Quantification of damage at different stages of cryopreservation of endangered North American bison (Bison bison) semen and the effects of extender and freeze rate on post-thaw sperm quality.

Semen cryopreservation is an important technique for the banking of animal germplasm from endangered species and exploitation of genetically superior sires through artificial insemination. Being a member of bovidae family, bison semen has poor freezing ability as compared to dairy and beef bulls' semen. This study was designed to quantify the damage to bison sperm at different stages of cryopreservation, and to determine the effects of extender (commercial Triladyl(®) vs. custom made tris-citric acid [TCA]) and freeze rate (-10, -25 and -40°C/min) on post-thaw quality of bison semen. Semen was collected from five bison bulls (three woods and two plains) via electroejaculation. In Experiment 1, semen was diluted in Triladyl® extender and frozen with freeze rate -10°C/min. Sperm motility characteristics were recorded in fresh, diluted, cooled (4°C) and freeze-thawed semen using computer-assisted sperm analyzer (CASA). In Experiment 2, semen was diluted in Triladyl® or TCA extender, and frozen with three different freeze rates, i.e. -10, -25 or -40°C/min. Thawing was performed at 37°C for 60s. Post-thaw sperm motility characteristics were assessed using CASA, and sperm structural characteristics (plasma membrane, mitochondrial membrane potential and acrosomes) were evaluated using flow cytometer, at 0 and 3h while incubating semen at 37°C. In Experiment 1, total and progressive motilities did not differ among pre-freeze stages of cryopreservation (P>0.05). However, sperm total and progressive motilities declined (P<0.001) in freeze-thawed semen by 35% and 42%, respectively, compared to after cooling (pre-freeze) semen. In Experiment 2, Triladyl®, as compared to TCA, yielded greater (P<0.05) post-thaw sperm total motility (41% compared to 36%) and progressive motility (34% compared to 29%) at 0h, respectively. The percent change in post-thaw sperm total and progressive motilities, VAP, VCL, VSL, IPM-high ΔΨm and IPM-IACR during 3h incubation at 37°C, was less (P<0.05) in TCA than in Triladyl®. There was an effect of freeze rate on post-thaw sperm average path velocity at 0h, and total motility, progressive motility, VCL, IPM and IPM-IACR at 3h were the greatest (P<0.05) when bison semen was frozen at -40°C/min. Likewise, the percent change in post-thaw sperm total and progressive motilities, during 3h incubation at 37°C, was less (P<0.05) in bison semen frozen at -40°C/min. All post-thaw bison sperm characteristics decreased (P<0.05) from 0h to 3h, during incubation at 37°C. In conclusion, the maximum damage to bison sperm occurred during freeze-thaw processes. Post-thaw total and progressive motilities of bison sperm were greater in Triladyl® at 0h whereas sperm survival was greater in TCA extender during 3h post-thaw incubation. Bison sperm had greater survival (P<0.05) when frozen at -40°C/min freeze rate.

Hussain SA ，Lessard C ，Anzar M 《-》

A strategy for improvement of postthaw quality of bison sperm.

The objective was to improve the postthaw quality of bison semen using zwitterion (ZI)-based extenders, glycerol addition at a lower temperature (4 °C), adding reduced glutathione (GSH) in extender, or treating bison sperm with cholesterol-loaded cyclodextrin (CLC) before freezing. Postthaw sperm motility and structural characteristics were analyzed using a computer-assisted sperm analyzer and flow cytometer respectively, at 0 and 3 hours postthaw incubation at 37 °C. In experiment 1, each ejaculate (N = 11) was diluted in Triladyl extender (control) or in ZI extenders (Tes-Tris or HEPES-Tris). In addition, glycerol in semen was added either at 37 °C or 4 °C before cryopreservation. Extenders had no significant effect on postthaw sperm motilities at 0 hour. However, sperm velocity parameters were higher (P < 0.05) in ZI extenders than in Triladyl. Sperm population with intact plasma membrane (IPM) and acrosomes (IACR) were higher in Triladyl than in ZI extenders (P < 0.05). Postthaw sperm total and progressive motilities, average path velocity, straight-line velocity, IPM, and IPM-IACR did not improve with the addition of glycerol at 4 °C. In experiment 2, semen was diluted (50 × 10(6) sperm per mL) in Triladyl extender containing 0 (control), 0.5, 1.0, or 2.0 mM GSH (an antioxidant) at 37 °C. Postthaw sperm motility and structural characteristics at 0 hour and percentage declined after 3 hour incubation, but did not differ because of GSH in the extender (P > 0.05). In experiment 3, fresh bison sperm (100 × 10(6) sperm in 1 mL) were pretreated with 0, 1, 2, or 3 mg/mL of CLC at 22 °C for 15 minutes and diluted to 50 × 10(6) sperm per mL in Tris-citric acid-egg yolk-glycerol extender before cryopreservation. The CLC pretreated sperm had higher (P < 0.05) postthaw total and progressive motilities, IPM, and IACR at 0 hour and less percentage of decline in these characteristics after 3 hour postthaw incubation. In conclusion, zwitterion extenders (Tes-Tris and HEPES-Tris), temperatures of glycerol addition, and GSH in extender did not significantly improve postthaw quality of bison sperm. However, pretreatment with CLC significantly improved postthaw quality of bison sperm, which should enhance its use in assisted reproductive technologies.

Hussain SA ，Lessard C ，Anzar M 《-》

Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes.

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.

Anzar M ，Kroetsch T ，Boswall L 《-》

Retained functional integrity of bull spermatozoa after double freezing and thawing using PureSperm density gradient centrifugation.

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.

Maxwell WM ，Parrilla I ，Caballero I ，Garcia E ，Roca J ，Martinez EA ，Vazquez JM ，Rath D ... -  《REPRODUCTION IN DOMESTIC ANIMALS》

Post-thaw viability of european bison (Bison bonasus) semen frozen with extenders containing egg yolk or lipids of plant origin and examined with a heterologous in vitro fertilization assay.

Pérez-Garnelo SS ，Oter M ，Borque C ，Talavera C ，Delclaux M ，Martínez-Nevado E ，Palasz AT ，De la Fuente J ... -  《JOURNAL OF ZOO AND WILDLIFE MEDICINE》