bluuebunny/crossref_metadata_2025 huggingface dataset:
130 THE EFFECT OF ALTERED ENERGY SUBSTRATE CONCENTRATIONS ON THE DEVELOPMENT OF DIPLOID PARTHENOGENETIC PORCINE EMBRYOS CREATED FROM OOCYTES FROM GILTS AND SOWS
Authors: Beebe L. , McIlfactrick S. , Nottle M.
Published: December 2004

There is building evidence that altering the concentration of energy substrates in the culture medium to more closely approximate the concentrations thought to be present in the reproductive tract improves porcine embryo development. The aim of this experiment was to examine the development of porcine parthenogenetic embryos in such a modified version of NCSU23. The embryos were created from both sow- and gilt-derived oocytes to see whether the source of oocytes influences how the embryos respond. Ovaries from slaughtered sows or prepubertal gilts were collected, follicles (3–6 mm) were aspirated and oocytes surrounded by at least three layers of compact cumulus cells were collected and matured in TCM199 containing cysteamine, insulin, FSH, EGF, and 10% sow follicular fluid, for approximately 40 h. Cumulus cells were removed and good quality mature oocytes with a visible polar body were electrically activated (approximately 44 h after the start of maturation) by 2 DC pulses (1.5 kVcm−1, 60 μs) applied 1 s apart, cultured in NCSU23 + 7.5 μg/mL cytochalasin B for 3 h and then placed into the treatment droplets. All long-term culture was in 50-μL droplets under mineral oil in an atmosphere of 5%CO2, 5%O2, 90%N2 at 38.5°C for a total of 6 days (144 h). The treatments were: NCSU23 (N23); a modified NCSU23 containing 0.6 mM glucose, 0.2 mM pyruvate, and 5.7 mM lactate (PLG); or the modified NCSU23 for the first 48 h and then NCSU23 for the remaining 4 days (dPLG). All treatments were transferred into fresh droplets of the appropriate medium at 48 h. Heat inactivated fetal bovine serum (10%) was added early on Day 5. On Day 6, the embryos were assessed for morphological development and those embryos judged to be good blastocysts were stained for cell count. Experiments using sow or gilt oocytes were conducted and analyzed separately. Morphological data were analyzsed by χ2 and cell number data by ANOVA with the LSD test used to determine differences between treatments. There were no statistical differences in the percentage of activated sow or gilt oocytes developing to blastocysts (sow: N23 51.3%, PLG 48.9%, dPLG 55.1%; and gilt: N23 25.6%, PLG 38.2%, dPLG 38.6%). The blastocysts produced in the dPLG treatment contained significantly more cells than those produced in the N23 treatment for both sow- (mean ± SEM; N23 47.1 ± 2.1; dPLG 53.8 ± 1.7; P < 0.001) and gilt (N23 39.3 ± 3.4; dPLG 50.5 ± 3.0; P < 0.05)-derived embryos. The number of cells per blastocyst produced in the PLG treatment did not differ significantly from the other treatments (sow: 49.9 ± 2.0; gilt: 43.4 ± 2.8). These data demonstrate that culturing for the first 48 h with the modified energy substrate concentrations improved the cell number of the resulting blastocysts but did not affect the proportion developing into blastocysts by day 6 of culture. This effect appeared to be consistent whether sow- or gilt-derived parthenogenetic embryos were used.
138 THE EFFECT OF SYNTHETIC HYALURONAN, BSA AND SERUM ON IN VITRO DEVELOPMENT AND GENE TRANSCRIPTION OF BOVINE BLASTOCYSTS
Authors: Gutiérrez-Adán A. , Rodriguez-Martinez H. , Beltrán Breña P. , De la Fuente J. , Palasz A.T.
Published: December 2004

The objective of this study was to examine the effect of synthetic hyaluronan (s-HA), BSA and fetal calf serum (FCS) on bovine embryo in vitro development, ultrastructure, and mRNA transcription of four developmentally important genes: apoptosis (BAX), oxidative stress (SOX), growth factor (IGF-II), and cell-to-cell adhesion (Ecad). A total of 1406 presumptive zygotes (7 replicates) were cultured initially in two Groups: 1, SOFaa + 4% BSA only, and 2, SOFaa + 4% BSA and 10% FCS. On Day 4 (96 h after insemination) of culture, the number of zygotes that developed to the <8-cell-stage were recorded, and 2.5 mg/mL of s-HA (MAP-5; Bioniche Inc, Belleville, ON, Canada) was added to half of the embryos from each group; the other half received an equivalent volume of corresponding SOF medium without s-HA. Embryos were cultured in 50-μL drops (25 zygotes per drop) under paraffin oil at 39°C and 5% CO2 in humidified air. Cleavage rates were recorded on Day 2 and the number of blastocysts on Days 7, 8, and 9. At least five blastocysts from each replicate and from each treatment were frozen for evaluation of gene expression patterns. Poly(A) mRNA was prepared from 4–5 groups of pools of 10 embryos. The quantification of all gene transcripts was performed by real time quantitative RT-PCR in three replicates. The fine structure of blastocysts was studied using transmission electron microscopy. Embryo developmental stages and blastocyst formation were analyzed by chi-square analysis, and data on mRNA expression were analyzed by one-way repeated-measures ANOVA. No differences in cleavage rates were observed between groups. There was no difference between the BSA group with or without s-HA in the percentages of embryos developed to the blastocyst stage at Days 7, 8, and 9 (38.3 and 38.1%, respectively). However, significantly (P < 0.05) less blastocysts developed in medium supplemented with BSA + FCS (18.3%) or with BSA + FCS + s-HA (27.4%). Synthetic HA added to the medium containing BSA significantly (P < 0.05) increased the level of expression of EGF-II and decreased (P < 0.05) the level of expression of BAX, SOX, and Ecad. On the other hand, presence of FCS significantly (P < 0.05) increased the level of SOX and decreased the level of IGF-II (P < 0.05), and the addition of s-HA to SOF containing FCS showed no effect on the level of transcription of any analyzed genes. The general fine structure of embryos cultured with s-HA regardless of protein supplement was conspicuously improved in comparison with the respective controls. It can be concluded that, within our culture system, addition of s-HA on Day 4 of culture to the SOFaa medium supplemented with BSA but not in combination with FCS showed a positive effect on embryo development and molecular composition of the embryos.
14 EFFECT OF TEMPORARY WEANING AND ECG TREATMENT ON PREGNANCY RATES IN POSTPARTUM BOS INDICUS COWS FOLLOWING TREATMENT WITH PROGESTERONE VAGINAL DEVICES AND ESTRADIOL BENZOATE
Authors: Cutaia L. , Balla E. , Rizzi C. , Peres L. , Bo G.A.
Published: December 2004

Although treatments with progesterone (P4)-releasing devices and estradiol benzoate (EB) have been extensively used in fixed-time AI (FTAI) programs in beef cattle, pregnancy rates in postpartum Bos indicus cows kept on pasture often have been lower than expected because of poor body condition score (BCS) and a high incidence of anestrus. Temporary weaning and eCG treatment have been shown to increase pregnancy rates in suckled beef cows. Therefore, this experiment was designed to compare the effect of eCG treatment and temporary weaning on pregnancy rates in postpartum cows in fair to poor BCS. Lactating multiparous crossbred Bos indicus cows (n = 393), 60 to 90 d postpartum with a BCS 2.0 (1 to 5 scale) were randomly allocated to 1 of 4 treatment groups, in a 2 × 2 factorial design. At the beginning of the experiment (Day 0), all cows received a P4 intravaginal device (DIB, Syntex, Argentina) and 2 mg EB i.m. (Syntex). On Day 8, DIB devices were removed and all cows received 150 μg D (+) cloprostenol i.m. (Ciclase, Syntex) and were randomly divided to receive 400 IU eCG (Novormon 5000, Syntex) or no treatment at the same time. Furthermore, half of the cows in each treatment group have their calves weaned temporarily, from the time of DIB removal until the end of the FTAI, whereas the other half remained with their calves all the time. Finally, all cows received 1 mg EB i.m. on Day 9 and were FTAI 52 to 56 h after DIB removal. Cows were examined by ultrasonography 42 d after FTAI to determine pregnancy status. Data were analyzed by logistic regression and the effects of treatment and semen used were considered in the model. Ovarian activity was estimated by rectal palpation on Day 0: there were 72/393 (18.2%) cows with a CL, 140/393 (35.6%) cows with palpable medium size follicles, and 181/393 (46.1%) cows with ovaries containing no detectable structures. There was no effect of ovarian status at the time of treatment (P = 0.91) or semen (P = 0.91) on pregnancy rates. Treatment with eCG tended (P = 0.08) to increase pregnancy rates (94/192, 48.9% and 79/201, 39.3% for cows treated or not treated with eCG, respectively). However, there was no effect of weaning or eCG by weaning interaction (P = 0.98) on pregnancy rates (86/191, 45.1% and 87/202, 43.1% for cows that have their calves weaned or not weaned for 56 h, respectively). Results confirm those of previous studies that demonstrated that the use of eCG in a P4/EB/FTAI program improved pregnancy rates in postpartum Bos indicus crossbred cows that were in fair-to-poor BCS. However, no improvement in pregnancy rates was observed after temporary weaning in the present study.
Research was supported by Syntex S.A., Estancia El Mangrullo S.A., and Agencia Cordoba Ciencia S.E.
152 IN VITRO AND IN VIVO DEVELOPMENT OF BOVINE IVP EMBRYOS FOLLOWING SINGLE-CELL BIOPSY ON DAY 4
Authors: Hartwich K. , Peachey B. , Cockrem K. , Marsh A. , Pugh A.
Published: December 2004

Maximum advantage can be gained from gene discovery programs, by screening embryos carrying the desired genes(s) prior to immediate transfer. This requires an efficient and reliable genotyping system and a method for biopsy preparation that does not compromise subsequent embryo or fetal development. The present study examined the effect of removing a single-cell from the developing 8–16 cell embryo on its subsequent ability to continue development to at least the late morula stage in vitro and then survive following triple transfer to recipients. Abattoir-sourced ovaries were obtained and subjected to IVP as previously described (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). Briefly, oocytes were matured in TCM199 +10% FCS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL estradiol, and 100 μM cysteamine under 5% CO2 in air at 38.5°C for 24 h. Percoll-separated sperm (1 × 106/mL) were then co-incubated with the matured oocytes (Day 0) for 24 h with the presumptive zygotes further cultured in mSOF medium under 5%CO2, 7% O2, 88% N2. On Day 4 embryos with a minimum of 8 cells were selected and held at 38.5°C in HEPES-buffered SOF (HSOF) until biopsy at ambient temperature. Embryo biopsy was performed in HSOF medium + 5 μg/mL cytochalasin B. A single cell was removed using a 30 μm biopsy pipette. Both biopsied and control embryos were then further cultured in mSOF in individual wells prepared in a 1% agarose matrix (Peura TT 2003 Cloning Stem Cells 5, 13–24). Embryos were scored for grade and stage of development reached on Day 7, and Grades 1 and 2 blastocysts and expanded blastocysts were transferred to synchronized recipients (three embryos of the same stage and grade to each recipient; n = 50). Fetal number was determined on Day 35 and 62 of gestation. A model for embryo survival was fitted to the data (McMillan WH et al. 1998 Theriogenology 50, 1053–1070) in order to estimate embryo (“e”) and recipient (“r”) contributions to embryo survival. Values were then compared to those determined for control embryos, produced using identical IVP methods (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). A total of 358 control and 561 biopsied embryos were cultured. Removal of a single cell did not significantly affect in vitro development (60.1% vs. 56.0%; control vs. biopsy). Day 35 survival of biopsied embryos was 44.7% with calculated “e” and “r” values of 0.48 and 0.94, respectively, which did not differ from control values (44.1%; 0.50 and 0.89). However, by Day 62 fetal survival had significantly decreased with a concomitant drop in “e” but not “r” (30.0%; 0.32 and 0.94, respectively; control “e” and “r” were unchanged). In conclusion, single-cell biopsy of the 8–16-cell embryo does not affect in vitro development or embryo survival to Day 35. However, significant fetal loss occurs by Day 62 that may limit commercial application. Further work is required to elucidate the cause of and overcome fetal loss.
163 DOES LH PLUS hCG, USED TO INDUCE OVULATION IN SUPERSTIMULATED COWS, IMPROVE PREGNANCY RATES AFTER TRANSFER OF EMBRYOS THAT ARE NOT EXCELLENT?
Authors: Nogueira M.F.G. , Buratini Jr J. , Barros C.M.
Published: December 2004

The LH receptor (LHr) present in ovarian follicle cells is essential for the biological response due to its binding to the ligand (LH). The events following the LH surge are related to the presence of LHr and its affinity to LH. Studies on LHr gene expression in theca and granulosa cells from bovine follicles have demonstrated the presence of at least four isoforms of LHr mRNA by alternative splicing. Two of the four detected isoforms have an open reading frame (ORF) and can be translated to functional proteins (receptors coupled with G protein) with different affinities to their ligands (Robert C et al. 2003 Reproduction 125, 437–446). From those two isoforms with ORF, one (full) has affinity to both LH and hCG molecules, whereas the second isoform (with deletion of exon 10) has affinity to hCG only. Recently we have detected the same four isoforms described by Robert et al. in both granulosa and theca cells from abattoir. Based on this information, the present study tested the hypothesis that, in cows superstimulated with FSH, administration of both LH and hCG, as an attempt to stimulate any variety of LH receptor present in the follicles, would result in improvement of oocyte quality and/or increase in ovulation rate. Nelore cows (Bos taurus indicus) were superstimulated according to the protocol termed P-36 (Barros CM et al. 2003 Theriogenology 59, 524 abst), and embryos were flushed 7 to 8 days after inducing ovulation. Ovulation was induced with LH (pLH, 12.5 mg, i.m.; Lutropin, Vetrepharm, London, Ontario, Canada; Group 1) or both LH (12.5 mg) and hCG (1500 IU, i.m.; Choragon, Ferring GmbH, Kiel, Germany; Group 2). Superstimulation protocol and embryo transfer were performed simultaneously on both groups. Mean (± SEM) of total structures, viable embryos, and viability rate were: 12.4 ± 2.36, 10.0 ± 2.38 and 80.8% (Group 1, n = 8 flushings) and 12.2 ± 2.03, 8.9 ± 1.66, and 73.1% (Group 2, n = 14); there was no significant difference between groups (P = 0.96; P = 0.71 (ANOVA), and P = 0.18 (Fisher's Exact Test), respectively). In a subset of embryos (excellent, good, fair, and poor qualities) transferred without freezing, the pregnancy rates for groups 1 and 2 were 41.7% (25/60) and 56.9% (37/65), respectively (P = 0.11, Fisher's Exact Test). The overall pregnancy rates from embryos of excellent, good, and fair qualities were 46.3% (25/54) and 58.9% (33/56), respectively for Groups 1 and 2 (P = 0.25). When only embryos of good, fair, and poor qualities were pooled, the pregnancy rates were 18.8% (06/32) and 60.0% (30/50), respectively for Groups 1 and 2 (P = 0.0003). It is concluded that simultaneous administration of LH and hCG to induce ovulation in superstimulated animals did not alter production of viable embryos or viability rate. However, the use of hCG in addition to LH administration could be beneficial for embryos of good, fair, and poor qualities.
Nogueira has a fellowship from FAPESP (São Paulo).
172 PRIMORDIAL GERM CELL DIFFERENTIATION FROM ES CELLS IN VITRO IN MOUSE
Authors: Kobolak J. , Deak E. , Dinnyes A.
Published: December 2004

The primordial germ cells (PGC) in the genital ridge of the embryo are the progenitors of sperm and eggs. The goal of the present study was to derive PGC cells from embryonic stem (ES) cells and compare their gene expression with that of primary PGC cultures. R1 (Nagy A et al. 1990 Development 110, 815–821) and Oct4-GiP (Ying QL et al. 2002 Nature 416, 545–548) ES cell lines were differentiated into PGCs. For in vitro differentiation, the modified method of Geijsen N et al. (2004 Nature 427, 148–154) was used. In brief, ES cell suspension was put into hanging drops (400 cells per drop) for two days, where they formed embryoid bodies (EBs). The medium consisted of Iscove's Modified Dulbecco's Media (Gibco) supplemented with 10% FBS (Hyclone, Logan, UT, USA), 30 μg mL−1 iron saturated transferrin (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Sigma, Hungary), non-essential amino acids (Sigma), 4.5 mM monothioglycerol (Sigma), 50 μg mL−1 ascorbic acid (Sigma), 2 mM glutamine (Gibco), and antibiotics. The EB clumps were differentiated in suspension culture for 2 or 5 days, and then dissociated with collagenase treatment. Cells positive for SSEA-1 were isolated from dissociated EBs by immunomagnetic bead sorting and plated into gelatinized plates in the presence of 2 μM retinoic acid (Sigma). After 7 days of culture, individual PGC colonies were isolated and subcloned. The subcloned PGCs were cultured in PGC medium consisting of Dulbecco's Modified Eagle Media (Gibco) supplemented with 15% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1000 U recombinant mouse leukaemia inhibitor factor (ESGRO®, Chemicon International, Inc., Temecula, CA, USA), 20 ng mL−1 basic fibroblast growth factor (Sigma), 60 ng mL−1 soluble mouse stem cell factor (Sigma), and antibiotics. The gene expression profile was monitored using semi-quantitative RT-PCR. The gene expression of Oct4, Nanog, Stella, Piwil2, Rnf17, and Tex14 were analyzed during the differentiation. Primary PGC cultures were also isolated from (C57BL/6 × DBA)F1 embryos of age 8.5 and 11.5 days post-coitum, and differentiated in vitro. The previously described PGC medium was used to proliferate the isolated cells. The gene expression profile of PGCs and ES-derived PGC lines were compared. There were no great differences between the gene expression profiles of PGCs and ES cell-derived PGC cells. SSEA-1 and alkaline phosphatase staining of cells did not show differences between the two cell populations. We have shown here the two PGC populations do not differ from each other in gene expression of the selected genes. Further investigation is needed to differentiate the PGCs into gametes and to analyze the gene expression of other genes involved in gamete differentiation.
The authors would like to acknowledge Gyorgyi Kungl for the technical help. This research was supported by OTKA T046171 grant.
240 GESTATION LENGTH AND BIRTH WEIGHT OF IN VITRO PRODUCED EMBRYOS FROM ZEBU DAIRY CATTLE
Authors: Camargo L.S.A. , Viana J.H.M. , Ramos A.A. , de Sa W.F. , Ferreira A.M. , Fonseca J.F. , Vale Filho V.R.
Published: December 2004

Gir cattle are a popular zebu dairy breed in tropical and subtropical regions because of their tolerance of heat stress and resistance to tick-borne disease. The use of in vitro embryo production (IVP) technology may help accelerate genetic improvement of this breed. However, in general, IVP systems have been implicated in the production of large offspring and a greater proportion of male calves. Natural breeding results in newborn Gir calves weighing around 25 kg despite the fact that dams may weigh over 500 kg. It is unknown whether in vitro-produced Gir embryos also result in large offspring. The aim of this work was to evaluate the effect of in vitro embryo production on gestation length, birth weight, and sex ratio in Gir cattle. COCs were harvested by oocyte pickup from mature non-lactating Gir cows and in vitro-matured in TCM 199 medium (Gibco, Sao Paulo, Brazil) with 10% inactivated estrous cow serum for 24 h under 5% CO2 at 38.5°C in air. Gir spermatozoa were obtained through the swim-up method and co-incubated with oocytes in Fert-TALP media (Parrish JJ et al. 1988 Biol. Reprod. 38, 1171–1180) with 10 μg/mL heparin (Sigma, Sao Paulo, Brazil) and 6 mg/mL fatty acid-free bovine albumin (Sigma) for 18 h in 5% CO2 at 38.5°C in air. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa medium (Wilkinson RF et al. 1996 Theriogenology 45, 41–49) with 10% fetal calf serum in humid atmosphere of 5% CO2 at 38.5°C in air. Fresh Day 7 blastocysts were transferred to synchronized B. indicus × B. taurus crossbred recipients. Data of gestation length, birth weight, and gender ratio from 26 IVP calves (IVP group) were recorded and compared to data obtained from Gir calves produced by artificial insemination or natural mating (n = 24; control group) using ANOVA or chi-square analysis. There was no difference (P > 0.05) in gestation length between pregnancies of the IVP and control groups (mean ± SEM, 285.4 ± 1.5 vs. 284.4 ± 1.1 days, respectively). IVP and control calves showed similar (P > 0.05) weight at calving (29.6 ± 0.9 vs. 26.9 ± 1.2 kg for IVP and control male calves, and 27.0 ± 2.5 vs. 25.2 ± 0.5 kg for IVP and control female calves, respectively). The percentage of male calves was greater (P < 0.05) in the IVP group than in the control group (76.9% vs. 43.4%, respectively). IVP calves did not show abnormalities associated with Large Offspring Syndrome, such as breathing difficulty and perinatal death. These data suggest that in vitro production may affect the development of Gir embryos, biasing the sex ratio in a manner similar to previously reported for in vitro-produced embryos from Bos taurus breeds.
This work was supported by FAPEMIG and CNPq.
260 REPLACEMENT OF PVA WITH FETAL BOVINE SERUM IMPROVES FORMATION AND HATCHING OF PORCINE BLASTOCYSTS PRODUCED IN VITRO
Authors: Yoshioka K. , Suzuki C. , Rodriguez-Martinez H.
Published: December 2004

Porcine embryos, derived from in vitro maturation and fertilization, were used to investigate the effects of timing of serum inclusion and PVA replacement in the medium for in vitro culture (IVC) on rates of blastocyst formation and hatching. In Experiment 1, presumptive zygotes at 20 h post-insemination (hpi) or cleaved embryos obtained by culture in porcine zygote medium (PZM-5) containing 3 mg mL−1 polyvinyl alcohol (PVA) at 48 or 96 hpi were further cultured in either PZM-5 containing PVA or PZM-5 where PVA was replaced by 1%, 5%, or 10% fetal bovine serum (FBS) until Day 6 (Day 0 = the day of in vitro insemination). Supplementation with 1% to 10% FBS at 20 and 48 hpi reduced (P < 0.05; by ANOVA and Fisher's PLSD test) blastocyst rates on Days 5 (0% to 1%) and 6 (3% to 6%) compared with PVA supplementation (4% and 22%, respectively). However, addition of 10% FBS at 96 hpi increased (P < 0.05) blastocyst rates (30%) on Day 5 compared with PVA (11%) and 1% FBS (15%); there was no significant difference among treatments in rates of blastocyst formation on Day 6 (24% to 40%). The total number of blastomeres in Day 6 blastocysts did not differ among treatments at any timing of serum supplementation (26.5 to 48.3 cells). In Experiment 2, presumptive zygotes were cultured from 20 to 96 hpi in PVA medium, and the cleaved embryos were later transferred into PZM-5 containing PVA, or 1%, 5%, or 10% FBS for another 4 days. Hatching rates of embryos on Days 7 and 8 were significantly higher (P < 0.05) in PZM-5 where PVA was replaced with 10% FBS (15% and 20%, respectively) than those in PZM-5 containing PVA (1% and 5%, respectively). Moreover, the total cell number in hatching/hatched blastocysts on Day 8 were significantly greater (P < 0.05) in medium containing 10% FBS (135.1 cells) than that in PVA medium (77.0 cells). In Experiment 3, at 130 hpi, blastocysts derived from IVC with PZM-5 containing PVA were transferred into PZM-5 containing PVA, 3 mg mL−1 bovine serum albumin (BSA) or 10% FBS for another 2 days. Hatching rates of blastocysts on Days 6, 7 and 8 were significantly higher (P < 0.05) in PZM-5 where PVA was replaced with 10% FBS (12%, 56%, and 64%, respectively) than those in PZM-5 containing PVA (0%, 12%, and 20%, respectively) and BSA (0%, 12%, and 20%, respectively). Moreover, the total cell number in hatching/hatched blastocysts on Day 8 were significantly greater (P < 0.05) in medium containing 10% FBS (138.7 cells) than that in PVA (71.7 cells) and BSA medium (70.7 cells). The results indicate that the timing of serum inclusion in the culture medium markedly affects porcine embryo development in vitro and that replacement of PVA with FBS in PZM-5 at 96 hpi or later improves the subsequent development of embryos to the hatching/hatched blastocyst stage.
This work was supported by MAFF, Japan, and STINT and FORMAS, Sweden.
275 THE EFFECTS OF MATURATION CULTURE PERIOD ON THE SEX RATIO OF BOVINE IVF EMBRYOS
Authors: Agung B. , Wongsrikeao P. , Murakami M. , Watari H. , Otoi T.
Published: December 2004

The maturational stage of oocytes at the time of insemination has been suggested as influencing the sex ratio of resulting embryos (Dominko and First, 1997 Theriogenology 47, 1041–1050). However, there are very few reports concerning the relation between the maturation culture period of oocytes and the sex ratio of resulting embryos. The objective of this study was to investigate the effects of maturation culture period of oocytes on the sex ratio of bovine IVF blastocysts, using a novel technique of loop-mediated isothermal amplification (LAMP). Cumulus-oocyte complexes (COCs) were collected from ovaries of slaughtered cows. The COCs were cultured for various times (16, 22, 28 and 34 h) in maturation medium (TCM 199 supplemented with 5% fetal cow serum (FCS), 0.02 mg mL−1 of follicle-stimulating hormone and 50 μg mL−1 of gentamicin). After maturation culture for each period, the oocytes were inseminated with frozen-thawed spermatozoa (4 × 106 spermatozoa mL−1). After incubation with spermatozoa for 5 h, oocytes were transferred into culture medium (TCM199 supplemented with 5% FCS, and 5 μg mL−1 of insulin) and cultured for 7 days at 38.5°C in a humidified atmosphere of 5% CO2 in air. The zona pellucida of blastocysts collected on Day 7 after insemination (Day 0) was removed by brief exposure to 0.2% pronase, and the sex determination of embryos was conducted by the LAMP method, using a bovine embryo sexing kit (Eiken Chemical Co., Ltd., Tochigi, Japan). The rates of blastocyst formation were analyzed by ANOVA, and the sex ratios of embryos were compared by chi-square analysis. As shown in Table 1, the rate of blastocyst formation after insemination was significantly higher (P < 0.05) in the oocytes matured for 22 h than in the oocytes matured for 16, 28, and 34 h. The proportion of male blastocysts derived from oocytes matured for 34 h was significantly higher (P < 0.05) than from oocytes matured for 16 and 22 h. Moreover, the proportions of male blastocysts increased with delaying insemination. These results indicate that skewing of the sex ratio of IVF blastocysts is apparently influenced by the maturation culture period of oocytes.

Table 1.
Effect of maturation culture period on the development of bovine oocytes after IVF and the sex ratio of blastocysts
301 EFFECT OF ENERGY SUBSTRATES ON METABOLISM, NUCLEAR MATURATION, AND DEVELOPMENT OF GILT AND SOW OOCYTES DURING IN VITRO MATURATION
Authors: Tubman L. , Peter A. , Krisher R.
Published: December 2004

Metabolic mechanisms control both nuclear and cytoplasmic maturation in oocytes. Elevated glucose metabolism is typically associated with improved developmental competence. The objective of this study was to compare nuclear maturation, oocyte metabolism, and subsequent embryonic development following the use of different energy substrates during in vitro maturation (IVM) and to determine the specific role of each substrate. Cumulus-oocyte complexes (20–50/treatment (Trt)/replicate) were placed into maturation medium for 42 h in 7% CO2 in air at 38°C. Maturation treatments included a negative control (NC; 0.01 mM pyruvate and 6 mM lactate), addition of 1:100 dilution of fatty acids (FA; Gibco, Grand Island, NY, USA), 1 × NEAA/0.5 × EAA/1 mM glutamine (AA), or 2 mM glucose (GLU) individually; and a positive control (PC; addition of all three substrates). For each of six replicates, metabolism of 10 denuded oocytes/treatment was measured in hanging drops containing labeled glucose (0.0125 mM 5-3H glucose, glycolysis; 0.482 mM 1-14C glucose, pentose phosphate pathway, PPP). Oocytes were then fixed and stained for determination of meiotic stage. Remaining oocytes were fertilized and cultured in vitro. Cleavage and blastocyst development were recorded at 30–40 and 144 h post-insemination, respectively. The Purdue Porcine Media system was used throughout (PPM; Herrick et al. 2003 Reprod. Fertil. Dev. 15, 249–254). All data were subjected to analysis of variance. Oocyte metabolism and embryonic development are presented In Table 1. Except for FA, energy substrate influenced the percentage of oocytes reaching metaphase II (NC, 1.37 ± 0.01; FA, 1.35 ± 0.01; AA, 33.33 ± 0.06; GLU, 25.81 ± 0.06; PC, 54.29 ± 0.06) but age of oocyte donor did not. Blastocyst metabolism and cell number were not affected by treatment. In general, sows were more responsive to treatment effects. These data demonstrate that exogenous fatty acids do not play a role in porcine oocyte maturation. Amino acids appear to promote meiosis and glycolysis, but do not support oocyte developmental potential. Elevated metabolism in this treatment may be due to a recovery effect when glucose-starved oocytes were placed into glucose containing metabolism medium. Glucose appears to be important for meiosis and cytoplasmic maturation leading to developmental competence with minimal effect on oocyte metabolism. The success of the positive control suggests that a combination of glucose and amino acids is beneficial to maturation and embryonic development of porcine oocytes.

Table 1.
Metabolism and development of oocytes after IVM
31 CONSTRUCTION OF A TARGETING VECTOR SPECIFIC FOR THE BOVINE BETA-CASEIN GENE
Authors: Chang M. , Oh K.-B. , Wee G. , Koo D.-B.n , Shin S.-T.e , Lee K.-K. , Han Y.-M.
Published: December 2004

Although using livestock as animal bioreactors is a powerful tool to produce valuable therapeutic proteins in the milk, there are still some limitations to the technology such as a low frequency of transgenesis and a low expression of the transgenes by random integration. In this study, we constructed gene-targeting vectors for a mammary gland-specific gene and then obtained two homologous recombinant cell clones after transfection of the vectors into bovine somatic cells. Two targeting vector cassettes, BCKI I and BCKI II, which have homology regions for a bovine beta-casein gene, were constructed. The beta-casein gene is expressed only in the mammary gland and is the most abundant milk protein in the cow. The targeting sequence lengths of the BCKI I and BCKI II vector cassettes were 13.1 kb and 9.1 kb, respectively, and contained different long arm lengths. A neo gene was inserted into the vectors as a selection marker, and a few restriction enzyme sites were made in front of the neo gene. The human thrombopoietin (TPO) gene was inserted into the restriction enzyme sites of the vector cassettes, named BCTPOKI I and BCTPOKI II vectors. The BCTPOKI I and BCTPOKI II vectors were transfected into bovine embryonic fibroblasts (bEF) and ear skin fibroblasts (bESF) using Lipofectamine™ 2000 reagent (Invitrogen, Seoul, South Korea). In order to determine the highest transfection efficiency, a variety of factors such as DNA concentration, lipid volume, and exposure time to DNA-liposome complexes based on the manufacturers' guideline, was optimized. The 2:1 and 1:2 ratios of DNA (μg) to transfection reagent (μL) were efficient for bEF and bESF, respectively, under overnight exposure to DNA-Lipofectamine™ 2000 reagent. Seventeen percent (51/304) of bESF clones and 6% (9/149) of bEF clones were normally expanded into passage 8. PCR and Southern blotting indicated that 6.3% (2/32) of the clones carrying with BCTPOKI II vectors was homologously targeted at the beta-casein gene. However, none (0/60) of the clones carrying BCTPOKI I was targeted. Additionally, both of the targeted clones were from bESF. When the targeted cells were transferred into enucleated oocytes and cultured, 83% (43/52) of the cloned embryos were transgenic. Thus, we found that homologous recombinant events using gene-targeting vectors might be dependent on cell types, vector sizes, and transfection procedures. In conclusion, mammary gland-specific gene-targeting vectors coupled with somatic cell nuclear transfer technology will be very useful for developing animal bioreactors that produce therapeutic proteins in milk.
33 PRODUCTION OF TRANSGENIC-CLONED PIGS CARRYING HDAF, GnT-III AND HETEROZYGOUSLY DISRUPTED ±-1,3-GALACTOSYLTRANSFERASE GENES
Authors: Fujimura T. , Takahagi Y. , Nagashima H. , Miyagawa S. , Shigehisa T. , Murakami H.
Published: December 2004

In pig-to-human xenotransplantation, transplants are rapidly rejected by binding of human natural antibodies to porcine xenoantigen, mostly Gal α-1-3Gal oligosaccharides, and subsequent complement attack. To overcome this rejection, we so far have produced transgenic pigs expressing both human CD55/DAF (decay-accelerating factor, a complement-regulatory protein) and GnT-III (N-acetylglucosaminyltransferase III, a sugar chain modifying enzyme). In the present study, we heterozygously disrupted the α-1,3-galactosyltransferase (GT) gene, which catalyses the biosynthesis of Gal α-1-3Gal epitopes, in the fetal fibroblast cells from the DAF/GnT-III transgenic pigs by homologous recombination, and successfully produced GT-knockout pigs by nuclear transfer. Fibroblast cells isolated from Day 30 fetuses of DAF/GnT-III transgenic pigs were transfected with a GT-targeting vector. The targeting event in drug-resistant colonies was confirmed by PCR analysis, and targeted cells were used as nuclear donors. The reconstructed embryos were electrically activated and transferred to estrus-synchronized recipient pigs. At pregnancy Day 27 of gestation, fetuses were collected and their fibroblast cells were isolated for secondary nuclear transfer. The genomic DNA of live-born piglets produced by the secondary nuclear transfer were analyzed for the presence of DAF and GnT-III genes as well as the heterozygous disruption of the GT gene. From a total of 5.5 × 107 cells transfected with the GT-targeting vector, 2,749 drug-resistant colonies were obtained. Eighteen colonies were judged positive for targeting events by PCR analysis. After transfer of 321 cloned embryos reconstructed with the knockout cells to three recipients, four knockout fetuses were obtained from one recipient. Transfer of 633 cloned embryos reconstructed with the knockout fibroblast cells from one knockout fetus to six recipients gave rise to two live knockout piglets. PCR analysis of genomic DNA confirmed that the cloned piglets carried both DAF and GnT-III transgenes as well as the heterozygously disrupted GT gene.
332 ENHANCEMENT OF GROWTH PERFORMANCE IN DOUBLE TRANSGENIC MICE WITH	GROWTH HORMONE RECEPTOR AND IGF-1 RECEPTOR GENES
Authors: Ju Kim H. , Naruse K.i , Choi W.S. , Im K.S. , Park C.S. , Jin D.I.
Published: December 2004

The effect of amplifying growth-related receptor signaling, through overexpression of receptors, on growth regulation in animals was examined. Transgenic mice lines were produced by DNA microinjection using the metallothionein promoter ligated to either the growth hormone receptor (GHR) or IGF-1 receptor (IGF-1R) genes (3 GHR founders and 3 IGF-1R founders). Transgenic mouse lines were estimated to contain approximately 4 to 20 copies of transgenes per cell by Southern blot analysis. Founder mice of each transgenic line transmitted transgenes into F1 and F2 pups with Mendelian ratio. Double transgenic (IGF-1R/GHR) mice were produced by the mating between nine pairs of IGF-1R and GHR hemizygous transgenic F1 mice. The transmission patterns in the 78 F2 pups produced from these matings were 20 with no transgene (25.6%), 17 with the IGF-1R gene (21.8%), 25 with the GHR gene (32.1%), and 16 with both GHR and IGF-1R genes (20.5%). The mRNA expression of transgenes using RT-PCR with the specific primers for IGF-IR and GHR genes was checked in tissues of transgenic mice. Double transgenic mice with IGF-IR and GHR genes expressed more mRNAs of transgenes than non-transgenic or single transgenic mice. Growth of double transgenic mice was fastest compared with single transgenic mice containing IGF-1R or GHR genes. And GHR transgenic mice grew faster than IGF-1R transgenic mice. When body weights of 15 transgenic mice for each transgenic line were measured at 4, 10, and 14 weeks after birth, double transgenic mice were significantly heavier compared with non-transgenic control mice at each stage (24 to 30% heavier in double transgenic mice; 15 to 20% heavier in single transgenic mice, P < 0.05). These results suggest that overexpression of growth-related receptor genes could promote the growth of transgenic animals with an additive effect.
42 PRODUCTION OF CLONED PIGS FROM SOMATIC STEM CELLS DERIVED FROM SALIVARY GLAND
Authors: Kurome M. , Ueda H. , Tomii R. , Nakamura K. , Okumura K. , Matsumoto S. , Endo F. , Nagashima H.
Published: December 2004

The aim of the present study was to investigate whether a somatic stem cell derived from the salivary gland can be an efficient donor cell for pig cloning. Somatic stem cells (salivary gland progenitor cells, SGP) were isolated from the salivary gland of a 4-month old male pig (Matsumoto et al. 2004). Briefly, tissue sections of salivary gland were gently digested by collagenase/hyaluronidase and dispersed into single cells. Isolated cells (5 × 104) were cultured on a type I collagen-coated dish in William's medium E; then colonies having epithelial-like morphology were picked to establish the primary culture of SGP cells (CD49, intracellular laminin-positive) which have the potential to differentiate in vitro into hepatic and pancreatic endocrine cells after spherical body formation in vitro. SGP cells to be used as nuclear donors were cultured for 2 days under serum starvation. Fetal fibroblast (FF) cells were used as control nuclear donors. IVM oocytes were obtained from abattoir ovaries and matured in NCSU23. Donor cells were fused with the enucleated recipient oocytes by a single DC pulse of 190 V/mm for 10 μs in 0.28 M mannitol + 0.15 mM MgSO4. Reconstructed embryos were electrically activated by DC pulse of 150 V/mm for 100 μs in 0.28 M mannitol + 0.05 mM CaCl2 + 0.1 mM MgSO4 at 1–1.5 h after the NT, followed by cytochalasin B treatment for 3 h. Development of the NT embryos was assessed in vitro by fixing and staining at either 2 h post NT or after culture for 7 days in NCSU23, or in vivo by transfer to the oviducts of estrus-synchronized recipient gilts. Incidence of premature chromosome condensation was similar regardless of donor cell type. Development of the NT embryos reconstructed with SGP to the blastocyst stage was significantly higher compared to that of the FF group (38/137, 27.7% vs. 19/168, 11.3%, respectively; P < 0.05). Transfer of 278 cloned embryos reconstructed with SGP to two recipients resulted in the production of three live piglets. Production efficiency of piglets from the cloned embryos reconstructed with FF was 2/263. Based on the in vitro development of the reconstructed embryos, SGP is a promising nuclear donor cell for pig cloning; further transfer experiments are to be carried out.
This study was supported by PROBRAIN.
52 NUCLEAR REMODELING AFTER SOMATIC CELL NUCLEAR TRANSFER (SCNT) IN THE RHESUS MONKEY
Authors: Mitalipov S. , Byrne J. , Sparman M. , Ramsey C. , Wolf D.
Published: December 2004

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. MPF degradation, caused by fertilization or artificial activation, is in turn required for pronuclear formation and subsequent embryonic cleavage, and involves cyclin B catalyzed proteolysis by the proteasome system. In the rhesus monkey, SCNT results in the production of cleaving embryos, but development arrests at the morula stage presumably because of abnormal or incomplete reprogramming. We undertook this pilot study to examine the timing and extent of nuclear remodeling events (NEBD, PCC) in monkey SCNT embryos. The proteasome inhibitor MG-132 was employed to avoid or delay premature MPF degradation (Zhou et al. 2003 Science 302, 1179). Monkey fetal fibroblasts employed as nuclear donor cells were fused with nonactivated cytoplasts and incubated in the presence (n = 20) or absence (control; n = 35) of MG-132. Embryos were fixed and co-labeled with DAPI (DNA) and monoclonal antibody against lamin A/C (nuclear envelope). In monkey germinal vesicle-stage oocytes (n = 5) and zygotes (n = 6), a lamin A/C signal was detected at the nuclear periphery while matured MI (n = 6) and MII (n = 12) oocytes were negative for lamin A/C staining, consistent with the absence of a nuclear membrane. Donor fetal fibroblasts arrested at the G1 stage of the cell cycle exhibited a lamin A/C signal. Minimal or no changes were observed in donor nuclei within 1 h after fusion. The majority of control SCNT embryos sampled 4 h after fusion exhibited only slight chromatin condensation; however, they failed to form metaphase chromosomes. Positive lamin A/C staining indicated the presence of intact nuclear membranes. Following activation these SCNT embryos cleaved, but arrested at the 8–16 cell stage. In initial experimentation we determined the minimal efficient concentration of MG-132 to be 5 μM, that is capable of inhibiting first polar body extrusion during the MI–MII transition. Subsequent in vitro development to the blastocyst stage (53%) of fertilized oocytes treated with 5 μM MG-132 for up to 4 h was similar to that of nontreated controls. Incubation of SCNT embryos (n = 20) for 4 h with MG-132 resulted in robust chromosome condensation, spindle formation, and weak or partial lamin A/C signal. Our observations suggest that incomplete nuclear remodeling events in monkey SCNT embryos may be due to premature MPF inactivation perhaps caused by the fusion pulse. Future studies will address the developmental potential of monkey SCNT embryos exposed to MG-132.
This work was supported by NIH grant NS04330, Core Grant RR00163 and a product donation from Ares Advanced Technology, Inc.
55 NUCLEUS CHANGES AND DEVELOPMENT OF PORCINE RECONSTRUCTED (NT) AND PARTHOGENETICALLY ACTIVATED (PA) EMBRYOS
Authors: Mtango N.R. , Kono T.
Published: December 2004

Nuclear reprogramming is characterized by functional modification(s) of the transferred nucleus that allows it to direct normal embryo development with the potential to grow to term. The aim of our study was to investigate the process of nuclear changes in reconstructed and activated embryos as well as their developmental competence. All chemicals used were from Sigma Chemicals (St. Louis, MO, USA). Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries of prepurbetal gilts and matured for 42 h in vitro. The cumulus cells were removed by adding in 1 mg mL −1 hyaluronidase in TLP-HEPES. For the NT experiment, oocytes with first polar body were cultured in 0.4 μg mL−1 demecolcine for 1 h. A protruding membrane was removed by micromanipulator and a single donor nucleus from fetal fibroblast was injected subzonally. Fusion was conducted immediately after transfer in 0.3 M mannitol, 0.5 mM HEPES, 0.1% PVA, and 0.1 mM MgCl2 in a fusion chamber with parallel electrodes set 1 mm apart using a singe DC pulse of 125 V mm−1 for 80 s. Activation was done 2–4 h after fusion in the same medium as fusion but with 0.1 mM CaCl2 added; embryos were cultured in 5 μg mL−1 cytochalasin B and 10 μg mL−1 cyclohexamide for 6 h. The embryos were cultured in glucose-free NCSU-37 containing 4 mg mL−1 BSA as basic medium supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2, and then in basic medium with 5.55 mM D-glucose from Days 2–6 (Kikuchi K et al. 2002 Biol. Reprod. 66, 1033–1041). Non-manipulated oocytes (PA) were electrically activated as stated above. For observing the changes of donor cells, some reconstructed oocytes were fixed 2 h after fusion, prior to activation, and some 12 h after activation in acetic acid:ethanol (1:3) and stained in 1% orcein. The activated oocytes were fixed at 12 h and stained as stated above. There were 47.5% (38/80) of reconstructed oocytes with premature chromosome condensation (PCC), and 23.7% (19/80) with nuclear swelling two hours after fusion. Pronuclear like formation 12 h after activation was 45% (27/60) and 83.3% (50/60) in NT and PA, respectively. The blastocyst rate was 8.3% (5/60) and 46% (69/150) for NT and PA, respectively. The results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of NT embryos to the blastocyst stage.
61 CLONED MOUSE PRODUCED USING A ZONA FREE METHOD OF NUCLEAR TRANSFER
Authors: Ribas R. , Oback B. , Taylor J. , Maurício A. , Sousa M. , Wilmut I.
Published: December 2004

Mice have been cloned from somatic and embryonic cells; however, only 0–3% of the reconstructed embryos develop into viable offspring. In addition, the piezo microinjection method widely used for mouse nuclear transfer (NT) is difficult to master. Our objective was to compare cumulus and ES cells as nuclear donors using a simplified method of zona-free NT. In cattle, zona-free NT is simpler, faster, easier to learn and more reproducible than zona-intact NT (Oback et al. 2003 Cloning Stem Cells 5, 3–12). Oocytes were recovered at metaphase II stage (13 h after hCG injection) from the oviducts of C57BL/6J × DBA/2 F1 females (8–10 weeks of age). Cumulus cells were removed with hyaluronidase (300 units/mL) and the zona pellucida digested with pronase (0.5%) at 37°C for 3 min. Oocytes were then enucleated under UV light in cytochalasin B (5 μg/mL) after a 5-min staining with Hoechst (5 μL/mL). The metaphase DNA was removed in an enucleation pipette (16–20 μm, perpendicular break) by separating karyoplast and cytoplast with a simple separation pipette (60–80 μm, perpendicular break, closed round tip). Embryonic stem (ES) cells were cultured for 3 days and serum-starved for 16 h before use. Cells from this line had yielded offspring by the piezo procedure. Cumulus cells were used freshly. Donor cells were attached to the cytoplasts with phytohemagglutinin (10 μg/mL) and couplets were electrically fused in 0.2 mM mannitol buffer. Reconstructed embryos were activated 1–2 h after fusion for 5–6 h in CZB medium containing 10 mM strontium chloride and 5 μg/mL of cytochalasin B. Embryos were cultured individually in 5-μL droplets in CZB. Morulae and blastocysts were transferred into the uteri (Day 2.5) of pseudopregnant surrogate mothers (C57BL/6J × CBA/2J). Recipient mothers were sacrificed at 19.5 days postcoitum and pups removed. Airways were cleaned to remove fluid and the pups were held in a warm box before being fostered by a lactating mother. During development of the technique, we assessed the frequency of fusion, cleavage of reconstructed embryos, and development to morula/blastocyst stage. Fusion (58.1 ± 6.7% vs. 24.2 ± 1.7%, P < 0.001) and cleavage (66.4 ± 4.2% vs. 50.5 ± 5.4%, P < 0.05), all respectively, were higher when cumulus cells were used as donors, as compared with ES cells. However, the percentage of embryos developing to morula/blastocyst stage was greater when ES cells were used (22.2 ± 4.2% vs. 5.3 ± 2.7%, P < 0.01). Using ES cells as donors, 19/94 (20.2%) reconstructed embryos reached compacted morula/blastocyst stage. After transfer to five recipients, one pup was born (5.2%). It was larger and heavier than uncloned pups of the same age. The pup is healthy and now 12 weeks old. Genotype was confirmed by microsatellite analysis. The birth of a healthy cloned mouse pup from zona-free NT provides “proof of principle” of a technology that promises to increase throughput, ease of operation, and reproducibility of mouse cloning.