A rapid method for sex determination of bovine and buffalo embryos

Furthermore, selective sex predetermination together with the multiple ovulation and embryotransfer (MOET) procedures (Colleau, 1991) can effectively aid in improvement of the geneticgain of the herd. Progress in the field of in vitro embryo production has opened up new horisonsfor supporting the bovine industry on both research and economic levels, in terms of embryomanipulation, genetic enhancement and production of sexed embryos (Bavister, 2002; Faber etal., 2003). Buffalo species is considered an important farm animal with respect to its productiverole (Suzuki et al., 1992). Improvement of reproductive technologies and genetic approachesrepresents a significant aim of interest with the possibility of an integrated wide-spread geneticupgrading in such species (Gasparrini, 2002). Several methods, such as cytogenetic analysis ofchromosomes (Kitiyanant et al., 2000), immunological methods (Utsumi and Iritani, 1993)immunocytochemical hybridization techniques (Bondioli et al., 1989) and X-linked enzymeactivities as a biochemical assay (Monk and Handyside, 1988) have been presented for sexdetermination of preimplantation embryos. However, most of these approaches were found to betime consuming, little accurate and unreliable to cover potentially the daily practice of embryotransfer programs. Polymerase chain reaction (PCR) was introduced as a molecular approach forembryo sexing using a few biopsied embryonic cells (Peura et al., 1991; Faber et al., 2003;Manna et al., 2003) depending up on amplification of Y-chromosome specific DNA sequence asa specific indicator for male embryos and an autosomal repeated fragment common for both maleand female sexes. In order to augment the DNA amplification of a single copy embryonic biopsy,sex of bovine embryos was determined by PCR with some modifications, like the use of primerextension pre-amplification (PEP-PCR) using random oligonucleotides (Hassun et al., 1999), coamplificationof fragments that are present on both X and Y chromosomes using nested andallele-specific amplification (Kirkpatrick and Monson, 1993) or the use of fluorogenic probes in apre-amplification nested PCR (Virta et al., 2002) that does not require post-PCR electrophoresis.Moreover, loop-mediated isothermal amplification (LAMP) was recently used for sexing ofbovine blastomeres depending on the visual estimation of the reaction turbidity, (Hirayama et al.,2004). However, these multi-step molecular methods are often time consuming and requiretechnical skill. Beside this, a risk of contamination may take place during the duplicate PCRprocedures or post-PCR electrophoresis. Development of a rapid sensitive molecular method forsexing bovine embryo biopsies remains essential. In addition, little is known about the moleculartools for sexing of buffalo embryos. Therefore, the objective of this study was to develop a rapid2

and sensitive method for sex determination of bovine embryo blastomeres using real time PCR.We also established a preliminary sexing of single copy female gametes of buffalo and buffaloembryos using one step real time PCR assay.Material and methodsIn vitro embryo productionOvaries of slaughtered cows and buffaloes were aspirated with a 18 gauge needle and the oocyteswere matured up to 24 hours in bicarbonate-buffered TCM 199 supplemented with FSH/LH (0.05U.I./ml, Pergovet, Serono, Rome, Italy) and 10% FCS, in 5% CO 2 and 95% humidified air at38.5°C. Frozen-thawed semen was selected by 40 min centrifugation at 800 × g on discontinuousPercoll gradients (45-90%) and then washed by 10 min centrifugation at 500 × g in HEPESbuffered TALP medium. The pellet was resuspended in bicarbonate buffered TALP IVFcontaining penicillamine, hypotaurine, epinephrine (PHE) and 1µg/ml heparin, to the desiredconcentration. The sperm-containing medium was put into 4-well plates in 50µl microdropsunder mineral oil (Sigma) and 20 matured oocytes per drop were added and incubated in 5%CO 2 , 5% O 2 and 95% humidified air at 38.5°C. After 18 to 20 hours, the presumptive fertilisedeggs were vortexed in Hepes-buffered h-SOF medium to remove the cumulus cells. Denudedzygotes were cultured in 300µl of bicarbonate-buffered SOF medium and at the day 4 half of themedium was renewed. At day 6 of culture, half volume of the culture medium was replaced withTCM 199 supplemented with BSA. Blastocysts were evaluated at day 7 and day 8.Preparation of embryonic cells for PCRBovine morulae at 4-8 cells stage were washed in PBS and incubated for 2-3 min with 0.05%pronase E (P5147, Sigma) for degradation of zona pellucida. Blastomeres were then separated bymouth pipetting using a fine glass pipette pulled on flame to approximately the size of ablastomere. Under a stereomicroscope, each single, double or triple blastomeres from eachbovine morula (as shown in table 1) were transferred individually in a volume of 1µl PBS + 0.1%BSA into 0.2 ml eppendorf tubes and stored at –20 °C until PCR analysis. For buffalo, matureoocytes and single blastocysts were washed and stored as described for bovine embryos (eachsingle or two oocytes and each individual blastocyst in 0.2 ml micro-tube).3

Table 1. Number of bovine morulae (M1 to M14) and number of the separated cells in the form of single,double or triple blastomeres.MorulaeM1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14Number of samplesSingle blastomere 4 2 6 9 4 7 7 2 9 9 2 6 6 2Two blastomeres 4 2 2 1 2 5 1 1 4 1Three blastomeres 1 2DNA extraction from the embryonic cellsDNA was extracted from the embryonic cells (bovine blastomeres, buffalo oocytes andblastocysts) according to the method described by Peura et al. (1991) with some modifications.Briefly, the total lysis mixture consisted of 10 µl of 1 X PCR-buffer containing 25 mM MgCl2,0.1 % triton X-100 and 150 µg/ml proteinase K (Applied Biosystems). The tubes were incubatedfor 60 min at 37 °C followed by 8 min at 99 °C and then stored at – 4 °C.DNA extraction from bloodGenomic DNA was extracted from blood samples of male and female bovine and buffalo species(positive controls) using DNeasy tissue kit (Qiagen) according to the manufacturer’s instructions.DNA of the positive controls was prepared as 10 fold serial dilution (10, 1, 0.1, 0.01, 0.001 ng/µl)in order to test the sensitivity of PCR at the lowest target concentration.Primers and probes designTwo different sets of primers and fluorogenic TaqMan probes were designed using PrimerExpress software 2.1 (PE Applied Biosystems). The male-specific set (Y) was designed on aconserved region of Y chromosome-linked BBYS 1 DNA locus of water buffalo (GenBankaccession no. X93551). The second set is common for both sexes and used for bovine and buffalospecies (BOV) was designed on the bovine locus 1.715 repeated satellite DNA (GenBankaccession no. V00125). Screening of primers and probes alignment was verified by Blastsoftware (www.ncbi.nlm.gov). Primers and probes sequences are shown in table 2.4

Table 2. Oligonucleotide primers and probes sequences used in the real time PCR assayAmpliconY-specificBOVPrimers and probes sequencesForward: 5’-CCCCTGAGCTTGCCATGA-3’Reverse: 3’-CTGCAGTGCTGGTCCATTTCT-5’Probe (FAM-labelled): 5’-ATGAACATCCTTTGCCTTTTTTCTGAGGTT-3’Forward: 5’-TCTCGAATTGTGACGGGTATCTC-3’Reverse: 3’-GACCAATCTCGCGACCTCTCT-5’Probe (VIC-labelled): 5’-TGGAGCTCACTGGGTGGACTCAAGG-3’Real time PCR for sex determination of bovine and buffalo embryonic cellsReal time PCR was carried out by ABI PRISM 7900HT Sequence Detection System (SDS)(Applied Biosystems). The PCR mixture (25 µl total volume) contained 300 nM forward primer,300 nM reverse primer, 200 nM TaqMan probe for Y amplicon and BOV amplicon, and 12.5 µlTaqMan 2 X universal PCR Master Mix (Applied Biosystems). For bovine blastomeres, PCRwas conducted in two different wells using 7.5 µl DNA lysate of each blastomere for Y ampliconand 2.5 µl for BOV amplicon. For buffalo samples (oocytes and blastocysts) the reaction wasperformed in a multiplex real time PCR utelising all the 10 µl DNA lysate. For the positivecontrols, 1 µl of the target DNA of each dilution (10, 1, 0.1, 0.01, 0.001 ng/µl) was amplified.The amplification protocol consisted of two initial steps, the first at 50 °C for 2 min (AmpEraseUNG activation) and the second at 95 °C for 10 min (Amplitaq Gold DNA Polymeraseactivation) followed by 50 repeated cycles of two steps (95 °C for 15 sec and 60 °C for 1 min).The fluorescence emitted from hydrolysis of TaqMan probes was detected and analysed by SDSsoftware (Applied Biosystems). The cycle threshold (Ct) at which the fluorescence emission risesabove a baseline is recorded. For male embryos, two amplification plots were detected for bothprimer sets, while for females, only the BOV plot was detected. Results are shown asamplification plots for each target amplicon (Fig.1)Polymerase chain reaction (PCR)For confirming the results obtained by the real time PCR, bovine embryonic blastomeres weresubjected to a classical PCR analysis using two different primers (Peura et al., 1991) with somemodifications. The total PCR volume was adjusted to 25 µl containing 10 µl DNA lysate of eachblastomere.5

CtFig.1. An example of the amplification plots of the two primer sets, showing the plot of eachtarget amplicon.ResultsSensibility of the primers and TaqMan probes has been tested through amplifying genomic DNAof the male and female positive controls (bovine and buffalo) as 10 fold serial dilution (from 10to 0.001 ng/µl). For bovine male control, the Ct of Y primers ranged from 33.83 to 41.34, while,for BOV primers, Ct ranged from 16.79 to 27.00 and from 16.46 to 28.33 for male and femalebovine control respectively. Ct values of buffalo male control ranged from 22.31 to 37.53 for Yprimers, while for BOV primers ranged from 21.11 to 35.49 and from 27.75 to 33.23 for maleand female buffalo control respectively. A total number of 71 blastomeres, originating from 14bovine morulae , were analysed. A number of 57 blastomeres were analysed using real time PCRassay (one to four replicates from each embryo) and 14 blastomeres (one replicate from each ofthe 14 morulae) were analysed by classical PCR. After real time PCR analysis, 5 morulae weredetected as males and 9 morulae were detected as females. The analisys of the 14 blastomeres byclassical PCR, confirmed the 9 females and 4 males to be correctly sexed, while for one morula(M 8) the signal was not the same as seen after real time PCR analysis. Overall, 56 out of 57bovine blastomeres were succesfully sexed by real time PCR and the results were confirmed bythe classical PCR analysis of the same embryo replicate (98.24 % of correct sex). Results ofblastomeres analysis by the real time PCR assay are shown in table 3.6

Table 3. Real time PCR sex determination results of bovine one to three embryo blastomeresNumber of cells Analysed Females Males Incorrect sex1 blastomere 45 30 15 02 blastomeres 9 6 3 13 blastomeres 3 3 0 0Total 57 39 18For buffalo, a number of 5 blastocysts and 23 oocytes (5 single and 18 double) were analysed in amultiplex real time PCR. Three out of 5 blastocysts were detected as males and two blastocystsas females. The analysis of the oocytes has shown a correct normal female (XX) amplificationplot and no amplification of the Y-specific primers was reported in any sample.DiscussionThe connection between the scientific research and the economical needs of the society remainsan important basic target. In particular, our study supports potentially some of the needs of theanimal breeding market disciplines and can help in drawing specific strategies for a dairy or beeflivestock. Over recent years, different methods have been proposed for sex determination ofembryonic blastomeres, but most of these methods are multi-step, lacking the full accuracy,sensitivity and may require a technical skill. In this study, we presented a rapid sensitive methodin one step by real time PCR for sex determination of bovine embryo blastomeres and buffalopreimplantation embryos. Real time PCR possesses the advantages of being sensitive throughdetection of the amplification in a real time after release of the fluorescence from the specificprobes, and because there is no need for post-PCR electrophoresis the contamination risk isminimised. Beside this, the reaction was conducted in one step without needing duplicate PCRprocedures or PEP-PCR reaction. Moreover, the reaction could be done in 96-well plate thusallowing the analysis of a large number of samples within few hours and this strongly supportsthe daily need of ET industry. Male and female genomic DNA (bovine and buffalo) were used aspositive controls (10 fold serial dilution) in order to test the sensibility of the assay at lower targetconcentrations. It is obvious that each new primer pair and probe combination needs a series ofcareful optimisation steps. For bovine blastomeres as well as the controls, the two primes wereused separately due to the unsuccessful amplification when they are used in a multiplex.Moreover, Ct detection for bovine DNA required a large number of amplification cycles for theY-specific primers at the lowest concentration (Ct 41.34) that might require an improvement of7

the fluorogenic probe design in the future throughout changing its region on the Y-specific locusor a careful optimisation. Sex determination was successful in 56 out of 57 bovine blastomeresdemonstrating an accuracy of more then 98% with the same results obtained by the classical PCRfor the same embryo replicates. Real time PCR has reflected a high efficiency degree in sexing ofblastomeres as little as a single copy embryonic cell in one step. For buffalo, this preliminarystudy shows for the first time the usefulness and the efficiency of a multiplex real time PCRassay for sexing in one step. A preliminary sexing of some buffalo blastocysts as well as buffalooocytes that representing single copy female gametes (XX) was performed successfully. Ct valueof Y primers in buffalo DNA positive controls was earlier than bovine DNA at the lowestconcentration (Ct 37.53) that may indicate the more sensitivity of the probe to the Y locus ofbuffalo DNA. Our preliminary sexing of buffalo blastocysts as well as single copy buffalooocytes will open the gate for a future novel sexing assay of single buffalo blastomeres. In thisstudy, we developed a novel, rapid and sensitive method for embryo sexing as a single copyblastomere by using real time PCR. This will provide a powerful tool for embryo sexing in theanimal breeding market and should be supported by other future studies to enhance and toimprove the sexing and the genetic efficiency of some economically and agriculturally importantspecies.References1.2.3.4.5.6.7.8.9.Bavister, B. D. Early history of in vitro fertilisation. Reprod. 2002, 124, 181-196.Bondioli, K. R., Ellis, S. B., Pryor, J. H., Williams, M. E., Harpold, M. M. The use of malespecificchromosomal DNA fragments to determine the sex of bovine preimplantation embryos.Theriog. 1989, 31, 95-104.Colleau, J. J. Using embryo sexing within closed mixed multiple ovulation and embryo transferschemes for selection on dairy cattle. J Dairy Sci. 1991, 74, 3973-3984.Faber, D. C., Molina, J. A., Ohlrichs, C. L., Vander Zwaag, D. F., Ferre, L.B. Commercializationof animal biotechnology. Theriog. 2003, 59, 125-138.Gasparrini, B. In vitro embryo production in buffalo species: state of the art. Theriog. 2002, 57,237-256.Hassun, P. A., Mello, M. R. B., Porto, L. P. C., Garcia, J. F. Bovine embryo sexing by primerextension preamplification polymerase chain reaction (PEP-PCR). Theriog. 1999, 51, 398Herr, C. M., Reed, K. C. Micromanipulation of bovine embryos for sex determination. Theriog.1991, 35, 45-54.Hirayama, H., Kageyama, S., Moriyasu, S., Sawai, K., et al. A. Rapid sexing of bovinepreimplantation embryos using loop-mediated isothermal amplification: Theriog. 2004, 62, 887-896.Kirkpatrick, B. W., Monson, R. L. Sensitive sex determination assay applicable to bovineembryos derivedfrom IVM and IVF. J Reprod. Fertil. 1993, 98, 355-340.8

10.11.12.13.14.15.16.Kitiyanant, Y., Saikhun, J., Siriaroonrat, B., Pavasuthipaisit., K. Sex determination by polymerasechain reaction and karyotyping of bovine embryos at first cleavage in vitro. ScienceAsia 2000, 26,9-13.Manna, L., Neglia, G., Marino, M., Gasparrini, B., et al. Sex determination of buffalo embryos(Bubalus bubalis) by polymerase chain reaction. Zygote 2003, 11, 17-22.Monk, M., Handyside, A. H. Sexing of preimplantation mouse embryos by measurement of X-linked gene dosage in a single blastomere. J Reprod. Fertil. 1988, 82, 365-368.Peura, T., Hyttinen, J. M., Turunen, M., Janne, J. A reliable sex determination assay for bovinepreimplantation embryos using the polymerase chain reaction. Theriog. 1991, 35, 547-555.Suzuki, T., Singla, S. K., Sujata, J., Madan, M. L. In vitro fertilization of water buffalo follicularoocytes and their ability to cleave in vitro. Theriog. 1992, 38, 1187-1194.Utsumi, K., Iritani, A. Embryo sexing by male specific antibody and by PCR using male specific(SRY) primer. Molec. Reprod. Develop. 1993, 36, 238-241.Virta, J., Markola, J., Peippo, J., Markkula, M., Vilkki, J. Sex determination of bovine embryoblastomeres by fluorogenic probes. Theriog. 2002, 57, 2229-2236.9