letter C represents allylic hydrogens, and letter F represents the methyl hydrogens of linolenic acid [¹⁸]. The oxidative state of oils and fats can be assessed by observing changes in these signals.

Figure 2
H¹ NMR spectrum of the flaxseed cake oil used in this study. A: methylene hydrogens between two double bonds (diallylic hydrogens). B: α carbonyl hydrogens of all fatty acids esterified to the glycerol moiety. C: methylene α olefin hydrogens of all unsaturated fatty acids (allylic hydrogens). D: β carbonyl hydrogens from all fatty acids esterified to the glycerol moiety. E: methyl hydrogens of linolenic acid. F: methyl hydrogens of the other fatty acyl chains [¹⁸].

The data of the 1H NMR spectra are presented in Table 3. A significant reduction (P<0.01) of 3.4% in linolenic acid content was observed by the third day of storage of flaxseed cake. This reduction is due the greater the degree of unsaturation of the fatty acid, the higher its susceptibility to oxidation reactions, with consequent changes in its structure that are detected in the NMR spectrum.

Table 3
Fatty acid composition (g/100 g) of flaxseed cake oil obtained by ¹H NMR.

Day	Linolenic acid	Linoleic acid	Oleic acid	Saturated fatty acids
0	52.01 ± 0.30a	7.18 ± 0.29c	30.68 ± 0.35a	10.13 ± 0.56
3	50.85 ± 0.16b	10.29 ± 0.31a	29.46 ± 0.47b	9.38 ± 0.33
6	50.72 ± 0.23b	9.21 ± 1.04ab	29.27 ± 0.60b	10.80 ± 1.82
9	50.25 ± 0.86b	8.68 ± 0.98b	29.62 ± 0.50b	11.45 ± 0.64
p-value	0.01	<0.01	<0.05	0.16

• ¹Means in the same column followed by different letters differ by the Fisher’s test.

Milk characteristics

Table 4 shows the mean milk yield of cows fed a diet with or without flaxseed cake. The addition of flaxseed cake to the diet did not cause any changes in the milk volume produced (p>0.05). However, regarding the period evaluated, cows exhibited higher daily milk yield in March compared to April and May (p<0.05). The silo change that occurred in the present study in April may explain this variation in the milk yield of the animals.

Table 4
Mean milk yield (L/cow/day) according to test-day month in each treatment.

Month	Without flaxseed	With flaxseed	Mean
March	25.02A	26.74A	25.9a
April	21.50A	21.93A	21.72b
May	20.40A	21.74A	21.10b
Mean	22.30A	23.47A

• ¹ Means in the same column followed by different letters differ by the Fisher’s test.

The inclusion of flaxseed cake did not influence (p>0.05) the fat, protein, lactose or total solid content of milk (Table 5). The results agree with those found by Stoffel et al. [²⁴], Toral et al. [²⁵], Meignan et al. [⁶] and Zubiria et al. [²⁶].

Table 5
Mean and standard deviation of fat, protein, lactose, and total solid content of milk from cows fed a diet with or without the addition of flaxseed cake according to month of supplementation.

Month	Without flaxseed	With flaxseed	Mean
Fat (%)
March	3.66 ± 0.52	3.70 ± 0.37	3.68
April	3.91 ± 0.46	4.05 ± 0.51	3.98
May	3.51 ± 0.66	3.94 ± 0.24	3.72
Mean	3.69	3.90
Protein (%)
March	3.23 ± 0.26	3.25 ± 0.26	3.24b
April	3.29 ± 0.27	3.34 ± 0.29	3.32ab
May	3.46 ± 0.20	3.48 ± 0.24	3.47a
Mean	3.33	3.36
Lactose (%)
March	4.48 ± 0.24	4.55 ± 0.11	4.51
April	4.53 ± 0.22	4.58 ± 0.10	4.55
May	4.44 ± 0.24	4.56 ± 0.08	4.50
Mean	4.48	4.56
Total solids (%)
March	12.40 ± 0.64	12.54 ± 0.62	12.47
April	12.70 ± 0.56	12.96 ± 0.75	12.83
May	12.52 ± 0.94	13.11 ± 0.58	12.81
Mean	12.54	12.87	12.70

• ¹ Means followed by different letters differ by the F test (p<0.05).

The saturated, unsaturated and monounsaturated fatty acids, linoleic acid (18:2), and linolenic acid (18:3) in the milk was analyzed on day 40 of the experiment and the results showed that the addition of 4.5% flaxseed cake to the diet of dairy cows did not cause changes (p>0.05) in the profile of the fatty acids evaluated (Table 6).

Table 6
Linolenic acid, linoleic acid, and monounsaturated and saturated fatty acid content of milk from cows fed a diet with or without inclusion of flaxseed cake.

Fatty acid	Without flaxseed	With flaxseed	p-value
Linolenic acid (C18:3) (% of total fatty acids)	0.094 ± 0.043	0.111 ± 0.02	0.306
Linoleic acid (C:18:2) (% of total fatty acids)	0.296 ± 0.11	0.294 ± 0.04	0.978
Monounsaturated (% of total fatty acids)	21.93 ± 1.98	21.57 ± 1.54	0.673
Saturated (% of total fatty acids)	68.99 ± 1.63	69.17 ± 1.58	0.814
Butyric acid (% of total fatty acids)	8.69 ± 0.72	8.91 ± 0.02	0.518

• ¹A p-value less than 0.05 indicates a significant difference between treatments by the F test.

Blood parameters in dairy cattle

Table 7 shows the blood parameters of cows fed a diet with or without flaxseed cake evaluated on day 82 of the experiment. The addition of flaxseed cake to the diet did not cause changes in the blood parameters (p>0.05).

Table 7
Blood parameters of cows fed a diet with or without the addition of flaxseed cake.

Parameter	Without flaxseed	With flaxseed
Total cholesterol (mg/dL)	165.67 ± 37.9	163.67 ± 23.2
HDL (mg/dL)	81.33 ± 8.5	80.56 ± 4.6
LDL (mg/dL)	83.07 ± 31.0	82.00 ± 20.9
Triglycerides (mg/dL)	6.33 ± 1.4	5.56 ± 1.6

• ¹ Means in the same row followed by different letters differ by the F test (p<0.05).

Figure 3 shows the SCC according to treatment. There was no interaction effect (p>0.05) between assessment period and the addition or not of flaxseed cake to the diet on milk SCC. Likewise, no difference (p>0.05) in SCC was observed between treatments. Despite the lack of a significant difference, there was trend towards a linear decrease in SCC after March in animals fed flaxseed cake. The same trend was not observed in animals of the control group, with a reduction in SCC in April (138.4 × 10³ cells/mL) and a significant increase in May (449.8 × 10³ cells/mL).

Figure 3
Mean somatic cell counts (SCC) in milk from cows fed a diet with or without inclusion of flaxseed cake. February: test-day records obtained before the beginning of the experiment.

DISCUSSION

Oxidative stability of Flaxseed cake

Despite the increase in the AI and PI of flaxseed cake oil after 9 days of accelerated oxidation, the indices are within the standards established by the Codex Alimentarius for cold-pressed oils, which determines an upper limit of 15 mEq kg^-1 for PI and of 4.0 mg KOH g^-1 for AI [²⁷]. Thus, the increase in these indices observed over the storage period was not sufficient to compromise the quality of the oil.

The antioxidant activity of flaxseed cake can be attributed to the phenolic compounds that remain as glycoside derivatives or are attached to the cell wall after oil extraction [²⁸]. Ecoisolariciresinol diglucoside (SDG) is the main lignan in flaxseed seed coat and is recognized for high antioxidant activity [²⁸,²⁹]. Furthermore, flaxseed cake may contain carotenoids, chlorophylls, tocopherols and proteins and other compounds responsible for antioxidant protection [³⁰-³²]. The concentration of these compounds may vary depending on the process used to extract the oil [³⁰,³²]. The way the oil is presented may also have influenced this result, since linseed oil is adhered to the cake particles, reducing its exposure to oxidation.

Polyunsaturated fatty acids are more vulnerable to oxidation than monounsaturated fatty acids since the rate of oxidation depends on the number of double bonds and their positions [³³]. Linolenic acid, which contains three double bonds, oxidizes 2.4 times faster than linoleic acid with only two double bonds [³⁴]. Lipid oxidation comprises a series of reactions that primarily form hydroperoxides. Due to their low stability, these hydroperoxides decompose to form low molecular weight short-chain compounds such as alcohols, aldehydes, ketones, esters, and other hydrocarbons [³⁵,³⁶]. The relative stability of acidity observed in flaxseed cake is an important factor since a high level of acidity would indicate extensive lipid oxidation, which can compromise the absorption of proteins and folic acid by the body. This impaired absorption, in turn, can cause pathological changes in the digestive tract. This inhibits enzymatic activity, increase blood cholesterol and peroxide levels, activate atherosclerosis, and induce carcinogenic activity [³⁷].

The hydrogen in PUFAs requires less activation energy to induce the formation of free radicals. Oxidation usually starts in the diallyl position of the chain since the diallylic carbon atom is more likely to donate a hydrogen atom, resulting in a diallyl radical that consequently forms hydroperoxides [³⁸]. Thus, the oxidation rates of linoleic and linolenic acids are higher than the oxidation rate of oleic acid since the fatty acid chain of the former contains more reactive sites that are particularly susceptible to free radical attack [³⁹].

As a monounsaturated fatty acid, oleic acid remained practically stable because of its low oxidation rate. There was no significant difference (p=0.16) in saturated fatty acids according to storage time, indicating greater oxidative stability. Due to its antioxidative mechanisms, flaxseed was found to withstand long storage periods without undergoing significant lipid oxidation-related changes.

Milk characteristics

The addition of 4.5% flaxseed cake to the diet of dairy cows did not cause changes in the milk volume and profile of the fatty acids. The fatty acid composition of milk is mainly determined by interactions between dietary factors and rumen metabolism [⁶,²⁶]. Regarding dietary factors, changes in the composition of milk nutrients due to the use of lipids in the diet also depend on the nature of the lipid source (degree of unsaturation, length of the lipid chain), content and form of inclusion [⁶].

Despite the high linolenic acid content of flaxseed oil (50 to 55%), it was not sufficient to increase the concentration of this fatty acid in milk fat of supplemented animals. The inclusion of soybean and linseed oil at 2.5% in the diet of Holstein cows can modify the fatty acid profile in milk, with a reduction in the content of saturated fatty acids and an increase in unsaturated fatty acids [⁴⁰].

About the somatic cell count of milk, this numerical reduction observed in cows treated with flaxseed cake may thus reflect the effect of the high α-linolenic acid content of flaxseed oil (50 to 55%), which is a source of omega-3 PUFAs [⁴⁰]. The latter is important for the immune system. One factor that may explain the lack of a significant effect is the small amount of flaxseed cake used, i.e., 4.5% of the concentrate (DM basis).

Fatty acids of the omega-3 and omega-6 family are important in the human diet since they are not synthesized by the body and are precursors of long-chain PUFAs such as eicosapentaenoic, docosahexaenoic, and arachidonic acids. These fatty acids play important roles in the body, for example in the synthesis of eicosanoids, which are directly involved in the immune system and inflammatory responses [⁴¹]. Thus, part of these fatty acids that do not undergo biohydrogenation and are absorbed intact may exert a similar function in the animal’s body, enhancing immune system function and reducing the occurrence of mastitis.

Blood parameters in dairy cattle

The susceptibility of blood lipids to oxidation is an indication of oxidative stress in animals, a situation characterized by an imbalance between the production of free radicals and antioxidant levels. Its important balancing lipid immunomodulators that are involved in inflammatory responses [⁴²]. During the imbalanced immunometabolic response, signals from metabolic tissues affect the functioning of immune cells, promoting inflammation. Oxidative stress can cause damage to important lipids and macromolecules and modify metabolic pathways, with consequent physiological changes that can trigger the development of diseases such as placenta retention, mastitis, and udder edema [⁴³]. Lipid supplementation of cows aims to improve the quality of milk fat; however, this supplementation causes an increase in the blood concentrations of PUFAs which, by nature, are susceptible to lipoperoxidation and expose animals to the risks of oxidative stress.

CONCLUSIONS

The flaxseed cake oil analyzed exhibited satisfactory oxidative stability. Despite the increase in the formation of peroxides and free fatty acids, their levels remained within the range recommended by the Codex Alimentarius. The storage period of flaxseed cake evaluated in this study (corresponding to a period of 9 months) did not cause significant changes in the fatty acid profile or in the formation of compounds derived from oxidation. Supplying 4.5% flaxseed cake in concentrate over a period of 90 days was not sufficient to promote changes in milk yield, milk chemical composition, milk fatty acid profile, SCC, or blood cholesterol and triglyceride levels.

REFERENCES

• ¹
  Paramasivam C, Kendra V, Tiruppur. Potential and Utilization of By-Products of Oilseeds in Animal Feed Industry. Biotica Res Today 2021;3:655-7.
• ²
  Karelakis C, Zevgitis P, Galanopoulos K, Mattas K. Consumer Trends and Attitudes to Functional Foods. J Int Food Agribus Mark. 2019;32:1-29.
• ³
  Campos JR, Severino P, Ferreira CS, Zielinska A, Santini A, Souto SB, Souto EB. Linseed Essential Oil - Source of Lipids as Active Ingredients for Pharmaceuticals and Nutraceuticals. Curr Med Chem. 2019;26(24):4537-58.
• ⁴
  Deme T, Haki GD, Retta N, Woldegiorgis A, Geleta M. Fatty Acid Profile, Total Phenolic Content, and Antioxidant Activity of Niger Seed (Guizotia abyssinica) and Linseed (Linum usitatissimum). Front Nutr (8)674882.
• ⁵
  Moallem U. Invited review: Roles of dietary n-3 fatty acids in performance, milk fat composition, and reproductive and immune systems in dairy cattle. J Dairy Sci. 2018;101(10):8641-61.
• ⁶
  Meignan T, Lechartier C, Chesneau G, Bareille N. Effects of feeding extruded linseed on production performance and milk fatty acid profile in dairy cows: A meta-analysis. J Dairy Sci. 2017;100(6):4394-408.
• ⁷
  Swanepoel N, Robinson PH. Impacts of feeding a flaxseed based feed supplement on production and health of mid through late lactation multiparous Holstein cows on a commercial dairy farm. Anim Feed Sci Technol. 2019;258:114318.
• ⁸
  Orlova Y, Harmon RE, Broadbelt LJ, Iedema PD. Review of the kinetics and simulations of linseed oil autoxidation. Prog Org Coat. 2021;151:106041.
• ⁹
  Osorio JAC, Daniel JLP, Cabral JF, Almeida KV, Guimarães KL, Sippert MR, et al. Annatto seeds as Antioxidants Source with Linseed Oil for Dairy Cows. Animals. 2021;11(5):1465.
• ¹⁰
  Zhang B, Wang H, Wang Y, Yu Y, Liu J, Liu B, et al. Identification of antioxidant peptides derived from egg-white protein and its protective effects on H2O2-induced cell damage. Int J Food Sci Technol. 2019;54(6):2219-27.
• ¹¹
  Domínguez R, Pateiro M, Gagaoua M, Barba FJ, Zhang W, Lorenzo JM. A Comprehensive Review on Lipid Oxidation in Meat and Meat Products. Antioxidants. 2019;8(10):429.
• ¹²
  Baron LF, Pazinatto R, Baron CP. [Lipid oxidation and implications for the nutrition and health of production animals] Cad Ciênc Tecnol. 2020;37(1):26597.
• ¹³
  Salzano A, Neglia G, D’Onofrio N, Balestrieri ML, Limone A, Cotticelli A, et al. Green feed increases antioxidant and antineoplastic activity of buffalo milk: A globally significant livestock. Food Chem. 2021;344:128669.
• ¹⁴
  Malcicka M, Visser B, Ellers J. An Evolutionary Perspective on Linoleic Acid Synthesis in Animals. Evol Biol. 2018;45(1):15-26.
• ¹⁵
  Official methods of analysis of AOAC international. 16th ed. Washington, DC: Association of Official Analytical Chemists; 1995. 2 p.
• ¹⁶
  Abou-Gharbia HA, Shahidi F, Adel A, Shehata Y, Youssef MM. Effects of processing on oxidative stability of sesame oil extracted from intact and dehulled seeds. J Am Oil Chem Soc. 1997;74(3):215-21.
• ¹⁷
  Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37(8):911-7.
• ¹⁸
  Barison A, Grandizoli C, Campos F, Simonelli F, Lenz C, Ferreira A. A simple methodology for the determination of fatty acid composition in edible oils through 1H NMR spectroscopy. Magn Reson Chem MRC. 2010;48:642-50.
• ¹⁹
  Nutrient Requirements of Dairy Cattle: Seventh Revised Edition, 2001 [Internet]. Washington, D.C.: National Academies Press.
• ²⁰
  Brescia M, Mazzilli V, Sgaramella A, Ghelli S, Fanizzi F, Sacco A. 1H NMR characterization of milk lipids: A comparison between cow and buffalo milk. J Oil Fat Ind. 2004;81:431-6.
• ²¹
  Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18(6):499-502.
• ²²
  Silverstein, Webster, Kiemle: Spectrometric Identification of Organic Compounds, 7th Edition - Student Companion Site [Internet]. [cited on July 4, 2024]. Available at: https://bcs.wiley.com/he-bcs/Books?action=index&itemId=0471393622&itemTypeId=BKS&bcsId=2174
  » https://bcs.wiley.com/he-bcs/Books?action=index&itemId=0471393622&itemTypeId=BKS&bcsId=2174
• ²³
  Guillén MD, Cabo N. Relationships between the Composition of Edible Oils and Lard and the Ratio of the Absorbance of Specific Bands of Their Fourier Transform Infrared Spectra. Role of Some Bands of the Fingerprint Region. J Agric Food Chem [Internet]. 1998,46(5). [Cited on July 4, 2024]. Available at: https://doi.org/10.1021/jf9705274
  » https://doi.org/10.1021/jf9705274
• ²⁴
  Stoffel CM, Crump PM, Armentano LE. Effect of dietary fatty acid supplements, varying in fatty acid composition, on milk fat secretion in dairy cattle fed diets supplemented to less than 3% total fatty acids. J Dairy Sci. 2015;98(1):431-42.
• ²⁵
  Toral PG, Bernard L, Belenguer A, Rouel J, Hervás G, Chilliard Y, et al. Comparison of ruminal lipid metabolism in dairy cows and goats fed diets supplemented with starch, plant oil, or fish oil. J Dairy Sci. 2016;99(1):301-16.
• ²⁶
  Zubiria I, Garcia-Rodriguez A, Atxaerandio R, Ruiz R, Benhissi H, Mandaluniz N, et al. Effect of Feeding Cold-Pressed Sunflower Cake on Ruminal Fermentation, Lipid Metabolism and Bacterial Community in Dairy Cows. Animals. 2019;9(10):755.
• ²⁷
  Codex Alimentarius. Standard for edible fats and oils not covered by individual standards (CXS 210-1999). 1999.
• ²⁸
  Ma G, Wang J, Wang X, Gan S, Yang F, Dong G. Assessing the influence of Secoisolariciresinol diglucoside multi‐antioxidants on flaxseed oil’s oxidative stability. Eur J Lipid Sci Tech. 2024;126(5):2300159.
• ²⁹
  Tavarini S, Castagna A, Conte G, Foschi L, Sanmartin C, Incrocci L, et al. Evaluation of Chemical Composition of Two Linseed Varieties as Sources of Health-Beneficial Substances. Mol Basel Switz. 2019;24(20):3729.
• ³⁰
  Zhang T, Zhang Z, Wang X. Composition and Antioxidant Ability of Extract from Different Flaxseed Cakes and Its Application in Flaxseed Oil. J Oleo Sci. 2023;72(1):59-67.
• ³¹
  Barthet VJ, Klensporf-Pawlik D, Przybylski R. Antioxidant activity of flaxseed meal components. Can J Plant Sci. 2014;94(3):593-602.
• ³²
  Mannucci A, Castagna A, Santin M, Serra A, Mele M, Ranieri A. Quality of flaxseed oil cake under different storage conditions. LWT. 2019;104:84-90.
• ³³
  Else PL. The highly unnatural fatty acid profile of cells in culture. Prog Lipid Res. 2020;77:101017.
• ³⁴
  Gong M, Wei W, Hu Y, Jin Q, Wang X. Structure determination of conjugated linoleic and linolenic acids. J Chromatogr B Analyt Technol Biomed Life Sci. 2020;1153:122292.
• ³⁵
  Gavahian M, Chu YH, Barba F, Misra NN. A critical analysis of the cold plasma induced lipid oxidation in foods. Trends Food Sci Technol. 2018;77.
• ³⁶
  Hematyar N, Rustad T, Sampels S, Kastrup Dalsgaard T. Relationship between lipid and protein oxidation in fish. Aquac Res. 2019;50(5):1393-403.
• ³⁷
  Karpińska M, Borowski J, Danowska-Oziewicz M. The use of natural antioxidants in ready-to-serve food. Food Chem. 2001;72(1):5-9.
• ³⁸
  Cao H, Xiong SF, Dong LL, Dai ZT. Study on the Mechanism of Lipid Peroxidation Induced by Carbonate Radicals. Molecules. 2024;29(5):1125.
• ³⁹
  Karavalakis G, Stournas S, Karonis D. Evaluation of the oxidation stability of diesel/biodiesel blends. Fuel. 2010;89(9):2483-9.
• ⁴⁰
  Oliveira MXS, Palma ASV, Reis BR, Franco CSR, Marconi APS, Shiozaki FA, et al. Inclusion of soybean and linseed oils in the diet of lactating dairy cows makes the milk fatty acid profile nutritionally healthier for the human diet. PloS One. 2021;16(2):e0246357.
• ⁴¹
  Alagawany M, Elnesr SS, Farag MR, El-Sabrout K, Alqaisi O, Dawood MAO, et al. Nutritional significance and health benefits of omega-3, -6 and -9 fatty acids in animals. Anim Biotechnol. 2022;33(7):1678-90.
• ⁴²
  Zachut M, Tam J, Contreras GA. Modulating immunometabolism in transition dairy cows: the role of inflammatory lipid mediators. Anim Front. 2022;12(5):37-45.
• ⁴³
  Ayemele AG, Tilahun M, Lingling S, Elsaadawy SA, Guo Z, Zhao G, et al. Oxidative Stress in Dairy Cows: Insights into the Mechanistic Mode of Actions and Mitigating Strategies. Antioxidants. 2021;10(12):1918.

• Funding:

  This research received no external funding

Edited by

Editor-in-Chief:

Paulo Vitor Farago

Associate Editor:

Paulo Vitor Farago

Publication Dates

• Publication in this collection
  08 Nov 2024
• Date of issue
  2024

History

• Received
  22 July 2024
• Accepted
  26 Aug 2024