Impact of vitamin D3 Nanoemulsion on spermatogenesis and antioxidant enzymes in Vitamin D deficient induced albino male rats.

Diyar Hamid Karim; Sulaf Mustafa Mohammed; Hoshyar Abdullah Azeez

doi:10.21271/ZJPAS.33.1.7

Authors

Diyar Hamid Karim Department of Biology, college of Science, University of Sulaimani, As-Sulaymaniyah, Kurdistan Region, Iraq.
Sulaf Mustafa Mohammed Department of Biology, college of Science, University of Sulaimani, As-Sulaymaniyah, Kurdistan Region, Iraq.
Hoshyar Abdullah Azeez College of pharmacy, University of Sulaimani, As-Sulaymaniyah, Kurdistan Region, Iraq.

DOI:

https://doi.org/10.21271/ZJPAS.33.1.7

Keywords:

Vitamin D, Testosterone, Nanoemulsion, Catalase, Glutathione Peroxidase, Sperm morphology, Sperm count.

Abstract

Vitamin D deficiency is common with several effects that include nonskeletal impacts. Many studies showed associations between Vitamin D deficiency and oxidative statues with disturbed testicles in male rats (Rattus norvegicus) which might be improved by vitamin D. The aim of this work is to find the effects of nanoemulsion pea protein isolates and novel vitamin D carrier, developed by sonication and pH shifting of pea protein isolate nanoemulsion on the spermatogenesis in induced vitamin D deficient male rats. Thirty male albino rats distributed into 5 groups, sufficient control was fed with normal diet, deficient control was treated with 100 mg/kg Rimfapicin and 50 mg/kg Isoniazed for three weeks, pea protein isolate group :vitamin D deficient rats treated daily with 60 mg/ml/kg nanoemulsion pea protean isolate, Vitamin D deficient rats were treated with vitamin D (54 mcg/ml/ kg), and pea protean isolate + vitamin D group: Vitamin D deficient rats were treated by nanoemulsion pea protein isolate (60 mg/dl/kg) plus Vitamin D (54 mcg/ml/ kg) for four weeks. The serum used for Vitamin D and total testosterone assay, after estimation of sperm count and morphology. Homogenized testes used for determination of Catalase and Glutathione peroxidase. The results revealed that vitamin D and supplementations each one alone have a positive significant effects on spermatogenesis. The nanoemulsion pea protein isolate +vitamin D resulted in a significant increase in the levels of vitamin D of total testosterone more than the supplementation of vitamin D and nanoemulsion pea protein isolate alone. Also sperm count, normal sperm morphology, as well as catalase and Glutathione peroxidase improved significantly in this group. In conclusion we demonstrated vitamin D nanoemulsion as more efficient formulation with more prominent effects on spermatogenesis in induced vitamin D deficient rats.

References

Almajwal, A. M., Abulmeaty, M. M. A., Feng, H., Alruwaili, N. W., Dominguez-Uscanga, A., Andrade, J. E., Razak, S., & Elsadek, M. F. (2019). Stabilization of vitamin D in pea protein isolate nanoemulsions increases its bioefficacy in rats. Nutrients, 11(1). https://doi.org/10.3390/nu11010075

Aquila, S., Guido, C., Middea, E., Perrotta, I., Bruno, R., Pellegrino, M., & Andò, S. (2009). Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1, 25 (OH) 2D3) regulates different aspects of human sperm biology and metabolism. Reproductive Biology and Endocrinology, 7(1), 140.

Atmaca, G. (2004). Antioxidant effects of sulfur-containing amino acids. In Yonsei Medical Journal (Vol. 45, Issue 5, pp. 776–788). https://doi.org/10.3349/ymj.2004.45.5.776

BELL, S. J. (2000). Whey protein concentrates with and without immunoglobulins: a review. Journal of Medicinal Food, 3(1), 1–13.

Blomberg Jensen, M., Jørgensen, A., Nielsen, J. E., Bjerrum, P. J., Skalkam, M., Petersen, J. H., Egeberg, D. L., Bangsbøll, S., Andersen, A. N., Skakkebæk, N. E., Juul, A., Rajpert-De Meyts, E., Dissing, S., Leffers, H., & Jørgensen, N. (2012). Expression of the vitamin D metabolizing enzyme CYP24A1 at the annulus of human spermatozoa may serve as a novel marker of semen quality. International Journal of Andrology, 35(4), 499–510. https://doi.org/10.1111/j.1365-2605.2012.01256.x

Blomberg Jensen, Martin, Bjerrum, P. J., Jessen, T. E., Nielsen, J. E., Joensen, U. N., Olesen, I. A., Petersen, J. H., Juul, A., Dissing, S., & Jørgensen, N. (2011). Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa. Human Reproduction, 26(6), 1307–1317. https://doi.org/10.1093/humrep/der059

Blomberg Jensen, Martin, Nielsen, J. E., Jørgensen, A., Rajpert-De Meyts, E., Kristensen, D. M., Jørgensen, N., Skakkebaek, N. E., Juul, A., & Leffers, H. (2010). Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract. Human Reproduction, 25(5), 1303–1311. https://doi.org/10.1093/humrep/deq024

Boye, J. I., Aksay, S., Roufik, S., Ribéreau, S., Mondor, M., Farnworth, E., & Rajamohamed, S. H. (2010). Comparison of the functional properties of pea, chickpea and lentil protein concentrates processed using ultrafiltration and isoelectric precipitation techniques. Food Research International, 43(2), 537–546. https://doi.org/10.1016/j.foodres.2009.07.021

Brodie, M. J., Boobis, A. R., Hillyard, C. J., Abeyasekera, G., Stevenson, J. C., MacIntyre, I., & Park, B. K. (1982). Effect of rifampicin and isoniazid on vitamin D metabolism. Clinical Pharmacology and Therapeutics, 32(4), 525–530. https://doi.org/10.1038/clpt.1982.197

Caprio, M., Infante, M., Calanchini, M., Mammi, C., & Fabbri, A. (2017). Vitamin D: not just the bone. Evidence for beneficial pleiotropic extraskeletal effects. Eating and Weight Disorders-Studies on Anorexia, Bulimia and Obesity, 22(1), 27–41.

Chen, Y., & Zhi, X. (2020). Roles of Vitamin D in Reproductive Systems and Assisted Reproductive Technology. Endocrinology, 161(4). https://doi.org/10.1210/endocr/bqaa023

Çınar, K. (2017). A review on nanoemulsion: Preparation methods and stability. Trakya Üniversitesi Mühendislik Bilimleri Dergisi, 18(1), 73–83.

D’Aniello, A., Di Cosmo, A., Di Cristo, C., Annunziato, L., Petrucelli, L., & Fisher, G. (1996). Involvement of D-aspartic acid in the synthesis of testosterone in rat testes. Life Sciences, 59(2), 97–104.

Dahl, W. J., Foster, L. M., & Tyler, R. T. (2012). Review of the health benefits of peas (Pisum sativum L.). British Journal of Nutrition, 108(SUPPL. 1). https://doi.org/10.1017/S0007114512000852

de Angelis, C., Galdiero, M., Pivonello, C., Garifalos, F., Menafra, D., Cariati, F., Salzano, C., Galdiero, G., Piscopo, M., Vece, A., Colao, A., & Pivonello, R. (2017). The role of vitamin D in male fertility: A focus on the testis. Reviews in Endocrine and Metabolic Disorders, 18(3), 285–305. https://doi.org/10.1007/s11154-017-9425-0

Dong, H.-J., Wu, D., Xu, S.-Y., Li, Q., Fang, Z.-F., Che, L.-Q., Wu, C.-M., Xu, X.-Y., & Lin, Y. (2016). Effect of dietary supplementation with amino acids on boar sperm quality and fertility. Animal Reproduction Science, 172, 182–189.

Erdmann, K., Cheung, B. W. Y., & Schröder, H. (2008). The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. Journal of Nutritional Biochemistry, 19(10), 643–654. https://doi.org/10.1016/j.jnutbio.2007.11.010

Farhangi, M. A., Nameni, G., Hajiluian, G., & Mesgari-Abbasi, M. (2017). Cardiac tissue oxidative stress and inflammation after vitamin D administrations in high fat- diet induced obese rats. BMC Cardiovascular Disorders, 17(1), 1–7. https://doi.org/10.1186/s12872-017-0597-z

Fattah, Y. M., & Mustafa, S. I. (2012). Effects of the pesticide cyren on chromosomes and sperms of albino males mice.

Forrest, K. Y. Z., & Stuhldreher, W. L. (2011). Prevalence and correlates of vitamin D deficiency in US adults. Nutrition Research, 31(1), 48–54.

Fu, L., Chen, Y. H., Xu, S., Ji, Y. L., Zhang, C., Wang, H., Yu, D. X., & Xu, D. X. (2017). Vitamin D deficiency impairs testicular development and spermatogenesis in mice. Reproductive Toxicology, 73, 241–249. https://doi.org/10.1016/j.reprotox.2017.06.047

Grossmann, R. E., & Tangpricha, V. (2010). Evaluation of vehicle substances on vitamin D bioavailability: A systematic review. Molecular Nutrition and Food Research, 54(8), 1055–1061. https://doi.org/10.1002/mnfr.200900578

Guttoff, M., Saberi, A. H., & Mcclements, D. J. (2015). Formation of vitamin D nanoemulsion-based delivery systems by spontaneous emulsification: Factors affecting particle size and stability. Food Chemistry, 171, 117–122. https://doi.org/10.1016/j.foodchem.2014.08.087

Hammoud, A. O., Wayne Meikle, A., Matthew Peterson, C., Stanford, J., Gibson, M., & Carrell, D. T. (2012). Association of 25-hydroxy-vitamin D levels with semen and hormonal parameters. Asian Journal of Andrology, 14(6), 855–859. https://doi.org/10.1038/aja.2012.77

Han, D., Sen, C. K., Roy, S., Kobayashi, M. S., Tritschler, H. J., & Packer, L. (1997). Protection against glutamate-induced cytotoxicity in C6 glial cells by thiol antioxidants. American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 273(5 42-5), 1771–1778. https://doi.org/10.1152/ajpregu.1997.273.5.r1771

Hasanvand, E., Fathi, M., & Bassiri, A. (2018). Production and characterization of vitamin D 3 loaded starch nanoparticles: effect of amylose to amylopectin ratio and sonication parameters. Journal of Food Science and Technology, 55(4), 1314–1324.

Haussler, M. R., Jurutka, P. W., Mizwicki, M., & Norman, A. W. (2011). Vitamin D receptor (VDR)-mediated actions of 1α,25(OH) 2 vitamin D 3 : Genomic and non-genomic mechanisms. Best Practice and Research: Clinical Endocrinology and Metabolism, 25(4), 543–559. https://doi.org/10.1016/j.beem.2011.05.010

He, W., Tan, Y., Tian, Z., Chen, L., Hu, F., & Wu, W. (2011). Food protein-stabilized nanoemulsions as potential delivery systems for poorly water-soluble drugs: preparation, in vitro characterization, and pharmacokinetics in rats. International Journal of Nanomedicine, 6, 521.

Hosseini, M., Pourganji, M., Khodabandehloo, F., Soukhtanloo, M., & Zabihi, H. (2012). Protective effect of l-arginine against oxidative damage as a possible mechanism of its Bene. cial properties on spatial learning in ovariectomized rats. Basic and Clinical Neuroscience, 3(5), 36–44.

Jaiswal, M., Dudhe, R., & Sharma, P. K. (2015). Nanoemulsion: an advanced mode of drug delivery system. 3 Biotech, 5(2), 123–127. https://doi.org/10.1007/s13205-014-0214-0

Jameson, J. L. (2018). Harrison’s principles of internal medicine. McGraw-Hill Education,.

Jensen, M. B. (2014). Vitamin D and male reproduction. Nature Reviews Endocrinology, 10(3), 175–186. https://doi.org/10.1038/nrendo.2013.262

Jiang, J., Zhu, B., Liu, Y., & Xiong, Y. L. (2014). Interfacial structural role of pH-shifting processed pea protein in the oxidative stability of oil/water emulsions. Journal of Agricultural and Food Chemistry, 62(7), 1683–1691. https://doi.org/10.1021/jf405190h

Jiang, S. (2015). Enhanced physicochemical and functional properties of pea (Pisum sativum) protein by pH-shifting and ultrasonication combined process [University of Illinois at Urbana-Champaign]. http://hdl.handle.net/2142/88229

Jiang, S., Yildiz, G., Ding, J., Andrade, J., Rababahb, T. M., Almajwalc, A., Abulmeatyc, M. M., & Feng, H. (2019). Pea Protein Nanoemulsion and Nanocomplex as Carriers for Protection of Cholecalciferol (Vitamin D3). Food and Bioprocess Technology, 12(6), 1031–1040. https://doi.org/10.1007/s11947-019-02276-0

Johnson, J. A., Grande, J. P., Roche, P. C., & Kumar, R. (1996). Immunohistochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat reproductive tissues. Histochemistry and Cell Biology, 105(1), 7–15. https://doi.org/10.1007/BF01450873

Kadappan, A. S., Guo, C., Gumus, C. E., Bessey, A., Wood, R. J., McClements, D. J., & Liu, Z. (2018). The Efficacy of Nanoemulsion-Based Delivery to Improve Vitamin D Absorption: Comparison of In Vitro and In Vivo Studies. Molecular Nutrition and Food Research, 62(4), 1–24. https://doi.org/10.1002/mnfr.201700836

Karefylakis, C., Särnblad, S., Ariander, A., Ehlersson, G., Rask, E., & Rask, P. (2018). Effect of Vitamin D supplementation on body composition and cardiorespiratory fitness in overweight men—A randomized controlled trial. Endocrine, 61(3), 388–397.

Kentish, S., & Feng, H. (2014). Applications of power ultrasound in food processing. Annual Review of Food Science and Technology, 5(1), 263–284. https://doi.org/10.1146/annurev-food-030212-182537

Kinuta, K., Tanaka, H., Moriwake, T., Aya, K., Kato, S., & Seino, Y. (2000). Vitamin D is an important factor in estrogen biosynthesis of both female and male gonads. Endocrinology, 141(4), 1317–1324. https://doi.org/10.1210/endo.141.4.7403

Korhonen, H., & Pihlanto, A. (2005). Food-derived Bioactive Peptides - Opportunities for Designing Future Foods. Current Pharmaceutical Design, 9(16), 1297–1308. https://doi.org/10.2174/1381612033454892

Korish, A. A. (2010). Multiple antioxidants and L-arginine modulate inflammation and dyslipidemia in chronic renal failure rats. Renal Failure, 32(2), 203–213.

Kwiecinski, G. G., Petrie, G. I., & DeLuca, H. F. (1989). Vitamin D is necessary for reproductive functions of the male rat. The Journal of Nutrition, 119(5), 741–744.

Lerchbaum, E, Pilz, S., Trummer, C., Rabe, T., Schenk, M., Heijboer, A. C., & Obermayer‐Pietsch, B. (2014). Serum vitamin D levels and hypogonadism in men. Andrology, 2(5), 748–754.

Lerchbaum, Elisabeth, & Obermayer-Pietsch, B. (2012). Vitamin D and fertility: a systematic review. Eur J Endocrinol, 166(5), 765–778.

Liang, H. N., & Tang, C. H. (2013). PH-dependent emulsifying properties of pea [Pisum sativum (L.)] proteins. Food Hydrocolloids, 33(2), 309–319. https://doi.org/10.1016/j.foodhyd.2013.04.005

Liu, G., Zhou, Y., & Chen, L. (2019). Intestinal uptake of barley protein-based nanoparticles for β-carotene delivery. Acta Pharmaceutica Sinica B, 9(1), 87–96.

Lucotti, P., Monti, L., Setola, E., La Canna, G., Castiglioni, A., Rossodivita, A., Pala, M. G., Formica, F., Paolini, G., & Catapano, A. L. (2009). Oral L-arginine supplementation improves endothelial function and ameliorates insulin sensitivity and inflammation in cardiopathic nondiabetic patients after an aortocoronary bypass. Metabolism, 58(9), 1270–1276.

Luzi, F., Maertens, L., Mijten, P., & Pizzi, F. (1996). Effect of feeding level and dietary protein content on libido and semen characteristics of bucks. Proc. 6. World Rabbit Congress, Toulouse, Proc. 2, 87–92.

Macchia, G., Topo, E., Mangano, N., D’Aniello, E., & Boni, R. (2010). dl-Aspartic acid administration improves semen quality in rabbit bucks. Animal Reproduction Science, 118(2–4), 337–343. https://doi.org/10.1016/j.anireprosci.2009.07.009

Mahmoudi, A. R., Zarnani, A. H., Jeddi-Tehrani, M., Katouzian, L., Tavakoli, M., Soltanghoraei, H., & Mirzadegan, E. (2013). Distribution of vitamin D receptor and 1α-hydroxylase in male mouse reproductive tract. Reproductive Sciences, 20(4), 426–436.

Menegaz, D., Rosso, A., Royer, C., Leite, L. D., Santos, A. R. S., & Silva, F. R. M. B. (2009). Role of 1α,25(OH)2 vitamin D3 on α-[1-14C]MeAIB accumulation in immature rat testis. Steroids, 74(2), 264–269. https://doi.org/10.1016/j.steroids.2008.11.015

Merke, J., Hügel, U., & Ritz, E. (1985). Nuclear testicular 1, 25-dihydroxyvitamin D3 receptors in Sertoli cells and seminiferous tubules of adult rodents. Biochemical and Biophysical Research Communications, 127(1), 303–309.

Mohammed, S. M. (2015). Effect of Broccoli (Brassica oleracea) on Some Physiological Variables and Reproductive System on Lead Acetate Exposed Adult Male Albino (Rattus rattus). Tikrit University-Iraq.

Mortimer, D., Barratt, C. L. R., Björndahl, L., De Jager, C., Jequier, A. M., & Muller, C. H. (2013). What should it take to describe a substance or product as ‘sperm-safe.’ Human Reproduction Update, 19(suppl_1), i1–i45.

N.W., A., M.M.A., A., A.M., A., M.F., E., & S., R. (2017). Novel vitamin D-nanoemulsion improves testicular function of vitamin D deficient rats. In FASEB Journal (Vol. 31, Issue 1 Supplement 1). http://www.fasebj.org/content/31/1_Supplement/lb389.abstract?sid=53411162-021c-472f-9a4c-10f9113c3b2a%0Ahttp://ovidsp.ovid.com/ovidweb.cgi?T=JS&PAGE=reference&D=emed18&NEWS=N&AN=616959246

Nizza, A., Di Meo, C., & Taranto, S. (2000). Effect of lysine and methionine on libido and semen characteristics of bucks. World Rabbit Science, 8(4), 181–184.

O’Donnell, L., Stanton, P., & de Kretser, D. M. (2017). Endocrinology of the male reproductive system and spermatogenesis. In Endotext [Internet]. MDText. com, Inc.

O’sullivan, J., Murray, B., Flynn, C., & Norton, I. (2016). The effect of ultrasound treatment on the structural, physical and emulsifying properties of animal and vegetable proteins. Food Hydrocolloids, 53, 141–154.

Overduin, J., Guérin-Deremaux, L., Wils, D., & Lambers, T. T. (2015). NUTRALYS® pea protein: Characterization of in vitro gastric digestion and in vivo gastrointestinal peptide responses relevant to satiety. Food and Nutrition Research, 59, 1–9. https://doi.org/10.3402/fnr.v59.25622

Park, S. J., Garcia, C. V, Shin, G. H., & Kim, J. T. (2017). Development of nanostructured lipid carriers for the encapsulation and controlled release of vitamin D3. Food Chemistry, 225, 213–219.

Peña-Ramos, E. A., Xiong, Y. L., & Arteaga, G. E. (2004). Fractionation and characterisation for antioxidant activity of hydrolysed whey protein. Journal of the Science of Food and Agriculture, 84(14), 1908–1918. https://doi.org/10.1002/jsfa.1886

Pilz, S., Frisch, S., Koertke, H., Kuhn, J., Dreier, J., Obermayer-Pietsch, B., Wehr, E., & Zittermann, A. (2011). Effect of vitamin D supplementation on testosterone levels in men. Hormone and Metabolic Research, 43(3), 223.

Pludowski, P., Holick, M. F., Grant, W. B., Konstantynowicz, J., Mascarenhas, M. R., Haq, A., Povoroznyuk, V., Balatska, N., Barbosa, A. P., Karonova, T., Rudenka, E., Misiorowski, W., Zakharova, I., Rudenka, A., Łukaszkiewicz, J., Marcinowska-Suchowierska, E., Łaszcz, N., Abramowicz, P., Bhattoa, H. P., & Wimalawansa, S. J. (2018). Vitamin D supplementation guidelines. Journal of Steroid Biochemistry and Molecular Biology, 175(2016), 125–135. https://doi.org/10.1016/j.jsbmb.2017.01.021

Pownall, T. L., Udenigwe, C. C., & Aluko, R. E. (2010). Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.) Enzymatic protein hydrolysate fractions. Journal of Agricultural and Food Chemistry, 58(8), 4712–4718. https://doi.org/10.1021/jf904456r

Pownall, T. L., Udenigwe, C. C., & Aluko, R. E. (2011). Effects of cationic property on the in vitro antioxidant activities of pea protein hydrolysate fractions. Food Research International, 44(4), 1069–1074. https://doi.org/10.1016/j.foodres.2011.03.017

Prusik, K., Kortas, J., Prusik, K., Mieszkowski, J., Jaworska, J., Skrobot, W., Lipinski, M., Ziemann, E., & Antosiewicz, J. (2018). Nordic walking training causes a decrease in blood cholesterol in elderly women supplemented with Vitamin D. Frontiers in Endocrinology, 9, 42.

Ramlau-Hansen, C. H., Moeller, U. K., Bonde, J. P., Olsen, J., & Thulstrup, A. M. (2011). Are serum levels of vitamin D associated with semen quality? Results from a cross-sectional study in young healthy men. Fertility and Sterility, 95(3), 1000–1004. https://doi.org/10.1016/j.fertnstert.2010.11.002

Schachter, A., Goldman, J. A., & Zukerman, Z. (1973). Treatment of oligospermia with the amino acid arginine. The Journal of Urology, 110(3), 311–313.

Seminotti, B., Leipnitz, G., Amaral, A. U., Fernandes, C. G., da Silva, L. de B., Tonin, A. M., Vargas, C. R., & Wajner, M. (2008). Lysine induces lipid and protein damage and decreases reduced glutathione concentrations in brain of young rats. International Journal of Developmental Neuroscience, 26(7), 693–698.

Shand, P. J., Ya, H., Pietrasik, Z., & Wanasundara, P. (2007). Physicochemical and textural properties of heat-induced pea protein isolate gels. Food Chemistry, 102(4), 1119–1130.

Sharma, A., & Sharma, U. S. (1997). Liposomes in drug delivery: progress and limitations. International Journal of Pharmaceutics, 154(2), 123–140.

Sharpe, R. M., Maddocks, S., Millar, M., Kerr, J. B., Saunders, P. T. K., & McKinnell, C. (1992). Testosterone and Spermatogenesis Identification of Stage‐Specific, Androgen‐Regulated Proteins Secreted by Adult Rat Seminiferous Tubules. Journal of Andrology, 13(2), 172–184.

Solans, C., & Solé, I. (2012). Nano-emulsions: Formation by low-energy methods. Current Opinion in Colloid and Interface Science, 17(5), 246–254. https://doi.org/10.1016/j.cocis.2012.07.003

Sood, S., Reghunandanan, R., Reghunandanan, V., Marya, R. K., & Singh, P. I. (1995). Effect of vitamin D repletion on testicular function in vitamin D-deficient rats. Annals of Nutrition and Metabolism, 39(2), 95–98.

Spiro, A., & Buttriss, J. L. (2014). Vitamin D: An overview of vitamin D status and intake in Europe. Nutrition Bulletin, 39(4), 322–350. https://doi.org/10.1111/nbu.12108

Stone, A. K., Karalash, A., Tyler, R. T., Warkentin, T. D., & Nickerson, M. T. (2015). Functional attributes of pea protein isolates prepared using different extraction methods and cultivars. Food Research International, 76(P1), 31–38. https://doi.org/10.1016/j.foodres.2014.11.017

Sung, C. C., Liao, M. T., Lu, K. C., & Wu, C. C. (2012). Role of vitamin D in insulin resistance. Journal of Biomedicine and Biotechnology, 2012(Figure 1). https://doi.org/10.1155/2012/634195

Tartagni, M., Matteo, M., Baldini, D., Tartagni, M. V., Alrasheed, H., De Salvia, M. A., Loverro, G., & Montagnani, M. (2015). Males with low serum levels of vitamin D have lower pregnancy rates when ovulation induction and timed intercourse are used as a treatment for infertile couples: Results from a pilot study. Reproductive Biology and Endocrinology, 13(1), 1–7. https://doi.org/10.1186/s12958-015-0126-9

Uhland, A. M., Kwiecinski, G. G., & DeLuca, H. F. (1992). Normalization of serum calcium restores fertility in vitamin D-deficient male rats. The Journal of Nutrition, 122(6), 1338–1344.

Wallner, S., Hermetter, A., Mayer, B., & Wascher, T. C. (2001). The alpha‐amino group of l‐arginine mediates its antioxidant effect. European Journal of Clinical Investigation, 31(2), 98–102.

Wang, H., Wolosker, H., Morris, J. F., Pevsner, J., Snyder, S. H., & Selkoe, D. J. (2002). Naturally occurring free D-aspartate is a nuclear component of cells in the mammalian hypothalamo-neurohypophyseal system. Neuroscience, 109(1), 1–4.

Yang, B., Sun, H., Wan, Y., Wang, H., Qin, W., Yang, L., Zhao, H., Yuan, J., & Yao, B. (2012). Associations between testosterone, bone mineral density, vitamin D and semen quality in fertile and infertile Chinese men. International Journal of Andrology, 35(6), 783–792.

Zanatta, L., Zamoner, A., Zanatta, A. P., Bouraïma-Lelong, H., Delalande, C., Bois, C., Carreau, S., & Silva, F. R. M. B. (2011). Nongenomic and genomic effects of 1α,25(OH)2 vitamin D3 in rat testis. In Life Sciences (Vol. 89, Issues 15–16, pp. 515–523). https://doi.org/10.1016/j.lfs.2011.04.008

Zhu, C. L., Xu, Q. F., Li, S. X., Wei, Y. C., Zhu, G. C., Yang, C., & Shi, Y. C. (2016). Investigation of serum vitamin D levels in Chinese infertile men. Andrologia, 48(10), 1261–1266. https://doi.org/10.1111/and.12570