The influence of age and gender on emu (Dromaius novaehollandiae) fat

Studies were carried out to determine the influence of age and sex on two types of fat (back fat and abdominal fat) in the emu, as these are factors that influence the composition of animal tissues. The material involved 26 emus at the age 1 (6 males), 3 (6 males) and 15 years (8 females and 6 males), kept on the same farm and fed the feed of the same nutritional value. The basic chemical composition, cholesterol and mineral content, as well as fatty acid profile of back and abdominal fat of emu were determined. Abdominal fat was characterized by higher content of fat and ash, as well as Mn and Ba. Back fat, on the other hand, showed a higher level of protein, cholesterol, C16:1 and the elements K, P, Si, Na, Ca, Mg, Fe, Zn, Se and Cu. With age, regardless of the type of fat tissue, fat content decreased and water content increased. The highest content of protein, ash, cholesterol, some fatty acids (C18:0, C18:1n9c, C18:2n6c), generally higher content of MUFA, PUFA and the elements K, P, Ca, Mg, Fe, Zn, Pb, Se, Cr, Cd, were found in the fatty tissue of 15-year-old emus. Sex did influence the content of Si, Ca, Cu, Sr, which was higher in the fatty tissue of males. The composition of emu storage fat is determined by factors such as age, sex and the location of the fat tissue in the body.

Cholesterol level. The extracts obtained for determining the cholesterol content in the examined fats are described in the section Fatty acids content and profile (see text below). As with fatty acids, cholesterol was determined by the gas chromatography-mass spectrometry (GC-MS) method. The next steps of sample preparation and appropriate chromatographic analyses were made in accordance with the procedure described by Cunha et al. 34 GC parameters for cholesterol: Capillary column COL-ELITE-5MS 30 m × 0.25 mm × 0.25 μm; carrier gas helium (He) 6.0; gas flow rate 1 ml/min; injection volume 1 μl; split injection ratio (split) 200:1; injector temperature 280 °C; p column temperature programme 100 °C for 5 min; temperature gradient 20 °C/min to 280 °C, 280 °C for 25 min; transfer line temperature 280 °C.
Fatty acids profile. GC-MS was used to determine both the level of cholesterol and the profile of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs).
Extraction: Before lipid extraction, all fats of a single bird specimen were placed in a polypropylene container and then, for easy homogenisation, a small, specific volume of demineralised water was added. Homogenisation in a laboratory blender was performed until the obtained mass showed complete homogeneity thereby providing the averaged samples to be collected for analysis. Thereafter, 2 g of homogenate taken from a number of points were placed in amber glass screw-capped vials (7.5 ml) with Teflon seal. Into each vial, 4 ml of chloroform were added and nitrogen was introduced. Then they were closed under continuous stream of nitrogen and vigorously shaken for 2 h. In order to separate the chloroform phase from the non-lipid residue, the vials were centrifuged at 2000 rpm for 20 min. The extraction process was repeated three more times to elute all the lipids completely, and extracts were combined together and filled up with chloroform to 20 ml.
Hydrolysis: The chloroform phase was placed into amber glass vials (4 ml) in an amount corresponding to 5 mg of extracted lipids and chloroform was evaporated under a stream of nitrogen. The vials were then closed with a screw cap equipped with a vent allowing neutral gas and reagents to be introduced without air entering. The vials were immediately filled up with nitrogen, and 400 μl of 0.5 M KOH in methanol solution were added and incubated in a heating block at 80 °C for 20 min.
Esterification: After cooling, 500 μl of 14% boron trifluoride (BF3) in methanol solution were introduced into each vial and incubated at 80 °C for 35 min. In order to extract fatty acid methyl esters (FAME), 1 ml of saturated NaCl solution and 2 ml of isooctane were added to each cooled vial as an extractant, vigorously shaken Scientific RepoRtS | (2020) 10:11082 | https://doi.org/10.1038/s41598-020-68103-1 www.nature.com/scientificreports/ for 1 h, and left for 0.5 h until the phases separated. The upper isooctane layer was collected to separate vials containing about 0.6 g of anhydrous sodium sulphate (Na2SO4). The vials were then filled with nitrogen and left for 2 h. The dried FAME extracts were placed in vials into an automatic sample changer (autosampler) of the gas chromatograph. GC-MS: The determination of fatty acid methyl esters in liver lipids was made by the GC-MS method on a PerkinElmer CLARUS ® 600 GC/MS with a capillar y column COL-ELITE-5MS 60 m × 0. 25

Minerals content.
The levels of minerals in fats were determined by inductively coupled plasma optical emission spectrometry using the Optima 2000 DV ICP-AES (PerkinElmer Inc., Germany) following digestion in a microwave oven type Multiwave (Anton Paar GmbH, Austria) equipped with a system of continuous temperature and pressure control in each quartz vessel. The weighed amount of tissue homogenate (ca. 1 g) was transferred to a pressure quartz glass vessel, into which 5.0 ml of 65% HNO 3 and 0.5 ml of 30% H 2 O 2 (both Suprapur, Merck) were successively added. Mineralisation was conducted according to the equipment application mode MEAT: 0-5 min-linear gradient of power 100-600 W; 6-10 min-600 W (constant); 11-20 min-1,000 W or less after reaching 75 MPa or 300 °C; 21-35 min-vessel cooling. The cooled and degassed mineralisate was filled up to 10 ml in volumetric flasks.
Microelements (As, Ba, Cd, Cr, Cu, Fe, Mn, Se, Si, Sr, Pb and Zn) were directly determined in the solutions prepared this way, whereas mineralisates were diluted 10-or 100-fold for the determination of macroelements (Ca, Mg, Na, K and P) in order to obtain the range of linear dependence of emission signal on the concentration of a given mineral component. Measurements of the intensity of emitted radiation for micro-elements were made selecting a longer, axial optical path (along plasma), whereas macroelements were analysed radially (across plasma). As standard, a certified multi-element solution for ICP (ICP Multielement Standard IV, Merck) was used. Standard solutions were supplemented with the addition of acid used in mineralisation, in the concentration which occurred in mineralised samples. In order to minimise potential interferences in sample introduction to plasma and other physical disturbances, analyses were made using the internal standard method by yttrium (Y) introduction into sample and standard solutions in a concentration of 0.5 mg Y/l.

Statistical analyses.
The results were statistically analysed using the Statistica 13.1 PL software package (IBM Corp.; SPSS Statistics for Windows; Version 23.0; 2016). Two-way ANOVA was used to determine if two different factors: fat type and emu age and fat type and emu sex have an effect on a measured variable. Least Squares Means were obtained using the Tukey test. The significance was calculated at a 5% confidence level.
Ethics approval. All procedures were performed according to the guidelines for the care and use of research animals and were approved by the Local Ethics Committee on Animal Experimentation in Szczecin (ZUT Szczecin) (resolution number 12/2014, Szczecin, PL).

Consent for publication.
All authors they express their consent for publication.

Results
Proximate composition. When analysing the basic chemical composition of back fat and abdominal fat in emu male depending on the age (Table 2), it was noted that the place of fat deposition influenced the content of protein, fat and ash (P ≤ 0.05). More than twice as much protein content was found in back fat (1.13%). Abdominal fat was characterized by a higher content of fat (96.6%) and ash (0.081%). The content of all the studied basic Table 1. Selected abundances (m/z*) and retention time (RT) for fatty acid methyl ester (FAME) GC MS analysis. *Ion mass to charge ratio. www.nature.com/scientificreports/ components depended on the age of emu (P ≤ 0.05). With age, the amount of water in the fatty tissue increased, regardless of its type, with decreasing fat content. These changes were linear and concerned the 1-year, 3-year and 15-year-old male emus. Fat of the 15-year-old male emus was characterized by a higher proportion of water (2.66%), protein (1.14%) and ash (0.081%), and a lower presence of fat (95.7%). Significant interactions (type of fat × age of emu) were found for water and fat content (P ≤ 0.05). The highest water content was recorded in fat of the 15-year-old male birds, especially in abdominal fat (2.74%). In comparison, abdominal fat of the 1-yer-old emus contained 1.02% water. Also in this fat a higher level of fat was detected (98.0%), while its lowest share was in fat from the 15-year-old male emus (95.7%).
The analysis of the 15-year-old female and male emus' adipose tissue (Table 3) revealed the influence of its location in the emu body upon protein, fat and ash content (P ≤ 0.05). Back fat was characterized by a higher protein content (1.44%) while abdominal fat that of fat (95.9%) and ash (0.083%). No influence of gender and interaction fat type × gender on the basic chemical composition was observed in 15-year-old female and male emus (P ≥ 0.05). Table 2. Basic chemical composition (%) of back fat and abdominal fat of emu males depending on age (mean ± SE). *The same symbol in the superscript indicates statistically significant differences (P ≤ 0.05) for the type of fat. abc Statistically significant differences (P ≤ 0.05) for the emu age were marked with different letters in the subscript. abc Statistically significant differences (P ≤ 0.05) for interaction type of fat × age of emu were marked with different letters in the upper index.
Comparing the cholesterol content in fat of the 15-year-old females and males of emu (Table 5), only a significant effect of the type of fat was observed (P ≤ 0.05). The amount of cholesterol in back fat was higher by an average of 3.4 mg/100 g compared to abdominal fat (62.1 mg/100 g).
No gender or fat type × emu age interaction (P ≥ 0.05) was found to affect the fatty acid profile of 15-yearold female and male emu fat ( Table 5). The effect of fat type was proved in case of other SFA (P ≤ 0.05; back fat − 0.29%, depot fat − 0.34%) and C16:1 (P ≤ 0.05), where its greater share was found in back fat (3.87%).

Minerals content.
It was found that the type of fat did not affect the content of Pb, Cr, Cd and Sr (P ≥ 0.05; Table 6). For other elements, except Ba and Mn, back fat contained more of them than abdominal fat (P ≤ 0.05). The age of emu male affected the content of all studied elements (P ≤ 0.05). Fat of 15-year-old male emus con-  (Table 7) shows that the type of fat does not affect Pb, Cu, Ba, Cr, Cd and Sr (P ≥ 0.05). A higher amount of other mineral components was found in back fat (P ≤ 0.05). Gender influence was observed in the case of Si, Ca, Cu and Sr (P ≤ 0.05), with a higher content of these elements in 15-years-old male fat. The interaction between fat type and sex of 15-years-old emu was observed for P, Ca and Fe (P ≤ 0.05). Most P (119 mg/kg) was found in the back fat of females and Ca (18.8 mg/kg) and Fe (4.09 mg/kg) in the back fat of males.

Discussion
Our research revealed that adipose tissue water and protein content increased with age, while the fat content decreased. As is well known, the higher water content in the tissue fat contributes to its greater susceptibility to the development of pathogenic microflora and an increased tendency to rancidity. Such fat has a lower melting point, therefore its technological usefulness is limited. In turn, a higher protein content may have a reducing effect on the storage stability of the product. The scientific literature lacks information on the basic chemical composition of fatty tissue of emu and other economically used ratites. For poultry, only the basic composition of goose fat is known. Bełkot and Pyz-Łukasik 28 studied the basic chemical composition of back fat and abdominal fatty tissue in Biała Kołudzka goose aged 16-18 weeks and 3 years. The authors of those studies stated that age significantly differentiated the amount of basic components in back fat. Higher water (15.28%), protein (2.60%) and ash (0.14%) content was found in younger geese while fat (91.5%) in older geese, i.e. contrary to our own studies ( Table 2). In the studies of the quoted authors, back fat and abdominal fat differed significantly in respect of the content of all chemical components in [16][17][18] week old geese, whereas in 3-year-old geese only for the level of protein. Less fat (82.6%) and more water (15.3%), protein (young geese − 2.60%, older geese − 1.20%) and ash (0.14%) were found in back fat. A similar dependence on protein and fat content in back fat was found in our own studies (Tables 1 and 2). It should also be noted that in comparison to the study by Bełkot and Pyz-Łukasik 28 , the back fat of emu was characterized by a lower content of water, protein and ash, and a higher content of fat. Abdominal fat, on the other hand, contains less water, more fat and similar level of protein and ash.
In the study of Okruszek 35 on 17-week-old Rypin geese and swan geese no influence of the breed on the fat content in abdominal fat was found, its content (97.1-97.4%) was similar to our own results. However, in subsequent studies by the same author 36 on older 24-week-old females of the same breeds, higher fat levels were recorded (98.2-98.7%), which may suggest the opposite effect of age on fat content than in our own studies.
Fukushima et al. 37 analysed the content of cholesterol in emu oil and recorded a value similar to that obtained in our own study for 1-year and 3-year-old male birds (Table 4). It should be added, however, that the authors did not provide the age and sex of the examined birds. www.nature.com/scientificreports/ Table 7. Mineral content (mg/kg) of back fat and abdominal fat of 15-years-old emu depending on sex (mean ± SE). *The same symbol in the superscript indicates statistically significant differences (P ≤ 0.05) for the type of fat. abc Statistically significant differences (P ≤ 0.05) for the emu age were marked with different letters in the subscript. abc Statistically significant differences (P ≤ 0.05) for interaction type of fat × age of emu were marked with different letters in the upper index. www.nature.com/scientificreports/ In case of 14-month-old ostriches 38 differences in cholesterol content were found depending on the type of fat. Back fat contained more cholesterol (74.3 mg/100 g) than breast fat (49.5 mg/100 g). Those results differ from ones obtained in our own studies (Tables 3 and 4), which is probably due to species differences.
Haraf et al. 29 noted the influence of the breed on the content of cholesterol in the abdominal fat of 17-weekold geese. The content of this sterol was 70.2 mg/100 g for Kartusian geese and 79.9 mg/100 g for Lublin geese. Comparing those results to our own studies we can conclude that emu fat has a lower cholesterol content (Tables 4 and 5).
Wang et al. 39 did not report any significant differences in the fatty acid profile in abdominal fat and back fat of emu. Then, studies carried out by Shimizu and Nakano 40 on back fat, peritoneal cavity fat and kidney leaf fat of emu were not supported by statistical analysis. In those studies SFA (29.8-31.5%) and MUFA (56.0-58.8%) were diametrically different from the results of our own studies (Table 4) (Table 5) had a higher SFA share of about 13% and a lower MUFA share of about 17%. It should be noted that Wang et al. 39 and Shimizu and Nakano 40 did not specify the birds age, sex, feeding habits and feed composition, which have a significant effect on the fatty acid profile. The content of PUFA (11.3-13.5%) and particular fatty acids (C18:2n6: 10.3-11.4%; C18:3n3: 0.90-1.86%) in the studies of the above mentioned authors was similar to the results obtained in our own studies (Tables 4 and 5).
Similar studies were conducted by Shimizu and Nakano 40 , Horbańczuk et al. 38 , Hoffman et al. 41 and Majewska et al. 30  In back fat of rheas, 36.9% SFA, 40.2% MUFA and 22.9% PUFA were found 40 . Comparing those results to the results obtained in our own studies, a conclusion can be drawn that the fatty acid profile of rheas is more similar to that of the 15-year-old emus rather than that of the 1-year and 3-year-old birds (Tables 4 and 5).
Numerous studies on goose fat have shown that the content of fatty acid groups can vary over a wide range: SFA-19.45-33.41%; MUFA-41.90-67.32%; PUFA-11.27-32.95% 28,29,31,36,[42][43][44][45] . These differences are due to the fact that the research was carried out on geese of various breeds and ages. This also explains the apparent differences in SFA and MUFA content compared to our own research, in particular to 1-year and 3-year emus (Tables 4 and 5). Additionally, it should be mentioned that the changes in the fatty acid profile of goose fat are influenced by nutrition 31,42,43 , breed 29,35,36 , packing time and conditions 44 , age 28,42 and type of fat 28 .
Although the content of elements in animal raw materials is very important, the scientific literature lacks information on their content in the spare tissue of animals. In general, too high mineral content in fats is undesirable because of the reduction of their durability due to the catalytic effect of metals on the process of fat oxidation during storage and processing. The quality of fat and oil in terms of their storage and toxicity can be assessed by determining the content of certain elements. This applies especially to Fe, Cu, Mg, Co, Zn, Mn and Ni 46,47 . According to our research, back fat contains more of the metals responsible for the catalytic processes of fat oxidation. The content of most of these elements increased with age of the emus. On the other hand, elements such as As, Cr, Cd and Pb are undesirable due to their toxicity and adverse role in the body metabolism 46,47 . Our own research shows that the accumulation of these elements is not affected by the type of body fat. However, the effect of age is noticeable only during longer production cycle (15 years).
Codex Standard for Named Animal Fats CODEX-STAN 211-1999 (FAO/WHO) gives the permitted limit for certain elements in edible animal oils. It shows that the tested emu fat (Tables 6 and 7) did not exceed the permissible limit for Cu (max 0.4 mg/kg) and As (max 0.1 mg/kg), which was below the detection level. Fat from 15-year-old emus exceeded the maximum limit for Pb (max 0.1 mg/kg) twice, which is most likely related to the age of the bird. The content of Pb in the 1-year and 3-year-old emus was within the permissible standard. In case of Fe (max 1.5 mg/kg) only the 3-year-old emus did not exceed the maximum permissible level of this element. It should be noted, however, that emu fat is only the raw material from which the oil is produced and it can be assumed that only part of the minerals will enter the oil, which is related to the method of its extraction. Ponphaiboon et al. 48 proved that the content of iron and polyunsaturated fatty acids in ostrich oil and its antioxidant activity were dependent on the method of oil extraction. Therefore, we propose that an appropriate extraction method could also eliminate the excess of undesirable elements from the fat of birds kept longer, so that they meet the acceptable standards and could also be used.

Conclusions
The conducted research is a source of knowledge on the chemical composition of emu fat, which is a valuable raw material in the pharmaceutical, cosmetic and food industries. Slight differences in the basic chemical composition, fatty acid profile depending on the location of adipose tissue in the bird's body indicate that emu both dorsal and abdominal fats are a valuable raw material for processing and there is no need for their separation in the technological process.
Fat of 15-year-old emu, regardless of the type, was characterized by the largest share of unsaturated fatty acids (MUFAs and PUFAs). From a nutritional point of view, this is a desirable trait, though it can be problematic for the technological process, especially in terms of accelerating rancidification. In addition, 15-year-old emu fat had a higher content of heavy metals. To sum up, fat obtained from young, 1-and 3-year-old birds seems to be Scientific RepoRtS | (2020) 10:11082 | https://doi.org/10.1038/s41598-020-68103-1 www.nature.com/scientificreports/ most recommendable for use in the food and cosmetics industries. Research on the quality of this raw material should continue, with attention to oxidative stability and sensory quality.