R.S. Canseco

Embryo Density and Medium Volume Effects on Early Murine Embryo Development

BackgroundOne-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro.MethodsEmbryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-µl drops of CZB under silicon oil at 37.5°C in a humidified atmosphere of 5% CO2and 95% air.ResultsDevelopment score for embryos cultured in 10 µl was higher than that of embryos cultured in 20 or 40 µl. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-µl drop. The percentage of live embryos in 20 or 40 µl was lower than that of embryos cultured in 10 µl. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups.ConclusionsOur results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

Gene transfer efficiency during gestation and the influence of co-transfer of non-manipulated embryos on production of transgenic mice

Litter size of DNA microinjected zygotes is lower than for non-manipulated zygotes. The rate of embryonic and fetal survival in early, mid and late gestation was determined to assess whether DNA integration was responsible for embryonic losses. Also, the effect of including non-microinjected embryos with injected embryos on pregnancy rate and transgenic pup production was determined. In Experiment 1, one-cell embryos from immature CD-1 mice were microinjected with a whey acidic protein promoter-human protein C gene construct. One hour after microinjection embryos were transferred to pseudopregnant recipients (45 transfers of 30 embryos each). Fifteen recipients were sacrificed on day 4, 12 and 18 of gestation and the embryos/fetuses analysed for the transgene. The percentage of embryos or fetuses that were positive for the transgene was not significantly different at any day. However, the number of viable embryos at day 4 was significantly greater than fetuses on days 12 or 18. In addition, a high degree of mosaicism was observed in day 18 fetuses and placentae recovered. In Experiment 2, one-cell embryos from CD-1 mice were microinjected and co-transferred with non-manipulated embryos (C57BL/6). Pregnancy rate and the total number of pups born were improved by addition of non-injected embryos. However, the number of transgenic mice produced was similar whether non-injected embryos were included or not. There were 32.2% (15/46) transgenic pups when 0 non-injected embryos were transferred compared with 15.1% (13/86) transgenic pups when 4 or 8 non-injected embryos were added to the transfers. In summary, a high degree of embryonic and fetal mortality occurs among microinjected embryos. Furthermore, since the percentage of transgenesis did not change throughout pregnancy, DNA integration does not appear to account for all of the embryonic losses. other factor(s) related to the microinjection procedure may be involved in the embryonic and fetal failure of microinjected embryos. Addition of non-injected embryos, although it increased pregnancy rate and the number of pups born from microinjected embryos, actually decreased the number of transgenic pups obtained per pregnancy.

Effect of low dose of FSH given at the beginning of the estrous cycle and subsequent superovulatory response in Holstein cows

A total of 47 superovulations were conducted on forty non-lactating cows to evaluate two different schemes using follicle stimulating hormone (FSH) for superovulating cattle. Cows randomly assigned to treatment A (26 collections) were superovulated beginning on days 9 to 13 of the estrous cycle by giving FSH at decreasing doses of 6, 6, 5, 5, 3, 3, and 2, 2 mg for 4 consecutive days at 12-h intervals while those in treatment B (21 collections) also received 2.5 mg of FSH on days 3 and 4 of the estrous cycle. Animals in both treatments were each given 12.5 mg of prostaglandin F2alpha (PGF2alpha) at 60 and 72 h after the initiation of superovulatory treatment. Cows were artificially inseminated at 0, 12, and 24 h after the onset of estrus. Embryos were recovered nonsurgically on d 6 and morphologically evaluated. Ovaries of the cows were palpated at the end of flushings to assess the number of corpora lutea (CL). The mean interval from PGF2alpha to the onset of estrus was not different (P>0.05) for treatments A (56.6 h) and B (50.0 h). Also, mean duration of standing estrus was not different for either treatment (13.4 h vs 12.8 h). The mean number of CL palpated (7.3 vs 12.9) and ova recovered (5.5 vs 14.2) were significantly greater (P<0.05) for treatment B. The mean number of excellent and good embryos recovered was lower for treatment A animals, but not significant (P>0.05). Therefore, low doses of FSH given at the beginning of the cycle increased ovulation rate and embryo recovery in non-lactating cows.

Effect of gossypol on bovine embryo development during the preimplantation period.

The purpose of this study was to evaluate the effect of varying doses of gossypol acetic acid on early bovine embryo development in vitro. One hundred and forty-eight excellent and good quality bovine morulae were randomly cultured in 0, 1.0, 5.0, 10.0, 30.0 mug gossypol acetic acid (GAA) in normal steer serum and Hams F-10 media. Bovine embryo development was assessed at 12-h intervals for 96 h. Sixty-seven percent of embryos developed in 0 mug GAA to the hatched blastocyst stage, while 43, 19, 4 and 0% had comparable development in 1.0, 5.0, 10.0 and 30.0 mug GAA, respectively. Embryos in 5.0 mug GAA had a delayed development to the blastocyst stage compared to embryos in 1.0 mug GAA. Development time to expanded blastocyst stage was longer for 10.0 mug GAA embryos than 0, and 1.0 GAA-treated embryos. No embryo cultured in 30.0 mug GAA advanced past the morula stage. Final developmental scores were highest for embryos in 0 mug GAA (4.06) and lowest for embryos cultured in 10.0 and 30.0 mug GAA (0.44 and -0.02, respectively). Embryos cultured in higher doses of GAA degenerated sooner than embryos cultured in 0 mug GAA. These data show a dose-dependent detrimental action of GAA on early bovine embryo development and suggest a direct action on the embryo itself.

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos.

The effect of DNA microinjection at various times afterin vitro insemination on DNA detection and survival rates of bovine embryos was investigated. Oocytes were inseminated 24 h after maturation with frozen/thawed semen prepared with a Percoll separation procedure. At 11, 15 and 19 h after insemination, embryos were centrifuged to visualize pronuclei and microinjected with a murine whey acidic protein-human protein C genomic DNA construct. After culture for 7 days on Buffalo Rat Liver cells, embryos were assessed for stage of development and assayed for the presence of the transgene by polymerase chain reaction. Of zygotes in the 11h after insemination treatment, 16% (25/152) of non-injected and 7% (11/161) of injected embryos developed to the morula or blastocyst stage. Comparable development of non-injected and injected embryos treated at 15h after insemination was 15% (23/158) and 4% (6/159) and treated at 19 h after insemination was 14% (23/162) and 1% (1/165), respectively. Development of injected embryos was greater (p<0.05) when injection was performed at 11 h after insemination compared to 19 h after insemination. Development of non-injected embryos was greater (p<0.01) than that of injected embryos. There was no difference in transgene detection frequency in embryos of all developmental states between treatments (53% at 11; 50% at 15; 48% at 19h after insemination). Injected embryos testing positive for the presence of the transgene exhibited increased development over negative embryos (p<0.01). Greater development efficiencies can be obtained in microinjected bovine embryos when injection is performed early in pronuclear formation.

Ovulation rate, zygote recovery and follicular populations in FSH-superovulated goats treated with PGF2α and/or GnRH

Follicular development and ovulation were examined in superovulated Nubian and Nubian-cross dairy goats following prostaglandin F(2alpha) (PGF(2alpha)) and/or gonadotropin releasing hormone (GnRH) treatment. Estrus was synchronized with Synchromate-B((R)) implants. Superovulation was induced with follicle stimulating hormone (FSH) and augmented with GnRH and/or PGF(2alpha). The PGF(2alpha) treatment was administered on Day 2 of superovulation. Implants were removed from all goats on Day 3 of superovulation. The GnRH treatment was administered 24 h after implant removal. All does were exposed to fertile males for 48 h at the time of GnRH injection. Surgical embryo recovery and ovarian response evaluation were conducted 64 to 78.5 h after implant removal. The number of ovulations was higher with GnRH treatment (18.5 +/- 7; x +/- SEM) than that in the controls (5.3 +/- 4.1; P < 0.05). There were fewer follicles in the GnRH-treated does than in the untreated does (10.9 +/- 2.9 vs 22.1 +/- 3.2; P < 0.05). The number of follicles smaller than 4 mm in diameter (5.8 +/- 0.8) did not differ between treatments. The GnRH-treated does had fewer 4- to 8-mm follicles (4.2 +/- 2.0 vs 9.1 +/- 1.6; P < 0.05) and fewer follicles larger than 8 mm (0.7 +/- 1.4 vs 7.3 +/- 1.6; P < 0.01) than the controls. Predicted times for 1- and 2-cell embryo recoveries were 68.5 and 73.7 h following implant removal, respectively. This study demonstrates that GnRH is an effective supplement used with FSH superovulation regimens in dairy goats. Moreover, GnRH provides for enhanced early embryo collection for DNA microinjection studies.

In vitro and in vivo development of mouse morulae encapsulated in 2% sodium alginate or 0.1% poly-l-lysine.

In Experiment 1, development of zona pellucida-intact (ZPI) morulae was measured every 24 hours for 120 hours after encapsulation in 2% sodium alginate (ALG) or 0.1% poly-L-lysine (PLL). Encapsulation significantly reduced development to hatched blastocysts at 48 and 72 hours. Developmental stages and diameters of ZPI and zona pellucida-free (ZPF) unencapsulated and encapsulated morulae were measured every 24 hours for 72 hours in Exeriment 2. At 72 hours, the percentage of ZPI embryos developing to expanded blastocysts, their diameters and their nuclear counts were not different from each other or from ZPF embryos. In Experiment 3, ZPI morulae encapsulated in ALG or PLL were transferred into recipients. Five of six recipients that received unencapsulated embryos (n=71) delivered 16 live pups. None of the recipients of encapsulated embryos delivered offspring; therefore, a final experiment was performed to examine fetal development on Day 10 of gestation. The percentage of pregnant recipients was similar for all 3 treatments: unencapsulated (71.4%), ALG (87.5%) and PLL (87.5%). However, the presence of viable fetuses was higher for unencapsulated embryos (42.1%) than for ALG (17%) and PLL (14.6%) embryos. These results suggest that encapsulation did not detrimentally affect embryonic size or cellular development in vitro; however, mortality occurred in vivo due to an asynchronous condition between the uterine environment and the embryos.

Transgene detection during early murine embryonic development after pronuclear microinjection

The polymerase chain reaction (PCR) technique was used to detect a whey acidic protein (WAP) gene and transgene presence in mouse ova cultured to various stages of development after pronuclear microinjection at the one-cell stage. The PCR technique detected an endogenous 442 bp WAP DNA sequence in 78% of one-cell, 88% of two-cell and 94% of four-cell ova, and in 95% of morulae and 97% of blastocysts. The heterologous WAP-human protein C transgene was detected in 88% of one-cell, 88% of two-cell and 44% of four-cell ova, and in 40% of morulae and 29% of blastocysts. For comparison, the integration frequency for transgenic mouse production using the same DNA construct was 22%. After five days ofin vitro culture, embryos that were either developmentally arrested or fragmented were tested for the presence of the transgene. The injected construct was detected in 83% of arrested one-cell, 85% of arrested two-cell, and 85% of fragmented ova. In culture, only 28% of zygotes microinjected with DNA developed to the blastocyst stage compared to 74% of noninjected zygotes, while 63% of zygotes developed to the blastocyst stage after injection of buffer alone. Pronuclear injection of the transgene at concentrations of 1.5, 15 and 50 μg ml−1 resulted in 28, 11 and 9% development to blastocysts and 29, 86 and 88% transgene detection, respectively. Transgene detection was 85, 96 and 97% in degenerate embryos at the respective doses of DNA. These data show that pronuclear microinjection of the transgene is detrimental to subsequent embryonic development. Also, unintegrated copies of the transgene probably exist at least until the blastocyst stage, and thereafter are degraded to the extent that they can no longer be detected by PCR.

Observations on in vitro development of bovine morulae in Ham's F-10 and Dulbecco's phosphate buffered saline supplemented with normal steer serum

This study was conducted to compare in vitro development of bovine morulae in Hams F-10 and Dulbeccos phosphate buffered saline (D-PBS) media supplemented with 10% (v/v) normal steer serum. Fifty-three excellent and good embryos were obtained by superovulating 15 non-lactating Holstein cows. Embryos were placed randomly in culture with Hams F-10 or D-PBS media and development was recorded at 12-h intervals for the duration of culture. All embryos reached early blastocyst, blastocyst and expanded blastocyst stage. Nineteen of 27 embryos (70.1%) cultured in Hams F-10 developed to hatched blastocyst stage in contrast to three out of 26 in D-PBS (11.5%). The mean developmental scores at 24, 48, 72, 96 and 120 h of culture were significantly (P<0.001) higher for embryos cultured in Hams F-10. Also, the mean times to reach early blastocyst (25.84 +/- 6.65 vs 46.67 +/- 9.99 h), blastocyst (44.57 +/- 11.45 vs 61.89 +/- 16.62 h) and expanded blastocyst stage (65.00 +/- 13.20 vs 73.41 +/- 15.80 h) were significantly (P<0.001) shorter for embryos cultured in Hams F-10. No difference was observed in the mean time to reach hatching (90.00 +/- 10.85 vs 84.00 +/- 16.97 h) and hatched blastocyst stage (97.26 +/- 18.71 vs 96.00 +/- 0.00 h). The results obtained support the concept that Hams F-10 and normal steer serum provide for optimal bovine embryo development and suggest that 10% normal steer serum could be used as a protein supplement with D-PBS for short term storage and culture of bovine embryos.

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.