Analysis of the Fatty Acid Profile of the Tissues of Hunted Mallard Ducks (Anas platyrhynchos L.) from Poland

Simple Summary The contemporary human diet contains many times more saturated fatty acids than that of our Palaeolithic ancestors, and the ratio of n-6 to n-3 polyunsaturated fatty acids (PUFAs) is nearly 20 times higher. Lifestyle changes and an unsuitable diet have led to the rapid development of civilization diseases and sudden deaths. This is clearly evident to contemporary consumers, who look for foods produced with respect for the natural environment, which also have an original flavor and health-promoting properties, such as game meat. Abstract The aim of the study was to analyse the fatty acid profile of selected tissues of mallard ducks (Anas platyrhynchos L.), in relation to where they were obtained and their sex, with regard to the human diet. The study was carried out on material obtained from mallard ducks from two study areas: the Siedlce hunting district and the Leszno hunting district. The research material was the breast and leg muscles of 28 mallards. The samples were frozen and stored at −20 °C. The fatty acid profiles in the biological samples were determined by selected ion recording (SIR). The results showed significantly (p < 0.05) lower average levels of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) and significantly higher (p < 0.05) average levels of polyunsaturated fatty acids (PUFAs), n-6 PUFAs, and n-3 PUFAs in the breast muscles of the mallards obtained in the Siedlce hunting district. This in conjunction with the higher (p < 0.05) hypocholesterolaemic/hypercholesterolaemic index (h/H) in the leg muscles and lower (p < 0.05) atherogenic and thrombogenic indices (AI and TI) in the leg and breast muscles of mallards in the Siedlce hunting district indicate the higher health-promoting value of the meat of ducks from this region. The average n-6/n-3 PUFA ratio in the breast muscles was significantly (p < 0.05) higher in mallards obtained in the Leszno hunting district. Males of the species had a significantly (p < 0.05) higher average n-6/n-3 PUFA ratio in the breast muscles than females. The PUFA/SFA ratio was significantly (p < 0.05) higher in the leg muscles of the female mallards than in the males.


Introduction
The mallard duck (Anas platyrhynchos L.) is a game species hunted in Poland for its lean, tasty meat. Its nutritional, culinary, and processing value derives from the chemical composition of the muscle tissue, connective tissue, and fat in the meat. The high nutritional value of mallard meat is linked to the ducks' wild origin and behaviour. The innate activity of these birds results in high content of haem pigments and a low-fat content. The carcass contains on average 73% muscle and 2.3% adipose tissue. Mallards have lower fat content and higher protein content than domesticated livestock [1]. According to the Regulation of the Minister of the Environment of 11 March 2005 on establishment of the list of game species [2], the hunting period for the species is from 15 August to 21 December. Hunters are authorized by law to manage game populations [3]. According to estimates [4][5][6], the average number of mallard ducks culled annually in recent years is 75,000. Therefore per

Animals and Sample Collection
The study was carried out on material from mallard ducks reared in two study areas: the Siedlce hunting district, located in the Masovian Voivodeship, and the Leszno hunting district in the Greater Poland Voivodeship (Figure 1). In both the Voivodeships, cereals (wheat, barley, and maize) are dominant in the crop structure [18]. Therefore, it is likely that grains of cereals made up a large percentage of the mallards' diet. The two regions were distinguished by different environmental resources and different degrees of intensification of agriculture. In the Greater Poland Voivodeship, there are many large farms with intensive crop production, including high levels of mineral fertilizers. Farms in the Masovian Voivodeship have a smaller area and are managed more sustainably, so their environmental impact is smaller [19].

Animals and Sample Collection
The study was carried out on material from mallard ducks reared in two study areas: the Siedlce hunting district, located in the Masovian Voivodeship, and the Leszno hunting district in the Greater Poland Voivodeship (Figure 1). In both the Voivodeships, cereals (wheat, barley, and maize) are dominant in the crop structure [18]. Therefore, it is likely that grains of cereals made up a large percentage of the mallards' diet. The two regions were distinguished by different environmental resources and different degrees of intensification of agriculture. In the Greater Poland Voivodeship, there are many large farms with intensive crop production, including high levels of mineral fertilizers. Farms in the Masovian Voivodeship have a smaller area and are managed more sustainably, so their environmental impact is smaller [19]. The research material was obtained by hunters who are authorized by law to manage populations of game animals [3]. Mallards were culled in accordance with the Annual Hunting Plan, which included harvesting of wild ducks for the hunting districts analysed in the study, drawn up for the period from 1 April to 31 March of the following year, i.e., for the 2018/2019 hunting season. Ducks were hunted by individuals and groups, always with hunting dogs trained for this type of hunting. This practice guaranteed that all birds shot down would be found, and thus was ethical according to the rules and regulations in force in the Polish Hunting Association. Each person hunting mallards had a permit issued by the head of the relevant Hunting Club, which contained information on the number of individuals the hunter may harvest. Ducks were hunted in three ways: evening blind hunting for birds flying in to feed, morning blind hunting for birds returning from feeding, and stalking during the day in the birds' preferred places, always in compliance with safety principles. Acquisition of mallards was limited to the first two months of the hunting period for the species, i.e., from 15 August to 15 October, before the birds had begun their migration. The carcasses obtained by the hunters were dissected. The research material was the breast and leg muscles of 28 mallards, including 6 females and 6 males The research material was obtained by hunters who are authorized by law to manage populations of game animals [3]. Mallards were culled in accordance with the Annual Hunting Plan, which included harvesting of wild ducks for the hunting districts analysed in the study, drawn up for the period from 1 April to 31 March of the following year, i.e., for the 2018/2019 hunting season. Ducks were hunted by individuals and groups, always with hunting dogs trained for this type of hunting. This practice guaranteed that all birds shot down would be found, and thus was ethical according to the rules and regulations in force in the Polish Hunting Association. Each person hunting mallards had a permit issued by the head of the relevant Hunting Club, which contained information on the number of individuals the hunter may harvest. Ducks were hunted in three ways: evening blind hunting for birds flying in to feed, morning blind hunting for birds returning from feeding, and stalking during the day in the birds' preferred places, always in compliance with safety principles. Acquisition of mallards was limited to the first two months of the hunting period for the species, i.e., from 15 August to 15 October, before the birds had begun their migration. The carcasses obtained by the hunters were dissected. The research material was the breast and leg muscles of 28 mallards, including 6 females and 6 males in the Siedlce hunting district and 8 females and 8 males in the Leszno hunting district. The meat samples were frozen and stored at −20 • C.
The composition of the mallards' diet was probably similar in both hunting districts. It consisted of both animal and plant material, including cereal grains, which could be seen in their stomachs during dissection. It may also have included aquatic plants, aquatic invertebrates, gastropods, and insect larvae. Variation in the composition of the diet of mallards between hunting districts may have been linked to the level of intensification of agriculture.

Statistics
Statistical analysis of the results was performed using Statistica 13.0 software. The numerical data were characterized by means of arithmetic mean (x), extreme values (min., max.), standard deviation (s), and coefficient of variation (V%). In addition, two-way non-orthogonal analysis of variance (Fisher-Snedecor F test) was performed. A detailed comparison of means was performed using the Tukey test for a significance level of p < 0.05. Correlations between features were determined using Pearson's correlation coefficient (r), and the significance of these relationships was verified for p < 0.05 and p < 0.01.
The sex of mallards was shown to influence the TI index; the males had a significantly higher TI (0.83) than the females (0.82). The differences for the other indices were statistically non-significant. The location where the mallards were obtained was shown to influence the fatty acid indices. The h/H index in the leg muscles of the birds from the Siedlce hunting district (3.45) was statistically significantly higher than in the Leszno hunting district (3.21); whereas the AI and TI indices were statistically significantly higher in birds from the Leszno hunting district (0.30 and 0.84, respectively) in comparison with the Siedlce hunting district (0.29 and 0.80, respectively).

Discussion
Differences in the diet of the mallards from the two hunting districts may have significantly influenced the fatty acid profile of the muscles of these birds. The nutritional value of the ducks' diet in a given region may have been affected by differences in environmental resources and in the degree of impact of agricultural production on the environment. Many authors have reported a positive effect of the inclusion of various cereal species in the diet on the fatty acid profile of various animal species [20][21][22][23].
If the proportion of insect larvae in the diet of mallards is too high, it may affect the fatty acid profile in their muscles. Daszkiewicz et al. [24] reported that ≥50% inclusion of full-fat black soldier fly (Hermetia illucens) larva meal as an alternative protein source in the diet of broiler chickens is too high, as it negatively affects the fatty acid profile of the meat. Gastropods in the diet of mallards may also affect the fatty acid profile of the muscles. Özogul et al. [25] and Milinsk et al. [26] showed a high proportion of unsaturated fatty acids in the total fat fraction of the meat of gastropods.
In all groups of mallards, the SFA level was higher in the breast muscles than in the leg muscles, which is confirmed by [27]. The SFA level in the breast muscles of mallards ranged from 30.05% to 33.59%. A higher average SFA level in the breast muscles of mallards than in the present study was obtained by [28], and a lower level by [27]. A higher average SFA level in the breast muscles of other poultry breeds and species than the level obtained in the present study was observed in broiler chickens and in Cherry Berry ducks [29,30], as well as in Pekin ducks (AP54, PP54, PP45 and Star H.Y.) [31], which given recommendations to reduce SFAs in the human diet may indicate that the meat of wild mallards is of higher nutritional value. The SFA level in the leg muscles of mallards ranged from 29.94% to 31.73%. A lower average concentration of SFAs in the leg muscles of other breeds and species has been observed by [32] in Pekin and Muscovy ducks and by [33] in Korean native ducks, whereas higher concentrations were reported by [31] in Pekin Star 53 H.Y., AP54, PP54, and PP45 ducks and by [34] in ring-necked pheasants. Wołoszyn et al. [35] showed a higher average level of SFAs in the leg muscles of Pekin P33 and A3 ducks and Miniduck K2 ducks than that observed in the present study in male mallards from both hunting districts. Kijanko et al. [36] reported a higher average SFA level in the liver of mallards (from 43.05 to 47.03%) than that observed in the present study in ducks obtained in both hunting districts.
Organs, including the liver, contain lower levels of MUFAs and higher levels of PUFAs than the skeletal muscles [37]. The breast muscles and legs of the groups of mallards had a higher level of MUFAs than of SFAs, which is beneficial for the consumer. A higher average level of MUFAs was shown in the leg muscles than in the breast muscles, which is confirmed by [27]. The MUFA level in the breast muscles of mallards ranged from 37.16% to 43.46%. A lower average MUFA level in the breast muscles of mallards (23.7%) was reported by [27]. A lower average MUFA level in the breast muscles of mallards than that recorded in the present study was also noted by [28,38]. Compared to the mallards from the Siedlce hunting district, a higher average MUFA level in the breast muscles was found by [39] in White Koluda geese, by [32] in Muscovy ducks, and by [38] in Greenlegged partridge and Rhode Island Red capons, turkeys, and chicken broilers. Ali et al. [29] reported a lower average MUFA level in the breast muscles of broiler chickens and Cherry Berry ducks than in the mallards from the Leszno hunting district. The MUFA concentration in the leg muscles of mallards ranged from 45.00% to 48.32%. A lower average MUFA level in the leg muscles compared to that obtained in the present study in mallards was observed in Pekin and Muscovy ducks [32], Pekin Star 53 H.Y., AP54, and PP54 ducks [31], Korean native ducks [33], and farmed ring-necked pheasants [34], which may indicate that the leg muscles of mallards have higher nutritional value.
The average level of PUFAs was higher in the breast muscles of mallards than in the leg muscles, which is consistent with results obtained by [27]. The PUFA concentration in the breast muscles of mallards ranged from 25.68% to 31.74%. [27,28,38] noted a higher average PUFA level in the breast muscles of mallards than in the present study. In other breeds and species of poultry [32,33,39,40], a lower PUFA level was shown in these muscles, which may have been due to differences in the birds' diet as well as to genotypic differences. The PUFA concentration in the leg muscles of mallards ranged from 20.43% to 23.91%. A higher average PUFA concentration in the leg muscles compared to the mallards from both hunting districts was observed in Pekin and Muscovy ducks [32] and in Korean native ducks [33], whereas a lower level was observed in White Koluda geese [39] and in ring-necked pheasants [34]. Another study showed a higher average level of PUFAs in the leg muscles of male ducks than in the male mallards in the present study [35].
In the present study, all groups of mallards had a higher average level of n-6 PUFAs in the breast muscles than in the leg muscles. The level of n-6 PUFAs in the breast muscles of mallards ranged from 22.359% to 27.685%, whereas in the leg muscles it ranged from 18.09% to 20.75%. A higher average level of n-6 PUFAs in the breast muscles of mallards of both sexes (33.06% and 32.97% in males and females, respectively) was reported by [28]. The present study showed a higher average level of n-6 PUFAs in the breast muscles of mallards obtained in the Siedlce and Leszno hunting districts than that observed in Pekin and Muscovy ducks [32]. Korean native ducks had a higher average level of n-6 PUFAs in the breast and leg muscles than that shown in mallards [33].
In the present study, all groups of mallards had a higher average level of n-3 PUFAs in the breast muscles than in the leg muscles. The n-3 PUFA concentration ranged from 1.88% to 3.56% in the breast muscles of mallards from 1.51% to 2.40% in the leg muscles. In both the present study and in [28], a higher average concentration of n-3 PUFAs was shown in the breast muscles of female mallards than in males. The average level of n-3 PUFAs shown in the breast muscles of male mallards in the present study was higher than that reported by [41] in all groups of birds tested. A higher share of n-3 PUFAs in the breast and thigh muscles of Pekin ducks than that shown in the present study in the mallards from both hunting districts was observed by [32].
The n-6/n-3 PUFA ratio in the breast muscles of mallards ranged from 7.76 to 12.47. For human health, the value of this ratio should be as low as possible. A lower average n-6/n-3 PUFA ratio in the breast muscles of male and female mallards (3.12% and 3.10%, respectively) than that observed in the mallards of both sexes in the present study was reported by [28], which may indicate a higher proportion of foods containing greater amounts of n-6 PUFAs in the diet of these birds. Pekin A44 ducks had a lower n-6/n-3 PUFA ratio than was shown in the present study in mallards from both hunting districts [42]. Wołoszyn et al. [35] observed a lower average n-6/n-3 PUFA ratio in the breast muscles of male Pekin P33 and Miniduck K2 ducks. The results of the present study showed a higher average n-6/n-3 PUFA ratio in the muscles of mallards than that shown in Pekin and Muscovy ducks [32] and in the breast muscles of broiler chickens [30]. Differences in the average n-6/n-3 PUFA ratio between farmed breeds and species of poultry and mallards may have been due to dietary differences, i.e., feed rich in n-3 fatty acids given to farmed poultry. A higher average value for this ratio in the breast muscles and liver of chickens receiving a sunflower oil supplement was reported by [43]. Mallards obtained in the Leszno hunting district had a significantly higher n-6/n-3 PUFA ratio in the leg muscles than birds from the Siedlce hunting district. The values were 10.225 and 9.168, respectively, which are nutritionally favourable. The n6/n3 PUFA ratio in meat is mainly regulated by diet [44]. The significantly higher average n-6/n-3 ratio in the meat of mallards from the Leszno hunting district may have been due to the high intensity of crop production, including high levels of mineral fertilizers. Male mallards had a significantly higher average value for this ratio (10.16) than females (9.42). The n-6/n-3 PUFA ratio in the leg muscles of mallards ranged from 8.64 to 11.96. Wołoszyn et al. [35] reported a lower average n-6/n-3 PUFA ratio in the leg muscles of male ducks compared to the male mallards in the present study.
In all groups of mallards, the average PUFA/SFA ratio was higher in the breast muscles than in the leg muscles. The PUFA/SFA ratio should be at least 0.40 [45]. The ratio in the breast muscles of mallards exceeded this value, ranging from 0.75 to 1.02. The present study showed a lower PUFA/SFA ratio in the breast muscles of female and male mallards obtained in both hunting districts than that reported by [28] for both sexes. Compared to the mallards from both hunting districts, a lower, less favourable PUFA/SFA ratio was found in the breast muscles of chicken broilers and Cherry Berry ducks by [29], in ring-necked pheasants by [34], and in Cherry Valley and Spent Layer ducks by [30]. A highly favourable PUFA/SFA ratio was obtained in the present study in the leg muscles of mallards, ranging from 0.70 to 0.78. Kokoszyński [31] showed a lower average PUFA/SFA ratio in the leg muscles of mallards than in the female and male mallards in the analysed hunting districts. Wołoszyn et al. [35], on the other hand, showed a higher average value for this ratio in the leg muscles of male ducks from four conserved flocks (Miniduck K2, Polish Pekin P33, Pekin population-type A3, and synthetic Polish flock SB) and two breeding strains (A55 and P66).
A relatively high h/H ratio and low AI and TI values reduce the incidence of cardiovascular disease [46]. The higher h/H ratio in the leg muscles and lower AI and TI indices in the leg and breast muscles of mallards from the Siedlce hunting district in comparison to the Leszno hunting district indicate that the meat of ducks from the Siedlce hunting district has higher health-promoting value. The h/H, AI, and TI indices can be indicators of the influence of the fatty acid profile on cardiovascular disease [17]. The more favourable fatty acid profile, high h/H ratio, and low AI and TI in the meat of mallards from the Siedlce hunting district may have been linked to the diet of birds obtained in a region with sustainable management.
The limitations of this study include drought in the hunting districts during the study period and the associated difficulties with obtaining enough mallards to allow for reliable inferences. Therefore, there is a need for further study to confirm the research hypotheses.

Conclusions
The meat of mallards is of high nutritional value. This is evidenced by the high average proportion of essential fatty acids and low proportion of saturated fatty acids in the breast muscles and the higher proportion of MUFAs than of SFAs in the breast and leg muscles of ducks from the analysed hunting districts. Given that it is recommended to limit the amount of SFAs in the human diet, this is beneficial for consumer health. The significantly higher average concentrations of EFAs, linoleic acid, and α-linolenic acid in the breast and leg muscles and the significantly lower n-6/n-3 ratio in the breast muscles of mallards from the Siedlce hunting district compared to the Leszno hunting district may have been influenced by the diet of the ducks in the Siedlce district, which improved the fatty acid profile of the meat.