3.2. Milk Fatty Acid Profile
Fatty acids in milk originate from mammary uptake of preformed FA from blood and de novo synthesis in the mammary gland. All C4:0 to C12:0 FA, the majority of C14:0 (ca. 95%), and approximately half of C16:0 in milk fat derives from de novo synthesis, whereas half of C16:0 and all C18 and very-long chain FA originate from feed, ruminal metabolism, and body fat reserves [
3]. De novo synthesis in the mammary gland decreases when the availability of these preformed FA increases. The milk fat of dairy cows contains high proportions of SFA and consequently lower proportions of monounsaturated FA (MUFA) and polyunsaturated FA (PUFA), due to ruminal lipolysis and biohydrogenation of dietary unsaturated fatty acids and de novo synthesis [
3]. In addition, the PUFA absorbed in the small intestine are incorporated in phospholipids and cholesterol esters that are not as readily available FA sources for the mammary gland as FA in triacylglycerols [
24].
The fatty acid composition of milks is shown in
Table 3. Compared with control and SO, C4:0, C14:1
cis-9, C18:1
trans-11, C18:1
cis-9, C18:2
cis-9,
trans-11, C20:5n-3, C24:1n-9, and C22:6n-3 were higher (
p < 0.05) in FO. SO increased (
p < 0.05) C18:2
cis-9,
cis-12, and C18:3
cis-9,
cis-12, and
cis-15 in milk fat compared to control and FO. Both SO and FO increased (
p < 0.05) C18:0, and decreased (
p < 0.05) C6:0, C8:0, C10:0, and C14:0 compared with control. The most abundant FA in milk fat across all diets was C16:0, followed by C18:0 and C18:1
cis-9. C18:1
cis-9, the most abundant
cis-MUFA in milk fat, originates directly from the ruminal escape of C18:1
cis-9 as well as Δ
9 desaturation of C18:0 in the mammary gland. Desaturation of C18:0 is responsible for 60% of C18:1
cis-9 in milk, and in ruminants, around 40% of C18:0 taken up by the mammary gland is desaturated to C18:1
cis-9 by the Δ
9 desaturase enzyme [
25]. In this study, C18:0 and C18:1
cis-9 were increased (
p < 0.05) by both SO and FO compared with control. The results regarding the effects of SO diet on milk FA composition were as expected, as plant oil supplements and oilseeds enriched with C18:2
cis-9,
cis-12 in dairy cow feeding typically increase the availability of C18:0 for the mammary gland and elevate
cis-MUFA levels (mainly C18:1
cis-9) in milk fat [
3]. However, we observed an increase (
p < 0.05) in both C18:0 and C18:1
cis-9 in milk fat from FO diet, although marine oil supplementation in dairy cow diets usually decreases both of these FA in milk fat [
26]. In humans, dietary C18:1
cis-9 can lower total blood cholesterol and low-density lipoprotein [
27].
Both SO and FO increased (
p < 0.05) levels of C18:2
cis-9,
trans-11 when compared with control, but FO diet resulted in the highest (
p < 0.05) proportions which is in line with the results from earlier studies. Plant oils containing abundant amounts of C18:2
cis-9,
cis-12, and C18:3
cis-9,
cis-12,
cis-15 increase C18:2
cis-9,
trans-11 concentrations in milk fat [
3]. C18:2
cis-9,
trans-11 in milk fat originates directly from ruminal biohydrogenation of C18:2
cis-9,
cis-12, and C18:3
cis-9,
cis-12,
cis-15 and from increased outflow of C18:1
trans-11, a biohydrogenation intermediate, which is further desaturated in the mammary gland by Δ
9 desaturase [
28]. In accordance with previous studies [
3], FO high in n-3 PUFA induced higher proportions of C18:2
cis-9,
trans-11 in milk fat compared with SO. It has been reported that supplementation of moderate amounts of FO as Ca salt (2.7% DM; [
7] or as unrefined salmon oil (2.6% DM; [
5] in dairy cow diets increases the contents of C18:1
trans-11 and C18:2
cis-9,
trans-11 in milk FA. This is because the very long-chain n-3 PUFA in marine lipid supplements inhibit the last biohydrogenation step of 18-carbon FA, i.e., the conversion of C18:1
trans-11 to C18:0, resulting in accumulation of C18:1
trans-11 in the rumen, which provides more C18:1
trans-11 to the mammary gland that can be converted to C18:2
cis-9,
trans-11 by the Δ
9 desaturase [
24].
Because the transfer rate of C20:5n-3 and C22:6n-3 into milk is extremely limited even in high levels of supplementation due to their extensive ruminal biohydrogenation and utilization in other functions than milk fat synthesis, marine lipid supplements in dairy cow feeding are mainly studied for their role as inhibitors of the conversion of C18:1
trans-11 to C18:0 in the rumen [
3,
26]. In addition, fish oil and marine algae supplements may decrease milk fat synthesis, especially if fed in high amounts to dairy cows [
3,
26]. Nevertheless, C20:3n-3, C20:5n-3, C22:6n-3, and total omega-3 (n-3) FA were higher (
p < 0.05) in milk from FO treatment compared with control and SO (
Table 4). Higher n-3 levels in blood or on the erythrocyte membrane are associated with reduced cardiovascular mortality [
29]. Even though C20:5n-3 and C22:6n-3 were increased in FO milk, their concentrations were not enough to markedly contribute to the human intake recommendations (approx. 1 g/d) of n-3 to reduce the risk of cardiovascular disease [
30]. Even though the n-3 levels were increased in FO, the total PUFA were not changed relative to control. However, the total PUFA in milk fat from SO diet were increased (
p < 0.05) compared with other diets. This reflects the higher intake and ruminal escape of C18:2
cis-9,
cis-12 in SO, although transfer efficiency of C18:2
cis-9,
cis-12 from plant oils and oilseed supplements to milk is low, as it is for the very long-chain PUFA [
3].
There were some time effects which may reflect possible rumen microbial adaptations to the oil treatments [
31]. In some cases, SO and FO decreased individual SFA and increased some unsaturated FA. For example, from 21 to 63 days of supplementation with SO and FO, the following milk FA were decreased: C6:0, C14:0, C16:0, and C18:0, whereas C14:1
cis-9, C18:1
trans-10, C18:1
trans-11, C18:1
cis-9, C18:3
cis-6,
cis-9, and
cis-12 were increased. Interestingly, with the exception of C20:0 and C24:0, FA with more than 20 carbons had a quadratic effect: they increased from day 21 to 42 and then decreased in day 63 with FO.
Main fatty acid groups in milk fat, calculated fatty acid ratios, health lipid indices, and product/substrate concentration ratios as estimates for Δ
9 desaturase activity in the mammary gland are shown in
Table 4. In general, SFA concentrations of milk were reduced (
p < 0.05) with SO and FO treatments compared with control. According to Ulbricht and Southgate (1991), C12:0, C14:0, and C16:0 are SFA that can promote atherosclerosis and coronary thrombosis. The reduction of SFA content in milk has been reported previously when plant oils such as SO [
3] or FO alone [
3,
5,
7], or in combination with vegetable oils [
5,
24], are incorporated into dairy cow diets. As dairy products are a major source of C12:0, C14:0, and C16:0 in the human diet [
3], the reduction in the level of 14:0 in milk fat from cows fed SO and C14:0 and C16:0 in milk fat of cows fed FO in this study is a favourable change. Contrary to the previous studies with SO or other plant oils [
3], SO supplement did not induce a reduction in the proportion of C12:0 nor C16:0 in the present study. Generally, according to the comprehensive literature review of Kliem and Shingfield [
3], fish oil and marine algae induce more subtle changes in C12:0, C14:0, and C16:0 proportions in milk fat compared with vegetable oils, and marine lipids generally cause a decrease in the proportion of 18:0, whereas vegetable oils elevate its levels in milk fat. Thus, the results of the present study regarding changes induced by FO are different than expected, but more favourable to human health. Naturally, decreases in the levels of SFA in milk fat are accompanied by increases in unsaturated FA. For the SO diet, the total PUFA in milk fat increased compared with control, whereas the FO diet induced increases in MUFA. When dietary SFA were replaced with
cis-unsaturated FA, the level of serum lipid biomarkers were improved, thus reducing risk factors of CVD [
32].
Total SFA, PUFA, n-3 PUFA, n-6/n-3, atherogenicity index, and thrombogenicity index were decreased (
p < 0.05) from day 21 to 63 by SO and FO. These results reflected findings from individual milk FA as they are used for the calculation of the aforementioned groups of lipids, ratios, or indices. The ratio C14:1
cis-9/C14:0 was higher (
p < 0.05) in FO compared with control and SO. The C14:0 is synthesized in the mammary gland, and therefore C14:1
cis-9 can only be produced by desaturation through Δ9-desaturase enzyme. The average Δ9-desaturase activity for C14:1
cis-9/C14:0 was 0.03 in this study. However, Bu et al. [
20] reported that this index ranges from 0.048 to 0.085 depending on the fat supplement.
The increases observed in milk C18:2
cis-9,
trans-11 can be partially attributed to the increased index of C18:2
cis-9,
trans-11/C18:1
trans-11 in the FO and SO over the control (
Table 4). Increasing contents of C18:2
cis-9,
trans-11 (also known as rumenic acid) is desirable in dairy products. Human studies have reported the health advantages of consuming C18:2
cis-9,
trans-11, since it might improve cancer prevention, cardiovascular diseases, immune and inflammatory responses, and bone health [
33]. It should be noted that the primary dietary sources of C18:2
cis-9,
trans-11 for humans are dairy products and meat products from ruminant animals.
3.3. Physicochemical Properties of Ice Cream
This study focused on the interface of animal production and food science, and thus, our approach was to improve milk FA profile for ice cream production by modifying the cow’s diet.
In order to analyze the effect of dietary lipids, all ice cream treatments were standardized to obtain a final fat content of 16%. It has been reported that a reduction in the fat content in the ice cream particularly to or below 30 g/kg can result in the loss of textural and sensory properties [
34]. The protein content of ice cream was slightly higher (
p < 0.05) in SO compared with control, whereas FO did not differ from other treatments. However, those changes were minimal (<0.2%), and therefore they were unlikely to affect the overall nutritional and functional properties of ice cream. Fat, lactose, and sucrose were not affected by treatments and there was not treatment × time interactions (
Table 5).
Physical parameters of ice creams are shown in
Table 5. Draw temperature was higher (
p < 0.05) in control and SO compared with FO. The lower draw temperature in FO ice creams could be associated with their higher contents of monounsaturated FA in the milk used for ice cream manufacturing. Overrun was lowered in FO ice creams compared with control. Ice creams that have more saturated FA may form solidified fat at a higher temperature during ice cream manufacturing, leading to a higher efficiency on retaining air in the matrix [
35], which is reflected in increased overrun in the control and SO ice creams.
Firmness was similar in control and SO but lower (
p < 0.05) in FO. Melting rate was higher (
p < 0.05) in FO compared to control and SO. Firmness and melting rate results can be explained because FO had an increased proportion of unsaturated FA compared with control and SO that may have more liquid-like matrix behaviour, in contrast with saturated FA which will have a more solid-like form at low temperatures [
36]. Firmness of ice cream can be influenced by numerous factors such as ice crystal content, ice crystal size, extent of fat destabilization, overrun, and the rheological properties of the mix [
37]. Studying the above-mentioned factors would be highly recommended in future experiments where milks with high-unsaturated FA profile for ice cream manufacturing are used.
The lowered (
p < 0.05) yellowness (b *) intensity found in SO and FO may be explained by the FA unsaturation that those ice creams had. Previously, it has been reported that the b value increases as the fat content of samples increases [
38].