# Lipotoxic Effects of Gossypol Acetate Supplementation on Hepatopancreas Fat Accumulation and Fatty Acid Profile inCyprinus carpio

Authors: Yuru Zhang; Junmei Zhang; Yanhua Ren; Mengdan Zhang; Junping Liang
Journal: Aquaculture Nutrition
 (2022)

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## Abstract

Cottonseed meal is an excellent protein source with low price and abundant nutrients, but the existence of free gossypol restricts its wide application in animal feeds. To explore the toxic effects of free gossypol, the effects of gossypol acetate on the growth performance, hepatopancreatic fat accumulation, and muscle fatty acid composition of the common carp (Cyprinus carpio) were investigated in the present study. Carp were randomly divided into four groups, which were fed a diet supplemented with 0, 170, 340, and 680 mg/kg gossypol acetate, respectively. After 60 days of feeding, the gossypol residue in the tissue, growth performance, blood biochemical indices, hepatopancreas histology, fatty acid components, and expression of key genes involved in lipid metabolism pathways were measured. It was found that the concentrations of free gossypol were increased significantly in the serum, bile, and hepatopancreas of carp fed 340 mg/kg and 680 mg/kg gossypol acetate (P<0.05). Dietary gossypol acetate had a limited effect on growth performance, and high concentrations of gossypol acetate induced excessive hepatopancreatic fat deposition and hepatocyte injury in C. carpio. In addition, gossypol acetate decreased total polyunsaturated fatty acids (PUFAs) and docosahexaenoic acid in muscle. In the hepatopancreas, decreased HSL, PPARα, and CD36 may result in fat accumulation. In the muscle, decreased FADS2 may be the key reason for the decrease in PUFAs. Taken together, these findings indicated that dietary gossypol acetate can cause lipid metabolism disorders, such as hepatopancreatic damage and significant changes in tissue fatty acid profile, which are regulated by the key lipid metabolic genes in common carp.


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## Body

## 1. Introduction

Cottonseed meal has been considered as an alternative protein in terrestrial animals and fish diets [1–3] because it is low cost and rich in nutrients, such as proteins, carbohydrates, and minerals. However, because cottonseed meal contains toxic free gossypol, improper use can cause animal health problems, which restricts its widespread application in animal feed ([4–6] [7, 8]).Previous animal research has suggested that dietary gossypol produces adverse effects, including growth depression, reduced fertility, lipid metabolic disorder, and pathological changes in the liver and other tissues [9, 10]. For example, when dairy cows were fed with a diet supplemented with 717 mg/kg and 951 mg/kg of free gossypol for 170 days, the pregnancy rate decreased and the incidence of abortion increased [11]. Dietary gossypol can significantly reduce body weight in sheep and cause liver cell swelling, nuclear lysis, and kidney cell necrosis [12]. Dietary gossypol also decreases blood glucose and insulin levels, increases insulin sensitivity, and improves lipid metabolism in rats with type 2 diabetes [13]. Furthermore, the acute toxicity of gossypol can cause liver cell damage and increase fat accumulation in the liver of laying hens [14]. Unlike terrestrial animals, fish have a strong tolerance to gossypol [2], but high levels of gossypol can cause various problems, such as slow growth, decreased appetite, and reduced reproductive performance [10, 15, 16]. For instance, the growth and survival of adult rainbow trout were not affected by consuming cottonseed meal at gossypol supplementation levels of up to 619 mg/kg over 10 months, but there was reduced reproductive performance in females [17]. In addition, gossypol can reduce growth and cause hepatopancreatic fibrosis and hepatopancreatic injuries in turbot (Scophthalmus maximus L.) [18]. At supplementation levels of 1200 mg/kg of dietary gossypol acetate, the hepatopancreatic function can potentially be damaged in tilapia (Oreochromis niloticus×O. aureus) [19].The above studies showed that free gossypol in the feed can affect lipid metabolism in various ways in livestock, poultry, mammals, and fish. However, the specific roles of gossypol in lipid metabolism in common carp remain unclear. Thus, in the present study, the diet of common carp was supplemented with different concentrations of gossypol acetate (0, 170, 340, and 680 mg/kg) to analyse the effects of gossypol on carp growth performance; fat accumulation in the hepatopancreas; fatty acid (FA) composition in the brain, hepatopancreas, intestine, and muscle; and mRNA expression of key genes involved in lipid metabolism. The results of this study will provide further insights into the optimal proportions of cottonseed meal in common carp feed and clarify the effects of gossypol on lipid mentalism.


## 2. Material and Methods

### 2.1. Feed Preparation

The basal feed, which did not contain free gossypol, was purchased from Tongwei Group Co., Ltd. (China). The content of crude protein, crude fat, ash, and moisture in the basal feed was 39.81%, 5.87%, 9.17%, and 6.68%, respectively. Gossypol acetate (CAS NO.12542-36-8) was purchased from Dalian Meilun Biotechnology Co., Ltd. (China), and the purity of gossypol was greater than 97.5%. Following the methodology previously published by our team [20], the basal feed was mixed thoroughly with gossypol acetate to obtain four different experimental feeds containing 0, 170, 340, and 680 mg/kg gossypol acetate. The diets were converted into 3 mm pellets using a granulator (F-26, Guangzhou Huagong Optical Mechanical Electrical Technology). After natural air drying, the diets were stored at 4°C until required. Finally, the free gossypol concentration in the experimental diets was determined as 0, 161.70±3.56, 342.07±15.67, and 670.35±34.03 mg/kg, respectively (Table S1), which was basically consistent with the theoretical values.


### 2.2. Feeding Trial and Samples Collection

Healthy common carp (Cyprinus carpio) (60±2.5g) were purchased from Zhongmu National Agricultural Park (Zhengzhou, Henan, China) and cultured in an indoor recirculating aquaculture system of Henan Normal University and acclimatised for two weeks. Then, 360 healthy fish were randomly divided into 12 plastic tanks (four groups) with 30 carp in each tank (200 L). The duration of the breeding experiment was eight weeks. The fish were fed three times a day (9 : 00, 12 : 30, and 17 : 00). One-third of the feeding water was replaced every two days, and the cotton filter was cleaned every day. Water quality was monitored weekly using a multifunctional water quality detector (Shandong Gelanpu Technology Co., Ltd., China). Throughout the experiment, the average water temperature was 25±1°C, the pH was 7.0–7.5, the dissolved oxygen was above 5 mg/L, the ammonia nitrogen was less than 0.01 mg/L, and the light cycle was 12 L : 12D.After the breeding experiment, all carp were anaesthetized with MS-222 (100 mg/L) (Aladdin, Shanghai, China) prior to sampling. Five fish were randomly selected from each tank for tail vein blood collection. The blood was maintained at 4°C for 12 h, after which it was centrifuged at 3000 rpm for 15 min, and the supernatant obtained was used to determine the serum enzyme activity. Meanwhile, 12–14 fish were randomly chosen from each tank to detect the FA content (3–5 fish/tank) and gene expression levels (9 fish/tank). The tissues, including the brain, intestine, hepatopancreas, and muscle, were dissected and frozen using liquid nitrogen and then stored at −80°C for detecting the FA content and gene expression levels.All the procedures involving fish followed the NIH Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, revised 1996) and were approved by the Laboratory Animal Care and Use Committee of Henan Normal University.


### 2.3. Growth Performance and Morphological Index Determination

The growth performance and morphological index were calculated using the following formulas:(1)WGRweightgainrate=Wt−W0/W0×100,SGRspecificgrowthrate=lnWt−lnW0/t×100,FCRfeedconversationratio=F/Wt−W0,SRsurvivalrate=FN/IN×100,CFconditionfactor=Wt/L3×100,VSIvisceralindex=Wv/Wt×100,HSIhepatosomaticindex=Wh/Wt×100,where Wt is the final body weight (g), W0 is the initial body weight (g), F is the food intake (g), FN is the survival quantity (tail), IN is the initial quantity (tail), Wv is the wet weight of the visceral mass (g), Wh is the wet weight of the hepatopancreas (g), L is the body length (cm), and t is the culture time (d).


### 2.4. Analysis of Gossypol Concentration in Tissues

Following previous research procedures to extract gossypol present in serum, bile, and the hepatopancreas, acetone was used to extract the gossypol [21–23]. High-performance liquid chromatography (HPLC) was used to detect gossypol content. An HPLC system (Agilent 1260, Germany) equipped with a TC-C 18 column (250mm×4.6mm) was used. The samples were detected at 235 nm, with a flow rate of 1.0 ml/min. Using gossypol-acetic acid (Sigma) as a standard, the external standard curves of each enantiomer were generated for the analyses.


### 2.5. Analysis of Serum Biochemistry Index

Serum triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the serum were determined using a test kit purchased from Nanjing Jiancheng Biological Reagent Company, China.


### 2.6. Histological Analysis of Hepatopancreas

Three carp were randomly selected from each tank, and their hepatopancreas was placed in 4% paraformaldehyde solution for 24 h. The paraffin and frozen sections were prepared following standard histological procedures for staining with HE and oil red O, respectively. Histological images were obtained using a digital slice scanning system (3DHIESTECH, Panoramic MIDI, Hungary).


### 2.7. Crude Fat and FA Content Analyses

Three to five fish were randomly selected from each tank. Exactly 2.0 g of hepatopancreas from each fish was dried to a constant weight in a vacuum freeze dryer. The crude fat content of the hepatopancreas was determined using the Soxhlet method [24].Fatty acids were extracted from the brain, intestinal tract, hepatopancreas, and muscle ofC. carpio using chloroform-methanol (2 : 1 volume ratio) [25]. The FAs were analysed by gas chromatography (GC) using an Agilent 7890B series gas chromatographer equipped with a 100m×250μm×0.2 μm HP-88 column (Agilent) with helium as the carrier gas at 1.4 mL/min and a flame ionization detector. The GC had an initial temperature of 120°C for 1 min, followed by an increase of 10°C/min to 190°C for 19 min, and then an increase of 2°C/min to 200°C for 28 min. The injector temperature was 250°C, pressure was 41.8 psi, flow rate was 3 mL/min, and split ratio was 3 : 1.


### 2.8. Gene Expression Analysis

The total RNA was extracted from the brain, intestine, hepatopancreas, and muscle using a TaKaRa RNAiso PLUS kit. RNA reverse transcription was performed using the PrimeScript™ RT reagent kit (Perfect Real Time) from Takara. Specific primers for real-time PCR amplification were designed using primer 5.0 using the existing gene sequences of carp (Table1). PCR amplification was performed using the TB Green® Premix Ex Taq™ II kit (Tli RNaseH Plus) from Takara, and the relative expression level was calculated using the 2−△△Ct method.Table 1
Specific primers used in the real-time PCR amplification.
Metabolism functionGene namePrimer(5′-3′)Length/bpGene ID (accession number)Transcription factorPPARαF GCATGAAGCCTACCTCAGACACTT231FJ849065.1R ACCGAGGCGTACTGGCAGAAPPARβF GCAGCACAGCCAGTCCTGAA128XM_019110518.1R AACGCCGTAGTGGAATCCTGACPPARγF GCAAGGCAGTGGAGGACAAGAAC121LOC109050306R ACGCAACACAGCACCATAAGAGGSREBP-1F CACGGCTCTGCTCAACGACAT294XM_019073316.1R TGCGGAGGAGACTGCTGGAASREBP-2F CAGATGAGTGTGAGTCCGCAGAAG174XM_019064582.1R CCATAGGCTGGTCCTCAGAGTCALipogenesisFASNF TCTGTGCTGTGCGGACTGGAA253KY378913.1R GCAACATCGGCTGGATATTGAGGAGACACβF ACTGGCTGGCTAGATCACCTTATTG207XM_019106587.1R GCACCGCATACCACACCTAACATLipolysisATGLF CACCAACACCTCCATTCAGTTCACA227KY906167.1R ACTCTTCATCCTCCTCACCGTCAGHSLF TTGATGCCTATGCTGGTACGAGTTG182MF061228.2R TGATGTGGTTGGAGAGGATGATGCTPUFA biosynthesisFADS2aF CTGTAACATTGAGCAATCCGCCTTC236MK852165.1R GGTACGCATCCAGCCAGAGTTCFADS2bF AAGTAAGCAGCAGTCAGTCAGAGTT242MK852166.1R CCGTGGCATCTTCTCCAGCATAGLipid transportCD36F CCGTAGGCACAGAGGAGGACATAT116KM030422.1R GAGTGTGGAATTGGAGCGTTGGAFABP1bF ATCGAATCCCTGACCGGAGA156XM_019124255.1R CCTCTTGTAGACGAGGCTGCFABP10aF AACCCCTGGAAAAACCGTCA176EU363800.1R GGGTCTCCACCATCTCTCCTFABP10bF AACTCCTTCACCATCGGCAA166EU363801.1R CTCCTACCGTCAGGGTCTCCFABP11F TTGAGCTGTTGCTGGAGTGAC235XM_019119618.1R GTTTTGAAGGTGCTCTGCGAControl18SF CTGAGAAACGGCTACCACATC101FJ710827.1R CAGACCTGTTATTGCTCCATCT


### 2.9. Statistical Analysis

Not all our data were balanced, for unbalanced data, including FAs content and gene expression, a least-squares analysis was used to provide theLSMEAN±SEM, and the data were processed using SAS 9.0 (SAS Institute, Cary, NC, USA). For the balanced data, the significant differences in the growth performance, gossypol concentration, and serum biochemistry index were analysed using one-way ANOVA and LSD multiple comparisons using SPSS 22.0. Data are presented as the mean±SD, unless specifically indicated. The results were considered statistically significant if P<0.05. The regression curve was analysed using Excel. All figures were created using GraphPad Prism 6.02 (GraphPad Software, La Jolla, CA, USA) and Adobe Photoshop CC 2017 (Adobe Systems Incorporated, USA).


## 2.1. Feed Preparation

The basal feed, which did not contain free gossypol, was purchased from Tongwei Group Co., Ltd. (China). The content of crude protein, crude fat, ash, and moisture in the basal feed was 39.81%, 5.87%, 9.17%, and 6.68%, respectively. Gossypol acetate (CAS NO.12542-36-8) was purchased from Dalian Meilun Biotechnology Co., Ltd. (China), and the purity of gossypol was greater than 97.5%. Following the methodology previously published by our team [20], the basal feed was mixed thoroughly with gossypol acetate to obtain four different experimental feeds containing 0, 170, 340, and 680 mg/kg gossypol acetate. The diets were converted into 3 mm pellets using a granulator (F-26, Guangzhou Huagong Optical Mechanical Electrical Technology). After natural air drying, the diets were stored at 4°C until required. Finally, the free gossypol concentration in the experimental diets was determined as 0, 161.70±3.56, 342.07±15.67, and 670.35±34.03 mg/kg, respectively (Table S1), which was basically consistent with the theoretical values.


## 2.2. Feeding Trial and Samples Collection

Healthy common carp (Cyprinus carpio) (60±2.5g) were purchased from Zhongmu National Agricultural Park (Zhengzhou, Henan, China) and cultured in an indoor recirculating aquaculture system of Henan Normal University and acclimatised for two weeks. Then, 360 healthy fish were randomly divided into 12 plastic tanks (four groups) with 30 carp in each tank (200 L). The duration of the breeding experiment was eight weeks. The fish were fed three times a day (9 : 00, 12 : 30, and 17 : 00). One-third of the feeding water was replaced every two days, and the cotton filter was cleaned every day. Water quality was monitored weekly using a multifunctional water quality detector (Shandong Gelanpu Technology Co., Ltd., China). Throughout the experiment, the average water temperature was 25±1°C, the pH was 7.0–7.5, the dissolved oxygen was above 5 mg/L, the ammonia nitrogen was less than 0.01 mg/L, and the light cycle was 12 L : 12D.After the breeding experiment, all carp were anaesthetized with MS-222 (100 mg/L) (Aladdin, Shanghai, China) prior to sampling. Five fish were randomly selected from each tank for tail vein blood collection. The blood was maintained at 4°C for 12 h, after which it was centrifuged at 3000 rpm for 15 min, and the supernatant obtained was used to determine the serum enzyme activity. Meanwhile, 12–14 fish were randomly chosen from each tank to detect the FA content (3–5 fish/tank) and gene expression levels (9 fish/tank). The tissues, including the brain, intestine, hepatopancreas, and muscle, were dissected and frozen using liquid nitrogen and then stored at −80°C for detecting the FA content and gene expression levels.All the procedures involving fish followed the NIH Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, revised 1996) and were approved by the Laboratory Animal Care and Use Committee of Henan Normal University.


## 2.3. Growth Performance and Morphological Index Determination

The growth performance and morphological index were calculated using the following formulas:(1)WGRweightgainrate=Wt−W0/W0×100,SGRspecificgrowthrate=lnWt−lnW0/t×100,FCRfeedconversationratio=F/Wt−W0,SRsurvivalrate=FN/IN×100,CFconditionfactor=Wt/L3×100,VSIvisceralindex=Wv/Wt×100,HSIhepatosomaticindex=Wh/Wt×100,where Wt is the final body weight (g), W0 is the initial body weight (g), F is the food intake (g), FN is the survival quantity (tail), IN is the initial quantity (tail), Wv is the wet weight of the visceral mass (g), Wh is the wet weight of the hepatopancreas (g), L is the body length (cm), and t is the culture time (d).


## 2.4. Analysis of Gossypol Concentration in Tissues

Following previous research procedures to extract gossypol present in serum, bile, and the hepatopancreas, acetone was used to extract the gossypol [21–23]. High-performance liquid chromatography (HPLC) was used to detect gossypol content. An HPLC system (Agilent 1260, Germany) equipped with a TC-C 18 column (250mm×4.6mm) was used. The samples were detected at 235 nm, with a flow rate of 1.0 ml/min. Using gossypol-acetic acid (Sigma) as a standard, the external standard curves of each enantiomer were generated for the analyses.


## 2.5. Analysis of Serum Biochemistry Index

Serum triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the serum were determined using a test kit purchased from Nanjing Jiancheng Biological Reagent Company, China.


## 2.6. Histological Analysis of Hepatopancreas

Three carp were randomly selected from each tank, and their hepatopancreas was placed in 4% paraformaldehyde solution for 24 h. The paraffin and frozen sections were prepared following standard histological procedures for staining with HE and oil red O, respectively. Histological images were obtained using a digital slice scanning system (3DHIESTECH, Panoramic MIDI, Hungary).


## 2.7. Crude Fat and FA Content Analyses

Three to five fish were randomly selected from each tank. Exactly 2.0 g of hepatopancreas from each fish was dried to a constant weight in a vacuum freeze dryer. The crude fat content of the hepatopancreas was determined using the Soxhlet method [24].Fatty acids were extracted from the brain, intestinal tract, hepatopancreas, and muscle ofC. carpio using chloroform-methanol (2 : 1 volume ratio) [25]. The FAs were analysed by gas chromatography (GC) using an Agilent 7890B series gas chromatographer equipped with a 100m×250μm×0.2 μm HP-88 column (Agilent) with helium as the carrier gas at 1.4 mL/min and a flame ionization detector. The GC had an initial temperature of 120°C for 1 min, followed by an increase of 10°C/min to 190°C for 19 min, and then an increase of 2°C/min to 200°C for 28 min. The injector temperature was 250°C, pressure was 41.8 psi, flow rate was 3 mL/min, and split ratio was 3 : 1.


## 2.8. Gene Expression Analysis

The total RNA was extracted from the brain, intestine, hepatopancreas, and muscle using a TaKaRa RNAiso PLUS kit. RNA reverse transcription was performed using the PrimeScript™ RT reagent kit (Perfect Real Time) from Takara. Specific primers for real-time PCR amplification were designed using primer 5.0 using the existing gene sequences of carp (Table1). PCR amplification was performed using the TB Green® Premix Ex Taq™ II kit (Tli RNaseH Plus) from Takara, and the relative expression level was calculated using the 2−△△Ct method.Table 1
Specific primers used in the real-time PCR amplification.
Metabolism functionGene namePrimer(5′-3′)Length/bpGene ID (accession number)Transcription factorPPARαF GCATGAAGCCTACCTCAGACACTT231FJ849065.1R ACCGAGGCGTACTGGCAGAAPPARβF GCAGCACAGCCAGTCCTGAA128XM_019110518.1R AACGCCGTAGTGGAATCCTGACPPARγF GCAAGGCAGTGGAGGACAAGAAC121LOC109050306R ACGCAACACAGCACCATAAGAGGSREBP-1F CACGGCTCTGCTCAACGACAT294XM_019073316.1R TGCGGAGGAGACTGCTGGAASREBP-2F CAGATGAGTGTGAGTCCGCAGAAG174XM_019064582.1R CCATAGGCTGGTCCTCAGAGTCALipogenesisFASNF TCTGTGCTGTGCGGACTGGAA253KY378913.1R GCAACATCGGCTGGATATTGAGGAGACACβF ACTGGCTGGCTAGATCACCTTATTG207XM_019106587.1R GCACCGCATACCACACCTAACATLipolysisATGLF CACCAACACCTCCATTCAGTTCACA227KY906167.1R ACTCTTCATCCTCCTCACCGTCAGHSLF TTGATGCCTATGCTGGTACGAGTTG182MF061228.2R TGATGTGGTTGGAGAGGATGATGCTPUFA biosynthesisFADS2aF CTGTAACATTGAGCAATCCGCCTTC236MK852165.1R GGTACGCATCCAGCCAGAGTTCFADS2bF AAGTAAGCAGCAGTCAGTCAGAGTT242MK852166.1R CCGTGGCATCTTCTCCAGCATAGLipid transportCD36F CCGTAGGCACAGAGGAGGACATAT116KM030422.1R GAGTGTGGAATTGGAGCGTTGGAFABP1bF ATCGAATCCCTGACCGGAGA156XM_019124255.1R CCTCTTGTAGACGAGGCTGCFABP10aF AACCCCTGGAAAAACCGTCA176EU363800.1R GGGTCTCCACCATCTCTCCTFABP10bF AACTCCTTCACCATCGGCAA166EU363801.1R CTCCTACCGTCAGGGTCTCCFABP11F TTGAGCTGTTGCTGGAGTGAC235XM_019119618.1R GTTTTGAAGGTGCTCTGCGAControl18SF CTGAGAAACGGCTACCACATC101FJ710827.1R CAGACCTGTTATTGCTCCATCT


## 2.9. Statistical Analysis

Not all our data were balanced, for unbalanced data, including FAs content and gene expression, a least-squares analysis was used to provide theLSMEAN±SEM, and the data were processed using SAS 9.0 (SAS Institute, Cary, NC, USA). For the balanced data, the significant differences in the growth performance, gossypol concentration, and serum biochemistry index were analysed using one-way ANOVA and LSD multiple comparisons using SPSS 22.0. Data are presented as the mean±SD, unless specifically indicated. The results were considered statistically significant if P<0.05. The regression curve was analysed using Excel. All figures were created using GraphPad Prism 6.02 (GraphPad Software, La Jolla, CA, USA) and Adobe Photoshop CC 2017 (Adobe Systems Incorporated, USA).


## 3. Results

### 3.1. Effects of Gossypol Acetate on the Growth Performance and Morphometric Parameters ofC. carpio

The WGR (157.26–162.16%), SGR (1.57–1.60%), FCR (1.57–1.61), VSI (7.78–8.62%), HSI (1.87–1.95%), and CF (2.21–2.36) did not vary significantly among the treatment groups (P>0.05) (Table 2). The SR in all groups was 100%, meaning there was no mortality throughout the experiment.Table 2
Effect of gossypol acetate on growth performance ofCyprinus carpio.
GroupGossypol acetate supplementation (mg/kg)Parameter0170340680Initial body weight (g)57.02±0.9657.47±0.4357.64±0.657.84 ± 0.45Final body weight (g)149.42±7.9147.87±12.62148.23±12.49151.29±14.71WGR (%)162.16±15.80157.26±21.29157.32±24.00161.59±25.53SGR (%)1.60±0.101.57±0.141.57±0.151.60±0.16FCR1.57±0.111.59±0.181.61±0.171.58±0.17SR(%)100100100100VSI (%)8.62±0.388.32±0.677.78±0.487.80±0.35HSI (%)1.95±0.261.89±0.031.90±0.141.87±0.17CF(g/cm3)2.31±0.082.35±0.052.21±0.112.36±0.06Data are shown asmeans±SD (n=3). Dietary gossypol acetate had little effect on the contents of WGR, SGR, VSI, HSI, and FCR in C. carpio (P>0.05). WGR: weight gain rate; SGR: specific growth rate; SR: survival rate; VSI: visceral index; HSI: hepatosomatic index; FCR: feed conversation ratio.


### 3.2. Gossypol Acetate Deposition in Tissues ofC. carpio

No free gossypol was found in the serum, bile, and hepatopancreas of fish from the control and 170 mg/kg groups. The higher levels of dietary gossypol acetate caused the deposition of gossypol acetate in the serum, bile, and hepatopancreas of common carp and increased with increasing levels in the diet (Table3). In the 680 mg/kg group, the concentration of free gossypol was 197.62 μg/g (hepatopancreas), 0.47 μg/mL (bile), and 0.22 μg/mL (serum), which were significantly higher than those in other groups (P<0.05). The deposition order of free gossypol in the fish was hepatopancreas > bile > serum.Table 3
Free gossypol in the tissues ofCyprinus carpio.
GroupGossypol acetate supplementation (mg/kg)Tissue0170340680Serum (μg/mL)0.00±0.00b0.00±0.00b0.21±0.02a0.22±0.03aBile (μg/ml)0.00±0.00c0.00±0.00c0.35±0.03b0.47±0.06aHepatopancreas (μg/g)0.00±0.00c0.00±0.00c102.01±24.09b197.62±23.22aData are shown asmeans±SD (n=3). Lowercase letters in the same row indicate significant differences from the control group (P<0.05). Increasing the dietary gossypol acetate content also increased the deposition of gossypol acetate in the hepatopancreas (F), bile (G), and serum (H) in common carp. The free gossypol content was the highest in the hepatopancreas.


### 3.3. Effects of Gossypol Acetate on Serum Biochemical Parameters ofC. carpio

With increasing gossypol acetate concentration, the serum ALT content showed an increasing trend, and the level in the 680 mg/kg group was significantly higher than that in other groups (P<0.05). Similarly, the serum AST level in the 340 mg/kg and 680 mg/kg groups was significantly higher than that in the control group (P<0.05). In contrast, the serum levels of LDL-C, HDL-C, and TG showed a decreasing trend. In the 680 mg/kg group, the serum LDL-C level was significantly lower than that in the other three groups (P<0.05). The HDL-C and TG content in the 680 mg/kg gossypol acetate supplementation group was significantly lower than that in the control group (P<0.05) (Table 4).Table 4
Effects of gossypol acetate on serum biochemical parameters and liver crude fat.
GroupGossypol acetate supplementation (mg/kg)Parameter0170340680ALT (U/mL)4.91±0.65b5.08±0.61b5.87±0.29b10.79±1.38aAST ((U/mL)34.74±0.65b35.56±1.14b39.69±1.25a38.18±1.33aTG (mmol/L)1.59±0.03a1.13±0.07c1.26±0.02b1.09±0.02cLDL-C (mmol/L)3.58±0.09a3.57±0.17a3.49±0.17a2.99±0.07bHDL-C (mmol/L)2.57±0.19a2.57±0.06a2.35±0.26ab2.32±0.06bCrude fat in hepatopancreas (%)13.17±0.74b12.71±0.95bc11.61±0.86c22.6±1.11aIn the serum, dietary gossypol acetate increased the ALT and AST but decreased the LDL-C, HDL-C, and TG content and increased crude fat within the hepatopancreas. Data are shown asmeans±SD (n=3). Different superscript letters denote significant differences between the groups fed the different diets as determined by a one-way ANOVA with Tukey and Duncan’s comparison tests (P<0.05).


### 3.4. Effects of Gossypol Acetate on Hepatopancreas Lipids Content and Hepatopancreas Morphology in Common Carp

Our analysis of the lipid content in the hepatopancreas showed that the crude fat content in the hepatopancreas of the 680 mg/kg group was significantly higher than that of the other groups (P<0.05) (Table 4). Quadratic regression analysis showed that crude fat content in the hepatopancreas was the lowest when the gossypol acetate supplemental level was 198 mg/kg (Figure 1(a)). The HE staining of the hepatopancreas (Figure 1(b)) showed that an increase in gossypol acetate concentration had a corresponding significant increase in the fat vacuoles in the hepatopancreas cells. In the control group, the hepatocyte nucleus was located near the center of the cell. In the 170 mg/kg group, the hepatopancreas cells in the carp showed lipid vacuolation. In the 340 mg/kg group, the carp hepatocyte nucleus was significantly shifted with increased lipid vacuolization. In the 680 mg/kg group, the hepatopancreas cell nucleus was squeezed to one side with hyperaemia in the hepatic sinus. Similarly, the oil red staining showed that the fat content in the hepatopancreas gradually increased with increasing gossypol acetate supplementation in the diet (Figure 1(b)).Figure 1
Dietary gossypol acetate increased the lipid accumulation in the hepatopancreas and caused hepatocellular injury inCyprinus carpio. (a) Quadratic regression analysis showing the value of dietary gossypol acetate for increased hepatopancreas crude fat in carp for eight weeks. (b) Hepatopancreas tissue sections stained by HE and oil red. HC: hepatocytes; HS: hepatic sinusoids; LD: lipid droplet. Bar: 50 μm.
(a)(b)


### 3.5. Effects of Gossypol Acetate on FA Profile in Different Tissues of Common Carp

Total saturated FA (SA) decreased at the lower concentrations of gossypol acetate (170 mg/kg and 340 mg/kg) and increased when higher levels were provided (680 mg/kg) in both the brain and intestine. In contrast, the total SA content was significantly higher in the hepatopancreas of the 170 mg/kg group and significantly lower in that of the 340 mg/kg and 680 mg/kg groups (P<0.05). The total SA content was significantly lower in the muscle of all carp from the groups fed gossypol acetate. The variations in the stearic acid (C18:0) content in the four tissues of the different groups were consistent with the results of total SA (Figure 2(a) and Table S2). Conversely, the total n6 polyunsaturated FA (PUFA) in the brain was increased in carp fed with low doses of gossypol acetate (170 mg/kg and 340 mg/kg) and decreased in carp fed with the highest dose (680 mg/kg). The results were the opposite in the hepatopancreas. The n6-PUFA contents were significantly lower in the intestinal and muscular tissue of the gossypol acetate treatment groups (P<0.05) (Figure 2(b) and Table S2). The contents of docosahexaenoic acid (DHA, C22:6n3) and total n3-PUFA in the hepatopancreas of the 170 mg/kg and 680 mg/kg groups were significantly higher. Conversely, the contents of DHA and n3-PUFA in the muscle were significantly lower in all gossypol acetate supplement groups (Figure 2(c) and Table S2). The total PUFA content in the muscle was significantly lower in the gossypol acetate treatment groups (170, 340, and 680 mg/kg) (P<0.05) (Figure 2(d) and Table S2).Figure 2
Gossypol acetate altered the SA (a), n3-PUFA (b), n6-PUFA (c), and PUFA (d) contents in the brain, intestine, hepatopancreas, and muscle ofCyprinus carpio. Data are presented as LSMEAN±SE using at least three independent replicates. Letters indicate significant differences in the FAs between groups treated with different gossypol acetate concentrations in the same tissue (P<0.05)∗. When gossypol acetate was not added to the dietary feed (0 mg/kg gossypol acetate), the content of specific FA in the different tissues was significantly different from the brain tissues. ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. SA = C14:0 + C16:0 + C18:0 + C20:0; PUFA = C18:2n6 + C18:3n6 + C18:3n3 + C20:2 + C20:3n6 + C20:4n6 + C20:5n3 + C22:6; n3 = 18:3n3 + C20:5n3 + C22:6n3; n6 = C18:2n6 + C18:3n6 + C20:3n6 + C20:4n6.
(a)(b)(c)(d)In addition, regardless of the effect of gossypol acetate on FA composition, using the brain tissue as a control, the FA content in four tissues within the control group (0 mg/kg) was compared. Brain tissues were found to contain the highest total SA and the lowest total PUFAs and total n6-PUFA (P<0.001) (Figure 2 and Table S2).


### 3.6. Effect of Gossypol Acetate on the Expression of Key Genes Involved in the Lipid Metabolism Pathway

In the brain, the expression ofACACβ, PPARγ, SREBP-2, FADS2a, and FADS2b in the gossypol acetate treatment groups was significantly higher than that in the control group (P<0.05) (Figure 3(a)). The expression of HSL and PPARβ increased at low dosages (170 mg/kg and 340 mg/kg) and then decreased to the control levels in the high-dose group (680 mg/kg) (Figure 3(b)). The expression of CD36, FABP1b, FABP10a, and FABP10b showed a similar trend to that of the catabolic genes (P<0.05) (Figure 3(c)).Figure 3
Effects of gossypol acetate on the expression of genes related to lipid metabolism in the brain (a–c), intestine (d–f), hepatopancreas (g–i), and muscle (j–l). Data are presented asLSMEAN±SE using a minimum of three independent replicates. Different superscript letters denote significant differences between fish fed the different diets (P<0.05).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)In the intestine, the expression ofFASN and SREBP-1 in the gossypol acetate groups was significantly lower than that in the control group. The expression of ACACβ in the 170 mg/kg group was significantly higher than that in the other groups (Figure 3(d)). The expression of FADS2a and FADS2b was significantly higher in the 170 mg/kg and 340 mg/kg groups, but the highest dose (680 mg/kg) resulted in FADS2a and FADS2b expression levels as low as those in the control group (Figure 3(d)). The expression of ATGL, HSL, PPARα, and PPARβ showed a trend of increased levels at low doses and decreased levels at high doses of gossypol acetate (Figure 3(e)). The expression of CD36 and FABP1b was higher in carp fed 170 mg/kg of gossypol acetate and lower in those fed 340 mg/kg and 680 mg/kg of gossypol acetate. The expression of FABP10a and FABP10b was significantly higher in the gossypol acetate groups compared with that in the control group (P<0.05) (Figure 3(f)).In the hepatopancreas, the expression ofFASN, FADS2a, and FADS2b showed a trend of higher levels in carp fed lower doses of gossypol acetate and lower levels in those fed higher doses (Figure 3(g)). The expression of HSL and PPARα in the gossypol acetate-treated groups was significantly lower than that in the control group (P<0.05) (Figure 3(h)). The expression of FABP1b, FABP10a, and FABP10b in each experimental group was higher at low doses but lower as the dose of gossypol acetate increased. However, the expression of CD36 and FABP11 was significantly lower in the gossypol acetate-treated groups compared with that in the control group (P<0.05) (Figure 3(i)).In the muscle, the expression ofACACβ, SREBP1, CD36, FABP1b, FABP10a, ATGL, and PPARβ was significantly higher in the 680 mg/kg group than those in the control group (P<0.05) (Figures 3(j)–3(l)). However, the expression of FASN, FADS2a, and FABP10b was significantly lower in carp in the gossypol acetate treatment groups compared with that in the control group (P<0.05) (Figures 3(j) and 3(l)).


## 3.1. Effects of Gossypol Acetate on the Growth Performance and Morphometric Parameters ofC. carpio

The WGR (157.26–162.16%), SGR (1.57–1.60%), FCR (1.57–1.61), VSI (7.78–8.62%), HSI (1.87–1.95%), and CF (2.21–2.36) did not vary significantly among the treatment groups (P>0.05) (Table 2). The SR in all groups was 100%, meaning there was no mortality throughout the experiment.Table 2
Effect of gossypol acetate on growth performance ofCyprinus carpio.
GroupGossypol acetate supplementation (mg/kg)Parameter0170340680Initial body weight (g)57.02±0.9657.47±0.4357.64±0.657.84 ± 0.45Final body weight (g)149.42±7.9147.87±12.62148.23±12.49151.29±14.71WGR (%)162.16±15.80157.26±21.29157.32±24.00161.59±25.53SGR (%)1.60±0.101.57±0.141.57±0.151.60±0.16FCR1.57±0.111.59±0.181.61±0.171.58±0.17SR(%)100100100100VSI (%)8.62±0.388.32±0.677.78±0.487.80±0.35HSI (%)1.95±0.261.89±0.031.90±0.141.87±0.17CF(g/cm3)2.31±0.082.35±0.052.21±0.112.36±0.06Data are shown asmeans±SD (n=3). Dietary gossypol acetate had little effect on the contents of WGR, SGR, VSI, HSI, and FCR in C. carpio (P>0.05). WGR: weight gain rate; SGR: specific growth rate; SR: survival rate; VSI: visceral index; HSI: hepatosomatic index; FCR: feed conversation ratio.


## 3.2. Gossypol Acetate Deposition in Tissues ofC. carpio

No free gossypol was found in the serum, bile, and hepatopancreas of fish from the control and 170 mg/kg groups. The higher levels of dietary gossypol acetate caused the deposition of gossypol acetate in the serum, bile, and hepatopancreas of common carp and increased with increasing levels in the diet (Table3). In the 680 mg/kg group, the concentration of free gossypol was 197.62 μg/g (hepatopancreas), 0.47 μg/mL (bile), and 0.22 μg/mL (serum), which were significantly higher than those in other groups (P<0.05). The deposition order of free gossypol in the fish was hepatopancreas > bile > serum.Table 3
Free gossypol in the tissues ofCyprinus carpio.
GroupGossypol acetate supplementation (mg/kg)Tissue0170340680Serum (μg/mL)0.00±0.00b0.00±0.00b0.21±0.02a0.22±0.03aBile (μg/ml)0.00±0.00c0.00±0.00c0.35±0.03b0.47±0.06aHepatopancreas (μg/g)0.00±0.00c0.00±0.00c102.01±24.09b197.62±23.22aData are shown asmeans±SD (n=3). Lowercase letters in the same row indicate significant differences from the control group (P<0.05). Increasing the dietary gossypol acetate content also increased the deposition of gossypol acetate in the hepatopancreas (F), bile (G), and serum (H) in common carp. The free gossypol content was the highest in the hepatopancreas.


## 3.3. Effects of Gossypol Acetate on Serum Biochemical Parameters ofC. carpio

With increasing gossypol acetate concentration, the serum ALT content showed an increasing trend, and the level in the 680 mg/kg group was significantly higher than that in other groups (P<0.05). Similarly, the serum AST level in the 340 mg/kg and 680 mg/kg groups was significantly higher than that in the control group (P<0.05). In contrast, the serum levels of LDL-C, HDL-C, and TG showed a decreasing trend. In the 680 mg/kg group, the serum LDL-C level was significantly lower than that in the other three groups (P<0.05). The HDL-C and TG content in the 680 mg/kg gossypol acetate supplementation group was significantly lower than that in the control group (P<0.05) (Table 4).Table 4
Effects of gossypol acetate on serum biochemical parameters and liver crude fat.
GroupGossypol acetate supplementation (mg/kg)Parameter0170340680ALT (U/mL)4.91±0.65b5.08±0.61b5.87±0.29b10.79±1.38aAST ((U/mL)34.74±0.65b35.56±1.14b39.69±1.25a38.18±1.33aTG (mmol/L)1.59±0.03a1.13±0.07c1.26±0.02b1.09±0.02cLDL-C (mmol/L)3.58±0.09a3.57±0.17a3.49±0.17a2.99±0.07bHDL-C (mmol/L)2.57±0.19a2.57±0.06a2.35±0.26ab2.32±0.06bCrude fat in hepatopancreas (%)13.17±0.74b12.71±0.95bc11.61±0.86c22.6±1.11aIn the serum, dietary gossypol acetate increased the ALT and AST but decreased the LDL-C, HDL-C, and TG content and increased crude fat within the hepatopancreas. Data are shown asmeans±SD (n=3). Different superscript letters denote significant differences between the groups fed the different diets as determined by a one-way ANOVA with Tukey and Duncan’s comparison tests (P<0.05).


## 3.4. Effects of Gossypol Acetate on Hepatopancreas Lipids Content and Hepatopancreas Morphology in Common Carp

Our analysis of the lipid content in the hepatopancreas showed that the crude fat content in the hepatopancreas of the 680 mg/kg group was significantly higher than that of the other groups (P<0.05) (Table 4). Quadratic regression analysis showed that crude fat content in the hepatopancreas was the lowest when the gossypol acetate supplemental level was 198 mg/kg (Figure 1(a)). The HE staining of the hepatopancreas (Figure 1(b)) showed that an increase in gossypol acetate concentration had a corresponding significant increase in the fat vacuoles in the hepatopancreas cells. In the control group, the hepatocyte nucleus was located near the center of the cell. In the 170 mg/kg group, the hepatopancreas cells in the carp showed lipid vacuolation. In the 340 mg/kg group, the carp hepatocyte nucleus was significantly shifted with increased lipid vacuolization. In the 680 mg/kg group, the hepatopancreas cell nucleus was squeezed to one side with hyperaemia in the hepatic sinus. Similarly, the oil red staining showed that the fat content in the hepatopancreas gradually increased with increasing gossypol acetate supplementation in the diet (Figure 1(b)).Figure 1
Dietary gossypol acetate increased the lipid accumulation in the hepatopancreas and caused hepatocellular injury inCyprinus carpio. (a) Quadratic regression analysis showing the value of dietary gossypol acetate for increased hepatopancreas crude fat in carp for eight weeks. (b) Hepatopancreas tissue sections stained by HE and oil red. HC: hepatocytes; HS: hepatic sinusoids; LD: lipid droplet. Bar: 50 μm.
(a)(b)


## 3.5. Effects of Gossypol Acetate on FA Profile in Different Tissues of Common Carp

Total saturated FA (SA) decreased at the lower concentrations of gossypol acetate (170 mg/kg and 340 mg/kg) and increased when higher levels were provided (680 mg/kg) in both the brain and intestine. In contrast, the total SA content was significantly higher in the hepatopancreas of the 170 mg/kg group and significantly lower in that of the 340 mg/kg and 680 mg/kg groups (P<0.05). The total SA content was significantly lower in the muscle of all carp from the groups fed gossypol acetate. The variations in the stearic acid (C18:0) content in the four tissues of the different groups were consistent with the results of total SA (Figure 2(a) and Table S2). Conversely, the total n6 polyunsaturated FA (PUFA) in the brain was increased in carp fed with low doses of gossypol acetate (170 mg/kg and 340 mg/kg) and decreased in carp fed with the highest dose (680 mg/kg). The results were the opposite in the hepatopancreas. The n6-PUFA contents were significantly lower in the intestinal and muscular tissue of the gossypol acetate treatment groups (P<0.05) (Figure 2(b) and Table S2). The contents of docosahexaenoic acid (DHA, C22:6n3) and total n3-PUFA in the hepatopancreas of the 170 mg/kg and 680 mg/kg groups were significantly higher. Conversely, the contents of DHA and n3-PUFA in the muscle were significantly lower in all gossypol acetate supplement groups (Figure 2(c) and Table S2). The total PUFA content in the muscle was significantly lower in the gossypol acetate treatment groups (170, 340, and 680 mg/kg) (P<0.05) (Figure 2(d) and Table S2).Figure 2
Gossypol acetate altered the SA (a), n3-PUFA (b), n6-PUFA (c), and PUFA (d) contents in the brain, intestine, hepatopancreas, and muscle ofCyprinus carpio. Data are presented as LSMEAN±SE using at least three independent replicates. Letters indicate significant differences in the FAs between groups treated with different gossypol acetate concentrations in the same tissue (P<0.05)∗. When gossypol acetate was not added to the dietary feed (0 mg/kg gossypol acetate), the content of specific FA in the different tissues was significantly different from the brain tissues. ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001. SA = C14:0 + C16:0 + C18:0 + C20:0; PUFA = C18:2n6 + C18:3n6 + C18:3n3 + C20:2 + C20:3n6 + C20:4n6 + C20:5n3 + C22:6; n3 = 18:3n3 + C20:5n3 + C22:6n3; n6 = C18:2n6 + C18:3n6 + C20:3n6 + C20:4n6.
(a)(b)(c)(d)In addition, regardless of the effect of gossypol acetate on FA composition, using the brain tissue as a control, the FA content in four tissues within the control group (0 mg/kg) was compared. Brain tissues were found to contain the highest total SA and the lowest total PUFAs and total n6-PUFA (P<0.001) (Figure 2 and Table S2).


## 3.6. Effect of Gossypol Acetate on the Expression of Key Genes Involved in the Lipid Metabolism Pathway

In the brain, the expression ofACACβ, PPARγ, SREBP-2, FADS2a, and FADS2b in the gossypol acetate treatment groups was significantly higher than that in the control group (P<0.05) (Figure 3(a)). The expression of HSL and PPARβ increased at low dosages (170 mg/kg and 340 mg/kg) and then decreased to the control levels in the high-dose group (680 mg/kg) (Figure 3(b)). The expression of CD36, FABP1b, FABP10a, and FABP10b showed a similar trend to that of the catabolic genes (P<0.05) (Figure 3(c)).Figure 3
Effects of gossypol acetate on the expression of genes related to lipid metabolism in the brain (a–c), intestine (d–f), hepatopancreas (g–i), and muscle (j–l). Data are presented asLSMEAN±SE using a minimum of three independent replicates. Different superscript letters denote significant differences between fish fed the different diets (P<0.05).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)In the intestine, the expression ofFASN and SREBP-1 in the gossypol acetate groups was significantly lower than that in the control group. The expression of ACACβ in the 170 mg/kg group was significantly higher than that in the other groups (Figure 3(d)). The expression of FADS2a and FADS2b was significantly higher in the 170 mg/kg and 340 mg/kg groups, but the highest dose (680 mg/kg) resulted in FADS2a and FADS2b expression levels as low as those in the control group (Figure 3(d)). The expression of ATGL, HSL, PPARα, and PPARβ showed a trend of increased levels at low doses and decreased levels at high doses of gossypol acetate (Figure 3(e)). The expression of CD36 and FABP1b was higher in carp fed 170 mg/kg of gossypol acetate and lower in those fed 340 mg/kg and 680 mg/kg of gossypol acetate. The expression of FABP10a and FABP10b was significantly higher in the gossypol acetate groups compared with that in the control group (P<0.05) (Figure 3(f)).In the hepatopancreas, the expression ofFASN, FADS2a, and FADS2b showed a trend of higher levels in carp fed lower doses of gossypol acetate and lower levels in those fed higher doses (Figure 3(g)). The expression of HSL and PPARα in the gossypol acetate-treated groups was significantly lower than that in the control group (P<0.05) (Figure 3(h)). The expression of FABP1b, FABP10a, and FABP10b in each experimental group was higher at low doses but lower as the dose of gossypol acetate increased. However, the expression of CD36 and FABP11 was significantly lower in the gossypol acetate-treated groups compared with that in the control group (P<0.05) (Figure 3(i)).In the muscle, the expression ofACACβ, SREBP1, CD36, FABP1b, FABP10a, ATGL, and PPARβ was significantly higher in the 680 mg/kg group than those in the control group (P<0.05) (Figures 3(j)–3(l)). However, the expression of FASN, FADS2a, and FABP10b was significantly lower in carp in the gossypol acetate treatment groups compared with that in the control group (P<0.05) (Figures 3(j) and 3(l)).


## 4. Discussion

### 4.1. Dietary Gossypol Acetate Has Limited Effects on the Growth Performance ofC. carpio

Previous research has shown that gossypol can inhibit growth in poultry [26] and aquatic animals [2]. Fish are more tolerant of gossypol than terrestrial animals [2]. For example, O. aureus could tolerate dosages of gossypol up to 1,800 mg/kg in their diet [27]. Conversely, another study found that 900 mg/kg of dietary gossypol acetate could significantly reduce the growth performance of tilapia (O. niloticus×O. aureus) [19], whereas 198.14 mg/kg and 267.02 mg/kg of dietary gossypol caused no significant difference in the WGR and SGR of Nile tilapia (O. niloticus) [9]. Turbot fed with diets supplemented with 600 mg/kg gossypol showed no significant change in their growth performance when compared with fish fed the same diet without gossypol, but growth significantly decreased when the fish were fed diets supplemented with 1200 mg/kg gossypol [18]. The growth of rainbow trout was not affected by 200–600 mg/kg gossypol content in their diet, but growth was impeded when the gossypol content was increased to 1000 mg/kg [28]. In channel catfish (Ictalurus punctatus), slow growth was observed when gossypol was higher than 1400 mg/kg in the diet [15], but another study found that 300 mg/kg gossypol in the diet slowed catfish growth [29].Our results are in agreement with the previous research findings. When compared with the control group, there were no significant differences in growth performance among all gossypol acetate supplement groups (170, 340, and 680 mg/kg), indicating that the common carp can tolerate gossypol acetate. According to the Agricultural Industry Standard of the People’s Republic of China (NY 5072-200), the gossypol content in fish compound feed should be no more than 300 mg/kg. In the present study, the concentration of gossypol acetate did not significantly exceed the safe range, which may be within the tolerance range of carp. Therefore, there were no significant differences in WGR, SGR, FCR, SR, VSI, HSI, and CF among the different groups. Moreover, the species, diet composition, and environmental conditions in the different studies may account for the variations in the results.


### 4.2. Hepatopancreas as the Primary Gossypol Storage Tissue

Previous studies have suggested that the liver is the preferred storage site for gossypol. For example, residues of gossypol were found in chicken organs in the following order: liver > kidney > spleen > heart > muscle [11]. In lambs fed cottonseed, the residual levels of gossypol in the tissues were in the following order: liver > heart > muscle > spleen [30]. In rainbow trout, the gossypol concentration was highest in the hepatopancreas, followed by the kidney, intestine, testis, blood plasma, stomach, and muscle [31]. Similarly, the present study found that free gossypol mainly resided in the hepatopancreas.


### 4.3. Dietary Gossypol Acetate Increased the Content of ALT and AST but Decreased the Content of LDL-C, HDL-C, and TG in Serum

Studies in rats showed that the serum transaminase content (ALT and AST) significantly increased after gossypol acetate treatment [32]. In addition, a study conducted on tilapia found that the ALT content in fish fed with diets supplemented with 1200 mg/kg gossypol was significantly higher than that of the control group (P<0.05) [19]. In the present study, the levels of ALT and AST increased significantly in carp treated with 680 mg/kg gossypol acetate, which supports the previous findings. The increase in serum ALT and AST levels indicated that gossypol acetate may induce hepatic cell destruction. In addition, the levels of LDL-C, HDL-C, and TG in the 680 mg/kg gossypol acetate-treated group decreased significantly (P<0.05), which was consistent with the results of the previous studies [33]. These results suggest that the ability of lipids accumulated in the hepatopancreas to be transported to the peripheral tissues was blocked, restricting the transfer of excessive fat in the hepatopancreas, resulting in excessive fat accumulation in the hepatopancreas.


### 4.4. Dietary Gossypol Acetate Caused Excessive Lipid Accumulation and Cell Injury in the Hepatopancreas, Which Is Regulated by Genes Related to Lipid Metabolism

Studies on livestock, poultry, and mammals have found that gossypol can cause pathological changes, such as oxidative stress, hepatopancreas lipid accumulation, hepatopancreas tissue lesions, and other pathological changes in terrestrial animals [33, 34]. In addition, gossypol can inhibit FA desaturation and lead to an increase in cholesterol levels in the body [7]. Studies on fish also found that supplementing the diet with gossypol resulted in nuclear migration, loose arrangements, necrosis, and other damage in the hepatopancreas [6, 18]. In the present study, an increase in gossypol acetate concentration caused hepatopancreas tissue lesions to be aggravated and fat content to increase. Hepatopancreas sinusoid hyperaemia was observed in the 680 mg/kg group, and the hepatopancreatic nuclei were squeezed to the edge of the cell. Additionally, the crude lipid content in the hepatopancreas of the high gossypol acetate treatment group (680 mg/kg) was significantly higher than that in the control group. These results are supported by the previous studies and suggest that gossypol exposure could increase fat accumulation in the hepatopancreas and cause hepatopancreatic tissue damage in common carp.Lipid metabolism is a complex process involving synthesis, decomposition, and transport and is regulated by many genes and tissues. To elucidate the reasons for the increased hepatopancreatic fat accumulation after gossypol treatment, the expression of key genes was detected in the lipid metabolism pathways. Fat synthesis genes, includingFASN, ACACβ, PPARγ, SREBP-1, and SREBP-2; lipolysis genes, including ATGL, HSL, PPARα, and PPARβ; and FA-binding and transporter genes, including CD36, FABP1b, FABP10a, FABP10b, and FABP11 were further analysed. To understand the causes of the differences in the composition and PUFAs content, including DHA (C22:6n3) in the different tissues, the expression of the FA desaturase genes (FADS2a and FADS2b) was tested.In this study, the expression of most synthesis and decomposition genes showed similar trends in the intestine and muscle but displayed the opposite trends in the brain. For example, the gossypol acetate increased the expression of genes both in synthesis and catabolism in the brain but decreased the expression levels in the intestine, hepatopancreas, and muscle. The differences in gene expression among various tissues may account for metabolic physiological differences. However, the specific gene regulatory network across different tissues requires further research.In the present study, gossypol acetate inhibited the mRNA expression of genes involved with triglyceride decomposition (HSL and PPARα) in the hepatopancreas of carp. Low expression of these genes may lead to liver fat accumulation.FA translocase (CD36) is an integral membrane glycoprotein that mediates long-chain FA transport [35–37]. Numerous studies have shown that CD36 can enhance FA uptake in various tissues, including the skeletal muscle, heart, liver, and intestine [38–40]. In the present study, the expression of CD36 was significantly decreased in the hepatopancreas of the gossypol acetate-treated groups (170 mg/kg, 340 mg/kg, and 680 mg/kg). In contrast, the expression of CD36 was increased in the muscle with the increase of gossypol acetate. This result indicates that dietary gossypol acetate increases the uptake of FAs in the muscle, which may have been transported from the liver and intestine. However, the transport and balance of FAs between different tissues require further research.In vivo, free FAs are cytotoxic and need to bind to FA-binding proteins (FABPs) [41], such as the hepatopancreas-type binding protein (FABP1) and the liver-based FA-binding protein (FABP10), which are mainly expressed in the hepatopancreas [42–44]. In the present study, the expression of FABP1b, FABP10a, and FABP10a in the hepatopancreas of the gossypol acetate-treated groups was significantly higher than that in the control group, indicating that an increase in hepatopancreas lipid synthesis requires more FABPs to bind with the free FAs, thereby reducing the damage caused by hepatopancreas fat accumulation.


### 4.5. Dietary Gossypol Acetate Altered FA Composition

The abundant PUFAs in fish muscle improve the nutritional value. However, the synthesis and accumulation of PUFAs in muscle is a complex process that requires multiple tissues to regulate the uptake, absorption, transport, and metabolism of PUFAs in fish. However, the specific mechanisms remain unclear. Therefore, the composition and content of FAs in different tissues were analysed to provide a theoretical basis for further research on the deposition of PUFAs and other FAs in muscles.Studies investigating the effects of gossypol acetate on FA composition remain limited. Previous research has identified that supplementation with cottonseed oil can alter FA composition in hens. For example, dietary gossypol acetate decreased the content of C18:0 in the liver of laying hens [3]. Similarly, in the present study, gossypol acetate decreased the content of C18:0 in the hepatopancreas and muscle tissue. Gossypol acetate could regulate the metabolism of FAs to varying degrees in different tissues, and the SA and PUFA contents showed opposite trends in the brain and hepatopancreas. Additionally, dietary gossypol acetate significantly decreased the content of DHA, n3-PUFAs, n6-PUFAs, and total PUFAs in the muscle. These findings indicate that adding gossypol acetate to the diet reduced the muscle quality of common carp.Fatty acid desaturases (FADS2a and FADS2b) are key enzymes in the synthesis of PUFAs. In the present study, the contents of key PUFAs in the intestine and muscle were at levels consistent with those of the tissue expression of FADS2a and FADS2b. In particular, the expression of FADS2a was significantly decreased in the muscle of all gossypol acetate-treated groups, which may be the main cause for the decrease in PUFAs.


## 4.1. Dietary Gossypol Acetate Has Limited Effects on the Growth Performance ofC. carpio

Previous research has shown that gossypol can inhibit growth in poultry [26] and aquatic animals [2]. Fish are more tolerant of gossypol than terrestrial animals [2]. For example, O. aureus could tolerate dosages of gossypol up to 1,800 mg/kg in their diet [27]. Conversely, another study found that 900 mg/kg of dietary gossypol acetate could significantly reduce the growth performance of tilapia (O. niloticus×O. aureus) [19], whereas 198.14 mg/kg and 267.02 mg/kg of dietary gossypol caused no significant difference in the WGR and SGR of Nile tilapia (O. niloticus) [9]. Turbot fed with diets supplemented with 600 mg/kg gossypol showed no significant change in their growth performance when compared with fish fed the same diet without gossypol, but growth significantly decreased when the fish were fed diets supplemented with 1200 mg/kg gossypol [18]. The growth of rainbow trout was not affected by 200–600 mg/kg gossypol content in their diet, but growth was impeded when the gossypol content was increased to 1000 mg/kg [28]. In channel catfish (Ictalurus punctatus), slow growth was observed when gossypol was higher than 1400 mg/kg in the diet [15], but another study found that 300 mg/kg gossypol in the diet slowed catfish growth [29].Our results are in agreement with the previous research findings. When compared with the control group, there were no significant differences in growth performance among all gossypol acetate supplement groups (170, 340, and 680 mg/kg), indicating that the common carp can tolerate gossypol acetate. According to the Agricultural Industry Standard of the People’s Republic of China (NY 5072-200), the gossypol content in fish compound feed should be no more than 300 mg/kg. In the present study, the concentration of gossypol acetate did not significantly exceed the safe range, which may be within the tolerance range of carp. Therefore, there were no significant differences in WGR, SGR, FCR, SR, VSI, HSI, and CF among the different groups. Moreover, the species, diet composition, and environmental conditions in the different studies may account for the variations in the results.


## 4.2. Hepatopancreas as the Primary Gossypol Storage Tissue

Previous studies have suggested that the liver is the preferred storage site for gossypol. For example, residues of gossypol were found in chicken organs in the following order: liver > kidney > spleen > heart > muscle [11]. In lambs fed cottonseed, the residual levels of gossypol in the tissues were in the following order: liver > heart > muscle > spleen [30]. In rainbow trout, the gossypol concentration was highest in the hepatopancreas, followed by the kidney, intestine, testis, blood plasma, stomach, and muscle [31]. Similarly, the present study found that free gossypol mainly resided in the hepatopancreas.


## 4.3. Dietary Gossypol Acetate Increased the Content of ALT and AST but Decreased the Content of LDL-C, HDL-C, and TG in Serum

Studies in rats showed that the serum transaminase content (ALT and AST) significantly increased after gossypol acetate treatment [32]. In addition, a study conducted on tilapia found that the ALT content in fish fed with diets supplemented with 1200 mg/kg gossypol was significantly higher than that of the control group (P<0.05) [19]. In the present study, the levels of ALT and AST increased significantly in carp treated with 680 mg/kg gossypol acetate, which supports the previous findings. The increase in serum ALT and AST levels indicated that gossypol acetate may induce hepatic cell destruction. In addition, the levels of LDL-C, HDL-C, and TG in the 680 mg/kg gossypol acetate-treated group decreased significantly (P<0.05), which was consistent with the results of the previous studies [33]. These results suggest that the ability of lipids accumulated in the hepatopancreas to be transported to the peripheral tissues was blocked, restricting the transfer of excessive fat in the hepatopancreas, resulting in excessive fat accumulation in the hepatopancreas.


## 4.4. Dietary Gossypol Acetate Caused Excessive Lipid Accumulation and Cell Injury in the Hepatopancreas, Which Is Regulated by Genes Related to Lipid Metabolism

Studies on livestock, poultry, and mammals have found that gossypol can cause pathological changes, such as oxidative stress, hepatopancreas lipid accumulation, hepatopancreas tissue lesions, and other pathological changes in terrestrial animals [33, 34]. In addition, gossypol can inhibit FA desaturation and lead to an increase in cholesterol levels in the body [7]. Studies on fish also found that supplementing the diet with gossypol resulted in nuclear migration, loose arrangements, necrosis, and other damage in the hepatopancreas [6, 18]. In the present study, an increase in gossypol acetate concentration caused hepatopancreas tissue lesions to be aggravated and fat content to increase. Hepatopancreas sinusoid hyperaemia was observed in the 680 mg/kg group, and the hepatopancreatic nuclei were squeezed to the edge of the cell. Additionally, the crude lipid content in the hepatopancreas of the high gossypol acetate treatment group (680 mg/kg) was significantly higher than that in the control group. These results are supported by the previous studies and suggest that gossypol exposure could increase fat accumulation in the hepatopancreas and cause hepatopancreatic tissue damage in common carp.Lipid metabolism is a complex process involving synthesis, decomposition, and transport and is regulated by many genes and tissues. To elucidate the reasons for the increased hepatopancreatic fat accumulation after gossypol treatment, the expression of key genes was detected in the lipid metabolism pathways. Fat synthesis genes, includingFASN, ACACβ, PPARγ, SREBP-1, and SREBP-2; lipolysis genes, including ATGL, HSL, PPARα, and PPARβ; and FA-binding and transporter genes, including CD36, FABP1b, FABP10a, FABP10b, and FABP11 were further analysed. To understand the causes of the differences in the composition and PUFAs content, including DHA (C22:6n3) in the different tissues, the expression of the FA desaturase genes (FADS2a and FADS2b) was tested.In this study, the expression of most synthesis and decomposition genes showed similar trends in the intestine and muscle but displayed the opposite trends in the brain. For example, the gossypol acetate increased the expression of genes both in synthesis and catabolism in the brain but decreased the expression levels in the intestine, hepatopancreas, and muscle. The differences in gene expression among various tissues may account for metabolic physiological differences. However, the specific gene regulatory network across different tissues requires further research.In the present study, gossypol acetate inhibited the mRNA expression of genes involved with triglyceride decomposition (HSL and PPARα) in the hepatopancreas of carp. Low expression of these genes may lead to liver fat accumulation.FA translocase (CD36) is an integral membrane glycoprotein that mediates long-chain FA transport [35–37]. Numerous studies have shown that CD36 can enhance FA uptake in various tissues, including the skeletal muscle, heart, liver, and intestine [38–40]. In the present study, the expression of CD36 was significantly decreased in the hepatopancreas of the gossypol acetate-treated groups (170 mg/kg, 340 mg/kg, and 680 mg/kg). In contrast, the expression of CD36 was increased in the muscle with the increase of gossypol acetate. This result indicates that dietary gossypol acetate increases the uptake of FAs in the muscle, which may have been transported from the liver and intestine. However, the transport and balance of FAs between different tissues require further research.In vivo, free FAs are cytotoxic and need to bind to FA-binding proteins (FABPs) [41], such as the hepatopancreas-type binding protein (FABP1) and the liver-based FA-binding protein (FABP10), which are mainly expressed in the hepatopancreas [42–44]. In the present study, the expression of FABP1b, FABP10a, and FABP10a in the hepatopancreas of the gossypol acetate-treated groups was significantly higher than that in the control group, indicating that an increase in hepatopancreas lipid synthesis requires more FABPs to bind with the free FAs, thereby reducing the damage caused by hepatopancreas fat accumulation.


## 4.5. Dietary Gossypol Acetate Altered FA Composition

The abundant PUFAs in fish muscle improve the nutritional value. However, the synthesis and accumulation of PUFAs in muscle is a complex process that requires multiple tissues to regulate the uptake, absorption, transport, and metabolism of PUFAs in fish. However, the specific mechanisms remain unclear. Therefore, the composition and content of FAs in different tissues were analysed to provide a theoretical basis for further research on the deposition of PUFAs and other FAs in muscles.Studies investigating the effects of gossypol acetate on FA composition remain limited. Previous research has identified that supplementation with cottonseed oil can alter FA composition in hens. For example, dietary gossypol acetate decreased the content of C18:0 in the liver of laying hens [3]. Similarly, in the present study, gossypol acetate decreased the content of C18:0 in the hepatopancreas and muscle tissue. Gossypol acetate could regulate the metabolism of FAs to varying degrees in different tissues, and the SA and PUFA contents showed opposite trends in the brain and hepatopancreas. Additionally, dietary gossypol acetate significantly decreased the content of DHA, n3-PUFAs, n6-PUFAs, and total PUFAs in the muscle. These findings indicate that adding gossypol acetate to the diet reduced the muscle quality of common carp.Fatty acid desaturases (FADS2a and FADS2b) are key enzymes in the synthesis of PUFAs. In the present study, the contents of key PUFAs in the intestine and muscle were at levels consistent with those of the tissue expression of FADS2a and FADS2b. In particular, the expression of FADS2a was significantly decreased in the muscle of all gossypol acetate-treated groups, which may be the main cause for the decrease in PUFAs.


## 5. Conclusions

In conclusion, the findings indicate that dietary gossypol acetate has little effect on the growth performance of carp. However, high concentrations of gossypol acetate (680 mg/kg) induce hepatopancreatic injury and excessive lipid accumulation in hepatopancreas and decrease PUFAs in the muscle, which are regulated by the genes involved in lipid metabolism, including synthesis, decomposition, transport, and binding. The present study details the novel approach of using a product with toxic components as a food source in animal production. Furthermore, this product has previously been tested and will continue to be developed as a useful fish feed that will have long-term effects on the industry and environment.



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*Source: 2969246-2022-04-04.xml*