Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells.

Since the advent of procedures for cloning animals, conservation biologists have proposed using this technology to preserve endangered mammals. Here we report the successful cloning of a wild endangered animal, Ovis orientalis musimon, using oocytes collected from a closely related, domesticated species, Ovis aries. We injected enucleated sheep oocytes with granulosa cells collected from two female mouflons found dead in the pasture. Blastocyst-stage cloned embryos transferred into sheep foster mothers established two pregnancies, one of which produced an apparently normal mouflon. Our findings support the use of cloning for the expansion of critically endangered populations.

Effect of different diluents on goat semen fertility.

Skim milk (SM) is considered to be the most widely employed extender for goat sperm used for artificial insemination (AI). However, the fertilizing life span of sperm stored in milk or milk-based extenders does not exceed 12h. Besides some seminal plasma components, such as a protein fraction from the goat bulbourethral gland secretion (SBUIII), interacts with some milk fractions and inhibits the spermatozoa motility. The aim of this study was to prolong the survival of buck semen and its fertility. Buck ejaculates were diluted to a final concentration of 100x10(6)spermatozoa/ml with three different diluents: SM, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPOL+hyaluronic acid (TEMPOL+HA). At 7h from dilution 42 goats were inseminated with semen diluted with SM (short-term semen) while after storage for 24h, 44 and 45 goats were inseminated with semen diluted with TEMPOL and TEMPOL+HA (long-term storage), respectively. At day 50 from AI the percentages of pregnant goats were 71.4% (30/42) with SM, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, with significant differences between SM and TEMPOL+HA. The kidding rate was 66.7% (28/42) with SM diluent, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, without significant differences among treatment groups. In conclusion, it is possible to maintain good fertility in goats after AI with semen stored for 24h in TEMPOL.

Effect of recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo development and viability.

The objective of this work was to study the effect of a preparation of human recombinant gonadotrophins (r-FSH and r-LH) on the in vitro maturation (IVM) and development of sheep oocytes. In addition, the viability of fresh and vitrified blastocysts obtained after transfer was tested. Oocytes collected from slaughtered animals were divided into five different maturation groups. All groups were matured in a medium containing TCM199 with 4 mg/ml BSA, 100 microM cysteamine and 1 microg/ml estradiol-17beta. Each group was also treated with one of the following: 0.1 UI/ml r-FSH (r-FSH group), 0.1UI/ml r-LH (r-LH group), 0.1 UI/ml r-FSH and 0.1 UI/ml r-LH (r-FSH/r-LH group), 5 microg/ml FSH and 5 microg/ml LH hypophysial gonadotrophins (h-G group) as a control, or no gonadotrophins (no-G group). After in vitro fertilization with fresh ram semen, presumptive zygotes were cultured in vitro for 6-7 days and a total of 109 blastocysts were then transferred in pairs into synchronized ewes. To determine the viability of embryos after vitrification, 36 blastocysts from the r-FSH/r-LH group and 30 from the h-G group were vitrified in 10% ethylene glycol (EG) and 10% dimethylsulphoxide (DMSO) for 5min, followed by 20% EG, 20% DMSO and 0.5M Sucrose (S) for <45 s. They were loaded into open pulled straws (OPS) and plunged into LN(2). After warming, the blastocysts were transferred in pairs into synchronized ewes. The highest maturation rate was reached in the r-FSH/r-LH group (91.9%). However, no statistical difference was found when this group was compared with the h-G group (84.0%). Likewise, the cleavage rate of the r-FSH/r-LH group (81.4%) was not significantly different from that of the h-G group (82.3%). The cleavage rates of all other groups, however, were significantly lower than the r-FSH/r-LH and h-G groups. The blastocyst rate was highest in the h-G group (53.6%), and it was statistically higher than in the r-FSH/r-LH group (41.5%). The blastocyst rate was very similar between groups r-FSH and r-FSH/r-LH (42.0 and 41.5%, respectively). The lowest lambing rate (31.8%) was in the no-G group. The highest lambing rate was achieved in the r-FSH/r-LH group (66.6%). The vitrified embryos of h-G and r-FSH/r-LH groups had a very similar lambing rate (16.6% and 19.4%). In conclusion, these data provide support for the hypothesis that sheep oocytes respond to human recombinant gonadotrophins used for in vitro embryo production.

Membrane integrity and fertilizing potential of cryopreserved spermatozoa in European mouflon.

There is a pressing need to develop and use assisted reproductive techniques in wildlife species living in small and captive groups. We evaluated the effect of freezing on membrane integrity and fertilizing capacity of European mouflon (Ovis gmelini musimon) spermatozoa collected during the breeding season. After thawing, the percentage of live spermatozoa, stained with fluorescein isothiocynate labeled Pisum Sativum agglutinin and propidium iodide, was 47% of which 19% showed intact acrosomal membrane. After culture in TCM 199 + 10% FCS, the number of live spermatozoa was significantly (P < 0.01) lower than in a medium with oviductal epithelial cells. The absence of oviductal cells decreased significantly the fertilization rates (P < 0.05), 24.0 vs. 63.1 with oviductal epithelial cells and 59.1 in vivo of in vitro matured ovine oocytes. Polyspermic fertilization rate of oocytes was lower (P < 0.05) with oviductal epithelial cells (1.6) than in absence of cells (12.8). However, the percentage of embryos that reached blastocyst stage was significantly higher in vivo than in vitro. These results provide interesting preliminary data for the development of genetic resource banks for European mouflon.

Polyvinyl alcohol as a defined substitute for serum in vitrification and warming solutions to cryopreserve ovine embryos at different stages of development.

The purpose of this study was to assess the viability of ovine embryos after replacing fetal calf serum (FCS) with polyvinyl alcohol (PVA) in vitrification and warming solutions. Ovine embryos were obtained from superovulated Sardinian breed ewes at 4, 5, 6, and 7 days after insemination. All vitrification and warming solutions were prepared using buffered saline solution with 20% FCS (group a) or 0.1% PVA (group b). Embryos were vitrified in 20 microliters of glycerol 3.4 M + ethylene glycol 4.6 M and loaded into the centre of 0.25 ml straws between two columns of sucrose solution (0.5 M), and plunged immediately into liquid nitrogen. After being warmed in a water bath at 35 degrees C for 10 s, the vitrified embryos were moved to 0.25 M sucrose solution for 3 min. Embryos were cultured in TCM-199 after washing with 10% FCS and sheep oviductal epithelial cells up to hatching or re-expansion of the blastocoelic cavity. No significant difference in the viability rates was observed between embryos vitrified/warmed in PVA or FCS solutions. In both groups, the rate of in vitro viability was (P < 0.01) lower at the precompacted and compacted morula stages than at the expanded, hatching or hatched blastocyst stage. In both groups, early blastocysts were less viable than expanded (P < 0.01), hatching or hatched blastocyst (P < 0.05). There was no significant difference in survival rates at days 14 (79 and 76%) and 45 (63 and 59%) after transfer into sychronised recipients between vitrified expanded blastocysts of groups a and b, respectively. These results suggest that it is possible replace serum with PVA in vitrification and warming solutions without reducing in vivo and in vitro viability.

Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts.

Ovine blastocysts were produced by maturation, fertilization and in vitro culture (IVM/IVF/IVC) of oocytes from slaughtered adult and prepubertal ewes and collection from superovulated and inseminated adult animals. Dulbecco's PBS supplemented with 0.3 mM Na Pyruvate and 20% FCS was used as the basic cryopreservation solution. The embryos were exposed to the vitrification solution as follows: 10% glycerol (G) for 5 min, then 10% G +20% ethylene glycol (EG) for 5 min. Embryos were placed into 25% G + 25% EG in the center of 0.25- mL straws and plunged immediately into LN2. Warming was done by placing the straws into a water bath at 37 degrees C for 20 sec, and their contents were expelled into a 0.5 M sucrose solution for 3 min; the embryos were then transferred into 0.25 M and 0.125 M sucrose solution for 3 min each. Warmed blastocysts were transferred to the culture medium for 24 h. Survival was defined as the re-expansion of the blastocoele. All surviving blastocysts were transferred to synchronized recipient ewes, and the pregnancy was allowed to go to term. Of 68 vitrified in vitro produced blastocysts, 46 re-expanded (67.6%) and 10 lambs were born (14.7%). From the 62 in vivo derived and vitrified embryos, 52 re-expanded (83.8%) and 39 lambs were born (62.9%). The lambing rate of in vitro produced fresh transfer embryos was 40% (20 lambs/50 blastocysts transferred), and of the 32 in vivo derived blastocysts and transferred fresh, 26 lambs were born (81.2%). The results indicate that in vitro produced embryos can be successfully cryopreserved by vitrification.

Ovum pick-up in sheep: efficiency of in vitro embryo production, vitrification and birth of offspring.

The production of offspring involving available technologies like ovum pick-up, in vitro embryo production and cryopreservation has not been fully described in the sheep. We tested the overall efficiency of these procedures on 20 Sarda dairy ewes that were twice stimulated for recovery of follicular oocytes. In total, 415 oocytes were aspirated from 522 follicles (11.5 oocytes/ewe), and 328 of them (9.1 oocytes/ewe) were selected for in vitro embryo production procedure. Development into blastocysts occurred in 98 embryos (2.7 blastocysts/ewe), of which 64 were vitrified and 34 were transferred, in pairs, directly to recipients. The pregnancy rate, diagnosed at 80 d for fresh and vitrified embryos, did not differ significantly (47.1 vs 42.8%, respectively), but there were significant differences in lambing rates between the 2 groups (41.2 vs 23.8%, respectively). Overall, 24 lambs were born; all weighed within the range for the breed, but head deformities were observed in 2 cases. The results of this study show that with application of the above techniques, it is possible to obtain repeatedly embryos and viable offspring.

Superovulatory response and quality of embryos recovered from anestrus ewes after a single injection of porcine FSH dissolved in polyvinylpyrrolidone.

The effects of a single injection of porcine FSH (pFSH) administered in long acting vehicle on the superovulatory response of milk (Sarda breed) sheep were determined during the anestrous season. The sheep (n=42), synchronized with intravaginal sponges (40 mg fluorogestone acetate -FGA- for 14 d) were submitted 24 h before sponge removal to three different superovulatory treatments. Group 1 (n=16) was treated with a single intramuscular (im) injection of 16 mg of pFHS dissolved in 30 % polyvinylpyrrolidone (PVP); Group 2 (n=12) was injected im with 6, 5, 3 and 2 mg of pFSH every 12 h over 2 d; Group 3 (n=14) was given 800 IU of PMSG and 12 mg of pFSH. All sheep were mated with a fertile ram. Embryos were recovered surgically at Day 7 of sponge removal and graded for the quality according to their morphology. The percentage of good quality embryos recovered was 84% in Group 1, 68% in Group 2 and 77% in Group 3. Data for the onset of estrus, number of corpora lutea (CL), number of unovulated follicles, embryo recovery rate, embryo quality and fertilization rate were recorded for the 3 groups. The onset of estrus, number of CL, number of unovulated follicles, fertilization rate and number of good quality embryos did not differ significantly among the 3 groups. The embryo recovery rate was significantly lower in the group treated with PMSG-FSH (Group 3) than in the 2 other groups. It is concluded that during the anestrous season a single injection im of pFSH results on average in a superovulatory response as good as the more traditional treatments like multiple injections of pFSH and PMSG-pFSH combined.

Effect of biopsy and vitrification on in vitro survival of ovine embryos at different stages of development.

The objective of the present study was to assess the in vitro viability of ovine embryos at different stages of development after combining cell sampling and vitrification. Precompacted morulae, compacted morulae and blastocysts were obtained from superovulated Sarda ewes at 4, 5 or 6 d following insemination. Embryo cell biopsy was carried out in a 100-microl drop of PBS + 10% fetal calf serum (FCS) with 10 micromol nocodazole and 7.5 microg/ml cytochalasin-b by aspiration (3-5 cells). Embryos were cryopreserved at room temperature after exposure of 2 solutions for 5 min, transferred into a vitrification solution, loaded into the center of 0.25-ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. In Experiment 1, the in vitro viability of manipulated or vitrified embryos after in vitro co-culture in TCM 199 medium with 10% FCS and sheep oviductal epithelial cells (SOEC) in 5% CO2 humidified atmosphere in air at 39 degrees C was significantly lower (P < 0.05 and P < 0.01, respectively) at precompacted morula (60 and 30%) and compacted morula (62 and 39%) stages than intact embryos at the same stages (87 and 88%). No differences were found at the blastocyst stage. In Experiment 2, the in vitro survival rate of precompacted morulae which were manipulated and immediately vitrified was lower (P < 0.05) than in those manipulated and, after a temporary period of culture, vitrified at blastocyst stage (21 vs 48%); while no differences were found at compacted morula and blastocyst stages. The results show that 1) the stage of development influences the subsequent in vitro viability of manipulated and vitrified ovine embryos, 2) temporary culture after manipulation and before vitrification improves the in vitro viability of embryos, and 3) the hole in the zona pellucida resulting from biopsy does not affect blastocyst survival after subsequent vitrification.

Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived.

We compare different vitrification protocols on the pregnancy and lambing rate of in vitro produced (IVP) and in vivo derived (IVD) ovine embryos. Ovine blastocysts were produced by in vitro maturation, fertilization and culture of oocytes collected from slaughtered ewes or superovulated and inseminated animals. Embryos were cryopreserved after exposure at room temperature either for 5 min in 10% glycerol (G), then for 5 min in 10% G + 20% ethylene glycol (EG), then for 30 s in 25% G + 25% EG (glycerol group), or for 3 min in 10% EG + 10% dimethyl sulphoxide (DMSO), then for 30s in 20% EG + 20% DMSO + 0.3 M sucrose (DMSO group). One group of in vitro produced embryos was cryopreserved similarly to the DMSO group, but with 0.75 M sucrose added to the vitrification solution (DMSO 0.75 group). Glycerol group embryos were then loaded into French straws or open pulled Straws (OPS) while the DMSO group embryos were all loaded into OPS and directly plunged into liquid nitrogen. Embryos were warmed with either a one step or three step process. In the one step process, embryos were placed in 0.5 M sucrose. The three-step process was a serial dilution in 0.5, 0.25 and 0.125 M sucrose. The embryos of DMSO 0.75 group were warmed directly by plunging them into tissue culture medium-199 (TCM-199) + 20% foetal bovine serum (FBS) in the absence of sucrose (direct dilution). Following these manipulations, the embryos were transferred in pairs into synchronised recipient ewes and allowed to go to term. The pregnancy and the lambing rate within each group of IVP and IVD embryos indicated that there was no statistical difference among the vitrification protocols.

Embryo cloning in sheep: work in progress.

We summarize here the procedures for nuclear transfer using S-phase cytoplasts and describe a new method for avoiding loss of reconstructed embryos from the oviducts during in vivo culture. We obtained 2 clones of 5 genetically identical animals following the transfer of blastomeres from 16-cell embryos into enucleated preactivated cytoplasts. Metaphase II oocytes and embryos were surgically collected from superovulated Sarda breed ewes 54 and 120 h after sponge removal, respectively. Oocytes were exposed for 15 min to 5 mug/ml of Hoechst 33342 and were micromanipulated at room temperature. Efficiency in embryo reconstruction was 100% for enucleation and 98% for fusion. Embryos were embedded in agar as separate clones and transferred into the oviducts of temporary recipients. The fimbriae were closed with glass-nylon made filters. Embryo recovery from the temporary recipients was 97.3%, with a cleavage rate of 81.4%; development to morula-blastocyst stage was 70.6%. A total of 29 Grade 1 blastocysts corresponding to 5 clones were transferred into 13 naturally synchronous ewes, and scanning was performed at 30 and 90 d. Ten ewes were pregnant at the first scanning and nine at the second for a final pregnancy rate of 71.4%; the survival rate at term was 48%. Overall, we obtained 4 clones of identical lambs: two sets of 5 (one male set and one female set) and two sets of twins (both sets male). Pregnancy length in recipients carrying clones was longer than the standard period in Sarda breed (153 vs 150 d, respectively). Weight at birth was higher for male lambs obtained from nuclear transfer than for normal males (4.1 vs 3.6 kg), while the weight for females was normal.

Lambing rate using vitrified blastocysts is improved by culture with BSA and hyaluronan.

Fatty acid-free bovine serum albumin (BSA(FAF)) can be added to supplement medium used in the culture of sheep embryos. BSA(FAF) was able to support blastocyst and subsequent embryo development at rates equivalent to that of fetal calf serum (FCS)-supplemented medium when fresh embryos were transferred. Furthermore, culture with BSA(FAF) significantly increased development of vitrified blastocysts transferred into synchronized sheep. The addition of the glycosaminoglycan, hyaluronan (HA) to the culture medium in the third and fifth day also increased cryo-tolerance of blastocysts and in turn lambing rate was improved.

Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts.

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation.

Immunization with DNA vaccines encoding different mycobacterial antigens elicits a Th1 type immune response in lambs and protects against Mycobacterium avium subspecies paratuberculosis infection.

Paratuberculosis, or Johne's disease, is a disease of domestic and wild ruminants that culminate with a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis. The aim of this work was to evaluate the type of immune response, Th1 or Th2, induced by DNA vaccinations in lambs of Sarda breed. Twenty-five lambs, serum negative for M. paratuberculosis, were selected at birth from equally serum negative mothers. The lambs were inoculated at 5 months of age with three different mycobacterial antigens cloned into a mammalian expression vector as fusion protein with the enhanced green fluorescent protein (pEGFP-N1). The animals were divided in five groups containing each five lambs. Each group was vaccinated as following (A: physiological solution; B: Gudair; C: p-85A-Mav; D: p-85A-BCG; E: p-Hsp65). Immune response was evaluated by measuring the expression of INF-gamma (Th1 type response) and IL-10 (Th2 type response) by real-time PCR. Gene expression was estimated by comparing the results with that of beta-actin. INF-gamma expression level was increased in lambs vaccinated with plasmids codifying mycobacterial antigens, in particular with p-Hsp65, in comparison with the controls suggesting stimulation of a Th1 immune response similar to that supported by natural infection of M. paratuberculosis. Moreover, animals were infected orally with live M. paratuberculosis. Three months after vaccination and again INF-gamma and IL-10 expression was evaluated in order to verify in vivo the protection level of the vaccines. Plasmids p-85A-BCG and p-Hsp65 seem to elicit a stronger protective immune response against M. paratuberculosis by evaluating the expression level of INF-gamma and evaluating the presence of M. paratuberculosis and animal cell organ damage post-mortem.

In vitro viability of vitrified mouse morulae thawed at different temperatures.

The effect of thawing temperature on in vitro development of vitrified mouse morulae was investigated. The embryos were vitrified in a solution based on ethylene glycol as cryoprotectant, and Ficoll as macromolecule to assist vitrification. They were then thawed at 20 degrees, 37 degrees and 48 degrees C for 6 sec and at 48 degrees C for 2 sec. Among groups, there was no significant difference on the development at 72 h of culture when embryos were thawed at 20 degrees, 37 degrees C for 6 sec or 48 degrees C for 2 sec. At 48 h of culture the embryos thawed at lower temperature had a reduced resumption (69.5%) while the embryos thawed at 37 degrees and 48 degrees C for 2 sec had a higher resumption rate (80.0% and 82.5%). It was concluded that a high development in vitro of vitrified mouse morulae can be obtained at three different temperatures of thawing, although at higher temperatures there seems to be a tendency of an earlier resumption development.

Improved development to blastocyst of ovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes.

The timing of pronuclear formation and the initiation and duration of the DNA synthetic period (S-phase) were determined during the first cell cycle of electrically activated ovine oocytes matured in vivo. Reconstructed embryos were produced by electro-fusion-mediated nuclear transfer of unsynchronized single blastomeres. These were derived from embryos produced in vivo at the 16-cell stage (Day 4) and transferred to enucleated metaphase II oocytes at the time of activation or to enucleated activated oocytes during early, mid, and late stages of the presumptive S-phase. The frequency of development to blastocyst was greatest in embryos reconstructed during the presumptive S-phase of enucleated activated oocytes than in embryos reconstructed at the time of activation (mean 55.4% vs. 21.3%). No significant differences were observed when embryos were reconstructed during early, mid, or late stages of the presumptive S-phase (61.3%, 45.7%, and 57.7%, respectively). The results indicate that the use of enucleated activated oocytes as cytoplasts for embryo reconstruction can increase the frequency of development to blastocyst of embryos reconstructed from unsynchronized donor blastomeres.

Genomic imprinting in ruminants: allele-specific gene expression in parthenogenetic sheep.

Studies in the mouse have established that both parental genomes are essential for normal embryonic development. Parthenogenetic mouse embryos (which have two maternal genomes and no paternal genome), for example, are growth-retarded and die at early postimplantation stages. The distinct maternal and paternal contributions are mediated by genomic imprinting, an epigenetic mechanism by which the expression of certain genes is dependent on whether they are inherited from mother or father. Although comparative studies have established that many imprinted mouse (and rat) genes are allele-specifically expressed in humans as well (and vice versa), so far imprinting studies have not been performed in other mammalian species. When considering evolutionary theories of genomic imprinting, it would be important to know how widely it is conserved among placental mammals. We have investigated its conservation in a bovid ruminant, the domestic sheep, by comparing parthenogenetic and normal control embryos. Our study establishes that, like in the mouse, parthenogenetic development in sheep is associated with growth-retardation and does not proceed beyond early fetal stages. These developmental abnormalities are most likely caused by imprinted genes. We demonstrate that, indeed, like in mice and humans, the growth-related PEG1/MEST and Insulin-like Growth Factor 2 (IGF2) genes are expressed from the paternal chromosome in sheep. These observations suggest that genomic imprinting is conserved in a third, evolutionarily rather diverged group of placental mammals, the ruminants.

Two culture systems showing a biphasic effect on ovine embryo development from the 1-2 cell stage to hatched blastocysts.

This study compared the effect of using either CZB or TCM 199 media on both the development of 1-2 cell ovine embryos from superovulated ewes to the blastocyst stage (Experiment 1), and the hatching process of ovine blastocysts developed in vitro (Experiment 2). For the first 5 d, the CZB medium showed higher rates of embryo development than the TCM 199 medium (p < 0.001). The embryos reaching the > 16 cell stage were 79 vs 52% and 74 vs 20% with or without an oviductal monolayer, respectively, and those reaching the blastocyst stage were 71 vs 46% and 46 vs 13% with or without cells. The CZB medium was less able to support the hatching process of the blastocysts obtained in the first experiment than was the TCM-199 medium + 10% FCS (fetal calf serum) with cells (31 vs 92%; p < 0.001) or without cells (13 vs 66%; p < 0.001). No blastocysts completely escaped from the zona pellucida (ZP) in the CZB medium compared with 80 and 61% in the TCM 199 medium with or without cells, respectively. In Experiment 3, 47% of the blastocysts migrated through the artificial opening of the ZP and hatched completely. After 24 h of culture in the CZB medium, however, they showed blastocoelic cavity breakdown. During the preliminary cleavages, the ovine embryos developed better in CZB medium than in TCM 199, but the latter was more efficient in promoting the hatching process of the blastocysts.

[Use of a protein-free medium for the in vitro development of sheep embryos]. / Impiego di un medium privo di proteine per lo sviluppo in vitro di embrioni di ovino.

The employment of protein-free medium for the culture of ovine embryos collected at the 1-2 cell stage from superovulated ewes was investigated. For this purpose sheep zygotes were randomly allocated in four treatment groups: T1) CZB medium + bovine serum albumin (BSA) on sheep oviductal monolayer (SOM), T2) CZB + polyvinyl alcohol (PVA) + SOM, T3) CZB + PVA + SOM supplemented with inositol (I) and serine (S), T4) TCM 199 + 10% fetal calf serum + SOM. Standard culture conditions were 2 ml of medium in 35 mm Petri dishes, under 5% CO2 in air at 39 degrees C. The percentages of morulae and blastocysts were recorded after 4 and 7 days of culture. After 4 days of culture there was no significant difference (P > 0.05) in the percentage of morulae between embryos cultured in T1 (86%), T2 (85%), T3 (88.8%), and T4 (87.5%). After 7 days the percentages of blastocysts were T1 (70%), T2 (50%), T3 (55.5%) and T4 (46.8%). These data suggest that a protein-free medium, CZB + PVA and CZB + PVA + I + S, can support ovine preimplantation embryo development in vitro; however CZB medium supplemented with BSA enhances development to blastocyst.

[Microencapsulation in Na-alginate and in vitro development of sheep blastomeres]. / Microincapsulazione in Na-alginato e sviluppo in vitro di blastomeri di ovino.

Embryos at 4 cell stage obtained from Sarda ewes superovulated with FSHp (Sigma) were micromanipulated in order to obtain single blastomeres (1/4 E). The 1/4 E have been located randomly in two groups. In the first (Group A n. 30) the 1/4 E have been put back in empty zonae pellucidae; in the second (Group B n. 21) they have been microencapsulated in sodium alginate (1.1%) by dropping cell-alginate solution in a 1.5% CaCl2. Each capsule (1 mm diameter) contained four 1/4 E. The blastomeres have been co-cultured for 5 days in CZB medium on oviductal cell monolayer in a humidified incubator (5% CO2, 95% air, 38.5 degrees C). No differences were found between the groups reaching blastocyst stage after the end of the culture period (A 50%-B 47%).