Separation of ram spermatozoa bearing X and Y chromosome by centrifugal countercurrent distribution in an aqueous two-phase system.
The availability of reliable quantification techniques of X and Y chromosome-bearing spermatozoa in a given insemination dose would allow further approaches to their separation, which is a topic of unquestionable interest in animal production. The aim of the current work was the development of a combined approach of polymerase chain reaction and countercurrent distribution to address both objectives. First, using Sac/polymorphisms for ZFX/ZFYloci in sheep deoxyribonucleic acid, a linear correlation has been established between the densitometric quantification of the restricted fragment length polymorphisms corresponding to the amplified loci ZFX/ZFY by polymerase chain reaction and the theoretical proportions of X and Y chromosomes in standard solutions. The method, subsequently applied to semen samples, estimated an equal proportion of spermatozoa bearing each chromosome. Second, by using centrifugal countercurrent distribution in a sensitive-charge aqueous two-phase system, we achieved the separation of a sperm population enriched in Y chromosome-bearing ram spermatozoa (75%) with a high viability rate (57%).
Ollero
M
M
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Universidad de Zaragoza, Spain.
Pérez-Pé
R
R
Gargallo
I
I
Morlanes
S
S
Osada
J
J
Muiño-Blanco
T
T
Cebrián-Pérez
J A
JA
D000818:Animals; D001483:Base Sequence; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D003377:Countercurrent Distribution / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific; D005260:Female; D051741:Kruppel-Like Transcription Factors; D008297:Male; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D019849:Sex Determination Processes; D012756:Sheep; D013094:Spermatozoa / Q000166:cytology*; D014157:Transcription Factors; D014960:X Chromosome; D014998:Y Chromosome
D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D014157:Transcription Factors; C062076:zinc finger protein, X-linked; D004247:DNA; C024408:endodeoxyribonuclease SacI; D015252:Deoxyribonucleases, Type II Site-Specific