L-glutamine Supplementation Enhances Sperm Motility and Viability in Low-count Samples during Cryopreservation: Evidence from a Nonrandomized In vitro Study

A BSTRACT Introduction: Sperm cryopreservation is a cornerstone of assisted reproductive technologies, yet the process often induces reactive oxygen species (ROS) formation, which compromises plasma membrane integrity and subsequently reduces motility and viability. L-glutamine has emerged as a promising in vitro supplement to mitigate these deleterious effects, particularly of oligozoospermic samples. This study aimed to assess the effectiveness of in vitro L-glutamine supplementation in maintaining the motility and viability of low-count sperm during cryopreservation. Methods: Eight samples were collected from volunteers attending the Medical Biology Laboratory, Faculty of Medicine, Universitas Airlangga, in September and October 2023. Each sample was divided into two cohorts: the treatment group, which received 10 mM L-glutamine supplementation, and the control group, which received no supplementation. Individual sperm (10–15 per sample) were isolated using a micromanipulator and transferred onto cryo straws containing sperm freezing media before undergoing freezing and subsequent thawing. The postthaw sperm motility and viability were compared between groups using paired t -test, with a statistical significance level set at 5%. Results: The L-glutamine supplementation group indicated markedly enhanced sperm motility relative to the control group (9.54 ± 4.55% versus 1.75 ± 4.95%, respectively; P < 0.05). While sperm viability was also higher in the L-glutamine group, the difference lacked statistical significance (65.25 ± 8.48% versus 52.63 ± 10.47%, respectively; P > 0.05). Conclusion: In vitro L-glutamine supplementation during cryopreservation substantially preserves greater motility of low-count sperm and offers a modest improvement in viability. This approach holds promise for optimizing outcomes in sperm cryopreservation protocols.