P.J. Booth

HIGH BOVINE BLASTOCYST DEVELOPMENT IN A STATIC IN VITRO PRODUCTION SYSTEM USING SOFAA MEDIUM SUPPLEMENTED WITH SODIUM CITRATE AND MYO-INOSITOL WITH OR WITHOUT SERUM-PROTEINS

We describe a bovine embryo culture system that supports repeatable high development in the presence of serum or BSA as well as under defined conditions in the absence of those components. In the first experiment, embryo development in SOF with amino acids (SOFaa), sodium citrate (SOFaac) and myo-inositol (SOFaaci) and with BSA or polyvinyl alcohol (PVA) was compared with that in a M199 granulosa cell co-culture (M199 co-culture). Subsequently, development and cell numbers of blastocysts cultured under defined conditions in SOFaaci with PVA (SOFaaci-PVA), or under undefined conditions in SOFaaci with 5% cow serum (SOFaaci-CS) or M199 co-culture were compared. The repeatability of culture results in SOFaaci-CS was checked by weekly replicates (n = 30) spread over 11 months. The viability of embryos developed in SOFaaci-PVA was estimated by transfer of morphologically good blastocysts (n = 10) to synchronized recipients. In the second experiment, the effect of omitting CS or BSA from IVM and IVM-IVF on subsequent embryo development in SOFaaci-PVA or in SOFaaci-CS was investigated. Blastocyst development in SOFaa-PVA, SOFaac-PVA, SOFaa-BSA and M199 was 16 +/- 3b, 23 +/- 2ab, 30 +/- 8a and 36 +/- 7a%, respectively (Pab < 0.05). Additional inclusion of myoinositol resulted in 42 +/- 1a% blastocysts in SOFaaci-PVA vs 19 +/- 3b% in SOFaac-PVA, 47 +/- 7a% in SOFaac-BSA, and 36 +/- 7a% in M199 co-culture, respectively (Pab < 0.01). In 30 replicates, the average cleavage and blastocyst rates of oocytes in SOFaaci-CS were 87 +/- 4 and 49 +/- 5%, respectively. Five normal calves were produced after transfer of 10 blastocysts developed in defined culture medium (i.e., SOFaaci-PVA). Defined IVM or IVM-IVF (i.e., in absence of CS and BSA) reduced cleavage rates (83 +/- 3 and 55 +/- 3% vs 90 +/- 1% in presence of CS; P < 0.01). Subsequent embryo development in SOFaaci-CS was not affected in either of these defined conditions. However, cleavage and blastocyst rates under completely defined IVP conditions were 54 +/- 7 and 19 +/- 4%, respectively. It was concluded that under defined culture conditions, addition of citrate and myo-inositol improved blastocyst development to rates comparable to those obtained with serum, BSA or co-culture and that the quality of blastocysts was not affected by the absence of serum or BSA. However, serum was essential during IVM/IVF for normal fertilization and subsequent high blastocyst development.

Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique.

Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods. The technique comprises the bisection of zona-free oocytes and the reconstruction of embryos comprising two half cytoplasts and a somatic cell by adherence using phytohaemagglutinin-P (PHA) followed by an electropulse and subsequent culture in microwells (termed WOWs--well of the well). The development of the system was based on results using parthenogenetic and in vitro fertilized zygotes in order to (a) select the optimal primary activation agent that induced the lowest lysis rate but highest parthenogenetic blastocyst yield, (b) evaluate the quantity and quality of zona-free blastocysts produced in WOWs, and (c) establish any potential embryotoxic effects of PHA-P. The initial data indicated that, of calcium ionophore A23187, ionomycin, and electropulse treatments as primary activation agents, the two former were equally efficient even with reduced exposure times. WOW-culture of zona-free versus zona-intact zygotes were not different in either blastocyst yield (44.6 +/- 2.4% versus 51.8 +/- 13.5% [mean +/- SEM]) or quality (126.3 +/- 48.4 versus 119.9 +/- 32.6 total cells), and exposure of zygotes to PHA-P did not reduce blastocyst yields compared to vehicle control (40.8 +/- 11.6% versus 47.1 +/- 20.8% of cultured oocytes). Subsequent application of the optimized technique for nuclear transfer using nine different granulosa cell primary cultures (cultured in 0.5% serum for 5-12 days) generated 37.6 +/- 3.9% (11 replicates; range, 16.4-58.1%) blastocysts per successfully fused and surviving reconstructed embryo (after activation), and 33.6 +/- 3.7% blastocysts per attempted reconstructed embryo. Mean day 7 total blastocyst cell numbers from 5 clone families was 128.1 +/- 15.3. The ongoing pregnancy rate of recipients each receiving two nuclear transfer blastocysts is 3/13 (23.1%) recipients pregnant at 5 months after transfer. These results suggest that the zona-free nuclear transfer technique generates blastocysts of equivalent quantity and quality compared to conventional micromanipulation methods, requires less technical expertise, is less time consuming and can double the daily output of reconstructed embryos (even after taking into consideration the rejection of the half oocytes containing the metaphase plate).

Numerical Chromosome Errors in Day 7 Somatic Nuclear Transfer Bovine Blastocysts

Abstract Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulation method: half-cytoplasts were made from zona-free oocytes by bisection, after which two half-oocytes and one granulosa cell (serum-starved primary culture) were fused together and activated. The NT embryos were cultured in modified synthetic oviductal fluid containing essential and nonessential amino acids, myoinositol, sodium citrate, and 5% cattle serum in microwells for 7 days, at which time nuclei from all blastocysts were extracted and chromosome aberrations were evaluated using dual-color fluorescent in situ hybridization with bovine chromosome 6- and 7-specific probes. Five embryo clone families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 ± 3.7% (mean ± SEM) diploid, 2.6 ± 0.5% triploid, 10.0 ± 3.1% tetraploid, and 0.5 ± 0.2% pentaploid or greater; the vast majority (>75%) of the abnormal nuclei were tetraploid. Completely diploid and mixoploid embryos represented 22.1 ± 4.5% and 73.7 ± 5.5%, respectively, of all clones. Six totally polyploid blastocysts, containing ≤91 nuclei, were recorded. The ploidy distributions (classified as 2N, 3N, 4N, and ≥5N chromosome complements, respectively) between two clone families were different (P < 0.01), as were blastocyst yields between other clone families (P < 0.01). Blastocyst yield was not correlated to % total ploidy error between clone families, but an inverse relationship (P < 0.01) between blastocyst total cell number and total % chromosome abnormality was observed within embryos. Categorization of the blastocysts into three quality grades (good, medium, and poor) and comparison of the distribution of ploidies when classified into 0%, 0.1–5.0%, 5.1–10.0%, 10.1–15.0%, and 15.1–100% errors within embryos indicated that medium- and poor-grade embryos were different (P < 0.05) from good-quality, in vitro-produced embryos. In a separate study, 11 different granulosa cell cultures (that did not correspond to those used for NT) were evaluated and found to possess only 0.23 ± 0.12% ploidy errors. These results demonstrate that 1) the percentage of ploidy errors in bovine NT blastocysts is inversely related to total blastocyst cell number, 2) the mixoploid condition is representative of the majority of embryos, 3) 100% polyploid NT blastocysts can exist, and 4) the ploidy errors seem not to be derived from the donor cells.

Application of the Zona-Free Manipulation Technique to Porcine Somatic Nuclear Transfer

The recent demonstration of a successful zona-free manipulation technique for bovine somatic nuclear transfer (NT) that is both simpler and less labor intensive is of considerable benefit to advance the applications of this technology. Here, we describe that this method is also applicable to porcine somatic NT. Porcine cumulus oocyte complexes were matured in TCM-199 medium before sequential removal of the cumulus and zonae. Zona-free oocytes were bisected using a microknife, and the halves containing the metaphase plate (as determined by Hoechst 33342 staining) were discarded. Each half cytoplast was agglutinated to a single granulosa cell (primary cultures grown in 0.5% serum for 2-5 days prior to use) in phytohaemagglutinin-P. Subsequently, each half cytoplast-granulosa cell couplet was simultaneously electrofused together and to another half cytoplast. Reconstructed embryos were activated in calcium ionophore A23187 followed by DMAP and were then individually cultured in microwells in NCSU-23 medium. On day 7 after activation, blastocyst yield and total cell numbers were counted. Of 279 attempted reconstructed NT embryos, 85.0 +/- 2.8% (mean +/- SEM; n = 5 replicates) successfully fused and survived activation. The blastocyst rate (per successfully fused and surviving embryo) was 4.8 +/- 2.3% (11/236; range, 0-12.8%). Total blastocyst cell count was 36.0 +/- 4.5 (range, 18-58 cells). The blastocyst rate and total cell numbers of parthenogenetically activated and zona-free control oocytes propagated under the same conditions was 11.6 +/- 3.9% (35/335 embryos; n = 3 replicates) and 36.8 +/- 5.2, respectively. Developmentally halted embryos that could still be evaluated on day 7 possessed 54.4 +/- 2.3% (53/96 embryos; n = 3 replicates) anucleate blastomeres, the latter representing 53.5 +/- 6.6% of the blastomeres in such embryos. In conclusion, blastocyst yield was independent of activation efficiency and was likely reduced by insufficient nuclear remodeling, reprogramming, imprinting, or other effects. The data also suggest that fragmentation was a considerable problem that could conceivably contribute to halted development in a high proportion of embryos. The results indicate that the zona-free manipulation technique can be successfully applied to pig somatic NT. Although such zona-free early cleavage stage embryos cannot be transferred to recipients at present, this technique permits simplification of the NT technique for application in basic research, until pig nonsurgical blastocyst transfer becomes a realistic option.

Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts: Work in progress

Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 microM, 5 min) and cycloheximide (10 microg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified,in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% +/- SEM) were not affected (83.7+/-9.2 vs. 79.8+/-4.6; P>0.05), but cleavage (55.7+/-2.9 vs. 92.8+/-3.9; P = 0.0002) and blastocyst rates (7.2+/-5.0 vs. 32.6+/-7.8; P = 0.0025, vitrified vs. nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos.

Developmental Kinetics of Bovine Nuclear Transfer and Parthenogenetic Embryos

Early developmental kinetics of nuclear transfer (NT) embryos reconstituted with blastomeres and parthenogenones produced by ionophore activation followed by either dimethylaminopurine (DMAP) or cycloheximide (CHX) treatment was studied. In vitro produced (IVP) embryos served as controls. Embryos were cultured to the hatched blastocyst stage, and images were recorded every 0.5 h throughout the culture period. The longest cell cycle shifted from 4th to 5th cycle (26 +/- 4 and 44 +/- 5 h) in NT-embryos compared to IVP-embryos (41 +/- 2 and 20 +/- 3 h) and showed greater asynchrony between blastomeres than any other embryo category. Compared to DMAP, CHX prolonged the 1(st) (23 +/- 1 vs. 33 +/- 1 h) and shortened the 3(rd) cell cycle (17 +/- 2 vs. 13 +/- 1 h). Moreover, though cytoskeleton activity was initialised, a larger proportion of CHX embryos was unable to accomplish first cleavage. The parthegenones differed from IVP embryos with respect to the lengths of the 1st, 3rd, and 4th cell cycles and time of hatching. The findings are discussed in relation to known ultrastructural, chromosomal and genomic aberrations found in NT embryos and parthenogenones. We hypothesize that the shift of the longest cell cycle in NT embryos is associated with a shift in the time of major genomic transition.

Ploidy of Bovine Nuclear Transfer Blastocysts Reconstructed Using In Vitro Produced Blastomere Donors

The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro development. Because little is known about ploidy errors in nuclear transfer embryos, this was examined using embryos reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6-dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blastomeres from day 4-5 in vitro produced donors. The embryos were cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 +/- 20.2 (mean % +/- SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8%) were potentially abnormal because in these, 1.1%, 1.4%, 5.3%, 7.5%, 26.3%, 30.4%, and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors comprised mainly triploid (8.2 +/- 10.3 [0-26.3]: % +/- SD [range]) and tetraploid (10.6 +/- 19.9 [0-54.9]) nuclei with other ploidy combinations accounting for only 0.9 +/- 2.1 [0-2.1]% of deviant nuclei. The proportion of completely normal nuclear transfer embryos was no less than those produced by in vitro fertilization but the distribution of chromosome abnormalities was different (p = 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chromosome abnormalities to embryonic loss in the remaining embryos deserves further investigation.

Post-thaw in vitro survival of vitrified cloned bovine embryos

Heat-induced illness in dogs HEATSTROKE is a well recognised condition in dogs, particularly in hot, humid conditions, but clinical information is limited. In reviewing the medical records of 42 cases, it was found that factors associated with a poor outcome included coma or hypothermia at the initial examination, ventricular arrhythmias, low serum concentrations of albumin, total protein and cholesterol, and high serum concentrations of total bilirubin and creatinine. The median age of the dogs was three years and age did not affect their chance of survival. The median period for which they were kept in hospital was 40-5 hours, but some were discharged immediately after having been examined and others were kept in for up to 10 days. Of the 42 dogs, 15 died or were euthanased and 27 survived. Some of the dogs had exercised in the heat for as little as 20 to 30 minutes, but died in spite of intensive medical treatment. DROBATZ, K. J. & MACINTIRE, D. K. (1996) Journal of the American Veterinary Medical Association 209, 1894