Sperm cryodamage occurs after rapid freezing phase:flow cytometry approach and antioxidant enzymes activity at different stages of cryopreservation

Background： In order to improve the efficiency of bovine sperm cryopreservation process, it is important to understand how spermatozoa respond to differences in temperature as well as the ability to recover its own metabolism. The combination between flow cytometry approach and antioxidant enzymes activity allows a more sensible evaluation of sperm cell during cryopreservation. The aim of this study was to evaluate sperm attributes and antioxidant enzymes activity during different stages of cryopreservation process. Semen samples from Holstein bulls （n = 4） were separated in 3 treatments： fresh （37 ℃）; cooled （5 ℃）; and thawed. Evaluation occurred at 0 h and 2 h after incubation. Membrane integrity, mitochondrial membrane potential （MMP） and DNA damages were evaluated by flow cytometry; activities of antioxidant enzymes such as catalase, superoxide dismutase and gluthatione peroxidase were measured by spectrofotometry. Results： There was an increase in the percentage of sperm with DNA damage in the thawed group, compared to fresh and cooled, and for 2 hs of incubation when compared to 0 h. Considering MMP, there was an increase in the percentage of cells with medium potential in thawed group when compared to fresh and cooled groups. Opposingly, a decrease was observed in the thawed group considering high mitochondrial potential. Also in the thawed group, there was an increase on cells with damaged acrosome and membrane when compared to fresh and cooled groups. Significant correlations were found between antioxidant enzymes activity and membrane or mitochondrial parameters. Conclusion： Based on our results, we conclude that cryopreservation affects cellular and DNA integrity and that the critical moment is when sperm cells are exposed to freezing temperature. Also, our study indicates that intracellular antioxidant machinery （SOD and GPX enzymes） is not enough to control cryodamage.

<i>In Vivo</i>9.4T MRI and ¹H MRS for Evaluation of Brain Structural and Metabolic Changes in the Ts65Dn Mouse Model for Down Syndrome

In the present study we investigated structural and metabolic modifications of the brain in the Ts65Dn mouse model of Down syndrome(DS)using both in vivo magnetic resonance imaging(MRI)and proton magnetic resonance spectroscopy(MRS). MRI was performed for further texture analysis and changes in texture parameters, including mean grey levels, contrast and homogeneity, and they were found in Ts65Dn compared to diploid littermates (2n). These phenotypic changes were different in the hippocampus and cerebellum, since in Ts65Dn mean grey levels increased in the cerebellum and decreased in the hippocampus. In addition, proton NMR spectra revealed differences in metabolite ratios. Levels of N-acetylaspartate(NAA)and glutamate(Glu), were lower compared to total creatine levels (CX), in the Ts65Dn brain. However, the most striking finding was an increase in the concentration of myo-inositol(Ins)and choline(Cho)in the hippocampus, whereas the Ins concentration was reduced in the cerebellum. Overall, these data illustrate that MRI and MRS are valuable assesment tools sufficiently sensitive to detect associated changes in different brain areas, thus providing new insight into the causative role of dosage-sensitive genes in the Ts65Dn DS mouse model.

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals：A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species.Moreover,these novel tools have become easier to use and have resulted in a great increase of applications.Whilst gene knockout（KO） or knockin（KI） animal models are relatively easy to achieve,there is a bottleneck in the detection and analysis of these mutations.Although several methods exist to detect these targeted mutations,we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process.The HMA-CE method uses a simple PCR amplification of genomic DNA（gDNA） followed by an automated capillary electrophoresis step which reveals a heteroduplexes（HD） signature for each mutation.This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.