Sexing of embryos before transfer and implanting has great potential for the livestock industry. Manipulating the sex of offspring has been a dream of the cattle industry for decades. A number of approaches to the sexing of semen have been attempted, and several have been reported as successful. However, the only method of semen sexing that has shown any promise has been the sorting of spermatozoa according to the DNA content, by means of flow cytometry (Johnson et al. 1988). This methodology is severely limited by the small number of cells that can be sorted per hour (recently claimed 2 millions per hour) and the high cost of the equipment.
The widespread use of embryo transfer technology has created an alternative approach to controlling the sex of offspring. Embryo sexing has been attempted by a variety of methods, including cytogenetic analysis, assays for X-linked enzyme activity, analysis of differential development rates, detection of male-specific antigens, and the use of Y-specific DNA probes. In DNA amplification techniques, the probe itself is not used. The sequence information generated from the identification and cloning of the probe is used instead.
Since the development of DNA amplification techniques, particularly the polymerase chain reaction (PCR), these techniques have been applied to numerous situations in which the analysis of rare sequences is desired (Saiki et al. 1988). It was the identification of bovine Y-chromosome specific DNA probes, and the subsequent development of DNA amplification techniques by PCR, that made the possibility of sexing embryos into a reality.
The first part of this Bulletin presents the technology of embryo transfer in dairy cattle as currently used in Taiwan. The technical parameters are summarized. The second part presents the technology of sampling and sex determination as it is applied on dairy farms in Taiwan.
Embryo Transfer in Dairy Cattle
The best donor in embryo transfer (ET) is generally a young cow, lactating but not at the peak lactation stage, with no history of reproductive problems. Too much body fat will decrease the success of ET. Fat cows generally respond poorly to superovulation treatments. A suitable recipient is a heifer or a cow that will cycle normally and have a good conception rate. At the time of ET, the recipient must be in adequate heat synchrony with the donor, and have a palpable corpus luteum.
The most common method used is a two-shot prostaglandin regime. Prostaglandin is given twice, with the shots eleven days apart. The second injection is timed two days before the donor is due to come into heat. Recipients take 2-3 days to come into heat after prostaglandin, if they respond at all.
Superovulation is the process of stimulating multiple follicular development and ovulation by administering gonadotrophic hormones, followed by a prostaglandin injection. There are two main gonadotrophins in common use for the superovulation of donor cows. PMSG has a half-life of up to five days, and is administered as a single injection. FSH-P has a half-life of 2 - 5 hours, and is administered twice daily for 3.5 days. Heat should occur 48 hours after the PG injection. PG may be given on the third day of a 3.5-day FSH-P stimulation. A total dosage of 36-40 mg of FSH-P with a decline dose is administered.
At the present time, non-surgical embryo recovery is the technique in world-wide use. The base of the tail of the donor is clipped, and an epidural injection of 6 mL of 2% procaine is given. The French IMV three-way collection equipment with an extendible head catheter then is manipulated into the selected horn, so that the inflatable balloon is in the right place. The balloon is inflated quickly with 20 mL of air. Approximately 600 to 800 mL of Dulbecco's phosphate buffered saline, plus 1% bovine serum medium warmed to 37° C, are placed for infusion.
It is important that the culture medium and all equipment used to handle the embryos is sterile.
The embryos are frozen in a medium containing Dulbecco's phosphate buffered saline solution with 1.5M ethylene glycol, 0.4% BSA and 0.1M sucrose. The freezing procedure is as follows. Embryos are put into a 1.5M EG solution, and loaded into a 0.25 mL straw. After five minutes, they are placed in a 6°C freezing chamber, and kept at -6°C for 10 minutes. The cooling rate then continues at 0.5°C/minute until a temperature of -32°C is reached. The embryo is then plunged into liquid nitrogen.
The direct transfer procedure is as follows. The straw is removed from the liquid nitrogen, and held in the air for five seconds. It is then immersed in a water bath at 25°C for 25 seconds. The straw is then removed, wiped dry, loaded into an ET gun, and transferred directly into the cow. For optimal results, the transfer should take place within six minutes as the embryo is loaded into the ET gun.
Embryo Sexing Technology
Amplification of Y chromosome-specific DNA by means of the PCR technique seems to be the most reliable and practical method of sexing bovine embryos (Herr et al. 1991, Peura et al. 1991). In Taiwan, Chen et al. (1999) have cloned and characterized both male-specific and gender-neutral DNA. Primers, namely bE5, for the PCR were synthesized on the basis of this DNA to provide PCR products which differentiate between males and females. In our tests, less than 10 cells were sufficient to discriminate males from females.
Preparation of Samples
The embryos for sexing are collected on day 6.5 after first AI. Only embryos graded as excellent or good from the stage of compact morula to early blastocyst are biopsied. The biopsy sample is washed three times with PBS. Two µl of this solution is placed in a 0.5 mL tube. Eighteen µl of double distilled water is added. The test tube is spun for a few seconds by centrifuge, then kept it at 97°C for two minutes on a PCR apparatus. Finally, 30 µl of reaction mixture is added, so that each sample is around 50 µl. For the negative control, 10 µl of distilled water is put into a 0.5 mL test tube. For the positive control, each 10 µl of purified male and female bovine genomic DNA is put into a 0.5 mL test tube.
Preparation of Reaction Mixture
Reaction mixture was a preparation which combined 5 µl PCR buffer, 4 µl of dNTP (2.5 mM), 6 µl of MgC1 2 (25mM), 0.4 µl of BE5(100 pM), 0.2 µl of X-498 (100 pM), 0.2 µl of Y-368 (100 pM) and double distilled water (13.7 µl). Another solution which can be used is Taq enzyme (0.5 µl).
The PCR cycle is made of step 1, 2, 3 and 4, and the second to the 50 th cycle, of step 2, 3 and 4. The timing of this system of thermal cycles is as follows. Step 1 is 1 min at 94°C for denaturation. Step 2 is 10 seconds at 94°C. Step 3 is 15 seconds at 56°C for the annealing of primers. Step 4 is 10 seconds at 72°C for amplification. After completing amplification, the sample is kept for two minutes at 72°C.
Analysis of PCR Products by Electrophoresis
The interpretation of the results is as follows. The embryo is male if the 518 bp and 498 bp are seen. The embryo is female if the 498 bp is absent.
Transfer of Sexed Embryos in Field Trials
Eighty embryos were flushed out on day 6.5 after the first AI. Between one and four blastomeres were aspirated from the embryo at the morula and early blastocyst stage. These were used for sex determination. Results indicated that 45% of the embryos were sex determined (36/80). Aspirated embryos were cultured overnight. Of these, 92.5% (74/80) showed a normal development rate. Twenty-five sexed embryos were transferred into recipients, and 13 of the recipients became pregnant (52.0%, 13/25). The pregnancy rate when questionable embryos were used was 38.5% (10/26). The pregnancy rate when fresh embryos were transferred without sexing was 41.8% (23/55). The accuracy of embryo sex determination in the field trials was shown at calving to be 85% (11/13).
Using DNA analysis to analyse the sex of embryos has been shown to be reliable. The removal of a few cells caused very little trauma to the embryos. It did not alter developmental potential in vitro. The procedure has been shown to be sensitive, accurate and efficient, and pregnancy rates have not been affected, compared to those after transfer of fresh embryos without sexing. However, techniques that require embryo biopsy still need to be cautious, in case they reduce embryo viability. When successful techniques of embryo transfer and sexing become available, the demand for these techniques is likely to grow considerably. Research to improve sexing and embryo transfer technology is ongoing, while at the same time, the application of sexing through embryo transfer is being developed.
- Chen, C.M., C.L. Hu, C.H. Wang, C.M. Hung, H.K. Wu, K.B. Choo, and W.T.K. Cheng. 1999. Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene. Mol. Reprod. Dev. 54: 209-214.
- Herr, C.M., and K.C. Reed. 1991. Micromanipulation of bovine embryos for sex determination. Theriogenology 35: 45-54.
- Johnson, L.A. 1988. Flow cytometric determination of sperm sex ratio in semen purportedly enriched for X- or Y-bearing sperm. Theriogenology 29: 265.
- Peura, T., J-M. Hyttinen, M. Turunen, and J. Janne. 1991. A reliable sex determination assay for bovine preimplantation embryos using the polymerase chain reaction. Theriogenology 35: 547-555.
- Saiki, R.K., D.H. Gelfand, S. Stoffel, S.J. Scharf, R. Higuchi, G.T. Horn, K.B. Mullis, and H.A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487.
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