Effect of L-proline supplementation on long-term cryopreservation of Piaractus brachypomus sperm: post-thaw motility, viability, and morphological assessment of sperm

Mariammal S.; J. Jaculine Pereira; V. Anix Vivek Santhiya; R. Shalini; R. Somu Sunder Lingam

doi:10.17017/j.fish.1322

Mariammal S. Department of Aquaculture, Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Thoothukudi - 628 008, Tamil Nadu, India
J. Jaculine Pereira Department of Aquaculture, Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Thoothukudi - 628 008, Tamil Nadu, India
V. Anix Vivek Santhiya Parakkai Centre for Sustainable Aquaculture, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Kanyakumari - 629 601, Tamil Nadu, India
R. Shalini Department of Fish Quality Assurance and Management, Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Thoothukudi - 628 008, Tamil Nadu, India
R. Somu Sunder Lingam Krishnagiri- Barur Centre for Sustainable Aquaculture, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Krishnagiri-635 201, Tamil Nadu, India

DOI: https://doi.org/10.17017/j.fish.1322

Keywords: L-proline, liquid nitrogen storage, membrane stability, modified cortland medium, pacu, sperm cryobiology

Abstract

Sperm cryopreservation is widely used in aquaculture for long-term preservation of genetic resources and broodstock management; however, prolonged storage can adversely affect sperm quality due to structural damage and ionic imbalance. This study evaluated the influence of L-proline supplementation, with and without dimethylsulfoxide (DMSO), on the cryopreservation efficiency of Piaractus brachypomus sperm. Milt from hormonally induced males was cryopreserved in Modified Cortland Medium (MCM) supplemented with 2, 5, 10, and 30 mM L-proline, either in the absence of DMSO or in combination with 10% DMSO, and maintained under liquid nitrogen for 90 days. Sperm quality after thawing was evaluated at 15-day intervals. Significant differences were observed in sperm motility and viability among treatments. Among the MCM supplemented with L-proline treatments, 10 mM L-proline maintained the highest post-thaw sperm motility (58.05–53.40%) and viability (62.50%), whereas the MCM without cryoprotectant showed the lowest values. Among the MCM containing 10% DMSO treatments, 10% DMSO and 10% DMSO + 2 mM L-proline maintained comparatively higher post-thaw sperm motility (54.36–51.04% and 56.44–49.75%, respectively), whereas other groups exhibited higher post-thaw sperm viability (>52%) than the other DMSO-based treatments. Although a progressive decline in sperm quality was observed over the 90-day storage period, L-proline-supplemented groups maintained superior functional performance. Morphological assessment showed better preservation of sperm head integrity and fewer structural abnormalities in the 10 mM L-proline treatment compared to other cryopreserved treatments. The findings indicate that supplementation with 10 mM L-proline enhances sperm membrane stability and overall cellular function during cryostorage.

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