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).     

Respiratory transfer impedances with pressure input at the mouth and chest

Two methods of measuring respiratory transfer impedance (Ztr) were compared in 14 normal subjects, from 4 to 30 Hz, 1) studying the relationship between transrespiratory pressure (Prs) and flow at the chest when varying pressure at the mouth (Ztrm) and 2) studying the relationship between Prs and flow at the mouth when varying pressure around the chest wall (Ztrw). The similarity of the two relationships was expected on the basis of a T-network model. Almost identical phase responses were obtained from the two methods. Pressure-flow ratios were slightly larger for Ztrw than for Ztrm, but differences did not exceed 2% on average in 11 of 14 subjects. When the data were analyzed with the six-coefficient model proposed by DuBois et al. (J. Appl. Physiol. 8: 587-594, 1956), similar values were found for tissue compliance and tissue inertance but slightly different values for gaseous inertance in the airways (1.97 +/- 0.35 X 10(-2) cmH2O X l-1 X s2 for Ztrw vs. 1.73 +/- 0.26 for Ztrm; P less than 0.01). Similar results were also found for total respiratory resistance but with a slightly larger contribution of airway resistance for Ztrw (64 +/- 14 vs. 57 +/- 10%; P less than 0.05). As a practical conclusion it is recommended to measure Ztrw, which is technically much easier.     

Comparison of electro-fusion and intracytoplasmic nuclear injection methods in pig cloning

This paper methodologically compares the electro-fusion (EF) and intracytoplasmic injection (ICI) methods, as well as simultaneous fusion/activation (SA) and delayed activation (DA), in somatic nuclear transfer in pigs using fetal fibroblast cells. Comparison of the remodeling pattern of donor nuclei after nuclear transfer by ICI or EF showed that a high rate (80-100%) of premature chromosome condensation occurred in both cases whether or not Ca2+ was present in the fusion medium. Formation of pseudo-pronuclei tended to be lower for nuclear transfer performed by the ICI method (65% vs. 85-97%, p < 0.05). In vitro developmental potential of nuclear transfer embryos reconstructed with IVM oocytes using the EF method was higher than that of those produced by the ICI method (blastocyst formation: 19 vs. 5%, p < 0.05), and it was not improved using in vivo-matured oocytes as recipient cytoplasts. Embryos produced using SA protocol developed to blastocysts with the same degree of efficiency as those produced under the DA protocol (11 vs. 12%). Use of the EF method in conjunction with SA was shown to be an efficient method for producing cloned pigs based on producing a cloned normal pig fetus. However, subtle differences in nuclear remodeling patterns between the SA and DA protocols may imply variations in their nuclear reprogramming efficiency.     

A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72-78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.     

Developmental potential and transgene expression of porcine nuclear transfer embryos using somatic cells

We examined whether porcine nuclear transfer (NT) embryos carrying somatic cells have a developmental potential and NT embryos carrying transformed fibroblasts express transgenes in the preimplantation stages. In Experiment 1, different activation methods were applied to NT embryos and the development rates were examined. Relative to A23187 only or A23187/6-DMAP, electrical pulse made a significant increase in both cleavage rate (58.1+/-13.9 or 60.7+/-6.3 vs. 74.9+/-7.5%) and development rate of NT embryos to the blastocyst stage (2.2+/-2.8 or 2.2+/-1.5 vs. 11.0+/-4.1%). In Experiment 2, in vitro developmental competence of NT embryos was investigated. The developmental rate to the blastocyst stage of NT embryos (9.9+/- 2.4% for cumulus cells and 9.8+/-1.6% for fibroblast cells) was significantly lower than that (22.9+/-3.5%) of IVF-derived embryos (P<0.01). NT blastocysts derived from either cumulus (28.9+/-11.4, n = 26) or fibroblast cells (30.2+/-9.9, n = 27) showed smaller mean nuclei numbers than IVF-derived blastocysts (38.6+/-10.4, n = 62) (P<0.05). In Experiment 3, nuclear transfer of porcine fibroblasts expressing the GFP (green fluorescent protein) gene resulted in green blastocysts without losing developmental potential. These results suggest that porcine embryos reconstructed by somatic cell nuclear transfer are capable of developing to preimplantation stage. We conclude that somatic cells expressing exogenous genes can be used as nuclei donors in the production of NT-mediated transgenic pig.     

Cryopreservation of channel catfish sperm: effects of cryoprotectant exposure time, cooling rate, thawing conditions, and male-to-male variation

The purpose of this study was to extend previous work on the cryopreservation of channel catfish (Ictalurus punctatus) sperm. The objectives were to compare the effects of freezing and thawing on motility of sperm for: (1) 1 or 48-h exposure before freezing to 5% methanol and use of 0.5 or 0.25 mL straws; (2) 1 h or 5-day exposure before freezing to 5% methanol; (3) cooling at 45 or 3 degrees C/min; (4) thawing at 30, 40 or 50 degrees C using 5 or 10 s duration, and (5) cryopreservation with 5 or 10% methanol of samples from 50 males to analyze male-to-male variation. No differences were found in motility reduction for 1 or 48 h exposure times in 5% methanol, for use of 0.5 or 0.25 mL straws, or for 1 h or 5-day exposures in 5% methanol. A cooling rate of 45 degrees C/min resulted in lower motility reduction (33+/-9%) than a rate of 3 degrees C/min (83+/-13%) (P=0.002). A thawing temperature of 50 degrees C resulted in lower motility reduction (25+/-14%) than 30 degrees C (51+/-21%) or 40 degrees C (59+/-11%) (P=0.001). A thawing duration of 10 s resulted in lower motility reduction (38+/-12%) than a duration of 5 s (52+/-12%) (P=0.005), and there was an interaction between thawing temperature and duration (P=0.050). A concentration of 5% methanol resulted in lower motility reduction (43+/-17%) than 10% methanol (67+/-14%) (P=0.001). Regression analysis showed no relationship between motility before freezing and after thawing for 5% methanol (r2=0.012) or 10% methanol (r2=0.011).     

Application of the rat thymus receptor in a specific, sensitive and simplified assay of 1,25-dihydroxyvitamin D in blood serum

A simple method has been employed to prepare crude nuclear extract from rat thymus, using hypertonic buffer after previous treatment with hypotonic buffer. The preparation is free from serum vitamin D-binding protein and contains a 3.7 S receptor molecule, which specifically binds 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3). The receptor is of high affinity (KD = 0.85 X 10(-11) M at O degrees C) and low capacity (260-460 fmol/g tissue). The Scatchard analysis of ligand binding results in a concave downward curve. The Hill analysis of the same data gives good linear fitting (r = +0.971) with the Hill coefficient nH = 1.63. These facts indicate positive cooperativity between two ligand binding sites of the rat thymus 1,25-(OH)2D3 receptor. The preparation was used in a competitive protein binding assay of 1,25-(OH)2D in serum extracts, purified on Sep-Pak C18 followed by silica Sep-Pak cartridges. The method was sensitive to 0.5 pg/tube (2.0 ng/l) when 1 ml of serum was extracted. Intra- and interassay coefficients of variation were 9% and 14%, respectively. The serum 1,25-(OH)2D concentration estimated in 33 children (mean age 6.5 +/- 3 years) was 46.6 +/- 18.4 ng/l (mean +/- S.D.).     

Nuclear transfer in cattle using colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells

To assess the developmental potential of nuclear transfer embryos in cattle using mammary gland epithelial (MGE) cells derived from the colostrum, we compared the effectiveness of cloning using those cells and fibroblast cells derived from the ear. The fusion rate of the enucleated oocytes with fibroblast cells (75 +/- 4%) was significantly higher than that with MGE cells (56 +/- 7%, P<0.05). There were no significant differences in the cleavage rate (85 +/- 3% vs. 91+/- 2%) or in the developmental rate to the blastocyst stage (35 +/- 6% vs. 35 +/- 5%) using MGE cells vs. fibroblast cells as donor nuclei (P>0.05). After transfer of blastocysts derived from nuclear transfer embryos produced using MGE cells and fibroblast cells, 13% (4/31) and 16% (6/37) of recipient heifers were pregnant on Day 42 as assessed by ultrasonography, respectively. Two of the 4 and 4 of the 6 recipients of embryos with MGE cell- and fibroblast cell-derived nuclei, respectively, aborted within 150 days of pregnancy. Four live female calves were obtained from MGE cells or fibroblast cells. However, one died from internal hemorrhage of the arteria umbilicalis. The other three calves were normal and healthy. There were no differences in the pregnancy rate or calving rate when using MGE cells vs. fibroblast cells. Microsatellite DNA analyses confirmed that the cloned calves were genetically identical to the donor cows and different from the recipient heifers. We conclude that colostrum-derived MGE cells have the developmental potential to term by nuclear transfer, and the efficiency of development of those cloned embryos was the same as that of embryos obtained using fibroblast cells as donor nuclei, although there was a significant difference in the fusion rate. This method using MGE cells derived from colostrum, which is obtained easily and safely from live adult cows, is more advantageous for cloning with somatic cells.     

In vitro and in vivo survival of frozen-thawed bovine oocytes after IVF,nuclear transfer,and parthenogenetic activation

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus. Mol. Reprod. Dev. 51:281–286, 1998. © 1998 Wiley-Liss, Inc.     

Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidanceof parthenogenesis, and successful embryogenesis

Manipulations of DNA and cellular structures are essential for the propagation of genetically identical animals by nuclear transfer. However, none of the steps have been optimized yet. This study reports a protocol that improves live dynamic imaging of the unfertilized bovine oocyte's meiotic spindle microtubules with microinjected polymerization-competent X-rhodamine-tubulin and/or with vital long-wavelength excited DNA fluorochrome Sybr14 so that the maternal chromosomes can be verifiably removed to make enucleated eggs the starting point for cloning. Suitability of the new fluorochromes was compared to the conventional UV excitable Hoechst 33342 fluorochrome. Enucleation removed the smallest amount of cytoplasm (4-7%) and was 100% efficient only when performed under continuous fluorescence, i.e., longer fluorescence exposure. This was in part due to the finding that the second metaphase spindle is frequently displaced (60.7 +/- 10%) from its previously assumed location subjacent to the first polar body. Removal of as much as 24 +/- 3% of the oocyte cytoplasm underneath the polar body, in the absence of fluorochromes, often resulted in enucleation failure (36 +/- 6%). When labeled oocytes were exposed to fluorescence and later activated, development to the blastocyst stage was lowest in the group labeled with Hoechst 33342 (3%), when compared to Sybr14 (19%), rhodamine-tubulin (23%), or unlabeled oocytes (37%). This suggests that longer wavelength fluorochromes can be employed for live visualization of metaphase spindle components, verification of their complete removal during enucleation, and avoidance of the confusion between artifactual parthenogenesis versus "cloning" success, without compromising the oocyte's developmental potential after activation.     

A Comparative Study on the Efficiency of Two Enucleation Methods in Pig Somatic Cell Nuclear Transfer: Effects of the Squeezing and the Aspiration Methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72–78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.     

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.     

Chemically assisted handmade enucleation of porcine oocytes

The purpose of our work was to find an efficient and reliable chemically assisted procedure for enucleation of porcine oocytes connected to the handmade cloning (HMC) technique without the potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection. After 41-42 h in vitro maturation, porcine oocytes were incubated with 0.4 microg/mL demecolcine for 45 min. Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with extrusion cones or oocytes only with polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm with the extrusion cone or adjacent to the PB was removed with a microblade. The remaining putative cytoplasts, containing the major part of the cytoplasm, were used as recipients for reconstruction with porcine fetal fibroblasts as nuclear donors. The overall efficiency achieved with chemically assisted enucleation was higher compared to oriented bisection without demecolcine incubation (90 +/- 3% vs. 81 +/- 4%, respectively; mean +/- absolute deviation [AD]). Reconstructed and activated embryos were cultured in vitro for 7 days. Fusion, cleavage and blastocyst rates were 87 +/- 7%, 97 +/- 6%, and 28 +/- 9%, respectively. These rates are at least as good as those achieved with normal HMC (81 +/- 4%, 87 +/- 8%, and 21 +/- 9%, respectively). For traditional, micromanipulator-based cloning, fusion and blastocyst rates were similar (81 +/- 10% and 21 +/- 6%, respectively), but the cleavage rate was lower (69 +/- 9%). In conclusion, chemically assisted handmade enucleation seems to be a simpler and potentially superior alternative to more conventional methods used for somatic cell nuclear transfer in pigs.     

Comparison of methods for estimating percent body fat in distance-runners]

A comparative study was conducted between two independent methods to estimate body fat in 13 college male distance-runners (20.2 +/- 1.1 yrs) and 11 male college students (19.6 +/- 0.7 yrs) as control group. The methods dealt with different body component parameters. Body fat was estimated (1) in terms of total body water based on the analysis of dilution of orally ingested deuterium oxide (D2O) in urine, and (2) in terms of body density based on underwater weighing. The results were as follows: 1) The skinfold thickness at 14 sites in distance-runners were thinner than those in control group. The mean values for subcutaneous fat in distance-runners were 4.3 +/- 0.7 kg (7.2 +/- 1.1%), which were lower than those (8.3 +/- 2.7 kg & 13.3 +/- 3.4%) in control group significantly. Oh the other hand, the mean values for internal fat in distance-runners were 8.7 +/- 1.4 kg (14.4 +/- 1.6%), which were larger than those (6.5 +/- 3.1 kg & 10.2 +/- 4.3%) in control group. 2) The mean values for percent body fat in control group were 16.1 +/- 1.6% by skinfolds method, 20.2 +/- 5.1% by body density method and 23.5 +/- 4.6% by total body water method. The estimated values of percent body fat by the methods of body density and total body water were approximately the same. 3) The mean values for percent body fat in distance-runners were 11.9 +/- 1.4% by skinfolds method, 11.8 +/- 1.7% by body density method and 21.5 +/- 1.9% by total body water method. The estimated values of percent body fat in distance-runners were lower than those in control group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatial, temporal and muscle action patterns of Tai Chi gait.

This study was to quantitatively characterize the spatial, temporal, and neuromuscular activation patterns of Tai Chi gait (TCG). Ten healthy young subjects were tested. The kinematics of TCG and normal gait (NG) were measured using a marker-based motion analysis system and two biomechanical force plates. Surface electromyography (EMG) was recorded from six left-side muscles: tibialis anterior, soleus, peronaeus longus, rectus femoris, semitendinosus, and tensor fasciae latae. The results showed that TCG had (1) a longer cycle duration (11.9+/-2.4 vs. 1.3+/-0.2 s) and a longer duration of single-leg stance time (1.8+/-0.6 vs. 0.4+/-0.05 s); (2) a larger joint motion in ankle dorsi/plantar flexion (40+/-9 degrees vs. 20+/-8 degrees), knee flexion (82+/-8 degrees vs. 53+/-10 degrees), hip flexion (81+/-7 degrees vs. 24+/-4 degrees), and hip abduction (20+/-8 degrees vs. 0+/-3 degrees); (3) a larger lateral body shift (>25% vs. 5% body height); and (4) significant involvement of ankle dorsiflexors, knee extensors/hip flexors and hip abductors, as indicated by significantly higher peak (88+/-14%, 80+/-18% and 83+/-17% vs. 35+/-10%, 14+/-8% and 28+/-19% peak amplitude, respectively) and root-mean-square values of their EMG (37+/-6%, 32+/-7% and 33+/-7% vs. 23+/-7%, 11+/-8% and 22+/-11% peak amplitude, respectively), longer proportions of action (76+/-19%, 68+/-8% and 65+/-19% vs. 59+/-23%, 16+/-23% and 40+/-32% gait cycle duration, respectively), longer proportions of isometric and eccentric actions, and longer proportions of co-activations. These results demonstrate that the biomechanical characteristics of TCG can be quantified. The quantification of TCC movements is important for understanding its effect on balance, flexibility, strength, and health.     

Morphometric quantification of mitochondria in the two steroidogenic ovine luteal cell types

Progesterone secretion is regulated by different mechanisms in large and small steroidogenic ovine luteal cells. Large cells secrete approximately 7-fold more progesterone in an unstimulated state than small cells. Since cholesterol side-chain cleavage, which is catalyzed by an inner mitochondrial membrane enzyme complex, is a major rate-limiting step in progesterone synthesis, mitochondrial components were quantified in the two steroidogenic cell types throughout the estrous cycle. Corpora lutea collected on Days 4 (n = 4), 8 (n = 4), 12 (n = 5), and 16 (n = 6) of the estrous cycle were prepared for electron microscopy. Volume densities of cell types within corpora lutea and mitochondrial densities within cell types were estimated by point-counting; nuclear and cytoplasmic volume densities were estimated by planimetric analysis. A total of 570 micrographs (magnification 5300 X) were analyzed. Large cell volume density was unchanged during the cycle (35 +/- 1%) while small cell volume density increased (p less than 0.05) from 13 +/- 1% on Day 4 to 20 +/- 3% on Day 12. Large cell mitochondrial volume density increased (p less than 0.05) from 13 +/- 1% on Day 4 to 23 +/- 1% on Day 16 accompanied by an increase in cytoplasmic volume density such that nuclear to cytoplasmic ratio increased (p less than 0.05) from 1:14 to 1:34 between Days 4 and 16. Small cell mitochondrial volume density increased from 11 +/- 1% on Day 4 to 14 +/- 1% (p less than 0.05) for the rest of the cycle while the nuclear to cytoplasmic ratio remained at 1:14.(ABSTRACT TRUNCATED AT 250 WORDS)     

VO2, VCO2, and RQ in a respiratory chamber: accurate estimation based on a new mathematical model using the Kalman-Bucy method.

A respiratory chamber is used for monitoring O(2) consumption (Vo(2)), CO(2) production (Vco(2)), and respiratory quotient (RQ) in humans, enabling long term (24-h) observation under free-living conditions. Computation of Vo(2) and Vco(2) is currently done by inversion of a mass balance equation, with no consideration of measurement errors and other uncertainties. To improve the accuracy of the results, a new mathematical model is suggested in the present study explicitly accounting for the presence of such uncertainties and error sources and enabling the use of optimal filtering methods. Experiments have been realized, injecting known gas quantities and estimating them using the proposed mathematical model and the Kalman-Bucy (KB) estimation method. The estimates obtained reproduce the known production rates much better than standard methods; in particular, the mean error when fitting the known production rates is 15.6 +/- 0.9 vs. 186 +/- 36 ml/min obtained using a conventional method. Experiments with 11 humans were carried out as well, where Vo(2) and Vco(2) were estimated. The variance of the estimation errors, produced by the KB method, appears relatively small and rapidly convergent. Spectral analysis is performed to assess the residual noise content in the estimates, revealing large improvement: 2.9 +/- 0.8 vs. 3,440 +/- 824 (ml/min)(2) and 1.8 +/- 0.5 vs. 2,057 +/- 532 (ml/min)(2), respectively, for Vo(2) and Vco(2) estimates. Consequently, the accuracy of the computed RQ is also highly improved (0.3 x 10(-4) vs. 800 x 10(-4)). The presented study demonstrates the validity of the proposed model and the improvement in the results when using a KB estimation method to resolve it.     

Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos

The yield and quality of (a) parthenogenetic blastocysts produced by two activation treatments (cycloheximide [CHX] or 6-dimethylaminopurine [DMAP]) and (b) nuclear transfer blastocysts generated using these two activation treatments and three different ages of karyoplast derived from day 3, 4, or 5 in vitro produced donor embryos, were examined in order to define an optimal nuclear transfer protocol. The two activation protocols comprised calcium ionophore followed by either CHX or DMAP. Parthenogenetic blastocyst yields were greater (P < 0.001) following activation with DMAP than CHX (59.7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P < 0.0001) using cytoplasts activated with DMAP. The individual rates using day 3, 4, and 5 donors and using CHX and DMAP activation treatments were 31.9 +/- 5.0, 31.7 +/- 6.2, 20.4 +/- 7.3 and 27.8 +/- 4.7, 20.1 +/- 7.5, 12.7 +/- 8.3, respectively. Blastocyst rate per fused embryo was negatively correlated (P = 0.0091) with the total number of blastomeres per donor embryo. Despite this inverse relationship, the calculated potential blastocyst yield per donor embryo was positively correlated (P < 0.0048) to karyoplast age. The individual potential yields on days 3, 4, and 5 and for the two activation protocols (CHX and DMAP) were 4.7 +/- 0.8, 7.2 +/- 1.2, 10.1 +/- 2.1 and 3.8 +/- 0.8, 5.5 +/- 2.1, 7.3 +/- 4.1, respectively. One possible explanation for the observed inverse relationship is that differentiation events during early cleavage are able to reduce the ability of the cytoplast to reprogram the transferred karyoplast and hence reduce blastocyst yields. The mechanism that mediates the differential effect of the CHX and DMAP on blastocysts yields between parthenogenetic and nuclear transfer embryos remains to be elucidated. In conclusion, the results indicate that although activation of oocytes with DMAP can produce a higher percentage of blastocysts, CHX activation is superior for use in nuclear transfer.     

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.