Biopsy and sex determination by PCR of IVF bovine embryos

Macháty, Zoltán; Páldi, Andràs; Csáki, T; Varga, Zsolt; Kiss, István Z.; Bárándi, Zs.; Vajta, Gábor

doi:10.1530/jrf.0.0980467

Cited by 61 publications

(35 citation statements)

References 15 publications

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“…Cell sampling by biopsy has been applied to IVP bovine embryos at the 16-to 32-cell stage [47,48], morula-stage [49,50], and blastocyst-stage [51,52]. Since efficiency of sexing decreased significantly when less than five cells were subjected to PCR [52], the larger sampling size (8-10 cells) has been recommended for successful sexing of the embryos [53].…”

Section: Cryopreservation Of Sex-identified Embryosmentioning

confidence: 99%

Cryopreservation and Sexing of In Vivo- and In Vitro-Produced Bovine Embryos for Their Practical Use

Tominaga

2004

Journal of Reproduction and Development

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Abstract. My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GLTip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GLTip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex predetermination.

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Section: Cryopreservation Of Sex-identified Embryosmentioning

confidence: 99%

Cryopreservation and Sexing of In Vivo- and In Vitro-Produced Bovine Embryos for Their Practical Use

Tominaga

2004

Journal of Reproduction and Development

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“…When embryo sexing is performed with male-specific target sequences, one cannot distinguish the false-negative amplification caused by sampling errors or from lack of amplification in female samples. To solve this problem, various combination of two different PCR primer sets, one for a male-specific sequence and another for a sequence common to both sexes, have been examined as a multiplex PCR to obtain internal bovine control for embryo sexing [12][13][14][15][16][17].…”

mentioning

confidence: 99%

Multiplex Polymerase Chain Reaction Used for Bovine Embryo Sex Determination

Rattanasuk

Parnpai

Ketudat-Cairns

2011

Journal of Reproduction and Development

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Abstract. Widely used bovine sexing primers were compared in terms of suitability in determining the sex of bovine embryos. Under optimized multiplex PCR conditions, the ConBV/ConEY couple primers did not show accurate results when combined together in multiplex PCR, but worked well when the couple primers were used separately. The S4BF/ S4BR primers showed accurate results; however, some unexpected bands were detected. When the BY/BSP couple primers were used to determine one-cell, two-cell, four-cell and eight-cell stage embryos of known sexed SCNT-derived embryos, the results showed 100% accuracy. The BY/BSP couple primers were also able to identify the sex of one-cell and two-cell IVF-derived embryos. Key words: Bovine sex determination, Embryo sexing, Multiplex PCR (J. Reprod. Dev. 57: [539][540][541][542] 2011) ex determination of preimplantation embryos is important for manipulating the sex ratios of domestic animals at an early stage of embryo development [1,2]. A number of approaches have been used to determine the sex of bovine embryos but have failed to gain much popularity due to the lack of either accuracy or speed [3]. Simple and precise methods for embryo sexing are important for management and improvement of bovine species. Polymerase chain reaction (PCR) has been used for sexing of bovine embryos [4,5] with various target sequences, including male-specific repetitive sequences [6,7] and single-copy genes on the Y chromosome, such as amelogenin [8,9] and ZFY gene [10][11][12]. When embryo sexing is performed with male-specific target sequences, one cannot distinguish the false-negative amplification caused by sampling errors or from lack of amplification in female samples. To solve this problem, various combination of two different PCR primer sets, one for a male-specific sequence and another for a sequence common to both sexes, have been examined as a multiplex PCR to obtain internal bovine control for embryo sexing [12][13][14][15][16][17].In the present study, three widely used primer sets were compared using genomic DNA and embryo at various developmental stages. The embryos used in this study were somatic cell nuclear transfer (SCNT)-derived embryos of known sex and in vitro fertilization (IVF)-derived embryos of unknown sex. They were used as template in the multiplex PCR reactions.To compare the suitable couple primers used for bovine sex determination, 0.42 ng of high molecular weight DNA from male and female bovine fibroblast was amplified using three different sets of bovine sexing primers (Table 1) and subjected to gel electrophoresis. The PCR products were detected after ethidium bromide staining. The results in Fig. 1 suggested that the BY/BSP couple primers were able to amplify male bovine genomic DNA, which gave two fragments of 300 bp from Y-specific primers (BY) and 538 bp from bovine-specific primers (BSP) (lane 1) and a clear single band of 538 bp (lane 2) from BSP amplified female bovine genomic DNA. The S4BF/S4BR primers showed clear accurate results with bovine genomic DN...

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“…Takeuchi et al [17] reported that they collected human single blastomeres at the 4-and 8-cell stages by the enucleation, aspiration, or extrusion method, and the viability of embryos at the 4-cell stage was not affected by the aspiration or extrusion method, and the viability of embryos at the 8-cell stage was not affected by either method. In cattle, single blastomeres at the 16-to 32-cell stages collected by the aspiration method were transplanted, and fertility similar to that of the control subjects was obtained [6]. In this study, the 7/8 embryos obtained by the extrusion method developed to blastocysts without difference from the development of intact embryos at the 8-cell stage, showing that the collection of embryos by the extrusion method causes less inhibition of embryonic development.…”

Section: Discussionmentioning

confidence: 54%

“…They reported that PCR product common to both sexes was detected in 95% of the samples, and the sex ratio (male ratio) was 65.8%. Macháty et al [6] collected single blastomeres from embryos at the 16-and 32-cell stages, and determined the sex with male-specific and gender neutral primers. The detection rate for PCR product common to both sexes was 95%, but the sex ratio was not mentioned.…”

Section: Discussionmentioning

confidence: 99%

“…Among these methods, the method using a male-specific antigen and the method using sex difference in the rate of development are not always accurate, and the overall sexing rates of the method for measuring enzymes linked to the X chromosome and the method for detecting the Y chromosome are low. Therefore, the method for detecting Y chromosome-specific nucleotide sequences by amplifying the sequence by polymerase chain reaction (PCR method) is considered to be promising, and has been developed for cattle [3][4][5][6][7], pigs [8] and mice [9,10]. As an advantage of the PCR method, tests can be rapidly and accurately performed on only a few cells, and commercial use in cattle has begun, but with the current biopsy method, blastomeres are collected by excising mural trophectoderm cells with the zona pellucida at the blastocyst stage, and there is a risk of affecting the normal development of the fetus.…”

Section: Introductionmentioning

confidence: 99%

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Sex Determination by PCR in Single Bovine Blastomeres Biopsied by Extrusion Method at the 8-cell Stage.

et al. 2000

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Abstract:We attempted to determine the sex by polymerase chain reaction (PCR) in single bovine blastomeres at the 8-cell stage. PCR was performed on male-specific primers attached to a bovine embryonic sex determination kit, an XY Selector. Embryos at the 8-cell stage were isolated by the EDTA method, and one (1/8 embryo), two (2/8 embryo), and four (4/8 embryo) blastomeres were subjected to PCR. The detection rates for male-specific PCR product were 25.8, 25.8 and 45.2% for 1/8, 2/8 and 4/8 embryos, respectively. In some embryos, despite detection of the male-specific product in 4/8 embryos, the male-specific product was not detected in 1/8, 2/8 or both 1/8 and 2/8 embryos derived from the same embryo (3.2, 3.2 and 16.1%, respectively). Collecting single blastomeres by the extrusion method affected neither the rate of development to blastocysts nor the number of cells in blastocysts. PCR was performed in 1/8 embryos collected by the extrusion method, and the male-specific PCR product was detected. Nevertheless, in 27.5% of embryos, despite detection of male-specific PCR product in 7/8 embryos, the male-specific product was not detected in the 1/8 embryo from the same embryo. These findings indicated that collecting single blastomeres at the 8-cell stage allows the selection of the male embryo by PCR, and also the extrusion method is useful for biopsy of embryo at the 8-cell stage. Key words: Sexing, PCR, Bovine, Embryo, Biopsy As sex determination methods for early mammalian embryos, a method using male-specific antigen, a method using sex difference in the rate of development, a method which quantifies X chromosome-linked enzymes, a method for detecting the Y chromosome, and a method that amplifies Y chromosome-specific nucleotide sequences and analyzes the product have been investigated [1,2]. Among these methods, the method using a male-specific antigen and the method using sex difference in the rate of development are not always accurate, and the overall sexing rates of the method for measuring enzymes linked to the X chromosome and the method for detecting the Y chromosome are low. Therefore, the method for detecting Y chromosome-specific nucleotide sequences by amplifying the sequence by polymerase chain reaction (PCR method) is considered to be promising, and has been developed for cattle [3][4][5][6][7], pigs [8] and mice [9,10]. As an advantage of the PCR method, tests can be rapidly and accurately performed on only a few cells, and commercial use in cattle has begun, but with the current biopsy method, blastomeres are collected by excising mural trophectoderm cells with the zona pellucida at the blastocyst stage, and there is a risk of affecting the normal development of the fetus. In mice, the extrusion method has been established, in which blastomeres at the 4-or 8-cell stage are biopsied by extruding from a small slit made in the zona pellucida [11,12]. Collecting samples for PCR by this method may reduce injury to the embryo.Therefore, in this study, we collected single blastomeres at the 8-cell s...

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Biopsy and sex determination by PCR of IVF bovine embryos

Cited by 61 publications

References 15 publications

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

Multiplex Polymerase Chain Reaction Used for Bovine Embryo Sex Determination

Sex Determination by PCR in Single Bovine Blastomeres Biopsied by Extrusion Method at the 8-cell Stage.

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