In vivo development of microinjected embryos from superovulated prepuberal slaughter lambs

This study was performed to investigate the developmental potential of microinjected embryos recovered from superovulated prepuberal lambs. Fifty-nine mixed-bred lambs (about 3 mo old) were superovulated either with 18 mg FSH-P with (Group FSH/+S) or without (Group FSH/-S) progestagen treatment, or with 10 mL Ovagene following progestagen treatment (Group OVA/+S). All animals received hCG to induce ovulation. Ovulation rates and the number of ova recovered per animal for the different groups were 8.7 and 4.7 (55%, FSH/+S); 7.3 and 3.2 (42%, FSH/-S); and 6.4 and 4.0 (65%, OVA/+S), respectively. No significant differences were seen in the ovulation and the recovery rates, but animals without progestagen treatment showed a significantly lower fertilization rate (44%) when compared with progestagen-treated groups (87%; P<0.001). Foreign DNA was microinjected into the pronuclei of fertilized ova (n = 155), which were transferred (n = 98) into synchronized recipient ewes (n = 21). Two animals were detected pregnant and both gave birth to a single lamb. Results of superovulation and embryo recovery from prepuberal lambs were promising, but the low rate of development to term indicates that ova recovered from prepuberal lambs have reduced developmental competence in vivo. Although 2 lambs were born, it seems that this is not a successful method for use in future gene transfer programs.     

In vitro development of bovine embryos cultured with activin A

The aim of this study was to investigate the effects of activin A on development, differential cell counts and apoptosis/necrosis rates of bovine embryos produced in vitro. Presumptive zygotes were cultured up to Day 8 in synthetic oviduct fluid containing aminoacids, citrate, myo-inositol and BSA. In Experiment 1, activin (10 ng mL⁻¹) was added: 1/from Day 1 to Day 3; 2/from Day 1 to Day 8; 3/from Day 3 to Day 8; or 4/absent (control). In Experiment 2, 10 ng mL⁻¹ activin were added either before (Day 3 to Day 5) or after (Day 5 to Day 8) the early morula stage. In Experiment 1, activin during the first 72 h of culture reduced Day 3 cleavage, 5-8 cell rates and blastocyst development, while hatching rates increased. No changes were observed within differential cell counts. In experiment 2, activin improved blastocyst development after, and had no effect before, the Day 5 morula stage. However, trophectoderm (TE) cell numbers decreased with activin both before and after the Day 5 morula stage, suggesting that activin inhibits TE differentiation. The presence of activin during the whole culture had no effect on TUNEL positive cells, but when added at shorter periods activin increased apoptotic rates. Effects of activin during in vitro bovine embryo development, depends on timing of its addition to the culture medium.     

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirmed an individual effect of the bull as well as development rates of embryos produced with sorted sperm lower than embryos with unsorted sperm, independent of the culture system used. The cryoresistance to vitrification of embryos produced with sexed sperm did not differ from that of conventionally produced embryos (re-expansion rates at 24 and 48 h: 74.6% vs. 75.5%, and 64.5% vs. 68.1% for embryos produced with conventional and sorted sperm, respectively; hatching rates at 48 h: 63.55% vs. 55.5% for embryos produced with conventional and sorted sperm, respectively). Finally, no significant differences were found in pregnancy rates after the embryo transfer of fresh and vitrified/warmed blastocysts (52.8% vs. 42.0%, respectively; P > 0.05). Male and female embryos produced with sorted sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. Our culture system, coupled with the vitrification in fiber plugs, provides good quality sex-known embryos which survive vitrification at similar rates than embryos produced with conventional unsorted sperm; also it produces good pregnancy rates after transfer of sexed embryos both fresh and after vitrification and warming.     

Glucose inhibits development of hamster 8-cell embryos in vitro

Relative preferences of energy substrates (glucose, pyruvate, and lactate) for in vitro development of hamster 8-cell embryos were investigated. Using protein-free modified Tyrode's medium (TLP-PVA) containing 10 mM lactate (L), 0.1 mM pyruvate (P), and amino acids (Phe, Ile, Met and Gln), we found that development of hamster 8-cell embryos to blastocysts was supported better in the absence of glucose than in medium containing (standard) 5 mM glucose (88.1% and 50%, respectively). Addition of even 0.25 mM glucose to the medium significantly inhibited blastocyst formation (54.1%). Medium T-PVA, containing 5 mM glucose as sole energy substrate (without pyruvate, lactate, and amino acids), very poorly supported embryo development (less than or equal to 7.9% blastocysts), but addition of 0.1 mM pyruvate enhanced blastocyst formation (52%). Elimination of pyruvate in TL-PVA medium containing 5 mM glucose and amino acids markedly reduced blastocyst formation by 4-fold (13.5%); the optimal pyruvate concentration was 0.2 mM. However, if the same medium was devoid of glucose, blastocyst formation was high both in the absence (71.1%) and presence (83.3%) of 0.1 mM pyruvate. Similarly, in glucose-free T-PVA medium, addition of either 10 mM lactate or amino acids supported 8-cell embryo development to blastocysts (61.7% and 60.5%, respectively) as opposed to 18.8% and 30.6%, respectively, in the presence of 5 mM glucose. This augmented development in the absence of glucose is suggested to the due to the efficient conversion of lactate to pyruvate and of amino acids to amphibolic intermediates and hence their utilization via the Krebs cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro development of bovine embryos in Buffalo rat liver- or bovine oviduct-conditioned medium

A culture system for bovine embryos was developed using Buffalo rat liver cell (BRL) line-conditioned medium without serum. Zygotes, obtained by in vitro maturation and fertilization of oocytes, were cultured either in unconditioned medium (TCM 199 or DMEM/F12) or in the same medium conditioned by bovine oviduct or BRL cells. No serum was added during conditioning or during embryo culture. The DMEM/F12 medium was superior to TCM 199 for development of bovine embryos to the 5 to 8-cell stage: on average between 50 and 57% of the embryos reached this stage after 2 d of culture in DMEM/F12 or in conditioned medium, while 36% reached this stage in TCM 199. Further development to the blastocyst stage was enhanced by conditioning. The highest percentage of blastocysts was achieved in DMEM/F12 medium conditioned with BRL cells (30%). The yield of blastocysts was similar in TCM 199 and in DMEM/F12 media conditioned with bovine oviduct cells (22 versus 20%), but after conditioning with BRL cells, DMEM/F12 medium yielded a higher percentage of blastocysts than TCM 199 (30 versus 18%). This might be explained by the fact that viability of BRL cells was better in DMEM/F12 medium than in TCM 199 when serum was omitted. Blastocysts produced in BRL-conditioned medium had a higher number of cells than blastocysts obtained in bovine oviduct-conditioned medium, and their transfer to recipients led to pregnancies and birth of calves. In conclusion, culture of bovine embryos in DMEM/F12 medium conditioned with BRL cells without serum led to the development of good-quality blastocysts and is thus a promising method for producing embryos for the study of potential embryotrophic factors. The use of rat liver cell lines guarantees against bovine viruses and allows for better production of embryos.     

Detection of microinjected genes in bovine preimplantation embryos with combined DNA digestion and polymerase chain reaction

We have developed a simple digestion-polymerase chain reaction (PCR) assay for a simultaneous transgene detection and sexing of pronucleus-injected bovine preimplantation embryos. Bovine embryos were microinjected with dam-methylated gene construct and cultured in vitro for 6–7 days after the injections. The developed blastocysts and compact morulae were bisected and the embryonic biopsies representing mainly trophoblasts were subjected to the digestion-PCR, while the biopsied embryos remained in culture. Embryonic DNA was released with proteinase K and the samples were digested with a Dpnl-Bal31 mixture before the PCR amplification of the transgene, bovine αS1-casein, and bovine Y-chromosome fragments in the same reaction. The whole assay from biopsy to electrophoresis took less than 6 hr. The digestion removed up to 50 fg of dam-methylated transgene copies (unintegrated or contaminants) and also a few hundred copies of contaminating PCR products from the embryonic samples. The digestion-PCR assay eliminated all transgene contaminations from noninjected blastocysts, which were exposed to the microinjection DNA during the stay in injection chambers, and reduced the amount of transgene-positive embryos among pronucleus-injected blastocysts as compared with unmodified PCR. Analysis of 486 microinjected bovine embryo biopsies in 13 separate experiments revealed that we were able to sex 398 (82%) of the biopsies and 77 (19%) of the biopsies were scored as transgene positive and 57 (14%) as transgene questionable. Upon reanalysis of 41 of the biopsied embryos, 38 (93%) of the embryos were observed to be transgene negative and 2 questionable in both assays and uneven distribution of transgene copies was observed in one embryo. The results from sexing were in accordance with biopsies and remaining embryos in 38 (93%) of the embryos. © 1996 Wiley-Liss, Inc.     

Histological comparison between wheat embryos developing in vitro from isolated zygotes and those developing in vivo

There is currently great interest shown in understanding the process of embryogenesis and, due to the relative inaccessibility of these structures in planta, extended studies are carried out in various in vitro systems. The culture of isolated zygotes in particular provides an excellent platform to study the process of in planta embryogenesis. However, very few comparisons have been made between zygotic embryos grown entirely in cultures and those grown in vivo. The present study analyses the differences and similarities between the in vitro and in vivo development of wheat zygotic embryos at the level of morphology and histology. The study was possible thanks to an efficient culture system and an appropriate method of preparing isolated wheat zygotes for microscopy. The in vitro embryos were fixed, embedded and sectioned in the two-celled, globular, club-shaped and fully differentiated stages. Embryos developing in vitro closely followed the morphology of their in planta counterparts and their cell types and tissues were also similar, demonstrating the applicability of the present culture system for studying the process of zygotic embryogenesis. However, some important differences were also detected in the case of in vitro development: the disturbance of or lack of initial polarity led to changes in the division symmetry of the zygotes and subsequently to the formation of uniform cells in the globular structures. Presumably, differences between the in vitro and in planta environments resulted in a lower level of differentiation and maturation in in vitro embryos and in abundant starch and protein accumulation in the scutellum.     

Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality

The objectives of this study were to examine the effect of culture system on bovine blastocyst formation rates and quality. Presumptive IVM/IVF bovine zygotes were cultured either in vitro in synthetic oviduct fluid (SOF, 25 embryos/25 microL in 5% CO2, 5% O2, 90% N2 at 39 degrees C) or in vivo in the ewe oviduct (approximately 100 embryos per oviduct). The recovery rate after in vivo culture was 53% (813/1,530). The blastocyst rate on Day 7 was significantly higher for the in vitro system (28%, 362/1,278 vs 17%, 37/813; P< 0.0001). However, after culture in vitro for a further 24 h, there was no difference in Day 8 yields (36%, 457/1,278 vs 32%, 258/813, for in vitro and in vivo culture, respectively). There was no difference in blastocyst cell number between treatments (Day 7: 96 vs 103; Day 8: 78 vs 85 for in vitro and in vivo culture, respectively). Irrespective of culture system, Day 7 blastocysts had a significantly higher cell number than those appearing on Day 8. There was no difference in pregnancy rate at Day 35 after fresh transfer of a single Day 7 blastocyst (37.5%, 21/56 vs 45.3%/, 24/53 for in vitro and in vivo culture, respectively). After cryopreservation by freezing in 10% glycerol, VS3a vitrification or solid surface vitrification, the survival of in vitro cultured embryos was significantly lower than survival of embryos cultured in the ewe oviduct or those produced by superovulation of donors. In conclusion, these findings demonstrate that while bovine zygotes cultured in vitro are capable of rates of development similar to those of their in vivo cultured counterparts (in terms of Day 8 blastocyst yield, cell number and early pregnancy rate), there are significant differences in embryo cryosurvival. This suggests that current in vitro culture systems need to be improved to optimize embryo quality and pregnancy rates.     

Fate of genetically marked mitochondrial DNA from spermatocytes microinjected into mouse zygotes.

Cytoplasts from single spermatocytes of NZB/BinJ mice were separated from the nuclei and individually microinjected into B6D2F1 (C57BL/6 x DNBA/2J) hybrid embryos at the pronuclear stage (20 h after hCG injection). Of 363 zygotes injected, 311 (86%) survived and developed. From these experiments, we transferred 222 embryos into 20 pseudopregnant recipients. Eighteen (90%) became pregnant and 82 pups were born (37% of transfers). Mitochondrial DNA (mt DNA) from the NZB/BinJ strain lacks a RsaI restriction site and can thus be distinguished from the host embryo following PCR amplification. We were unable to detect the transferred mtDNA in blastocysts on day 4-5 after injection. Nor could we detect NZB/BinJ mtDNA in placentae, nor in tissues from mice born to host mothers following the transfer of blastocysts that developed from injected zygotes. Rejection of paternal mitochondria by the embryo normally occurs at the 4- to 8-cell stage in mice and is apparently dependent on mutual recognition between the mitochondria and the nuclear genome. We conclude that this mechanism has probably already developed by the time the germ cells have become committed to meiosis.     

In vitro development to blastocysts of early porcine embryos produced in vivo or in vitro

The objective of this study was to compare the development of porcine embryos from the 2- and 4-cell stages to the blastocyst stage after in vivo or in vitro fertilization and in vivo or in vitro culture. Early-stage embryos were collected either from superovulated gilts 36 h after the second mating or after in vitro fertilization (IVF) of in vivo-matured oocytes, both followed by in vitro culture to the blastocyst stage. Blastocysts collected from superovulated donors served as controls. In the first experiment, a total of 821 2- and 4-cell embryos derived from in vivo-fertilized oocytes was cultured either in medium NCSU 23, modified Whittens' medium or modified KRB for 5 d. Significantly (P < 0.05 and P < 0.001) more embryos overcame the 4-cell block and developed to the blastocyst stage in medium NCSU 23 than in the 2 other culture media. Hatching was only observed in medium NCSU 23. In the second experiment, embryos derived from in vivo-matured oocytes fertilized in vitro were cultured in medium NCSU 23. Of 1869 mature oocytes 781 (41.8%) cleaved within 48 h after in vitro fertilization. A total of 715 embryos was cultured to the morula and blastocyst stages, and 410 (57.3%) overcame the developmental block stage, with 358 embryos (50.1%) developing to the morula and blastocyst stages. None of the embryos hatched, and the number of nuclei was significantly (P < 0.05) lower compared with that of in vivo-fertilized embryos (18.9 +/- 9.8 vs 31.2 +/- 5.8). In the third experiment, 156 blastocysts derived from in vitro fertilization and 276 blastocysts derived from in vivo fertilization and in vitro culture were transferred into synchronized recipients, while 164 blastocysts were transferred immediately after collection into 6 recipients, resulting in a pregnancy rate of 83.3%, with 35 piglets (on average 7.0) born. From the in vitro-cultured embryos, 58.3% (7/12) of the recipients remained pregnant at Day 35 after transfer, but only 33.3% maintained pregnancy to term, and 14 piglets (on average 3.5) were born. In contrast, the transfer of embryos derived from in vitro-fertilized oocytes did not result in pregnancies. It is concluded that 1) NCSU 23 is superior to modified Whittens' medium and modified KRB and 2) blastocysts derived from in vitro fertilization have reduced viability as indicated by the lower number of nuclei and failure to induce pregnancy upon transfer into recipients.     

Environmental variables influencing in vitro development of hamster 2-cell embryos to the blastocyst stage

This study is a systematic analysis of environmental variables influencing development of 2-cell hamster embryos to the blastocyst stage in vitro. Experiments were done using a chemically defined (protein-free) culture medium (HECM-2). Physicochemical variables examined were temperature, the concentrations of CO2, HCO3-, Ca2+, Mg2+, K+, and O2, the presence of a silicone oil overlay, and osmotic pressure. The optimal temperature for development in vitro was 37.5 degrees C; lower temperatures were inhibitory. There was no significant effect on blastocyst development of alterations in the concentrations of NaHCO3, CaCl2, MgCl2, and KCl, or in the ratios of Ca2+:Mg2+ and Na+:K+, over the ranges tested. Development to the blastocyst stage was significantly stimulated by increased CO2 concentrations (7.5% and 10%), by reduced O2 concentrations (10% and 5%), and by the presence of silicone oil overlying the culture medium. Reduction of blastocyst development in the absence of an oil overlay was not caused by increased osmotic pressure. Cleavage stage embryos were not affected by osmolalities ranging from 250 to 350 mOsmols, but blastocyst development was inhibited at greater than or equal to 325 mOsmols. Under optimized conditions (37.5 degrees C, 10% CO2, 25 mM HCO3-, 2.0 mM Ca2+, 0.5 mM Mg2+, 3.0 mM K+, 10% O2, 250-300 mOsmols, with silicone oil overlay), 51-57% of 2-cell hamster embryos developed to the blastocyst stage. This represents a significant improvement over previous "standard" culture conditions, which supported development of 26% blastocysts from 2-cell hamster embryos. The culture system described here provides an adequate baseline response to support detailed investigations into the regulation of embryo development in the hamster.     

Fluorescence microscopy of nuclear DNA in oocytes and zygotes during in vitro fertilization and development of early embryos in mice

Fluorescence microscopic investigations of nuclear DNA stained with mithramycin or 4',6-diamidino-2-phenylindole (DAPI) were undertaken in oocytes and zygotes during in vitro fertilization (IVF) and subsequent early embryo development in mice. Mithramycin and DAPI bind stoichiometrically with G-C and A-T pairs of DNA, respectively. In the present study, it was possible to visualize the germinal vesicle, the nucleolus, and the chromosome in the oocyte. After IVF, the nuclei, male and female pronuclei, polar bodies, nucleolus-associated chromatins, and chromosomes were successively observed in the zygote, the blastomere during cleavage, and the blastocyst until the hatching stage. The formation of male and female pronuclei, the process of their fusion, the appearance of the chromosome, and the commencement of the first cleavage of the zygote to form two blastomeres were all clearly demonstrated. The number of blastomeres was easily counted in the morula in spite of the compaction of the blastomeres. The present study indicates that the methods reported herein are useful for the determination of exact functional stages of the oocyte and the zygote during IVF and early embryo development, and thus contributes to the advancement of reproductive and developmental biomedical research.     

Progesterone enhances in vitro development of bovine embryos

Increased pregnancy rates in cattle given progesterone (P₄) prior to 5 d after breeding have recently been reported. The objective was to determine if this increase in pregnancy rate could be attributed to a direct positive effect of P₄ on the developing embryo. In Experiment 1, 280 bovine oocytes were inseminated in vitro and at Day 3 (insemination = Day 0), good quality 8 cell embryos (n = 206) were randomly allocated to be cultured in either CR1aa+serum with 0 or ∼15 ng/mL (n = 102 and n = 104, respectively). In Experiment 2, 881 bovine oocytes were used; on Day 3, good quality 8 cell embryos (n = 511) were randomly allocated to either the control (CR1aa+FCS, n = 168), vehicle (CR1aa + FCS + ethanol, n = 170), or P₄ treatment (CR1aa + FCS + ∼15 ng/mL P₄ in ethanol, n = 173). On Day 7, in both experiments, there were increased numbers of blastocysts developing in the P₄ group (Experiment 1, 59% and Experiment 2, 71%) compared to the vehicle (Experiment 2, 53%) or control (40 and 62% in Experiments 1 and 2, respectively). The addition of P₄ (8%) stimulated the rate of embryo development (early blastocysts or more advanced stages on Day 6) compared to vehicle (3%) and control (0%) and the P₄ group had more hatched or hatching blastocysts (33%) on Day 9 compared to the control or vehicle group (21 or 22%). Additionally, the P₄ group had greater embryo diameter and significantly more Grade 1 blastocysts on Day 7. In conclusion, P₄ had a direct, positive effect on developing bovine embryos cultured in vitro.     

Persistence of human mitochondrial DNA throughout the development to the blastocyst of mouse zygotes microinjected with human mitochondria

The conditions for transfer of human mitochondria into fertilised mouse ova were elaborated. Species-specific primers were designed to discriminate human mitochondrial DNA (mtDNA) and the endogenous mtDNA in the preimplantation embryos. Human mitochondria isolated from the HepG2 cell line were microinjected into murine zygotes, and the latter cultured for 96 h to the blastocyst stage. The polymerase chain reaction allowed the detection of human mtDNA at every stage of embryo cleavage. In some cases a clear disparity in distribution of human mtDNA among blastomeres was evident.     

In vitro development and post-thaw survival of blastocysts derived from delipidated zygotes from domestic cats

The ability to cryopreserve in vitro-produced feline embryos was investigated. To improve the survival rate of cryopreserved embryos, first the developmental ability of in vitro fertilized feline zygotes (after removal of intracellular lipids) was determined, followed by the post-thaw survival of cryopreserved blastocysts derived from delipidated zygotes. More than 67% of the delipidated zygotes cleaved and 36% of them developed to the morula stage. The developmental ability of delipidated zygotes to the blastocyst stage (26%) was similar to that of sham-operated (30.5%) or control embryos (31.3%). Although the survival rate of delipidated blastocysts (81.8%) after freezing and thawing tended to be higher than that of control embryos without delipidation (60.6%), rates were not significantly different between the both groups. In conclusion, in vitro-produced feline blastocysts were successfully frozen, removal of the cytoplasmic lipid content in feline zygotes did not impair their in vitro developmental competence (up to the blastocyst stage), and reduction of cytoplasmic lipids by aspiration had no apparent effects on the survival of in vitro-derived blastocysts after cryopreservation.     

In vitro development of bovine embryos as affected by different lots of bovine serum albumin and citrate

The effect of bovine serum albumin (BSA) lots on the development of in vitro-derived bovine embryos in synthetic oviductal fluid was investigated. Citrate concentration was determined for each lot of BSA, and then correlated with differences noted in the ability of BSA lots to support embryo development. Development of bovine embryos to the blastocyst stage was also compared after culture in chemically-defined medium with varying levels of citrate. There were distinct differences in the ability of the different BSA lots to support embryo development to the blastocyst stage (P相似文献   

In vitro maturation and fertilization of bovine oocytes and in vitro culture of presumptive zygotes in the presence of bovine pestivirus

A pathogen which has been shown to commonly contaminate in vitro bovine embryo production system is bovine pestivirus (bovine viral diarrhea virus). Three experiments were designed to evaluate the in vitro maturation (experiment I), fertilization (experiment II) and embryo development (experiment III) of immature oocytes, inseminated oocytes and presumptive zygotes in the presence of a bovine pestivirus (non-cytopathic, nCP type 1). The virus inoculum used was derived from a persistently infected cow. In experiment I, follicular oocytes (n=1257) recovered from slaughterhouse derived ovaries were randomly assigned to either a control group (n=578) which did not become exposed to bovine pestivirus and a treatment group (n=679) which was inoculated with bovine pestivirus (2.20-3.69 log(10) TCID(50)/50 microl) at the time of commencement of in vitro maturation. Overall, there was no significant difference between the control and pestivirus inoculated oocytes in either the cumulus cell expansion rate (79+/-7.5% versus 74+/-10.7%) or the nuclear maturation rate (89+/-4.8% versus 85+/-7.4%), respectively. In experiment II, in vitro matured oocytes (n=607) were inseminated either in the absence (control; n=301) or the presence of bovine pestivirus (4-4.6 log(10) TCID(50)/50 microl; n=306). A significant (P<0.01) reduction in the overall number of fertilized oocytes with two well formed male and female pronuclei was observed in the treatment group compared to the control group (58.5+/-5.8% versus 73.3+/-3.6%, respectively). In experiment III, after in vitro maturation and fertilization, presumptive zygotes were randomly assigned to either a control group (n=139) which was not exposed to bovine pestivirus or a treatment group which was inoculated with bovine pestivirus (2.97-4.47 log(10) TCID(50)/30 microl; n=139). The zygotes were then cultured under mineral oil in an atmosphere of 88% N(2), 7% O(2) and 5% CO(2) at 39 degrees C. The morphologic appearance of the embryos was assessed 48 h after the commencement of culture, and then every 48 h up to days 7-8 after insemination. The 22% (31/139) and 3.6% (5/139) of the presumptive zygotes developed to the morula or blastocyst stage in the control and the bovine pestivirus inoculated groups, respectively (P<0.001). This study demonstrates that bovine pestivirus has a significant detrimental effect on in vitro fertilization and early in vitro embryo development.