Cloning and sequencing of buffalo male-specific repetitive DNA: sexing of in-vitro developed buffalo embryos using multiplex and nested polymerase chain reaction

Buffalo Y-chromosome specific repetitive DNA (BuRY.I) was cloned and sequenced in order to develop a sensitive method for sexing of buffalo preimplantation stage embryos using polymerase chain reaction (PCR). A highly sensitive and reliable sex determination assay using a primary (BRY.I), nested (BuRYN.I) and multiplex (BuRYN.I, ZFX/ZFY) PCR was developed. The BRY.I and BuRYN.I primers are targeted to amplify Y-specific sequences, while the ZFX/ZFY loci was amplified to serve as a positive control for both male and female samples. Accuracy of the sex determination assay was initially verified with genomic DNA obtained from blood of known gender. Further sensitivity and reproducibility of the assay was examined using DNA obtained from 1 or 2 blastomeres to demi embryos. Altogether, 80 IVF-derived embryos ranging from the 2 to 4 cell to the blastocyst stage were used for sex determination. Definite and clear signals following PCR amplification were obtained from all embryo samples. Accuracy of assays was determined by comparing results from a single cell with those of blastocyst stage embryos, thereby indicating that 1 or 2 blastomeres from a preimplantation buffalo embryo is sufficient for sex determination by PCR. No misidentification was observed within the embryo samples using nested (BuRY.I), primary (BRY.I) and multiplex (BuRYN.I; ZFX/ZFY) PCR, suggesting that this technique is a highly reliable method for sexing buffalo embryos.     

PCR-sexing of bovine embryos: a simplified protocol

To make bovine embryo sexing under farm conditions more feasible we developed a simplified protocol utilizing manual biopsy and detection of the Y chromosome directly from polymerase chain reaction (PCR) reaction tubes. Twenty-four embryos (morulae and blastocysts) were biopsied manually into 2 to 4 samples. One sample of each original embryo was diagnosed for sex, based on restriction fragment length polymorphism of PCR-amplified DNA of the ZFX/ZFY locus. The remaining 44 samples were diagnosed using the tube detection assay. In this assay the biopsies were pipetted into 0.5 -ml reaction tubes containing lysis mixture, incubated 10 to 60 min at 37 degrees C and inactivated 10 min at 98 degrees C. Then the PCR mixture was added containing buffer, DNA polymerase, ethidium bromide and primers designed to amplify the highly repeated btDYZ-1 region of the bovine Y chromosome. After 50 cycles of PCR, the reaction tubes were examined under UV illumination for pink fluorescence indicating the presence of Y-chromosomal DNA. All sexing results from the replicates were in agreement with the ZFX/ZFY assay, with 12 of the original embryos diagnosed as females and 12 as males. We conclude that highly efficient and accurate PCR-sexing of embryos can be accomplished without the use of micromanipulators, control primers and electrophoresis. The 2 reaction mixtures needed for sex diagnosis can be stored at -20 degrees C and -196 degrees C, respectively. The tube detection assay minimizes the risk of carryover contamination by previously amplified products as there is no need to open the tubes following PCR.     

Sex determination of bovine embryo blastomeres by fluorogenic probes

One of the major challenges of using genetic information in marker assisted selection (MAS) is the detection of multiple marker loci from a small biopsy sample of a preimplantation stage embryo. The objective of this study was to develop a fast, nested, multiplex preamplification, polymerase chain reaction (PCR) method for the determination of sex in bovine embryo blastomeres. For this aim, ZFX/ZFY sequences were preamplified simultaneously with other genomic regions. The preamplification product was used as a template in an allelic discrimination assay, with nested primers and sex specific fluorogenic probes for ZFX and ZFY. Fluorogenic probes were used to eliminate the need for time consuming electrophoresis. Compared to sexing with Bovy/kappa-casein co-amplification method and other replicates from the same embryo, the accuracy of sexing with the use of fluorogenic probes after preamplification was 99% (112/113 blastomeres). The amplification efficiency was 96% (113/117 blastomeres).     

Interfamilial characterization of a region of the ZFX and ZFY genes facilitates sex determination in cetaceans and other mammals

Sequence polymorphism of homologues ZFX and ZFY, in a 604-base pair exon region, was examined in 10 known males and 10 known females across seven cetacean families and used to design a simple, highly sensitive and widely applicable fluorescent 5' exonuclease assay for gender determination in cetaceans. Multiplex amplification, cloning, and sequencing of these previously uncharacterized regions revealed (i) eight fixed differences between ZFX and ZFY, (ii) 29 variable sites between ZFX and ZFY and (iii) very low interspecific nucleotide diversity for both ZFX and ZFY across all families examined. We developed a 5' exonuclease assay that produces a small (105 bp) polymerase chain reaction (PCR) product from both the X and the Y chromosome orthologs, and used double-labelled fluorescent probes to distinguish between the two genes in a real-time PCR assay that is highly reproducible and sensitive. We demonstrated sex specificity for 33 cetacean species in nine families. Given the availability of conserved primers and sequence information for many mammalian species, this approach to designing sexing assays for a wide range of species is both practical and efficient.     

Sexing and detection of gene construct in microinjected bovine blastocysts using the polymerase chain reaction

We present a polymerase chain reaction (PCR)-based procedure for rapid bovine embryo sexing and classifying embryos for the presence of exogenous DNA. Fourteen bovine blastocysts microinjected with gene construct DNA at the pronuclear stage were divided into quarters and subjected to amplification with construct-specific and sex gene-specific (ZFY/ZFX) primers in the same initial PCR reaction. Blastocysts carrying microinjected construct DNA could be identified by the presence of construct-specific PCR product in approximately 4 h. Approximately half of the microinjected and two of 16 non-microinjected blastocysts typed PCR-positive for the construct DNA. Owing to erroneous amplifications in the two non-microinjected control blastocysts, and the inability of the system to distinguish integrated from non-integrated copies of the microinjected construct, the number of construct-positive blastocysts determined in our assay most likely overestimates the number of true transgenic embryos. Nevertheless, using this assay, we were able to determine that approximately half of the microinjected embryos were negative for the transgene construct and thus could be eliminated from transfer to a recipient cow. Embryo sexing was achieved in less than 6 h by restriction fragment length polymorphism analysis of nestedZFY/ZFXPCR products reamplified from initial PCR reactions. In 11/14 microinjected blastocysts all sections assayed unambiguously as the same sex. In one embryo, only one section was analysed, while two other blastocysts whowed some discrepancies of sexing results between the sections analysed. The approach employed here to determine the sex and presence of microinjected construct DNA in bovine preimplantation embryos is rapid, accurate among different sections of an embryo and can be used to increase the efficiency of current transgenic cattle production procedures.     

Molecular Method for Determining Sex of Walruses

Abstract: We evaluated the ability of a set of published trans-species molecular sexing primers and a set of walrus-specific primers, which we developed, to accurately identify sex of 235 Pacific walruses (Odobenus rosmarus divergens). The trans-species primers were developed for mammals and targeted the X- and Y-gametologs of the zinc finger protein genes (ZFX, ZFY). We extended this method by using these primers to obtain sequence from Pacific and Atlantic walrus (O. r. rosmarus) ZFX and ZFY genes to develop new walrus-specific primers, which yield polymerase chain reaction products of distinct lengths (327 and 288 base pairs from the X- and Y-chromosome, respectively), allowing them to be used for sex determination. Both methods yielded a determination of sex in all but 1–2% of samples with an accuracy of 99.6–100%. Our walrus-specific primers offer the advantage of small fragment size and facile application to automated electrophoresis and visualization.     

Sex determination of in vitro developed buffalo (Bubalus bubalis) embryos by DNA amplification

This study was conducted to determine the sex of buffalo embryos produced in vitro by amplifying male specific DNA sequences using the polymerase chain reaction (PCR). This method uses three different pairs of bovine Y-chromosome specific primers and a pair of bovine satellite specific primers. Buffalo in vitro fertilized embryos at the 4-cell to blastocyst stage were collected at days 3, 4, 6, and 8 postinsemination, and the sex of each embryo was determined using all three different Y-chromosome specific primers. The bovine satellite sequence specific primers recognize similar sequences in buffalo and are amplified both in males and in females. Similarly, Y-chromosome specific primers amplify the similar Y-chromosome specific sequences in male embryos of buffalo. Upon examining genomic DNA from lymphocytes of adult males and females, and embryos, the results demonstrate the feasibility of embryo sexing in buffaloes. Furthermore, sex determination by PCR was found to be a rapid and accurate method. © 1993 Wiley-Liss, Inc.     

Sex diagnosis of equine preimplantation embryos using the polymerase chain reaction

A rapid and reliable method for sex determination of preimplantation-stage equine embryos has not been available. The aim of the present study was to find an enzyme which would distinguish sexes in the horse by finding a polymorphic restriction site between the ZFY and ZFX homologues amplified by the polymerase chain reaction (PCR). Altogether, 38 different restriction enzymes were tested using female and male DNA extracted from blood. The primers used for amplification were selected from conserved sequences between human ZFY and ZFX genes and mouse Zfy-1 and Zfy-2 genes. Nine enzymes cut the PCR product of approximately 450 basepairs, but only Bsm I yielded different banding patterns in female and male DNA. All blood samples were correctly diagnosed. To test the method on embryonic cells, 17 horse demi-embryos were obtained from expanding blastocysts 220 to 950 mum in diameter. Demi-embryos were further cut into 3 to 7 parallel samples which were analyzed individually to test the repeatability of the method. Eight of the original embryos were diagnosed as females and 9 as males. No misidentifications were observed within the embryonic samples, suggesting that this sexing method is highly reliable. This study provides a rapid and accurate method to sex horse embryos.     

An ARMS-based technique for sex determination of red panda (Ailurus fulgens)

Molecular sexing is a key component in the investigation of wild populations. In this study, we developed a fast, accurate and reliable amplification refractory mutation system (ARMS) technique for sex determination of red panda based on the exon 4 of the ZFX/ZFY gene. The amplicons were distinguished simply by agarose gel electrophoresis, exhibiting one fragment in females (X: 300 bp) and two in males (X: 300 bp, Y: 166 bp). Robustness of this ARMS system was confirmed by testing both 43 captive red pandas using DNA samples with known-sex and 10 wild red pandas using faecal DNA samples with unknown sex.     

A rapid sex-identification test for the forest musk deer (Moschus berezovskii) based on the ZFX/ZFY gene

We describe a rapid sex-identification method for the forest musk deer (Moschus berezovskii) using PCR based on zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes. Fragments of the ZFX and ZFY genes were amplified and sequenced. The ZFX and ZFY fragments were identical in length and 94% similar in nucleotide sequence. Specific primers for forest musk deer sex identification were designed on the basis of sequence differences between ZFX and ZFY. All the primers were multiplexed in single-tube PCR. Both male and female forest musk deer showed amplification bands of 447 bp and 212 bp separated in agarose gels. A sex-specific 278-bp band was amplified only from males. These results show that testing by PCR for the presence of the 278-bp sequence is a rapid and reliable method for sex identification.     

Multiple genotype analysis and sexing of IVF bovine embryos

Twenty-one in vitro-fertilized bovine blastocysts were quartered, lysed and subjected to primer elongation preamplification (PEP) procedure, allowing for the analysis of up to 40 genotypes per quarter embryo. The quarter-embryos were sexed by polymerase chain reaction (PCR) using BRY.1, Bov97M and ZFX/ZFY loci, and then genotyped for k-casein, bovine leukocyte adhesion deficiency (BLAD) and microsatellite D9S1. The mitochondrial cytochrome B locus was used as an internal control with a 95% success rate. The PEP procedure amplified genomic fragments in 93% of all cases. The embryos were identified to be 11 males and 10 females. Sexing accuracy was 87% for BRY.1, 97% for ZFX/ZFY and 100% for Bov97M. False genotyping was due mostly to amplification of BRY.1 in the female embryos and to the nonamplification of the ZFY locus in the male embryos. The results indicate that the combined use of Bov97M and ZFX/ZFY loci is a highly accurate procedure for sexing bovine embryos. Genotyping for kappa-casein, D9S1 and BLAD was successful in 94, 99 and 91% of assays, respectively. Sex ratios and allele frequencies of embryos for gk-casein, BLAD and D9S1 were all close to the observed frequencies in the Israeli Holstein population. These results support the conclusion that the genotyping of embryos is as accurate as that of mature animals. Thus, marker-assisted selection can be efficiently applied at the preimplantation embryo level for loci of economic importance.     

A PCR assay for gender assignment in dugong (Dugong dugon) and West Indian manatee (Trichechus manatus)

Gender assignment for some aquatic mammals in the field is difficult. Molecular sexing from tissue biopsies is possible as males are heterogametic. Here we describe a multiplex PCR assay that amplifies the male specific SRY gene and differentiates ZFX and ZFY gametologues in two sirenian species, dugong (Dugong dugon) and West Indian manatee (Trichechus manatus). The assay was validated with animals of known gender and proved accurate and robust to experimental failure.     

毛冠鹿ZFY、ZFX基因片段的克隆与性别鉴定

根据人和鼠性别分化相关的ZFY、ZFX基因序列设计引物,以雌雄毛冠鹿的基因组DNA为模板进行PCR扩增,将扩增产物克隆到pMD18T上,获得ZFY、ZFX重组克隆,并测定了ZFY、ZFX基因片段的序列,序列比较显示两者同源性达 91%,仅在少数位点有差异,以此确定AvaⅡ为ZFX上特异酶切位点,通过PCR扩增和AvaⅡ特异酶切对毛冠鹿性别进行鉴定。 Abstract: According to the human sex differentiation related ZFY and ZFX genes, a pair of primers were designed , and fragments were amplified from the genomic DNA of male or female tufted deer. Subsequently the amplified fragments were cloned into the vector pMD18T and were sequenced. It is found that the sequences of ZFY gene and ZFX gene have 91% homology. Based on the different nucleotides, restriction site of AvaⅡ was found to be specific to ZFX gene. The results show that the combination of PCR with AvaⅡ digestion is a simple and sensitive way to identify the tufted deer sex.     

Determination of sex and scrapie resistance genotype in preimplantation ovine embryos

The aim of this study was to test the accuracy of genotype diagnosis after pre-amplification of DNA extracted from biopsies obtained by microblade cutting of ovine embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer to recipients. Sex and PrP genotypes were determined. Sex diagnosis was done by PCR amplification of ZFX/ZFY and SRY sequences after PEP-PCR while PrP genotype determination was performed after specific pre-amplification of specific target including codons 136, 154 and 171. Embryos were collected at Day 7 after oestrus. Blastocysts and expanded blastocysts were biopsied immediately after collection whereas compacted morulae were biopsied after 24 hr of in vitro culture. Eighty-nine biopsied embryos were frozen by vitrification. Fresh and vitrified whole embryos were kept as control. DNA of biopsies was extracted and pre-amplified. Sex diagnosis was efficient for 96.6% of biopsies and PrP genotyping was determined in 95.8% of codons. After embryo transfer, no significant difference was observed in lambing rate between biopsied, vitrified control and fresh embryos (54.5%, 60% and 66.6%, respectively). Embryo survival rate was not different between biopsied and whole vitrified embryos (P = 0.38). At birth, 96.7% of diagnosed sex and 95.4% of predetermined codons were correct. Lamb PrP profiles were in agreement with parental genotype. PEP-PCR coupled with sex diagnosis and nested PCR coupled with PrP genotype predetermination are very accurate techniques to genotype ovine embryo before transfer. These original results allow planning of selection of resistant genotype to scrapie and sex of offspring before transfer of cryopreserved embryo.     

PCR-CTPP: a rapid and reliable genotyping technique based on ZFX/ZFY alleles for sex identification of tiger (<Emphasis Type="Italic">Panthera tigris</Emphasis>) and four other endangered felids

An inexpensive, time-saving and reliable method, polymerase chain reaction with confronting two-pair primers (PCR-CTPP), was developed for sex identification in tiger (Panthera tigris) based on zinc finger alleles (ZFX/ZFY). A site of “C/G” transversion representing fixed differences that discriminated between ZFX and ZFY exons among felids was identified for primers designing. This primer set was successfully tested on samples including blood, shed hairs, dried skin, and stool which contained potential contamination caused by prey DNA. Cross species tests shown that this primer set was also useful for sex identification in four other endangered felids.     

A PCR-RFLP assay for gender assignment in the three-toed sloths (Bradypus, Pilosa, Bradypodidae)

The three-toed sloths (Bradypus) are slow-moving arboreal neotropical mammals. Understanding demographic variables (such as sex ratio) of populations is a key for conservation purposes. Nevertheless, gender assignment of Bradypus is particularly challenging because of the lack of sexual dimorphism in infants and in adults, particularly B. torquatus, the most endangered of the three-toed sloths, in which sex is attributed by visual observation of the reproductively active males. Here, we standardized a method for sexing Bradypus individuals using PCR-RFLP of sex-linked genes ZFX/ZFY. This assay was validated with known-gender animals and proved accurate to assign gender on three Bradypus species.     

Identification of sex in Cetaceans by multiplexing with three ZFX and ZFY specific primers

We sequenced 540 nucleotides of the last exon in the ZFY/ZFX gene in two males and two females for eight cetacean species; four odontocetes (toothed whales) and four mysticetes (baleen whales). Based upon the obtained nucleotide sequences, we designed two sets of oligonucleotide primers for specific amplification of the ZFX and the ZFY sequence in odontocetes and mysticetes, respectively. Each primer set consisted of three oligonucleotides; one forward-orientated primer, which anneals to the ZFY as well as the ZFX sequence, and two reverse-orientated primers that anneal to either the ZFX or the ZFY sequence. The resulting two amplification products (specific for the ZFY and ZFX sequences) can be distinguished by gel-electrophoresis through 2% NuSieve™. The accuracy of the technique was tested by determination of gender in 214 individuals of known sex. Finally we applied the technique to determine the sex of 3570 cetacean specimens; 2284 humpback whales, 315 fin whales, 37 blue whales, 7 minke whales, as well as 592 belugas, 335 narwhals and 25 harbour porpoises.     

Cloning the swamp buffalo SRY gene for embryo sexing with multiplex-nested PCR

The polymerase chain reaction (PCR) is an efficient method for sexing embryos. The objective of this study was to develop an accurate and reliable method for sexing swamp buffalo (Bubalus bubalis) embryos. The SRY gene from swamp buffalo genomic DNA was amplified by PCR, using primers based on the sequence of the Holstein SRY gene. This fragment was sequenced based on a BLAST search; the SRY gene was highly conserved. Using a Southern blot, there was a strong signal in genomic DNA only from male swamp buffalo. Two pairs of nested primers, targeted to amplify the swamp buffalo SRY conserved region, were designed for sex identification. Simultaneously, the G3PDH gene was co-amplified to serve as an internal control. A multiplex-nested PCR system was optimized by varying the following individually: concentrations of Mg(2+) and dNTPs, ratio of concentrations of primers and numbers of cycles. Biopsies of 27 IVF-derived embryos and 24 embryos fertilized with Y-chromosome-bearing sperm were examined. Using optimized procedures, clear signals following PCR amplification were obtained from all embryo samples; PCR amplification accuracy was further verified by comparing PCR and dot blots. We concluded that this PCR technique was highly reliable for sexing swamp buffalo embryos.