Kianen Sekiita1 , Emily Kiridi G.E2 , Solomon M. Uvoh3 , Bonnie K. Goodhope4 , Leghemo, Ebifaghe K5
1Department of Human Physiology School of Basic Medical Sciences, College of Health Sciences University of Benin, Benin City Edo State, Nigeria
2Department of Human Physiology, Faculty of Basic Medical Sciences, College of Health Sciences Niger Delta University Amassoma Bayelsa State, Nigeria
3Department of Human Physiology, Faculty of Basic Medical Sciences, College of Health Sciences University of port Harcourt Rivers State, Nigeria
4Department of Chemistry, Faculty of Science University of Lagos, Lagos State, Nigeria
5Bayelsa State College of Health Sciences, Nigeria
Corresponding Author Email: Solomonu31@gmail.com
DOI : https://doi.org/10.51470/AMSR.2025.04.02.73
Abstract
Aim/Objective: The aim of this research was to determine the effects of starvation-induced stress on reproductive parameters of Wistar rats fed with ketogenic diets. Materials/methods: Forty healthy male Wistar rats acclimatized for 14 days, weighing 110-200g when weighed with Golding meter (USA) digital weighing scale. Group A was the normal control while Group B was also a control; they were fed with ketogenic diet and water ad libitum daily without stress induction. Group C&D were administered 15hrs ketogenic diet, 9 hours starvation daily and 6 hours KD, 18 hours starvation daily. The hormonal profiles were determined using Tiet and Layman methods. Results: A significant decrease was observed in all three major hormones among the test groups considered in this study compared with the control group that was not subjected to starvation- induced stress. Comparing GP-B (control 2) fed with ketogenic diet and water ad libitum daily with GP-A (normal control 1) fed with standard diet and water ad libitum also shows a dcline in serum male hormones among KD-fed group with a significant p value in (LH) 18hours stress induced group. Regarding serum FSH, there was a significant (p-value ≤ 0.05) decrease among 9 and 18hrs stress-induced groups compared with the control while testosterone decrease was non-significant. Conclusion: Starvation-induced stress with ketogenic-diet possesses antigonadotrophic consequences. We therefore recommend that further research on this starvation-induced stress should not be limited to reproductive parameters only but to wide range of parameters involving other body systems.
Keywords
Download this article as: 
INTRODUCTION
Ketogenic diets have high fat concentration, low protein, and very low carbohydrates. They enhance ketone body production due to the breakdown of fat to produce energy [ 1]. KD is are effective treatment for most metabolic diseases, including tumour growth [2]. Studies have shown that KD has curative benefits. It has been recommended as a supplementary remedy for acne, conventional therapy, etc [3]. [26-4]. KD also assists in reducing cholesterol levels in obese patients and maintaining mood stability in bipolar disorder [5-6]. Clinical trial, findings indicate that KD consumption for 20 days significantly reduced deposits of CO2 in the body [3]. Irrespective of its popular use, there have been some concerns on some possible consequences of the ketogenic diet on the whole body system. Since the ketogenic diet substitutes glucose with fat as the main energy source, the body is induced to activate a series of fat metabolic processes for energy acquisition [7]. Fat metabolic processes produce acetyl coenzyme A (Acetyl-CoA) as the principal product, which then enters the tricarboxylic acid cycle and becomes oxidized to produce ATP [8]. Acetyl-CoA that exceeds the activity of the citric acid cycle and/or the availability of oxaloacetate leads to a rise in ketone bodies (acetoacetate, β-hydroxybutyrate, and acetone). This process is known as ketogenesis [8].Ketone bodies from KD are acidic; hence rapid removal of acids via the kidneys may decrease bicarbonate ions (HCO3–) [9]. As a consequence, the significance of the KD reduced blood pH, resulting in ketoacidosis [10]. The effect of a high-fat diet on the function of important organs, such as the liver and kidneys, has been effectively studied using several animal models [11,12-13].High KD has been shown to alter renal lipid metabolism in mice, especially the balance between lipolysis and lipogenesis, resulting in the accumulation of lipid in the kidneys and, hence, renal dysfunction [14].
MATERIALS AND METHODS
Ketogenic Diets
The Ketogenic diet preparation and the duration of administration were in accordance with [15]. Ketogenic diets are composed of 60% fats, 25% proteins, and 15% carbohydrates.
Preparation of the Ketogenic Diets
The ketogenic diet was prepared with a mixture of the Standard Diet and Margarine. 1kg of Margarine was added to 1kg of Standard Diet. Both components were hand-mixed thoroughly until no trace or sample of Margarine was noticed in the homogenous mixture.
LABORATORY ANIMALS USED
The animals used for this research were 40 healthy male Wistar rats with weights ranging from 110 to 200g using a Golden Meter USA scale calibrated in grams. They were acclimatized for two weeks and sheltered in 4 cages of 10 per group, having access to natural light, air, rat feeds, and water ad libitum (for the control group, while the test-groups were subjected to hours of fasting before the administration of ketogenic diets). [16)
RESEARCH DESIGN
▪ Group A (Control 1): 24 hours SD and wateronly. No Starvation
▪Group B (Control 2): 24 hours KD and water. No Starvation
▪ Group C: 15 hours KD and water. 9 hours’ starvation daily
▪Group D: 6 hours KD and water.18 hours’ starvation daily
SAMPLE COLLECTION AND ANALYSIS
Five rats were sacrifice from each group with a 5ml of blood collected via cardiac puncture for hormonal analysis.
Effect of hours of starvation on the testosterone level of Wistar rats on ketogenic diets
There were no significant differences in 9 hours and 18 hours’ starvation group compared with control.
Effect of hours of starvation on FSH of Wistar rats on ketogenic diets
There were significant decreases in 9 hours and 18 hours starvation group compared with control, but there were no significant changes in 18 hours starvation compared with 9 hours starvation.
Effect of hours of starvation on LH in Wistar rats on ketogenic diets
There was a significant decrease in 18 hours starvation compared with control and 9 hours starvation group, but there was no significant difference in 9 hours starvation compared with control
Effect of hours of starvation on the luteinizing hormone of Wistar rats on standard and ketogenic diets
There was a significant decrease in ketogenic diet group compared with the standard diet group at 18 hours’ starvation, though there were no significant changes among of ketogenic diet for the control and 9 hours’ starvation group compared with standard diet.
Effect of hours of starvation on the follicle-stimulating hormone of Wistar rats on standard and ketogenic diets
There were significant decreases in the ketogenic diet group compared with the standard diet group at 9 hours and 18 hours of starvation, though there was no significant change in the ketogenic diet group for the control group compared with the standard diet.
Effect of hours of starvation on the testosterone level of Wistar rats on standard and ketogenic diets
There were no significant changes in among group of the ketogenic diet for control, 9 hours, and 18 hours’ starvation group compared with the standard diet.
Effect of hours of starvation on the weight change of Wistar rats on standard and ketogenic diets
There were no significant changes in the group of the ketogenic diet for control, 9 hours, and 18 hours’ starvation group compared with the standard diet.
DISCUSSION
Body Weights in Wistar Rats Fed with Ketogenic Diet
There were significant decreases in the Body Weight Change of both the 9- and 18-hour starvation when compared with the Control in rats fed with Ketogenic diets. This shows that both prolonged starvation and high-fat (ketogenic) diet consumption contribute to the hypoglycemic effects of the rats’ tissue and hence the significant loss of body weight, as it is the case with this study.
Mean Values of Body Weights in Wistar Rats fed with Standard Diet and Ketogenic Diet
The fact that there was no significant difference in mean Body Weight Change for the Control, the 9- and 18-hour starvation when the Standard diet was compared to the Ketogenic diet shows that both moderate and prolonged starvation have no significant effect on the mean Body Weight when rats fed with the Standard diet were compared to those that were fed with Ketogenic diets. This is in contrast with the work of [17]. whose findings revealed that rats treated with the ketogenic diet had a significant weight loss as a result of induced ketosis.
Hormonal Assays in Wistar Rats fed with Ketogenic Diet
Observation from the results of the present study shows that there is a unanimous decrease in the serum level of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) after 42 days of intermittent starvation and feeding with ketogenic diets.LH and FSH are known as gonadotropins that attain stimulatory affinity with the Gonads, the male testes [18].
Testosterone acts on the Sertoli and peritubular cells of the seminiferous tubules and directly enhances spermatogenesis stimulation [19-21].In this light, both LH and FSH inhibit the gonads either from producing sperm or synthesizing a sufficient quantity of testosterone. FSH stimulates the various developmental phases from spermatogonia to spermatocytes and also maintains the spermatogenic processes, while both LH and FSH are necessary for meiotic development of the spermatids [22].Testosterone is necessary for meiosis and sperm development, in response to FSH [23-24].The significant decrease in serum LH in the group of 18 hours of intermittent starvation and ketogenic diet suggest that high-fat diet and prolonged starvation have inhibitory effects on the hypothalamic-pituitary-testicular axis. This will, in turn, lead to a concomitant decrease in Testosterone production by the Leydig Cell and hence a suppressive decrease in sperm cell maturation. [25]. Also, the significant decrease in serum FSH concentration in the 9 and 18hours- starved rats also portrays the inhibitory effect that the prolonged starvation and high-fat (ketogenic) diet exert on the hypothalamic-pituitary axis to decrease FSH secretion.
The decrements in the concentration of these gonadotrophic hormones suggest a direct inhibition of the anterior pituitary homogenesis. Testosterone is the major hormone of the gonads in males, produced by the Leydig cells in the testis. Also, in addition to LH and FSH, it is the major hormonal marker of androgenicity [26].The development and maintenance of male reproductive organs are also enhanced by testosterone [27-28].).In this study, although the testosterone of the starvation-induced stressed rats with the ketogenic diet is decreased but are not significant when compared with the control. The decrease in Testicular protein, cholesterol, and glycogen are not more marked [29].
CONCLUSION
Long-term starvation exerts appreciable decreases in the reproductive hormones, especially FSH and LH, in starved rats fed with a ketogenic diet for a prolonged period of time. This study is among the first to successfully delve into investigations on starvation-induced stress, standard diet, and ketogenic diet on reproductive function. Prolonged starvation exerts significant detriment on body weight, even though standard or ketogenic feeds were given without limitation after the starvation period. This was the case because although starved rats tend to eat more vigorously than their un-starved counterparts, nutrients lost during extensive starvation could not be completely recovered.
Conflict of Interest: None
REFERENCES
1 Kosinski, C., andJornayvaz, F. R. (2017). Effects of Ketogenic Diets on Cardiovascular Risk Factors: Evidence from Animal and Human Studies. Nutrients, 9(5), 517.https://doi.org/10.3390/nu9050517
2. Barañano, K. W., & Hartman, A. L. (2008). The ketogenic diet: uses in epilepsy and other neurologic… illnesses. Current treatment options in neurology, 10(6), 410–419. https://doi.org/10.1007/s11940-008-0043-8
3. Alessandro R., Gerardo B., Alessandra L. (2015) “Effects of twenty days of the ketogenic diet on metabolic and respiratory parameters in healthy subjects,” Lung, vol. 193, no. 6, pp. 939–945
4. Rho J.M. (2017) “How does the ketogenic diet induce anti-seizure effects?” Neuroscience Letters, vol. 637, pp. 4–10
5. Mak S.C., Chi C.S., and Wan C.J. (1999) “Clinical experience of ketogenic diet on children with refractory epilepsy,” ActaPaediatricaTaiwanica, vol. 40, pp. 97–100.
6. Dashti, H.M, Mathew T.C., Khadada M. (2007).“Beneficial effects of ketogenic diet in obese diabetic subjects,” Molecular and Cellular Biochemistry, vol. 302, no. 1-2, pp. 249–256.
7. Nazarewicz R. R, Ziolkowski W., Vaccaro P.S., and Ghafourifar P. (2007) “Effect of short-term ketogenic diet on redox status of human blood,” Rejuvenation Research, vol. 10, no. 4, pp. 435–440.
8. Alessandro R., Gerardo B., Alessandra L. (2015) “Effects of twenty days of the ketogenic diet on metabolic and respiratory parameters in healthy subjects,” Lung, vol. 193, no. 6, pp. 939–945
9. Bough K.J. and Rho J.M. (2007) “Anticonvulsant mechanisms of the ketogenic diet,” Epilepsia, vol. 48, pp. 43–58.
10. Hall, J.E. (2015) Guyton and Hall Textbook of Medical Physiology E-Book, Elsevier Health Sciences, Amsterdam, Netherlands.
11. Puchalska P. and Crawford P.A. (2017) “multi-dimensional roles of ketone bodies in fuel metabolism, signalling, and therapeutics,” Cell Metabolism, vol25, no. 2, pp 262-284
12. Lagua R.T. and Claudio V.S. (2012).Nutrition and Diet therapy Reference Dictionary, Springer, Berlin, Germany.
13. Fukao T., Lopaschuk G. D., and Mitchell G. A. (2004) “Pathways and control of ketone body metabolism: on the fringe of lipid biochemistry,” Prostaglandins, Leukotrienes and Essential Fatty Acids, vol. 70, no. 3, pp. 243–251
14. Noeman S.A., Hamooda H.E., and Baalash A.A. (2011) “Biochemical study of oxidative stress markers in the liver, kidney and heart of high fat diet induced obesity in rats,” Diabetology& Metabolic Syndrome, vol. 3, p. 17.
15. Raubenheimer P.J., Nyirenda M.J., and Walker B.R. (2006) “A choline- deficient diet exacerbates fatty liver but attenuates insulin resistance and glucose intolerance in mice fed a high fat diet,” Diabetes, vol. 55, no. 7, pp. 2015-2020
16. Vial G., Dubouchaud, H., Couturier K. (2011) “Effects of a highfat diet on energy metabolism and ROS production in rat liver,” Journal of Hepatology, vol. 54, no. 2, pp. 348–356.
17. Kume S., Uzu T., Araki, S.I. (2007). “Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet,” Journal of the American Society of Nephrology, vol. 18, no. 10, pp. 2715–2723.
18. Arsyad A., Idris I., Rasyid A. A., Usma R. A., Faradillah K. R., Latif W. U., Lubis Z. I.,
Yustisia A. I., Djabir Y. Y., (2020) “Long-Term Ketogenic Diet Induces Metabolic Acidosis,
Anaemia, and Oxidative Stress in Healthy Wistar Rats”, Journal of Nutrition and Metabolism,
Article ID 3642035, 7 pages, https://doi.org/10.1155/2020/3642035
19. Adienbo O. M., Nwafor A., Dapper D. V. (2015). Impairments in testicular function indices in male wistar rats: a possible mechanism for infertility induction by Xylopiaaethiopica fruit extract. Int J ReprodContraceptObstetGynecol;4:71-5.
20. Singh J, O’Neill C, Handelsman D. J. (1995). Induction of spermatogenesis by androgens in gonadotropin-deficient (hpg) mice.Endocrinology; 136:5311-21.Bhasin, S., Fielder, T. J., Swerdloff, R. S. (1987). Testosterone selectively increases serum follicle-stimulating hormonal (FSH) but not luteinizing hormone (LH) in gonadotropin-releasing hormone antagonist-treated male rats: evidence for differential regulation of LH and FSH secretion. Biology of reproduction, 37(1), 55–59.37.1.55
22. O’Donnell L., McLachlan R. I., Wreford N. G., Robertson D. M. (1994). Testosterone promotes the conversion of round spermatids between stages VII and VIII of the rat spermatogenic cycle. Endocrinology135:2608 2614
23. Lostroh A. J. (1963). Effect of Follicle-Stimulating Hormone and Interstitial Cell-Stimulating Hormone on Spermatogenesis in Long-Evans rats hypophysectomised for six months. Acta Endocrinologica, Volume 43, Issue 4, Pages 592–600.
24. Chemes H. E., Dym M., Raj H. G. M. (1979).The role of gonadotrophins and testosterone on the initiation of spermatogenesis in the immature rat.Biol Reprod.21:241-249.
25. Haneji, T., Maekawa, M.,Nishimune, Y. (1984). Vitamin A and follicle hormone synergistically induced differentiation of type Aspermatogonia in adult mouse cryptorchid testes in vitro. Endocrinology 114,801-805.
26 Cheng C. Y., Mruk D. D. (2002). Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development.
27. Walton S., Cunliffe W. J., Keczkes K., Early A. S., McGarrigle H. H. G., Katz M. (1995).Clinical, ultrasound and hormonal markers of androgenicity in acne vulgaris.Br.J.Dermatol.133(2):249–53. doi: 10.1111/j.1365- 2133.1995.tb02623.x
28. Mooradian, A. D., Morley, J. E., Korenman, S. G. (1987). Biological actions of androgens. Endocrine reviews, 8(1), 1–28.
29. Woode, E.,Abass, A.,Abaidoo C. (2012). Effect of Xylopic Acid on Sex Hormones and Spermatogenesis in Male Rats.Al Ame en J Med Sc. 5.
30. Guyton A. C., Hall H. E. (2011). Textbook of Medical Physiology, 12th Edition.