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The effect of alanine (1 mM) and glycine (10 mM) supplementation on bovine embryo development in vitro was investigated. Presumptive bovine zygotes, produced by in vitro maturation and insemination of oocytes, were cultured for 144 h in CR1aa medium in the absence (Experiments 1 and 2) or presence of cumulus cells (Experiment 3). In Experiment 1, the proportion of morulae and blastocysts of cleaved embryos in glycine-supplemented medium was not different from that of the control medium (34% in both media)glycine-enriched medium (69.5 vs. 53.3, P = 0.016). In Experiment 2, addition of alanine did not improve the formation of morulae and blastocysts (13% vs. 21% in control medium), and the mean cell numbers in morulae and blastocysts were lower than those in the control group (34.3 vs. 68.7, P = 0.007). In the presence of cumulus cells, the combined supplementation of glycine and alanine increased the proportion of morulae and blastocysts over that in the control medium (31% vs. 14%, P = 0.003).;

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CR1-PVP, a modification of CR1aa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CR1-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production.

The aims of this study were to evaluate the amount of amniotic fluid required for diagnosis of sex, milk protein and microsatellite variants by polymerase chain reaction (PCR) and to review methods for isolating DNA from the amniotic cells. Uterine and foetal tissues were used as controls, and milk protein and microsatellite variants to check contamination of maternal cells in the PCR. The results showed that the samples do not need to be purified after DNA release from the amniotic cells and that as little as 0.5-1.5 ml of amniotic fluid is sufficient for reliable diagnosis by PCR.

Morula and blastocyst stage embryos recovered from B6D2F 1 mice were bisected with a metal microlabde in M2 medium with or without sucrose and/or cytochalasin B supplementation. Cell lysis was determined by staining the embryos with Hoechst 33258 and propidium iodide. Lysed cells take up both stains but non-lysed cells only the Hoechst 33258 stain, resulting in pink fluorescence for lysed cells and blue fluorescence for non-lysed cells under UV excitation. During bisection of morulae, the presence of cytochalasin B decreased the proportion of lysed cells in both the absence (P=0.0001) and presence of sucrose (P=0.001). During bisection of blastocysts the average proportion of lysed cells was slightly lower in the presence of cytochalasin than that in the control medium, but the effect was not statistically significant (P=0.34). No effect of sucrose was observed in either demi-morulae or demi-blastocysts. These results are essentially similar to those obtained in simultaneous experiments with cattle embryos, suggesting that the simpler mouse model might be useful for developing less traumatic bisection protocols for cattle embryos.;

Informativeness of eleven microsatellite markers suggested for parentage control in cattle by the International Society for Animal Genetics (ISAG) was studied in Finnish Ayrshire and Holstein-Friesian populations. Calculations were based on a sample of 100 non-sib artificial insemination bulls. Assuming one known parent the nine loci suggested for routine testing exhibited exclusion probabilities of 99.84% in the Ayrshires and 99.91% in the Holstein-Friesians. The addition of markers INRA23 and TGLA53, recommended for further investigations, increased the attained values to 99.94% in Ayrshires and to 99.98% in Holstein-Friesians. The recommended core set of six microsatellites provided a combined exclusion probability of 98.25% in Ayrshires and 99.32% in Holstein-Friesians. Although the combined values were high in general, a relatively low level of polymorphism was detected in some instances.;

Continuous observation of embryonic growth can improve understanding of the early developmental events and allow us to use parametric statistical analyses with time as a parameter. A cinematographic study such as that reported here utilizes time-lapse video recording. Previously published methods for time-lapse video recording have involved building an incubator around a microscope, a process that is both expensive and laborious. Here we present a simplified method for time-lapse video recording of early bovine embryo development. The embryos were cultured during a 24-hour period in a standard pregassed tissue culture bottle, which was darkened and placed on the heating stage of an inverted microscope for recording through a red filter. The control embryos were cultured in a con-ventional CO 2 incubator. After 10 replicates we could not find a statistically significant difference between the cell numbers of these two treatments (P=0.95), suggesting that the culture setup is ap-propriate for continuous observation of early cleavage of the cattle embryo.;

The viability of microblade-bisected Day-7 embryos transferred to recipients without zona pellucidae directly after splitting or after overnight storage at 4°C was investigated in two experiments. In Experiment 1, 26 demi-embryos of excellent or good quality and two of fair quality were transferred to 28 heifer recipients 1-3 h after splitting. The transfers resulted in 20 pregnancies (71.4% pregnancy rate), and 19 calves were born (one pregnant heifer was slaughtered). Another 14 demi-embryos were cultured 1 to 3 h at room temperature, transported for 3 h at 8-10°C and stored for 12 h in a refigerator at 4°C. Twelve of the 14 demi-embryos were considered transferable after storage and nine of these were transferred to nine recipients, of which five became pregnant. Four live calves were born. In Experiment 2, 21 excellent or good quality demi-embryos were transferred into 21 heifer recipients to produce 12 pregnancies (57.1%) and 10 live calves. Another 19 demi-embryos (17 excellent to good quality and two fair quality) were transferred after storage for 1-2 h at 8°C and 18 h at 4°C. Five recipients became pregnant, of which four delivered live calves. It is concluded that a high pregnancy rate can be achieved after the transfer of fresh demi-embryos produced by a simple method of embryo bisection under farm conditions. Although the transfer of demi-embryos stored overnight at 4°C results in decreased pregnancy rates, refrigeration of demi-embryos may be useful in certain practical situations. However, further experiments are needed to determine the optimal conditions for overnight storage of demi-embryos.;

4',6'-diamidino-2-phenylindole (DAPI) is a fluorescent dye that binds only to the DNA of dead blastomeres of an embryo. The use of DAPI for assessing viabilities of equine Day 6 to 7 embryos was studied. The pregnancy and normal development of the embryo until 20 days after transfer (day of pregnancy termination) was used as an indicator of embryonic viability. Eleven embryos were stained with DAPI at room temperature for 15 min. They were then exposed to UV light to visualize staining, and cultured for 2 h before non-surgical transfer to recipient mares. Eight control embryos were cultured for 2 h before transfer to recipient mares. The recipient mares were scanned for pregnancies every other day starting 6 days after transfer. Twenty days after the transfer the pregnant recipient mares received luprostiol to induce abortion of the foetus. The pregnancy rate 6 days after transfer was 82% in the treatment group, and 75% in the control group (P>0.05). One DAPI-stained embryo resorbed and was no longer visible 12 days after transfer. However, this particular recipient mare had a uterine inflammation as shown by examination in the oestrus following embryo transfer. Although the number of embryos is insufficient to demonstrate lack of treatment effect, the high pregnancy rates following DAPI staining should encourage further studies on this subject.;

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production.

The effect of alanine (1 mM) and glycine (10 mM) supplementation on bovine embryo development in vitro was investigated. Presumptive bovine zygotes, produced by in vitro maturation and insemination of oocytes, were cultured for 144 h in CRlaa medium in the absence (Experiments 1 and 2) or presence of cumulus cells (Experiment 3). In Experiment I, the proportion of morulae and blastocysts of cleaved embryos in glycine-supplemented medium was not different from that of the control medium (34% in both media); however, the cell numbers of morulae and blastocysts were significantly higher in the glycine-enriched medium (69.5 vs. 53.3, P = 0.016). In Experiment 2, addition of alanine did not improve the formation of morulae and blastocysts (13% vs. 21% in control medium), and the mean cell numbers in morulae and blastocysts were lower than those in the control group (34.3 vs. 68.7, P = 0.007). In the presence of cumulus cells, the combined supplementation of glycine and alanine increased the proportion of morulae and blastocysts over that in the control medium (31% vs. 14%, P = 0.003).

Continuous observation of embryonic growth can improve understanding of the early developmental events and allow us to use parametric statistical analyses with time as a parameter. A cinematographic study such as that reported here utilizes time-lapse video recording. Previously published methods for time-lapse video recording have involved building an incubator around a microscope, a process that is both expensive and laborious. Here we present a simplified method for time-lapse video recording of early bovine embryo development. The embryos were cultured during a 24-hour period in a standard pregassed tissue culture bottle, which was darkened and placed on the heating stage of an inverted microscope for recording through a red filter. The control embryos were cultured in a conventional CO2 incubator. After 10 replicates we could not find a statistically significant difference between the cell numbers of these two treatments (P=0.95), suggesting that the culture setup is appropriate for continuous observation of early cleavage of the cattle embryo.

The viability of microblade-bisected Day-7 embryos transferred to recipients without zona pellucidae directly after splitting or after overnight storage at 4°C was investigated in two experiments. In Experiment 1, 26 demi-embryos of excellent or good quality and two of fair quality were transferred to 28 heifer recipients 1-3 h after splitting. The transfers resulted in 20 pregnancies (71.4% pregnancy rate), and 19 calves were born (one pregnant heifer was slaughtered). Another 14 demi-embryos were cultured 1 to 3 h at room temperature, transported for 3 h at 8-10°C and stored for 12 h in a refigerator at 4°C, Twelve of the 14 demi-embryos were considered transferable after storage and nine of these were transferred to nine recipients, of which five became pregnant. Four live calves were born. In Experiment 2, 21 excellent or good quality demi-embryos were transferred into 21 heifer recipients to produce 12 pregnancies (57.1%) and 10 live calves. Another 19 demi-embryos (17 excellent to good quality and two fair quality) were transferred after storage for 1-2 h at 8°C and 18 h at 4°C. Five recipients became pregnant, of which four delivered live calves. It is concluded that a high pregnancy rate can be achieved after the transfer of fresh demi-embryos produced by a simple method of embryo bisection under farm conditions. Although the transfer of demi-embryos stored overnight at 4°C results in decreased pregnancy rates, refrigeration of demiembryos may be useful in certain practical situations. However, further experiments are needed to determine the optimal conditions for overnight storage of demi-embryos.

The aims of this study were to evaluate the amount of amniotic fluid required for diagnosis of sex, milk protein and microsatellite variants by polymerase chain reaction (PCR) and to review methods for isolating DNA from the amniotic cells. Uterine and foetal tissues were used as controls, and milk protein and microsatellite variants to check contamination of maternal cells in the PCR. The results showed that the samples do not need to be purified after DNA release from the amniotic cells and that as little as 0.5-1.5 ml of amniotic fluid is sufficient for reliable diagnosis by PCR.

Morula and blastocyst stage embryos recovered from B6D2F_1 mice were bisected with a metal microblade in M2 medium with or without sucrose and/or cytochalasin B supplementation. Cell lysis was determined by staining the embryos with Hoechst 33258 and propidium iodide. Lysed cells take up both stains but non-lysed cells only the Hoechst 33258 stain, resulting in pink fluorescence for lysed cells and blue fluorescence for non-lysed cells under UV excitation. During bisection of morulae, the presence of cytochalasin B decreased the proportion of lysed cells in both the absence (P=0.0001) and presence of sucrose (P=0.001). During bisection of blastocysts the average proportion of lysed cells was slightly lower in the presence of cytochalasin than that in the control medium, but the effect was not statistically significant (P=0.34). No effect of sucrose was observed in either demi-morulae or demi-blastocysts. These results are essentially similar to those obtained in simultaneous experiments with cattle embryos, suggesting that the simpler mouse model might be useful for developing less traumatic bisection protocols for cattle embryos.

4’,6’-diamidino-2-phenylindole (DAPI) is a fluorescent dye that binds only to the DNA of dead blastomeres of an embryo. The use of DAPI for assessing viabilities of equine Day 6 to 7 embryos was studied. The pregnancy and normal development of the embryo until 20 days after transfer (day of pregnancy termination) was used as an indicator ofembryonic viability. Eleven embryos were stained with DAPI at room temperature for 15 min. They were then exposed to UV light to visualize staining, and cultured for 2 h before non-surgical transfer to recipient mares. Eight control embryos were cultured for 2 h before transfer to recipient mares. The recipient mares were scanned for pregnancies every other day starting 6 days after transfer. Twenty days after the transfer the pregnant recipient mares received luprostiol to induce abortion of the foetus. The pregnancy rate 6 days after transfer was 82% in the treatment group, and 75% in the control group (P>0.05). One DAPI-stained embryo resorbed and was no longer visible 12 days after transfer. However, this particular recipient mare had a uterine inflammation as shown by examination in the oestrus following embryo transfer. Although the number of embryos is insufficient to demonstrate lack of treatment effect, the high pregnancy rates following DAPI staining should encourage further studies on this subject.

A PCR-based sex determination assay for sheep and cattle embryos was developed using mouse embryos for optimizing the protocol. Samples were lysed either enzymatically or by alkaline treatment followed by enzymatic amplification of DNA from the ZFY/ZFX locus. Sex diagnosis could be done after the digestion of the amplified product by restriction endonucleases. Ovine and bovine embryos could be sexed from biopsies as small as 1-4 cells. Some embryos were split into 2-4 sections, which were amplified separately. Blind trials with such samples demonstrated that the method was highly accurate, even when embryo biopsy was done under farm conditions. The protocol involves an in-built control. This eliminates the need for autosomal control primers, which often inhibit the amplification of the Y-chromosome-specific DNA, especially when a small amount of template is used.