353 IN VITRO FERTILIZATION WITH FLOW-SORTED BUFFALO SPERM
M. Zhang A B , X. W. Liang A B , Y. Q. Lu A B and K. H. Lu A BA Guangxi Key Laboratory for Subtropical Bio-Resource Conservation and Utilization
B Animal Reproduction Institute, Guangxi University, Nanning, Guangxi 530005, China
Reproduction, Fertility and Development 18(2) 283-284 https://doi.org/10.1071/RDv18n2Ab353
Published: 14 December 2005
Abstract
Flow cytometrically sorted X and Y sperm have been successfully used for IVF and the production of offspring in cattle and sheep (Maxwell et al. 2004 Anim. Reprod. Sci. 82–83, 79–95).The objective of this study was to test the feasibility of flow sorted buffalo sperm used in IVF systems and to establish a suitable IVF system for sorted buffalo sperm. Oocytes were aspirated from 2–6 mm follicles on the buffalo ovaries from a slaughterhouse and matured for 22–24 h in a 1-mL dish containing TCM199 + 10% OCS + 3% BFF (bovine folliciular fluid) + hormones at 38.5°C, 5% CO2 in air with maximum humidity. Semen was sorted by a flow-sorter (Clontech, Mountain View, CA, USA) into X- and Y-chromosome bearing sperm at 90% accuracy and stored at 4°C for later use with IVF. Sorted sperm were prepared for IVF by centrifugation (700g, 20 min) through a Percoll gradient (90%:45%), and washed (centrifugation at 700g, 5 min) in mTALP-BSA. For IVF, groups of 10–15 oocytes were transferred to 40-μL drops of mTALP-BSA and incubated with motile sperm at a concentration of 2 × 106 sperm mL−1 in each fertilization drop for 8–10 h under mineral oil. Presumptive zygotes were cultured until Day 8 in 25-µL drops of TCM–199 supplemented with 0.33 mM pyruvate and 10% NCS with granulosa cells at 38.5°C under 5% CO2 in air. Cleavage and blastocyst rates per oocyte insemination were recorded on Day 2 and Days 6–8 after insemination, respectively. Data were analyzed by ANOVA procedures with replicates and treatments in the model. There were significant differences in cleavage rate (42.23% vs. 52.28%) and blastocyst rate (20.62% vs. 27.67%) between sorted and unsorted sperm, respectively (Table 1). There were no significant differences in the proportions of blastocyst development rates on Days 6, 7, or 8 after insemination with sorted and unsorted sperm. The results indicate that sorted buffalo sperm from two bulls have been successfully used in an IVF system to produce sex-controlled embryos.
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This research was supported by grants from the National Natural Science Foundation of China (30360073) and the Guangxi Department of Science and Technology (0330004–13). The authors (M. Z. and X.W. L.) contributed equally to this work.
