en Medical Biology Medical Biology مقاله پژوهشی Original Research Article <p style="text-align: justify;"><span style="font-size:16px;"><span style="font-family:Times New Roman;"><b><span style="background:#2d7f8f"><span style="color:white">Background & Objective:</span></span><b> </b></b>Infertility is a widespread health issue, with male factors solely responsible in approximately 20% of cases and contributing to an additional 30&ndash;40%. <em>In vitro</em> sperm preparation using motility-enhancing agents prior to assist reproductive techniques (ART) has shown potential in improving semen quality and increasing pregnancy rates. This study aimed to evaluate the effects of a novel combination of motility stimulants&mdash;Maca, L-carnitine, and pentoxifylline&mdash;on sperm function, particularly the DNA fragmentation index (DFI), and to compare the effectiveness of the swim-up and density gradient (layering) techniques for sperm activation.<br> <b><span style="background:#2d7f8f"><span style="color:white"><span style="letter-spacing:-.1pt">&nbsp;Materials & Methods:</span></span></span></b> <span style="letter-spacing:-.1pt">Semen samples were collected from 90 asthenozoospermic men. Samples were divided into two groups: a control group using Ham&rsquo;s F-12 medium and a treatment group using medium supplemented with Maca powder (1 mg/mL), pentoxifylline (10 mg/mL), and L-carnitine (0.5 mg/mL). Sperm activation was performed using both the swim-up and Layering techniques. Sperm parameters were evaluated according to WHO guidelines (1999 and 2021). Data were analyzed using SPSS version 23.0 with Student&rsquo;s t-test, ANOVA, and the LSD post-hoc test.</span><br> <b><span lang="EN-IN"><span style="background:#2d7f8f"><span style="color:white">Results: </span></span></span>&nbsp;</b><span style="letter-spacing:-.1pt">The treatment group demonstrated significant improvements (<em>P</em><0.05) in sperm concentration, motility (grades A and B), morphology, and reduced DNA fragmentation index. Both preparation techniques showed comparable improvements, although the Layering (density gradient) method yielded slightly better outcomes in DNA integrity.</span><br> <b><span style="background:#2d7f8f"><span style="color:white">Conclusion: </span></span>&nbsp;</b>The combination of pentoxifylline, L-carnitine, and Maca significantly enhanced sperm function. The Layering technique was marginally more effective than swim-up in selecting spermatozoa with intact DNA.</span></span></p> L-carnitine, Pentoxifylline, Maca (Lepidium meyenii), Sperm Preparation, Asthenozoospermia, DNA Fragmentation Index 192 201 http://journal.zums.ac.ir/browse.php?a_code=A-10-7291-1&slc_lang=en&sid=1 Israa Saleem Abdulzahra israasaleem@uomustansiriyah.edu.iq 5200319475328460084984 5200319475328460084984 Yes Department of Science, College of Basic Education, Mustansiriyah University, Baghdad, Iraq Saad Salih Al-Dujaily aldujaily8@yahoo.com 5200319475328460084985 5200319475328460084985 No Department of Physiology, High Institute of Infertility Diagnosis and ART, Al-Nahrain University, Baghdad, Iraq Areej Abbas Zabbon areejabbas@uomustansiriyah.edu.iq 5200319475328460084986 5200319475328460084986 No Department of Biology, College of Science, Mustansiriyah University, Baghdad, Iraq