Recipient Embryos Research Articles

Nontransmission of porcine circovirus 2 (PCV2) by embryo transfer

Two experiments were conducted to determine the association of porcine circovirus type 2 (PCV2) with embryos and the risk of viral transmission by embryo transfer. In the first experiment, 240 embryos from uninfected donors were exposed to PCV2a 104TCID50/mL in vitro before transfer to seronegative recipients; in the second experiment, 384 embryos recovered from infected donors, 10 days after donor inoculation with PCV2, were transferred to seronegative recipients. In total, 1120 embryos and/or ova were collected from 37 viral-free donors (experiment 1) and 1019 from 59 PCV2-infected donors (experiment 2) (P < 0.01). The washing and/or disinfection procedure recommended by the International Embryo Transfer Society was applied to embryos in both experiments. Transfer of embryos experimentally exposed in vitro to high titers of virus caused seroconversion of recipients (58%; N = 7/12) and their piglets (81%; N = 13/16). Postmortem, PCV2 DNA was detected in various organs of embryo transfer recipients and their embryo transfer-derived piglets. In contrast, the transfer of embryos recovered from infectious PCV2 donors did not result in the seroconversion of embryo recipients (N = 24) or their embryo transfer-derived piglets (N = 76). Neither PCV2 DNA nor infectious virus was detected in the tissues of either recipients or embryo transfer-derived piglets collected postmortem in the second experiment. The results obtained in this study indicate that the transmission of PCV2 from infected donors by embryo transfer is unlikely if the sanitary recommendations of the International Embryo Transfer Society are followed. In practical terms, this means that embryo transfer can be successfully used for the intentional elimination of PCV2 and to create virus-free offspring for the safe exchange of swine genetic materials.

Production of chimeras between the Chinese soft-shelled turtle and Peking duck through transfer of early blastoderm cells

Chimeras are useful models for studies of developmental biology and cell differentiation. Intraspecies and interspecies germline chimeras have been produced in previous studies, but the feasibility of producing chimeras between animals of two different classes remains unclear. To address this issue, we attempted to produce chimeras between the Chinese soft-shelled turtle and the Peking duck by transferring stage X blastoderm cells to recipient embryos. We then examined the survival and development of the PKH26-labeled donor cells in the heterologous embryos. At early embryonic stages, both turtle and duck donor cells that were labeled with PKH26 were readily observed in the brain, neural tube, heart and gonads of the respective recipient embryos. Movement of turtle donor-derived cells was observed in the duck host embryos after 48 h of incubation. Although none of the hatchlings presented a chimeric phenotype, duck donor-derived cells were detected in a variety of organs in the hatchling turtles, particularly in the gonads. Moreover, in the hatched turtles, mRNA expression of tissue-specific duck genes MEF2a and MEF2c was detected in many tissues, including the muscle, heart, small and large intestines, stomach and kidney. Similarly, SPAG6 mRNA was detected in a subset of turtle tissues, including the gonad and the small and large intestines. These results suggest that duck donor-derived cells can survive and differentiate in recipient turtles; however, no turtle-derived cells were detected in the hatched ducks. Our findings indicate that chimeras can be produced between animals of two different classes.

Production of Functional Gametes from Cryopreserved Primordial Germ Cells of the Japanese Quail

The Japanese quail (Coturnix japonica) is a valuable bird as both an experimental animal, for a wide range of scientific disciplines, and an agricultural animal, for the production of eggs and meat. Cryopreservation of PGCs would be a feasible strategy for the conservation of both male and female fertility cells in Japanese quail. However, the effects of freeze-thaw treatment on viability, migration ability and germline transmission ability of quail PGCs still remain unclear. In the present study, male and female PGCs were isolated from the blood of 2-day-old embryos, which were cooled by slow freezing and then cryopreserved at –196 C for 77–185 days, respectively. The average recovery rate of PGCs after freeze-thawing was 47.0%. The viability of PGCs in the frozen group was significantly lower than that of the control group (P<0.05) (85.5% vs. 95.1%). Both fresh and Frozen-thawed PGCs that were intravascularly transplanted into recipient embryos migrated toward and were incorporated into recipient gonads, although the number of PGCs settled in the gonads was 48.5% lower in the frozen group than in the unfrozen control group (P<0.05). Genetic cross analysis revealed that one female and two male recipients produced live progeny derived from the frozen-thawed PGCs. The frequency of donor-derived offspring was slightly lower than that of unfrozen controls, but the difference was not significant (4.0 vs. 14.0%). These results revealed that freeze-thaw treatment causes a decrease in viability, migration ability and germline transmission ability of PGCs in quail.

Transgenic chickens

The development of transgenic chicken technology has lagged far behind that of mammalian species. Two reasons for this are that only a one-cell-stage oocyte can be obtained from a sacrificed hen and that the yolk prevents high-magnification microscopic observation of oocytes. Recently, several new methods have been developed that will enable the successful establishment of transgenic chickens. Retroviral vectors are used in many cases because of their ability to incorporate transgenes into host cell chromosomes in a highly efficient manner. These viral vectors are injected directly into the embryos, usually at the blastodermal stage. In some cases, primordial germ cells (PGCs) are infected in vitro and then implanted into recipient embryos. Methods that do not rely on retroviral vectors are also available for creating transgenic chickens. Long-term culture of PGCs permits the selection of stably transfected cells and implantation of the manipulated PGCs. In addition, embryonic stem (ES) cell systems are available; however, the induction of functional gametes from ES cells has not, to our knowledge, been successful. It is clear that recent developments suggest that chickens may be used as a valuable experimental genetic system.

Effects of Donor Fibroblast Cell Type and Transferred Cloned Embryo Number on the Efficiency of Pig Cloning

Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a significantly higher birth rate of healthy clones (0.5009% vs. 0.3362% and 0.2433%) than that resulting from P,D,L,Y-AFBs and P,D,L,Y-FFBs. This suggests that using LW-AFBs as donor cells results in a higher cloning efficiency in pigs, compared with the other two donor fibroblast cell types. The birth rate of healthy clones was significantly improved when the number of transferred cloned embryos was increased from 150-199 to 200-450 per recipient. However, increase of the number of transferred embryos from 200-249 to 250-450 per surrogate did not change the birth rate of healthy clones. This suggests that transfer of excessive (250-450) cloned embryos to an individual surrogate is not necessary for increasing the cloning efficiency in pigs, and the relatively optimal number of reconstructed embryos transferred to individual recipient is 200-249. Furthermore, our results indicated that the numbers of total born clones, clones born alive, and clones born healthy per litter have a significantly high positive correlation with each other. The present study provides useful information for improving SCNT efficiency in pigs.

Evaluation of the effectiveness of semen processing techniques to remove bovine viral diarrhea virus from experimentally contaminated semen samples

The aim of this study was to evaluate the capacity of three semen processing techniques, Percoll gradient centrifugation, Swim-up and a combination of Swim-up and Percoll gradient centrifugation, to reduce the viral load of bovine viral diarrhea virus (BVDV) in experimentally infected semen samples. The evaluation was performed using two approaches: first, searching for the presence of virus in the processed samples (via virus titration and RT-PCR) and second, ascertaining the possible interference on in vitro embryo production. The sperm count and DNA integrity (Comet assay) of the processed samples were analyzed (Experiment 1). The amount of virus in the processed samples was determined by titration in cell culture (Experiment 2). The samples processed by Swim up/Percoll gradient centrifugation were utilized for in vitro embryo production, and the embryos produced were tested for BVDV by RT-PCR (Experiment 3). Sperm concentration, Comet assay and embryo production were analyzed by chi-squared tests (P<0.05). There was a significant difference between sperm separation techniques when the sperm count and Comet assay were analyzed. The sperm count obtained from the Swim up/Percoll gradient centrifugation group was lower than that obtained in either of the two other groups (Swim up and Percoll gradient centrifugation), and the Comet assay showed that the combination of the two semen processing techniques (Swim up/Percoll gradient) produced a 1.1% prevalence of Comet level 2, which was not observed in the other groups. The BVDV titer (10(6.68)TCID(50)/mL) added to experimentally infected semen samples decreased after Percoll gradient centrifugation to 10(2.3)-10(1)TCID(50)/mL; for the Swim up group, the titer range was 10(3.3)-10(1.87)TCID(50)/mL, and in the Swim up/Percoll gradient centrifugation group, BVDV was undetectable. The decreases in titer varied from 99.9% in the Swim up-processed group to 100% in the Swim up/Percoll gradient centrifugation group. In vitro embryo production displayed similar blastocyst development rates among all groups, and RT-PCR was negative for the produced embryos. The data showed that the combination of Swim up/Percoll gradient centrifugation promoted the elimination of BVDV from the semen samples without damaging spermatozoa cells and also allowed successful in vitro embryo production free of BVDV. Hence, the risk of BVDV contamination is negligible for the embryo recipient.

160 CONCEPTION RATES FOLLOWING TREATMENT OF LACTATING HOLSTEIN RECIPIENTS WITH A GONADOTROPIN RELEASING HORMONE (GnRH) ANALOGUE AT THE TIME OF TRANSFER OF OVUM PICK-UP/IVF-DERIVED EMBRYOS

The use of lactating dairy cows as ovum pick-up (OPU)/IVF-derived embryo recipients in large-scale embryo transfer programs may be limited by the effects of low progesterone concentrations on the uterine environment and subsequent embryo development. It was hypothesized that the injection of a GnRH analogue on the day of embryo transfer would induce ovulation and formation of an accessory corpus luteum, increasing progesterone concentrations and, as a result, conception rates. An experiment was designed to evaluate the effects of two different GnRH analogues injected IM on the day of embryo transfer on conception rates of lactating Holstein recipients. Oestrus was not synchronized and was detected continuously by multiple observers during the day and in the evening until midnight. Recipient days in milk varied from 45 to 150, daily milk production ranged from 20 to 45 kg, body condition scores and age ranged from 2.5 to 4.5 and 2.5 to 5.5 years, respectively. Herd pregnancy rates after the first artificial insemination ranged from 15% to 70%, considering the period the experiment was conducted. Embryos were produced by standard in vitro techniques following OPU on Holstein donors. Lactating Holstein recipients (n = 224) were allocated randomly to one of three treatment groups 7 days after detected oestrus: Control (n = 77) = vehicle; Buserelin (Sincroforte®, Ouro Fino, Sao Paulo, Brazil; n = 75) = 10 µg; and Deslorelin (Sincrorrelin®, Ouro Fino; n = 72) = 750 µg. Embryos were transferred nonsurgically 6 to 8 days after observed oestrus. Pregnancy was detected by ultrasonography between 62 and 72 days after embryo transfer. Percent pregnant data were analyzed as a binomial distribution under the GENMOD procedure of SAS (SAS Institute Inc., Cary, NC, USA). Conception rates were similar (P = 0.13) between Control (28%, 22/77), Buserelin (24%, 18/75), and Deslorelin (38%, 28/72) treatment groups. These data demonstrate that conception rates from in vitro produced embryos transferred to lactating dairy cows are not improved by treatment with a GnRH analogue on the day of transfer. Although not critically tested, results indicate that adequate conception rates may be obtained in commercial dairy operations from OPU/IVF-derived embryos, adding an important management tool for reproduction and animal breeding enhancement.

Transgenic sheep generated by lentiviral vectors: safety and integration analysis of surrogates and their offspring

The safety of HIV-1 based vectors was evaluated during the production of transgenic sheep. Vectors were introduced into the perivitelline space of in vivo derived one-cell sheep embryos by microinjection then transferred into the oviducts of recipient females. At 60-70days of gestation, a portion of the recipients were euthanized and tissues collected from both surrogates and fetuses. Other ewes were allowed to carry lambs to term. Inadvertent transfer of vector from offspring to surrogates was evaluated in 330 blood and tissue samples collected from 57 ewes that served as embryo recipients. Excluding uterine contents, none of the samples tested positive for vector, indicating that that the vector did not cross the fetal maternal interface and infect surrogate ewes. Evaluating ewes, fetuses and lambs for replication competent lentivirus (RCL); 84 serum samples analyzed for HIV-1 capsid by ELISA and over 600 blood and tissue samples analyzed by quantitative PCR for the VSV-G envelopes revealed no evidence of RCL. Results of these experiments provide further evidence as to the safety of HIV-1 based vectors in animal and human applications.

Effect of breed and corpus luteum on pregnancy rate of bovine embryo recipients

The objective of this study was to evaluate pregnancy rates of recipients of different breed groups (Nellore and crossbreed), as well as the effects of size and type of the corpus luteum (CL) on plasmatic concentrations of progesterone and pregnancy rates of embryo recipients. A total of 152 heifers were synchronized with progesterone implants and on the day of embryo transfer, previously obtained by superovulation and frozen in ethylene glycol, the diameter and type of the corpus luteum (cavitary and compact) was measured and blood was collected for progesterone measurement. The pregnancy rate was 44.1%, with a diameter of corpus luteum higher in recipients that became pregnant (2.03±0.41) compared with non-pregnant ones (1.86±0.34 cm). Plasmatic concentrations of progesterone did not differ between pregnant (1.50±1.05) and non-pregnant (1.31±0.91 ng/mL) animals. The type of corpus luteum did not influence the pregnancy rates. Only Angus and crossbred Marchigiana differ among themselves in pregnancy rates (33.3 and 59.2%, respectively). The pregnancy probability was affected only by CL diameter, but not by P4 plasmatic concentration. Selection of the corpus luteum size at the time of embryo transfer is an important factor to increase pregnancy rates in recipients, and compact and cavitary corpora lutea do not influence the pregnancy rates of bovine embryo recipients. Nellore recipients have pregnancy rates that are satisfactory and comparable to crossbred (Bos taurus × Bos indicus) recipients.

Ectopic formation of primordial germ cells by transplantation of the germ plasm: Direct evidence for germ cell determinant in Xenopus

In many animals, the germ line is specified by a distinct cytoplasmic structure called germ plasm (GP). GP is necessary for primordial germ cell (PGC) formation in anuran amphibians including Xenopus. However, it is unclear whether GP is a direct germ cell determinant in vertebrates. Here we demonstrate that GP acts autonomously for germ cell formation in Xenopus.EGFP-labeled GP from the vegetal pole was transplanted into animal hemisphere of recipient embryos. Cells carrying transplanted GP (T-GP) at the ectopic position showed characteristics similar to the endogenous normal PGCs in subcellular distribution of GP and presence of germ plasm specific molecules. However, T-GP-carrying-cells in the ectopic tissue did not migrate towards the genital ridge. T-GP-carrying cells from gastrula or tailbud embryos were transferred into the endoderm of wild-type hosts. From there, they migrated into the developing gonad. To clarify whether ectopic T-GP-carrying cells can produce functional germ cells, they were identified by changing the recipients, from the wild-type Xenopus to transgenic Xenopus expressing DsRed2. After transferring T-GP carrying cells labeled genetically with DsRed2 into wild-type hosts, we could find chimeric gonads in mature hosts. Furthermore, the spermatozoa and eggs derived from T-GP-carrying cells were fertile. Thus, we have demonstrated that Xenopus germ plasm is sufficient for germ cell determination.

Biological Characteristics of Fish Germ Cells and their Application to Developmental Biotechnology

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.

Embryo Transfer in Anovulatory Recipient Mares Treated with Estradiol Benzoate and Long-Acting Progesterone

This study aimed to prepare anovulatory mares in anestrus or in the transitional period as embryo recipients. Ninety embryo-recipient mares were divided into two groups (G). G1 (n = 45) comprised animals in anestrus or in the transitional period; these animals were treated for 3 days (D) with 5, 3, and 2 mg of estradiol benzoate (intramuscular) on D0 (day of the donor's ovulation), D1, and D2 (after ovulation), respectively, followed by weekly application of 400 mg of long-acting progesterone (intramuscular) from D3 after ovulation (donor) until the 120th day of gestation. G2 (n = 45) comprised mares with normal estrous cycles. Plasma levels of progesterone (P4) were measured on days D1, D2, D8, and D14. Sixty percent of the animals in G1 and 71.1% in G2 (P > .05) completed the pregnancy. On D8, there was no difference in P4 levels between G1 and G2 animals, but there was a difference in P4 levels on D14 (P < .05). It was concluded that anovulatory mares in anestrus or in the transitional period could be used as embryo recipients. The protocol was efficient and also considered an appropriate alternative to prepare the uterine environment for embryo transfer; long-acting progesterone administration kept P4 levels high enough to maintain pregnancy until the 120th day and provided recipients during the time of the year when fewer mares were cycling and ovulating.

Data of three first seasons of embryo transfer from two clones of the same mare

underwent serial pregnancy ultrasound exams per rectum following transfer and 35-day pregnancy rates were recorded. Pregnancy rates were compared between recipient mares that ovulated 8-12 versus 1317 days postpartum. Study 2: Blastocysts (n 1⁄4 18) recovered from mares following insemination on their first PP ovulation were transferred transcervically into recipient mares that were either nonfoal-heat mares or nonlactating mares. The recipient mares underwent ultrasound examination per rectum and 35-day pregnancy rates were recorded. The Chi-square test was used to compare values. The results in Table I show that the 35-day pregnancy rate of recipient mares that had embryos transferred following their first postpartum ovulation was 65% (151 pregnancies/ 232 embryos transferred). There was no difference in pregnancy rates between recipient mares that ovulated 8-12 days versus 13-17 days after ovulation. The results in Table II show that the 35-day pregnancy rate of recipient mares was 83% (15 pregnancies/18 transferred foal heat embryos). In conclusion, mares on their foal heat can serve as embryo recipients. Furthermore, when embryos are recovered from donor mares inseminated on the first postpartum estrus, recipient pregnancy rates are high.

Optimal utilization of modern reproductive technologies to maximize the gross margin of milk production

In this study, a linear programming model was developed to maximize the gross margin of milk production by determining the optimal use of different reproductive technologies in a dairy herd. The model has the potential to vary the use of conventional artificial insemination, insemination with X-sorted sperm, and the use of unselected or sex-selected embryo recovery and transfer. Data from Finnish dairy herd recording systems were used to parameterize the model. This paper presents the results of 6 scenarios for a herd size of 60 dairy cows. In the basic scenario, the optimum economic combination for Finnish conditions was to inseminate 10 heifers and 22 cows with unsorted semen, 8 heifers with X-sorted sperm, and to use 20 cows as embryo donors which was the upper constraint for this technique. The embryo donors were inseminated with conventional semen for both embryo production and their subsequent pregnancy. Without restriction on embryo recovery, the optimum combination was to use all heifers as donors of sex-selected embryos and all cows as donors of unselected embryos. It was more profitable to produce female embryos with X-sorted sperm than by sorting embryos. Embryo recipients were not economically justified in any scenario. In practice, the optimal strategy is herd-specific depending on the input costs, output values and the technical success of each reproductive technology in that herd. This single-year linear programming model adequately differentiates between breeding technologies within a herd, but further research is needed to develop dynamic models to consider genetic improvement and herd expansion.

Maternal transfer and protective role of antibodies in zebrafish Danio rerio

Maternal transfer of antibodies from mother to eggs has been reported in various species of fishes, and these antibodies have been proposed to play immune roles in developing embryos and larvae. However, firm evidence supporting this remains lacking. In this study, we clearly demonstrated that immunization of female zebrafish with the hapten–carrier complex, trinitrophenylated bovine serum albumin (TNP-BSA), induced a significant increase in anti-TNP antibody production in the mothers, which in turn induced a marked increase in anti-TNP antibody level in their eggs. Microinjection of anti-zebrafish IgM antibody into early embryos (to neutralize endogenous antibody action) resulted in a remarkable decrease in the resistance of recipient embryos to pathogenic Aeromonas hydrophila, whereas injection of BSA or anti-β-actin monoclonal antibody into the same stage embryos had little effect on their resistance to the pathogen. Moreover, the content of endogenous antibodies in eggs was clearly correlated with their antibacterial activity against A. hydrophila. This is the first report showing that maternally transferred antibodies in fish can protect early embyros/larvae from the attack of pathogens like A. hydrophila.

Use of long-acting progesterone to acyclic embryo recipient mares

The objective of this study was to evaluate the efficiency of cyclic and acyclic progesterone-treated recipient mares in a commercial embryo transfer program. Acyclic recipients were treated with decreasing amounts of estradiol cypionate (ECP® - Pfizer Saude Animal, Sao Paulo, Brazil), followed by 1500 mg of a sustained-release progesterone injection (P4LA150® - Laboratorios B.E.T., Rio de Janeiro, Brazil). Out of all 1506 embryo recovery attempts performed, 59.36% were positive (894/1506). Pregnancy rates of cyclic and acyclic mares were, respectively, 44.12% (319/723) and 57.68% (139/241) - higher in acyclic recipients under progesterone treatment. Embryo loss was similar between groups (9.72% 31/319 vs. 11.51% - 16/139). When fertility data on acyclic mares treated with long-action progesterone were grouped in relation to the interval of the beginning of the treatment with progesterone to the embryo transfer, there was no difference in pregnancy rates at 12 days (51.28% - 20/39; 58.92% - 33/56; 60.52% - 46/76 and 58.57% - 41/70 for embryos transferred 3 (P3), 4 (P4), 5 (P5) and 6 (P6) days after the beginning of the treatment with progesterone). Groups P3, P4, P5 and P6 showed embryo loss rates of 20.00% (4/20), 12.12% (4/33), 2.17% (1/46) and 17.07% (7/41), respectively. These rates were similar in groups P3, P4 and P6. Pregnant recipients submitted to an embryo transferring procedure on P5 showed lower embryo mortality rates than on P3 and P6. P4 recipients tended to suffer higher embryo mortality rates than P5. Acyclic recipient mares under progesterone treatment can be satisfactoryly used in embryo transfer programs.

Pregnancy outcomes in oocyte donation recipients: vaginal gel versus intramuscular injection progesterone replacement

Compare outcomes with vaginal gel versus intramuscular progesterone replacement in donor oocyte recipients. A single-center retrospective analysis (January 2004-December 2006) evaluated pregnancy outcomes (serum human chorionic gonadotropin, implantation, clinical pregnancy, delivery, total pregnancy loss rates) for 225 recipients of embryos from donor (aged <32 years) oocytes. Vaginal progesterone gel (Crinone® 8%; 90 mg twice daily; n = 105) or intramuscular progesterone (50 mg once daily; n = 120) was started the afternoon of oocyte retrieval and continued until a negative pregnancy test or 10 weeks' gestation. There were no statistically significant differences between groups for the five pregnancy outcomes; numerical results favored vaginal progesterone in all cases. Confidence intervals showed vaginal gel was within, or <1% from, a noninferiority limit of 10% versus intramuscular progesterone for four of five pregnancy outcomes. Pregnancy outcomes were comparable for progesterone replacement with vaginal gel and intramuscular progesterone in an oocyte donation program.

Disclosure patterns of embryo donation mothers compared with adoption and IVF

Attitudes towards information sharing in donor conception have changed in recent years in some parts of the world, with a move towards openness. This study follows up a sample of embryo donation mothers, examining their current disclosure patterns and comparing them with adoptive and IVF mothers to investigate any influence of the method of family creation. This study compared 17 mothers with an embryo donation child aged 5–9years with 24 mothers with an adopted child and 28 mothers with a genetically related IVF child. Embryo donation mothers were far less likely to share information with the child; 43% were inclined towards disclosing, compared with all adoptive mothers and nearly 90% of IVF mothers. Furthermore, embryo donation and IVF mothers who had disclosed had often only given partial explanations of the child’s conception. Differences between embryo donation and adoption in particular should be taken into account when advising embryo recipients.

Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

BackgroundDimeric human erythropoietin (dHuEPO) peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg) mice expressing dHuEPO and to investigate the characteristics of these mice.MethodsA dHuEPO-expressing vector under the control of the goat beta-casein promoter, which produced a dimer of human EPO molecules linked by a 2-amino acid peptide linker (Asp-Ile), was constructed and injected into 1-cell fertilized embryos by microinjection. Mice were screened using genomic DNA samples obtained from tail biopsies. Blood samples were obtained by heart puncture using heparinized tubes, and hematologic parameters were assessed. Using the microarray analysis tool, we analyzed differences in gene expression in the spleens of tg and control mice.ResultsA high rate of spontaneous abortion or death of the offspring was observed in the recipients of dHuEPO embryos. We obtained 3 founder lines (#4, #11, and #47) of tg mice expressing the dHuEPO gene. However, only one founder line showed stable germline integration and transmission, subsequently establishing the only transgenic line (#11). We obtained 2 F1 mice and 3 F2 mice from line #11. The dHuEPO protein could not be obtained because of repeated spontaneous abortions in the tg mice. Tg mice exhibited symptoms such as short lifespan and abnormal blood composition. The red blood cell count, white blood cell count, and hematocrit levels in the tg mice were remarkably higher than those in the control mice. The spleens of the tg mice (F1 and F2 females) were 11- and -21-fold larger than those of the control mice. Microarray analysis revealed 2,672 spleen-derived candidate genes; more genes were downregulated than upregulated (849/764). Reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were used for validating the results of the microarray analysis of mRNA expression.ConclusionsIn conclusion, dHuEPO tg mice caused excessive erythrocytosis that led to abnormal blood composition, short lifespan, and abnormal splenomegaly. Further, we identified 2,672 genes associated with splenomegaly by microarray analysis. These results could be useful in the development of dHuEPO-producing tg animals.

Lack of organ specific commitment of vagal neural crest cell derivatives as shown by back-transplantation of GFP chicken tissues

Neural crest cells (NCC) are multipotent progenitors that migrate extensively throughout the developing embryo and generate a diverse range of cell types. Vagal NCC migrate from the hindbrain into the foregut and from there along the gastrointestinal tract to form the enteric nervous system (ENS), the intrinsic innervation of the gut, and into the developing lung buds to form the intrinsic innervation of the lungs. The aim of this study was to determine the developmental potential of vagal NCC that had already colonised the gut or the lungs. We used transgenic chicken embryos that ubiquitously express green fluorescent protein (GFP) to permanently mark and fate-map vagal NCC using intraspecies grafting. This was combined with back-transplantation of gut and lung segments, containing GFP-positive NCC, into the vagal region of a second recipient embryo to determine, using immunohistochemical staining, whether gut or lung NCC are competent of re-colonising both these organs, or whether their fate is restricted. Chick(GFP)-chick intraspecies grafting efficiently labelled NCC within the gut and lung of chick embryos. When segments of embryonic day (E)5.5 pre-umbilical midgut containing GFP-positive NCC were back-transplanted into the vagal region of E1.5 host embryos, the GFP-positive NCC remigrated to colonise both the gut and lungs and differentiated into neurons in stereotypical locations. However, GFP-positive lung NCC did not remigrate when back-transplanted. Our studies suggest that gut NCC are not restricted to colonising only this organ, since upon back-transplantation GFP-positive gut NCC colonised both the gut and the lung.