Pre-freeze sperm quality
All four extenders maintained high initial sperm motility of 81-88% and durations around 70-75 secs before freezing (p>0.05) (Table 3), indicating standardized collection and handling protocols ensured uniform sample quality prior to cryopreservation.
Differential cryo-protection conferred by extenders
However post-thaw, the extenders differentially preserved sperm motility indicating distinct cryoprotective capacities intrinsic to their bioactive compositions. Across all thawing temperatures, E1 containing NaCl, KCl and NaHCO
3 salts buffered with NaH
2PO
4 markedly outperformed all other extenders in retaining motility-conferring 29-39% higher protection than E2 and enormous 39-42% greater viability over E3/E4 post-freeze (p≤0.05). For example, at 37°C thawing, while E1 maintained 42% motility with 63 sec durability, E2 only achieved 31% viability with 57 sec sustainability. More dramatically, E3 and E4 rendered sperm almost entirely non-motile (~3-5%) despite optimal thawing rates-highlighting their inability to prevent cryo-injuries
(Cabrita et al., 2005).
E1’s exceptional effectiveness for cryopreserving Golden Mahseer spermatozoa aligns with previous findings by
Basavaraja and Hegde (2004) that a similar formulation conferred maximal post-thaw retention of motility (~55%) and duration (~80 secs) in the related Deccan Mahseer. Collectively, the salts, buffers and osmolytes in E1 likely cooperatively stabilize cells during thermal and osmotic stresses of freeze-thaw by maintaining structural integrity. Specifically, balanced salt solutions prevent excessive dehydration or swelling by retaining optimal osmolality
(Cabrita et al., 2005). Bicarbonate buffers stabilize pH homeostasis during temperature and liquid-solid phase transitions
(Glogowski et al., 2002). Phosphate augments membrane architecture conservation and repairs cryo-damage. Glucose supplements provide energetic substrates to sustain motility machinery
(Cabrita et al., 1998; Basavaraja and Hegde, 2004;
Cabrita et al., 2005). The exact bioprotective concentrations and interactions maximizing viability in E1 warrants further investigation.
Conversely, the poor cryo-protection offered by E3 and E4 indicates suboptimal composition, concentration or absence of key osmolytes that left cells vulnerable to marked freeze-thaw injuries despite standardized cooling/warming protocols. Both formulations omitted essential salts and metabolic substrates. Furthermore, the egg yolk and milk powders in E3 seemingly failed to prevent viability loss. Their cholesterol and milk fat globule membrane supplements were clearly inadequate to offset cryo-damage compared to E1’s synergistic bioactives. The organic components in E3 and E4 may have had inherent properties or interactions that compromised their ability to protect sperm cells during cryopreservation, potentially interfering with the protective mechanisms conferred by other components in the extender solution
(Galeati et al., 2011).
While E2 containing NaCl, KCl and NaHCO3 salts performed better than E3/E4, its simpler salt-bicarbonate formulation still proved far less effective than E1’s more comprehensive composition. Absent phosphates, glucoses and lower NaCl in E2 likely compromised its capacity to balance osmolality, energize motility and repair injured architecture post-thaw
(Cabrita et al., 1998).
Overall, Extender E1, which contained a balanced salt solution along with buffer and an energy source, demonstrated remarkably better performance in retaining post-thaw sperm motility compared to other extenders. All extenders contained the same concentration (10% v/v) of the permeating cryoprotectant DMSO. However, the presence or absence of other components like salts, buffers and osmolytes influenced the overall cryoprotective ability, suggesting their critical role in complementing the permeating cryoprotectant. The findings revealed that extender salt composition, balance and permeating cryoprotectant supplementation critically govern cryopreservation success by shielding cells from osmotic, oxidative and structural damage during phase transition stresses.”
Thawing temperature dramatically influences post-thaw viability
For all extenders, elevating thawing warmth from 37®40®45°C progressively halved motility at each transition - collapsing from 31-42%®8-12%®1-11% respectively (p≤0.05). Even under E1’s optimal buffering, increasing thaw temperature curtailed motility 3-fold from 42% at 37°C to 12% and 11% at 40°C and 45°C respectively. Faster warming intensified thermal shock which likely disrupted sperm membrane architecture and organelle ultrastructure
(Alizadeh et al., 2016). Sudden ambient temperature shifts can fracture phospholipid arrangements and cytoskeletal dynamics necessary for motility by exceeding membrane fluidity thresholds
(Cerolini et al., 2000). Intracellular ice re-crystallization also potentially disrupts mitochrondrial and axoneme continuity. Moreover, amplified oxidative stress from warmer-induced biochemical fluctuations possibly overwhelmed endogenous antioxidant systems - permitting reactive oxygen species to accumulate and damage proteins, lipids and DNA to further affect viability (
Bansal and Bilaspuri, 2010).
Effect of activation delay on post-thaw spermatozoa motility
The effect of activation delay on post-thaw spermatozoa motility was investigated by delaying the activation of spermatozoa for 5, 10 and 20 minutes. The results revealed a gradual decrease in motility with increasing storage time (the time elapsed between thawing the samples and activating them, not the duration of cryostorage), as depicted in Fig 3. Specifically, a delay in activation by 10 minutes led to a sharp decline in spermatozoa motility when extenders E1 and E2 were used. An extremely low level of spermatozoa motility, approximately 12% for E1 and 10% for E2, was observed when the activation was delayed by 20 minutes. These findings emphasize the importance of minimizing activation delay for successful cryopreservation, particularly when using extenders E1 and E2.
Therefore, independent of extender composition, strictly regulated thawing rates are imperative to peacefully rehydrate cells after cryostorage without creating thermal shock. Gentle warming around body temperature (37°C) seems optimal for smoothly transitioning vitrified samples back to liquid state without overt disruption of intracellular architecture
(Cabrita et al., 2001). Controlling re-warming likely allows gradual permeation of external cryoprotectants to safely rehydrate cells without osmotic injury. Thus, both extending medium formulation and thawing temperature critically control post-thaw viability - highlighting target areas for strategic improvements.
Despite conferring maximal protection, E1 retained only ~50% baseline motility indicating enormous potential for further bio-enhancement by supplementing additional cryoprotectants and antioxidants. Anti-apoptotics like caspase inhibitors could also potentially amplify fertilizing capacity by blocking intrinsic cell death pathways triggered by freeze-thaw stress (
Martínez-Páramo et al., 2009). Nanocarrier delivery systems enabling timed-release of molecular shields may also minimize cryo-losses
(Moraes et al., 2010). Selective breeding approaches that isolate hardy cryo-tolerant sperm phenotypes based on biomarkers could ultimately generate resilient lines where >90% viability becomes achievable after biobanking (
Martinez-Pastor et al., 2004). Overall, the findings deliver a robust starting point for advancing reproducibility and amplifying the scale of biobanking for this endangered iconic species. Options now exist for efficiently regenerating selective bloodlines or resurrecting regionally extinct populations if needed to restore aquatic biodiversity. As wild stocks continue dwindling from escalating anthropogenic pressures, having an indefinite genome repository through biobanking serves as insurance against irreversible genetic erosion or extinction.
