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PubMed1994

Pregnancies following transfer of equine embryos cryopreserved by vitrification.

The objective of this study was to investigate the in vitro and in vivo developmental abilities of equine embryos cryopreserved by vitrification. Twenty-eight embryos were recovered from Native pony and Thoroughbred mares at Days 5 to 7 by nonsurgical uterine flushing (detection of ovulation=Day 0). The vitrification solution contained 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in PBS. The embryos were placed for 1 to 2 min in vitrification solution (Group 1) or following exposure to 20% ethylene glycol in PBS for 10 to 20 min (Groups 2 and 3). Single embryos were loaded in 0.25-ml straws, cooled for 1 min in liquid nitrogen vapor and immersed in liquid nitrogen. Straws were warmed in water (20 degrees C, 20 sec), and the contents were expelled with 0.5 M sucrose in PBS. Then the sucrose was diluted in 1-step (Groups 1 and 2) or 4-steps (Group 3). Embryos (n=21) were cultured for 120 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO2 in air and evaluated morphologically. Development to the hatching or hatched blastocyst stage was obtained in 0 7 , 4 7 and 4 7 embryos in Groups 1, 2 and 3, respectively. An additional 7 embryos were vitrified-warmed according to the treatment of Group 2 (4 embryos) and Group 3 (3 embryos). Five embryos were selected after in vitro culture for 4 h and were transferred nonsurgically into the uterine horn of Day-4 recipient mares. Transfer of 2 embryos (both Day-6 blastocysts: Group-2 treatment) resulted in pregnancies with a viable fetus at Day-60 of the gestation period.

Journal Article↗
PubMed1995

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.

Journal Article↗
PubMed1995

Interspecific transfer of IVM IVF-derived red sheep (Ovis orientalis gmelini ) embryos to domestic sheep (Ovis aries ).

Two embryo production methodologies were investigated to generate Red sheep embryos for use in an interspecific embryo transfer program. In Experiment 1, 4 multiparous female Red sheep (Ovis orientalis gmelini ) were implanted with CIDR type G devices for 11 d. Forty-eight hours prior to CIDR removal, a total of 22.5 mg bid of FSH-P was administered over a 3-d period. Laparoscopic embryo collection was performed 5 d post breeding, and embryos were transferred to domestic recipient ewes (Ovis aries and Ovis orientalis musimon ). In Experiment 2, 7 nulliparous female Red sheep were implanted with CIDR devices and injected with 200 IU of PMSG and 25 mg of FSH-P on the 8th day of implant insertion. At 60 to 70 h post PMSG/FSH-P treatment, follicular oocytes were aspirated laparoscopically. The recovered oocytes were matured in M199 (with fetal calf serum, FSH, LH, penicillin and streptomycin) at 39 degrees C in a humidified atmosphere containing 5% CO(2). At 24 h oocytes were fertilized with frozen-thawed semen at a concentration of 1.6 x 10(6) sperm/ml. The ova/embryos were placed in CR2 or BOEC culture medium at 20-22 h post IVF. Following 3 to 4 d in culture, embryos were transferred laparoscopically to the uterine horn of synchronized recipients. In Experiment 1, 4 embryos and 6 UFO were collected from 2 embryo donors, respectively. Two embryos were transferred with the aid of a laparoscope to each of 2 Rambouillet recipients, one of which gave birth to a healthy Red sheep lamb at 158 d of gestation. In Experiment2, a total of 62 oocytes was collected from 7 oocyte donors; 16 developed to the 16- to 32-cell stage and were transferred to 8 recipients. Three of these IVM-IVF embryos were transferred laparoscopically to 2 Mouflon recipients, resulting in no pregnancies. Thirteen IVM-IVF embryos were transferred to 6 Rambouillet recipients. Each of these gave birth to a single healthy Red sheep lamb. Gestation lengths of the 3 IVM-IVF lambs ranged from 152 to 162 d. This research demonstrates that when using compatible species IVM-IVF technology in conjunction with interspecific ET can lead to the production of live offspring and can be used to propagate exotic ovine species.

Journal Article↗
PubMed1995

Media alternatives for the collection, culture and freezing of mouse and cattle embryos.

The replacement of biological products in media for the collection, culture and freezing of mammalian embryos was studied. To test the hypothesis that chemically defined surfactants can replace bovine serum albumin (BSA) or serum in embryo media, morula-stage mouse and cattle embryos were collected, cultured, and/or frozen in the surfactant compound, VF5. Collection efficiency of mouse and cattle embryos did not differ whether the medium contained serum or surfactant. In addition, morula-stage mouse and cattle embryos developed and hatched at similar rates in culture media containing either BSA or surfactant. Although the freeze/thaw survival and development in culture of bovine embryos was not significantly different in any of the media, there was a significantly lower hatching rate of mouse embryos frozen with serum or surfactant than with cryoprotectant alone or with cryoprotectant plus albumin-free serum. However, the absence of serum or surfactant in embryo freezing media resulted in embryo loss, presumably due to stickiness. The data suggest that serum can be replaced by a chemically defined surfactant in mouse and cattle embryo transfer systems for the collection, culturing and freezing of embryos. It is likely that the beneficial effects of serum are due to its surfactant properties.

Journal Article↗
PubMed1995

Sex chromosome complement and developmental diversity in pre-and post-hatching porcine embryos.

To examine sex and development relationships in porcine embryos in early gestation, 10 gilts were killed on Day 4, 5, or 6 post mating (first day of standing estrus = Day 0). Embryos recovered immediately after slaughter were cultured in Medium 199 with colcemid (0.05mug/ml), fixed on slides, and stained with 4% Giemsa. The number of cells in each specimen was counted from the slides, and, whenever cell dispersion allowed, sex was determined by presence or absence of the Y-chromosome in at least 2 spreads from each embryo. Three gilts slaughtered on Day 4 yielded 2- and 4-cell stage embryos (n = 38), but no data on sex could be obtained due to lack of mitosis or readable metaphase spreads. Three Day 5 litters had individual specimens ranging from 8 to 14 cells (n = 8), 32 to 64 cells (n = 10), and 13 to 31 cells (n = 11), with the sex determined in 15 of these. Cell numbers ranged from 18 to 165 (n = 14), 16 to 32 (n = 9), 36 to 82 (n = 12), and 16 to 30 (n = 9) in the 4 gilts slaughtered on Day 6, with the sex determined in 26 of these. Embryos within each litter were divided into low, medium and high cell numbers by 3 equal divisions of the range of cell numbers. Three Day-5 embryos and 1 Day-6 embryo were lost during preparation; neither the cell numbers nor the sex could be determined in 4 Day-5 and in 3 Day-6 embryos. The overall sex ratio approximated 1:1, but on Day 5, the ratios for males to females were 0:5, 1:3 and 6:0 for the low, medium and high cell number groups, respectively. Embryos of undetermined sex in these same groups numbered 3, 1 and 3, respectively. On Day 6 the distribution was 1:11, 4:2 and 8:0 in favor of the males, while embryos of undetermined sex in the low, medium and high cell number groups numbered 5, 7 and 2, respectively. Chi-square analysis of the combined Day-5 and Day-6 results indicated the presence of significantly more females among embryos with low cell numbers and more males in the high cell number group (P < 0.01).

Journal Article↗
PubMed1996

Reducing the amount of cytoplasm available for early embryonic development decreases the quality but not quantity of embryos produced by in vitro fertilization and nuclear transplantation.

The effect of reducing the amount of cytoplasm available for early embryonic development was investigated in embryos produced by in vitro fertilization (IVF) and nuclear transplantation. In Experiment 1, approximately 1/2 or 1/20 of the cytoplasm was removed from bovine embryos at the pronuclear-stage of development. The percentage of embryos developing to the compact morula or blastocyst stage was significantly higher in non-manipulated controls (26%) than in embryos with 1/20 of the cytoplasm removed (16%), and those with 1/2 of the cytoplasm removed (10%; P < 0.05). There was also a significant difference in the average number of cells between blastocysts in which 1/20 of their cytoplasm was removed (67), those with 1/2 of their cytoplasm removed (55), and nonmanipulated controls (77; P < 0.05). In Experiment 2, nuclear transfer embryos were produced in which approximately 1/2 or 1/20 of the cytoplasm was removed during oocyte enucleation. The percentage of embryos developing to the blastocyst stage was 17% for both groups of nuclear transfer embryos compared to 44% for control embryos (P < 0.05). The mean number of cells in blastocysts produced by nuclear transfer in which 1/20 of the cytoplasm was removed during oocyte enucleation (61) was no different than that in control embryos (66), but significantly higher than the mean number of cells in blastocysts produced by nuclear transfer in which 1/2 of the cytoplasm was removed (42; P < 0.05). There was no indication that altering the amount of cytoplasm available for early embryonic development of IVF embryos affected the timing of differentiation events, including those of embryo compaction and blastocyst formation.

Journal Article↗
PubMed2006

Cryopreservation of somatic embryos of the herbaceous peony (Paeonia lactiflora Pall.) by air drying.

This study was carried out to establish a suitable method for the cryopreservation of somatic embryos of the herbaceous peony. The somatic embryos were obtained from cotyledon and anther cultures on a MS medium supplemented with abscisic acid (ABA) and phenylacetic acid (PAA), respectively. The frequency of somatic embryo formation was the greatest (61%) from the cotyledons cultured on a MS medium supplemented with 1.0 mg l(-1) of ABA. Embryos were also obtained directly from anthers cultured on a MS medium with or without 2.0 mg l(-1) of PAA. For the cryopreservation of peony somatic embryos, the embryos were dried under a stream of sterile air and frozen by immersion in liquid nitrogen. Thawed embryos were germinated into plantlets after placing on a medium containing 0.3 mg l(-1) of gibberellic acid (GA(3)). The frequency of the post-thaw regrowth of cryopreserved somatic embryos was related to their size and desiccation time, the latter ranging from 0 to 2 h. When the somatic embryos were desiccated for 1 h, the frequency of post-thaw regrowth was greater than 66%. The frequency of post-thaw regrowth of the cryopreserved somatic embryos from anthers and cotyledon tissues was generally high when they were 2-3 mm in size. Desiccation may be a suitable method for the cryopreservation of somatic embryos of the herbaceous peony.

Abscisic Acid↗
PubMed2004

The effect of oxygen tension on porcine embryonic development is dependent on embryo type.

Reducing oxygen concentration from atmospheric levels during in vitro culture generally, but not invariably, improves embryonic development across a range of species. Since the few published reports of such an action in the pig are contradictory--perhaps a consequence of the derivation of the embryos prior to culture--a study was performed to examine the effect of O2 tension during culture on three different types of porcine embryos, namely: in vivo flushed embryos, and in vitro matured oocytes either fertilized in vitro or parthenogenetically activated. In vivo embryos (n=208) were flushed at the 2-8 cell stage. Cumulus oocyte complexes (COCs) destined for IVF or parthenogenetic activation were derived from 2 to 6 mm, post-pubertal ovarian follicles and matured for 48 h in TCM-199. Parthenogenones were generated by activating denuded oocytes (n=573) with 10 mM calcium ionophore, followed by 2 mM DMAP prior to culture. The IVF embryos (n=971) were produced by fertilizing COCs (day 0) with fresh ejaculated semen in modified tris-based medium for 6 h before cumulus removal. All embryos were cultured in BECM-3 containing 12 mg/mL fatty-acid-free BSA up to day 4, followed by BECM-3 supplemented with 10% calf serum until day 7. The gas environment for IVM/IVF was 5% CO2 in air, while that for IVC was either 5% CO2 in air or 5% O2, 5% CO2 and 90% N2. Low O2 tension increased both day 7 blastocyst rates (high versus low O2, respectively; 9.3+/-2.9%: 26/280; 23.9+/-4.2%: 71/293; P<0.001) and total cell numbers (39.3+/-2.9, n=24 versus 61.2+/-7.7, n=61; P=0.01) of parthenogenetically activated embryos. In contrast, such a treatment neither affected blastocyst rates (89.3+/-6.9 versus 87.8+/-7.5) nor cell numbers (87.4+/-4.5 versus 87.7+/-4.8) of in vivo flushed embryos. The effect of reduced O2 concentration on IVF embryos was intermediate, since only cell numbers were improved (69.8+/-3.5, range=17-204, n=49; 88.5+/-5.8, range=28-216; n=66; P<0.01), equivalent to that recorded in in vivo flushed embryos. However, blastocyst rates were unaffected (10.7+/-1.4%: 51/486; 12.9+/-2.2%: 67/485). The effect, when present, of reducing O2 concentration from 20 to 5% was beneficial for pig in vitro embryonic development. The responses are apparently dependent on firstly, the manner by which the embryonic cell cycle is activated and secondly, the derivation of the tissue prior to placement into culture, if the observed resilience of in vivo embryos is independent of treatment duration.

Animals↗
PubMed2005

Anomalous mRNA levels of chromatin remodeling genes in swamp buffalo (Bubalus bubalis) cloned embryos.

The swamp buffalo (Bubalus bubalis) is a multi-purpose animal in agriculture that is challenged by extinction due to low reproductive efficiency. Nuclear transfer (NT) has been used to preserve special breeds of buffalo, as well as to increase the number of animals. However, cloned buffalo embryos have impaired development, as in other species. To understand the chromatin remodeling activities in cloned embryos and to improve NT technology, we examined the expression profiles of five genes involved in DNA and histone modifications, DNMT1, DNMT3A, DNMT3B, HAT1 and HDAC1, in single swamp buffalo metaphase II oocytes, NT and in vitro fertilized (IVF) embryos from the two-cell to the blastocyst stage, by quantitative real time RT-PCR. We observed similar expression dynamics for all genes studied in the NT and IVF embryos: relatively constant levels of expression for all genes were found from the MII oocyte up to the eight-cell stage; the levels of mRNA for HAT1 and DNMT3B continued to be stably expressed up to the blastocyst stage; while dramatic increases were seen for DNMT3A and HDAC1. Alternatively, the levels of DNMT1 started to decrease at the eight-cell stage. Despite the similarity in the dynamics of gene expression, dramatic differences in the relative levels of these genes between NT and IVF embryos were observed. The expression levels of all DNA modifying genes were higher in the NT embryos than in the IVF embryos at the eight-cell and blastocyst stages. The genes HDAC1 and HAT1 were also expressed significantly higher at the blastocyst stage in the NT embryos. Our results suggested differences in chromatin remodeling between NT and IVF embryos and that lower levels of DNA passive demethylation and higher levels of DNA de novo methylation occurred in the NT embryos. These observations are novel in the species of buffalo, and may be associated with developmental failure of cloned buffalo embryos due to the transcriptional repression effect of most genes studied here.

Acetyltransferases↗
PubMed1994

Embryo survival after pronuclear microinjection and trophectoderm biopsy.

OBJECTIVE: Our purpose was to compare murine embryo development after pronuclear microinjection of a gene construct, followed by trophectoderm biopsy at the blastocyst state, with development after a single micromanipulation, and with cultured controls. STUDY DESIGN: alpha-Myosin heavy-chain gene sequence was microinjected into the murine embryo pronucleus and cultured to blastocyst. After trophectoderm biopsy the embryos were allowed to re-expand. Re-expanded embryos were transferred to pseudopregnant females; implantation and live birth rates were recorded. In this study group the rates were compared with three control groups of embryos simultaneously cultured after (1) pronuclear microinjection only, (2) trophectoderm biopsy only, and (3) non-micromanipulated, culture only. RESULTS: A total of 1222 embryos were divided among the four groups. In the study group 472 embryos underwent pronuclear microinjection and trophectoderm biopsy. Of these, 203 (43%) reached the blastocyst stage and underwent biopsy; 183 (38.8%) re-expanded after biopsy. Of 275 pronuclear microinjected only (control 1) embryos, 113 (41.1%) reached the blastocyst stage. Of 336 embryos 148 (44.0%) reached the blastocyst stage and underwent trophectoderm biopsy only (control 2); 129 (39.2%) survived biopsy. The cultured only group (control 3) consisted of 139 pronuclear embryos; 67 (48.2%) developed to the blastocyst stage. CONCLUSIONS: Murine embryos can survive two micro-manipulations, pronuclear microinjection followed by trophectoderm micro-biopsy. Although there is minimal effect of these procedures on embryonic development in vitro, the live birth rate is tenuous.

Animals↗
PubMed1993

Sera from women with unexplained infertility inhibit both mouse and human embryo growth in vitro.

OBJECTIVES: To compare the effect of sera from women with reproductive disorders on the in vitro growth of mouse and human embryos up to the blastocyst stage and to determine the influence on human pregnancy outcome. DESIGN: The growth of mouse embryos and in vitro fertilized human embryos up to the blastocyst stage was compared in sera from women with unexplained infertility, and these results were correlated with pregnancy outcome. Also the growth of mouse embryos in sera from women with a history of recurrent abortions was correlated with their pregnancy outcome. PATIENTS, SETTING: Women with unexplained infertility were attending the IVF Unit at the John Radcliffe Hospital, Oxford, United Kingdom. Women with a history of recurrent abortions were attending the high risk pregnancy unit at the same hospital. MAIN OUTCOME MEASURES: Human embryo growth was monitored by daily morphological assessment and mouse embryo growth by both morphological assessment and tritiated thymidine uptake. RESULTS: In 15 women with unexplained infertility, poor mouse embryo development correlated with poor human embryo quality and impaired blastocyst formation when cultured in the same serum, as well as failure to achieve a pregnancy. In 11 women with a history of recurrent abortion, inhibition of mouse embryo growth correlated with unsuccessful pregnancy outcome. DISCUSSION: Assessment of both morphological development and cell proliferation in mouse embryos may be a useful test to determine the suitability of maternal sera for human IVF embryo culture, predicting pregnancy outcome and for categorizing women with reproductive disorders for future clinical management.

Abortion, Habitual↗
PubMed1999

Expression of leukemia inhibitory factor and its receptor in preimplantation embryos.

OBJECTIVE: To examine the expression of leukemia inhibitory factor (LIF) and its receptor (LIF-R) transcripts in human and murine preimplantation embryos. DESIGN: Prospective study. SETTING: University medical center. PATIENT(S): Human oocytes were obtained from patients undergoing IVF treatment. Two-cell murine embryos were obtained from ICR strain mice. INTERVENTION(S): Second-day intracytoplasmic sperm injection procedures were performed on oocytes that failed to be fertilized by IVF. Embryos were cultured to various stages and collected for study. MAIN OUTCOME MEASURE(S): The transcript levels of LIF and LIF-R in these embryos were examined and semiquantitated using single-cell reverse transcription-polymerase chain reaction methodology. RESULT(S): Leukemia inhibitory factor and LIF-R transcripts were detectable in most human preimplantation embryos (30 of 34 and 31 of 34 embryos showed LIF and LIF-R messenger RNA, respectively). There was a trend toward decreased expression of both transcripts in embryos at the four-cell stage and in embryos in which growth had been arrested for 24-48 hours. The expression of LIF and LIF-R genes in murine embryos was inconsistent. CONCLUSION(S): Preimplantation human embryos express LIF and LIF-R messenger RNA. It is suggested that LIF may be able to affect embryo development through its action at stages before implantation in an autocrine or paracrine manner.

Animals↗
PubMed2002

Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM-IVF oocytes.

The aim of this study was to analyze different culture systems on embryo development of prepubertal goat oocytes. We compare (i) the effect of the age of donor (goat) of oocytes on in vitro maturation, fertilization and subsequent embryo development, (ii) the effect of the origin of oviduct cells from coculture of prepubertal goat embryo development, and (iii) the effect of in vivo culture in rabbit oviducts for 1, 2 and 3 days on the development of prepubertal goat embryos produced in vitro. In Experiment 1, at 24 h post-insemination (hpi), oocytes from adult goats were allocated in TCM199 with oviduct cells from adult goats, and oocytes from prepubertal goats were randomly placed in drops with oviduct epithelial cells from adult (aOEC) or prepubertal (pOEC) goats. Cleavage rate and embryo development were evaluated at 48 hpi and after 7 days coculture, respectively. In Experiment 2, at 24 hpi, prepubertal oocytes were allocated in TCM 199 with pOEC. At 40-42 hpi, a group of embryos remained in the coculture (control group), and the rest were transferred to rabbit oviducts (three rabbits for replicate) for culturing in vivo for 24, 48 and 72 h. After these in vivo cultures, embryos were recovered, evaluated and placed in TCM199 with pOEC until Day 8 post-insemination. The maturation, fertilization and blastocyst rates did not differ significantly between oocytes obtained from adult and prepubertal goats. The percentage of blastocysts obtained from prepubertal goat embryos cocultured with aOEC or pOEC was also similar (12.1% versus 12.2%). The transfer of prepubertal goat embryos to rabbit oviducts for 1, 2 and 3 days did not improve the blastocyst rate compared to the control group (9.7, 10.9, 4.1 and 11.5%, respectively). In conclusion, in our conditions, there were no significant differences in embryo development between oocytes obtained from prepubertal and adult goats, and the embryo development from prepubertal goat oocytes were similar in the different culture systems compared.

Aging↗
PubMed1997

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (</= 2 microm) or micropipette (</= 30 microm). The procedure resulted in expulsion of the blastocyst through the perforation and formation of an incomplete demi-embryo configuration, connected by a very thin cell bridge (figure eight in shape). To separate the parts of the embryo, the cell bridge was cut using a glass microneedle. During the separation only a few cells were damaged. As a result of the procedure 4 20 (20.0%), 48 144 (33.3%) and 3 40 (7.5%) middle, late and expanded blastocysts hatched according to the pattern described. The developed procedure could be considered as a non-invasive alternative to conventional embryo splitting.

Journal Article↗
PubMed1999

Determining the sex of bovine embryos using polymerase chain reaction results: a six-year retrospective study.

Knowing the sex of embryos produced for use in an embryo transfer program can assist the dairy producer in managing his resources more effectively. A reliable procedure for accomplishing this goal is to apply PCR technology to the biopsy of an embryo. A description is provided of how the technique has been applied on a large scale in a commercial setting in western Canada between 1992 and 1997. A total of 4,183 embryos was biopsied over a 6-yr period. The sex was determined with more than 90% of the embryos. The results showed that there was a seasonal variation in the sex ratio, with more females being recorded in the period of least light (October to March), than in April to September. While both sire and embryo quality affected the sex ratio, the differences were too small to be of value in most breeding programs. Pregnancy rates with fresh sexed embryos (58 to 71%) were comparable to those with fresh unsexed embryos. The results following freezing and thawing of sexed embryos were low (37 to 66%) but sufficient to be viable commercially. When the sex assigned by PCR was verified by fetal sexing at 60 d of gestation, the error rate was 7%. This study demonstrates that sexing of embryos can be carried out on a large scale. Demand for quick, reliable determination of sex can be met in a cost effective manner. The pregnancy rates achieved with embryos after biopsy are suitable for use in a commercial setting.

Animal Husbandry↗
PubMed2003

Comparison of hybrid and purebred in vitro-derived cattle embryos during in vitro culture.

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P<0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P<0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.

Animals↗
PubMed1988

In vitro culture of postimplantation hamster embryos.

In vitro culture of intact rat and mouse embryos has been described extensively, but information on the culture of other species is sparse. The present study examined some culture requirements of early somite stage hamster embryos and assessed the embryotoxic effects of sodium salicylate (SS), a direct acting chemical and cyclophosphamide (CP), a proteratogen, on these embryos. Hamster embryos explanted on gestation days (GD) 8 and 9 were cultured in Waymouth's embryo-hepatocyte co-cultivation medium (WEHC), 70% McCoy's 5A medium-30% male rat serum (MMRS) or 100% male rat serum (MRS) for 24 hours under various oxygen concentrations. Embryos cultured GD 8 to 9 in the various media grew and differentiated much as they did in vivo, while embryos cultured GD 9 to 10 grew best in MMRS as compared to embryos at the same stage in vivo. Embryos exposed to SS in MMRS at concentrations of 250, 300, or 400 micrograms/ml showed dose related embryotoxicity which included CNS defects, absence of hind limb bud formation, and lack of axial rotation. Hamster embryos co-cultivated with pregnant hamster hepatocytes and treated with 2.5, 6.25 and 12.5 micrograms/ml of CP, showed dose-dependent toxicity when compared to co-cultivated controls. Hamster embryos develop extensively in culture over a 24 hour period. This system may therefore provide a valuable tool for evaluating the species differences of a variety of potential teratogens and embryotoxins and allow the comparison of these embryotoxic effects between rat, mouse and hamster during similar stages of organogenesis.

Abnormalities, Drug-Induced↗
PubMed2000

Embryo recovery and transfer for the production of transgenic goats from Korean native strain, Capra hircus aegagrus.

During the breeding season in Korea (September 1997 to April 1998), a goat embryo recovery and transfer program using a Korean native strain (Capra hircus aegagrus) was performed for the production of transgenic goats. Donors were synchronized with norgestomet implants and superovulated by a combined treatment with FSH and hCG. The treatment regime induced a consistent and efficient superovulation rate of 90% from donors with an ovulation rate of 12.1+/-0.5. 50.5% of the recovered oocytes/embryos were fertilized and most of them were at the 1-cell stage. After DNA microinjection, a total of 188 embryos were immediately transferred to naturally cycling or hormonally synchronized recipients with two or three embryos per animal. There was a tendency for the pregnancy rate of naturally cycling recipients to be higher (38.9%) than that of hormonally synchronized recipients (25.7%). When the recipients were classified into a two or three embryo-received group, the embryo viability was markedly decreased from 58.3% in two embryo-received group to 35.3% in three embryo-received group, without an increase in the kidding rate. This resulted from a high occurrence of abortions or stillbirths from the multiple-pregnant recipients which had received three embryos. This indicated that the transfer of two embryos per recipient is highly recommendable for an optimal embryo survival in Korean goats. Altogether, 188 embryos were transferred to 71 recipients and two transgenic Korean goats were produced from 25 offspring.

Journal Article↗