Ultra-Low Temperature Cryopreservation of Sperm from a New Type of Hybrid Bream

Sperm cryopreservation is crucial for artificial propagation and the long-term preservation of valuable germplasm resources. This study aimed to establish an effective ultra-low temperature sperm cryopreservation protocol for Hefang bream (HFB), a novel hybrid bream variety developed through distant hybridization between blunt snout bream (Megalobrama amblycephala) and topmouth culter (Culter alburnus) followed by two rounds of backcrossing. Different combinations of extenders and cryoprotectants were evaluated based on post-thaw sperm motility and motion parameters to identify the optimal cryoprotective formulation for HFB sperm, with untreated fresh sperm as the control. Fresh sperm exhibited a total motility (MOT) of 98.87 ± 1.40%, curvilinear velocity (VCL) of 118.87 ± 5.38 μm/s, straight-line velocity (VSL) of 86.18 ± 5.67 μm/s, and average path velocity (VAP) of 107.48 ± 4.23 μm/s. The optimal formulation consisted of D15 extender (composed of 8 g/L NaCl, 0.5 g/L KCl and 15 g/L glucose) supplemented with 10% dimethyl sulfoxide (DMSO). Using a fresh sperm-to-cryoprotective medium ratio of 1:5 and a stepwise cooling procedure, post-thaw MOT, VCL, VSL, and VAP were 43.77 ± 13.85%, 37.54 ± 3.06 μm/s, 29.82 ± 1.98 μm/s, and 31.99 ± 2.14 μm/s, respectively. Further analyses revealed that the plasma membrane integrity (PMI), DNA integrity (DI) and mitochondrial activity (MA) of cryopreserved sperm were 43.40 ± 2.01%, 57.10 ± 4.79%, and 42.00 ± 1.65%, respectively, all of which were significantly lower than those of fresh sperm (p < 0.05). Ultrastructural analyses using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated that cryopreserved spermatozoa exhibited structural abnormalities, including plasma membrane disruption, flagellar fragmentation, and mitochondrial damage. Artificial insemination experiments showed that the fertilization and hatching rates of the cryopreserved sperm group were 55.10 ± 3.30% and 81.50 ± 3.29%, respectively, indicating that the established protocol could effectively support artificial reproduction in HFB. Furthermore, proteomic analysis was performed to investigate cryopreservation-induced molecular damage, identifying 7 cryopreservation-associated leakage proteins that were mainly enriched in pathways related to energy metabolism, cytoskeletal organization, and oxidative stress response. This study establishes an effective sperm cryopreservation protocol for HFB and provides insights into the cellular and molecular mechanisms underlying cryopreservation-induced sperm damage, offering a valuable reference for the development of sperm cryopreservation technologies in other economically important fish species.