Abstinence period: Impact on seminal citric acid and sperm motility. Dale Goss, Bashir Ayad, Gerhard van der Horst, Bongekile Skosana, Stefan du Plessis Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University Introduction Basic semen parameters including sperm concentration, motility, morphology and viability remain the primary predictive factors for male infertility diagnosis despite inter-individual variations in semen characteristics. Ejaculatory abstinence (EA) has been suggested to be a cost-effective approach to improve these parameters thereby improving semen quality. A significant increase in the amount of citric acid available per sperm may be an indication of the mechanism which allows sperm to display increased motility parameters. After spermatogenesis, immature spermatozoa must pass through the length of the epididymis where they undergo a series of biochemical and physiological changes which is essential for the acquisition of motility. The variation in total and progressive motility of sperm after different periods of EA has been attributed to the accessory sex gland secretions which may vary over time (Elzanaty, Malm, & Giwercman, 2005). Citric acid, a prominent prostate gland secretion is believed to play a prominent role in sperm energy metabolism however there has been no conclusive studies on its direct relationship to motility and abstinence in human spermatozoa (Medrano et al., 2006). Therefore, this study aims to determine the effect of abstinence periods on citric acid secretion and the subsequent effect on sperm motility. Results Parameters Long Abstinence Short abstinence P - value Citric acid (ug/uL) 8.016 ± 5.408 5.958 ± 4.93 < 0.01 Citric acid (ug/per sperm) 1.01x10-4 ± 1.2x10-4 2.0x10-4 ± 3.4x10-4 Total Motility (%) 64.71 ± 13.94 71.84 ± 3.85 < 0.02 Progressive Motility (%) 47.73 ± 14.33 58.54 ± 13.38 < 0.002 pH 7.68 ± 0.12 7.8 ± 0.17 < 0.05 Materials and methods Freshly ejaculated semen samples were collected from 16 healthy donors. The first sample from each donor was collected after 4 days of EA and the second after 4 hours of EA. The sperm concentration and motility/kinematic parameters were determined with Computer-Aided Sperm Analysis (CASA) and the Sperm Class Analyser version 5.4 - SCA®, (Microptic, S.L, Barcelona, Spain). Citric acid concentration was measured with a Citric Acid Assay Kit (FertiPro N.V., Belgium) and the absorbance was read on a FLUOstar Omega micro plate reader (BMG Labtech, Ortenberg, Germany). Comparisons between parameters measured after long and short EA were performed using the Students Paired t-test on GraphPad Prism© version 7.00 for Windows, (GraphPad Software, La Jolla California, USA). Statistical significance was set at p≤0.05. Figure 2: Polar plot displaying the basic semen parameters and citric acid measurements from long versus short ejaculatory abstinence. * P < 0.05, ** P < 0.01, ***P < 0.001 Discussion & Conclusion The main function of the prostate gland is to produce and release large amounts (up to 180 mM) of ionized derivatives of citric acid in the form of citrate into prostatic fluid (Kavanagh, 1994). Prostatic citrate acts primarily as an energy substrate for sperm by being converted to malate in order to be utilized in the citric acid cycle in sperm mitochondria (Visconti, 2012). Apart from the above cited work, most previous studies have not systematically assessed citric acid and its ability to manipulate sperm characteristics directly during abstinence, suggesting that its significance may have been underestimated. References Elzanaty, S., Malm, J., & Giwercman, A. (2005). Duration of sexual abstinence: Epididymal and accessory sex gland secretions and their relationship to sperm motility. Human Reproduction (Oxford, England), 20(1), 221-225. doi:deh586 [pii] Kavanagh, J. (1994). Isocitric and citric acid in human prostatic and seminal fluid: Implications for prostatic metabolism and secretion. The Prostate, 24(3), 139-142. Medrano, A., Fernández‐Novell, J. M., Ramió, L., Alvarez, J., Goldberg, E., Rivera, M., .Rodríguez‐Gil, J. E. (2006). Utilization of citrate and lactate through a lactate dehydrogenase and ATP‐regulated pathway in boar spermatozoa. Molecular Reproduction and Development, 73(3), 369-378. Visconti, P. (2012). Sperm bioenergetics in a nutshell. Biology of Reproduction, 87(3), 72, 1-4. Figure 1: Experimental workflow