Supplementation of extender with chamuangone-enriched Garcinia cowa leaf extract improves quality parameters and longevity of cold-stored cat semen.

BACKGROUND: Semen preservation by cooling is less expensive, simpler and results in less sperm damage than freezing does. However, spermatozoa can only be preserved for a short period due to the excessive formation of reactive oxygen species (ROS). Although several antioxidants can protect sperms from ROS damage during storage at low temperatures, the use of natural antioxidants derived from plants would be a better alternative. OBJECTIVE: To assess the effects of chamuangone, which can reduce oxidation reactions in cells, on cat semen quality after preservation at 4 degree C for 15 days. MATERIALS AND METHODS: Epididymal sperm samples were collected before being diluted with tris-citric-fructose-egg yolk (TCFE) extender containing different concentrations of chamuangone (0, 50, 100, 150 and 200 ug/mL) and preserved at 4 degree C. Semen samples were evaluated before chilling and then every 3 days after chilling for up to 15 days. Each sample was assessed for sperm motility, viability, DNA integrity, plasma membrane integrity and percentage of spermatozoa with intact acrosomes. RESULTS: A significantly higher sperm motility was observed in the group supplemented with 100 ug/mL chamuangone compared to the control after 6 days of storage. However, the chamuangone concentration at 200 ug/mL did not significantly increase the sperm motility when compared to the control for the entire storage period. CONCLUSION: 100 µg/mL chamuangone can improve sperm characteristics during 15 days of preservation at 4 degree C, keeping sperm alive (49.3 ± 5.2%) and moving (7.1 ± 2.4%). These results can be used for the development of breeding programs using technologically advanced reproductive procedures in domestic and wild cats. https://doi.org/10.54680/fr24110110212.

In vitro Development of Zona Pellucida-free Porcine Zygotes Cultured Individually after Vitrification.

BACKGROUND: Cryopreservation of zona pellucida (ZP)-free embryos provides more options for somatic cell nuclear transfer, particularly during handmade cloning. OBJECTIVE: This study investigated whether the removal of the ZP affects the development of porcine zygotes after vitrification and warming. MATERIALS AND METHODS: We determined the appropriate volume of the corresponding medium for the individual culture of ZP-intact and -free embryos and evaluated the protection effect of ZP during cryopreservation on the resulting development of the vitrified-warmed zygotes. RESULTS: The volume of culture medium influenced the development of ZP-intact zygotes, and a volume of 15 µL was most suitable for their development. However, the volume of culture medium did not modify the development of ZP-free zygotes. The removal of the ZP before vitrification did not adversely affect embryonic development or quality of the resulting blastocysts. CONCLUSION: Our results suggest that the removal of the ZP does not cause detrimental effects to the development of vitrified-warmed zygotes.

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 µs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 µs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.

Chelation of trace elements in preservation medium influences the quality of boar spermatozoa during liquid preservation at 5°C for 4 weeks.

BACKGROUND: The addition of a metal chelator, ethylenediaminetetraacetic acid (EDTA), to semen extender has the purpose of capturing trace element ions. OBJECTIVE: This study was conducted to evaluate the effects of EDTA on the quality and in vitro fertilisability of liquid-preserved boar spermatozoa. METHODS: In Experiment 1, semen samples were preserved in the semen extender supplemented with 0, 3, 6, or 12 mM of Na-EDTA at 5 degree C for 4 weeks. In Experiment 2, semen samples were preserved in the extender supplemented with 3 mM of Na-EDTA, Ca-EDTA, or Zn-EDTA and without chelator EDTA. RESULTS: When Na-EDTA was used as a chelating substance in the extender, 3 mM was a most suitable concentration for sperm motility and viability after cold preservation. The supplementation of 3 mM Ca-EDTA had advantages regarding sperm motility, viability and plasma membrane integrity. CONCLUSION: Our findings indicate that 3 mM Ca-EDTA is the most suitable metal-chelating substance for the liquid preservation of boar semen.

Cell cycle synchronization of skin fibroblast cells in four species of family Felidae.

This study was examined whether the species of felid affects synchronization accuracy at the G0/G1 stage of the cell cycle and the occurrence of apoptosis by different protocols, such as serum starvation, confluent and roscovitine treatment. Skin fibroblast cells were obtained from the Asian golden cat, marbled cat, leopard and Siamese cat. The cells from each animal were treated with either serum starvation for 1-5 days, cell confluency-contact inhibition for 5 days or roscovitine at various concentrations (7.5-30 µm). Flow cytometric analysis revealed that serum starvation for 3 days provided the highest cell population arrested at the G0/G1 stage, irrespective of the felid species. In all species, 100% confluency gave a significantly higher percentage of cells arrested at the G0/G1 stage compared with the non-treated control cells. The effects of roscovitine treatment and the appropriate concentration on the rates of G0/G1 cells differed among the felid species. Serum starvation for more than 4 days in the marbled cat and Siamese cat and roscovitine treatment with 30 µm in the Asian golden cat and leopard increased the rates of apoptosis. In conclusion, different felid species responded to different methods of cell cycle synchronization. Asian golden cat and Siamese cat fibroblast cells were successfully synchronized to G0/G1 stage using the serum starvation and roscovitine treatment, whereas only confluency-contact inhibition treatment induced cell synchronization in the leopard. Moreover, these three methods did not successfully induce cell synchronization of the marbled cat. These findings may be valuable for preparing their donor cells for somatic cell nuclear transfer in the future.

Effect of sericin on preimplantation development of bovine embryos cultured individually.

The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 µm H(2)O(2)), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 µm H(2)O(2). However, the protective effect of sericin was not observed in development of embryos exposed to 50 µm H(2)O(2). When embryos were exposed to 100 µm H(2)O(2) during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress.