scholarly journals An X-Y paint set and sperm FISH protocol that can be used for validation of cattle sperm separation procedures

Reproduction ◽  
2001 ◽  
pp. 541-546 ◽  
Author(s):  
W Rens ◽  
F Yang ◽  
G Welch ◽  
S Revell ◽  
PC O'Brien ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Y Chromosome ◽  
Close Agreement ◽  
Fish Method ◽  
Sperm Fish ◽  
Sperm Separation ◽  
Sperm Decondensation

X and Y chromosome paints were developed from sorted yak chromosomes for sexing cattle spermatozoa. Clear hybridization signals were obtained for every spermatozoon using a modified sperm decondensation protocol and fluorescence in situ hybridization (FISH). The procedure was evaluated using the established Beltsville sperm sexing technology, which separates spermatozoa by flow cytometry into X- and Y-bearing fractions. Close agreement was found between the assessment of sperm separation by flow cytometry and by FISH with the X-Y paint set. The FISH method is a simple, reliable and robust procedure for assessing the effectiveness of separation of X and Y spermatozoa.

268 IDENTIFICATION OF X- AND Y-BEARING SPERMATOZOA IN SORTED BUFFALO (BUBALUS BUBALIS) SEMEN BY FLUORESCENCE IN SITU HYBRIDIZATION

2009 ◽  
Vol 21 (1) ◽  
pp. 231
Author(s):  
M. Zhang ◽  
X. J. Zhuang ◽  
Y. Q. Lu ◽  
C. H. Hu ◽  
S. S. Lu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Y Chromosome ◽  
X Chromosome ◽  
Chromosome Painting ◽  
Fish Method ◽  
Painting Probes ◽  
Flow Cytometry Sorting

Flow cytometry sorting technology has been successfully used to sort the X- and Y-chromosome bearing sperm. Previous studies showed that fluorescence in situ hybridization (FISH) method was a simple and reliable procedure for assessing the effectiveness of separation of X- and Y-sperm in the swine (Kawarasaki T et al. 1998 Theriogenology 50, 625–635) and the bovine (Rens W et al. 2001 Reproduction 121, 541–546). Reports of sex-preselection by flow-cytometry sorting of the X- and Y-sperm were also seen in the buffalo (Presicce GA et al. 2005 Reprod. Dom. Anim. 40, 73–75; Lu YQ et al. 2006 Anim. Reprod. Sci. 100, 192–196). There was, however, no report to date for using the FISH method to assess the purity of the sorted buffalo sperm. The objective of the present study was to verify the purity of flow cytometrically-sorted buffalo X- and Y-sperm by FISH using bovine X- and Y- chromosome painting probes prepared by microdissection. The X- and Y- chromosomes of bovidea were microdissected respectively from the metaphase spreads of Holstein blood cells with a glass needle controlled by a micromanipulator and amplified by degenerate oligo-nucleotide primer-PCR (DOP-PCR) (Mariela N et al. 2005 Genet. Mol. Res. 4, 675–683). The DOP-PCR products of X- and Y- chromosome were labeled with CY3-dUTP and Biotin-11-dUTP, respectively. The buffalo X- or Y-sperm DNA from unsorted semen and sorted semen were hybridized to the labeled probes, respectively. The results showed that the hybridized signals were clearly visible in the metaphase karyotype of bovine and buffalo semen samples. About 47.7% (594/1246) and 48.9% (683/1396) of the unsorted buffalo sperm emitted strong fluorescent signals when assessed by Y- and X-chromosome painting probes, respectively, which was conformed to the sex ratio in normal buffalo sperm (50%:50%). About 86.1% (1529/1776) hybridization signals of the sperm in the sorted X-semen assessed by X-chromosome painting probes were detected, while 82.2% (2232/2716) of the Y-sorted buffalo sperm emitted strong fluorescent signals when assessed by Y-chromosome painting probe. The results of the flow cytometer re-analysis revealed that the proportions of X- and Y-bearing sperm in the sorted semen were 89.6% and 86.7%, respectively. There were no apparent differences between the two assessment methods of sperm separation by flow cytometry re-analysis and by FISH with the X-Y paint probe. In conclusion, bovine X- and Y-chromosome painting probes prepared using microisolation method could be used to verify the purity of the sorted sperm in the buffalo. This study was supported by the Guangxi Department of Science and Technology (0626001-3-1) and National Key Technology R&D Program, The People’s Republic of China (2006BAD04A18). The authors (M. Zhang, X.J. Zhuang, and Y.Q. Lu) contributed equally to this work.

scholarly journals Fluorescence in situ hybridization in diluted and flow cytometrically sorted boar spermatozoa using specific DNA direct probes labelled by nick translation

Reproduction ◽  
2003 ◽  
pp. 317-325 ◽  
Author(s):  
I Parrilla ◽  
JM Vazquez ◽  
M Oliver-Bonet ◽  
J Navarro ◽  
J Yelamos ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Y Chromosome ◽  
Internal Control ◽  
Dna Probes ◽  
Chromosome 1 ◽  
Nick Translation ◽  
Fluorescent Signal ◽  
Sperm Separation

Successful evaluation of X- and Y-chromosome-bearing sperm separation technology using flow cytometry-cell sorter is of great importance. Fluorescence in situ hybridization (FISH), which allows for the detection of specific nucleic acid sequences on morphologically preserved spermatozoa, is an ideal method for quantitatively and qualitatively assessing the purity of sorted sperm samples. In this study specific pig DNA direct probes for small regions of chromosomes 1 and Y were used. Chromosome 1 was labelled in green and used as internal control to detect a lack of hybridization, whereas chromosome Y was labelled in red. Nick translation was used as the labelling method for the preparation of these probes. Spermatozoa, unsorted and sorted for high and low Y-chromosome purity from ejaculates of five boars, were fixed on slides and two-colour direct FISH was performed for chromosomes 1 and Y. About 500 non-sorted and 200 sorted spermatozoa per sample were scored. The proportion of Y-chromosome-bearing spermatozoa was determined by the presence of a red fluorescent signal on the sperm head and the proportion of X-chromosome-bearing spermatozoa was determined by subtraction. The efficiency of the hybridization procedure was established as near 98% on sorted and unsorted samples. The results of this study confirm that direct FISH using specific pig DNA probes labelled by nick translation provides a useful tool for laboratory validation of sperm separation by flow sorting technology. Moreover, the ease of nick translation and the quality of the fluorescent signal obtained using this method makes this procedure the most appropriate method for labelling pig DNA probes to be used for direct FISH on pig spermatozoa.

scholarly journals Telomere analysis by fluorescence in situ hybridization and flow cytometry

1998 ◽  
Vol 26 (16) ◽  
pp. 3651-3656 ◽  
Author(s):  
M. Hultdin ◽  
E. Gronlund ◽  
K.- F. Norrback ◽  
E. Eriksson-Lindstrom ◽  
G. Roos ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization

Localization of the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei by fluorescence in situ hybridization

Chromosoma ◽  
1993 ◽  
Vol 102 (8) ◽  
pp. 546-552 ◽  
Author(s):  
Ron Hochstenbach ◽  
Monique Wilbrink ◽  
Ron Suijkerbuijk ◽  
Wolfgang Hennig
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Y Chromosome ◽  
Drosophila Hydei
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