bluuebunny/crossref_metadata_2025 huggingface dataset:
59IN VITRO AND IN VIVO SURVIVAL OF NELORE NUCLEAR TRANSFER EMBRYOS
RECONSTRUCTED WITH ADULT AND FETAL FIBROBLASTS
Authors: Mello M.R.B. , Caetano H.V.A. , Padilha M.S. , Marques M.G. , Nicacio A.C. , Gerger R.P.C. , Mendes C.M. , Oliveira V.P. , Visintin J.A.
Published: February 2004

Adult skin and fetal fibroblasts are the most frequently used donor cell types for bovine cloning by nuclear transfer (NT) but there are few reports concerning Nelore cattle. The aim of this study was to evaluate in vitro and in vivo viability of Nelore nuclear transfer embryos reconstructed with Metaphase II oocytes and differentiated somatic cells (adult ear and fetal fibroblasts). Oocytes from ovaries collected at slaughterhouse were matured in vitro for 17h. Enucleation was conducted by aspiration of the first polar body (PB) and small volume of cytoplasm containing metaphase plate. For NT, each nucleus donor cell was inserted under the zona pellucida of each enucleated oocyte and the enucleated oocyte-nuclei donor cell complexes were electrofused (2 pulses of 4KVcm−1 for 20s). After electrical activation, the couplets were incubated in TCM199 plus 7.5% FCS supplemented with cycloheximide (10gmL−1) and cytochalasin D (2.5gmL−1) for 1h and ciclohexemide alone for 4 additional hours. Immediately after activation, reconstructed embryos were co-cultured with granulosa cells in SOF + 5% FCS for 7–9 days. At 7th day of culture, some blastocysts were fixed for counting cells and some transferred into recipients. A total of 377 couplets were reconstructed from fetal and 457 from adult fibroblasts. After electrofusion, 138 (fetal cells) and 166 (adult cells) embryos were incubated, and 24 (17.4%) and 26 (15.7%) reached blastocyst stage, respectively. The blastocyst cell number means were 101.3, 120 and 114.3, respectively, for adult, fetal and IVF (control) embryos. There were no significant differences in the numbers of cells of blastocysts among the groups. After transferring 18 (fetal cells) and 21 (adult cells) blastocysts, pregnancy rates at day 90 were 16.7% (3) and 19% (4). There were no significant differences between pregnancy rates. The first pregnancy from fetal cells delivered a healthy male calf weighing 34kg at Day 290. One of the remaining recipients died with hydrallantois at Day 229 and the other aborted at Day 252. There are four 5-month-pregnancies of adult fibroblast reconstructed embryos. These results indicate that NT embryos produced by fetal and adult fibroblasts of Nelore breed show similar rates of in vitro and in vivo developments. This work was supported by FAPESP (99/07377-3).
67ORGAN WEIGHT VARIATION IN CLONED PIGS
Authors: Piedrahita J.A. , Walker S. , Zaunbrecher G.
Published: February 2004

Previously, we compared adult clones and naturally bred control females using a series of physiological and genetic parameters and found considerable variation in certain traits in clones. In order to more fully understand this clonal variation we examined the organ weight differences among a group of clones from a single litter. Briefly, in vitro-matured oocytes were stripped of their cumulus cells at 46–48h postmaturation by vortexing in TCM 199 containing 5% FCS with 0.1% hyaluronidase, and placed in Ca-free NCSU-23 with 4mgmL−1 BSA. Oocytes were stained in Ca-free NCSU-23 containing 4mgmL−1 BSA and 5μgmL−1 Hoechst 3334 for 10min, and enucleated in Ca-free, salt buffered, NCSU-23 containing 5% FCS and 7.5μgmL−1 of cytochalasin B, Puromycin-resistant fetal fibroblasts grown for 35 days in D-MEM/F-12 containing 15% FCS and 1.65μgmL−1 puromycin were synchromized by contact inhibition for 1-2 days. Cells were trypsinized, resuspended in salt buffered NCSU-23 with 10% FCS and placed into the perivitelline space. Couplets wer fused by 2V AC for 2s followed by 2 pulses DC of 1.4kvcm−1 for 50μs. Following fusion, the oocytes were placed in NCSU-23 containing 4mgmL−1 BSA for 1.5–2h. The oocytes were then electroactivated by 2 pulses of 1.2kgcm−1 for 50μs, allowed to recuperate for 10–15min, and transferred into the oviduct of a naturally synchronized recipient. A total of 286 doublets were transferred into 4 recipients one of which became pregnant and gave birth to 9 clones (9/81). One clone died soon after birth. The remaining 8 clones were weaned at three weeks and kept as a group until Day 120 at which time the animals were euthanized for organ recovery. Weights were determined for major organs, and coefficients of variation (CV) calculated for both absolute weights, and corrected total weight. Corrected weights were calculated as weight of organ divided by weight of clone. CV for absolute and corrected weights, respectively, were: uterus, 15.1% and 5%; left kidney, 13.2% and 6.9%; right kidney, 11% and 6.8%; spleen, 29.7% and 27.1%; liver, 20.6% and 19.7%; lung, 37.5% and 32%; heart, 20.2% and 15.5%; and brain, 5.6% and 9.5%. Both absolute and corrected weights indicated that there is a high degree of variation in some of the organs among a group of identical clones. To determine whether the variation we observed is typical of non-cloned swine, we compared the variations in the clones to those observed in a group of 3 age-matched, sex-matched, breed-matched controls that had been kept with the clones for approximately 90 days. Relative coefficients of variation of corrected organ weights between clones and non-cloned animals were: uterus, 0.18; left kidney, 0.34; right kidney, 0.34; spleen, 2.58; liver, 1.08; lung, 1.06; heart, 0.59; and brain, 0.7. In summary, these results indicate that some organs in cloned animals have a high degree of weight variability, most likely due to epigenetic aberrations of overriding environmental effects.
68EFFECT OF ROSCOVITINE ON FIBROBLASTS' ABILITY TO FORM BLASTOCYSTS AND
ESTABLISH PREGNANCIES AFTER BOVINE NUCLEAR TRANSFER
Authors: Powell A.M. , Graininger P. , Talbot N. , Wall R.J.
Published: February 2004

Cloning efficiency of fibroblast nuclear transfer is dependent on donor cell chromatin status. Chromatin status is commonly regulated by serum starvation or contact inhibition. We have tested 3 methods of synchronizing chromatin activity, roscovitine exposure (in MEM + 10% serum) for 24h, with serum starvation (0.5% serum) for 5 days or growth to confluence in 10% serum prior to nuclear transfer. Roscovitine, a specific cyclin-dependent kinase (CDK)2 inhibitor, provides a means of precisely synchronizing bovine fetal fibroblasts (BFF) at G0/G1 cell cycle stage. Fibroblasts were from 100-day-old Jersey fetuses. Cells, frozen at passage 2, from fetus 10 are known to produce calves. Fetus 13 cells, frozen at passages 1 and 2, were compared for their ability to serve as nuclear donor cells. Oocytes, either purchased from Bomed or harvested from ovaries obtained from a local slaughterhouse and matured in Ham’s F10, were enucleated between 18–21h post-maturation initiation. Couplets were produced and fused by standard techniques. Embryos were activated 2 to 4 hours after fusion by exposure to ionomycin for 4min and DMAP for 4h. Embryos were then held in CR1aa for 12h before being cultured in G1 media for 3 days and then G2 media for another 3 days (38.5°C and 5% O2 + 5% CO2 + 90% N). On Day 7, good quality blastocysts were transferred to synchronized recipient heifers. The remaining embryos were evaluated after another day in culture. Blastocyst development [(100) X (total blastocysts/fused couplets)] was not influenced by fetus (BFF10, 31±3%; BFF13, 26±2%, P=0.126). However, a higher proportion of blastocysts were produced when fibroblasts were cultured in 0.5% serum (38±3%) compared to culture in 10% serum (29±3%) or in roscovitine (23±2%, P=0.001). Time in culture, as measured by passage, had a variable effect on the fibroblast’s ability to product blastocysts from the three fibroblast culture conditions tested. Passage 1 and 2 fibroblasts responded similarly to the 0.5% and 10% serum treatments (P>0.80). When cultured in roscovitine, passage 1 fibroblasts performed better then passage 2 fibroblasts (29±4% v. 16±3% blastocysts, P=0.010). Embryos have been transferred to 51 recipients to date. Ten recipients have given birth or are still pregnant. The 60-day non-return rate for those animals was 29%, 50%, and 31% for serum starvation, 10% serum, and roscovitine treatments, respectively. BFF10 and BFF13 cells have generated the same non-return rates (33%). In this study, of the 3 methods of synchronizing fibroblast chromatin, serum-starvation had an in vitro advantage. Cells cultured for different lengths of time (passages) responded differently to synchronization treatments. This may reflect a heterogeneous population of cells at early passages. Current non-return rates seem to favor synchronization by contact inhibition. Any advantage roscovitine offers may not be revealed until calving.
80DEVELOPMENTAL POTENTIAL OF BOVINE NUCLEAR TRANSFER EMBRYOS DERIVED
FROM FETAL FIBROBLASTS TRANSDUCED WITH SIMIAN VIRUS 40 LARGE T ANTIGEN
Authors: Zakhartchenko V. , Yang F. , Vogg B. , Pfeifer A. , Wolf E.
Published: February 2004

Primary cultured cells with limited lifespan are generally used as donors for nuclear transfer (NT). Immortalization or even extension of their proliferative capacity would provide an additional time-window for various genetic manipulations prior to NT. Previously, we reported that a spontaneously immortalized mammary epithelial cell line failed to support development of reconstructed embryos into blastocysts [Zakhartchenko et al., 1999 Mol. Reprod. Dev. 54, 264–272]. In contrast, telomerase-immortalized sheep fibroblasts could be substantially reprogrammed, although they were not fully competent for NT since no fetuses survived beyond 40 days of development [Cui et al., 2003 Biol. Reprod. 69, 15–21]. Simian virus 40 large T antigen (SV40Tag), a viral oncoprotein, is known to immortalize human diploid fibroblasts by soaking up the cellular negative growth regulators, pRb, p53, and some related factors to enable cells to grow continuously [Kim et al., 2001 Exp. Mol. Med. 33, 293–298]. In this study we examined the developmental competence of bovine NT embryos derived from SV40Tag-transduced fetal fibroblasts. Primary fetal fibroblasts (BFF) obtained from a 49-day-old fetus were first transduced with the green fluorescent protein (GFP) gene and then with SV40Tag using a replication-defective retrovirus. GFP-SV40Tag-positive cells (BFF-GFP-Tag), which can proliferate over 50 passages, were cultured until confluence and then used for nuclear transfer at passages 27–30. As control, confluent GFP-positive cells (BFF-GFP) and BFF, which stopped proliferation after 28 passages, were used at passages 4–6 and 1–2, respectively. Nuclear transfer and embryo culture procedures were essentially as described previously [Shi et al., 2003 Biol. Reprod. 69, 301–309]. Data were compared using chi-square test; differences were considered significant for P<0.05. There were no significant differences in the fusion and cleavage rates between all three groups [BFF-GFP-Tag: 233/248 (94%) and 167/233 (72%); BFF-GFP: 274/294 (93%) and 216/274 (79%); BFF: 129/136 (96%) and 97/129 (75%)]. However, development to the blastocyst stage was significantly lower (P<0.05) in the BFF-GFP-Tag group [5/233 (2%)] than in the BFF-GFP and BFF groups [110/275 (40%) and 53/129 (41%), respectively)]. From our results we conclude that SV40Tag-transduced bovine fetal fibroblasts with high proliferative potential may have lost the ability to respond to the normal cell cycle controls and, as a consequence, have low developmental potential for nuclear transfer.
81IN VITRO MATURATION, FERTILIZATION AND DEVELOPMENT OF BOVINE IMMATURE
OOCYTES CRYOPRESERVED BY VITRIFICATION WITH STEPWISE EXPOSURE USING A NYLON MESH
Authors: Abe Y. , Hara K. , Matsumoto H. , Sasada H. , Ekwall H. , R-Martinez H. , Sato E.
Published: February 2004

Vitrification of bovine immature oocytes has been reported using an open pulled straw, but with limited success. In a previous report, we developed an alternative material (nylon mesh) for vitrification of large quantities of oocytes and embryos. This study was conducted to demonstrate effects of components of a cryoprotectant and a protocol of exposure for bovine immature oocytes on their subsequent in vitro maturation, fertilization and development after cryopreservation by vitrification using a nylon mesh. Bovine oocytes at the germinal vesicle stage were collected from 2–5mm follicles in ovaries, and cumulus-oocytes complexes (COCs) were randomly assigned to treatment groups. Before vitrification, COCs were exposed to the cryoprotectant, which was composed of 40% ethylene glycol, 18% ficoll and 0.3M sucrose (EFS40) or 0.3M trehalose (EFT40) by single step or stepwise exposure. Forty COCs were transferred onto a nylon mesh (0.5cm2), which was then plunged directly into liquid nitrogen. After thawing in warm medium, vitrified COCs were in vitro-matured, fertilized and cultured. After culture for in vitro maturation, the rates in the oocytes reaching to metaphase II were 64.1% and 63.1% in the stepwise exposure to EFS40 or EFT40, respectively, which was significantly higher (P<0.05) than the corresponding rates after a single step (22.6% and 10.0%, respectively). There was no significant effect of the two sugars on in vitro maturation after single or step-wise equilibration. Transmission electron microscopy revealed that the cytoplasm of oocytes equilibrated in a single step had many vacuoles and broken mitochondria, while oocytes equilibrated in a step-wise manner had significantly fewer abnormalities and were similar to untreated controls. Cleavage rate of thawed oocytes after IVMFC was significantly higher after stepwise exposure to EFS40 or EFT40 than that after single step exposure (37.7% and 22.2% v. 20.8% and 0%, respectively, P<0.05). Step-wise equilibration of oocytes in EFT40 was dramatically detrimental: no cleaved embryos developed to blastocysts after a single step exposure to either vitrification solution, or stepwise exposure to EFT40. However, blastocysts were obtained following stepwise exposure to EFS40 (8%). These results suggest that stepwise equilibration and vitrification on a nylon mesh minimizes structural damage to the organelles of immature oocytes and facilitates successful cryopreservation.
87ONE-STEP VERSUS TWO-STEP VITRIFICATION AND IN-STRAW DILUTION OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Campos-Chillon L.F. , Walker D.J. , De La Torre-Sanchez J.F.
Published: February 2004

Slow-cooling techniques are widely used in cryopreservation of bovine embryos. We have previously developed a simple, two-step vitrification technique for direct transfer in the field; however, simplification to one-step vitrification would be attractive. Therefore, factorial combinations of two techniques (one-step and two-step) and two post-thaw temperatures until culture (24 and 37°C) were studied. Blastocysts (n=220) sired by two bulls were obtained in vitro in four replicates. Briefly, oocytes were aspirated from 2–8-mm follicles of ovaries obtained at a slaughterhouse, matured, fertilized and cultured in vitro with standard procedures using chemically defined media (CDM1/2 or G1/2). Two-step embryos were transferred in 1μL into 1mL of V1-CDM (5M ethylene glycol (EG) in HEPES-buffered holding medium (HCDM2)), and one-step embryos into a 7-μL droplet of V2-CDM (7M EG, 0.5M galactose and 18% w/v Ficoll 70 in HCDM2) for 3min at 24°C. Next, embryos for the two-step method were moved in 1μL into a 7μL droplet of V2-CDM at 24°C. Droplets containing embryos (one or two-step) were loaded into 0.25-mL straws preloaded with a 1-cm column of D-HCDM (0.5M galactose in HCDM2), then 0.5cm air, and then 7cm of D-HCDM followed by 0.5cm air. The column containing the embryos (0.5cm (7μL)) was followed with 0.5cm air and 1cm of D-HCDM. Straws were heat-sealed and plunged vertically, sealed end first, into liquid nitrogen just covering the embryo, and the rest of the straw was then slowly immersed. The time from loading to plunging was 40–50s. Straws were thawed in air (24°C) for 10s and then in water horizontally at 37°C until ice disappeared. Straws were gently shaken to mix the columns; then, after 5min at 24 or 37°C, embryos were expelled and cultured in CDM2+5% FCS. Re-expansion and hatching rates were evaluated 48 h post thaw. Data (Table 1) were calculated as a percentage of non-vitrified controls for respective replicates (control means: re-expansion 87%; hatching 74%) and analyzed by ANOVA. There were no main effects of post-thawing temperature (P>0.1), indicating that, after thawing, embryos can be kept at room or body temperature. Also, main effect means for re-expansion and hatching for one-step or two-step addition of cryoprotectant were similar (P>0.1), but there was a tendency for higher survival for the two-step procedure. Further refinements of the one-step technique including EG concentrations, embryological stages and equilibration times should be studied.

Table 1
Main effect means (least-square means±SEM) of vitrified embryos (% of non-vitrified controls)
89EFFECT OF PRE-EQUILIBRATION PROCEDURES ON THE DEVELOPMENT POTENTIAL OF
VITRIFIED BOVINE OOCYTES AFTER IVF
Authors: Chang W.C. , Xu J. , Jiang S. , Tian X.C. , Yang X. , Du F.L.
Published: February 2004

This experiment was designed to improve oocyte developmental potential after droplet vitrification and IVF by different equilibration procedures as reported by Papis K. et al. (2000 Theriogenology 54, 651–658). Bovine oocyte-cumulus complexes were collected from slaughterhouse ovaries, and matured in vitro for 24 hours in TCM199 medium supplemented with 7.5% FBS, 0.5μgmL−1 FSH, 5μgmL−1 LH and 2μgmL−1 estrodial under 5% CO2 in humidified air at 39°C. Oocytes were then partially stripped of most expanded cumulus cells with only 3–5 inner layers left by a short exposure to 0.1% hyaluronidase and careful pipetting. Oocytes were randomly assigned to the following pre-equilibration treatments (39°C): Group 1, oocytes were pre-equilibrated in medium 1 consisting of holding medium (HEPES-buffered TCM199 supplemented with 20% FBS)+10% ethylene glycol (EG) (v/v)+10% dimethylsulphoxide (DMSO) (v/v) for 30–45s; Group 2, oocytes were pre-equilibrated in medium 1 for 3min; and Group 3, oocytes were pre-equilibrated in medium 2 (holding medium+3% EG+3% DMSO) for 20min. Oocytes were then equilibrated in 1.0mL of vitrification medium (holding medium+20% EG and 20% DMSO) as described by Vajta G et al. (1998 Mol. Reprod. Dev. 51, 53–58). Each droplet contained 8–10 oocytes in about 2μL vitrification medium and was dropped into liquid nitrogen immediately after 25–30s exposure to the vitrification solution. Vitrified oocytes were subsequently warmed by transfer into 3mL holding medium containing 0.25M sucrose (39°C). After standard BO IVF procedure, presumptive zygotes were cultured in CR1-aa medium supplemented with 6mgmL−1 BSA at 39°C in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 for eight days. Non-vitrified oocytes were used as a control. Data were analyzed with General Linear Model, SPSS 11.0. As shown in Table 1, there was a significant low survival rate (P<0.05) in Group 3 compared to other treatments. The cleavage rates of Groups 1 and 2 were significantly (P<0.05) higher than that of Group 3, but lower than that in the Control. Furthermore, blastocyst rate on Day 8 in Group 2 was significantly (P<0.05) higher than in the other two experimental groups, but still lower than the Control. This study suggests that the developmental capacity of vitrified oocytes can be improved by a duration of equilibration for 3 minutes in holding medium plus 10% EG and 10% DMSO via droplet vitrification.

Table 1
In vitro development of vitrifield bovine oocytes following warming and IVF
9THE USE OF ECG TO INCREASE PREGNANCY RATES IN POSTPARTUM BEEF COWS FOLLOWING TREATMENT WITH PROGESTERONE VAGINAL DEVICES AND ESTRADIOL BENZOATE AND FIXED-TIME AI
Authors: Bo G.A , Cutaia L. , Chesta P. , Moreno D.
Published: February 2004

Treatments with progesterone (P4) releasing devices and estradiol benzoate (EB) have been extensively used in fixed-time AI (FTAI) programs in beef cattle. However, pregnancy rates in postpartum cows kept on pasture often have been lower than expected because of poor body condition score (BCS) and a high incidence of anestrous. A recent study (Cutaia L et al., 2003 Theriogenology 59, 216) suggested that the addition of eCG to a P4/EB FTAI program may improve pregnancy rates in cows with fair to poor BCS, but results were not conclusive. Therefore, this experiment was designed to further investigate the effect of eCG treatment on pregnancy rates in postpartum beef cows in those conditions. The experiment was performed at 2 locations with lactating Angus cows (n=93), 60 to 90d postpartum with a BCS 1.9 (1 to 5 scale; Location 1), and crossbred Zebu cows (n=290), 60 to 90d postpartum with a BCS 2.0 (Location 2). In each Location, cows were randomly allocated to 1 of the following treatment groups: Control, eCG Day6, or eCG Day8. All cows received a P4 intravaginal device (DIB, Syntex, Argentina) and 2mg EB i.m. (Syntex) on Day0, 500mg cloprostenol (Estroplan, Syntex) at the time of DIB removal (Day8), 1mg EB i.m. on Day9 and FTAI 52 to 56h after DIB removal. Cows in the Control group received no further treatment, whereas cows in the eCG-treated groups received 400 IU eCG (Novormon 5000, Syntex) on Day6 or Day8. Cows were examined on Day0 by rectal palpation (Location 1) or by ultrasonography (US; Location 2) and were classified as those with a CL or without a CL, with either large (>8mm) or small (<8mm)small follicles. Pregnancy was determined by US 45d after FTAI. Data were analyzed by logistic regression and the effects of location, treatment, ovarian status, AI technician and semen were considered in the model. There was no effect of location (P=0.3), AI technician (P=0.2) or semen (P=0.8) on pregnancy rates. However, there was an effect of treatment (P=0.02), attributed to higher pregnancy rates in the eCG Groups than in the Control Group (Table). Furthermore, cows with a CL or without a CL but with large follicles on Day 0 had higher pregnancy rates than those with small follicles (P=0.04). It was concluded that the use of eCG in a P4/EB FTAI program improved pregnancy rates in postpartum beef cows that were in fair topoor BCS.
93COMPUTER-ASSISTED ANALYSIS OF BOVINE SPERM MOTILITY BEFORE AND AFTER CRYOPRESERVATION
Authors: Defoin L. , Granados A. , Clos M. , Donnay I.
Published: February 2004

Sperm cryopreservation causes various types of damage, including membrane injury, oxidative stress, and loss of the acrosome. In cattle, the mortality rate after sperm cryopreservation reaches roughly 50%, and surviving sperm cells have a lower motility and lower fertility than their fresh counterparts. Large variations are also observed between bulls. The aim of this study was to analyse different motility parameters before and after freezing in order to establish correlations. The final objective is to determine, before freezing, parameters that could predict the characteristics of motility after freezing. A computer-assisted sperm analyser (Hobson Sperm Tracker) was used. We analyzed one ejaculate from 30 different bulls before and after freezing (minimum 300 spermatozoa/analysis). Reliable parameters (<10% variation for the same ejaculate) were then selected and included VCL (curvilinear velocity), VAP (average path velocity), MAD (mean angular head displacement), ALH (amplitude of lateral head displacement), STR (straightness of path), and the percentage of motility (%Mot). Linear regressions were established between those parameters before and after freezing. Results are shown in Table 1. The velocity parameters (VAP, VCL, and STR) of the motile sperm were conserved after freezing. Moreover, ejaculates with a high proportion of motile sperm before freezing have, on average, better values for velocity parameters after freezing, while no correlation was found between the percentage of motile sperm before and after freezing. The only parameter of fresh sperm that seems to be correlated with the proportion of motile sperm cells after freezing is MAD (inverse correlation). This could mean that an ejaculate with a high proportion of spermatozoa showing important lateral displacements of the head is more sensitive to cryopreservation. Similarly, a high MAD before freezing was related to a low velocity after thawing. A high MAD could result from a high proportion of capacitated spermatozoa, which is detrimental to their survival and motility. In conclusion, few parameters related to the motility can predict the proportion of motile sperm after freezing. However, by combining several parameters, it seems possible to predict the characteristics of motility of the sperm. Although further investigations are needed, the present evaluation could be of interest to evaluate the freezability of ejaculates, to understand variations between bulls, or to set up new freezing protocols.

Table 1
Coefficients of correlation (r2) before and after freezing, calculated from 30 ejaculates from 30 different bulls
94MEIOSIS INHIBITION OF BOVINE OOCYTES: EFFECTS ON SURVIVAL AFTER
VITRIFICATION BY OPEN PULLED-STRAW METHOD
Authors: Diez C. , Carbajo M. , Fernandez L. , Hidalgo C.O. , de la Varga S. , Fernandez A. , Facal N. , Gomez E.
Published: February 2004

Mammalian oocytes remain one of the most difficult cell types to successfully cryopreserve. The in vitro-maturation protocols (IVM) have a large impact on the oocyte maturation. Consequently, inhibition of meiosis has been used to improve developmental competence of oocytes without reducing blastocyst rates. Moreover, the meiotic stage influences the ability of oocytes to survive cryopreservation. This work analyzes the effect of the inhibition of meiosis (prematuration) on the freezability of the bovine oocyte. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries. Inmature oocytes (I) with compact cumulus and evenly granulated cytoplasm were selected. Prematuration (PM) was performed by incubating COCs for 22h in TCM199 NaHCO3 and roscovitine 25μM. IVM was accomplished in TCM199 NaHCO3, 10% FCS, FSH-LH and 17β-estradiol. Oocytes were subjected to 5 treatments prior the vitrification (see table). COCs were partially denuded from cumulus cells and vitrified/warmed using the OPS system (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). Warmed oocytes were fertilized (Day 0) and presumptive zygotes having a normal morphological appearance were cultured in SOFaa+5% of FCS (Day 3); elements with degenerated appearance were discarded and recorded. Fresh oocytes submitted to IVM (c-M) or prematured and matured (c-PM+M) were fertilized and cultured as controls. Data were analyzed by ANOVA and Duncan’s multiple range test and expressed as LSM±SE. Developmental data are referred to the zygotes cultured. Only oocytes vitrified after IVM reached the blastocyst stage, but at lower rates than fresh controls. However, no differences were found between treatments at any developmental stage. Oocytes vitrified both as prematured+matured and immature oocytes showed increased proportions (P<0.01) of degenerated oocytes (37.3±5.9 and 49.9±5.9, respectively), as compared with oocytes matured before vitrification (17.6±5.9). These results show that effects induced by incubation with roscovitine (Lonergan et al. 2003 Mol. Reprod. Dev. 64, 369–378) in combination with cryodamage compromise the oocyte developmental ability. Supported by CICYT, AGL2001-379.
97EFFECT OF RAPID TEMPERATURE CHANGE ON VIABILITY OF FROZEN-THAWED
IVM/IVF BOVINE EMBRYOS
Authors: Goda S. , Narita M. , Miyamura M. , Hamano S. , Dochi O. , Koyama H.
Published: February 2004

In on-farm conditions, frozen bovine embryos are frequently thawed at various environmental temperatures. Thawing temperature is an important factor affecting the viability of frozen-thawed bovine embryos. The present study investigated the effects of rapid temperature change on the viability of frozen IVM/IVF bovine embryos after thawing. Day 7- and 8- (Day 0=day of insemination) expanded blastocysts were used in this study. Embryos were produced as previously described by Hamano & Kuwayama (1993 Theriogenology 39, 703–712,). Embryos were frozen in TCM-199 supplemented with 1.4M glycerol, 20% calf serum (CS), and 0.25-M sucrose. The embryos were loaded into 0.25mL straws. After equilibration, the straws were placed directly into a precooled alcohol chamber of a freezer at −6°C, seeded 1min later, held at −6°C for 10min, cooled to −25°C at a rate of 0.33°C/min, and then plunged into liquid nitrogen. Embryos were thawed by holding the straws in room temperature air for 10s, and then immersing them in a 35°C water bath for 10s. The thawed straws were randomly assigned to one of two groups. Some thawed straws were held for 5min at −15, −5, 0, 5, or 15°C, and were then transferred directly into a water bath at 35°C for 5min (Group 1). The remaining straws were subjected to the same post-thaw cooling step procedures as Group 1 two times (Group 2). The embryos were then directly rehydrated in PBS supplemented with 5% CS at 35°C, and cultured in TCM-199 supplemented with 5% CS and 0.1mM β-mercaptoethanol. The morphology and hatching of embryos was assessed 72h later. Data were analyzed using the chi-square method and Fisher’s exact test. The results are presented in the Table. There were no significant differences in the hatching rate among 5 temperatures in Group 1. Although there were no differences in the hatching rate of embryos held at −5, 0, 5, or 15°C after thawing, the rate for embryos held at −15°C was significantly lower than those of the other treatments in Group 2 (P<0.05). The straws held at −15°C twice (Group 2), showed refreezing. These results suggest that exposing thawed straws to a broad range of environmental temperatures (−5 to 15°C) had no effect on the viability of frozen-thawed IVM/IVF bovine embryos. However, embryos might be irreversibly damaged when held at −15°C.

Table 1
Effect of rapid temperature change on the viability of frozen-thawed IVM/IVF bovine embryos
Digenetic trematodes, Acanthatrium sp. and Lecithodendrium sp., as vectors of Neorickettsia risticii, the agent of Potomac horse fever
Authors: Pusterla N. , Johnson E.M. , Chae J.S. , Madigan J.E.
Published: February 2004

Neorickettsia (formerly Ehrlichia) risticii, the agent of Potomac horse fever (PHF), has been recently detected in trematode stages found in the secretions of freshwater snails and in aquatic insects. Insectivores, such as bats and birds, may serve as the definitive host of the trematode vector. To determine the definitive helminth vector, five bats (Myotis yumanensis) and three swallows (Hirundo rustica, Tachycineta bicolor) were collected from a PHF endemic location in northern California. Bats and swallows were dissected and their major organs examined for trematodes and for N. risticii DNA using a nested polymerase chain reaction (PCR) assay. Adult digenetic trematodes, Acanthatrium sp. and/or Lecithodendrium sp., were recovered from the gastrointestinal tract of all bats and from one swallow. The intestine of three bats, the spleen of two bats and one swallow as well as the liver of one swallow tested PCR positive for N. risticii. From a total of seven pools of identical digenetic trematodes collected from single hosts, two pools of Acanthatrium sp. and one pool of Lecithodendrium sp. tested PCR positive. The results of this investigation provide preliminary evidence that at least two trematodes in the family Lecithodendriidae are vectors of N. risticii. The data also suggest that bats and swallows not only act as a host for trematodes but also as a possible natural reservoir for N. risticii.
Parasitic helminths of the wild rabbit, Oryctolagus cuniculus, in different bioclimatic zones in Tenerife, Canary Islands
Authors: Foronda P. , Castillo A. Del , Abreu N. , Figueruelo E. , Piñero J. , Casanova J.C.
Published: February 2004

Faunistic and ecological analyses of the wild rabbit helminth fauna were undertaken in Tenerife island (Canary Islands). Rabbits were collected between 1998 and 2000 in seven bioclimatic zones in Tenerife selected by orientation and altitude. Five parasite species were identified, three cestodes (Taenia pisiformis (larvae), Andrya cuniculi and Mosgovoyia ctenoides) and two nematodes (Trichostrongylus retortaeformis and Passalurus ambiguus). Taenia pisiformis presented an irregular distribution with significant differences in prevalences between the zones. Andrya cuniculi was only found in two zones and there were no significant differences in prevalence values. Mosgovoyia ctenoides presented a wide distribution with significant prevalences, which were higher in northern compared to southern zones. Trichostrongylus retortaeformis was absent in the low southern zones of the island. Passalurus ambiguus was found in all zones with no significant difference in the prevalence of infection. The differences in prevalences are likely to be explained by abiotic factors in the case of T. retortaeformis, and by the absence of definitive and intermediate hosts in the case of T. pisiformis and A. cuniculi, respectively. All parasite species in Tenerife are common helminths in the Iberian Peninsula, from which their rabbit hosts originated. No significant differences were recorded in the mean intensities of infection of any of the parasite species identified.
The microhabitat distribution of two Dactylogyrus species parasitizing the gills of the barbel, Barbus barbus
Authors: Kadlec D. , Šimková A. , Gelnar M.
Published: February 2004

The microhabitat distribution of two congeneric species Dactylogyrus carpathicus and D. malleus (Monogenea) parasitizing the gills of the barbel (Barbus barbus L.) was investigated. We tested whether congeneric species exhibited microhabitat preference and whether interspecific interactions could be attributed to the microhabitat segregation of congeners. The outlying mean index method was used to evaluate species microhabitats. Gill variables (different microhabitats within gills) were used as environmental factors characterizing the gills. When abundances of both species were highest, and no significant difference was found between the abundance of the two species, the gill segments and gill areas were the most important factors segregating the Dactylogyrus species on the gills. Niche overlap was low within each of the four gill arches, and parasites were segregated in the same microhabitats within each gill arch. When abundances of both species were low, each monogenean species was segregated at the level of the gill arches. When abundances of both species increased, the niche and overlap between species increased. The distribution of both congeneric species confirmed microhabitat preference within the gills. The results suggest that microhabitat preference is dependent on species abundances, species being segregated in the case of low abundance, possibly to increasing mating opportunities. Both niche and overlap between species increased with species abundance. In the case of the high abundance of both species, microhabitat preference seems to be related to interspecific interactions between monogenean species, as previously found for endoparasitic species.
Milbemycin oxime in a new formulation, combined with praziquantel, does not reduce the efficacy of praziquantel against Echinococcus multilocularis in cats
Authors: Jenkins D.J. , Romig T.
Published: February 2004

Twenty European domestic cats were each infected with 15,000 protoscoleces of Echinococcus multilocularis extracted from metacestodes grown in experimentally infected common voles (Microtus arvalis). Sixteen days after infection, ten cats were treated with a broad-spectrum anthelmintic and acaricide comprising praziquantel and milbemycin oxime. Five days later treated and untreated cats were euthanized and the intestine examined for E. multilocularis. Five of ten untreated cats were infected with E. multilocularis with worm burdens ranging from 235 to 1920 worms per cat. No E. multilocularis were recovered from any of the treated cats. This study has demonstrated that this new combination broad spectrum anthelmintic and acaricide for cats is highly efficacious against E. multilocularis and the relevance of this is discussed.
Inhibition of the Collapse of the Shaker K+ Conductance by Specific Scorpion Toxins
Authors: Gómez-Lagunas Froylan , Batista Cesar V.F. , Olamendi-Portugal Timoteo , Ramírez-Domínguez Martha E. , Possani Lourival D.
Published: February 2004

The Shaker B K+ conductance (GK) collapses when the channels are closed (deactivated) in Na+ solutions that lack K+ ions. Also, it is known that external TEA (TEAo) impedes the collapse of GK (Gómez-Lagunas, F. 1997. J. Physiol. 499:3–15; Gómez-Lagunas, F. 2001. J. Gen. Physiol. 118:639–648), and that channel block by TEAo and scorpion toxins are two mutually exclusive events (Goldstein, S.A.N., and C. Miller. 1993. Biophys. J. 65:1613–1619). Therefore, we tested the ability of scorpion toxins to inhibit the collapse of GK in 0 K+. We have found that these toxins are not uniform regarding the capacity to protect GK. Those toxins, whose binding to the channels is destabilized by external K+, are also effective inhibitors of the collapse of GK. In addition to K+, other externally added cations also destabilize toxin block, with an effectiveness that does not match the selectivity sequence of K+ channels. The inhibition of the drop of GK follows a saturation relationship with [toxin], which is fitted well by the Michaelis-Menten equation, with an apparent Kd bigger than that of block of the K+ current. However, another plausible model is also presented and compared with the Michaelis-Menten model. The observations suggest that those toxins that protect GK in 0 K+ do so by interacting either with the most external K+ binding site of the selectivity filter (suggesting that the K+ occupancy of only that site of the pore may be enough to preserve GK) or with sites capable of binding K+ located in the outer vestibule of the pore, above the selectivity filter.
Coupling Interactions between Voltage Sensors of the Sodium Channel as Revealed by Site-specific Measurements
Authors: Chanda Baron , Asamoah Osei Kwame , Bezanilla Francisco
Published: February 2004

The voltage-sensing S4 segments in the sodium channel undergo conformational rearrangements in response to changes in the electric field. However, it remains unclear whether these structures move independently or in a coordinated manner. Previously, site-directed fluorescence measurements were shown to track S4 transitions in each of the four domains (Chanda, B., and F. Bezanilla. 2002. J. Gen. Physiol. 120:629–645). Here, using a similar technique, we provide direct evidence of coupling interactions between voltage sensors in the sodium channel. Pairwise interactions between S4s were evaluated by comparing site-specific conformational changes in the presence and absence of a gating perturbation in a distal domain. Reciprocity of effect, a fundamental property of thermodynamically coupled systems, was measured by generating converse mutants. The magnitude of a local gating perturbation induced by a remote S4 mutation depends on the coupling strength and the relative equilibrium positions of the two voltage sensors. In general, our data indicates that the movement of all four voltage sensors in the sodium channel are coupled to a varying extent. Moreover, a gating perturbation in S4-DI has the largest effect on the activation of S4-DIV and vice versa, demonstrating an energetic linkage between S4-DI and S4-DIV. This result suggests a physical mechanism by which the activation and inactivation process may be coupled in voltage-gated sodium channels. In addition, we propose that cooperative interactions between voltage sensors may be the mechanistic basis for the fast activation kinetics of the sodium channel.
How Properties Emerge
Authors: Humphreys Paul
Published: February 2004

A framework for representing a specific kind of emergent property instance is given. A solution to a generalized version of the exclusion argument is then provided and it is shown that upwards and downwards causation is unproblematical for that kind of emergence. One real example of this kind of emergence is briefly described and the suggestion made that emergence may be more common than current opinions allow.
Nonlocality and the Aharonov-Bohm Effect
Authors: Healey Richard
Published: February 2004

At first sight the Aharonov-Bohm effect appears nonlocal, though not in the way EPR/ Bell correlations are generally acknowledged to be nonlocal. This paper applies an analysis of nonlocality to the Aharonov-Bohm effect to show that its peculiarities may be blamed either on a failure of a principle of local action or on a failure of a principle of separability. Different interpretations of quantum mechanics disagree on how blame should be allocated. The parallel between the Aharonov-Bohm effect and violations of Bell inequalities turns out to be so close that a balanced assessment of the nature and significance of quantum nonlocality requires a detailed study of both effects.
[Explanation] Is Explanation Better
Authors: Hardcastle Valerie Gray
Published: February 2004

Robert Wilson (1994) maintains that many interesting and fundamental aspects of psychology are non-individualistic because large chunks of psychology depend upon organisms being deeply embedded in some environment. I disagree and present one version of narrow content that allows enough reference to the environment to meet any wide challenge. I argue that most psychologists are already this sort of narrow content theorist and that these narrow content explanations of psychological phenomena meet Wilson's criteria for being a good explanation better than any wide explanation of the same event.
Duhem's Problem, the Bayesian Way, and Error Statistics, or “What's Belief Got to Do with It?”
Authors: Mayo Deborah G.
Published: February 2004

I argue that the Bayesian Way of reconstructing Duhem's problem fails to advance a solution to the problem of which of a group of hypotheses ought to be rejected or “blamed” when experiment disagrees with prediction. But scientists do regularly tackle and often enough solve Duhemian problems. When they do, they employ a logic and methodology which may be called error statistics. I discuss the key properties of this approach which enable it to split off the task of testing auxiliary hypotheses from that of appraising a primary hypothesis. By discriminating patterns of error, this approach can at least block, if not also severely test, attempted explanations of an anomaly. I illustrate how this approach directs progress with Duhemian problems and explains how scientists actually grapple with them.
Learning Theory and the Philosophy of Science
Authors: Kelly Kevin T. , Schulte Oliver , Juhl Cory
Published: February 2004

This paper places formal learning theory in a broader philosophical context and provides a glimpse of what the philosophy of induction looks like from a learning-theoretic point of view. Formal learning theory is compared with other standard approaches to the philosophy of induction. Thereafter, we present some results and examples indicating its unique character and philosophical interest, with special attention to its unified perspective on inductive uncertainty and uncomputability.
Quantities, Magnitudes, and Numbers
Authors: Kyburg Henry E.
Published: February 2004

Quantities are naturally viewed as functions, whose arguments may be construed as situations, events, objects, etc. We explore the question of the range of these functions: should it be construed as the real numbers (or some subset thereof)? This is Carnap's view. It has attractive features, specifically, what Carnap views as ontological economy. Or should the range of a quantity be a set of magnitudes? This may have been Helmholtz's view, and it, too, has attractive features. It reveals the close connection between measurement and natural law, it makes dimensional analysis intelligible, and explains the concern of scientists and engineers with units in equations. It leaves the philosophical problem of the relation between the structure of magnitudes and the structure of the reals. What explains it? And is it always the same? We will argue that on the whole, construing the values of quantities as magnitudes has some advantages, and that (as Helmholtz seems to suggest in “Numbering and Measuring from an Epistemological Viewpoint”) the relation between magnitudes and real numbers can be based on foundational similarities of structure.
Placebo Control Treatments and the Evaluation of Psychotherapy: A Reply to Grünbaum and Erwin
Authors: Greenwood John D.
Published: February 2004

In this paper, I respond to some criticisms of Greenwood (1996) advanced by Grünbaum (1996) and Erwin (1996). I argue that Grünbaum's problematic account of “placebo effects” and placebo control treatments does not really address, far less resolve, the problems with experimental evaluations of psychotherapy documented in my original paper.
On an Inconsistency in Constructive Empiricism
Authors: Bandyopadhyay Prasanta S.
Published: February 2004

I show that van Fraassen's empiricism leads to mutually incompatible claims with regard to empirical theories. He is committed to the claim that reasons for accepting a theory and believing it are always identical, insofar as the theory in question is an empirical theory. He also makes a general claim that reasons for accepting a theory are not always reasons for believing it irrespective of whether the theory is an empirical theory.
Between the Fundamental and the Phenomenological: The Challenge of ‘Semi-Empirical’ Methods
Authors: Ramsey Jeffry L.
Published: February 2004

Philosophers disagree how abstract, theoretical principles can be applied to instances. This paper generates a puzzle for law theorists, causal theorists and inductivists alike. Intractability can force scientists to use a “semi-empirical” method, in which some of an equation's theoretically-determinable parameters are replaced with values taken directly from the data. This is not a purely deductive or inductive process, nor does it involve causes and capacities in any simple way (Humphreys 1995). I argue the predictive successes of such methods require us to reanalyze our views about the nature of prediction, the status of models, and the goal(s) of science. When laws and experimental evidence are neither individually nor jointly sufficient for prediction, models become the locus of understanding. I analyze an historically important debate about the use of semi-empirical methods to construct potential energy surfaces.
Some Questions about Identifying Individuals: Failed Intuitions about Organisms and Species
Authors: Horvath Christopher D.
Published: February 2004

Treating species as individuals and not classes has been crucial to the integration of evolutionary theory with modern systematics. Despite the theoretically important role the concept of individuality plays in modern phylogenetic systematics and in evolutionary theory more generally, many have been content to rely on common-sense intuitions about what counts as an individual. One of the most often cited intuitions is that individuals should be defined intrinsically. Unfortunately, common-sense intuitions like this one have proven to be inadequate for identifying and characterizing historical individuals (like species). An examination of real-world biological examples shows that our common-sense intuitions are equally inadequate when applied to at least some biological organisms—the paradigm individuals.
Old Evidence and New Explanation
Authors: Wagner Carl G.
Published: February 2004

Jeffrey has devised a probability revision method that increases the probability of hypothesis H when it is discovered that H implies previously known evidence E. A natural extension of Jeffrey's method likewise increases the probability of H when E has been established with sufficiently high probability and it is then discovered, quite apart from this, that H confers sufficiently higher probability on E than does its logical negation H̄.