In vitro effects of alpha-ketoglutarate and folic acid supplementation on bull sperm exposed to ammonia stress

Document Type : Original Article

Authors

1 Department of Clinical Science, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran

2 Department of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

3 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Selcuk University, Konya, Türkiye

4 Connective Tissue Diseases Research Center, Tabriz University of Medical Science, Tabriz, Iran

Abstract
This study examined the effects of alpha-ketoglutaric acid (AKG; 2.00, 4.00, and 8.00 mM L-1) and folic acid (FA; 50.00 nM L-1) on bull sperm cells under ammonia stress induced by 175 µM L-1 ammonium chloride. Sperm parameters including kinematic motility, survival rate, membrane integrity, DNA integrity, superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), total antioxidant capacity (TAC) activities, and apoptosis were assessed in the groups. Sperm motility indices, survival rate, plasma membrane integrity, SOD, CAT, TAC, and GPx enzymes activity, DNA damage, and apoptosis in the treated groups were significantly differed from those in the control groups. The AKG concentrations of 4.00 and 8.00 mM L-1 co-supplemented with 50.00 nM L-1 FA mitigated the negative effects of ammonia on sperm cells. This study indicated that supplementation with AKG and FA at the desired concentrations counteracted the adverse effects of ammonia toxicity that preceded clinical signs. Further studies are needed to evaluate the fertility of these sperms, either in vitro or in vivo.

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1.     Butler WR. Review: effect of protein nutrition on ovarian and uterine physiology in dairy cattle. J Dairy Sci 1998; 81(9): 2533-2539.
2.     Constable PD, Hinchcliff KW, Done SH, et al. Veterinary medicine: a textbook of the diseases of cattle, horses, sheep, pigs and goats. 11th ed. Missouri, USA: Elsevier Health Sciences 2016; 618-620.
3.     Hogan JP. The absorption of ammonia through the rumen of the sheep. Aust J Biol Sci 1961; 14: 448-460.
4.     Sinclair KD, Kuran M, Gebbie FE, et al. Nitrogen metabolism and fertility in cattle: II. Development of oocytes recovered from heifers offered diets differing in their rate of nitrogen release in the rumen. J Anim Sci 2000; 78(10): 2670-2680.
5.     Allahkarami S, Atabakhsh M, Moradi MN, et al. Correlation of uric acid, urea, ammonia and creatinine of seminal plasma with semen parameters and fertilization rate of infertile couples. Avicenna J Med Biochem 2017; 5(2): 76-80.
6.     Godden SM, Kelton DF, Lissemore KD, et al. Milk urea testing as a tool to monitor reproductive performance in Ontario dairy herds. J Dairy Sci 2001; 84(6): 1397-1406.
7.     Ye N, Lv Z, Huang Z, et al. Dietary folic acid supplementation improves semen quality and spermatogenesis through altering autophagy and histone methylation in the testis of aged broiler breeder roosters. Theriogenology 2022; 181: 8-15.
8.     Montazeri R, Golshan-Iranpour FA, Dashti GR, et al. Effects of biotin and folic acid on motility, viability, morphology, chromatin density and integrity of cryopreserved and thawed sperm in normozoospermic men. SJKU 2021; 26(4): 38-49.
9.     Lipovac M, Nairz V, Aschauer J, et al. The effect of micronutrient supplementation on spermatozoa DNA integrity in subfertile men and subsequent pregnancy rate. Gynecol Endocrinol 2021; 37(8): 711-715.
10. Kennedy EP, Lehninger AL. Intracellular structures and the fatty acid oxidase system of rat liver. J Biol Chem 1948; 172(2): 847. PMID: 18901210.
11. Stoll B, McNelly S, Buscher HP, et al. Functional hepatocyte heterogeneity in glutamate, aspartate and alpha-ketoglutarate uptake: a histoautoradiographical study. Hepatology 1991; 13(2): 247-253.
12. Espat NJ, Watkins KT, Lind DS, et al. Dietary modulation of amino acid transport in rat and human liver. J Surg Res 1996; 63(1): 263-268.
13. Kitamura SS. Ammonia poisoning in cattle and rats: renal performance in detoxification and the use of alternative treatments [Portuguese]. PhD Thesis. University of São Paulo, Brazil: 2002.
14. Foote RH. Fertility of bull semen at high extension rates in tris-buffered extenders. J Dairy Sci 1970; 53(10): 1475-1477.
15. Rarani FZ, Golshan-Iranpour F, Dashti GR. Correlation between sperm motility and sperm chromatin/DNA damage before and after cryopreservation and the effect of folic acid and nicotinic acid on post-thaw sperm quality in normozoospermic men. Cell Tissue Bank 2019; 20(3): 367-378.
16. Li SF, Liu HX, Zhang YB, et al. The protective effects of alpha-ketoacids against oxidative stress on rat spermatozoa in vitro. Asian J Androl 2010; 12(2): 247-256.
17. Neeley WE, Phillipson J. Automated enzymatic method for determining ammonia in plasma, with 14-day reagent stability. Clin Chem 1988; 34(9): 1868-1869.
18. Amann RP, Waberski D. Computer-assisted sperm analysis (CASA): capabilities and potential developments. Theriogenology 2014; 81(1): 5-17 e1-3.
19. Swanson EW, Bearden HJ. An Eosin-Nigrosin stain for differentiating live and dead bovine spermatozoa. J Anim Sci 1951; 10(4): 981-987.
20. Revell SG, Mrode RA. An osmotic resistance test for bovine semen. Anim Reprod Sci 1994; 36(1-2): 77-86.
21. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, et al. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984; 70(1): 219-228.
22. Tejada RI, Mitchell JC, Norman A, et al. A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril 1984; 42(1): 87-91.
23. Koopman G, Reutelingsperger CP, Kuijten GA, et al. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84(5): 1415-1420.
24. Wheeler CR, Salzman JA, Elsayed NM, et al. Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity. Anal Biochem 1990; 184(2): 193-199.
25. Johansson LH, Borg LA. A spectrophotometric method for determination of catalase activity in small tissue samples. Anal Biochem 1988; 174(1): 331-336.
26. Webb DW, Bartley EE, Meyer RM. A comparison of nitrogen metabolism and ammonia toxicity from ammonium acetate and urea in cattle. J Anim Sci 1972; 35(6): 1263-1270.
27. Hussain I, Cheeke PR. Effect of dietary Yucca schidigera extract on rumen and blood profiles of steers fed concentrate- or roughage-based diets. Anim Feed Sci Technol 1995; 51(3-4): 231-242.
28. McPherson RA, Pincus MR. Henry's clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia, USA: Elsevier Health Sciences 2021; 238.
29. Storey BT. Mammalian sperm metabolism: oxygen and sugar, friend and foe. Int J Dev Biol 2008; 52(5-6): 427-437.
30. Zhang Z, He C, Gao Y, et al. α‐ketoglutarate delays age‐related fertility decline in mammals. Aging Cell 2021; 20(2): e13291. doi: 10.1111/acel.13291.
31. Asadi Shahmirzadi A, Edgar D, Liao CY, et al. Alpha-ketoglutarate, an endogenous metabolite, extends lifespan and compresses morbidity in aging mice. Cell Metab 2020; 32(3): 447-456. e6.
32. Bayliak MM, Gospodaryov DV, Lushchak VI. Mimicking caloric restriction for anti-aging effects: the pro-oxidant role of alpha-ketoglutarate. Curr Opin Toxicol 2022; 30. 100339. doi: 10.1016/j.cotox.2022.02.012.
33. Li T, Liu J, Liu K, et al. Alpha-ketoglutarate ameliorates induced premature ovarian insufficiency in rats by inhibiting apoptosis and upregulating glycolysis. Reprod Biomed Online 2023; 46(4): 673-685.
34. He K, Luo X, Wen M, et al. Effect of acute ammonia toxicity on inflammation, oxidative stress and apoptosis in head kidney macrophage of Pelteobagrus fulvidraco and the alleviation of curcumin. Comp Biochem Physiol C Toxicol Pharmacol 2021; 248: 109098. doi: 10.1016/j.cbpc.2021.109098.
35. Lamond S, Watkinson M, Rutherford T, et al. Gene-specific chromatin damage in human spermatozoa can be blocked by antioxidants that target mitochondria. Reprod Biomed Online 2003; 7(4): 407-418.
36. Mahfouz RZ, du Plessis SS, Aziz N, et al. Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress. Fertil Steril 2010; 93(3): 814-821.
37. Hadjihambi A, Cudalbu C, Pierzchala K, et al. Abnormal brain oxygen homeostasis in an animal model of liver disease. JHEP Rep 2022; 4(8): 100509. doi: 10.1016/ j.jhepr.2022.100509.
38. Wang L, Xu Q, Chen D, et al. Effects of dietary α-ketoglutarate supplementation on liver glutamine content, antioxidant capacity and the expressions of growth hormone and insulin-like growth factor I genes of juvenile hybrid sturgeon fed different protein source diets. Chin J Anim Nutr 2016; 28(2): 3917-3924.
39. Chen D, Wang L, Xu Q. Effects of α-ketoglutarate supplementation on morphology, digestive enzyme activity and antioxidant capacity in intestine of hybrid sturgeon [Chinese]. J Dalian Fish Coll 2015; 30(4): 363-368.
40. Wallock LM, Tamura T, Mayr CA, et al. Low seminal plasma folate concentrations are associated with low sperm density and count in male smokers and nonsmokers. Fertil Steril 2001; 75(2): 252-259.
41. Bazrafkan M, Amir Jannati N, Gilany K, et al. P-055 Evaluation of seminal plasma targeted metabolomics (amino acids) following untargeted antioxidant therapy in asthenozoospermia patients: a proposed approach for Personalized Medicine. Hum Reprod 2022; 37 (Suppl 1): deac107. 051. doi: 10.1093/ humrep/deac107.051.
42. Ghadhban RF, Alwan NA. The role of folic acid on some physiological parameters and efficiency of sperm in male rabbits. Sys Rev Pharm 2020; 11(9): 1003-1007.
43. Joshi R, Adhikari S, Patro BS, et al. Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity. Free Radic Biol Med 2001; 30(12): 1390-1399.
44. Wong WY, Merkus HM, Thomas CM, et al. Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial. Fertil Steril 2002; 77(3): 491-498.
45. Ardestani Zadeh A, Arab D, Kia NS, et al. The role of vitamin E - selenium - folic acid supplementation in improving sperm parameters after varicocelectomy: a randomized clinical trial. Urol J 2019; 16(5): 495-500.
46. Yaris M, Akdogan N, Öztürk M, et al. The effects of two different antioxidant combinations on sperm parameters. Urologia 2022; 89(4): 629-635.
47. Wang W, Peng M, Yuan H, et al. Studying the mechanism of sperm DNA damage caused by folate deficiency. J Cell Mol Med 2022; 26(3): 776-788.
48. Zhang Y, Yuan H, Peng M, et al. Folic acid deficiency damages male reproduction via endoplasmic reticulum stress-associated PERK pathway induced by Caveolin-1 in mice. Syst Biol Reprod Med 2021; 67(5): 383-394.
49. Reuter S, Gupta SC, Chaturvedi MM, et al. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 2010; 49(11): 1603-1616.
50. Sathyasaikumar KV, Swapna I, Reddy PV, et al. Fulminant hepatic failure in rats induces oxidative stress differentially in cerebral cortex, cerebellum and pons medulla. Neurochem Res 2007; 32(3): 517-524.
51. Li X, Zeng YM, Luo YD, et al. Effects of folic acid and folic acid plus zinc supplements on the sperm characteristics and pregnancy outcomes of infertile men: a systematic review and meta-analysis. Heliyon 2023; 9(7): e18224. doi: 10.1016/j.heliyon.2023. e18224.
52. Cheng D, Liu X, Gao Y, et al. α-Ketoglutarate attenuates hyperlipidemia-induced endothelial damage by activating the Erk-Nrf2 signaling pathway to inhibit oxidative stress and mitochondrial dysfunction. Antioxid Redox Signal 2023; 39(10-12): 777-793.
Volume 16, Issue 11
November 2025
Pages 629-638

  • Receive Date 26 August 2024
  • Accept Date 11 September 2024