4.2. Feed Intake, Milk Yield, and Milk Composition
In the present study, no significant differences were noticed related to the DMI between the TMR and pTMR cows. However, milk production was significantly lower in the pTMR cows, probably due to the low energy intake of pasture and energy costs implied by the walk to pasture, and by grazing. Opposite to our results, studies conducted by De La Torre-Santos et al. [
33] emphasized an increment of DMI and milk production in the cows fed using a partial mixed ration (TMR + pasture), compared to those fed using the TMR. These differences probably rely on the fact that legume silages (peas and beans, respectively) were used in TMRs’ compositions, instead of the corn silage used by us. Steinshamn [
34] reported that the DMI of legume silage is higher based on the lower neutral detergent fiber (NDF) concentration of legumes and the higher rate of NDF degradation than in grasses, enabling a better balance between the metabolizable energy and the absorbed amino acids, therefore supporting a higher milk production. The TMR intake in the cows from the pTMR group based on the grazing period showed significant differences; the lowest TMR intake was recorded in the first grazing period (
p ˂ 0.05), due to the higher pasture intake, probably caused by the lower crude fiber content of grass.
In our study, the significantly lower energy content of the pasture compared to the TMR ration (1.44 vs. 1.66 NE
L Mcal/kg DM,
Table 1) limited milk production, also in agreement with the weight loss registered in the cows from this group (pTMR cows) compared to those fed with the TMR, which gained weight. Other studies also reported a decrease in milk yield and body weight in the grazing cows compared to those fed indoors using TMR [
35,
36].
Although a higher mean of daily milk yield in the TMR cows was recorded, compared to the pTMR cows (24.86 vs. 22.75 kg/d), no significant differences were registered related to fat, protein, and lactose production; these results being in line with those of other studies comparing TMR vs. pTMR [
7,
12,
37].
Many studies proved a lower fat content of milk yielded by cows fed using TMR compared to that yielded in grazing-based feeding systems [
38,
39]. This statement was assigned to the high fiber content in the pasture diet, unlike the TMR diets which show a high starch content and low dietary fiber, inducing the production of high amounts of propionic acid in rumen bacteria. Such diets (high in starch and low in fiber) have been associated with fat concentration decrement in milk due to the suppression of the de novo synthesis of short- and medium-chain FAs in milk [
40].
Additionally, it is well known that lactose concentration in milk is less influenced by the type of feeding [
41]. In our study, a higher concentration of lactose was noticed in the milk from cows fed indoors, using the TMR. A potential explanation could reside in the high starch concentration of the TMR, resulting in a higher propionic acid throughout the rumen fermentation processes. The increment of propionate concentration in the rumen is crucial to reach higher glucose and lactose synthesis in milk [
42].
Additionally, in our study, the significantly higher urea levels in the pTMR cows’ milk could be the result of a protein excess, showing a high degradability in rumen and an inadequate energy/protein ratio in pasture [
11]. On the other hand, the high amounts of fermentable compounds (soluble sugars) from the pasture (especially at the beginning of the grazing—P
1), negatively impact the normal ruminal microbial activity. Thus, nitrogen ammonia resulted from the degradation of high soluble proteins is not used and converted into microbial protein, but is mainly converted to urea nitrogen in milk and urine. A higher urea level in the milk yielded by the pTMR cows indicates a higher protein intake by using grass, but also an energy deficit [
43]. O’Callaghan et al. [
4] reported higher urea concentrations in the milk obtained using pastures (perennial ryegrass with white clover) rather than in that from cows fed using TMR.
Our results, concerning the influence of the grazing period on milk chemical composition, endorse those previously reported by Radonjic et al. [
29] and Bohacová et al. [
30], which claimed that milk’s fat content increased according to the pasture biomass maturation. The lower milk yield achieved in the P
2 and P
3 grazing periods is in line with the lower NE
L content, lower crude protein, and higher crude fiber content of grass from the pasture compared to the P
1 period. The highest urea content in milk was reported along the P
1 period, maybe caused by the higher protein intake, as a result of the higher legumes percentage in the pasture floristic composition, but also of the content rich in high-soluble proteins [
6].
4.3. Fatty Acid Composition
The FA profile of milk’s fats specify its nutritional qualities. The type of feeding influenced milk fat content in OA and total MUFAs, which showed higher values in the milk yielded by the cows having access to pasture (pTMR group) compared to the TMR type of feeding. This may be the result of a higher ALA intake from grass, which is further biohydrogenated in the rumen, leading to SA formation. The SA and other FAs resulting from ALA biohydrogenation, such as VA could be unsaturated to OA and RA, respectively, in the mammary gland [
43]. In our study, the higher SA and OA content of milk fat in cows having access to pasture (pTMR), and associated with their body weight loss in the experimental period, compared to the TMR cows, indicates an energy deficit in the pTMR cows’ feed and an increased mobilization of body fat reserves [
7]. OA is an indicator for the intensive mobilization of body fat [
44].
The OA (C18:1
c9) results by rumen biohydrogenation of feed’s PUFAs (mainly ALA and LA), as well as by SA desaturation (C18:0) in the mammary gland in the presence of the stearate desaturase enzyme, which is also present in the mammary tissues [
45]. The fact that the Δ9-desaturase activity, in our study, does not show differences in the two groups of cows ascertains the hypothesis that the higher C18:1
c9 ratio in the milk fat of pTMR cows is the outcome of the mobilization of body fat reserves.
Lower concentrations of SFAs (C12:0, C14:0, and C16:0) in the milk of cows having limited access to pasture is in agreement with other authors’ statements [
46,
47]. This decrease is probably partly due to the higher PUFAs intake from grass, but also to the fact that the products resulted by rumen biohydrogenation of ALA from the grass are potent inhibitors of the synthesis of FAs with 10 to 14 carbon atoms in the mammary gland [
48].
ALA (C18:3 n-3) content was 1.3 times higher in milk yielded by cows having access to pasture (pTMR) compared to those housed in shelter (TMR). These results are in line with the previous studies conducted by Morales-Almaraz et al. [
12] and Barca et al. [
7] proving a 44% and 2.64 times, respectively, higher ALA content in the milk of cows fed using pasture and TMR, compared to those fed using only TMR.
The omega-6 FA, mainly C18:2 n-6 (LA) were found in a higher concentration in the milk of cows fed using only TMR (without pastures), which was also previously reported by Morales-Almaraz et al. [
12] and Barca et al. [
7].
ALA (C18:3 n-3) is the upmost component in the fat of pasture, while LA (C18:2 n-6) is in the corn silage’s and concentrates’ fats [
4]. Thereby, in our study, the higher ALA concentration in the milk of pTMR cows and the higher LA concentration in the milk of TMR cows, is due to the different intakes of these FAs from feed, which egressed rumen biohydrogenation.
Most of the CLA from milk is synthesized in the mammary gland based on C18:1
t11 throughout desaturation processes in the presence of ∆-9 desaturase [
49]. The VA amount in milk was 1.48 times higher in pTMR cows. This main CLA precursor results from LA (C18:2 n-6) and linolenic acid (C18:3 n-3) incomplete rumen biohydrogenation [
50], which are common in pasture fat. The lower enzymatic desaturation activity in the mammary gland of the cows having access to pasture (0.58 vs. 0.73) is consistent with the higher long-chain FA amounts in milk, resulting from body fat (mainly C18:1
c9) mobilization and with the higher PUFA amounts from the grass, which escaped rumen biohydrogenations and reached the duodenum, thus reducing the activity of specific enzymes in the mammary gland (∆-9 desaturation, acetyl-coA carboxylase) [
7].
A higher VA and CLA
c-9,
t-11 content in the milk of pTMR cows suggests a healthier profile of this milk for consumers, since VA can be converted into CLA in human tissues, which shows anticancer effects [
2].
The main FA group (PUFA/SFA, n-6/n-3 FA, and h/H) ratios, the AI, the TI, and the HPI are commonly used to assess milk fats quality related to the effect on consumers’ health. In general, a PUFA/SFA ratio above 0.45 and an n-6/n-3 FA ratio below 4.0 are recommended for human nutrition [
51,
52]. In the present study, the PUFA/SFA ratio showed considerably lower values (0.08–0.11) in both groups, than the recommended ones, while the n-6/n-3 FA ratios were within the recommended values, but only in the pTMR group of cows (1.75), based on the FA n-3 increment and the FA n-6 decrement in milk, compared to the cows fed using the TMR diet.
The consumption of milk and dairy products showing lower AI and TI values and higher h/H FA and HPI ratios, indicate benefic health effects of the cardiovascular system of consumers, but no organization has yet made recommendation in terms of values for these fat quality indices [
53].
In our study, we found that cows fed on pasture (pTMR group) had significantly improved h/H FA ratio, and AI, TI, and HPI value, as a result of ALA intake increases throughout the grass, that enabled the FA n-3 content augmentation in milk. Feeding cows exclusively based on TMR revealed a negative effect on these quality indices of milk fat, due to the SFAs and LA increment and the decrement FAs considered benefic for human health.
The SFA content in milk gradually increased during the grazing period, with the highest SFA content in the third period (P
3), mainly based on the C18:0 and C16:0 FAs’ increase. These findings support those of Radonjic et al. [
29] and Bohacová et al. [
30], even if Ferlay et al. [
54] reported a slight decrease in the C16:0 content in milk yielded from spring to autumn, which is not consistent with our results. This could reside in pasture composition differences.
The total MUFA content decrease, including that of the individual FAs (mainly OA and VA), during the grazing period could be explained by the fact that at the same time with plants’ vegetative growing stages, C18:2 n-6 and C18:3 n-3 decreases and it is converted into C18:1 (C18:1
c9 and C18:1
t11) by rumen biohydrogenation, thereby influencing their content in milk. Similar results were reported by Radonjic et al. [
29], Bohacová et al. [
30], and Ferlay et al. [
54].
Based on the high fiber content of grass in the third grazing period (P
3), the milk fat content increased, next to PUFA decrease, meanwhile SFA content achieved high levels, which is an unwanted event in terms of impact on human health. Consequently, even if the milk yielded at the beginning of the grazing period shows a lower fat content, it also contains higher levels of FAs considered benefic for human health (VA, ALA, CLA). The early vegetative stage of grass showing a high ALA content (50–75%) could be the cause of the higher PUFA content in the milk produced in the first grazing period [
55]. A part of the PUFAs are directly transferred into milk, whereas another part are converted into VA by rumen biohydrogenation, and further into CLA (
c9,
t11) in the mammary gland by desaturation [
56]. Similar results of the total PUFA and individual FA content in cows’ milk, depending on the phenological vegetative phase of pasture, were disclosed also by Radonjic et al. [
29] and Coppa et al. [
57].
Milk fat showed a ratio of n-6/n-3 FAs, AI, and TI lower values at the beginning of grazing, whereas the most critical values of sanogenous lipid indices were registered at the end of the grazing period, maybe as a result of the chemical composition change of grass and probably of the energy deficit recorded as a consequence of decreased intake and digestibility of grass nutrients.
4.4. Fat-Soluble Vitamins and Antioxidant Capacity
The results of this study reveal significantly higher α-tocopherol, β-carotene, and retinol concentrations in the milk of cows that grazed and received TMR-supplemented feed in the shelter; similar results were also reported by other researchers [
33,
58]. A four times higher concentration of fat-soluble vitamins and β-carotene in the milk of cows fed on pasture, compared to those fed indoors using TMR, was reported by Butler et al. [
59]. In our study, the increase in fat-soluble antioxidant concentrations in the grazing cows’ milk (pTMR group) had lower values (2–2.5 times), since the grazing time was limited to 8 h/day.
The highest milk antioxidant activity (μM TE/mL) was associated with the highest concentration of α-tocopherol (0.74 mg/100 g), retinol (125.62 μg/100 g), and β-carotene (0.69 μg/100 g) determined in the cows’ milk of the pTMR group, which had access to pasture. These results backup the conclusions of Stobiecka et al. [
60] claiming that the increase in the fat-soluble antioxidants content showed the same trend as milk antioxidant activity, which increased too.
Additionally, in our study, the α-tocopherol and retinol concentrations insignificant diminution in the pasteurized milk could be explained by the low temperature used at pasteurization (65 °C) [
15]. The lowering of natural antioxidants concentration in the milk stored for four days in the refrigerator could be justified by the fact that this period accounts for the formation of reactive oxygen species, which are inactivated by milk antioxidants. By means of this function, α-tocopherol, retinol, and β-carotene are oxidized, thus conducting to a decrease in their concentration in milk [
61].
TAC (µmol TE/mL) showed lower values in heat-treated milk rather than in raw milk, significant differences being observed only in the case of the pTMR group. Calligaris et al. [
62] stated that the pasteurization process (at 90 °C for 10 min.) led to an increased pro-oxidant activity, probably because of the breakup of some natural antioxidants (tocopherols, vitamin A, carotenes) and the formation of new oxidative molecules during Maillard reactions. Furthermore, Yilmaz-Ersan et al. [
63] noted that heat treatment conditions (temperature and time) and milk composition in natural antioxidants influence milk antioxidant capacity, which could endorse the results of our study, in view of a lower lipophilic antioxidants content in TMR milk. These results suggest that the milk obtained from pTMR cows had higher nutritional qualities than the one yielded by cows fed indoors, using TMR.
The antioxidant activity does not change significantly during storage, although it tends to decrease slightly. Accordingly, consumers may enjoy the benefits of milk’s nutritional qualities and bioactive compounds during the four days of storage of the pasteurized milk in the refrigerator.
The mean values of milk samples TAC in the present study were higher than those reported by other authors [
64,
65], probably as outcome of the higher fat concentration of the Jersey cows’ milk, used in our study. Moreover, it is well known that milk TAC is influenced by heat treatment, but also by fat concentration [
66].