Abstract
There is a dearth of experimental evidence available as to whether the consumption of fermented pork fat (FPF) food has any harmful effects on metabolism and reproduction due to its excessive calories, high fat content, and fatty acid methyl ester (FAME) levels. We hypothesized that exposure to a FPF-diet with excessive calories, a high fat content, and high FAME levels alters testicular physiology and metabolism, leading to permanent damage to the testicular system and its function. Thirteen-week-old male rats (n = 20) were assigned to a high-calorie, high-fat diet (FPF-H, fat-60%, 23 kJ/g), a moderate-calorie, moderate-fat diet (FPF-M, fat-30%, 17.5 kJ/g), a low-calorie and low-fat diet (FPF-L, fat-15%, 14.21 kJ/g) compared to the standard diet (Control, fat-11%, 12.56 kJ/g) orally for 90 days. GC–MS analysis of the three FPF-diets showed high quantities of saturated fatty acids (SFAs) and polyunsaturated fatty acids-ω6 (PUFA-ω6) and low levels of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids-ω3 (PUFA-ω3) compared to the control diet. Consequently, the levels of serum FAMEs of the FPF-diet fed rats were significantly increased. In addition, a high level of n-6:n-3 PUFA towards PUFA-ω6 was observed in the serum of FPF-diet fed rats due to the high content of linoleic, γ-linolenic, and arachidonic acid. Long-term consumption of FPF-diets disturbed the anthropometrical, nutritional, physiological, and metabolic profiles. Furthermore, administration of FPF-diets generated metabolic syndrome (dyslipidemia, leptinemia, insulin resistance, obesity, hepato-renal disorder and function), increased the cardiovascular risk factors, and triggered serum and testis inflammatory markers (interleukin-1↑, interleukin-6↑, interleukin-10↓, leukotriene B4↑, prostaglandin↑, nitric oxide↑, myeloperoxidase↑, lactate dehydrogenase↑, and tumor necrosis factor-α↑). Activated testis oxidative stress (conjugated dienes↑, lipid hydroperoxides↑, malondialdehyde↑, protein carbonyl↑, and fragmented DNA↑) and depleted antioxidant reserve (catalase↓, superoxide dismutase↓, glutathione S-transferase↓, reduced glutathione↓, glutathione disulfide↑, and GSH:GSSG ratio↓) were observed in FPF-diet fed rats. Disrupted testis histoarchitecture, progressive deterioration of spermatogenesis, poor sperm quality and functional indices, significant alterations in the reproductive hormones (serum and testis testosterone↓, serum estradiol↑, serum luteinizing hormone↓, and follicle-stimulating hormone↑), were noted in rats fed with FPF diets than in the control diet. Severe steroidogenic impairment (steroidogenic acute regulatory protein, StAR↓; 3β-hydroxysteroid dehydrogenase, 3β-HSD↓; and luteinizing hormone receptor, LHR↓), deficiency in germ cells proliferation (proliferating cell nuclear antigen, PCNA↓), and abnormally enhanced testicular germ cell apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL assay↑; B-cell lymphoma-2, BCL-2↓; Bcl-2-associated X protein, BAX↑; and BAX/BCL-2 ratio↑) were remarked in the FPF-diet administered rats in comparison with the control diet. In conclusion, the long-term feeding of an FPF-diet with excessive calories, a high fat content, and high FAME levels induced oxidative stress, inflammation, and apoptosis, resulting in metabolic syndrome and hampering male reproductive system and functions. Therefore, the adoption of FPF diets correlates with irreversible changes in testis metabolism, steroidogenesis, germ cell proliferation, and apoptosis, which are related to permanent damage to the testicular system and function later in life.
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The authors declare that all necessary data supporting the findings of this study are available within the article.
Abbreviations
- 3β-HSD:
-
3β-Hydroxysteroid dehydrogenase
- AI:
-
Apoptotic index
- ALP:
-
Alkaline phosphatase
- ALT:
-
Alanine aminotransferase
- ANOVA:
-
Analysis of variance
- AO:
-
Acridine orange
- AOCS:
-
Association of Official Analytical Chemists
- Apaf-1:
-
Apoptotic protease activating factor-1
- ARRIVE:
-
Animal research: reporting of in vivo experiments
- AST:
-
Aspartate aminotransferase
- AtI:
-
Atherogenic index
- ATP:
-
Adenosine triphosphate
- BAX:
-
Bcl-2-associated X protein
- BCL-2:
-
B-cell lymphoma 2
- BMI:
-
Basal metabolic index
- BUN:
-
Blood urea nitrogen
- B:C ratio CAI:
-
BUN:Creatinine ratio Coronary artery index
- cAMP:
-
Adenosine 3',5'-cyclic monophosphate
- CAT:
-
Catalase
- CI:
-
Cardiac index
- DCA:
-
Detrended correspondence analysis
- DNA:
-
Deoxyribonucleic acid
- dsDNA:
-
Double stranded DNA
- DSP:
-
Daily sperm production
- EI:
-
Electron bomb ionization mode,
- FAME:
-
Fatty acid methyl esters
- FID:
-
Flame ionization detector
- FPF:
-
Fermented pork fat
- FPF-H:
-
Fermented pork high fat diet (high calorie, high fat and high FAME levels)
- FPF-L:
-
Fermented pork low fat diet (low calorie, low fat and low FAME levels)
- FPF-M:
-
Fermented pork medium fat diet (moderate calorie, moderate fat and moderate FAME levels)
- FSH:
-
Follicle-stimulating hormone
- GC-MS:
-
Gas chromatography mass spectrometry
- GSH:
-
Reduced glutathione
- GSSG:
-
Glutathione disulfide
- GST:
-
Glutathione S transferase
- H&E:
-
Hematoxylin and eosin
- HDL:
-
High density lipoprotein
- HOMA-IR:
-
Homeostasis model assessment of insulin resistance index
- HOMA-β:
-
Pancreatic β-cell function
- HPG:
-
Hypothalamic–pituitary–gonadal axis
- HRP:
-
Horse radish peroxidase
- IL-1:
-
Interleukin 1
- IL-10:
-
Interleukin-10
- IL-6:
-
Interleukin 6
- IS:
-
Interstitial space
- JTBS:
-
Johnsen’s mean testicular biopsy score
- LDH:
-
Lactic dehydrogenase
- LDL:
-
Low density lipoprotein
- LH:
-
Luteinizing hormone
- LHR:
-
Luteinizing hormone receptor
- LLL:
-
Trilinolein
- LPO:
-
1-Linolein-2-palmitin-3-olein
- LPS:
-
Lipopolysaccharide
- LTB4:
-
Leukotriene B4
- MANOVA:
-
Multivariate analysis of variance
- MPO :
-
Myeloperoxidase
- MSTD:
-
Mean seminiferous tubule diameter
- MUFA:
-
Monounsaturated fatty acid
- MZU-IAEC:
-
Mizoram university: Institutional animal ethics committee
- NAFLD:
-
Non-alcoholic fatty liver disease
- NIH:
-
National institute of health
- NNN:
-
Trinonadecanoyl-glycerol
- NO:
-
Nitric oxide
- OLL:
-
1,2-Linoleoyl-3-oleoyl-sn-glycerol
- OOL:
-
1,2-Dioleyl-3-linolenoylglycerol
- OOO:
-
1,2,3-Trioleylglycerol
- OOP:
-
1,2-Oleoyl-3-sn-palmitoylglycerol
- OOSt:
-
1-Stearoyl-2-oleoyl-3-oleoyl-glycerol
- OPO :
-
1,3-Oleoyl-2-palmitoyl-sn-glycerol
- PARP:
-
Poly (ADP-ribose) polymerase
- PAST:
-
PAleontological Statistics
- PCA:
-
Principal component analysis
- PCNA:
-
Proliferating cell nuclear antigen
- PLL:
-
Palmitodilinolein
- PLP:
-
1,3-Palmitoyl-2-linoleoyl-sn-glycerol
- POP:
-
1,3-Palmitoyl-2-oleoyl-sn-glycerol
- PPL:
-
1,2-Palmitoyl-3-linolein-sn-glycerol
- PPO:
-
Dipalmitoyl-oleoyl-glycerol
- PPP:
-
Tripalmitoyl-glycerol
- PPS:
-
1,2-Palmitoyl-3-stearoyl-sn-glycerol
- PUFA:
-
Polyunsaturated fatty acid
- ROS:
-
Reactive oxygen species
- SFA:
-
Saturated fatty acid
- SOD:
-
Superoxide dismutase
- ssDNA:
-
Single stranded DNA
- StAR:
-
Steroidogenic acute regulatory protein
- STEH:
-
Seminiferous tubule epithelial height
- TAG:
-
Triacylglycerols
- TAT:
-
Tunica albuginea thickness
- TC:
-
Total cholesterol
- TDI:
-
Tubule differentiation index
- TL:
-
Tubular lumen
- TNF-α:
-
Tumor necrosis factor α
- TUNEL:
-
Terminal deoxynucleotidyl transferase dUTP nick end labeling
- VLDL :
-
Very low-density lipoprotein
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Acknowledgements
The authors thank Mizoram University for providing infrastructural facilities and administrative support to carry out this research work.
Funding
This work was supported by the instrumentation facility in the Department of Zoology, Mizoram University through Government of India funded projects by the University Grants Commission (UGC STRIDE, No.F.2–2/2019 (STRIDE-I), 3 December 2019) and the Department of Science and Technology (DST-FIST, No. SR/FST/LS-I/2017/15(C), 6 September 2018), New Delhi to GG and VKR.
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Sailo Lalrinzuali: Investigation, Data analysis, Data acquisition, Supervision, Writing, Reviewing and editing, Formal analysis, Revision of the manuscript. Maurya Khushboo: Investigation, Data analysis, Data acquisition, Supervision, Writing, Reviewing and editing, Formal analysis, Revision of the manuscript. Roy Dinata: Data analysis, Data acquisition, Visualization, and formal analysis. Baishya Bhanushree: Data analysis, Data acquisition, Visualization, and formal analysis. Nisekhoto Nisa: Data analysis, Data acquisition, Visualization, and formal analysis. Rema Momin Bidanchi: Investigation, Data analysis, Data acquisition, Supervision, Writing, Reviewing and editing, Formal analysis, Revision of the manuscript. Saeed-Ahmed Laskar: Data analysis, Data acquisition, Visualization, and formal analysis. Bose Manikandan: Data analysis, Data acquisition, Visualization, and formal analysis. Giri Abinash: Data analysis, Data acquisition, Visualization, and formal analysis. Buragohain Pori: Data analysis, Data acquisition, Visualization, and formal analysis. Vikas Kumar Roy: Conceptualization, Funding acquisition, Designing the experiments, Supervision, Original draft preparation, Reviewing, Editing, Formal analysis, Visualization, and Investigation. Guruswami Gurusubramanian: Conceptualization, Funding acquisition, Designing the experiments, Supervision, Original draft preparation, Reviewing, Editing, Formal analysis, Visualization, and Investigation.
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All experiments performed followed the guidelines of Laboratory Animal Research of the Institutional Animal Care and Use Committee of the Mizoram University, Mizoram, India, ARRIVE and NIH guidelines for the care and use of laboratory animals (Approval Number: MZU-IAEC/2018/15 dt. 26/03/2018).
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Lalrinzuali, S., Khushboo, M., Dinata, R. et al. Long-term consumption of fermented pork fat-based diets differing in calorie, fat content, and fatty acid levels mediates oxidative stress, inflammation, redox imbalance, germ cell apoptosis, disruption of steroidogenesis, and testicular dysfunction in Wistar rats. Environ Sci Pollut Res 30, 52446–52471 (2023). https://doi.org/10.1007/s11356-023-26018-0
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DOI: https://doi.org/10.1007/s11356-023-26018-0