bluuebunny/crossref_metadata_2025 huggingface dataset:
312THE EFFECTS OF ECG AND ESTRADIOL ON CANINE OOCYTES DURING IN VITRO
MATURATION
Authors: Hanna C.B. , Westhusin M.E. , Kraemer D.C.
Published: February 2004

The effects of eCG and estradiol (E2) were evaluated on canine oocytes during in vitro maturation (IVM). Canine ovaries were obtained from routine ovariohysterectomies, and oocytes with one or more intact cumulus layers and a consistently dark and smooth cytoplasm were collected by mincing the ovaries in TL HEPES warmed to 38°C. Oocytes were cultured for 72h at 38.5°C in 5% CO2 in humidified air in medium containing TCM 199, 2.92mM calcium lactate, 2.0mM pyruvic acid, 4.43mM HEPES, 10% fetal bovine serum, and 1% Pen/Strep solution (12166 GibcoBRL, Grand Island, NY, USA) and with no added hormones (control), 2ng/mL E2, 10IU/mL eCG, or both E2 and eCG. At the end of culture, oocytes were removed and denuded by aspiration in a medium containing 50% v/v Trypsin-EDTA 10X solution (T-4174Sigma, St. Louis, MO, USA) and 50% v/v PBS with 3mgmL−1 polyvinyl alcohol. Denuded oocytes were fixed in 3.7% paraformaldehyde for 1h and then transferred to a 1.9mM Hoechst 33342 solution in glycerol. Nuclear status was observed under UV light and recorded. Chi-square analysis was performed to determine statistical significance where P<0.05. Oocytes cultured with eCG were observed as having a significantly larger percentage of retained germinal vesicles (GV) at the end of culture (78%) compared to those cultured with E2 (62%) or in the control (66%). Addition of E2 with eCG was observed to increase the GV retention percentage (84%); however, it was not significantly greater than treatment with eCG alone. The eCG- and eCG with E2-treated oocytes also showed cumulus cell mass expansion where the cumulus oocyte complex diameter greatly increased and became ‘fluffy’ in appearance. The control and E2-supplemented media were the only treatments where MII oocytes were observed (5% and 9%, respectively), and the percentages of meiotic resumption for these treatments (15% and 17%, respectively) were significantly greater than those for eCG-(8%) and eCG with E2 (2%)-treated oocytes. The percentage of oocytes in which there was no observable DNA at the end of culture remained relatively constant between all four treatment groups and was also average compared to most reports of canine IVM in the literature. These data suggest that it is possible eCG promotes cumulus cell expansion and acts as a meiotic inhibitor for canine oocytes in vitro. The addition of E2 appears to augment the inhibitory effect of eCG, but in culture alone did not significantly increase the percentage of oocytes that resumed meiosis compared to the control. The authors would like to acknowledge Genetic Savings and Clone for making this work possible.

Table 1
Nuclear status of oocytes after culture
323OPTIMIZATION OF SERUM-FREE IVP PROTOCOL:
EFFECT OF SERUM-FREE IVM ON BOVINE EMBRYO DEVELOPMENT
Authors: Räty M.E. , Ketrja E.H. , Kananen-Anttila K.M. , Peippo J.M.H.
Published: February 2004

The use of serum in IVP medium may cause abnormalities, e.g. reduced post-thaw survival, in bovine IVP embryos (Abe H et al., 1999 Mol. Reprod. Dev. 53, 325–335). Moreover, serum may be a source of contamination and its composition is highly variable. Several reports have shown that serum-free IVF–IVC does not compromise embryo development (see, e.g. Keskinteipe L et al., 1996 Biol. Reprod. 55, 333–339). Here we studied the effect of serum-free IVM on bovine IVP embryo development, aiming for production of good quality Day 7 embryos for freezing.
In total, 11734 abattoir-derived bovine oocytes in 13 batches were washed with
emCare Complete Medium with BSA (1mgL−1) and matured for 24h in TCM-199 with glutamax-I (Gibco, Paisley, UK), 0.25mM Na-pyruvate,
100IUmL−1 penicillin, 100μgmL−1 streptomycin,
10μgmL−1 LH, 2μgmL−1 FSH and 1μg mL−1 β-estradiol supplemented either with 1) 10% FBS (Gibco, New Zealand), 2) 4mgmL−1 fatty acid-free albumin (FAF-BSA), 3) 4mgmL−1 FAF-BSA+growth factors (GF;; 100ngmL−1 IGF-I+100ngmL−1 EGF), 4) 4mgmL−1 polyvinylpyrrolidone (PVP), 5) 4mgmL−1 PVP+GF and 6) PVP+amino acids (10μLmL−1 MEM+20μLmL−1 BME). After 20h fertilization in FERT-TALP+2mgmL−1 BSA with semen of
pre-tested IVF-bull, the oocytes were denuded and cultured in modified
SOFaaci+6mgmL−1 FAF-BSA in 5% O2 (Holm P et al., 1999 Theriogenology 52, 683–700). The statistical analyses are based on logistic regression models with IVP batch and treatment as explanatory variables. The estimated probabilities (P) are shown in Table 1. The upper and lower values of 95% confidence intervals varied within P±0.05, P±0.03, and P±0.02 for cleavage, Day 7 embryo development and development of good quality Day 7 embryos, respectively. PVP+GF-IVM supported cleavage equally well as FBS-IVM (P=0.49), whereas the remaining serum-free IVM treatments had lower embryo cleavage rate than FBS-IVM (P<0.01). On Day 7 none of the serum-free IVM treatments supported embryo development and development of good quality embryos as well as FBS-IVM (P<0.01). Addition of GF in FAF-BSA-IVM reduced embryo cleavage, Day7 embryo development and development of good quality embryos compared to that of FAF-BSA alone (P<0.03). PVP-IVM resulted in lower embryo cleavage rate than PVP+GF (P<0.005), whereas according to the two other
criteria there were no differences between the treatments (P>0.13). In conclusion, these preliminary results indicate that replacing the FBS as a protein source in IVM needs more optimization.

Table 1
Estimated probability (proportion) for embryo cleavage at 34–38 hpi (Y1), Day 7 embryo development (Y2) and development of good quality Day 7 embryos (Y3) after serum-containing and serum-free IVM
324SPHINGOSINE-1-PHOSPHATE PROTECTS CULTURED BOVINE OOCYTES FROM PHYSIOLOGICALLY RELEVANT THERMAL STRESS
Authors: Roth Z. , Hansen P.J.
Published: February 2004

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P<0.01) and the number of oocytes developing to the blastocyst stage (P<0.04). There was a temperature x S1P interaction for cleavage rate (P<0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.
333PREGNANCY RATE OBTAINED WITH EMBRYOS COLLECTED AFTER INSEMINATION OF
SUPEROVULATED COWS WITH SEXED SORTED SEMEN
Authors: Panarace M. , Cattaneo L. , Caballero J.N. , Cerrate H.R. , Kaiser G.G. , Marfil M. , Medina M.J.
Published: February 2004

According to the production objectives of beef or dairy commercial farms, the possibility of gender selection of the offspring is an important tool to optimize the use of genetic and economical resources. The use of sexed sorted semen by flow cytometry to inseminate donors in a MOET program has successfully demonstrated a normal embryo production per cow (Theriogenology 59:513). The aim of this study was to evaluate the pregnancy rate and sex accuracy obtained after transferring embryos collected using sexed semen to inseminate the donors in MOET programs. The semen was provided by our commercial laboratory. Eighteen cows were inseminated with frozen female sexed semen and 22 with frozen male sexed semen. Embryos were collected using a commercial flushing medium with BSA and antibiotics. After the embryos were washed through 10 drops of holding medium, 50 embryos were transferred fresh;; 150 were frozen in 1.5M ethylene glycol, thawed 10 seconds in air;; and held for 30 seconds in a water bath at 35°C before transferring them. Fresh embryo stage and quality rates were: morula grade 1: 49%, grade 2: 13%; blastocyst grade 1: 34%, grade 2: 4%. Frozen embryo stage and quality rates were: morulae grade 1: 28%, grade 2: 18%; blastocyst grade 1: 47%, grade 2: 7%. The embryo transfers (ET) were performed in previously synchronized recipients injected with two doses of prostaglandin 12 days apart. Thirty days after ET, the diagnosis of pregnancy was done by transrectal ultrasonography (ALOKA 500 Scanner) to asses the pregnancy rate. Sixty days after the ET, fetal sex determination was done. The results are shown in the table 1 below. The pregnancy rates in both fresh and frozen-thawed preselected sex embryos are similar to those obtained with nonpreselected sex embryos (Theriogenology 56:1401). The high rate of sex accuracy shows the reliability of the technique of sexed sorted semen by flow cytometry to produce preselected sex embryos in superovulated cows. This research was supported by Fundación Margarita Perez Companc.

Table 1
337THE ONSET AND DURATION OF OVULATION IN DAIRY COWS SUPEROVULATED
FOLLOWING SYNCHRONIZATION OF FOLLICLE WAVE WITH CIDR AND ESTRADIOL BENZOATE
Authors: Kishida K. , Nishisouzu T. , Aoki S. , Iwata M. , Dochi O. , Koyama H.
Published: February 2004

Determining the optimal time for AI of superovulated cattle is important for yielding acceptable fertilization rates. The objectives of this study were to determine the interval to onset, and the duration of ovulation in dairy cows superstimulated with FSH following synchronization of follicular wave emergence with a CIDR and estradiol benzoate (EB). Holstein (n=8) and Jersey (n=1) cows received an intravaginal progesterone insert (CIDR-B, InterAg, Hamilton, New Zealand) combined with an injection of 2mg EB at a random stage of the estrous cycle (Day 0). Superstimulatory treatments were initiated on Day 6 with a total dose of 24 or 36mg FSH (Antrin, Denka, Kawasaki, Japan) via twice-daily i.m. injections in decreasing doses for 4 days. At 72h after the first FSH injection, 30mg PGF (Pronalgon F, Pharmacia, Tsukuba, Japan) was administered, and CIDR-B were removed at 80h. Transrectal ultrasonography of ovaries was performed at 3-h intervals from 40 to 77h after the PGF injection. The numbers of ovulations were counted as previously described (Purwantara et al. 1994 Anim. Reprod. Sci. 37, 1–5). All detected follicles were counted and classified as small, medium, or large (diameter 2–4mm, 5–9mm, or ≥10mm, respectively). The number of ovulations recorded after each scanning was defined as the change in the number of large- and medium-sized follicles observed since the previous scanning. Data were analyzed using ANOVA. Results are presented in Table 1. There were two patterns of ovulations: one was a concentrated pattern of ovulations (ovulatory period <10h); the other was a prolonged pattern of ovulations (ovulatory period >10h). In cows with a high superovulatory response (≥14 ovulations), the onset of ovulation was earlier (P<0.05) and the duration of the ovulatory period was longer (P<0.05) than in cows with lower (≤8 ovulations) responses. In conclusion, concentrated and prolonged ovulation patterns were seen in dairy cows superstimulated after synchronization of the follicle wave with a CIDR and EB. In cows with a large number of follicles, the onset of ovulation tended to be earlier and the duration of the ovulatory period longer than in cows with fewer follicles.

Table 1
The onset and duration of ovulation in dairy cows superstimulated following synchronization of the follicle wave with a CIDR and EB
339CORRELATION BETWEEN FOLLICLE SIZE AND QUALITY OF OOCYTES FROM THE
SUPEROVULATED CYNOMOLGUS MONKEY
Authors: Takenoshita M. , Ohta S. , Fujinami N. , Yamochi T. , Kunieda T. , Anzai M. , Matsumoto K. , Saeki K. , Hosoi Y. , Iritani A.
Published: February 2004

Variability in the superovulatory response continues to be one of the most frustrating problems with the application of assisted reproductive technologies in non-human primates. Superstimulation of donor animals with equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG) is widely used, but individual responses to these hormones vary widely. In human in vitro fertilization, follicle size is commonly used as a marker to determine the timing of hCG treatment in order to acquire oocytes matured in vivo. Incorporation of techniques designed to control follicular size in humans may improve superstimulatory response in non-human primates. In this study, we measured follicle size and examined oocyte quality in Cynomologus Monkeys superstimulated as described below. On the first day of spontaneous menses, monkeys were treated with long-acting GnRH (Luprin: Takeda Pharm, Co. Ltd., Osaka, Japan;; Day 0). A dose of 25IU/kg/day eCG (Serotoropin;; Teikokuzoki Pharm, Co. Ltd., Tokyo, Japan) was administered i.m. from Day 1 to Day 9, followed by 200IUkg−1 hCG (Puberogen;; Sankyo Pharm, Co. Ltd., Tokyo, Japan) i.m. on Day 10. Dominant follicle sizes were measured on Days 7 and 9 by ultrasonography. Oocytes were collected by laparoscopy in anesthetized monkeys 40h after the hCG injection. Oocytes were classified by nuclear status. Immature oocytes at the stages of germinal vesicles (GV) and metaphase (MI) were cultured until reaching the stage of Metaphase II (MII). Matured oocytes (MII) were fertilized by ICSI and cultured for 7 days. At the end of culture, the developmental stage of oocytes was examined. The ovaries with different-sized follicles on Day 7 were divided into two groups;; ovaries with large follicles (>4.5mm) were in the first group and ovaries with small follicles (<4.0mm) were in the second group. On Day 9, follicles in first group grew to more than 5.0mm and follicles in second group remained less than 5.0mm. Sixty-two percent of oocytes from follicles in first group were at MI or MII stage, while only 15% of oocytes in second group reached the MI or MII stage. After ICSI, 42% of MII oocytes from first group developed to the blastocyst stage, while no blastocyts were observed in second group. These results suggest that the size of dominant follicle was a limiting factor for the developmental ability of oocytes in vitro. For production of Cynomolgus monkey blastocysts derived from ICSI, the diameter of dominant follicle was required to be at least 5mm before hCG in order to collect MI and MII oocytes. Incorporation of hormonal treatments designed to optimize follicular size probably reduced the variability in quality of oocytes. Therefore, we expect that an adjustment of dose and duration of eCG and hCG treatment may improve developmental ability of oocytes from follicles that had not reached 5mm.
344ICSI-MEDIATED GENE TRANSFER SKEWS SEX RATIO
AGAINST FEMALE BIRTHS IN MICE
Authors: Moreira P.N. , Pintado B. , Montoliu L. , Gutiérrez-Adán A.
Published: February 2004

ICSI-mediated gene transfer has been used as an alternative method to pronuclear microinjection for the genomic modification of many species. With this method, transgenic embryos are produced by the microinjection of metaphase II oocytes with spermatozoa previously incubated with foreign DNA. Recently, it was shown in mice that the low percentage of transgenic animals produced from injected oocytes, results from the fact that the expression of foreign DNA is associated with paternal chromosome degradation (Szczygiel M.A. et al., 2003 Biol. Reprod. 68, 1903–1910). It is also known that sex chromosomes localize preferentially, at least in humans, on the periphery of the sperm nucleus on sub-acrosomal regions (Sbracia M. et al., 2002 Hum. Reprod. 17, 320–324), suggesting a high level of interaction with foreign DNA molecules with possible impact on the sex ratio of the offspring. In order to test this hypothesis we have compared ICSI (no DNA), and with ICSI-mediated EGFP (5Kb plasmid DNA from Clonetech, Spain) transfer, with ICSI-mediated YRT3 (a mouse tyrosinase gene derivative YAC-DNA with 100Kb; Montoliu L. et al., 1996 EMBO) transfer. Gametes were from 6–8 weeks old CD1 mice. ICSI-mediated gene transfer with post-thawed immotile spermatozoa, extended in M2 medium in the absence of ion chelators (EDTA and EGTA), was done as previously described (Szczygiel M.A. et al., 2003 Biol. Reprod. 68, 1903–1910). Table 1 below summarizes the data collected. Relative to our control, sex ratio deviation was a consequence of the coinjection of DNA. Forty-three percent of males were obtained with regular ICSI, whereas 64% and 65% were the respective percentages when EGFP or YRT3 DNA was coinjected with spermatozoa. This statistically significant (P<0.05, z-test, Sigma Stat, Jandel Scientific, USA) sex ratio deviation, favoring male ICSI offspring when foreign DNA is coinjected, may result from a higher female embryo susceptibility to parental sex chromosome fragmentation induced by the interaction with foreign DNA molecules. Possible impairment of X chromosome inactivation and dosage compensation resulting from the fragmentation of the sex chromosome on X-carrying spermatozoa could explain this female embryo degeneration. Supporting this view, it was recently shown in mice that sex ratio can be skewed against female births by a mutation in a single gene of the X chromosome (Tsix) involved in such mechanisms (Lee J.T., 2002 Nat. Genet.). In conclusion, mouse ICSI-mediated gene transfer induces sex ratio deviation favoring male offspring.

Table 1
Sex ratio of the offspring obtained with ICSI, ICSI-mediated EGFP transfer, and ICSI-mediated YRT3 transfer
40PREDICTORS OF CLONED CALF VIABILITY
Authors: Hill J. , Henderson B. , Cibelli J. , Page R.
Published: February 2004

The high rate of gestational loss in nuclear transfer (NT) pregnancies is a major economic and animal cost, preventing the widespread use of the technique. It is currently not possible to predict which early gestation fetuses will remain viable to term. As placental abnormalties are commonly associated with nonviable fetuses, placental proteins such as pregnancy specific protein (PSP 60, Heyman et al., 2002 Biol. Reprod. 63, 1787–1794; or PSPb Hill et al., 2000 Biol. Reprod. 66, 6–13) have been investigated as possible markers of viable pregnancy. To build upon these studies, we explored the predictive value of Day-35 maternal serum PSPb value to final cloned-calf viability; PSPb profiles throughout pregnancy; and the relationship of maternal plasma estradiol levels to calf viability. Maternal serum was sampled for PSPb at the time of the first ultrasound pregnancy examination (Day 35) to determine fetal number and viability (heartbeat). PSPb values were determined using an RIA by Dr. Garth Sasser (BioTracking, Moscow, ID). Median PSPb values were compared using the Kruskal-Wallis one-way ANOVA test on ranks. PSPb levels from failed single and twin pregnancies were significantly different from those of AI bred controls (P<0.05). PSPb levels from failed singles were not significantly different from those of viable single pregnancies although only nonviable cloned pregnancies had PSPb values >7.5. As PSPb levels at Day 35 were not highly predictive of viable outcome, we detailed the gestational PSPb profile for a different group of cows (n=40) that carried cloned pregnancies beyond Day 90. PSPb was determined at Days 35, 50, 65, and 90, then monthly to Day 240. Although there was no clear statistical significance, PSPb levels from nonviable cloned pregnancies were consistently higher at each time point than for viable pregnancies (14 cows gave birth to live calves) and significantly higher than those of controls. At term, plasma estradiol concentrations were assessed in a group of 5 NT pregnancies. Estradiol was investigated as cloned pregnancies often show a lack of readiness for parturition (e.g. lack of udder development, prolonged gestation, dysmature calves). These 5 pregnancies produced 3 highly viable NT calves: 1 with lowered viability that survived only with a high level of intensive care, and 2 nonviable despite a high level of assistance. In this small group, estradiol levels on the day of birth rose in parallel with viability (viable, 662pg; lowered viability, 170pg; nonviable, 76pg). This is a clinical observation rather than a statistical trend that suggests further investigation may be warranted. In summary, our results suggest maternal PSPb levels have value in identifying the outliers in a population of cloned pregnancies and that term-cloned pregnancies may show abnormally low maternal estradiol concentrations at term.

Table 1
44POSSIBILITY OF PRODUCTION OF A TRANSGENIC CLONED COW USING THE SOMATIC
CELL NUCLEAR TRANSFER OF HUMAN ALPHA 1-ANTITRYPSIN GENE-TRANSFECTED
CELLS
Authors: Jang G. , Bhuiyan M.M.U. , Park E.S. , Chun H.Y. , Cho J.G. , Park H.J. , Kang S.K. , Lee B.C. , Hwang W.S.
Published: February 2004

The present study was designed to produce, using a somatic cell nuclear transfer (SCNT), transgenic cloned cows that secreted a therapeutic protein into milk. Bovine cumulus cells were transfected with a plasmid that contained the alpha 1-antytrypsin DNA and green fluorescent protein (GFP) reporter gene using a Fugene 6 as a lipid carrier. In vitro-matured bovine oocytes were enucleated followed by confirmation of enucleation via nuclear staining. The GFP-expressing donor cells with the transgene were selected under blue fluorescent light just before injection of cells. The SCNT was performed according to the established system in our laboratory (Jang G et al., 2003 Reprod. Fertil. Dev. 15, 179–185). The couplets were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C in a humidified atmosphere of 5% CO2 in air for up to 7 days. The developmental ability (in Experiment 1) and number of inner cell mass (ICM) and trophectoderm (TE) cells (in Experiment 2) in transgenic and non-transgenic cloned embryos determined by differential staining as described by Thouas et al., (2001 Reprod. Biomed Online 3, 35–29) were compared. In Experiment 3, only GFP-expressing blastocysts were transferred to the uteri of recipient cows. A general linear model consisting of ANOVA and a least squares method in the SAS program was used for statical analysis. The percentage of blastocysts was lower (P<0.05) in transgenic cloned embryos compared to non-transgenic embryos (34% v. 20%). No difference (P<0.05) in the cell number of ICM and TE cells between two groups of embryos was observed. Out of 47 recipient cows, three pregnancies were detected by non-return to estrus and by rectal palpation. However, the pregnancies were not maintained to term; 2 fetuses were aborted at Days 60, and 150, and one fetus was aborted at Day 240. The genomic DNA from the fetus aborted at Day 240 was prepared and analyzed for the expression of GFP and alpha 1-antitrypsin gene by polymerase chain reaction. The expected PCR products (710 bp for alpha 1-antytrypsin DNA and 431 bp for GFP) were obtained and confirmed by sequence analysis. In conclusion, the present study demonstrated that developmental competence of cloned embryos derived from transgenic donor cells was lower compared with cloned embryos derived from non-transfected donor cells. Although we failed to produce a viable transgenic cloned calf, expression of alpha 1-antitrypsin in fetal tissue represented the possibility of production of a transgenic cloned cow by SCNT. This study was supported by Biogreen 21-1000520030100000.
45PRODUCTION OF CHIMERIC TRANSCHROMOSOMIC CALVES
Authors: Kasinathan P. , Nichols M.F. , Griffin J.E. , Robl J.M.
Published: February 2004

Chimeras have been used for investigating fundamental aspects of early embryonic development, and differentiation, and for introducing foreign genes into mammals (Robertson et al., 1986 Nature 323, 445–448; Cibelli et al., 1998 Science 280, 1256–1258). The main objective of this study was to determine if the transfer of blastomeres from in vitro-produced (IVP) embryos into cloned, transchromosomic embryos improved the efficiency of producing transchromosomic calves. Cloned embryos were produced using in vitro-matured bovine oocytes and bovine fetal fibroblasts containing a human artificial chromosome (HAC) (Kuroiwa et al., 2002 Nat Biotechnol 20, 889–894). IVP embryos were produced using standard procedures and blastomeres were harvested at the 8–16 cell stage by removing the zona pellucida with protease. Cloned embryos were randomly divided on Day 4 into two groups. One group received 3–4 IVP blastomeres while a second group served as a control (nonmanipulated cloned embryos). After transferring the blastomeres, the chimeric and cloned embryos were placed in culture (Kasinathan et al., 2001 Biol. Reprod. 64, 1487–1493) and on Day 7 development to the blastocyst stage was evaluated. Grades 1 and 2 embryos were transferred; two each per synchronized recipient. Pregnancy maintenance, calving, and calf survival were evaluated in both groups. Presence of a HAC in live calves was evaluated in both fibroblasts and peripheral blood lymphocytes (PBLs) using FISH analysis. Embryo development to the blastocyst stage, maintenance of pregnancy and number of calves born were analyzed using Chi-square. There were no differences in the rate of blastocyst development at day 7 or establishment of pregnancy at 40d (P>0.05). However, pregnancy rate at 120d, and number of calves that developed to term and were alive at birth (chimera 14/54 and clone 4/90), and at 1 month of age (chimera 13/54 and clone 1/90) were lower (P<0.01) for cloned embryos. The proportion of cells containing an HAC in PBLs, was higher in cloned calves (100%) compared to chimeric calves (26%). The HAC retension rates in PBLs in HAC-positive chimeric and cloned calves were 84% and 95%, respectively. These data indicate that, although the proportion of calves retaining an HAC was lower in chimeras compared to clones, more HAC-positive calves were produced in the chimeric treatment from fewer cloned embryos. We speculate that higher rates of development in the chimeras may be related to the normality of the placenta. Future studies will be required to determine the contribution of the IVP blastomeres to both the inner cell mass and trophectoderm. Therefore, a chimeric approach may be useful for improving the efficiency of producing cloned transchromosomic calves.
46DIFFERENTIAL PATTERNS OF PROTEIN SYNTHESIS BETWEEN NORMAL AND CLONED
PLACENTAE
Authors: Kim H.R. , Kang J.K. , Yoon J.T. , Seong H.H. , Park C.S. , Jin D.I.
Published: February 2004

Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by extremely low success rate. Most clones die before birth and survivors frequently display abnormalities. It is speculated that epigenetic reprogramming is somehow defective in reconstituted embryos (Reik W et al., 2003 Theriogenology 59 21–32; Han YM et al., 2003 Theriogenology 59, 33–44). It is likely that placental anomalies are directly or indirectly responsible for the death of cloned fetus and neonates. To address this question, we analyzed protein patterns of two placentae obtained after postnatal death of fetuses from SCNT of Korean Native Cattle and two normal placentae obtained after birth of AI fetuses. Global proteomics approach was employed by using 2-D gel electrophoresis and mass spectrometry to separate the different placenta proteins. Proteins within an isoelectric point range of 4.0 to 7.0 and a molecular weight range of 20–100kDa were analyzed by means of 2-D gel electrophoresis with three replications of each sample. The stained gels were scanned and calibrated at an optical resolution of 63.5μm/pixel using a GS-710 (Bio-Rad Laboratories, Hercules, CA, USA). Approximately 480 spots were detected in placental 2-D gel stained with coomassie-blue. Then, image analysis by Malanie III (Swiss Institute for Bioinformatics, Geneva, Switzerland) was performed to detect variations in protein spots between normal and SCNT placentae. In the comparison of normal and SCNT samples, at least 15 protein spots were identified as regulated differentially. Using MALDI-TOF-MS (PerSeptive Biosystems, Framinham, MA, USA), 10 spots were identified as up-regulated proteins in SCNT placentae including BPLP-I, Rho GDI 2, osteoclast stimulating factors, SM22, 60S Acidic Ribosomal and Protein P2, whereas five spots were down-regulated proteins such as Peroxiredoxin 2. Mass spectrometry with sequencing was used to further analyze the uncharacterized proteins. Most identified proteins in this analysis appeared to be related to cell proliferation and differentiation, fetal growth and development or metabolism. Further, specific functions of proteins in placenta have been investigated at the molecular levels during pregnancy.
47BOVINE OOCYTE CYTOPLASM SUPPORTS NUCLEAR REMODELING BUT NOT
REPROGRAMMING OF MURINE FIBROBLASTS
Authors: Kim N.-H. , Shin M.R. , Park S.H.
Published: February 2004

Nuclear transfer (NT) is used to elucidate fundamental biological functions such as cell differentiation reversibility and interactions between the cytoplasm and nucleus. In the present study, we compared nuclear and microtubule dynamics in bovine oocytes following NT of bovine and murine fibroblast cells. To clarify the nuclear reprogramming procedures, we additionally examined the expression of development-related genes (Octamer-binding transcription factors, Oct-4; E-cadherin, E-cad) and housekeeping genes (Heat shock protein 70.1, Hsp; Bos taurus apoptosis regulator box-a, Bax; Glucose transporter 1, Glut-1) in bovine embryos that had received nuclei from bovine and murine fibroblast cells. Bovine oocytes were matured in vitro and enucleated after 22h. The oocytes reconstructed with mouse embryonic fibroblast cells or bovine somatic fibroblast cells were cultured in CR1aa media. While the embryos that received nuclei from bovine fibroblast cells developed into blastocysts, those that received nuclei from murine fibroblasts did not develop beyond the 8-cell stage. Similar nuclear and microtubule dynamics were observed in oocytes reconstructed with murine and bovine fibroblast cells. A small microtubule aster-containing γ-tubulin spot was observed in association with decondensed chromatin following NT of mouse fibroblasts. Within 1h of fusion of enucleated, non-activated cytoplasm, most mouse fibroblast nuclei were transformed to premature chromosome condensation (PCC). Randomly arrayed microtubules were tightly associated with PCC and formed meiotic-like microtubular spindles in all cases. Condensed chromosomes were divided into two or three chromatin masses and developed into multiple pronuclear-like structures. Microtubule asters were observed near the pronuclear-like structures during apposition in the cytoplasm. Two poles of the γ-tubulin spot evident at the mitotic metaphase stage are involved in the formation of the astral microtubule spindle for initial mitosis. A number of housekeeping mouse genes (hsp70, bax and glt-1) were abnormally expressed in embryos that had received nuclei from mouse fibroblast cells. However, development-related genes, such as Oct-4 and
E-cad, were not expressed. The results collectively suggest that the bovine oocyte cytoplasm supports nuclear remodeling, but not reprogramming of mouse fibroblast cells.

Table 1
Relative abundance of mRNA expression (mean±SEM) in mouse and xenonuclear-transferred (X-NT) embryos
48EFFECT OF INSULIN-LIKE GROWTH FACTOR-1 SUPPLEMENT TO NCSU-23
MEDIUM ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS DERIVED FROM IN
VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER
Authors: Kim S. , Nam D.H. , Jung Y.W. , Kim H.S. , Lee S.H. , Lee G.S. , Hyun S.H. , Kim D.Y. , Kang S.K. , Lee B.C. , Hwang W.S.
Published: February 2004

The developmental potential of in vitro production of embryos is affected by various factors, including the culture system, oocyte quality, the presence of serum, and embryo paracrine and autocrine growth factors. Insulin-like growth factor is a good stimulator of oocyte maturation and embryo development. The present study investigated the effect of insulin-like growth factor-I (IGF-I) supplement on the preimplantation development of porcine embryos derived from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos and blastocysts at Days 2 and 7, respectively. The number of total cells and inner cell mass (ICM) cells in blastocysts were counted after differential staining at Day 7. All data were analyzed by ANOVA using a Generalized Linear Model (SAS). In Experiment 1, a total of 2,462 in vitro-matured oocytes (527, 458, 498, 481 and 498, respectively) were inseminated with frozen-thawed boar semen and subsequently cultured in North Carolina State University (NCSU)-23 medium supplemented with various concentrations of IGF-1 (0, 1, 10, 50 and 100ngmL−1). As a result, significant model effects on the development to the 2-cell stage (P=0.033) and to the blastocyst stage (P=0.0067) were found, and more blastocysts (16.9, 16.6, 17.5, 21.8 and 14.7 %, respectively) were obtained in medium supplemented with 50ngmL−1 of IGF-I. Moreover, increase in the total cell number (56.5, 53.2, 74.0, 76.4 and 58.4) and ICM (6.6, 5.8, 9.3, 9.4 and 6.1) cells was observed in IVF embryos cultured in NCSU-23 medium supplemented with 50ngmL−1 IGF-1. In Experiment 2, porcine cloned embryos were produced by our standard protocol using fetal fibroblasts as donor cells (Hyun SH et al., 2003 Theriogenology 59, 1641–1649) and cultured in NCSU-23 supplemented with the same concentration of IGF-1 as Experiment 1. As a result, a total of 501 reconstructed oocytes (99, 98, 102, 99 and 96, respectively) were cultured and significant model effects on the development to the 2-cell stage (P=0.0179) were found. More blastocysts (10.5, 11.2, 11.8, 20.8 and 10.1%) were produced when embryos were cultured in NCSU-23 medium supplemented with 50ngmL−1, even though no statistical significance was found (P=0.1182). Increases in the total cell number (42.7, 46.0, 45.9, 51.1 and 38.2) and ICM cells (3.8, 3.8, 5.6, 6.6 and 4.8, respectively) were observed in cloned embryos cultured in NCSU-23 medium supplemented with 50ngmL−1 of IGF-I. In conclusion, the present study demonstrated that IGF-1 at the concentration of 50ngmL−1 improves the development of preimplantion embryos derived from IVF and SCNT. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1).
49IN VITRO DEVELOPMENT OF PORCINE CLONED EMBRYOS FOLLOWING DIFFERENT
ACTIVATION TREATMENTS
Authors: Kim Y.S. , Ock S.A. , Lee S.L. , Choe S.Y. , Rho G.J.
Published: February 2004

The present study compared the development of cloned porcine embryos following different activation treatments. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 22h in NCSU#23 medium supplemented with 10% porcine follicular fluid, 0.57mM cysteine, 0.5μgmL−1 LH, 0.5μgmL−1 FSH and 10ngmL−1 EGF. The COCs were further cultured for an additional 22h in the same medium at 39°C in an atmosphere of 5% CO2 in air, without hormonal supplements. Primary cultures of fibroblasts from a female fetus on Day 40 of gestation were established in DMEM +15% FCS. For nuclear donation, cells at the 5th-6th passage were cultured in DMEM+0.5% FCS for 5 days in order to arrest the cells in G0/G1. Following enucleation, oocytes were reconstructed by transfer of donor cells and fusion by means of three DC pulses (1.4kVcm−1, 30μs) delivered by a BTX 200, in 0.28M mannitol containing 0.01mM CaCl2 and 0.01mMMgCl2. Eggs were then divided into three treatment groups; control (without further treatment, Group 1), eggs cultured in 10μgmL−1 cycloheximide (CHX) for 5h (Group 2), and eggs cultured in 1.9mM6-dimethylaminopurine (6-DMAP) for 5h (Group 3). The eggs were then cultured in sets of 30in 60μL drops of NCSU#23 supplemented with 4μgmL BSA (essentially fatty acid free) until Day 7 at 39°C in a humidified atmosphere of 5% CO2. On Day 4 the culture were fed by adding 20μL NCSU#23 supplemented with 10% FBS. All experiments were performed as 4 replicates and statistical analysis was performed by one-way ANOVA (P<0.05). Blastocyst development rates were significantly higher (P<0.05) in Group 3 embryos compared to Group 1 controls (27.6±2.7% v. 20.1±4.1%, respectively), but rates did not differ in Group 2 (23.8±5.7%) compared to control. Total cell number in Group 3 blastocysts was, however, significantly higher (P<0.05) than in Groups 1 and 2 (44.6±2.4 v. 19.9±1.9 and 21.9±2.1, respectively). These results suggest that 6-DMAP is more efficient than cycloheximide in the activation of electrically fused NT oocytes during in vitro production of cloned porcine embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010 and Cho-A Pharm. Co. LTD.]
50SPATIAL EXPRESSION OF OCT4 IS NORMALLY REGULATED IN PREIMPLANTATION
STAGE BOVINE SOMATIC CELL CLONES
Authors: Kurosaka S. , Eckardt S. , Friez M.K. , Leu N.A. , Reinbold R. , McLaughlin K.J.
Published: February 2004

Oct4 is a member of the POU family of transcription factors and is expressed in blastomeres, pluripotent embryonic cells and the germ cell lineage. In the mouse blastocyst, expression of Oct4 becomes restricted to the inner cell mass (ICM). In bovine, Oct4 has not been considered to be a marker for pluripotency because Oct4 protein is expressed in both the ICM and the trophectoderm (TE) of bovine blastocysts (van Eijk MJT et al., 1999 Biol. Reprod. 60, 1093–1103; Kirchhof N et al., 2000 Biol. Reprod. 63, 1698–1705). Oct4 has been used as a marker gene for nuclear reprogramming in cloned embryos. Aberrant spatial distribution and levels of Oct4 have been observed in the majority of mouse clone blastocysts and blastocyst outgrowths (Boiani M et al., 2002 Genes Dev. 16, 1209–1219), indicating reprogramming failure of mouse clones. Lack of or abnormal Oct4 protein expression was also observed in cloned monkey embryos (Mitalipov SM et al., 2003 Biol. Reprod. 68 (suppl 1), 159). The spatial distribution of Oct4 mRNA and protein in bovine clones has not been reported. Bovine oocytes were obtained from a commercial supplier (BOMED, Inc., Madison, WI, USA), and were matured in vitro. Enucleated oocytes were fused with fibroblasts from ear skin and then treated with 10μgmL−1 cycloheximide and 1.25μgmL−1 cytochalasin D for 6h. Embryos were cultured in SOF supplemented with 1% fetal calf serum (FCS) at 39°C under 5% CO2, 5% O2 and 90% N2 for 2 days. At Day 2, embryos were transferred to SOF supplemented with 5% FCS and cultured under the same conditions until Day 7. Blastocysts were analyzed at Day 7. Oct4 mRNA expression was visualized by whole-mount in situ hybridization using a bovine Oct4-specific antisense riboprobe. Oct4 protein was detected by immunocytochemistry. Control embryos were produced by IVF and were cultured under the same conditions to the blastocyst stage (Day 7). We found that Oct4 mRNA signal was restricted to the ICM in bovine blastocysts. Bovine clones were not different from control embryos in that distribution of Oct4 mRNA signal was typically restricted to the ICM (14 of 16). In contrast to our previous report on mouse clones (Boiani M et al., 2002 Genes Dev. 16, 1209–1219), ectopic expression of Oct4 mRNA in the TE was rarely detected in bovine clones (2 of 16). Distribution of Oct4 protein was also similar between clones and controls with distribution in both the ICM and TE (clones: 9 of 9; controls: 9 of 9). It is unclear why defects in Oct4 distribution should differ between bovine and other species tested including monkey (Mitalipov SM et al., 2003 Biol. Reprod. 68 (suppl 1), 159–160); however, the higher rate of normal Oct4 distribution is consistent with the generally higher rates of postimplantation development of bovine clones (Shi W et al., 2003 Differentiation 71, 91–113).
51DEVELOPMENT OF BOVINE CLONES CONSTRUCTED WITH CYTOPLASM ORIGINATING
FROM OOCYTES CULTURED IN RETINOL
Authors: Lawrence J.L. , Schrick F.N. , Godkin J.D. , Edwards J.L.
Published: February 2004

Incomplete reprogramming of a somatic nucleus by the oocyte cytoplasm may contribute in large part to inefficiencies of cloning procedures using somatic cell nuclear transfer. Predominant use of cytoplasts derived from in vitro-matured oocytes may further exacerbate problems. Addition of all-trans-retinol (Livingston TL et al. 2002 Biol. Reprod. 66, 104–105 abst) or 9-cis-retinoic acid (Duque P et al. 2002 Human Reprod. 17, 2706–2714) to culture medium improved developmental competence of oocytes to blastocyst stage. The objective of this study was to evaluate effects of retinol for improving developmental competence of cloned embryos constructed with retinol-treated cytoplasts. After removal from 3–8mm follicles, COC were matured in the presence of 0 (n=1005; diluent only) or 5μM all trans-retinol (n=1017). Beginning approximately 18h after placement into culture, oocytes with extruded polar bodies were enucleated of maternal chromatin. Ovarian/granulosa cells were aspirated from adult Jersey cows (n=2) using an ultrasound-guided transvaginal probe. Primary cell lines were established and, before nuclear transfer, cultured in the presence of 0.5% serum (5–8 days; passage =3). Ovarian/granulosa cells were fused with cytoplasts originating from control or retinol-treated oocytes using two electrical pulses of 2.2kVcm−1 for 20μs between 24–27h post-maturation (hpm). Cloned embryos were activated between 26.5 and 30hpm and then cultured in an atmosphere of 7% O2 and 5.5% CO2 in KSOMaa+BSA. Development of cloned embryos to morula and blastocyst stages was assessed on Days 6–7 post-activation. In 5 replicates, compact morulae and blastocysts were transferred to synchronous recipients. Establishment of pregnancy was confirmed 28 days post-estrus by presence of an embryonic heartbeat using ultrasound. With the exception of pregnancy, data were analyzed as a randomized block design using mixed models of SAS (2000) after testing for normality. Proportion of oocytes recovered after denuding (94.5 and 88.9%; SEM=2.2), those that had visibly lysed (6.4 and 7.3%; SEM=1.5), and extruded a polar body by 20hpm (61.7 and 62.5%; SEM=7.0) was similar for control and retinol-treated oocytes, respectively. In addition, ability of ovarian/granulosa cells to fuse with control or retinol-treated cytoplasts was similar (80.6 and 73.1%; SEM=4.5). Lysis of cytoplasts after electrical pulses (8.2 v. 13.6%; SEM=3.3) and activation (11.6 v. 5.0%; SEM=2.9) did not differ for control and retinol-treated cytoplasts. Development to morula and blastocyst stages was lower in cloned embryos constructed with retinol-treated cytoplasts (Table). However, ability of morulae and blastocysts to establish a pregnancy was comparable. One clone from each treatment developed to term and was born alive. Culture of oocytes in medium containing retinol during maturation did not improve developmental competence of clones.
55IN VITRO DEVELOPMENT OF ENUCLEATED DOMESTIC CAT OOCYTES RECONSTRUCTED
WITH SKIN FIBROBLASTS OF DOMESTIC AND LEOPARD CATS
Authors: Lorthongpanich C. , Laowtammathron C. , Muenthaisong S. , Vetchayan T. , Ketudat-Cairns M. , Likitdecharote B. , Parnpai R.
Published: February 2004

The domestic cat is a valuable model for studies in assisted reproductive technology in felid species. Therefore, in this experiment we evaluated the in vitro developmental potential of enucleated domestic cat oocytes reconstructed with somatic cells from domestic and leopard cats. Skin fibroblasts were isolated from female domestic and leopard cats. The oocytes were collected by aspiration of follicles from ovaries that were superovulated with 200IU PMSG. In vitro-matured oocytes were enucleated and individual donor cells (diameter 14–16μm) were inserted into the perivitelline space of the enucleated oocyte. Fusion was performed at 26–27h post-maturation by placing a cell-oocyte couplet between both tips of the needle electrode and electrostimulating with a 2-DC pulse (30V, 30μs) in fusion medium containing 0.3M Mannitol+0.1mM MgCl2. Activation was performed 1 to 2h post-fusion by incubation in 7% ethanol at room temperature for 5min followed by cultured in 10μgmL−1 cycloheximide and 1.25μgmL−1 cytochalasin D at 38°C in 5% O2, 5% CO2, 90% N2 conditions. After activation, the reconstructed embryos were cultured in 100-μL droplets of Tyrode’s medium (Gomez et al., 2003 Theriogenology 60, 239–251.) supplemented with 0.3% BSA at 38°C in a 5% O2, 5% CO2, 90% N2 environment for 2d. Then, 8-cell embryos were cultured in 100-μL droplets of Tyrode’s medium supplemented with 10% FCS at 38°C in a 5% O2, 5% CO2, 90% N2environment for 5d. The cleavage rates of oocytes reconstructed with either donor cell types were not different. The percentages of blastocyst formation from parthenogenotes and nuclear transfer embryos derived from domestic cat fibroblasts (8/56, 14.3% and 7/51, 13.7%, respectively) were significantly higher than that for nuclear transfer embryos constructed with leopard cat fibroblasts (3/45, 6.7%). These results indicate that enucleated domestic cat oocytes reconstructed with skin fibroblasts of leopard cats can develop to the blastocyst stage. This experiment was supported by Suranaree University of Technology.

Table 1
In vitro development of domestic cat oocytes reconstructed with domestic and leopard skin fibroblasts and parthenogenetic activation