Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification.
Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was <1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application.
Hirayama
Hiroki
H
Hokkaido Animal Research Center, Shintoku, 081-0038, Japan. h.hirayama@agri.pref.hokkaido.jp
Kageyama
Soichi
S
Moriyasu
Satoru
S
Sawai
Ken
K
Onoe
Sadao
S
Takahashi
Yoshiyuki
Y
Katagiri
Seiji
S
Toen
Keiko
K
Watanabe
Keiko
K
Notomi
Tsugunori
T
Yamashina
Hidenari
H
Matsuzaki
Sigenori
S
Minamihashi
Akira
A
D000818:Animals; D001757:Blastomeres / Q000737:chemistry; D002417:Cattle; D004247:DNA / Q000302:isolation & purification; D004624:Embryo Transfer / Q000662:veterinary; D004622:Embryo, Mammalian; D047108:Embryonic Development; D019286:Endopeptidase K; D005260:Female; D006358:Hot Temperature; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary*; D016133:Polymerase Chain Reaction; D011136:Polysorbates; D011247:Pregnancy; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis / Q000379:methods / Q000662:veterinary*; D012972:Sodium Hydroxide; D013997:Time Factors
D011136:Polysorbates; D012972:Sodium Hydroxide; D004247:DNA; D019286:Endopeptidase K