Comparison of overfed Xupu and Landes geese in performance, fatty acid composition, enzymes and gene expression related to lipid metabolism

Objective The aim of this study was to compare overfeeding performance, fatty acid composition, blood chemistry, enzymes and genes expression overfed Xupu and Landes geese. Methods Sixty male Xupu geese (80 d) and Landes geese (80 d) were selected. After a period of one-week of pre-overfeeding, Xupu and Landes geese were overfed three meals of 550 and 350 g/d, respectively, of a high-carbohydrate diet in the first week of the overfeeding period. The next week, geese were given four meals of 1,200 and 850 g/d, respectively, over 8 to 14 d. Finally, geese were given five meals of 1,600 and 1,350 g/d, respectively, for the last two weeks. Results After overfeeding for 28 d: Compared with Landes geese, Xupu geese liver weight and liver-to-body weight ratio decreased (p<0.05), while final weight, slaughter weight, total weight gain, abdominal fat weight, and feed-to-liver weight ratio increased (p<0.05). The levels of elaidic acid (C18:1t9), oleic acid (C18:1n-9), eicosenoic acid, and arachidonic acid in the liver of Xupu geese significantly increased (p<0.05), and the levels of myristic acid and stearic acid significantly decreased (p<0.05), while methyleicosanoate acid significantly increased (p<0.05). Xupu geese had higher plasma concentrations of triglyceride and very low density lipoprotein cholesterol (p<0.05), and decreased activities of alanine aminotransferase, aspartate aminotransferase, and lipase (LPS) (p<0.05). Landes geese had higher LPS activity (p<0.05), but lower cholinesterase activity (p<0.05) when compared with Xupu geese. The mRNA expression levels of fatty acid dehydrogenase (FADS) gene, elongase of long-chain fatty acid 1 (ELOVL1) gene, ELOVL5, and acyl-Co A: cholesterol acyltransferase 2 (ACAT2) gene were significantly upregulated (p<0.05) in Landes goose when compared with Xupu geese. Conclusion This study demonstrates that the liver production performance of Landes geese was better than that of Xupu geese to some extent, which may be closely related to LPS activity, as well as the expression of FADS, ELOVL1, ELOVL5, and ACAT2.


INTRODUCTION
With the occurrence of African swine fever in China, the consumption of safer poultry meat has increased dramatically [1,2]. When compared with other poultry meat, goose meat has beneficial characteristics including high protein and low fat content [3]. Moreover, goose liver has a high capacity of fat accumulation and is used for the production of "foie gras" in poultry production [4,5]. Unlike mammalian fatty liver, main components of foie gras are unsaturated fatty acids (UFA), which have been shown to help protect against car diovascular and cerebrovascular disease in humans [6,7].
The Landes goose (Anser anser) originated in the south western Landes province of France. Landes geese have a beneficial liver capacity in that it can increase 5 to 10fold in size over the course of a shortterm overfeeding proce dure. As a result, the Landes geese have become the world's most famous breed for producing fatty liver products [8]. The Xupu goose (Anser cygnoides domesticus) is an indige nous breed of western Hunan Province in China and has been included in the list of National Livestock and Poultry Genetic Resources Protection in China [9,10]. When com pared with other domestic goose breeds, the Xupu goose has the best capacity for fat accumulation in the liver. To this end, Fournier et al [11] showed that the difference of liver size between the two breeds of geese may be closely related to heredity. This difference may also be attributable to a wide range of other factors, including sex, nutrition, housing den sity, and housing environment.
At present, many studies have been conducted on Landes geese relative to Xupu geese; as a result, comparative studies between the two breeds remain scarce. Here, we sought to investigate overfeeding performance, plasma biochemistry indices, fatty acid composition, enzymes and gene expression related to lipid metabolism between Xupu and Landes geese. These data not only form the basis for the study regarding the mechanisms of liver fat deposition, but also provided a theoretical reference for the breeding of fatty liver geese.

MATERIALS AND METHODS
Experimental design, diets, and animal management All experiment were approved by the Institutional Animal Care and Use Committee of the Hunan Agricultural University (Hunan, China). All methods and procedures were performed in accordance with the approved guidelines and provided by the regional Animal Ethics Committee.
Sixty male Xupu geese (4,947.00±377.54 g) and sixty male Landes geese (4,751.00±244.73 g) were selected for this study. All geese were maintained under the same feeding and man agement conditions. Xupu geese were provided by the Hunan Hongyu Xupu Goose Industry Development Co., Ltd. (Huai hua City, Hunan Province, P. R. China) and Landes geese were provided by the Hunan Fugoose Industry Development Co., Ltd. (Chenzhou City, Hunan Province, P. R. China). At 80 d of age, a period of oneweek of preoverfeeding began. During this time, food intake was progressively increased to enlarge the volume of the digestive tract and to initiate metabolic adaptation to overfeeding. At the end of the preoverfeeding period, all geese were forcefed with a carbohydrate diet con sisted of 98% boiled maize, 1.0% plant oil, 0.5% salt, and 0.5% multivitamin (multivitamin provided per kilogram of diet: vitamin A, 80,000,000 IU; vitamin D, 6,000,000 IU; vitamin E, 40,000 IU; vitamin B 1 , 12,000 mg; vitamin B 2 , 50,000 mg; vitamin B 6 , 500 mg; vitamin B 12 , 2,000 mg; vitamin K 3 , 3,000  mg; vitamin C, 16,000 mg; pantothenic acid, 3,000 mg; folic  acid, 2,000 mg; nicotinic acid, 4,000 mg; biotin, 2,000 mg;  methionine, 10,000 mg; lysine, 8,000 mg; tryptophan, 800  mg; arginine, 1,800 mg; serine, 8,000 mg; alanine, 18,000  mg). In total, this intake amounted to 3,370 kcal/kg, with a compasition of 90 g of protein/kg and 4.5 g of fat/kg. Xupu geese, having a greater capacity for overfeeding ingestion, were fed by the operator to the maximum of their ingestion potential. The feed intake of Xupu and Landes geese were different because of their different body weights. Xupu and Landes geese were overfed three meals of 550 and 350 g/d, respectively, of a highcarbohydrate diet in the first week of the overfeeding period. The next week, geese were given four meals of 1,200 and 850 g/d, respectively, over 814 d. Finally, geese were given five meals of 1,600 and 1,350 g/d, respectively, for the last two weeks.
This overfeeding experiment was conducted from July to August in 2018. The overfeeding room had a constant tem perature (28°C to 34°C) and humidity (65% to 70%) range. All geese were divided into two groups according to the va rieties and reared on the ground (4 m×6 m). Each goose was labeled with a ring on its right foot. An overfeeding machine was used in our study, and operated by the same person each time overfeeding occurred. During the overfeeding period, all goose had ad libitum access to water.

Sample collection
Initial weight of each geese was recorded before the first meal of overfeeding. After 28 d of overfeeding, all geese were food deprived overnight for 12 h. During this time, geese had ad libitum access to water. On the following morning, geese were weighted and blood samples were taken by puncture of the occipital venous sinus. Blood sampling were maintained at room temperature for 1 h, and plasma was obtained by cen trifugation at 3,000×g for 20 min at 4°C. After blood sampling, the goose were killed by exsanguination and each individual liver was quickly removed and weighed. Liver sample were immediately taken from the ventromedial portion of the main lobe (right lobe) of eight Xupu geese and eight Landes geese with similar body weight gain. Liver samples were immedi ately frozen in liquid nitrogen and stored at -80°C until later analysis of enzyme activities and mRNA levels. Initial weight, final weight, slaughter weight, liver weight were recorded daily by each goose to calculate the total weight gain, body weight gain rate, livertobody weight ratio, and feedtoliver weight ratio. The indicator is calculated as follows:

Fatty acid composition
The fatty acid composition of the liver was determined ac cording to a previously described method [12]. Briefly, total lipids were extracted from the liver tissue using petroleum ether/anhydrous diethyl ether (1:1, v/v). Methyl esters of the lipids were prepared using saponification with a solu tion of KOH: methanol (4 mol:1 L). The organic layer was aspirated for fatty acid analysis using an Agilent 7890N gas chromatography equipped with a flame ionization detector (Agilent Technologies, Santa Clara, CA, USA) and a CPSil 88 fused silica open tube capillary column (100 m×0.25 nm; Agilent Technologies, USA). The gas chromatograph tem perature program was as follows: Initial temperature of 140°C for 5 min, temperature increase of 3°C/min to 220°C, 1 min temperature hold at 220°C, and then holding temperature at 220°C for additional 40 min. The injector and detector temperatures were maintained at 240°C and 260°C, respec tively. Hydrogen was used as the carrier gas at a flow rate of 40 mL/min. Individual fatty acid peaks were identified by comparing their retention times with those of the standards (Cat#: 189191AMP; Sigma Chemicals, St. Louis, MO, USA). The results were expressed as grams per 100 g of total iden tified fatty acids.

Lipid metabolism enzymes activities of liver and plasma
Approximately 0.5 g of liver sample was used to prepare the tissue homogenate. Tissues were diluted in 1:9 (w/v) using icecold 154 mmol/L sodium chloride solution, and homog enized using an UltraTurrax homogenizer (T10BS25, IKA, BadenWurttemberg, Germany). Resulting homogenates were then centrifuged at 3,500×g at 4°C for 10 min.
The supernatant and plasma were used to determine the activities of cholinesterase (CHE), lipase (LPS), lipoportein lipase (LPL), hepaticlipase (HL), and content of nonesterified free fatty acids (NEFA). All activities and content were deter mined according to corresponding, commercially available diagnostic kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions using a microplate reader (Multiskan GO; Thermo Fisher Scien tific, USA). Protein concentration in the supernatant of the liver homogenate was measured by using a protein assay kit (A0452; Nanjing Jiancheng Institute of Bioengineering, China).

Total RNA extraction, reverse transcription, and quantitative real-time polymerase chain reaction
Total RNA was isolated from liver tissues using a TaKaRa Min BEST Universal RNA Extraction Kit (Takara, Osaka, Japan) according to the manufacturer's protocol. The concentration and integrity of RNA were determined using a NanoDrop 2,000 Spectrophotometer (Thermo Scientific, Hudson, NH, USA) and 1% agarose gel electrophoresis, respectively. Only RNA specimens with an A260/A280 ratio of 1.8 to 2.0 and an A260/A230 ratio ≥2.0 were used for subsequent analyses. Total RNA from each sample was reverse transcribed into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser kit (Takara, Japan), and cDNA was then diluted 1:10 with nucleasefree water before being used for quantitative realtime polymerase chain reaction (PCR). The primer pairs for the amplification of adipocyte fatty acid binding protein (FABP4), stearoylCoA desaturase (SCD), fatty acid dehydro genase (FADS), elongase of longchain fatty acid 1 (ELOVL1), acylCo A: cholesterol acyltransferase 2 (ACAT2), LPL, fatty acid synthase (FASN), glyceraldehyde3phosphate dehydro genase (GAPDH), and betaactin (β-actin) gene were designed from GeneBank sequences using Primer Premier 5.0 and obtained from Shanghai ShengGong Biological Company (Shanghai, China), as shown in Table 1.
Quantitative realtime PCR (qPCR) was performed using SYBR Green Master Mix (Vazyme Biotech, Nanjing, China) in CFX96 Touch RealTime PCR Detection System (BioRad Laboratories, Hercules, CA, USA). The PCR systems con sisted of 2 μL of diluted cDNA template (1:9), 12.5 μL of SYBR Premix Ex Taq II, 1 μL PCR Forward Primer (10 μmol/L), 1 μL PCR Reverse Primer (10 μmol/L) and 8.5 µL of sterilized distilled water. The PCR programs was as fol lows: 95°C for 30 s, followed by 35 cycles of denaturation at 95°C for 5 s and 60°C for 30 s. Dissociation curves of the products were generated by increasing the temperature of samples incrementally from 55°C to 95°C as the final step of the PCR. GAPDH and β-actin genes were used as the dual in ternal standard for normalizing transcript abundance of mRNA expression. The relative expression levels of target genes were calculated by the 2 -ΔΔCt method as described by Livak and Schmittgen [13].

Statistical analyses
All data were analyzed using SPSS 21.0 (2015, IBMSPSS Inc., Chicago, IL, USA). Variability of all the data is expressed as standard error of the mean (SEM). Differences between mean values were compared using independent samples ttest, and considered significant at p<0.05.

Overfeeding performance
The results of overfeeding performance of Xupu and Landes geese are presented in Table 2 and 3. When compared with the Landes geese, final weight, total weight gain, slaughter weight, abdominal fat weight and feedtoliver weight ratio of Xupu geese significantly increased (p<0.05). In Xupu geese, the liver weight and livertobody weight ratio both decreased (p<0.05). There were no significant differences in the initial weight and body weight gain rate between Xupu and Landes geese (p>0.05).

Blood chemistry
As is shown in Table 5, the results of blood chemistry analyses for Xupu and Landes geese revealed that there were no signifi cant differences in either TC or LDLC (p>0.05). However, plasma levels of TG and VLDLC were significantly higher (p<0.05) in Xupu geese when compared with Landes geese. When compared with Landes geese, Xupu geese had a sig nificant decrease (p<0.05) in HDLC content, as well as ALT and AST activities.

Lipid metabolism-related enzymes activities
The results of lipid metabolismrelated enzymes activities in the plasma and liver of Xupu and Landes geese are presented in Table 6. When compared with the Landes geese, LPS ac tivity in the plasma and liver of Xupu geese both significantly decreased (p<0.05). However, a significant enhancement (p<0.05) of CHE activity in the liver of Xupu geese was ob served. There were no significant differences in either plasma or liver LPL or HL activities or in the NEFA content between Xupu and Landes geese (p>0.05).

Gene expression
The lipid metabolismrelated gene mRNA expression in livers of Xupu and Landes geese are shown in Table 7. The mRNA expression of FADS, ELOVL1, ELOVL5, and ACAT2 (p<0.05) in liver of Xupu goose were significantly downregulated than in liver of Landes goose. There were no significant differences in the mRNA expression of FABP4, SCD, LPL, FASN, and retinol binding protein 7 in liver between Xupu and Landes geese (p<0.05).

DISCUSSION
In our study, we found that the livers of Xupu geese were   smaller and the feedtoliver weight ratio of Xupu geese were higher than those of Landes geese. Since Xupu geese had a larger body shape, the index of livertobody weight ratio re vealed an opposite result. The abdominal fat weight of Xupu geese was higher than that of Landes geese. Possible reasons for this result include that a large amount of fat was trans ferred from the liver to extrahepatic (e.g., abdominal adipose tissue) in Xupu geese. The above results confirmed that Landes geese are the best breed globally to produce fatty liver products.
Overfeeding with a carbohydraterich diet results in high de novo lipogenesis. The present study was the first time to compare the fatty acid composition of the fatty liver in Landes and Xupu geese. There were both similarities and differences between the two breeds. The main products of fatty acid syn thesis were 16:0, 18:0, and above all 18:1. This fatty acid in particular accounted for more than 90% of the hepatic TG fatty acid content, and the proportions of 18:1 was more than 50% in both breeds. These results were consistent with the fatty liver content found previously in both geese and duck [14,15]. Relative to Landes geese, Xupu geese had an increase in the proportions of 18:1, but a decrease in the proportions of 18:0 in their fatty liver. This finding was in agreement with those found by both Cazeils et al [16] and Hatsugai et al [17]. Simultaneously, the proportions of monounsaturated fatty acids and polyunsaturated fatty acids (PUFA) in the fatty liver of Xupu geese were significantly higher than those in Landes geese. Moreover, the proportion of saturated fatty acids in the fatty liver of Xupu geese was significantly lower than that of Landes geese. These results were consistent with the pre vious study, which used 21 days of feeding for both Xupu and the Landes geese [18]. Given these findings, it is clear that the performance of overfeeding production and fat deposition in Xupu geese were inferior to that of Landes geese. However, the proportions of UFA in fatty liver of Xupu geese was signifi cantly higher than that of Landes geese, which may indicate that Xupu fatty liver is better for human health.
Fatty liver occurs in geese or duck when fat synthesis ex ceeds fat secretion. In response to overfeeding, de novo hepatic lipogenesis is dramatically increased, TG do not fully enter the secretion pathway, and a large proportion of TG remains stored in the liver [11,19]. During overfeeding, part of the newly synthesized TG in the liver are incorporated into he patic lipoprotein, mainly VLDL which can be secreted into blood and used (or stored) in extrahepatic tissues. We found that the plasma contents of TG and VLDL of Xupu geese were higher than those of Landes geese. These results were in agreement with previous studies, which showed higher VLDL concentration in Poland geese when compared with Landes geese, even though the liver weight of the former was less than the latter [11]. Xu et al [20] found that the plasma concentrations of TG and VLDL were higher in Sichuan white geese than in Landes geese. In the present study, overfeed ing with highenergy corn diet for 28 d induced elevations in the concentration of plasma HDL, which was in accor dance with findings of previous studies in broiler chickens and geese [21,22]. It clear the transfer of fat from the liver to the extrahepatic tissue is one of the reasons why the liver weight of Xupu geese was smaller, but their abdominal fat heavier than that of Landes geese. These results suggested that the mechanism behind geese fatty liver formation is mainly attributable to an imbalance between the storage and secretion (as plasma lipoproteins) of newly synthesized endogenous lipids and exogenous lipids in the cytoplasm.
When overfeeding using a highenergy corn diet, plasma ALT and AST are mainly from the liver, resulting in higher ALT and AST activities. Our study found that the plasma activities of ALT and AST in Xupu geese were lower than those of Landes geese, indicating that Landes geese had a higher degree of liver damage due to longterm overfeeding. The results in this experiment were in agreement with those of Zhu et al [21], which showed that longterm overfeeding induced liver cell inflammation. Therefore, we supported the opinion that goose hepatic adaptation to overfeeding is of notable importance. Our study found that the activity of LPS in the plasma and liver of Landes geese increased relative to that of Xupu geese. Pancreatic lipase plays an important role in fat absorption. Kobayashi et al [23] showed that lipase activity was high by using the higher fat content of diets. Krogdahl [24] also reported that when given a highfat diet, the lipase activity of birds was higher than that in birds fed a lowfat diet. Much past work has found that the activity of LPL was positively correlated with the weight of fatty liver in goose or duck, moreover, that LPL was useful in the se lection of Landes geese breeders with a higher susceptibility to liver steatosis [6,25,26]. Despite this, we found no differ

1963
Liu et al (2020) Asian-Australas J Anim Sci 33:1957-1964 ence in the activity of LPL in either plasma or liver between Xupu and Landes geese, suggesting further study is needed. Collectively, our previous study [18] and current data in dicate the development of severe steatosis was closely related to the genes involved in lipid synthesis, packaging, secretion, transportation, deposition or metabolism, including FADS, ELOVL1, ELOVL5, and ACAT2 [27,28]. It is well known that FADS gene plays a key role in the synthesis of longchain poly unsaturated fatty acid (LCPUFAs) and the metabolism of essential fatty acids [2933]. ELOVL1 plays an important role in the elongation of superlongchain saturated fatty acids and superlongchain monosaturated fatty acids. ELOVL5 is mainly responsible for the elongation of 18carbon fatty acids. ACAT2 catalyzes the conjugation of cholesterol and longchain fatty acids to form cholesterol esters, and plays an important role in the absorption, storage, transport, and apolipoprotein metabolism of cholesterol. Given this, we observed that the expression of FADS, ELOVL1, ELOVL5, and ACAT2 in the liver of Landes geese increased significantly relative to Xupu geese. Osman et al [34] showed that FADS expression levels were gradually increased after overfeeding, moreover, that the induction of FADS promoted the generation of LCPUFAs in goose fatty liver. This increase appeared to be well coordi nated with the size of fatty liver in Landes geese, which suggests that the FADS, ELOVL1, ELOVL5, and ACAT2 genes are im portant to the development of goose fatty liver.
In conclusion, the results of the present study showed that the overfeeding performance of Xupu geese was inferior to that of Landes geese, which may be related to the activity of LPS, and the expression of FADS, ELOVL1, ELOVL5, and ACAT2. However, the the proportions of UFA in Xupu fatty liver were significantly higher than those of Landes geese. Taken together, there results provide new insights into the cultivation of highquality fatty liver geese.