Effect of the addition of two superoxide dismutase analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation.

The main objective was to study the effects, on sperm function, of the addition of two superoxide dismutase (SOD) analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation. Twelve alpaca semen samples were collected using an artificial vagina and then diluted at a 1:3 ratio in an extender based on skim milk, egg yolk, and fructose. Each semen sample was divided into three equal parts to form the following groups: control, Tempo (1 mM), and Tempol (1 mM). Groups were cooled to 5 °C in 90 minutes (-1 °C in 3 minutes); when samples reached approximately 10 °C, SOD analogues were added to the respective groups. At 5 °C, ethylene glycol (final concentration, 0.1 M) was added to each group. After 30 minutes at 5 °C, samples were loaded in 0.25 mL plastic straws, placed in liquid nitrogen vapor for 15 minutes, and then plunged. Percentages of sperm motility, functional sperm membrane integrity, and viable sperm with intact acrosomes were evaluated before and after freeze-thaw using visual analysis, the hypoosmotic swelling test, and the double-stain trypan blue/giemsa technique, respectively. The Terminal deoxymucleotidyl transferase dUTP Nick End Labeling assay was performed for evaluation of sperm DNA fragmentation of frozen-thawed sperm. Sperm motility was higher (P < 0.05) in the Tempol and Tempo groups than in the control group (mean, 22.1%, 19.7%, and 11.2%, respectively), with similar results for functional sperm membrane integrity. Additionally, DNA fragmentation was lower (P < 0.05) in the Tempol group (16.7%) than in the control group (38.8%). Viable sperm with intact acrosomes were not affected by the use of SOD analogues. There was a negative correlation (r = -0.58) between DNA fragmentation of alpaca sperm and sperm motility after freeze-thawing, but DNA damage was neither related to functional membrane integrity nor viable sperm with intact acrosomes. We concluded that DNA fragmentation and loss of motility during cryopreservation of alpaca sperm could be partially prevented by supplementation of the semen extender with 1 mM Tempo or Tempol.

Santiani A ，Evangelista S ，Valdivia M ，Risopatrón J ，Sánchez R ... -  《-》

Can permeable super oxide dismutase mimetic agents improve the quality of frozen-thawed ram semen?

This study was carried out to assess the effects of MnTBAP, a cell permeable antioxidant, on motility, membrane integrity, capacitation status and in vitro fertilization ability of frozen-thawed ram semen. Fresh semen ejaculates were collected with artificial vagina from five rams, mixed and divided into five equal fractions, and diluted (1:20 v/v) with commercial extender, Bioxell®, containing 0 (control), 50, 100, 150 and 200 μM of MnTBAP. All diluted sperm suspensions were cooled to 5°C for 2h followed by transfer into 0.5 ml French straws before being stored in liquid nitrogen. The results showed that MnTBAP supplementation of extender improved ram semen quality in a dose-dependent manner. Accordingly, the extender supplemented with 150μM MnTBAP resulted in higher sperm motility and improved acrosomal membrane integrity compared to control. However, further supplementation (200μM) with MnTBAP not only did not improve the results but inversely affected motility and membrane integrity. The results of in vitro fertilization (IVF) indicated that the presence of MnTBAP in semen extender has a marginal beneficial effect on developmental competence of inseminated oocytes, though this improvement was not significant. In conclusion, this study demonstrated that semen extender supplemented with MnTBAP can reduce the oxidative stress provoked by freeze/thaw processes. Moreover, beneficial effect of 100 μM of MnTBAP on preservation of spermatozoa in a non-capacitated state post freezing, an important criterion for in vitro or in vivo fertilization, was observed. However, at 150 μM of MnTBAP, the harmful effects of cryopreservation on membrane integrity were decreased. Regarding to importance of non-capacitated spermatozoa during IVF or artificial insemination, the optimum MnTBAP concentration appears to be 100 μM for commercial ram semen extender tested here.

Forouzanfar M ，Fekri Ershad S ，Hosseini SM ，Hajian M ，Ostad-Hosseini S ，Abid A ，Tavalaee M ，Shahverdi A ，Vosough Dizaji A ，Nasr Esfahani MH ... -  《-》

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 《-》

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction.

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe(2+)) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n=13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe(2+), EE-CAT plus Fe(2+), EE-GPx plus Fe(2+) and EE-SOD plus Fe(2+)). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2h after thawing (P<0.05). Catalase supplementation, however, improved DNA integrity at 4h (P<0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6h, linear motility at 6h, mitochondrial activity at 6h, membrane integrity at 2 and 6h, and DNA integrity at 4h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2h after thawing (P<0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe(2+) negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P<0.05). After thawing, there were, however, no significant differences between the control plus Fe(2+) and the antioxidative enzymes supplementation plus Fe(2+) groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.

Thuwanut P ，Chatdarong K ，Johannisson A ，Bergqvist AS ，Söderquist L ，Axnér E ... -  《-》

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》