Co-culture of ovine eggs with oviductal cells and trophoblastic vesicles

Three experiments were conducted to determine whether coculture of early sheep eggs with oviductal cells would improve the ability of eggs to survive in culture. Eggs recovered from superovulated ewes were cultured in Ham's F-10 medium supplemented with 10% fetal calf serum (F10FCS) at 37.5 degrees C in 95% air:5% CO(2). In Experiment 1, eggs with one to eight cells were either transferred into recipient ewes immediately after collection or were cultured for 24 h in 5 ml Ham's F10 medium supplemented with 10% fetal calf serum (F10FCS), 5 ml F10FCS on a confluent monolayer of oviductal cells; in 25 ml of fresh F10FCS; or in 25 ml of F10FCS removed cultures of oviductal cells, 25 mul of fresh F10FCS or 25 mul of F10FCS removed from cultures of oviductal cells. After 24 h, the cultured eggs were transferred to recipient ewes synchronous with donors and subsequently recovered at necropsy on Day 8 post estrus. Coculture of sheep eggs with oviductal cells improved (P < 0.05) the development of transferred eggs compared to culture in F10FCS alone. In Experiment 2, eggs recovered from superovulated ewes on Days 3 to 6 after estrus had undergone 1.8 cleavages by Day 3 and 4.1 cleavages by Day 6. In Experiment 3, single-cell eggs were cultured for 3 d in 5 ml F10FCS, cocultured with ovine trophoblastic vesicles or cultured on a confluent monolayer of oviductal cells. Coculture of eggs in F10FCS on a monolayer of oviductal cells supported in vitro egg cleavage to a greater degree than did F10FCS alone or F10FCS with trophoblastic vesicles (P < 0.05). In Experiment 4, single-cell eggs were cultured for 3 d then transferred to recipients. Eggs were cultured in 5 ml F10FCS on confluent monolayers of oviductal cells from luteal or estrous ewes or on cells that had been frozen after recovery from a culture of oviductal cells. After culture, the eggs were transferred to oviducts of recipients and recovered 3 d later at necropsy. Coculture of eggs for 72 h with oviductal cell monolayers did not increase the in vitro, or subsequent in vivo, cleavage rate regardless of the type of oviductal cells.     

The effect of glutamine on the development of sheep embryos in vitro

The effects of glutamine (Gln) on the in vitro development of sheep embryos cocultured with sheep oviduct epithelial cells (SOEC) or cultured in medium alone were investigated. The in vitro development was evaluated after culture in synthetic oviduct fluid (SOF) medium to Day 6, and then the viability of some of the morula/blastocyst stage embryos was assessed by transfer into recipient ewes. In Experiment 1, sheep embryos were cultured from Day 2 to Day 6 in SOF containing 0 or 1 mM Gln with or without (SOEC) support in a 2 x 2 factorial design. An interaction was found between the effects of Gln and SOEC (P<0.001). The addition of Gln increased blastocyst formation (6%, 2 36 vs 35%, 11 31 ) and the rate of pregnancy (50%, 4 8 vs 100%, 5 5 ) when the embryos were cultured in medium alone, but had no beneficial effect in the presence of SOEC. In Experiments 2 and 3, sheep embryos were cultured from Day 1 to Day 6 in SOF supplemented with 1 mM Gln, with 1 mM alpha-ketoglutarate or without supplementation (control). In Experiment 2, no other amino acids were added, but in Experiment 3 SOF was supplemented with 19 other amino acids. In Experiment 2, when Gln was the only amino acid, the rate of blastocyst formation was increased by the addition of Gln (24%, 8 35 ), but alpha-ketoglutarate caused no increase in blastocyst formation (3%, 1 34 ) compared to the control group (6%, 2 34 ). In Experiment 3, when 19 other amino acids were added, neither Gln nor alpha-ketoglutarate affected the rate of blastocyst formation or the subsequent development of embryos in recipient ewes. These results showed that Gln, when used as a single amino acid, has a beneficial effect on the development of sheep embryos in serum free culture without somatic cells. The data suggest that Gln is used as a source of amino groups rather than as a source of energy since no beneficial effects were found when its deaminated carbon skeleton (alpha-ketoglutarate) was used or when other amino acids were present.     

Cloned mice derived from somatic cell nuclei

In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum.     

High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer

Successful cryopreservation of mammalian oocytes would provide a steady source of materials for nuclear transfer and in vitro embryo production. Our goal was to develop an effective vitrification protocol to cryopreserve bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. Bovine oocytes matured in vitro were placed in 4% ethylene glycol (EG) in TCM 199 plus 20% fetal bovine serum (FBS) at 39 degrees C for 12-15 min, and then transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose in TCM 199 and 20% FBS). Oocytes were vitrified in microdrops on a precooled (-150 degrees C) metal surface (solid-surface vitrification). The vitrified microdrops were stored in liquid nitrogen and were either immediately thawed or were thawed after storage for 2-3 wk. Surviving oocytes were subjected to 1) parthenogenetic activation, 2) in vitro fertilization, or 3) nuclear transfer with cultured adult fibroblast cells. Treated oocytes were cultured in KSOM containing BSA or FBS for 9 to 10 days. Embryo development rates were recorded daily and morphologically high-quality blastocysts were cryopreserved for nuclear transfer-derived embryos at Day 7 or Day 8 of culture. Immediate survival of vitrified/thawed oocytes varied between 77% and 86%. Cleavage and blastocyst development rates of vitrified oocytes following in vitro fertilization or activation were lower than those of the controls. For nuclear transfer, however, vitrified oocytes supported embryonic development as equally well as fresh oocytes.     

Development of screening systems for evaluation of materials used in mammalian embryo transfer

Embryo transfer units use a wide variety of materials that come in contact with embryos. Studies were conducted to evaluate procedures that could be utilized to determine the toxicity of some commonly used materials in embryo collection, culture and transfer. Forty-five female mice were sacrificed on Day 3 or 4 of gestation (Day 1 = vaginal plug), and the uterus and oviducts were removed and minced. A total of 522 embryos was collected (4-cell to blastocyst stages). Four to 16 cell embryos were cultured in Phosphate Buffered Saline (PBS) plus 20% fetal bovine serum. Morula to blastocyst stage embryos were cultured in Nutrient Mixture F10 (HAM) plus 20% fetal bovine serum gassed with 5% CO(2), 5% O(2) and 90% N(2). In Experiment I, embryos and culture media were placed in a covered embryological watch glass (EWG, control) or sealed in the lumen of a siliconized Foley catheter or a section of 1) latex tubing, 2) tygon tubing or 3) silastic tubing. In Experiment II, embryos were placed in EWG and cultured alone (control) or cocultured with sections of 1) tygon tubing, 2) silastic tubing or 3) latex tubing. In Experiment III, embryos were cultured in covered plastic petri dishes containing 15 ml of media, alone (control) or co-cultured with two new plunger tips from sterile Monoject syringes. All embryos were cultured at 32 to 34 degrees C for 24 h. The Criterion used for development was two or more cellular divisions within the 24-h period. Embryo development in Experiment I was lower (P<0.05) in latex (0%) and tygon (24%) tubing and in the siliconized Foley catheter (2%) than in silastic tubing (51%) and the EWG (46%), which did not differ. Experiment II embryos that were co-cultured with latex tubing (5%) showed very little development as compared with those co-cultured with tygon tubing (76%), silastic tubing (76%) and EWG (93%), the last of which were not significantly different. Embryos co-cultured with Monoject syringe plunger tips had a reduced embryo development rate compared to embryos in the control group (0% vs 52%). Although the embryos did not remain in contact with these seemingly toxic materials for prolonged periods, our results indicate that a significant reduction in embryo viability may occur due to this exposure.     

Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells

This study compared the nuclear transfer (NT) embryo development rates of adult and fetal cells within the same genotype. The adult fibroblast cells were obtained from a 21-yr-old Brahman bull. The fetal cells were derived from a Day 40 NT fetus previously cloned using cells from the Brahman bull. Overall, similar numbers of blastocysts developed from both adult (53 of 190; 28%) and fetal (39 of 140; 28%) donor cells. Improved blastocyst development rates were observed when fetal cells were serum-starved (serum-fed 12% vs. serum-starved 43%; P < 0.01) whereas there was no similar benefit when adult cells were serum-starved (both serum-fed and serum-starved 28%). Day 30 pregnancy rates were similar for blastocysts derived from adult (6 of 26; 23%) or fetal (5 of 32; 16%) cells. Day 90 pregnancy rates were 3 of 26 for adult and 0 of 32 for the fetal cell lines. One viable bull calf derived from a 21-yr-old serum-starved adult skin fibroblast was born in August 1999. In summary, somatic NT embryo development rates were similar whether adult or fetal cells, from the same genotype, were used as donor cells. Serum starvation of these adult donor cells did not improve development rates of NT embryos to blastocyst, but when fetal cells were serum-starved, there was a significant increase in development to blastocyst.     

Effect of transfer of one or two in vitro-produced embryos and post-transfer administration of gonadotropin releasing hormone on pregnancy rates of heat-stressed dairy cattle

Pregnancy rates following transfer of an in vitro-produced (IVP) embryo are often lower than those obtained following transfer of an embryo produced by superovulation. The purpose of the current pair of experiments was to examine two strategies for increasing pregnancy rates in heat stressed, dairy recipients receiving an IVP embryo. One method was to transfer two embryos into the uterine horn ipsilateral to the CL, whereas the other method involved injection of GnRH at Day 11 after the anticipated day of ovulation. In Experiment 1, 32 virgin crossbred heifers and 26 lactating crossbred cows were prepared for timed embryo transfer by being subjected to a timed ovulation protocol. Those having a palpable CL were randomly selected to receive one (n = 31 recipients) or two (n = 27 recipients) embryos on Day 7 after anticipated ovulation. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Heifers that received one embryo tended to have a higher pregnancy rate than those that received two embryos (41% versus 20%, respectively) while there was no difference in pregnancy rate for cows that received one or two embryos (57% versus 50%, respectively). Pregnancy loss between Day 64 and 127 only occurred for cows that received two embryos (pregnancy rate at Day 127=17%). Between Day 127 and term, one animal (a cow with a single embryo) lost its pregnancy. There was no difference in pregnancy rates at Day 127 or calving rates between cows and heifers, but females that received two embryos had lower Day-127 pregnancy rates and calving rates than females that received one embryo (P < 0.03). Of the females receiving two embryos that calved, 2 of 5 gave birth to twins. For Experiment 2, 87 multiparous, late lactation, nonpregnant Holstein cows were synchronized for timed embryo transfer as in Experiment 1. Cows received a single embryo in the uterine horn ipsilateral to the ovary containing the CL and received either 100 microg GnRH or vehicle at Day 11 after anticipated ovulation (i.e. 4 days after embryo transfer). There was no difference in pregnancy rate for cows that received the GnRH or vehicle treatment (18% versus 17%, respectively). In conclusion, neither unilateral transfer of two embryos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat stress.     

Developmental potential of early bovine zygotes submitted to centrifugation and microinjection following in vitro maturation of oocytes

This study was designed to investigate the potential use of in vitro matured, in vitro fertilized bovine zygotes for producing transgenic calves by microinjection of foreign DNA. In Experiment 1, the effect of centrifugation (4 min, 15,000 x g, 20 degrees C) on in vitro derived bovine zygotes was evaluated. In vitro development from 2 to 8 cells was not affected (80 vs 78%) when control zygotes (n = 211) were compared with zygotes treated (n = 210) 18 h post insemination. In Experiment 2, the influence of the centrifugation alone on the developmental potential of embryos was evaluated in rabbit oviducts for 120 h. The percentage of control and treated zygotes that developed to 1, 2 to 8, 8 to 32 and more than 32 cells were 7, 54, 10 and 10% vs 7, 40, 11 and 10%, respectively. In Experiment 3, the effect of pronuclear injection with plasmid containing CRF (corticotropin releasing factor) gene or pOCAT 330 Delta1 plasmids; 2 mug/ml in Tris 10 mM, EDTA 0.2 mM, 18 to 20 h post insemination was evaluated by in vivo development in the rabbit oviduct. The embryos submitted only to centrifugation and vortexing resulted in a morula-blastocyst (> 32 cells) rate of 25% (n = 226) compared with the injected zygotes of which only 5% (n = 206) achieved the same stage. We conclude that in vitro produced bovine zygotes have a reduced developmental potential following microinjection, and this effect is not due to the centrifugation process.     

Development of sheep preimplantation embryos in media supplemented with glucose and acetate

Two experiments were conducted to examine the effect of supplemental glucose (G; 1.5 mM) and/or acetate (A; 0.5 mM) on the development of early sheep embryos to blastocysts when cultured in vitro in glucose-free synthetic oviductal fluid (SOF) + sheep serum or bovine serum albumin (BSA). In Experiment 1, 2- to 4-cell, 8- to 16-cell and >16-cell embryos were cultured in SOF, SOF+G, SOF+A or SOF+G+A. All media were supplemented with 10% sheep serum. In addition, embryos were cultured in either microdrops under polysiloxane oil or in multiwell dishes. Overall, development to the blastocyst stage was 3%, 30% and 68% for 2- to 4-cell, 8- to 16-cell and >16-cell stages, respectively, suggesting that an 8-cell developmental block existed under our culture conditions. Glucose supplementation had little effect on embryo development, and no overall effect was observed from the addition of acetate. In Experiment 2, 8- to 16-cell embryos were cultured in SOF or SOF+G, both supplemented with BSA. Development to the blastocyst stage was 25% and 18%, respectively. The results show that the presence of glucose or acetate did little to enhance embryonic development in our incubation systems. Further work is required to evaluate fully the energy requirements for development of the early sheep embryo.     

Development of in vitro matured,in vitro fertilized domestic cat embryos following cryopreservation,culture and transfer

The ability of embryos to successfully survive cryopreservation is dependent on both morphological and developmental characteristics. Domestic cat oocytes matured in vitro exhibit alterations in nuclear and cytoplasmic maturation that may affect developmental competence, particularly after cryopreservation. In Experiment 1, we evaluated the developmental competence of in vitro produced (IVM/IVF) cat embryos after cryopreservation on Days 2, 4 or 5 of IVC. In Experiment 2, in vivo viability was examined by transfer of cryopreserved embryos into recipient queens. Oocytes recovered from minced ovaries were cultured in TCM-199 with hCG/eCG and EGF at 38 degrees C in 5% O(2), 5% CO(2), 90% N(2) for 24h. In Experiment 1, after IVM/IVF, on Day 2 (n=56), Day 4 (n=48) and Day 5 (n=42) of IVC, embryos were equilibrated for 10 min at 22 degrees C in HEPES (15m M) Tyrode's (HeTy) with 1.4M propylene glycol (PG), 0.125 M sucrose (S), 10% dextran and 10% FBS, loaded into 0.25 ml straws, cooled at 2.0 degrees C/min to -6.0 degrees C and held for 10 min. After seeding, cooling resumed at 0.3 degrees C/min to -30 degrees C and after a 10 min hold, straws were plunged into liquid nitrogen (LN(2)). Straws were thawed in air for 2 min and cryoprotectant was removed by a five-step rinse consisting of 3 min each in HeTY with 0.95 M PG/0.25 M S; 0.95 M PG/0.125 M S; 0.45 M PG/0.125 M S; 0 PG/0.125 M S; 0 PG/0.0625 M S. Contemporary IVM/IVF embryos were used as nonfrozen controls (Day 2, n=14; Day 4, n=26; Day 5, n=35). After 8 days of IVC, the number of embryos developing to blastocysts was recorded and blastocyst cell numbers were counted after staining with Hoechst 33342. In Experiment 1, developmental stage did not affect the survival rate after thawing (Day 2=79%, Day 4=90%, Day 5=98%) and was not different from that of controls (Day 2=89%, Day 4=88%, Day 5=96%). Blastocyst development was similar among days both after cryopreservation (Day 2=59%, Day 4=54%, Day 5=63%) and in controls (Day 2=55%, Day 4=54%, Day 5=58%). Mean (+/-S.D.) cell number of blastocysts was slightly lower (NS) in cryopreserved embryos (Day 2=152+/-19, Day 4=124+/-20, Day 5=121+/-24) than in controls (Day 2=141+/-25, Day 4=169+/-21, Day 5=172+/-19). In Experiment 2, embryos frozen on Day 2 (n=68), Day 4 (n=49) or Day 5 (n=73) were thawed and cultured for 3, 1, or 0 days before transfer by laparotomy to 5 (mean=12.6+/-2.4), 4 (mean=12.2+/-3.7) and 6 (mean=12.0+/-1.6) recipients, respectively. Four recipients were pregnant on Day 21; two from embryos frozen on Day 4 and two from Day 5. Two live kittens were born at 66 days, a third kitten died during parturition at 64 days and a fourth pregnancy aborted by Day 45. In summary, we have shown that a controlled rate cryopreservation technique can be successfully applied to cat embryos produced by IVM/IVF. In vitro development to the blastocyst stage was not affected by the age of embryos at cryopreservation. The births of live kittens after ET of cryopreserved embryos is additional validation of progress toward applying assisted reproductive technology to preservation of endangered felids.     

Developmental competence of nuclear transfer cow oocytes after direct injection of fetal fibroblast nuclei

We have examined the in vitro and in vivo development of cloned embryos produced by incorporation of fetal fibroblast into in vitro matured and enucleated cow oocytes by direct injection and by fusion. For injection, nuclei were either mechanically isolated using the microinjection needle or chemically isolated by treatment with NP-40 lysis buffer. Fetal fibroblasts were serum starved and treated with calcium ionophore before injection to induce chromatin condensation. A range of 8% to 16% of successfully injected oocytes developed to blastocysts in culture and a total of nine pregnancies resulted from transfer of cloned embryos produced by this method. Nuclear transfer by fusion resulted in 22% development to blastocysts. Unlike in mice, the embryos derived from injection did not result in viable pregnancies, which may suggest species differences. All pregnancies were terminated after 45 to 150 days from transfer. Two pregnancies resulted from transfer of cloned embryos obtained by fusion which produced two healthy female calves. The study proposes an alternative method for the production of cow cloned embryos. Further research, however, is required to optimize bovine cloning by injection.     

Failure to maintain interspecific pregnancy after transfer of Dall's sheep embryos to domestic ewes

Over a 3-year period, 32 Dall's sheep (Ovis dalli dalli) embryos were transferred into 24 domestic sheep (O. aries) recipients and 4 were transferred into 2 Dall's sheep recipients. In the first year, none of the 10 O. aries recipients was diagnosed pregnant. In the following 2 years, 9 (37%) of the domestic sheep recipients were pregnant on Day 18, 8 (33%) on Day 40, 6 (25%) on Day 90 and 4 (16%) on Day 120; 1 aborted at Day 125 and another at Day 145. Pregnancies were established only in ewes that had previously been recipients of Dall's sheep embryos. The 2 remaining pregnant sheep were treated with progesterone from Day 125 until the fetuses were determined to be dead at Day 145. Both of the Dall's sheep recipients (Year 2) established pregnancies; 1 live Dall's sheep lamb was born 174 days after mating. No differences in serum progesterone, oestrone, prostaglandin F-2 alpha metabolites or cortisol concentrations could be detected during pregnancy between recipients carrying Dall's sheep embryos, recipients receiving progesterone treatment or domestic ewes carrying domestic sheep pregnancies. Six fetuses were necropsied (1 at Day 125 and 5 at Day 145-146): all fetuses were premature and had various degrees of hydranencephaly. No significant differences were found when cotyledon numbers were compared among domestic ewes carrying Dall's sheep lambs. Dall's sheep ewes lambing naturally and domestic ewes lambing naturally. These results demonstrate that the transfer of Dall's sheep embryos to domestic ewes results in the establishment but subsequent loss of pregnancy and that these losses occur throughout gestation.     

The ovine uterus as a host for in vitro-produced bovine embryos

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.     

The effect of donor cell serum starvation and oocyte activation compounds on the development of somatic cell cloned embryos

Two experiments, one comparing nuclear transfer (NT) embryo activation compounds, the other donor cell treatments, were conducted with a goal of identifying factors that improve the in vitro development of cloned bovine embryos. In experiment 1, 539 NT embryos were produced by combining serum starved bovine fetal fibroblasts with enucleated in vitro matured oocytes, activated with ionomycin, then randomly allocated to be incubated for 4 hours in either Butyrolactone-I (BL-I) or 6-dimethylaminopurine (DMAP). There was no significant difference in development to blastocyst or compact morula of fused embryos at Day 6.5 between BL-I and DMAP activated embryos (22.4% vs. 20.2%; p = 0.18). Karyotyping of 20 blastocysts and compact morula from each group determined that 65% of BL1 and 63% of DMAP embryos were diploid with the remainder mixoploid (2n + 4n). In Experiment 2, the development of 389 NT embryos reconstructed from either serum starved or serum fed fetal fibroblasts was assessed. More Day 7 blastocysts and compact morula developed in the serum starved group (34.5% vs. 18.8%; p = 0.008). To verify the viability of BL-I activated embryos, 10 blastocytes from experiment 2 were transferred into 4 recipient cows. Two morphologically normal fetuses, genetically identical to the original fetal cell line, were surgically recovered at day 45 of gestation. In summary, serum starvation of bovine fetal fibroblasts prior to NT significantly improved development to blastocyst. Additionally, we have shown that BL-I is a novel alternative compound for use in combination with ionomycin to activate NT embryos.     

Development of porcine embryos from one- and two-cell stages to blastocysts in culture medium supplemented with porcine oviductal fluid

Oviductal fluid (OVF) was harvested chronically from 5 sows beginning on Day 1 of the estrous cycle (Day 0 of estrous cycle = day of detected estrus) and used for embryo culture (Day 3 OVF only). Two experiments were conducted to investigate in vitro development of 1-cell and 2-cell porcine embryos in a modified Kreb's Ringer bicarbonate medium (culture medium, CM), early luteal phase OVF or CM supplemented with OVF (CM-OVF, 25% OVF v/v in CM) with or without transfer to fresh CM. In Experiment 1, 1-cell and 2-cell embryos were harvested from sows (n = 7) approximately 44 h after detected estrus. In Experiment 2, 1-cell embryos were collected from 5 sows treated with altrenogest and gonadotropins, approximately 50 h after injection of human chorionic gonadotropin. The volume of OVF (ml) declined progressively throughout the 4 days of collection (24 h, 8.44 +/- 0.28; 48 h, 6.88 +/- 1.78; 72 h, 4.96 +/- 0.35; 96 h, 4.64 +/- 0.25 after onset of estrus; p less than .01). In both experiments, development to blastocyst stage was lowest among embryos cultured in OVF and highest among those cultured in CM-OVF (Experiment 1: CM, 27.3; OVF, 10; CM-OVF, 63.6; Experiment 2: CM, 26.7; OVF, 0; CM-OVF, 82.4; % blastocyst formation).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo development of vitrified rabbit embryos: Effects on prenatal survival and placental development

The aim of this work is to study the effect of the vitrification procedure on prenatal survival and on placental development at the end of gestation in rabbits (Oryctolagus cuniculus). One hundred eighty-one females were slaughtered at 72 h of gestation. Morphologically normal embryos recovered at 72 h of gestation were kept at room temperature until transfer or vitrification. Vitrified embryos (320 embryos) were transferred into a total of 24 does and fresh embryos (712 embryos) were transferred into a total of 43 does. Females were induced to ovulate 72 h before transfer when fresh embryos were transferred and 60 to 63 h before transfer when vitrified embryos were transferred. Each recipient doe received eight embryos into the left oviduct and eight embryos into the right oviduct. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at Day 14 of gestation. Recipient females were slaughtered by stunning and exsanguination 25 d after the transfer, and fetuses were classified according to their status. Live fetuses and fetal and maternal placenta were weighed Pregnancy rate was defined as the total number of females having at least one live fetus at Day 28 of gestation divided by the total number of females. Prenatal survival was estimated as live fetuses at Day 28 of gestation divided by the number of transferred embryos. The pregnancy rate after transfer of vitrified embryos (92%) was similar to that achieved with fresh embryos (86%), but prenatal survival was lower for vitrified than for fresh embryos (53% vs. 34%). We did not find differences in embryo survival from 72 h to implantation. Transfer of vitrified embryos reduced fetal survival from implantation to Day 28 (57% vs. 82%). Differences in the number of live fetuses at Day 28 of gestation were mainly due to the higher fetal mortality observed soon after implantation in pregnancies resulting from the transfer of vitrified embryos. A higher percentage of decidual reactions and atrophic maternal placentas (27.5% vs. 8.3%) and also of atrophic fetal and maternal placentas (12.1% vs. 5.3%) were observed after transfer of vitrified embryos. Both treatments showed similar percentage of dead fetuses (3.3% vs. 4%). Maternal placenta of the fetuses from fresh embryos was 15% heavier than maternal placenta of fetuses from vitrified embryos.     

Effects of membrane piercing and the type of pronuclear injection fluid on development of in vitro-produced bovine embryos

We studied the effects of mechanical damage of plasma and pronuclear membranes on the development of in vitro-produced bovine zygotes. The effects of the type of injection fluid and the presence of DNA in the zygotes were also studied. In the first experiment, either the plasma membrane or both the plasma and pronuclear membranes of zygotes were pierced with a capillary filled with DNA-buffer. Additionally, pronuclear microinjections with either MilliQ-water or buffer were performed. In the second experiment, pronuclear microinjections with buffer containing either none or 2 microg/ml of DNA were performed. Development of cleaved embryos to compact morulae and blastocysts at Day 7 was monitored. Results of Experiment 1 indicate that membrane piercing does not decrease development of cleaved embryos as compared with that of the controls (30.8, 28.8 and 29.9% compact morulae and blastocysts for controls, plasma membrane and pronuclear membrane pierced groups, respectively). Pronuclear microinjections decreased development significantly as compared with that of the controls, but no differences were observed between the effects of water and buffer (29.9, 18.4 and 15.5% compact morulae and blastocyst, respectively). Results of Experiment 2 showed that inclusion of DNA into the injection buffer decreased development even more drastically (36.7, 27.5 and 14.5% compact morulae and blastocyst in the control, buffer-injected and DNA-injected groups, respectively).     

Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos

Experiments were conducted to determine viability of equine embryos in vivo after vitrification. In a preliminary study (Experiment 1), embryos were exposed in three steps to vitrification solutions containing increasing concentrations of ethylene glycol and glycerol (EG/G); the final vitrification solution was 3.4 M glycerol + 4.6 M ethylene glycol in a base medium of phosphate-buffered saline. Embryos were warmed in a two-step dilution and transferred into uteri of recipients. No pregnancies were observed after transfer of blastocysts >300 microm (n = 3). Transfer of morulae or blastocysts < or = 300 microm resulted in four embryonic vesicles (4/6, 67%). In a second experiment, embryo recovery per ovulation was similar for collections on Day 6(28/36, 78%) versus Days 7 and 8(30/48, 62%). Embryos < or = 300 and >300 microm were vitrified, thawed and transferred as in Experiment 1. Some embryos < or = 300 microm were also transferred using a direct-transfer procedure (DT). Embryo development rates to Day 16 were not different for embryos < or = 300 microm that were treated as in Experiment 1(10/22, 46%) or transferred by DT (16/26, 62%). Embryos > 300 microm (n = 19) did not produce embryonic vesicles.     

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.     

Birth of piglets derived from porcine zygotes cultured in a chemically defined medium.

We evaluated the in vitro development of porcine zygotes that were cultured in a novel culture medium, porcine zygote medium (PZM), under different conditions and compared to in vivo development. The viability of these zygotes to full term after culture was also evaluated by embryo transfer to recipients. Porcine single-cell zygotes were collected from gilts on Day 2 after hCG injection. Culture of zygotes in PZM containing 3 mg/ml of BSA (PZM-3) produced better results in terms of proportion of Day 6 blastocysts, Day 8 hatching rate, and numbers of inner cell mass (ICM) cells and total cells in Day 8 embryos than that in North Carolina State University (NCSU)-23 medium. In culture with PZM-3, embryo development was optimized in an atmosphere of 5% CO2:5% O2:90% N2 compared to 5% CO2 in air. The ICM and total cell numbers in Day 6 embryos cultured in PZM-3 or in PZM-3 in which BSA was replaced with 3 mg/ml of polyvinyl alcohol (PZM-4) were also greater than those of NCSU-23 but less than those developed in vivo. However, no difference was found in the ratio of ICM to total cells among embryos developed in PZM-3, PZM-4, or in vivo. When the Day 6 embryos that developed in PZM-4 (99 embryos) or in vivo (100 embryos) were each transferred into six recipients, no difference was found in the farrowing rate (83.3% for both treatments) and in the number of piglets born (33 and 42 piglets, respectively). Our results indicate that porcine zygotes can develop into blastocysts in a chemically defined medium and to full term by transfer to recipients after culture.