Transgenic production from in vivo-derived embryos: effect on calf birth weight and sex ratio

We examined transgenic-cattle production by DNA microinjection into 1-, 2-, and 4-cell embryos, analyzing the impact on calf size and subsequent viability. Embryos were either collected at an abattoir by flushing oviducts from superovulated and artificially inseminated cows (in vivo-derived) or obtained by in vitro maturation and in vitro fertilization of oocytes aspirated from excised ovaries (in vitro-derived). A human serum albumin (hSA) milk-expression DNA construct was microinjected, either in one of the visible pronuclei of in vitro- and in vivo-derived 1-cell embryos or in the nuclei of two blastomeres of 2- and 4-cell in vivo-derived embryos. Microinjection-induced mortality (lysis and developmental block) was equivalent ( approximately 40%) for all microinjected embryos. Embryos were co-cultured with BRL cells in B-2 medium containing 10% fetal calf serum (FSC). Overall, embryo development to morulae/blastocysts was significantly greater for in vivo-derived ova (15.5%) than for in vitro-derived oocytes (9.3%). All morulae and blastocysts were transferred to synchronized recipient females on Days 6-8 post-fertilization. A total of 189 calves were delivered. Birth weights were significantly greater for calves generated from in vitro-derived oocytes compared with those generated from in vivo-derived oocytes. One transgenic bull calf was obtained from the microinjection of a 2-cell embryo. Fluorescence in situ hybridization (FISH) analysis of lymphocytes detected one transgenic integration site in all cells. Transmission frequency of the hSA transgene in embryos obtained through IVM/IVF/IVC utilizing the semen of the transgenic calf confirmed that it was not mosaic.     

Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium

In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves

Mature Brangus donor cows were superovulated with follicle stim-ulating hormone administered twice daily in intramuscular injections. On day 6.5 to 7 post-estrus, embryos were collected non-surgically using a phosphate-buffered saline medium. A total of 37 ova was collected, of which 28 were advanced morulae and early blastocysts. Twenty of these embryos were selected for micromanipulation with a radial-type Leitz micromanipulator. While the embryos were in a holding medium containing 10% fetal calf serum, three glass microinstruments were used to open the zona pellucida, remove the mass of blastomeres and bisect the embryo on a vertical plane. Halved embryos were inserted into bovine zonae and placed either as single half-embryos or twin half-embryos in 0.25 ml French straws with fresh holding medium. The micromanipulated embryos (demi-embryos) were then non-surgically transplanted, either as a single demi-embryo or as a twin demi-embryo pair, into the uterine horn of day 6.5 to 8 recipient beef females ipsilateral to the existing corpus luteum. Of the 14 micromanipulated embryos that were transplanted to recipients, pregnancy rates were 16.6% for the single demi-embryos and 62.5% for the twin demi-embryos. No pregnancies resulted from bisected blastocysts.     

Effect of human leukemia inhibitory factor on in vitro development of parthenogenetic bovine morulae

The present study was conducted to investigate the effect of human leukemia inhibitory factor (hLIF) addition to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HS) on the development of in vitro matured and parthenogenetically activated bovine oocytes. The oocytes matured for 30 h were exposured to ethanol (7%, 7 min) and cytochalasin B (5 mug/ml, 5 to 6 h). The treated oocytes were cultured for 5 d in SOFM supplemented with HS, and Day-5 morulae were cultured for 2 d in SOFM supplemented with HS and with or without hLIF (5000 U/ml) to investigate the subsequent in vitro development to the blastocyst stage. Of the 1531 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to 8-cell stage and 174 (13.8%) developed to the morula stage. The addition of hLIF at the morula stage resulted in a significantly (P<0.01) higher rate of development to the blastocyst stage in the medium with hLIF (55.9%) than without hLIF (28.9%). The mean cell number per blastocyst developed in the medium with hLIF was also significantly (P<0.01) higher than that developed in the medium without hLIF. To evaluate the viability, 6 parthenogenetically developed blastocysts were transferred to 3 recipient heifers (2 embryos per heifer), while in 2 other recipient heifers estrus was prolonged after transfer. The plasma progesterone levels of the 2 recipient heifers at the 28th day after transfer were 8.1 ng/ml and 9.0 ng/ml, but pregnancy was not observed by ultrasonic scanning. The present results indicate that the addition of hLIF to in vitro-produced, Day-5 parthenogenetic bovine morulae significantly improves the subsequent development to the blastocysts stage; however, the present method still does not promote for development of parthenogenetic fetuses in cattle.     

Blastomeres of the mouse embryo lose totipotency after the fifth cleavage division: expression of Cdx2 and Oct4 and developmental potential of inner and outer blastomeres of 16- and 32-cell embryos

Sixteen inner or outer blastomeres from 16-cell embryos and 32 inner or outer blastomeres from 32-cell embryos (nascent blastocysts) were reaggregated and cultured in vitro. In 24 h old blastocysts developed from blastomeres derived from 16-cell embryos the expression of Cdx2 protein was upregulated in outer cells (new trophectoderm) of the inner cells-derived aggregates and downregulated in inner cells (new inner cell mass) of the external cells-derived aggregates. After transfer to pseudopregnant recipients blastocysts originating from both inner and outer blastomeres of 16-cell embryo developed into normal, fertile mice, but the implantation rate of embryos formed from inner cell aggregates was lower. The aggregates of external blastomeres derived from 32 cell embryo usually formed trophoblastic vesicles accompanied by vacuolated cells. In contrast, the aggregates of inner blastomeres quickly compacted but cavitation was delayed. Although in the latter embryos the Cdx2 protein appeared in the new trophectoderm within 24 h of in vitro culture, these embryos formed only very small outgrowths of Troma1-positive giant trophoblastic cells and none of these embryos was able to implant in recipient females. In separate experiment we have produced normal and fertile mice from 16- and 32-cell embryos that were first disaggregated, and then the sister outer and inner blastomeres were reaggregated at random. In blastocysts developed from aggregates, within 24 h of in vitro culture, the majority of inner and outer blastomeres located themselves in their original position (internally and externally), which implies that in these embryos development was regulated mainly by cell sorting.     

Transfer of cow blastocysts after sexing

We obtained 174 Charolais cow blastocysts at day 13 (D(13)) after superovulation and slaughter of 24 donor heifers. Using de Wecker's scissors, we sampled 1 10 to 1 3 of the total blastocyst volume. After colcemid treatment, hypotonic shock, fixation and Giemsa staining, the sex of 149 blastocysts (60 %) was determined. The main difficulty in sexing was the absence of metaphase or the presence of incomplete metaphases on the preparations. During manipulation, the portion of the blastocyst to be transferred was stored at 37 degrees C in a medium of 20 % fetal calf serum containing 80 % B(2) under an atmosphere of 90 % nitrogen, 5 % O(2) and 5 % CO(2) for 6 to 7 hours. After sex determination, the blastocysts were put into an artificial insemination straw and placed with a catheter at the base of the uterine horns. Twenty-eight blastocysts were transferred into both horns of 14 recipient Charolais heifers; 4 heifers (28 %) had an extended cycle and 3 (21 %) were pregnant. When 2, 3 or 4 blastocysts were transferred into the ipsilateral horn with the corpus luteum, 20 to 25 % of the recipients had an extended cycle and 37, 40 and 75 %, respectively, were pregnant. The number of surviving embryos per recipient never exceeded 2.     

Effect of growth factors on morula and blastocyst development of in vitro matured and in vitro fertilized bovine oocytes

Growth factors were studied as a means of increasing the development of in vitro matured (IVM) and in vitro fertilized (IVF) oocytes into morulae or blastocysts. Cell numbers of blastocysts were also counted. In Experiment 1, 2- to 8-cell embryos derived from bovine IVM/IVF oocytes were randomly allotted to one of 3 culture groups: a) synthetic oviduct fluid (SOF); b) SOF + 10 ng/ml epidermal growth factor (EGF); or c) SOF + 100 ng/ml EGF; all 3 culture media contained 10% fetal bovine serum. Culture resulted in 12%, 23% and 14% (P>0.05), respectively, developing into morulae and blastocysts. In Experiment 2, 5 ng/ml of transforming growth factor B (1) (TGFB (1)) added to CR(1aa) medium containing BSA increased the percentage of blastocysts to 56% vs 40% for the control (P<0.05). In Experiment 3, EGF and TGFB(1), added singly and in combination to CR(1aa) did not produce a synergistic effect. More embryos developed into morulae and blastocysts (45%) in a bovine oviduct epithelial co-culture than in any other treatment except in CR(1aa) + EGF (34%; P>0.05). In Experiment 4, 0, 1 and 5 ng/ml of platelet derived growth factor (PDGF) added to CR(1aa) yielded 39%, 70% and 52% morulae and blastocysts, respectively (P<0.05). Cell number was not increased, indicating that growth factors can increase the proportion of embryos that develop into morulae and blastocysts without an increase in the cell number.     

Effects of culture systems on development of in vitro fertilized bovine ova into blastocysts

We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eight-cell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P<0.05) increased development into blastocysts (39.0 to 50.7%) when compared with co-culture with cumulus cells, control or rabbit oviducts(1.9 to 29.3%). Six of 16 recipients became pregnant with frozen embryos derived from co-culture with oviductal epithelial cells(1/2), trophoblastic vesicles(2/7) or amniotic sac cells(3/7). Eight calves, including two sets of twins, were obtained.     

Direct freezing of cattle embryos after partial dehydration at room temperature

A new and simple method for freezing of bovine morulae and blastocysts was developed. Embryos were predehydrated at room temperature, frozen at -30 degrees C (cooling rate = 12 degrees C/min), and plunged into liquid nitrogen. This method was compared in vitro and in vivo to the slow freezing method (0.3 degrees C/min to -30 degrees C). Predehydration of the embryos in 1.5M glycerol was achieved by sucrose solution that makes the cells osmotically shrink. After the predehydrated morulae and blastocysts were frozen and thawed, 6 .4% (33 52 ) were developed in vitro for 48h and 44.2% (23 52 ) were hatched. Development obtained with slowly frozen embryos were 70.8% (17 24 ) and 58.3% (14 24 ) respectively. After transfer to recipient heifers, 33.3% (7 21 ) of the embryos frozen according this new method developed normally into viable foetuses or calves. This was the case for 48.5% (16 33 ) of the slowly frozen embryos.     

Vitrification of rat embryos at various developmental stages

The effect of developmental stage on the survival of cryopreserved rat embryos was examined. Wistar rat embryos at various developmental stages were vitrified by a 1-step method with EFS40, an ethylene glycol-based solution, or by a 2-step method with EFS20 and EFS40. After warming, the survival of the embryos was assessed by their morphology, their ability to develop to blastocysts (or expanded blastocysts for blastocysts) in culture, or their ability to develop to term after transfer. Most (91-100%) of the embryos recovered after vitrification were morphologically normal in all developmental stages. However, the developmental ability of 1-cell embryos was quite low; exposing them to EFS40 for just 0.5 min decreased the in vitro survival rate from 76 to 9%. The survival rates of 2-cell embryos and blastocysts, both in vitro and in vivo, were significantly higher with a 2-step vitrification process than with a 1-step vitrification process. Very high in vitro survival rates (94-100%) were obtained in 4- to 8-cell embryos and morulae in the 1-step method. Although survival rates in vivo of 4-cell (40%) and 8-cell (4%) embryos vitrified by the 1-step method were comparatively low, the values were similar to those obtained in non-vitrified fresh embryos. When morulae vitrified by the 1-step method were transferred to recipients, the in vivo survival rate (61%) was high, and not significantly different from that of fresh embryos (70%). These results show that rat embryos at the 2-cell to blastocyst stages can be vitrified with EFS40, and that the morula stage is the most feasible stage for embryo cryopreservation in this species.     

Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration

Oocytes were recovered by laparoscopic aspiration from 3- to 8-week-old calves treated with follicle-stimulating hormone (FSH) followed by human chorionic gonadotropin (hCG) to induce follicular growth and oocyte maturation in vivo. Most of the recovered oocytes either had resumed meiotic maturation at the time of aspiration or were competent to undergo maturation during subsequent culture in vitro. Oocytes matured in vivo following FSH and hCG treatment underwent in vitro fertilization (70%) at rates not significantly different from those of control oocytes recovered from adult cow ovaries at abattoirs and matured in vitro (75%). Calf oocytes that were immature at aspiration exhibited lower fertilization rates after in vitro maturation (36%) but their rate of development to morulae and blastocysts did not differ from that of mature oocytes at aspiration. A total of 91% of the zygotes produced from calf oocytes developed to morula and 27% to blastocyst stages during 6 days of culture. The proportion developing to morulae was significantly higher (P<0.05) than that observed for zygotes resulting from in vitro maturation and fertilization of oocytes recovered from cow ovaries obtained at an abattoir and processed concomitantly (59% to morulae and 18% to blastocysts). Morulae or blastocysts developed from oocytes from 5 to 6-week-old calves, when transferred to synchronized recipient heifers, resulted in 2 confirmed pregnancies, one of which produced a single full-term live calf. The ability to produce embryos from oocytes recovered from newborn or prepubertal calves offers the potential for markedly reducing the generation interval in cattle, thereby substantially accelerating the rate of genetic gain that can be achieved through embryo transfer.     

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.     

Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis

The major obstacle in the extensive analysis of the embryonic genome is the small number of cells typically obtained after the embryo biopsy. The object of the present study was to develop a simple approach that would allow the collection of a sufficient number of cells from a single embryo for use in further analyses. A micromanipulator was used to make a hole in the zona pellucida of 28 compacted morulae, 27 early blastocysts and 31 expanded blastocysts. After further culture, the trophoblastic cells, which herniated through this hole, were cut and cultured in vitro for different periods and used for embryo sexing. The results showed that biopsies can be taken successfully from 96.3% of early blastocysts, compared with 67.7% of expanded blastocysts and 71.4% of compacted morulae. The trophoblastic vesicles contained 20.8 +/- 6.7 cells (mean +/- SEM) and, when cultured, formed a confluent monolayer. The sex of cells cultured was assayed by PCR and the 12 lambs born after transfer of biopsied embryos confirmed its 100% accuracy. Moreover, no significant differences were found in the viability rates in vitro among blastocysts vitrified immediately after biopsy (77.8%), blastocysts biopsied and vitrified after 24 h culture (76.9%) and blastocysts vitrified without manipulation (88.5%). In experiments in vivo, the lambing rate of biopsied and vitrified blastocysts was significantly (P < 0.05) lower (40.0%) compared with vitrified control embryos (68.7%). This new approach to the biopsy of preimplantation embryos is a useful good model in the assisted reproductive technologies of domestic, wild and human species.     

Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.     

Chimerism in cattle through microsurgical aggregation of morulae

A cattle chimera was produced by combining four halves of two parent embryos of different breeds (Brown-Swiss x Braunvieh plus Holstein-Friesian x Holstein-Friesian) in one zona pellucida. Parent embryos in the 32-cell morula stage were recovered non-surgically, were bisected, and the combined four halves were transferred non-surgically to recipient heifers. Chimerism of coat colour was used as evidence. Combining of only two half embryos from different parents resulted in five pregnancies carried to term but none of the calves born was a chimera.     

Addition of penicillamine, hypotaurine and epinephrine (PHE) or bovine oviductal epithelial cells (BOEC) alone or in combination to bovine in vitro fertilization medium increases the subsequent embryo cleavage rate

The cleavage rate of in vitro-matured bovine oocytes was compared after fertilization in 1) TALP medium alone (control); 2) in TALP + BOEC; 3) in TALP + PHE; or 4) in TALP + BOEC and PHE. The overall cleavage rate at 45 h post insemination was greater for embryos in Treatments 2 (52%), 3 (55%) and 4 (66%) than for Treatment 1 (32%). The oocyte cleavage rates for Treatments 2 and 3 were similar, but were lower than that of Treatment 4. Addition of PHE or BOEC, alone or in combination, to the fertilization medium resulted in more embryos at the 3- or 4-cell stage than the 2-cell stage by 45 h post insemination. After 5 d of co-culture with BOEC in M-199 medium, 21, 28, 25 and 35% of the cleaved embryos in Treatments 1, 2, 3 and 4, respectively, developed to the morula or blastocyst stage. The rate of development to morulae and blastocysts was similar among Treatments 1, 2 and 3, and between Treatments 2 and 4. Across treatments, a correlation of 0.98 was noted between the portion of embryos that had reached the 3- or 4-cell stage by 45 h post insemination and the percentage of embryos in each treatment that continued to develop to the morula or blastocyst stage in vitro.     

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.     

Pregnancies after in-vitro fertilization of cow follicular oocytes, their incubation in rabbit oviduct and their transfer to the cow uterus

Cow embryos fertilized in vitro (1-8-cell) (n = 113) were transferred surgically to the ligated oviduct of pseudopregnant female rabbits (31 +/- 4 h after 75 i.u. hCG). Rabbits were killed 99 +/- 5 h later and 67 embryos were recovered: 45 (67%) had cleaved at least once, 15 had reached the morula stage and 2 blastocysts were obtained. Transfer of cleaved embryos (2-8-cell) led to a high recovery rate (84%) compared to 39% for one-cell embryos. Of the embryos incubated for more than 99 h in the rabbit oviduct, 41% were at the morula stage. Embryos incubated in vivo (n = 21) (8-cell to blastocyst) were transferred to the uterus of 14 synchronized recipient heifers by a surgical (N = 5) or a non-surgical (N = 9) procedure: 6 pregnancies resulted (4 from the non-surgical procedures). In addition, 27 (2-8-cell) cow embryos developed in vitro were transferred to the oviduct of synchronized heifers by a paralumbar surgical approach on a standing animal, but no pregnancies resulted. It is therefore concluded that (1) the rabbit oviduct can be used to obtain cow eggs at an embryonic stage sufficiently advanced to permit transfer to the uterus of a synchronized recipient; and (2) the pregnancy rate after in-vitro fertilization and incubation in the rabbit oviduct are similar to results with fertilization in vivo.     

Development of bovine embryos in a cell-free culture medium: Effects of type of serum, timing of its inclusion and heat inactivation

Bovine embryos, derived from in vitro matured (IVM)/in vitro fertilized (IVF) ova, were used to investigate the effects of timing of serum inclusion in the culture medium and different types of blood sera and heat inactivation of the serum on embryo development. In Experiment 1, oocytes at 18 h post insemination were allocated to 1 of the following 4 treatments: 1) TCM-199 + 0.1 mg/ml polyvinylalcohol (PVA), 2) TCM-199 supplemented with 10% bovine calf serum (BCS), 3) PVA medium followed by BCS medium at 47 h, or 4) PVA medium followed by BCS medium at 82 h. Supplementation with BCS at 18 h post insemination suppressed (P<0.05) development of morulae/blastocysts (17.6%) when compared with PVA (30.5%) or with serum supplementation at 47 or 82 h post insemination (32.4 and 27.6%, respectively). However, inclusion of BCS at 18, 47 or 82 h post insemination produced more blastocysts (16.8, 29.3 and 22.1%, respectively; P<0.05) than medium +PVA (8.8%). In Experiment 2, ova were cultured from 18 h to 42 h post insemination in PVA-medium, then >/=2-cell embryos were transferred into serum-supplemented medium for another 168 h. Fetal bovine serum (FBS) +/- heat-inactivation (56 degrees C for 30 min, = heated FBS) suppressed morula/blastocyst development compared with medium + PVA, medium + BCS or medium + heated BCS (P<0.05). Bovine calf serum was superior to FBS in supporting blastocyst development (35.1 and 15.2%, respectively), but there was no difference between BCS and heated BCS. However, heated FBS increased the proportion of blastocysts/>/=8-cell embryos compared with that of FBS (51.0 and 31.4%, respectively; P<0.05). These results indicate that the type of serum supplementation and the timing of its inclusion in the culture medium markedly affect bovine embryo development in vitro, and that heat inactivation of serum with high embryotrophic properties is not necessary.