It is well known that ruminants’ diet greatly influences the milk nutritional characteristics, particularly fatty acid profile [1
]. This is of great importance because of the beneficial effects of some long-chain fatty acids for human health. Among the others, the conjugated linoleic acids (CLAs), a group of positional and geometric fatty acid isomers of linoleic acid, whose major isomer (up to 80% of total CLA) is cis-9, trans-11C18:2 or rumenic acid (RU), are considered to have immunomodulating, anti-carcinogenic and anti-artheriosclerosis properties [2
]. In milk, CLAs come from the activity of rumen bacteria on dietary unsaturated fatty acids but also from the action of mammary gland stearoyl-CoA desaturase (SCD) on trans-11 C18:1 (TVA, trans vaccenic acid), an intermediate product, obtained during polyunsaturated fatty acids biohydrogenation. SCD also acts on the biosynthesis of monounsaturated fatty acids by a cis double bond between carbons 9 and 10 in several saturated fatty acids (SFA), in particular, myristic (C14:0), palmitic (C16:0) and stearic (C18:0). Micro-RNAs (miRNAs) are a class of small non-coding RNAs (microRNAs), around 22 nucleotides in length, which are contained in exosomes, extracellular membranous organelles with pleiotropic biological functions [4
]. Exosomes are formed by the inward budding of multivesicular bodies (MVBs) and are released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane. They are contained in body fluids such as saliva, urine, plasma and milk, and they mediate the delivery of miRNAs to target cells. The double-layer lipid membrane makes functional and exceptionally stable exosomes both in human and cow milk. Indeed, milk-derived miRNAs are resistant to high temperature, low pH, multiple freeze-thaw cycles and RNase treatment [5
]. The remarkable stability of the endogenous milk-derived miRNAs implies that infants can intake miRNAs along the lactation according to Gu et al. [6
]. The effects of such RNA molecules on health of young and adult milk consumers represent a relevant aspect to explore. Bovine miRNAs are produced by mammary cells and are involved in milk fat metabolism along the lactation [7
]; according to Lin et al. [8
] the presence of some miRNAs, particularly 103 and 27a, in goats’ mammary cells affects the expression of the SCD
gene. Several factors influence the expression of genes involved in fat metabolism; within them, diet amino acids, lipids and vitamins modulate the miRNA transcriptome of mammary cells in goat milk [10
]. In addition, some studies indicated that dietary polyunsaturated fatty acids (PUFAs) might affect the miRNA profile in human [11
] and rats [12
]. On this basis, the aim of this trial was to study the influence of pasture, which is known to be rich in PUFAs, particularly α-linolenic and linoleic acids, on both the SCD
gene and miRNA 103 expression in goat milk. To date no data are available concerning the possible effects of pasture on miRNA expression in milk.
No refusals were found. Body weights (BW) were not different between the groups along the trial.
Milk yield was unaffected by dietary treatment while group G showed significantly higher fat (4.10% vs. 2.94%; p
< 0.01) and protein percentage (3.43% vs. 3.25%; p
< 0.01) than group S (Table 2
). Concerning milk fat (Figure 1
), the differences between groups were more evident in May and June; in July both groups showed a decrease of values, which was more severe in group G.
As depicted in Table 3
, group S showed significantly higher levels of SFA (74.86% vs. 71.42%; p
< 0.01) as well as of the C14:1 cis9/C14:0 ratio (0.018% vs. 0.009%; p
< 0.05), and lower values of monounsaturated fatty acids (MUFA, 21.76% vs. 24.39%; p
< 0.01). Total polyunsaturated fatty acids (PUFA, 3.38% vs. 4.19%; p
< 0.01), α-linolenic acid (C18:3, 1.35% vs. 0.80%; p
< 0.01) and total omega 3 (1.48% vs. 0.89%; p
< 0.01) were significantly higher in group G. In such group, total CLA were twice than in group S (0.646% vs. 0.311%; p
< 0.01) mainly because of the differences in cis9 trans 11 CLA (0.623% vs. 0.304%; p
Milk cis9 trans 11 CLA in grazing group was significantly (p
< 0.01) higher in August (Table 4
) according to the highest value of both linoleic and α-linolenic acids in the pasture (Table 5
The RNA yield extracted from 300 mL of milk was 4.05 ± 0.9 μg and 3.92 ± 0.6 μg for groups S and G, respectively. These values are considered sufficient for the following analysis [27
]. The purity of extracted RNA was measured by a UV spectrophotometer (A260/A280 ratio was 1.8; A260/A230 ratio was 2.1 for all the samples). The mean RNA Integrity Number (RIN) obtained for all extracted samples was 7.7 ± 0.5 and 7.9 ± 0.2, for groups S and G, respectively, thus higher than 6, the threshold to define the quality of RNA [28
The results of the PCR real time analysis showed that SCD
expression (AU: Arbitrary unit) was higher, although not significant, in grazing animals either as mean value (AU: 0.703 vs. 0.589) or at each sampling (Figure 2
). In this group, SCD
expression decreased from April to June, increased in July and decreased again in August, while it was almost unvaried along the trial in group S.
The mean yield of miRNA 103 extracted from 200 µL of milk was μg 19.2 ± 1.4 and 18.6 ± 1.1, for group S and G, respectively. The expression of miRNA 103 was higher, although not significant, in group G (AU: 0.417 vs. 0.390) with a similar trend for both groups: Decreasing from April to June, increasing in July and falling down in August (Figure 3
Milk yield was not different between groups while grazing goats showed significantly higher levels of milk fat. This was probably due to the higher intake of structural carbohydrates; indeed, pasture neutral detergent fiber (NDF) was higher than that of alfalfa hay (see Table 1
). The structural carbohydrates are fermented by the rumen cellulolytic bacteria with production of acetic acid, precursor of short and a large part of the medium chain milk fatty acids. Milk fat was similar between groups in July, when grazing group showed the lowest value, which, according to Loewenstein et al. [28
] could be due to a depression of pasture quality by the high temperatures.
Anyway, energy requirements were satisfied for both groups. As reported by Rubino [29
] the medium pasture ingestion of the local genotype goats is equal to 20 g DM/kg BW while energy needing for maintenance and milk synthesis are 0.0365 UFL/kg metabolic weight (MW = BW0.75
) and 0.41 UFL/kg fat-corrected milk (4% fat), respectively. In the present trial, the goats from the grazing group weighed 50 kg BW and the intake was 1 kg DM of pasture, equal to 0.76 UFL (see Table 1
). In addition, they yielded 1.793 kg milk with 4.1% fat thus their energy requirement was 1.42 UFL (0.69 UFL maintenance, plus 0.73 UFL milk synthesis). Energy deficiency was covered by the concentrate (1.03 UFL/kg DM). Similarly, group S met energy requirements by intake of 1.1 kg DM of alfalfa hay (0.75 UFL/kg DM) plus the concentrate.
Concerning milk fatty acid profile, α-linolenic acid was significantly higher in group G, probably due to the higher level of this acid in the pasture compared to the alfalfa hay (42.6% vs. 38.8%, see Table 1
). This result agrees with that reported by Shroeder et al. [30
] who found a significant increase of α-linolenic acid in milk of grazing cows compared to cows fed total mixed ration (g/100 g 0.57 vs. 0.07).
Milk CLA was significantly higher in the grazing group, according to the results reported by Dhiman et al. [31
] in cows, Nudda et al. [32
] in sheep and goats, D’Urso et al. [14
], Tudisco et al. [15
] and Zicarelli et al. [34
] in goats. Bergamo et al. [35
] and Secchiari et al. [36
] found higher levels of α-linolenic acid in milk from buffaloes housed in a stable but fed fresh forage rather than a total mixed ration. In contrast, Jahreis et al. [37
] did not observe differences in milk CLA between grazing and stabled goats.
Noteworthy, milk CLA in grazing group was significantly (p
< 0.01) higher in August (see Table 4
) according to the highest value of both linoleic and α-linolenic acids in the pasture (see Table 5
), as reported by other authors [38
On the contrary, pasture linoleic and α-linolenic acids did not similarly influence SCD
expression. Indeed, in the grazing group, SCD
expression decreased from April to June, increased in July and decreased again in August (see Figure 2
), showing an opposite trend to the pasture linoleic acid, while α-linolenic acid did not change along the experiment, except for an increase in August. These results could demonstrate a higher influence of omega 6 PUFA than omega 3 PUFA on the SCD
activity is measured comparing the ratio product/substrate of some fatty acids. As described by Lock and Garnsworthy [24
], the best marker of SCD
activity is the c9C14:1/C14:0 ratio since all the C14:0 in milk fat comes from the synthesis in the mammary gland; as a consequence, desaturation is the only source of C14:1. In the present trial, the values of SCD
activity showed an opposite trend compared to that of the expression of the gene encoding for the same enzyme. Accordingly, the supplementation of sunflower seed oil [41
] and linseed oil [42
] to maize silage-based diets for goats did not influence SCD
expression and activity, a similar supplement to a grass hay-based diet only reduced the SCD
]. Bernard et al. [44
] observed a similar phenomenon supplementing soya beans to lucerne hay-based diets. Bernard et al. [45
] underlined the importance of the interaction among ingredients of diet taking into account that rumen-bypass PUFA or bio-hydrogenation intermediates can inhibit SCD
activity via transcriptional or post-transcriptional mechanisms.
The trend of miRNA 103 expression was similar between the groups, thus no effect of dietary treatment was revealed. Indeed, in both groups, the miRNA 103 decreased from April to June, increased in July and decreased again in August. The miRNA expression was affected only by the stage of lactation according to Avril-Sassen et al. [46
], Chen et al., [47
] and Wang et al. [48
] who found a different expression of miRNAs in the mammary gland along the lactation in mouse and cow and mainly to Dong et al. [49
] who reported the lowest expression level of miRNAs at the peak of lactation in goats.
In addition miRNA and SCD gene expression showed similar trends as reported also by Lin et al. [8
] who found a high correlation between the overexpression of miRNA103 in mammary cells and the increased expression of genes involved in fat synthesis with accumulation of triglycerides and of a part of unsaturated fatty acids in milk. Indeed, fat synthesis in the mammary epithelial cells recognizes several metabolic processes: An initial neo-synthesis and a subsequent desaturation by SCD
with conversion to triglycerides [50
]. Importantly, further larger scale studies are in due course to confirm these preliminary results. In particular, to obtain a number of replicates that should allow a statistical analysis based on groups and to assess other aspects such as the relation between somatic cell count and miRNA expression as shown by Mura et al. [52
]. It would be also interesting to investigate, in subsequent studies, how different types of pasture, characterized by different forage essences, can influence milk characteristics. Indeed, to our knowledge this is the first observation of the effects of pasture on miRNA expression in milk from ruminant species.