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At least 19 recordsLinked to original sources

Livestock embryo sexing: A review of current methods, with emphasis on Y-specific DNA probes.

Control of the sex ratio of domestic species is potentially of great commercial importance to agriculture. While sexing of spermatozoa would be the most advantageous approach, studies to date suggest that this technology is unlikely to be available in the near future. As an alternative, four methods of sexing embryos have been developed. The use of X-linked enzymes and a serological assay involving H-Y antigen are noninvasive methods which have the advantage of allowing all embryos to be sexed, but these methods are not always accurate. Cytogenetic analysis and the use of Y-specific DNA probes are invasive methods which are limited by the accessibility of embryonic material for biopsy, but they are highly accurate. Each method is reviewed, with an emphasis on the use of Y-linked probes, and each is seen to have both advantages and limitations; the difficulty is in achieving a method that provides both an accurate sexing procedure and an acceptable pregnancy rate after embryo transfer. While no single method currently available fulfills all the criteria for a commercial method of embryo sexing, the potential for the development of an ideal method does exist.

Journal Article↗

Splitting and biopsy for bovine embryo sexing under field conditions.

Improvements on embryo micromanipulation techniques led to the use of embryo bisection technology in commercial embryo transfer programs, and made possible the direct genetic analysis of preimplantation bovine embryos by biopsy. For example, aspiration and microsection, allow bovine embryos sexing by detection of male-specific Y-chromosome in a sample of embryonic cells. We report on the application of the methodologies of splitting and biopsy of bovine embryos in field conditions, and on the results of embryo sex determination by the polymerase chain reaction (PCR). Pregnancy rates achieved with fresh bisected or biopsied embryos (50 to 60%) were similar to the fresh intact embryos (55 to 61%). The PCR protocol used for embryo sexing showed 92% to 94% of efficiency and 90 to 100% of accuracy. These results demonstrate these procedures are suitable for use in field conditions.

Animals↗

Using embryo sexing within closed mixed multiple ovulation and embryo transfer schemes for selection on dairy cattle.

Two types of multiple ovulation and embryo transfer schemes that included bull progeny testing were compared. In the juvenile schemes, embryos were collected at 16 to 18 mo of age without sexing, whereas, in the adult schemes, donors were chosen based on their first lactation record, and their embryos were systematically sexed. With the latter schemes, natural calves obtained at the first two calvings could compete with embryo transfer calves to be replacements. The optimal structure of this scheme was derived algebraically for the same number of transferred embryos as in the juvenile schemes. Predicted asymptotic annual genetic gains, after stabilization of genetic parameters taking into account the Bulmer effect, were found to be slightly in favor of the adult schemes for a given set of parameters (overall number of transferred embryos, number of embryos per collection, and embryo survival rate). In the adult schemes, the nucleus sizes were much larger than in the juvenile schemes, which allowed a higher selection differential on male paths, thus compensating for the longer generation interval. Asymptotic rate of genetic gain for Monte Carlo simulations were about 10 and 7% lower for juvenile and adult schemes, respectively, but still higher (20%) than the predicted value for the corresponding conventional scheme. Consequently, adult schemes with embryo sexing can be an efficient alternative to juvenile schemes without embryo sexing.

Animals↗

Embryo sex selection by a rat male-specific antibody and the cytogenetic and developmental confirmation in cattle embryos.

Embryos of mouse, rabbit, goat, sheep, and cattle were separated into 2 groups on the basis of their morphology when incubated with a male-specific antibody (qualified here as the H-Y antibody) prepared from newborn rat testis. When morula-stage embryos were cultured in the presence of this H-Y antibody, the development of roughly one half of the embryos was arrested at that stage, whereas the other half continued to develop to the blastocyst stage. The developmentally arrested group of embryos resumed their development into blastocysts when cultured in antibody-free medium. Eighty to 90% of cattle embryos whose development was unaffected by the antibody were shown to possess a female karyotype (XX), and close to 80% of those embryos whose development was arrested possessed a male karyotype (XY). Cattle embryos whose sex had been presumptively identified by development in the presence of the H-Y antibody were cryopreserved and transferred, and the sex of the calves was examined. The overt sex of the young born from sexed embryos was found to be the same as that determined by chromosomal analysis.

Animals↗

Ovine-specific Y-chromosome RAPD-SCAR marker for embryo sexing.

An accurate, sensitive, and quick (approximately 3 h) method for determining the sex of ovine embryos was developed using polymerase chain reaction (PCR) primers derived from an ovine-specific Y-chromosome random amplified polymorphic DNA marker (UcdO43). The accuracy and sensitivity of the assay were first tested using genomic DNA from 10 males and 10 females of five different sheep breeds, and then tested using serial dilutions of male-in-female DNA. The assay was 100% accurate in confirming the sex of the individuals and the ovine male-specific fragment was detected in dilutions containing as little as 10 pg of male DNA in 50 ng of female DNA. The assay was also confirmed to be specific for the ovine Y-chromosome as bovine, caprine, porcine, murine, and human DNA did not amplify. The ovine embryo sexing method is a duplex PCR system that also includes ZFY/ZFX primers. ZFY/ZFX provide an internal positive control for amplification as well as a means to confirm the results obtained with the UcdO43 primers. All embryo sexing results (36/36) from our method were in agreement with the ZFY/ZFX assay results. However, while our method requires an internal control to detect PCR failure, it has the advantages of not requiring nested PCR or restriction endonuclease digestion of the PCR product, and concerns about cross-species contamination are eliminated.

Animals↗

A novel repeated sequence located on the bovine Y chromosome: its application to rapid and precise embryo sexing by PCR.

A novel repeated sequence specific to male cattle was identified and named S4. S4 is a highly repetitive sequence and is a 1.5 kb repeating unit that contains various internal repeated sequences. FISH analysis showed that S4 is localized on the whole long arm and the proximal region of the short arm of the Y chromosome. We found that a PCR primer set for S4 amplified a male-specific 178 bp product in addition to a 145 bp product common to both male and female cells. Although the origin of the 145 bp product is unknown, it acts as a positive internal control in practical embryo sexing. Due to the high copy number of S4, PCR required only 0.5 pg purified DNA for accurate amplification. This made it possible to reduce the amount of biopsy sample required for embryo sexing and thus result in less damage to embryos manipulated. These studies indicate that embryo sexing based on the S4 sequence is accurate and sensitive.

Animals↗

Preimplantation embryo sexing by polymerase chain reaction amplification of the sry gene on single mouse blastomeres.

Accurate and rapid sex determination of preimplantation embryos has great potential both in animal breeding and in human pathology. In the past, sex determination has been accomplished by cytogenetic or immunologic means and by polymerase chain reaction amplification of Y-chromosome-specific repetitive sequences. More recently, amplification of the Y-specific single-copy ZFY gene has been used in humans for sex determination of preimplantation embryos. The experiments reported here indicate that another Y-chromosome-specific single-copy gene, the sex-determining region gene (sry) can be successfully amplified from single mouse blastomeres. Blastocysts positive for sry amplification were reimplanted to foster mothers, and six of six newborns were male. We conclude that sry gene amplification can represent a good marker for embryo sex determination.

Animals↗

[Detecting embryo sex by fluorescence in-situ hybridization in preimplantation genetic diagnosis].

OBJECTIVE: Applying fluorescence in situ hybridization(FISH) in preimplantation gender diagnosis. METHODS: Ovarian hyperstimulation was performed in 2 hemophilia A carriers and 2 patients with Y chromosome abnormality. Embryo sex was identified by single blastomere FISH after embryo biopsy. Female embryos were transferred into uterus. RESULTS: A total of 110 cumulus-oocyte complex were retrieved in 5 treatment cycles. Among them, 68.2% showed normal fertilized pronuclei and 55 embryos were available for embryo biopsy. The success rate of biopsy was 85.5%, with further cleavage rate of 61.7%. In FISH procedure, one cell was lost during fixation, led to 97.9% fixation rate. Totally, 18 female embryos were diagnosed and 16 were transferred into uterus. Two clinical and one biochemical pregnancies were achieved. The diagnosis was confirmed by the following analysis of aminocyte and embryonic buds after embryo reduction respectively. CONCLUSIONS: FISH is an efficient and accurate technique for determination of the sex of human preimplantation embryos. Selective abortion and birth of affected child can be avoided by preimplantation gender diagnosis.

Female↗

Recent developments in embryo sexing and its field application.

This review focuses on polymerase chain reaction (PCR) sexing of bovine embryos in commercial situations with emphasis on new developments. Simplifications of the biopsy technique is one of the major simplifications over the last few years. The stabilization of the embryo by means of protein-free medium or scratches produced on the bottom of the Petri dish makes it possible to perform a biopsy with a single microinstrument. The traditional PCR sexing approach utilizes electrophoresis, which involves the risk of deoxyribonucleic acid (DNA) contamination of subsequent assays. Such contamination, resulting in females misdiagnosed as males, is avoided efficiently by using a non-electrophoretic method in which the sex is determined based on fluorescence of unopened tubes. However, female samples cannot be distinguished from blank samples in the non-electrophoretic assay, which thus relies on accurate transfer of biopsy into tubes. Nevertheless, an accuracy of about 95% can be reached with both approaches. High pregnancy rates (50-70%) can be reached with biopsied Grade 1 embryos, but there is evidence that pregnancy rates with Grade 2 embryos is 15-20% lower. Recent data indicate that pregnancy rates of 50% can be achieved with frozen-thawed biopsied Grade 1 embryos. In conclusion, recent developments in biopsy techniques, detection systems and freezing should increase interest in PCR sexing.

Animals↗

Rapid CRISPR-based bovine embryo sexing to streamline genotype-informed cattle breeding.

Cattle in vitro fertilisation and embryo transfer programmes increasingly rely on embryo-level selection to accelerate genetic gain, but current sexing and genotyping workflows can be costly, slow and logistically demanding. This study developed an efficient, low-resource workflow for bovine embryo sexing that combines whole genome amplification (WGA) with recombinase polymerase amplification-CRISPR-Cas12a (RPA-Cas12a). It also assessed whether the same WGA biopsy products could be used for downstream single nucleotide polymorphism (SNP) microarray genotyping. A one-tube RPA-Cas12a assay targeting the bovine Y-chromosome S4 repeat was developed for fluorescence and lateral flow assay (LFA) readouts. Analytical sensitivity was assessed using serially diluted bovine genomic DNA (gDNA), and breed robustness was tested using male and female gDNA from five major beef breeds and Holstein cattle. The workflow was then applied to WGA products from 22 bovine blastocyst biopsies, with sex calls validated against an established real-time PCR melt curve assay and 100K SNP microarray genotyping. The assay detected male bovine gDNA down to 100 pg using both fluorescence and LFA readouts, with no signal from female gDNA. Male-specific detection was consistent across all breeds tested. All WGA-RPA-Cas12a sex calls from blastocyst biopsies were concordant with real-time PCR and SNP microarray sex calls, and WGA biopsy products produced genome-wide SNP call rates above 85%. This workflow provides a practical approach for rapid bovine embryo sex triage and could reduce unnecessary cryopreservation and genotyping while improving the efficiency of genotype-informed cattle breeding programmes.

Bovine embryo↗

Effect of cell cycle synchronization on the accuracy of murine and bovine embryo sex determination.

Different cell cycle synchronization methods were used to increase the mitotic index and accuracy of sex determination in murine and bovine embryos. For sexing purposes, colchicine treatment for 2, 4, 6 and 8 h and the FdU-thymidine-colchicine combination were tested in murine embryos. The best results were obtained with colchicine treatment for 8 h (96.88% accuracy) and with FdU-thymidine-colchicine (97.22% accuracy). Mitotic indexes differed significantly between the 2 treatments (21.71% for colchicine and 32.95% for FdU-thymidine-colchicine). For sex identification of murine and bovine demi-embryos, both treatments were demonstrated to be equally effective (nearly 90%). The mitotic index for the FdU-treated murine demi-embryos (19.04%) was higher than the one obtained for the 8-h colchicine treatment (15.62%).

Journal Article↗

Detection of embryo sex chromosome by dual color fluorescent in-situ hybridization.

In order to evaluate the effects of sex chromosomal mosaicism on the accuracy of single-cell gender diagnosis, sex chromosomes of 21 normal fertilized embryos were detected by dual color fluorescent in-situ hybridization (FISH). The results showed that 4 embryos had sex chromosomal mosaicism (19%) and the remaining 17 showed uniformly XX or XY signals in all blastomeres. In conclusion, identification of sex by dual color FISH analysis of a single cell was accurate and efficient, and sex chromosomal mosaicism would not affect preimplantation gender diagnosis.

Blastocyst↗

Polymerase chain reaction and its applications: special emphasis on its role in embryo sexing.

The polymerase chain reaction (PCR) has developed into one of the most promising methods for in vitro enzymatic amplification of DNA and has found widespread application in DNA cloning, sequencing and mutagenesis related studies. This innovative technique can selectively amplify a single target DNA molecule a billion-fold in a span of a few hours. Amplification of specific DNA sequences by PCR is useful in identification of sex, novel genes, pathogens and diseases. PCR has facilitated the establishment of evolutionary relationships among species and in revealing structural intricacies of single cells. In this article we review some of the major advances and applications of PCR, especially, its role in embryo sexing.

Journal Article↗

Effect of time of artificial insemination on embryo sex ratio in dairy cattle.

The objective of the present study was to examine whether different intervals between insemination and ovulation have an influence on the sex of seven-day-old embryos in dairy cattle. Cows were inseminated once with semen of one of two bulls of proven fertility between 36 h before ovulation and 12 h after ovulation. Time of ovulation was assessed by ultrasound at 4-h intervals. In total, 64 embryos were determined to be male or female. Of these 64 embryos, 51.6% were female. The sex ratio in the various insemination-ovulation intervals (early: between 36 and 20 h before ovulation; intermediate: between 20 and 8 h before ovulation; late: between 8 h before and 12 h after ovulation) did not significantly differ from the expected 1:1 sex ratio (50, 50 and 55% females, respectively). Bull (Bull A and B) and Parity (primiparous and multiparous) had no influence on the expected 1:1 sex ratio either. The number of cell cycles was similar for male and female (P = 0.23) embryos when quality of the embryo (P < 0.0001) was included in the model. The results of this study indicate that, in cattle, the interval between insemination and ovulation does not influence the sex ratio of seven-day-old embryos.

Animals↗

Generation of transgenic dairy cattle from transgene-analyzed and sexed embryos produced in vitro.

We have generated a transgenic calf from in vitro produced bovine embryos which had undergone transgene analysis and sexing prior to the embryo transfer. Bovine oocytes were isolated from slaughter-house-derived ovaries, matured and fertilized in vitro and subsequently microinjected with a dam-methylated gene construct consisting of genomic sequences encoding human erythropoietin and governed by bovine alpha S1-casein regulatory sequences. After 6 to 7 days in culture, the embryos were biopsied and while the embryo remained in culture, the biopsy was subjected to transgene analysis and sexing. The transgene analysis was accomplished with a combined treatment of the embryo lysates with DpnI restriction endonuclease and Bal31 exonuclease followed by polymerase chain reaction (PCR). The transgene analysis was based on the fact that DpnI only cleaves its recognition sequence if the adenine in the sequence is methylated. Pregnancy was induced by the transfer of three viable female embryos with a distinct transgene signal to a hormonally synchronized heifer recipient. Amniotic fluid analysis performed two months after the embryo transfer confirmed the presence of the transgene. The calf born was found to be transgenic by PCR analysis from blood, ear and fetal membranes. The presence of the transgene was also confirmed by Southern blotting.

Animals↗

Yolk androgens and embryo sex: maternal effects or confounding factors?

Maternal effects occur when offspring phenotype is affected by environmental factors experienced by the mother and, in egg-laying species, are often mediated via egg resources. There is currently great interest among behavioural ecologists in maternally allocated yolk androgens, especially their relationship with offspring sex and development. Such studies need embryonic tissue for sexing, however, requiring eggs to be incubated (usually for 3 days). Therefore, there are concerns about whether the androgen concentrations assayed reflect those allocated by the mother. In addition, studies showing sex biases in maternal allocation of androgens could be confounded if male and female embryos uptake or metabolise androgens at different rates. We ran a series of experiments using zebra finch (Taeniopygia guttata) eggs to address these potential confounding factors. First we showed, using eggs naturally incubated for up to 5 days, that eggs containing embryos had lower yolk androgen concentrations than eggs that had failed to form embryos. We then tested various hypotheses for this difference using controlled incubation treatments. Our results suggested that (a) embryo development causes the yolk to become progressively more diluted with albumin; and (b) between 3 and 5 days of incubation embryos start uptaking or metabolising androgens. Crucially, we found no decline in yolk androgen concentration at 3 days incubation, and no evidence for sex-specific rates of uptake or metabolism of androgens. This strongly suggests that yolk androgen levels up to 3 days incubation do reflect those allocated by the mother, and that studies of sex biased maternal allocation of yolk androgens are not confounded by sex differences in embryo development.

Animals↗

In vitro production of sexed embryos for gender preselection: high-speed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer.

The objectives for the present experiments were to apply sperm sexing technology to an in vitro production system with porcine oocytes obtained from slaughterhouse material. On six experimental days, ovaries were obtained from an abattoir, and cumulus-oocyte-complexes were matured in vitro. Semen was collected from mature boars of proven fertility and was sorted for X-chromosome-bearing sperm, using the Beltsville Sperm Sexing Technology incorporating the use of high-speed sorting. A total of 5,378 oocytes were submitted for in vitro fertilization (IVF). Of these, 559 ova were stained for cytogenetic analysis 18 h after IVF. From the remaining 4,819 ova, 1,595 cleaved, and 1,300 of the cleaved embryos were transferred into 26 synchronized recipients (5 control gilts for unsorted sperm, 21 gilts for X-sorted sperm). In a test of two fertilization media (FERT-A vs FERT-B) higher cleavage rates (P<.05) were obtained when FERT-B was used as a fertilization medium for unsorted (43.4+/-5.1%) and sorted sperm (43.1+/-1.1%;), whereas in FERT-A unsorted sperm gave a cleavage rate of 17.9+/-4.4% and sorted sperm gave 30.4+/-1.4%. Additionally, cleavage rates were higher (P<.05) after fertilization with sorted sperm vs unsorted sperm, independent of fertilization medium. Cytogenetic analysis of ova revealed that more oocytes with unsorted than with sorted sperm remained in Metaphase 2 arrest (P<.05). This was also independent of the fertilization medium. Monospermic fertilization rates were the same for IVF with unsorted or sorted sperm, independent of the fertilization system, except FERT-A with unsorted sperm (P<.05). Polyspermic fertilization rates were highest in FERT-B (37.6+/-6.6). A total of 57 pigs were born from nine litters. Six litters from sexed sperm (X-sorted) produced 33 females (97%) and one male. Three litters from control transfers produced 23 pigs, 11 of which were female (48%). The sex ratio of the offspring was predicted based on the sort reanalysis of the sorted sperm for DNA content.

Animals↗

Sex determination in sheep and goats using bovine Y-chromosome specific primers via polymerase chain reaction: potential for embryo sexing.

A simple and novel method, using polymerase chain reaction (PCR) has been standardized for accurate sex determination in sheep and goats. The assay utilizes a pair of bovine Y-chromosome specific primers and the genomic DNA isolated from blood samples of adult male and female sheep and goats. The primers recognize and amplify the Y-chromosome specific sequences in male goats and sheep. The assay is accurate, reliable and rapid.

Animal Husbandry↗