The use of gelatine in long‐term storage (up to 48 hr) at 5°C preserves the pre‐freezing and post‐thawing quality of brown bear sperm

Abstract

Sedimentation of spermatozoa occurs during long-term liquid storage and this may produce deleterious changes. Our aim was to apply gelatine supplementation during long-term pre-freezing storage of bear sperm, applying final dilution and 6% glycerol at room temperature and cool in straws. We tested four models of sperm storage using a 1:1 dilution in TTF-ULE-Bear extender (TesT-fructose-egg yolk-glycerol 6%): (i) second 1:1 dilution at room temperature (RT), cooling at 5°C in a tube and final dilution (100 × 10(6) sperm ml(-1) ) (Standard); (ii) final dilution at RT and cooling in a tube (FD-Tube); (iii) final dilution at RT and cooling in 0.25 ml plastic straw (FD-Straw); and (iv) final dilution at RT in extender supplemented with 1.5% gelatine (Gelatine) and cooling in a 0.25 ml plastic straw. A Standard sample was stored at 5°C for 1 hr (Control); the rest of the samples (Standard, FD-Tube, FD-Straw, Gelatine) were stored for 24 or 48 hrs before freezing (100 × 10(6) sperm ml(-1) , glycerol 6%). The quality of the samples was assessed for motility by CASA, and viability (SYBR-14/propidium iodide-PI-; VIAB), acrosomal status (PNA-FITC/PI; iACR) and apoptotic status (YO-PRO-1/PI; YOPRO-) by flow cytometry. At pre-freezing, after 48 hr, Gelatine showed significantly higher viability (for VIAB and YOPRO-) and progressiveness (PM, LIN and STR). At 48 hr, Gelatine showed similar YOPRO-, iACR, LIN, STR and ALH respect to Control. At both 24 and 48 h post-thawing, Gelatine sample had similar scores for YOPRO-, iACR, LIN, STR, WOB and VIAB (only 24 hr) when compared with Control, and lower for TM, PM, rapidPM, VAP and ALH. No differences were found among others experimental groups with respect to Control. In conclusion, gelatine could be a suitable alternative to preserve the viability and progressive motility of brown bear ejaculates during long-term pre-freezing storage at 5°C.

New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation

“…At present, the storage of organs by low temperatures is a widely used technology (Southard and Belzer 1995;Wang et al 2002). An absence of negative effect and even positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed before and is not new (Isachenko et al 2020;Lopez-Urueña et al 2016; b Fig. 2 Calcein-AM and PI staining data of fragments in control group a Profiler plot for two adherent staining fragments.…”

Freezability of Dog Semen after Collection in Field Conditions and Cooled Transport

“…Similar freezing protocols would also be interesting for wild species, whose semen could be collected in the field and then shipped to a specialized laboratory for freezing and storage. Encouraging results have been obtained, for instance, in the brown bear, whose semen, after storage at 5 °C for 24–48 h and freezing, could give satisfactory post-thawing outcomes [ 27 , 28 ].…”