Pomegranate is one of the most ancient cultivated trees traditionally used in folk medicine in the Middle East. Scientific investigations have provided evidence that the health properties of pomegranate mainly depend on the phytochemicals present in both the edible and non-edible portions of the fruit [1
]. The juice contains a valuable quantity of anthocyanins, flavonoids, and hydroxyl benzoic acids [2
] while the peel is rich in hydrolysable tannins, among which punicalagin is the dominant and peculiar compound [3
]. Also, pomegranate seeds are a source of polyunsaturated fatty acids (PUFA), among which punicic acid (PA; cis
-13 18:3) has been proven to be beneficial for human health [4
Pomegranate pulp is one of the by-products produced during pomegranate juice extraction and consists of variable proportions of peel, seeds, and residual pulp. The content of fiber, crude protein, and fat makes this by-product a suitable feed to be included at a high percentage in the diet of ruminants. Therefore, the recycling of pomegranate pulp as an ingredient of ruminant feeds could contribute to the reduction in the feed-to-food competition of animal production by replacing potentially human-edible feeds such as cereals and, at the same time, could represent a viable strategy to reduce the disposal costs for the industry. In addition, the residual bioactive compounds could improve rumen protein metabolism or impair the biohydrogenation of dietary PUFA [5
]. Also, most of these compounds are potent free radical scavengers that could favorably affect the antioxidant status of the animal [6
] and some of them can be absorbed and accumulated in milk and meat, improving their nutritive value and shelf life [5
]. These properties might be of particular interest in some areas, such as the Mediterranean, to attenuate the impact of the seasonal availability of good quality pasture on the yield and composition of sheep milk, or during late lactation when milk yield and quality physiologically degrade.
Agro-industrial by-products of pomegranate have been investigated to evaluate the effect on ruminal fermentation, performance, and product quality in large [7
] and small ruminants [8
]. However, recent research has only focused on the effect of pomegranate by-products obtained from single parts of the fruit. In contrast, the whole pomegranate pulp, where all the above-described phytochemicals are present at once, has been scarcely investigated. Therefore, only the effects of feeding PP silage has been investigated in cow milk and lamb meat production [10
]. In addition, to the best of our knowledge, pomegranate pulp has not been previously tested on dairy ewes. In the light of above, the aim of this study was to assess the effect of dietary pomegranate pulp on the yield and quality of milk from grazing ewes in advanced stage of lactation.
reports data on milk production, chemical composition, and protein profile of the milk. Dietary treatment did not affect milk yield, percentage of fat, protein and lactose, and the concentration of urea. Somatic cell count, expressed as linear score, was greater in the PP than the CTRL group (p
= 0.031). With respect to the milk protein profile, αs1
-CN) and β
-CN) were the most abundant proteins in the milk for both of the groups, representing more than 30% each, followed by β
-CN), and α
-Lac). The percentage of α
s1-CN tended to be greater in the PP than in the CTRL (p
= 0.056), while β
-CN and total casein were greater in the CTRL than the PP group (p
= 0.048 and p
= 0.002, respectively).
reports the effect of dietary treatment on milk fatty acid composition. The percentages of 14:0, 16:0, cis
-9 14:1, and cis
-9 16:1 were greater in the CTRL than in the PP milk. In contrast, the percentages of trans
-9 18:1, trans
-11 18:1 (VA), cis
-6 18:1, cis
-11 18:2 (RA), and cis
-15 18:3 (ALA) were greater in the PP than the CTRL group. Punicic acid was detected only in the milk of PP ewes, representing 0.19% of total milk fatty acids. Regarding the sums of fatty acids, the CTRL milk showed a greater percentage of SFA and a lower proportion of PUFA. Moreover, the CTRL milk had a lower percentage of total PUFA n-3. The dietary treatment did not affect the sum of MUFA, odd and branched chain fatty acids (OBCFA), and PUFA n-6.
reports the effect of dietary treatment on the milk antioxidant capacity. The results of the ORAC assay showed that the H-ORAC accounted for most of the TAC in milk as compared with the L-ORAC. Dietary treatment affected the TAC, with higher values found in the CTRL group as compared with the PP (p
= 0.006). These differences in the TAC values were due to the effect of the dietary treatment on the antioxidant capacity measured in the H-ORAC, which were higher for the CTRL group as compared with the PP group (p
= 0.002). Conversely, no difference between treatments was observed for L-ORAC.
In the present study, 648 g/kg DM dried pomegranate pulp was included in a pelleted feed given to grazing ewes in order to evaluate the effect of dietary pomegranate by-product on milk yield and quality traits during late lactation. According to Cannas et al. [12
], it was estimated that that the ingestion of the individual ewe was about 2.3 kg, and therefore the pomegranate pulp represented about 7% of total dry matter intake (DMI). The chemical composition of PP used in the present paper was in the range reported for similar pomegranate by-products [10
]. The crude fat of PP mostly arises from the seed oil, and therefore, it is not surprising that PA was the most abundant fatty acid in the fat of PP. The total phenolic compounds of pomegranate pulp were represented mainly by tannins, which is consistent with previous study [27
Tannins are water-soluble phenols characterized by their potential to create complexes with the proteins and, to a lesser extent, with metals and ions [28
]. Due to this chemical property, dietary tannins can have both adverse and beneficial effects on ruminants, depending on factors such as dose, chemical composition, and characteristics of the basal diet [29
]. Regarding dairy ruminants, tannins can increase milk protein percentage and reduce urea content [30
] by favoring the outflow of protein nitrogen from the rumen [31
], which is one of the limiting factors for milk production.
Controversial results are reported on the effect of dietary pomegranate by-products on milk yield and composition. Kotsampasi et al. [10
] did not observe a variation of DMI, milk yield, and gross composition when 75 or 150 g/kg of pomegranate silage were included in the diet of lactating cows. A similar result was observed by Modaresi et al. [8
] in lactating goats with the inclusion of 60 or 120 g/kg of pomegranate seed pulp. In contrast, Shaani et al. [7
] found that when 8% of ensiled PP replaced corn grain in a total mixed ration it reduced DMI and milk yield in cows, while lambs refused to consume a TMR containing more than 20% of pomegranate by-product [26
]. In the present trial, PP concentrate was completely consumed, despite the fact that the level of PP inclusion was greater than values reported in the literature and the tannins content was 61 g/kg DM. Moreover, no effects on milk yield, fat and protein percentage, and urea content were observed.
This inconsistency among the studies could be due to several factors. In their review, Jeronimo et al. [32
] stated that the species used in the different trials may show a variable sensitivity to the astringent sensation induced by the type and quantity of tannins in the feeds. Min et al. [33
] indicated that a dose of dietary tannins greater than 50 g/kg DM can reduce the voluntary feed intake and animal performance, however, this limit should be applied to the total ingested diet and not to a specific component. Finally, the potential of tannins to improve animal performance seems to depend also on the physiological status of the animals. Waghorn [34
] reported that the surplus of protein escaping the rumen, generated by the protective action of tannins, is better exploited by ewes which have greater protein requirements as compared with less productive animals. Interestingly, we observed that the dietary treatment affected individual milk proteins percentage. In particular, β
-CN and total casein were higher in the CTRL group as compared with the PP group.
The availability of essential amino acids (EAA) represents the most limiting factor for the endogenous biosynthesis of milk protein in the mammary gland. According to Orlandi et al. [35
], feeding tannins to ruminants increased the flux of EAA through the abomasum and to the duodenum [35
] with positive effects on milk protein yield. Surprisingly, our results contradict these observations. This is the first study reporting the effect of dietary pomegranate by-products on milk protein profile, and the lack of knowledge on the effect of dietary polyphenols on individual milk proteins does not allow a comparison with the literature. However, in a similar study, dietary chestnut hydrolysable tannins increased protein yield and casein index without affecting individual caseins in the milk from ewes during mid-lactation [30
]. A possible explanation for the lower percentage of β
-CN in the PP group could be the greater somatic cell count, expressed as LS, observed in PP milk. It is known that LS is positively correlated with the activity of plasmin, a protease that degrades β
-CN into γ-CN. Although the effect of plasmin is evident at values of LS greater than those recorded for both the groups in the present study, it cannot be excluded as a possible cause of the lower β
-CN and total casein in PP milk.
The antioxidant capacity of fresh pomegranate has been associated with the presence of tannins, anthocyanins, and other phenolic compounds in the different portions of the fruit [2
]. The potential of polyphenols to act as dietary antioxidants with the aim of improving the antioxidant properties of ruminant products has been investigated [5
], but it remains a controversial issue. In the present study, the ORAC test was used to evaluate the free radical-scavenging capacity of milk and it revealed that TAC was greater in the CTRL than in the PP milk. Specifically, this difference was due to a greater H-ORAC of CTRL milk, while the L-ORAC was comparable between the groups.
Both direct and indirect antioxidant mechanisms of phenolic compounds in vivo have been suggested. A direct effect is linked to the possibility that these compounds are absorbed in the gut in order to reach the tissues. It has been reported that phenols with a low molecular weight can be absorbed and transferred to the blood stream [5
]. Pomegranate phenols are mainly represented by hydrolysable tannins [3
], which, differently from the condensed tannins, can be degraded by rumen microbes. Therefore, it is likely that small products of degradation of pomegranate tannins can be absorbed. Consistently, Kotsampasi et al. [11
] showed that the total phenols in lamb meat were linearly correlated to the level of pomegranate silage inclusion in the diet of growing lambs. However, in the same paper, though the meat antioxidant capacity was greater in the groups receiving the pomegranate silage, it was not linearly correlated to the presence of phenols in meat, suggesting that the accumulation of antioxidant molecules is not the only mechanism responsible for the antioxidant capacity of the animal products.
Other authors found that the improvement in antioxidant capacity of different tissues was not related to a direct transfer of phenolic compounds from the diet [36
], suggesting that the antioxidant activity of dietary phenolic compounds could be indirectly mediated by an effect on the overall oxidative status of the animal organism. The balance between pro-oxidant and antioxidant components is an important factor affecting the antioxidant properties of animal products. In particular, the oxidation of susceptible compounds, such as PUFA, is prevented by endogenous defense systems and antioxidants of direct and indirect derivation from the diet. Zulueta et al. [24
] reported that the major peroxyl radical scavengers in milk are the caseins, among which β
-CN is the most prominent [37
]. On the one hand, this could partially explain the higher H-ORAC values found in CTRL milk, which had a greater percentage of β
-CN and total casein as compared with PP. On the other hand, PP milk had a higher percentage of both total PUFA and PUFA n-3. Therefore, it may also be supposed that, as compared with CTRL, anti-oxidants could have been consumed more in the PP milk, in order to counteract the higher susceptibility of PUFA to oxidation.
With respect to milk fatty acids, PP milk had a greater percentage of total PUFA and PUFA n-3 and a lower proportion of 14:0, 16:0, and total SFA. Similar results were reported when pomegranate seeds oil [9
] or seed pulp [8
] were added to the diet of goats. The reduction of total and individual SFA in milk and meat is a common result when dietary sources of PUFA are included in the diet of ruminants [38
]. A high quantity of 14:0 and 16:0 in foods is considered harmful for human health because they increase the atherogenic and the thrombogenic index. Therefore, the inclusion of PP in ruminant diet could be a valid strategy to improve the healthiness of milk by reducing these undesirable FA in favor of healthy FA such as PUFA and PUFA n-3.
In addition, PA was detected only in the PP milk. Punicic acid is a double-conjugated trienoic fatty acid (cis
-13 18:3) that represents the main fatty acid in the oil of pomegranate seeds [39
]. Its therapeutic and preventive properties against obesity, diabetes, and cardiovascular disease have been recently reviewed [4
]. In the present study, PA accounted for 60% of the total fatty acids in the PP concentrate, while it represented only 0.2% of milk fatty acids. Considering that PA in milk exclusively arises from the ingested diet, it is likely that PA was extensively biohydrogenated in the rumen of PP ewes. The ruminal biohydrogenation pathway of PA has not yet been reported. However, it is plausible that, similar to the biohydrogenation of linoleic acid (LA) and ALA [38
], VA and RA are intermediates of PA biohydrogenation. In line with this hypothesis, the proportion of VA and RA was higher in the PP milk.
There are many reports that tannins can protect dietary PUFA from the biohydrogenation [5
]. On the one hand, our results could lead to the conclusion that the protective effect of tannins on the dietary punicic acid occurred minimally. On the other hand, looking at the fatty acid composition of the experimental concentrates, an effect of tannins on the biohydrogenation cannot be excluded. The CTRL concentrate was richer in LA and ALA as compared with PP, and therefore a greater percentage of these two fatty acids would have been expected in the CTRL milk. However, ALA was greater in PP milk, while LA was comparable between the groups. According to Chilliard et al. [38
], LA and ALA represent the elective substrates for biohydrogenation. Thus, it could be supposed that the biohydrogenation of these compounds occurred normally in the rumen of CTRL ewes, while it was reduced in the PP group.