Liquid Chicken Oil Could Be a Healthy Dietary Oil

: Liquid chicken oil is similar to the human lipid ratio, and is similar to the ideal fatty acids ratio suggested by Hayes, but its benefits remain unclear (Hwang, Tung, H.P.; Shaw, H.M. J. Oleo. Sci. 69, 199-206 (2020)). Using soybean oil as a control, liquid chicken oil, coconut oil, lard oil, and olive oil, were tested on SD rats with the rodent diet 5001 plus 1% of high cholesterol addition and moderate 10 % of test oils. Positive results showed that a 10% liquid chicken oil diet reduced LDL and triglycerides, atherogenic index while increasing superoxide dismutase more than the soybean oil control (0.05 ≦ p < 0.10). Moreover, increment of hepatic endogenous glutathione peroxidase was found to be significantly different from the soybean oil control ( p < 0.05). In this study, liquid chicken oil had more benefits than vegetable soybean dietary oil, with little evidence of hyperlipidemia. Comparison of the test oils with categories of fatty acids to the idea ratio SFA : MUFA : PUFA = 1 : 1.5 : 1, scored by its average weight implied a parallel trend of lipidemia and hepatic antioxidant activity to its score. It is difficult to use the test of rat to reflect human physiology, it remain 19% different of the fatty acids ratio from human ratio, however, this study reveal that the healthiness of a dietary oil seems relate well to its compatibility to the idea ratio or the host oil ratio, in this case, it is the human ratio.

Chicken oil, which as an animal fat has been largely commercially abandoned, is usually used only as forage rather than as dietary oil for cooking or baking. Ahmad showed the most dominant fatty acid extracted from chicken adipose tissue is oleic acid 43.9 8 , yet the most abundant oleic acid in olive oil, effectively lowers LDL-C levels in normal triglyceridemic patients 9 . Moreover, a systematic review showed that a high amount 12 of MUFA in dietary regimens has the effect of reducing body fat and regulating blood pressure, which helps prevention of cardiovascular disease 10 .
We have previously reported that liquid chicken oil LK SFA : MUFA : PUFA 1 : 1.6 : 0.9 , harvested from cold recrystallization of crude chicken oil SFA : MUFA : PUFA 1: 1.4 : 0.5 , enriched with MUFA 43.5 C18:1 and PUFA 21.1 C18:2, 0.5 C18:3 n-3 , 0.2 C18:3 n-6 , has a unique property of conforming to Hayes suggested ideal oil ratio SFA : MUFA : PUFA 1 : 1.5 : 1 which believed to alleviate cardiovascular disease 11 . This LK rich in MUFA and PUFA n-3 and n-6 but contained less saturated fatty acid then general chicken oil. It not only mimicked the ideal fatty acid ratio but was also very compatible with human lipids SFA : MUFA : PUFA 1 : 1.8 : 0.6 , and was found to be the highest scored oil beyond camellia or olive oil on the base of the ideal oil ratio 12 . However, there has been little research on physiological activities of chicken oil and its relationship to lipid metabolism. In order to interpret the benefits of this liquid chicken oil, tests to investigate the effect of plasma lipids metabolism compared with other oils, and the hepatic antioxidant activities in SD rats were carried out.

Animals and diets
Male Sprague Dawley SD rats purchased from BioLAS-CO Co. Taipei, Taiwan, were housed individually in stainless-steel wire cages at 23 2 , with a controlled 12-hr light/dark cycle, and given free access to water and food. Protocols for animal care and handling were in accordance with guidelines of the Institutional Animal Care and Use Committee of Chia-Nan University of Pharmacy and Science.
The liquid chicken oil used was provided by Find Health Co. Ltd., Taiwan. The soybean oil from the President Co. Ltd., Taiwan. The lard from the I Mei food Co. Ltd., Taiwan. The olive oil from the self-brand of Carrefour Co. Ltd., Taiwan, and the coconut oil from the Kirkland Co. Ltd., Philippine. All test diets were prepared by blending the oils with the basic diet Laboratory Rodent diet 5001 Lab Supply Co. Ltd., Ford Worth, Texas, USA , which contained 5 fat, and an additional 1 cholesterol added. The rats were randomly divided into six groups: a control group fed soybean oil SC, 10 soybean oil , a liquid chicken oil group QK, 10 liquid chicken oil , a lard oil group LD, 10 lard oil , an olive oil group OL, 10 olive oil , and a coconut oil group CO, 10 coconut oil , each group with at least six rats, respectively. Body weight and food intake were routinely monitored and recorded during the 13-week test period.

Tissue preparation
On the last day of the 13-week feeding program, the rats were fasted overnight, and then sacrificed by carbon dioxide asphyxiation. Blood was collected from the abdominal vena cava, the liver, heart, kidney, spleen and testis, and the epididymis adipose tissue and peritoneal adipose tissue were collected and weighed. The EDTA-spiked blood was centrifuged with 1,000 x g to collect the plasma, and stored at 20 until analysis. One lobe of each liver was stored at 70 for the following analysis.

Plasma biochemical analysis
Plasma biochemical markers were assayed by an auto-mated chemical analyzer, AU5800, Beckman Coulter Inc., U.S.A. in brief; Plasma cholesterol was assayed by testing saponified cholesterol extracts using dehydrogenase 13

Hepatic antioxidant analysis
Glutathione peroxidase was assayed by glutathione peroxidase GPX catalyzing the oxidation of glutathione by cumene hydroperoxide 21 , using a Glutathione Peroxidase kit from RANSEL RANDOX Laboratories Ltd., Antrim, UK. Superoxide dismutase SOD activity in liver was measured by employing xanthine and xanthine oxidase to generate superoxide radicals, which react with 2-4-iodophenyl ω-4-nitrophenol -5-phenyltetrazolium chloride to form the red dye formazan 22 . This used a SD-25 kit, Superoxide Dismutase kit, from RANSOD RANDOX Laboratories Ltd., Antrim, UK.
The catalase activity was evaluated by enzyme catalase reaction with H 2 O 2 in methanol to aldehyde that reacts with a chromogen 4-amino-3-hydrazino-5-mercapto-1,2,4 triazole, forming a purple color that was measured colorimetrically 23 , using a catalase assay kit from Cayman Chemical Company, Ann Arbor, MI. Malondialdehyde MDA in the liver was measured by reacting malondialdehyde with thiobarbituruc acid TBA , forming a colorimetric MDA-TBA adduct 530-540 nm and measured 24 , using a commercial TBARS-assay kit from Cayman Chemical Company, Ann Arbor, MI.

Lipid analysis
The rats peritoneal adipose was collected, stewed at 125 for 15 min using Imarflex electronic pressure cooker, Imarflex Co. Ltd., Taiwan, removed of debris, washed with water, and vacuum dried as a rat oil. Fatty acids of the oil were analyzed according to the Taiwan FDA method, where Glycerolipids 40 mg were mixed with 2 mL of hexane and 0.2 mL of 2 M methanolic KOH at room temperature for 2 min. The fatty acid methyl esters in the hexane layer were analyzed by gas chromatography, using an HP-88 column Agilent, 100 m 0.25 mm I.D., 0.2 μm film thickness with a FID detector, and a Shimatzu GC Model-2010 25 .

Statistical analysis
All statistical calculations were done with SPSS ver. 12.0 SPSS Inc., Chicago, IL, USA and results are shown as mean SD. One-way analysis of variance used p 0.05 as statistically significant and Tukey-Kramer was used as post-hoc test.

Animal body weight, food intake and organs
The rats body weight, food intake, body weight gain, and feeding efficiency in Table 1 show insignificant differences among groups in these categories p 0.05 . Relative organ weights are shown in Table 2, with differences between groups not reaching significance p 0.05 . These results indicate that the efficiency of energy or nutrition usage of LK was similar to soybean and other commercial oils used. Table 3 show no significant difference between groups was found for plasma glucose, cholesterol and triglyceride. Glucose is slightly elevated higher but still within the normal range 26 . ALT and LDL are also slightly elevated, but not substantially above the normal range 27 29 . CO had a significant negative effect on TG regulation p 0.05 , and the 14 reduction of TG level in QK group was insignificant p 0.095 , showing that the QK had some positive effect on TG regulation. Neither HDL-C nor LDL-C showed significant difference from SC, but QK reduced 21 of the LDL compared to SC p 0.077 . In addition, the atherogenic index AI of QK and OL were 23 and 14 p 0.091 lower than SC, respectively. Thus QK showed a potentially significant effect in reducing atherogenic risk or obesity-related syndromes. Table 4 shows there was no significant difference in SOD activity among groups, but the SOD activity of QK was 19 higher than that of SC p 0.079 . The activity of GPX in QK was significantly 29 higher than SC p 0.05 . Histologic examination of non-alcoholic fatty liver disease activity by Balloon Score found a few rat livers with slight lymphocyte infiltration in some lobular parenchyma, especially in LD. But the average scored steatosis on each group was zero data not shown 30 .

Fatty acids analysis
Composition of the SD rats fatty acids is shown in Table  5. The rats fatty acid ratio and their weighting scores are compared with the Hayes ratio and the average ideal ratio of Hayes, Monica, and human ratios 12 . The results of Table  6 show that the rats fatty acid ratio differed from the ideal fatty acid ratio by 18 SFA : MUFA : PUFA 1 : 1.5 : 1 , and was 19 different from the human oil ratio of 1 : 1.8 : 0.6.

Discussion
As a macronutrient, fat is the densest source of food energy. Though the use of liquid chicken oil for human consumption is no longer common, Marx reported that the digestibility of poultry fat could be as high as 100 in dogs 31 . But we found no reports on the effects of chicken oil on plasma lipids, and its other possible health benefits remain unknown.
All rats fed with liquid chicken oil diet remained healthy during the study, with no significant differences in body weight or feeding efficiency did, compared to rats on a soybean oil diet. Table 3 shows that most of the tested values remain normal, there were insignificant differences between groups, and the ALT and Glu are higher among groups, indicating the possibility of hepatic inflammation or insulin resistance but within the normal range 26,31,32 .
Due to the wide variation in the normal range between reports, we surveyed all rats and noticed an occasional small lymphocyte infiltration on a few liver lobes, especially in LD. But the average score for fatty liver on the balloon test remained 0, indicating that under this moderately higher fat and cholesterol diet the tested fat seemed reach a threshold to trigger the non-alcoholic fatty liver syndrome, but values within this moderately high fat and cholesterol remained safe.
On the other hand, when rats were fed with the diet for 13 weeks the LDL was higher, perhaps due to the additional 1 cholesterol in the diet, but an insignificant difference remained between groups 28 . Although insignificant, QK showed a reduction of 21 in LDL over SC, consistent with many reports on benefits of oleic acid in LK 41 to reduce the level of LDL 33 35 . Moreover, if the index of ath-  erogenicity IA was evaluated by its composition of fatty acids, as Ulbritcht and Southgate proposed; IA C12:0 C14:0 x 4 C16:0 /Σ UFA, the IA of SC is 0.14, then the LD is 0.46, the QK is 0.35, the OL is 0.13, and the CO is 8.11 36 , showing that olive oil is the best oil, with the lowest IA. However, if the differences was evaluated by its atherogenic index AI as Amani suggested; AI Chol-HDL / HDL 17 , then QK lowered AI by 26 over SC p 0.05 and lowered by 40 over CO p 0.05 . Further exploration of the relationship between IA and AI requires further research, but QK performed significantly better AI than SC or better than even OL.
Soybean oil as the control in this study, has high levels of polyunsaturated fatty acids PUFA , which reduce the plasma cholesterol and reduce the incidence of CVD 37 . This study shows that the liquid chicken oil enriched with n-3 and oleic acid has a substantial benefit on lipid profile and AI, reducing levels to those of vegetable soybean oil better.
It is known that dietary lipid determines the structure and fatty acid composition of cell membranes, and the cellular susceptibility to peroxidation is dependent on its polyunsaturated fatty acid PUFA content and antioxidant status 38 . In addition, monounsaturated fatty acid is more stable against oxidation than PUFA. In this study, the liver MDA or the TBARS of soybean oil diet were not elevated as expected. This may be because previous tests were done with an abnormal AIN-76 diet, or high chemical carcinogen stress and, as such, may not reflect the normal nutrition physiology of commercial oils 38,39 . On the other hand, GPx, SOD, and catalase are well-known as cellular antioxidant enzymes. The changes in enzyme activity show its adaptation to oxidative stress 40 42 . This study finds a 12 decrease in catalase activity in normal rats fed with liquid chicken oil, while there was no increase in TBARS. This indicates that MUFA-rich liquid chicken oil may be less susceptible to peroxidation. The GPx and SOD activity were observed to be both elevated on QK see Table 4 . Moreover, the hepatic GPx activity was significant and 30 higher in QK than in SC. This may be due to the photosensitive yellow color of chicken oil, since chicken oil has high levels of vitamin A or beta-carotene antioxidant 43,44 . However, the antioxidant activity shown in this study was an endogenous enzyme, so our cold-harvested LK had some factors in addition to vitamin E or carotene that regulated hepatic antioxidant activity.
The effects of olive oil in the well-known Mediterranean diet are controversial. Abenavoli considered the Mediterranean diet as characterized by mainly plant-based foods and fish, with reduced consumption of meat and dairy products 45 . However, Khaw found little difference between olive oil, butter or coconut oil in trials 5 . Although olive oil is often considered a healthy oil, it has fewer n-3 fatty acids, and our test found the differences in activity of olive oil from soy bean oil to be insignificant, or with only a few positive effects.
On the other hand, Kang has stated that the endogenic hepatic antioxidant enzyme was regulated by a better SFA/ PUFA fatty acids ratio 1 1.5 46 , and the ratio of 0.9 for LK is similar, supporting LK s positive effect on endogenous hepatic antioxidant and reinforcing our hypothesis that the ideal fatty acid ratio for humans is SFA : MUFA : PUFA 1 : 1.5 : 1 12 . This may also reinforce the significance of AI and the insignificant but positive effects of SOD of QK in this study. Moreover, because the rat s fatty acid ratio showed  Table 6 shows that for all ideal ratios evaluated, liquid chicken oil always had the best score 12,47 . It seems that if there are health defects brought about by the dietary use of animal fat, then liquid chicken oil, should be regarded as an exception.
In conclusion, in this high cholesterol and moderate high fat condition, liquid chicken oil showed benefits similar to vegetable soybean dietary oil in both energy metabolism and feeding efficiency. In addition, QK showed a significant increase of the endogenous hepatic antioxidant GPx, and reduced AI.
This first evaluation of lipid by the host fatty acid composition, together with the relationship of oils scored, implying a sequential-like relationship between the oil scores for the benefits of hyperlipidemia and hepatic antioxidant activities, finds that, being the most similar oil to human lipids. It seems that the best oil for the soy bean plant is the soy oil, the best oil for the olive tree is the olive oil, and the best oil to us, homo sapiens, should be the human oil. However, as the dietary use of human oil is impossible, then the most human mimic or compatible lipid, liquid chicken oil could be a good choice for dietary consumption. Our test remain small, further studies are expected. A part of this study was presented at The Annual Symposium of The Nutrition Society of Taiwan, 2020 .

Authors' Contributions
K.-N. Hwang: material preparation, biochemical markers analysis, intelligent properties, logical analysis and manuscript processing; H.P. Tung: chicken oil preparation, anatomy and analysis; Y.H. Lu: fat analysis, antioxidant and data analysis. H.M. Shaw: experimental design, biochemical markers analysis, data processing and manuscript processing. Table 6 Comparison of the scored test oils. Coconut oil has the lowest score, and QK has the highest score among the test oils. Implying a sequential-like relationship between the oil scores for the benefits of hyperlipidemia and hepatic antioxidant activities.
Hayes ratio 1 : 1 S: saturated fatty acid, M: mono-unsaturated fatty acids, P: poly-unsaturated fatty acids, Avg ratio: the average of Hayes, Monnica, and human ratio. Relative scores were tested by the weighted method 12) .