The observed low average milk fat content is consistent with that reported in the literature for donkey’s milk[24, 25] and lower than that reported for mares, cows and humans.
The fat content was affected by the stage of lactation, showing a decreasing trend, which is in agreement with the results observed for the Ragusana breed[10, 28], Litoral-Dinaric donkeys, mares[9, 29, 30] and cows.
The low fat content of milk and its variations during the lactation period corresponded to a low energetic value with the same trend during lactation. The gross energy content of the milk was similar to that recorded by Salimei et al.. Compared with the energetic value of milk from different species, that of donkey milk is slightly lower than that of mare milk[29, 32] and lower than that of cow and human milk. This finding could be a limiting factor in its use in infant nutrition within a diet exclusively based on milk. Thus, it has been suggested that an appropriate modification in the composition of donkey’s milk be made for infant feeding by adding medium-chain triglycerides or sunflower oil[34, 35]. However, the low fat content and energetic value of this milk suggests its potential use in hypocaloric human diets.
The decreasing energy content of donkey’s milk during lactation agrees with the trend observed in mares[29, 32], although Salimei et al. did not observe any considerable variation in fat content or energy value during lactation, which is likely due to different experimental conditions.
The SFAs were the most representative fatty acids, in agreement with the results of other studies[36, 37] and comparable to the composition of mare and human milk; however, its content were observed to be lower than those observed in ruminant milk. The short-chain fatty acids and medium-chain fatty acids contents as in mares, were observed to be lower than those observed in cow’s milk and higher than those observed in human milk[26, 39]. The high fatty acid content < C16 content observed in this study, with respect to other monogastric species, agrees with the results from equine studies and could be attributed to their synthesis from acetate and 3-hydroxybutyrate, as observed in ruminants, and not from glucose as occurs in monogastrics[9, 40]. Furthermore, instead of ruminants, fatty acid de novo synthesis in equines includes parts of C18 next to C4 and C14 and parts of C16 fatty acids.
Among the observed SFA, butyric (C4:0) and caproic (C6:0) acids were observed in concentrations less than 1%, as has been observed in mares. The average amounts of caprylic (C8:0), capric (C10:0) and lauric (C12:0) acids were lower than those reported for Ragusana jennies (12.80%, 18.65%, and 10.67%, respectively;); this finding is likely related to differences in breed and/or body conditions. The caprylic (C8:0) acid content was higher compared with that reported for mares (3.1%), cows (1.3%) and human milk (traces). Among the observed SFAs, palmitic acid (C16:0) was observed at the highest concentration, in agreement with the results reported in other related studies[28, 37] and in studies on mares; however, the content was less than that of cow and human milk[26, 39].
During the early lactation period (15–30days), the milk was observed to contain more C4:0 to C14:0 than during the middle (day 120) and late (day 210) lactation periods, most likely due to an increased synthesis of C4 to C14 fatty acids in the mammary gland during the early lactation period, as has been observed in cows.
Long-chain fatty acids such as stearic acid (C18:0) were observed in modest amounts, consistent with the results reported for other breeds[37, 41] as well as for mares (1.55%)[9, 43], while it has been recorded at higher levels in cow and human milk (7-13%)[26, 27, 37]. As in equines, the low stearic acid content could be attributed to a low stearic acid content in the animals’ diets and to a high activity of Δ9-desaturase on the conversion of stearic acid (C18:0) into oleic acid (C18:1), which is further enhanced by a high palmitoleic acid (C16:1) content, as was observed in this study.
The SFA content decreased during lactation. A higher decrease was observed for lauric (C12:0, -59%) and myristic (C14:0, -54.5%) acids and a lower decrease for palmitic acid (C16:0, -17%), likely due to a decrease in the concentration of the precursors of de novo synthesis. A decrease in the short- and medium-chain fatty acid contents during lactation has also been reported in mares[9, 44], while small changes in the SFA content have been observed in human milk; additionally, increases in these fatty acid contents have been observed in cow’s milk.
From a nutritional standpoint, it has been reported that C18 SFA has a neutral health effect, while C4 to C10 SFAs have positive effects and C12 to C16 SFAs have negative health effects. In particular, SFAs C14 - C16 are considered dangerous because they are associated with high serum LDL-cholesterol concentrations in human subjects. Medium-chain fatty acids are characterised by better absorption than long-chain fatty acids. Furthermore, it has been suggested that equids’ milk is more digestible than cow’s milk, according to experiments performed on rats, and allows for faster evacuation of the stomach.
The UFA content, in agreement with other studies (50.69%), was similar to that of mare’s and human milk but higher than that reported for ruminant’s milk (23-32%)[27, 41].
The UFA/SFA ratio is comparable to mare’s milk and higher than that recorded in the Ragusana donkey (0.48) and that reported for ruminant’s milk (0.26-0.41)[41, 47].
The MUFA content and oleic (C18:1 n-9) and palmitoleic (C16:1 n-7) acid contents were higher than those observed in donkey’s milk by other researchers[11, 41], likely due to different experimental conditions; these contents were observed to be similar to those reported for mares but lower than those in human milk (C18:1, 46%).
The MUFA content increased during the lactation period, whereas Chiofalo et al. did not find significant variability throughout lactation in the Ragusana breed, most likely due to different experimental conditions.
In this study, the amount of desaturation, calculated from the ratio of Δ9-desaturase product/sum of Δ9-desaturase product and substrate, was relatively high for C18:1 and C16:1 and increased as lactation progressed, reaching higher values at 150 to 210 days of lactation. This result may explain the observed high value of these MUFAs in milk and their increased trend during lactation. Moreover, it has been reported that preformed fatty acids, such as oleic acid, could be derived from mobilisation from adipose tissue or from absorption from the digestive tract, with an increase being observed for a forage diet[26, 44], which likely occurred in this study for the asses fed on pasture.
It has been reported that in the human diet the high relative percentage of MUFA has beneficial effects with respect to arterial disease, i.e., it lowers plasma LDL cholesterol and total cholesterol as well as the fibrinolytic activity of circulating plasma by modifying vascular endothelial physiology, while the intake of SFA is a risk factor in contracting coronary heart disease.
Among PUFAs and EFAs, LA (C18:2 n-6) and ALA (C18:3 n-3) acid were the most representative of PUFA n-6 and PUFA n-3, respectively, exhibiting higher values than those of the Ragusana breed and comparable to the range recorded in mares but higher than that reported in ruminant’s milk. Furthermore, the n-3/n-6 fatty acid ratio was higher than that recorded in Ragusana asses (0.19) and higher than that reported in humans (0.012) or in cows (0.28). As in equines, according to other researchers[19, 30], the reasons for this difference could be related to the amounts of these acids in the animals’ diets and the absence of biohydrogenation of fatty acids in the digestive tract before absorption, unlike what occurs in ruminants.
In humans, LA has been reported to play a role in the prevention of gastric ulcers. It is a precursor of prostaglandin E that increases with stimulation by linoleic acid. Prostaglandins are involved in the prevention of gastric ulcers through a rise in mucosal protective factors; this prevention is important in foals, which have a certain incidence of gastric ulcers.
In agreement with the results of Gastaldi et al., the percentage of ALA was higher compared with that of human’s (1.14%) and cow’s milk (0.48%) whereas the LA content was similar to that reported for human milk.
The ALA content increased during the lactation period, consistent with the results observed for mares, and the maximum level was achieved late in the lactation period. This result reflects the higher PUFA n-3/n-6 ratio at the 7th month of lactation (day 210). This higher ratio may be explained by the increase in pasture feeding by jennies on richer grass during the late lactation period. Grass is rich in ALA content, and no biohydrogenation occurs before absorption. Furthermore, higher ALA contents have been reported in milk from asses and mares fed on herbage rather than hay.
AI and TI, which indicate the healthfulness of milk with respect to fatty acid content and their potential to prevent or cause atherosclerosis and thrombosis, were observed to be present in lower quantities than in cow’s milk (2.51 and 1.86, respectively).
It should be noted that LA is the precursor of PUFA n-6, and AA (C20:4 n-6) and ALA are the precursors of PUFA n-3, EPA (C20:5 n-3) and DHA (C22:6 n-3) acids. AA and EPA/DHA are substrates for the formation of eicosanoids. Prostaglandins and leukotrienes are important in controlling several cell activities, and DHA is important in the development of the central nervous system. The role of PUFA is important in the development of the neonatal brain as well as in the development of the retina and cognitive functions[62–64]. The high content of n-3 fatty acids in donkey milk could have a significant effect on the development of the neural system, vision and infant growth.
Among the minor PUFA observed in this study, small amounts of EPA, DHA and AA were observed, consistent with other studies on donkey’s milk[11, 37, 41] and mare’s milk. The percentage of EPA was higher compared with human milk (0.27%); in contrast, the DHA and AA contents were lower (0.40 and 0.59%). The reasons for this result are likely due to the different elongation and desaturation processes in mammary glands. A higher AA/EPA ratio in breast milk from the mothers of atopic infants compared with non-atopic infants and the relationship between lower levels of EPA and the early development of atopic disease have been reported. The low AA/EPA ratio observed in this study (0.18), with respect to those reported for human’s and cow’s milk (29.5 and 3.67), suggests that donkey milk could be used in childhood nutrition to prevent the risk of developing atopical diseases. Moreover, donkey’s milk could be useful in pre-term infants’ diets because premature infants seem able to form AA from linoleic acid and EPA and DHA from α-linolenic acid[66, 67]. The positive effects of donkey’s milk in infant nutrition has been demonstrated in the treatment of multiple food intolerances[34, 68, 69] and in selected cases of cow’s milk allergies[7, 70].
The high recorded amounts of PUFAs in donkey milk suggest that this milk may also be used adult human diets. From a nutritional point of view, it has been reported that the rapid changes in the diet of the Western industrialised society, mainly over the last 150 years with respect to the ancestor-constituted genetic profile, involve a particular increase in saturated fats and n-6 fatty acids and a decrease in n-3 fatty acids[71–74]. The levels of long-chain n-6 to n-3 (mainly LA compared with ALA) fatty acids are important, particularly n-3 fatty acids, in maintaining cardiovascular health[76, 77] and they influence the ratios of ensuing eicosanoids and metabolic functions. The dietary balance of the ratios of n-6 to n-3 PUFA affects the regulation of metabolic functions and the development of metabolic syndrome, including lipid profile and adiposity, insulin sensitivity and inflammation[79, 80]. It has been estimated that the ratio of n-6 to n-3 fatty acids in the Western diet is 15-20/1, and more n-3 than n-6 fatty acids have been suggested, with a ratio that tends towards 1:1.
In this study, the PUFA n-6 to n-3 ratio and the LA/ALA ratio, in particular, were approximately 2:1, with a lower value (< 1) during the 7th month of lactation due to a high increase in the ALA content, potentially suggesting the more optimal use of milk collected during this period.
In addition to the benefits related to coronary health disease, the beneficial health effects of n-3 fatty acids include those related to inflammatory disease, such as rheumatoid arthritis, dermatitis[83, 84], cancer, depression and dementia, and may be potentially used to treat late-onset Alzheimer’s disease[86, 87].
Long-chain PUFA n-3 has been shown to induce immunomodulatory activity on natural and acquired immunity[88, 89] through the synthesis of lipid mediators (pros-taglandins, leukotrienes), peptide mediators (cytokines), reactive oxygen species (superoxide), and enzymes. The metabolites of n-3 fatty acids are less inflammatory than those of n-6 (in particular, AA). The eicosanoid derived from AA, i.e, prostaglandin E2 and leukotriene B4, promote atopic inflammation; in contrast, PUFA n-3 and their derived eicosanoid influence AA metabolism, promoting anti-inflammatory properties[90, 91]. The high PUFA n-3 content in donkey’s milk could have a functional effect on the immunological system. In our study on an in vitro model of human peripheral blood mononuclear cells[6, 92], donkey milk exhibited the ability to induce IgG secretion and the release of interleukins (IL-12, IL-1β, IL-10) and TNF-α - important to the immunotreatment of immune-related disease - and a high release of nitric oxide (NO), a potent promoter in the prevention of atherosclerosis. Indeed, NO is a strong vasodilator of terminal vessels, improves blood flow, and is an effective antimicrobial agent in the development of atherosclerosis. In combination, these results support the concept that donkey’s milk, likely due to its high PUFA n-3 content, can prevent atherosclerosis via the production of NO and, at moderate intake level (200 mL/d;), can up-regulate the immune response in elderly hosts.