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Experiments on sexing yak spermatozoa by fluorescence in situ hybridisation using bovine Y-chromosome specific DNA probe

R. Tamás,1 Q. Xuebin,2 P.T. Erika,3 N. Szabolcs,4 H. Jianlin,2 K. András,5 T. András3 and S. András1

1. Department of Biochemistry and Food Technology, Budapest University of Technology and Economics, H-1111 Budapest Muegyetem rkp. 3., Hungary
2. Department of Animal Science, Gansu Agricultural University, Lanzhou 730070, Gansu, P.R. China
3. Department of Obstetrics and Gynaecology, Faculty of Health Sciences, Semmelweis University, Budapest, Hungary
4. Department of Animal Genetics, Institute of Animal Husbandry, University of West Hungary, Mosonmagyaróvár, Hungary
5. Research Institute for Animal Breeding and Nutrition, Herceghalom, Hungary

Summary

Y-bearing bovine spermatozoa may be distinguished by fluorescence in situ hybridisation (FISH) using specific DNA probe, offering an in vitro control of experiments for producing sex-oriented or sex-specific semen. Bovine Y-specific probe gave distinguishable signal also in yak spermatozoa. Specific binding of the probe to the corresponding sex chromosome was confirmed on yak chromosome preparations.

Keywords: Cattle, fluorescence in situ hybridisation, sexing, spermatozoa, yak

Introduction

Cattle (Bos taurus, L.) and yak (B./Poephagus grunniens, L.) are two related species having identical diploid chromosome number (2n = 60; Krallinger 1931; Zuitin 1935). The autosomes are all acrocentric, the X-chromosome is a large sub-metacentric in both species, the Y-chromosome of yak is small sub-metacentric (Popescu 1969; Kovács et al. 1982) demonstrating some polymorphism (Gang et al. 1991 cited by Jianlin 1996), similar to European cattle (B. taurus typicus).

Y-bearing cattle spermatozoa may be identified by fluorescence in situ hybridisation (Hassanane et al. 1998; Tardy et al. 1998; Hassanane et al 1999; Révay et al. 2000). Based on the morphologically similar sex-chromosomes, we tried to apply bovine Y-specific probes for sexing yak spermatozoa. Our goal was to elaborate a rapid and effective in vitro technique to control the experiments on producing sex-oriented yak semen (Cao et al. 1996).

Materials and methods

Sample preparation

Frozen/thawed yak semen doses were diluted with 0.9% NaCl, smeared, air-dried in China and mailed to Hungary. To facilitate the penetration of the probe and better signal visualisation, sperm heads were decondensed with papain-dithiothreitol solution (Schwerin et al. 1991; Hassanane et al. 1999).

Chromosome preparations were made from blood taken from a yak bull in Hungary (Moorhead et al. 1960).

Preparation of chromosome specific hybridisation probe

The Y-chromosome specific probe was prepared by polymerase chain reaction (PCR) method. The primers are specific to the BC 1.2 region on the bovine Y-chromosome (Cotinot et al. 1991; Schwerin et al. 1991). The reaction was carried out using bovine male genomic DNA as template. The sequences of the oligonucleotide primers were: 5'ATC AGT GCA GGG ACC GAG ATG 3' and 5'AAG CAG CCG ATA AAC ACT CCT T 3'. The PCR was performed as described by Kobayashi et al. (1998) with modified MgCl2 concentration (1.2 mM). The 250 bp product was labelled with biotin-16-dUTP during the PCR.

Fluorescent in situ hybridisation

FISH was performed as described by Pinkel et al. (1986) with slight modifications. The biotin labelled probe was detected with the Tyradmide Signal Amplification System (Direct FITC, NEN Life Sciences) according to the manufacturer. The slides were examined under a fluorescent microscope (Zeiss Opton) with simple microscopic observation.

Results and discussion

Decondensation of yak spermatozoa needed a prolonged treatment as compared to cattle; 1525 vs. 36 minutes. The bovine Y-probe produced strong signals in decondensed yak spermatozoa (Figure 1). Specificity of the probe was confirmed on yak metaphase chromosomes: the Y-chromosome was labelled with the probe, and there was no signal on the autosomes (Figure 2).

Figure 1. Detection of the bovine Y-probe on yak spermatozoa.

Figure 2. Detection of the bovine Y-probe on yak metaphase chomosome preparation.

We can conclude that it is possible to use the bovine Y-probe to control experiments on producing sex-oriented yak semen. Further effort will be needed to prepare bovine X-chromosome specific DNA probe, which is easy to produce and allow simultaneous detection of both the sex chromosomes on yak spermatozoa.

Acknowledgments

We would like to express our sincere thanks to Prof Lu Zhonglin for supplying yak semen for this study. This work was supported by the Hungarian Academy of Sciences (OTKA grant T 025300). We thank Prof Dohy János academician, head of the Agricultural Committee of the Hungarian Academy of Sciences for supporting our work from his grant from the Hungarian Ministry of Education (FKFP 0468/1997).

References

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