bluuebunny/crossref_metadata_2025 huggingface dataset:
312 CHEMICAL ACTIVATION OF IN VITRO-MATURED PORCINE OOCYTES
Authors: Bali Papp A. , Varga E.
Published: December 2006

Parthenogenetic oocyte activation is important for nuclear transfer and for the understanding of cell cycle regulation of oocytes. Several chemical agents, including ethanol, cycloheximide, strontium, cytochalasin B, 6 dimethylaminopurine, CaCl2 and ionophore A23187 can induce mammalian oocyte activation in vitro. The objectives of the present study were: (1) to assess the ability of strontium chloride (S), cytochalasin B (CB), cycloheximide (CX), and 6-dimethylaminopurine (D) to induce activation and parthenogenetic development in porcine oocytes; and (2) to verify whether the combinations of treatments (SB group = strontium combined with cytochalasin; SX group = strontium combined with cycloheximide, and SD group = strontium combined with 6-dimethylaminopurine) improves activation and parthenogenetic development rates. Oocytes from slaughterhouse ovaries were matured in vitro for 42 h at 39�C, in 5% CO2 in air. The basic medium used for oocyte maturation was TCM-199 supplemented with 10% pig follicular fluid, 1.25 mM L-glutamine, 0.9 mM Na-pyruvate, 100 �M cysteamine, 0.1 mg/mL streptomycin sulfate, 10 IU/mL pregnant mare serum gonadotropin (PMSG), and 10 IU/mL hCG (Werfft-Chemie GmbH, Vienna, Austria). Denuded MII oocytes were cultured in activation solution for 5 h. Thereafter the oocytes were cultured in NCSU37 for 6 days. At 48 h and 6 days after activation, oocytes, zygotes were fixed in acetic acid:alcohol (1/3 w/v), then stained with 0.1% (w/v) orcein in 45% (v/v) acetic acid, and evaluated under a phase contrast microscope. Each experiment was repeated four times. All data were analyzed by ANOVA, followed by Duncan's multiple range test (P < 0.05). A total of 2243 oocytes were activated in the different groups. In all groups, more than 45% of the oocytes were activated. No significant difference was observed in activation rate among SD (346/170, 49.13%), SX (302/164, 54.3%), and SB (318/182, 57.23%) groups. The activation rate for CB was significantly higher (P < 0.05) than for D or S (323/192, 59.44 � 6.84%; 366/176, 48.09 � 3.43%; and 319/183, 53.29 � 5.39%, respectively). The blastocyst rate for SX was significantly higher (P < 0.05) than that for D, SD, or SB (8.64 � 8.07%; and 0 � 0%; 0 � 0%; and 1.27 � 2.41%, respectively). In conclusion, this study suggests that chemical activation procedure is the most effective in strontium chloride combined with cycloheximide. The lowest oocyte fragmentation rates were in SX (28.40 � 1.26%) and CX (21.05 � 1.12%).
This work was supported by the the Hungarian Scientific T 43131 Research Foundation and the Hungarian Science on Technology Foundation E 14/04.
316 CHANGES IN MPF ACTIVITY IN PORCINE OOCYTES FOLLOWING ACTIVATION BY VARIOUS METHODS
Authors: Nanassy L. , Lee K. , Javor A. , Machaty Z.
Published: December 2006

In mammalian oocytes metaphase II arrest is maintained by high activity of the M-phase promoting factor (MPF), which is a heterodimer of cdc2 and cyclin B. The repetitive increase in the intracellular free calcium concentration at fertilization leads to the inactivation of MPF; low MPF activity will then stimulate the resumption of meiosis and entry into interphase. Most oocyte activation methods are able to trigger only a single calcium transient that may not be sufficient to keep MPF levels low enough to induce pronuclear formation. The objective of this study was to determine the profile of MPF activity after different oocyte activation protocols. Mature pig oocytes from prepubertal gilts were allocated into three groups: (1) oocytes activated by two consecutive electrical pulses; (2) oocytes electroporated and incubated for 4 h in 100 μM butyrolactone I, an inhibitor of cdc2; (3) oocytes electroporated and treated for 5 h with 10 μg/mL cycloheximide, a protein synthesis blocker. Some oocytes were removed from the medium at 2, 4, and 6 h after the electrical pulse and used to determine MPF activity using a cdk1/cdc2 kinase assay kit. The remaining oocytes were placed into PZM-1 medium and cultured for 7 days. Data were compared using ANOVA. After the electrical stimulus the initial activity of MPF (0.25 pmol/min/oocyte) dropped in all cases, i.e., the amount of phosphate incorporated into histones decreased. However, in oocytes activated with an electrical pulse only, MPF activity that by had 4 h dropped to 0.15 pmol/min/oocyte started to increase and by 6 h it reached 0.2 pmol/min/oocyte. In the cycloheximide-treated oocytes this increase was negligible (by 6 h the activity was 0.12 pmol/min/oocyte) whereas incubation in butyrolactone resulted in a continuous decrease in MPF activity which by 6 h was as low as 0.07 pmol/min/oocyte. Developmental data of the activated oocytes are presented in Table 1. The percent of embryos cleaved and the percent of oocytes that formed blastocysts were higher in the cycloheximide-treated group than in controls. In contrast, the total cell number per blastocyst was the highest in oocytes that were activated by electroporation only. Inhibiting cdc2 kinase or protein synthesis helps to maintain low MPF activity after stimulation and results in a high cleavage ratio and blastocyst formation in the pig. Such treatment, however, had a negative effect on total cell number at the blastocyst stage.

Table 1.
Developmental data of activated oocytes
329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT
Authors: Kananen-Anttila K. , Eronen M. , Matilainen J. , Kallio M. , Peippo J. , Halmekytö M.
Published: December 2006

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μÂ­m; serum+FSH-free: 12 ± 0.5 μÂ­m, P < 0.05; α-amanitin: 10 ± 0.6 μÂ­m, P < 0.001) and sperm asters (control: 86 ± 4 μÂ­m; serum+FSH-free: 82 ± 4 μÂ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription.

Table 1.
Average percentages ± (n) for nuclear maturation,
fertilization (min two pronuclei), embryo cleavage, and blastocyst development
332 EMBRYONIC DEVELOPMENT AFTER SOMATIC CELL NUCLEAR TRANSFER OF PORCINE OOCYTES MEIOTICALLY INHIBITED WITH ROSCOVITINE
Authors: Kim S. W. , Kim D. H. , Seo J. S. , Im G. S. , Yang B. C. , Park H. S. , Hwang I. S. , Cheong H. T. , Yang B. S.
Published: December 2006

Numerous factors affect on the developmental competence of cloned embryos, and one of the factors might be the disturbed synchronization of nuclear and cytoplasm maturation. Roscovitine, a purine known to specifically inhibit M-phase promoting factor (MPF) kinase activity by blocking the ATP in numerous cell systems, has been successfully used in maintaining porcine oocytes at GV stage without affecting their developmental potential. However, developmental ability of roscovitine treated porcine oocytes after nuclear transfer has not been evaluated. The purpose of this study was to examine the development of nuclear transferred porcine embryos after meiotic inhibition with roscovitine (ROS). Cumulus-oocyte complexes (COCs) were collected from antral follicles of slaughtered prepubertal gilts. COCs were cultured in pre-maturation medium (TCM-199 containing 50 �M Roscovitine) for 24 h, and then further cultured in conventional maturation medium for 44 h. A control group was cultured in the maturation medium for 44 h. Matured oocytes were enucleated and a porcine fetus cell was inserted into each enucleated oocyte. Couplets were simultaneously fused and activated with electric pulse of two 1.2 kV/cm for 30 �s. Nuclear transferred (NT) embryos were cultured in PZM-1 medium for 6 days (five replicates). Apoptotic cell death was analyzed by using a TUNEL assay and total cell number was examined by Hoechest 33342 counterstaining. At 3 h after fusion, NT embryos were fixed for microfilament staining. Data were analyzed by ANOVA and Student's t-test. The rates of fusion, cleavage, and blastocyst formation of the ROS-treated group (85, 68, and 18%, respectively) after nuclear transfer did not differ from control (78, 76, and 16%, respectively). The cell number in blastocysts of the ROS-treated group (30.8 � 10.6) was significantly lower than that of the control (42.3 � 13.7) (P < 0.01), but the mean proportion of apoptotic cells was not different between the two groups (6.9 � 7.1 and 4.8 � 4.9% for control and ROS group, respectively). Recovery of microfilaments after fusion was delayed in NT embryos derived from ROS-treated oocytes. This study demonstrated that porcine oocytes pre-cultured for 24 h in presence of roscovitine can be developed to blastocysts after somatic cell nuclear transfer. This could provide flexibility for studying porcine oocyte development and embryo cloning.
347 ARTIFICIAL INSEMINATION OF LACTATING HOLSTEIN COWS WITH SEXED SPERM
Authors: Crichton E. , Huffman S. , McSweeney K. , Schenk J.
Published: December 2006

The recommended use of sexed sperm has been limited to heifers. The objective of this field trial was to determine pregnancy rates in lactating dairy cows following artificial insemination of 2 or 10 � 106 sexed or 10 � 106 unsexed frozen/thawed sperm. Sperm were separated into X chromosome-bearing populations at rates of 3000-5000/s, on the basis of DNA content, with a flow cytometer/sperm sorter operating at 40 psi and using 150 mW of laser intensity. Sperm were sorted, collected and cryopreserved in a Tris-based medium. Holstein cows, averaging 2.4 lactations (range: 1-9), were presynchronized with prostaglandin F2� (PGF)2�; (25 mg) i.m. at 40-58 days in milk (DIM) (average, 44 days). A second dose of PGF2� (25 mg) i.m. was administered 14 days later. All cows were screened with ultrasound 14 days later, and those with normal ovarian and uterine status were used for this study. Qualifying cows received 100 �g gonadotropin-releasing hormone (GnRH) i.m. (average, 72 DIM) and 25 mg PGF2� i.m. 7 days later, and then 100 �g GnRH i.m. 2 days after the prostaglandin. Cows (n = 172) were fixed-timed inseminated 16-19 h after the last GnRH treatment, balanced over three sperm insemination treatments, two Holstein bulls, and two inseminators. Both 32-day pregnancy and 60-day fetal sex were determined using ultrasound. Cows carrying fetuses identified as dying at the 32-day ultrasound check were considered not pregnant. Data were subjected to ANOVA. Effects of sperm treatment, bulls, inseminators, and subsequent interactions on pregnancy rate were not significant (P > 0.05). The 32-day pregnancy rate and 95% confidence interval for 10 � 106 unsexed, 10 � 106 sexed, and 2 � 106 sexed inseminates were 55.6 (42.5-68.6), 43.9 (31.6-57.0), and 40.4% (28.4-53.6), respectively. Additionally, DIM or cow parity did not affect pregnancy rate (P > 0.05). Fetal death (<32 days) for unsexed inseminates was 5.5% (two pregnancies) compared to 9.5% (four pregnancies) for pooled sexed inseminates (P > 0.05). There were four confirmed pregnancies of sexed sperm that also aborted between Days 32-60. Fetal sex ratio was significantly altered (P < 0.05) for sexed (93% female fetuses) compared to unsexed (52% female fetuses) inseminates. Pregnancy rate in these selected lactating cows inseminated with sexed sperm was not significantly lower (P > 0.05) than that for unsexed sperm. However, these results should be viewed cautiously due to the limited numbers of cows used. Successful use of sexed sperm in dairy cows, which approached an elective waiting period of 80 DIM, was achieved with cows that were selected for reproductive status prior to insemination.
352 IMPROVING THE EFFICIENCY OF LAPAROSCOPIC INTRAOVIDUCTAL INSEMINATION WITH SEX-SORTED BOAR SPERMATOZOA
Authors: Vazquez J. M. , Martinez E. A. , Parrilla I. , Cuello C. , Gil M. A. , Garcia E. , Caballero I. , Almiñana C. , Roca J. , L. Vazquez J.
Published: December 2006

The insemination of a low number of sex-sorted spermatozoa is a critical issue that must be solved in order to enable the commercial application of this technology in pigs. A new procedure for laparoscopic intraoviductal insemination in sows has recently been reported (Vazquez et al. 2005 Reprod. Dom. Animals 40, 375 abst.). To improve the efficiency of this technique, this experiment was designed to determine the influence of insemination time, relative to the time of ovulation, on the number and quality of zygotes recovered after laparoscopic insemination of sows with sex-sorted spermatozoa. Spermatozoa were stained with Hoechst 33342 and sexed using the EPICS Altra flow sorter (Coulter Corporation, Miami, FL, USA) modified to operate at 42 psi for spermatozoa. Sorted spermatozoa were collected in tubes containing 1.5 mL of TEST-yolk (2%)-seminal plasma (10%). Post-weaning crossbred sows (n = 212; parity 2-4) were hormonally treated with eCG (Folligon; Intervet, Boxmeer, The Netherlands) and hCG (Veterin Corion, Divasa, Spain) and their ovaries were examined using transrectal ultrasonography at intervals of 4 h from 30 h after hCG injection to the laparoscopic insemination. Sows were allotted into three groups according to their ovarian status at insemination: preovulatory (P; n = 131), ovulating (O; n = 43), and ovulated (CL; n = 38) follicles. Follicle status was reconfirmed at insemination by direct observation using the laparoscope. Sows were inseminated in both oviducts with 0.3 million sex-sorted spermatozoa in 0.1 mL of extender. Eighteen hours later, putative zygotes were collected by washing the oviducts after laparotomy, fixed, stained with lacmoid, and examined by phase-contrast microscopy. Penetration rates were evaluated as numbers of monospermic and polyspermic oocytes per oocytes collected. Monospermic rates were evaluated as numbers of monospermic oocytes per oocytes penetrated. Data were analyzed by ANOVA. The number of putative zygotes collected were 2825, 957, and 736 for P, O, and CL groups, respectively. Penetration rates were not different (P > 0.05) among groups (90.4%, 94.5%, and 93.7% for P, O, and CL, respectively). However, the monospermic rate was significantly higher (P < 0.05) in the P group (97.4%) when compared to the O or CL groups (66.7% and 5.1% for O and CL, respectively). Moreover, percentages of sows with six or more zygotes, potentially able to carry the pregnancy to term, were 90.8% and 46.5% for P and O groups, respectively (P < 0.05). No sow of the CL group presented six or more zygotes. In conclusion, laparoscopic insemination should be performed only in sows with preovulatory follicles when sex sorted spermatozoa are inseminated using this technology.
This work was supported by CDTI and Fundacion Seneca.
362 EFFECT OF TIME OF REMOVAL OF A PROGESTERONE RELEASING DEVICE AND GnRH TREATMENT ON THE INTERVAL TO AND DISTRIBUTION OF OVULATIONS IN SUPERSTIMULATED BEEF COWS
Authors: Chesta P. , Maraña D. , Peres L. , Bo G. A.
Published: December 2006

An experiment was designed to evaluate the effect of the time of removal of an intravaginal progesterone (P4) releasing device and GnRH treatment on the interval to and the distribution of ovulations in beef cows superstimulated with Folltropin-V (Bioniche Animal Health, Belleville, Ontario, Canada). Red Angus donors (22 cows and 12 heifers) with a body condition score between 2.5 to 3.5 out of 5 were blocked by parity and randomly assigned to one of four treatment groups in a 2 × 2 factorial design. On Day 0, all donors received an i.m. DIB vaginal device (Syntex, Argentina) plus 2.5 mg estradiol-17β and 50 mg P4 (Laboritorio Rio de Janeiro, Argentina). Superstimulatory treatments were initiated on Day 4, with a total dose of 320 mg (cows) or 200 mg (heifers) NIH-FSH-P1 (Folltropin-V; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) in twice daily i.m. injections over 4 days. All cows received PGF treatment (150 μD(+)cloprostenol: Ciclase; Syntex) in the morning and afternoon of Day 6 and were randomly divided to have DIB devices removed at the time of the second PGF treatment (Day 6.5) or 12 h later (Day 7). On Day 8, donors were further subdivided to receive GnRH (0.050 mg Lecirelina, Gonasyn; Syntex) or no further treatment, and all cows were fixed-time AI either 60 and 72 h after the first PGF injection (0 h). All cows and heifers were examined by ultrasonography every 8 h for a period of 120 h commencing at the time of DIB removal, to determine the time and distribution of ovulations. For each cow, mean time of ovulation was used for the statistical analysis. The distributions of ovulations were compared among groups by Levene's test, and then by ANOVA (equal variances) or Friedman two-way non-parametric ANOVA (unequal variances). No significant differences were detected between cows and heifers (P > 0.13) for the parameters evaluated, and data were combined. There was no effect (P > 0.22) of treatment on the number of ovulations (GnRH: 12.6 ± 1.6 vs. no GnRH: 15.1 ± 1.1 and Day 6.5: 13.7 ± 1.3 vs. Day 7: 14.3 ± 1.5), or numbers of transferable embryos (GnRH: 5.1 ± 1.1 vs. no GnRH: 3.1 ± 0.8 and Day 6.5: 3.5 ± 0.8 vs. Day 7: 4.81 ± 0.1). However, there was a significant effect of GnRH treatment and a GnRH-by-time-of-DIB-removal interaction on the distribution of ovulations (P < 0.05; Table 1). The administration of GnRH in a superstimulatory protocol using P4 releasing devices results in earlier and more synchronous ovulations than when GnRH is not used.

Table 1.
Distribution of ovulations (hours after the first PGF) in superstimulated Red Angus donors treated with progesterone-releasing devices (DIB) for 6.5 or 7 days with and without GnRH treatment on Day 8
364 DISTRIBUTION OF OVULATION AND SUBSEQUENT EMBRYO PRODUCTION USING LUTROPIN AND ESTRADIOL-17β FOR TIMED AI OF SUPERSTIMULATED BEEF FEMALES
Authors: Larkin W. , Chesta P. , Looney C. , Bo G. , Forrest D.
Published: December 2006

This experiment was designed to compare timing of ovulation and embryo production between traditional superstimulatory and AI methods and the use of additional treatments of Estradiol-17β and pLH (Lutropin; Bioniche Animal Health, Belleville, Ontario, Canada) to facilitate timed AI in beef cows. A total of 31 mature cross-bred beef cows were randomly assigned three treatments. On Day 0, experimental cows were selected upon transrectal ultrasound evaluation for corpus leutum (>10 mm) prior to CIDR insertion plus 2.5 mg estradiol-17β and 50 mg Progesterone i.m. Superstimulatory treatments with Folltropin-V (Bioniche Animal Health) began on Day 4 for 3.5 days (236 mg NIH-FSH-P10) in decreasing dosages (7 injections at 0700 and 1900). All cows received prostaglandin F (PGF) treatments of 625 mcg D-cloprostenol i.m. (Estrumate; Schering-Plough, Union, NJ, USA) in both AM and PM on Day 6, and CIDRs were removed in AM of Day 7, followed by the final injection of Folltropin. In addition, Heat Watch patches (Heat Watch; Cow Chips, Denver, CO, USA) were applied to optimize heat detection. Donors selected as control treatment were AI at 12 and 24 h post-onset of estrus (first mount) with frozen semen from the same bull. Donors selected on the Estradiol-17β treatment received 1 mg i.m. 12 h after CIDR removal and timed AI at 24 and 36 h. Donors selected for Lutropin treatment received 12.5 mg (5cc i.m.) 24 h after CIDR removal and were timed AI at 12 and 24 h. All AI procedures used 2 units from the same bull. All cows were examined by ultrasonography every 8 h beginning 24 h post-CIDR removal and ending at 60 h to determine the time and distribution of ovulation. Ovulation was determined by the disappearance of follicles (>12 mm) from the prior examination. All AI and ultrasound procedures were performed by the same technician. There were no differences detected between the three treatments as evaluated by all the variables we studied (Table 1). However, donors treated with Lutropin tended to produced more viable embryos per collection and had a tighter degree of distribution of ovulations. Donors treated with Estradiol-17β tended to have fewer viable embryos. More research is needed to determine if donors can be timed AI without regard to estrus.

Table 1.
Mean onset of estrus, distribution of ovulation, and embryo production in superstimulated beef donors treated with Estradiol-17β, and Lutropin
367 INFLUENCE OF SUPERESTIMULATION AND HORMONAL DEPRIVATION PROTOCOLS ON IN VITRO PRODUCTION OF NELORE EMBRYOS (BOS TAURUS INDICUS)
Authors: Barros C. M. , Ferreira M. M. G. , Potiens J. R. , Eberhardt B. G. , Melo D. S. , Monteiro F. M.
Published: December 2006

There are indications in the literature that delaying the period between ovarian superestimulation and ovum pickup (OPU) will induce follicles to a condition of initial atresia, which could be beneficial to oocyte development. Blondin et al. (2002 Biol. Reprod. 66, 38–43) superstimulated (FSH) Holstein heifers and delayed OPU for 48 h, i.e. they induced initial atresia in these follicles deprived of FSH (starvation) for 48 h. Additionally, 6 h before OPU, LH was administered to accelerate follicular maturation. This protocol yielded a surprisingly high blastocyst rate (80.4 ± 9.4%). In the present work, Blondin’s protocol was simultaneously compared to other protocols used for OPU and in vitro production of embryos (IVP), in Nelore cattle. Nelore cows (n = 18) were randomly allocated in three groups: Group 1 (just OPU), Group 2 (superestimulation and OPU), and Group 3 (superestimulation associated with FSH deprivation and OPU). Three OPU were performed, and the animals were switched to a different group each time in such a way that at the end of the experiment all cows received the three protocols. At a random stage of the estrous cycle (D2), follicles ≥6 mm were aspirated to induce a new follicular wave 2 days afterward (D0). In Group 1, OPU was performed on D2 and oocytes were processed to IVP. In Group 2, starting on D0, cows were superestimulated (pFSH, Folltropin®, 30 mg administered daily, IM, during 3 consecutive days; Bioniche Animal Health, Belleville, Ontario, Canada), and 6 h after the last FSH dose they received exogenous LH (12.5 mg, Im, Lutropin®, D3; Bioniche Animal Health). OPU was performed 6 h after LH administration, i.e. 12 h after the last dose of FSH. Animals in Group 3 received the same treatment as in Group 2, except that LH was administered 36 h after the last dose of FSH, and OPU occurred 6 h later. Therefore, in this Group, follicles were deprived of FSH during 48 h. Both cleavage and blastocyst rates were similar (P > 0.05, logistic regression) among oocytes from Groups 1, 2, and 3, respectively: 77.4% (144/185) and 42.70% (79/185); 75.54% (105/139) and 31.65% (44/139); 63.52% (101/159) and 33.33% (53/159). However, hatched blastocyst rate was higher (P < 0.01) in Group 1 (30.27%, 56/185) when compared to Group 2 (11.51%, 16/139) or 3 (15.72%, 25/159). It is concluded that, contrary to previous work on European breeds (taurus), ovarian superestimulation associated with deprivation of FSH and OPU (Group 3) do not increase IVP of Nelore embryos (indicus). Additionally, the highest hatched blastocyst rates were observed in oocytes from non superstimulated cows (Group 1).
382 ULTRASONOGRAPHIC EVALUATION OF AMNIOTIC SAC SIZE DURING EARLY PREGNANCY IN COWS
Authors: Panarace M. , Garnil C. , Jauregui G. , Lagioia J. , Marfil M. , Medina M.
Published: December 2006

In ruminants, the embryonic vesicle is composed of the yolk and the allantoic and amniotic sacs. In cattle, the allantoic sac represents the largest fluid accumulation in the uterus from early pregnancy onward, but is irregular in shape as it conforms to the uterine horns. It is, therefore, difficult to ultrasonographically measure the allantoic compartment. Conversely, the amniotic sac remains ellipsoidal in shape and can be evaluated more easily. The aim of this study was to characterize the development of the amniotic sac in relation to embryo development. Thirteen multiparous, nonlactating Aberdeen Angus cows were examined weekly from 30 days to 72 days of gestation. Epidural anesthesia was induced (6 mL of 2% lidocaine) to obtain light relaxation of the rectum and prevent straining. Measurements were taken by ultrasonography (Toshiba Nemio 20, Tokyo, Japan) using a 5–10 MHz intraoperative finger probe from 30 days to 58 days of pregnancy, and a 3–6 MHz linear-array probe thereafter. Length (D1) was registered in a longitudinal section of the amniotic sac, whereas width (D2) and height (D3) were measured in a cross section. Volume (cm3) was calculated with the formula 4 ÷ 3 × Pi × (D1 ÷ 2) × (D2 ÷ 2) × (D3 ÷ 2) ÷ 1000 and mean amniotic diameter (MAD) was calculated as the average of the three measurements (Hellman et al. 1969 Obstet. Gynecol. 103, 789). Crown rump length (CRL) was measured in a longitudinal section of the embryo when it was in a neutral position. Results show that volume, MAD, and the CRL increase progressively during the first trimester (Table 1). In the first 50 days of gestation, the difference (MAD-CRL) changed from a negative to a positive value, probably as a reflection of increasing embryonic production of amniotic fluid (Wallenburg et al. 1988 Med. 5, 191). From 58 days of gestation onward, the difference became progressively more negative due to a faster relative growth rate of the embryo than of the amniotic sac. To conclude, the present table may be useful in distinguishing between normal and abnormal early pregnancy, including embryonic growth retardation and hydrops amnios (Wintour 1986 et al. Aust. Vet. J. 216, 221).
Table 1.
Mean amniotic diameter (MAD), crown rump length (CRL), and the difference (MAD-CRL) during pregnancy in cattle (mean ± SD; n = 13)
47 BLASTOCYST DEVELOPMENT RATE OF CLONED BOVINE EMBRYOS USING SERIAL NUCLEAR TRANSFER OF CELLS CONTAINING AN X-AUTOSOME-TRANSLOCATED CHROMOSOME t(Xp+;23q-)
Authors: King W. A. , Jeon B.-G. , Betts D. H.
Published: December 2006

Somatic cell nuclear transfer (SCNT) has been utilized to study various genetic and epigenetic contributions of specific biomedical diseases and developmental events by using various donor cell types such as mature lymphocytes, brain tumor cells, and other unique genotypes. Previously, we produced cloned fetuses and offspring derived from SCNT of adult ear skin fibroblasts obtained from a sub-fertile cow harboring an X-autosome translocation as a model to study X-inactivation and chromosome dynamics during female meiosis. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned calf with the X-autosome translocation t(Xp+;23q-) compared to the original adult fibroblast donor containing the same chromosome translocation. Primary cultures of cells were established in DMEM +15% fetal calf serum (FCS). To serve as nuclear donors, cells at 5-7 passages were cultured for 5 days until confluent. Oocytes matured for 18 h in TCM-199 with hormones were removed of their chromatin, and reconstructed by transfer of donor cells and fusion with two DC pulses (1.2 kV/cm, 15 �s), delivered by a BTX 2000 Electro Cell Minupulator (BTX, Inc., San Diego, CA, USA), in 0.28 M mannitol containing 0.01 mM MgCl2. After 1 h of fusion, the eggs were activated with 5.5 �M ionomycin for 5 min, followed by 11 �g/mL cyclohexamide for 5 h. The eggs were cultured for 9 days in L-SOF at 39�C in a humidified atmosphere of 5% CO2, 5% O2, 90% N2. Chi-square analysis revealed no significant (P > 0.05) differences in the rates of cleavage, blastocyst frequencies, and cell numbers between the 1st and 2nd generation cloned embryos. Cleavage rates were 87.4% and 85.4% for 1st and 2nd generation cloned embryos, respectively. The frequencies of blastocyst development and hatched blastocyst formation on Day 9 were 41.4% (91/220) and 38.7% (92/238), and 26.4% (58/220) and 22.7% (54/238) for the 1st and 2nd generation cloned embryos, respectively. The numbers of total cells and inner cell mass (ICM) cells of Day 9 blastocysts were 183 and 52, respectively, in the 1st generation embryos and 167 and 51 cells in the 2nd-generation cloned embryos. In summary, 2nd generation cloned embryos derived from fibroblasts of a cloned calf with an X-autosome translocated chromosome showed embryo development and cell numbers similar to those of the 1st generation clones. These results demonstrate that serial nuclear transfer does not improve the blastocyst development rate of cloned embryos containing the X-autosome translocation t(Xp+;23q-).
This work was funded by OCAG, OMAF, and CRC.
48 MORPHOLOGICAL ANOMALIES OF FETAL MEMBRANES DURING GESTATION IN BOVINE CLONES DERIVED BY SOMATIC NUCLEAR TRANSFER
Authors: Kohan-ghadr H. R. , Lefebvre R. , Fecteau G. , Smith L. C. , Durocher J.
Published: December 2006

High pregnancy loss and prenatal morbidity and mortality encountered in cloned animals might be explained by placental nutritional or steroidogenic insufficiency that would indirectly compromise fetal survival (Hashizume et al. 2002 Cloning and Stem Cells 4(3), 197-209). Our hypothesis is that ultrasonographic characterization of placenta in bovine clones could be used to predict anomalies in fetal development and well-being during pregnancy. We examined surrogate heifers bearing cloned fetuses (n = 37) and fetuses obtained by traditional embryo transfer (n = 5) in three stages of gestation (Day 70, 100 and 200). Morphological parameters based on direct ultrasonographic observations were collected: thickness and shape of amniotic membrane, utero-placental thickness, placentomes shape and length in fetal area, and umbilical cord shape and appearance. Analysis was done using repeated measures linear model, with day as a repeated factor. Of all clone pregnancies, 30 females were still pregnant at Day 70 and only 16 reached Day 100, from which 63% had different degrees of amniotic and/or umbilical cord anomalies. No placental anomalies or abortions were observed in the control group. The amniotic membrane abnormalities observed at Day 100 were irregularities and presence of nodules (rosary-like) in the cross-sectional view of the membrane. Hyperechogenic spikes or irregularities around the umbilical cord were observed at Day 100 in 38% of the clones (n = 6) and all of them also had amniotic anomalies. From the clone pregnancies with amniotic and/or umbilical cord problems, 30% aborted between Days 100 and 200, and for those that went to term (Days 264-278; n = 7), all calves but one died at birth or during the first month of life. Statistical analysis results showed that placentomes in cloned embryos were larger than those in control embryos (P = 0.003; least-squares mean (LSM) for clone = 4.34 cm, control = 2.97 cm) in all three stages of gestation. During the same period, amniotic membranes of clones became thicker compared to those of the control group (P = 0.0001; LSM for clone = 0.25 cm, control = 0.11 cm). Variation in utero-placental thickness was also observed. At Day 70, the utero-placental layer was thicker in clones in comparison to controls (LSM for clone = 0.32 cm, control = 0.30 cm) before it became thinner at Day 200 (LSM for clone = 0.38 cm, control = 0.40 cm). The present results showed that morphological anomalies of placentomes, amniotic membrane and umbilical cord appear during gestation of bovine cloned fetuses that could potentially compromise normal fetal development. Also, ultrasonographic monitoring of pregnancies of cloned animals is a useful tool to characterize the fetal membrane changes.
This work was funded by a Strategic Project Grant from the Natural Sciences and Engineering Research Council (NSERC) Canada.
49 INTRA-GENUS CLONED EMBRYOS PRODUCTION AND PREGNANCIES DERIVED FROM LEOPARD CAT NUCLEI AND DOMESTIC CAT ENUCLEATED EGGS
Authors: Kong I. K. , Lee H. S. , Kim N. H. , Kim L. H. , Shin H. D. , Yin X. J.
Published: December 2006

The leopard cat (Prionailurus bengalensis), a member of the felidae family, is currently listed as threatened by the Ministry of Environment in South Korea. In exotic or endangered species, the lack of oocytes and recipients precludes the use of traditional somatic cell nuclear transfer (NT), and an approach such as intragenus NT may be the only alternative for producing embryos and offspring. In the present study, we used the leopard cat (LC) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of LC fibroblast cell nuclei with domestic cat cytoplasts. A total of 412 enucleated domestic cat oocytes were reconstructed with either male (Treatment A) or female (Treatment B) adult LC fibroblasts. There was no significant difference in fusion rate (60.4 vs. 56.9%) between Treatment A and B. Of the fused couplets, the cleavage and blastocyst developmental rate in Treatment A were greater than those in Treatment B (69.5 vs. 60.9%; 8.3 vs. 7.8%; P < 0.05). In treatment A, in vivo developmental studies at 30-45 days postimplantation demonstrated 4.8% (21/435) of reconstructed embryos (n = 435) had entered into the uterine lining of recipients, but only 1.4% (6/435) formed fetuses. However, all of the reconstructed embryos failed to develop to term (65 days). Microsatellite analyses confirmed that the nuclear genome of the cloned fetuses were LC in origin.
5 ASSESSMENT OF HSP70.1 TRANSCRIPTION LEVELS IN BOVINE EMBRYOS AFTER CUMULUS REMOVAL BY DIFFERENT TECHNIQUES
Authors: Reeder A. , Schutzkus V. , Wiebelhaus-Finger J. , Khatib H. , Monson R. L. , Wheeler M. B. , Beebe D. , Rutledge J.
Published: December 2006

Previous results indicated that there was a difference in transcriptional activity depending on the method used to remove the cumulus cells after IVF (Zeringue et al. 2005 LabChip 5, 86-90). However, specific gene expression was not examined and therefore was the goal of the present study. The objective of this study was to compare the transcription levels of the HSP70.1 gene in bovine in vitro production (IVP) embryos that underwent cumulus removal by vortexing, hand stripping, or microfluidic treatment. Quantitative real-time PCR was used to estimate transcription levels of the HSP70.1 gene in bovine IVP zygotes and two-cell embryos. Transcription levels were compared between embryos that underwent three methods of cumulus removal. Presumptive zygotes were harvested at 2 or 24 h after cumulus removal by vortexing, by hand stripping, or by microfluidic means in order to compare the relative embryonic stress of these three treatments. Bovine in vitro embryos were produced by standard means with the only variable being the cumulus removal technique 24 h after fertilization. At 2 and 24 h post-cumulus removal, randomly selected presumptive zygotes were taken out of culture, preserved in RNAlater (Ambion, Inc., Austin, TX, USA) and stored at -20�C. RNA was extracted from single embryos via Qiagen's RNeasy Micro kit (Valencia, CA, USA). RNA was amplified and PCR products were detected with SYBR Green 1 (Applied Biosystems, Foster City, CA, USA) using an Opticon Monitor 3 real-time PCR machine (Bio-Rad Laboratories, Inc., Waltham, MA, USA). The threshold cycle (CT) numbers were determined for the amplified cDNA of the bovine HSP70.1 mRNA and for the housekeeping gene, acidic ribosomal phosphoprotein (PO), used as a reference. Then the amount of HSP70.1 was divided by the amount of PO to obtain a normalized HSP70.1 value expressed as the ratio of HSP70.1/PO. Ratios were analyzed by the GENMOD procedure in SAS (SAS Institute, Inc., Cary, NC, USA) accounting for replicates and treatments. Transcription levels of the HSP70.1 gene did not differ significantly between the microfluidically treated and vortexed groups of zygotes at 2 h post-cumulus removal (P = 0.1032) or between the hand-stripped and vortexed groups (P = 0.7567). In contrast, at 24 h post-cumulus removal, the embryos in the microfluidically treated group showed significantly higher levels of HSP70.1 transcription than the vortexed group (P = 0.0115). The transcription levels did not differ significantly between the hand-stripped and vortexed groups (P = 0.7875). This work strongly suggests that there is de novo RNA transcription in the early embryonic stages of the bovine. In addition to previously described improved developmental kinetics, the use of microfluidics in IVP leads to statistically significant differences in RNA transcription levels of HSP70.1.
50 PRODUCTION OF THIRD-GENERATION CLONES OF A PIG BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Kurome M. , Tomii R. , Ueno S. , Hiruma K. , Saito H. , Nakamura K. , Okumura K. , Matsumoto S. , Matsumoto M. , Kaji Y. , Endo F. , Nagashima H.
Published: December 2006

There has been a dominant view that serial cloning, i.e., cloning of a cloned animal, is only possible for a few generations. In this study, we examined the reproduction efficiency and normality of porcine offspring generated by serial somatic cell cloning. Salivary gland progenitor (SGP) cells were collected from a 4-month-old female cloned Landrace large white Duroc (LWD) pig (first generation, G1), which had been cloned from a fibroblast, and used as nuclear donors for second-generation clones (G2). The third generation of clones (G3) was produced by nuclear transfer using SGP cells from the G2 clones. Nuclear transfer was carried out by electric cell fusion using in vitro matured oocytes as recipients. Reconstructed embryos were electroactivated 1 to 1.5 hr after nuclear transfer, cultured for 1 to 2 days, and transplanted to the oviducts of estrus-synchronized surrogate gilts. A total of 391 embryos cloned from G1 animals were transplanted to three surrogates. All of the surrogates became pregnant and gave birth to a total of 13 (3.3%) of G2 clones (including two stillbirths). The average birth weight and size of eleven live piglets were 1203.6 � 113.5 g and 27.1 � 1.2 cm, both within the standard ranges of the original donor strain (LWD). Their growths until 8 months old were comparable to those of normal piglets of the same strain. For the generation of G3 clones, transplantation of 242 G2-derived embryos to two surrogate gilts resulted in one pregnant surrogate and three G3 clones (1.2%; average weight 1196.7 � 267.1 g and average size 35.7 � 2.3 cm), including a stillbirth. These results indicate that porcine serial cloning can efficiently generate up to three generations of apparently healthy clones, when SGP cells are used as nuclear donors.
This study was supported by PROBRAIN.
52 PRELIMINARY DATA ON PIG-BOVINE INTERSPECIES NUCLEAR TRANSFER EMBRYO DEVELOPMENT
Authors: Lagutina I. , Brunetti D. , Lazzari G. , Galli C.
Published: December 2006

Interspecies nuclear transfer (NT) is a very important tool for study of nuclear–cytoplasm interactions and somatic cell nucleus reprogramming. We constructed, by means of a zona-free method, NT embryos using bovine (Bo) or porcine (Po) oocytes matured in vitro and bovine fetal fibroblasts (BFF), pig adult fibroblasts (PAF), and pig fetal (PFF) green fluorescent protein (GFP)-positive fibroblasts. Constructs were fused by a double pulse of DC 1.2 kV/cm for 30 µs. At 3–4 h post-fusion, embryos with Bo were activated by 5 µM ionomycin for 4 min and incubated in 2 mM 6-DMAP in SOFaa for 4 h, whereas embryos with Po were activated by a double pulse of DC 1.2 kV/cm for 30 µs in the fusion medium with 1 mM Ca++ and incubated in SOFaa containing 5 µg/mL cytochalasin B in for 4 h. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The NT embryo development and GFP expression (D7) were checked. Our results (Table 1) showed that the blastocyst rate of control bovine and pig embryos was 74% and from 20 to 44%, respectively. ‘Pig fibroblasts into Bo’ embryos were arrested at the 8–21-cell stage while ‘BFF into Po’ embryos were arrested at the 4-cell stage. About 84% of ‘PFF GFP+ into Bo’ NT embryos started to express GFP, but only 3.2% (3/95) of the embryos were able to progress through the 16-cell stage suggesting insufficient embryonic genome activation. Overall significantly more ‘Pig fibroblast into Bo’ embryos were able to progress through the 4-cell stage pig developmental block than normal pig NT embryos (57.8 ± 3.5% vs. 47.1 ± 1.3%; t-test, P = 0.02). This study shows that early embryo development is driven by recipient cytoplasm up to the stage when genome activation should occur. The arrest of interspecies NT embryos at the stage of embryonic genome activation suggests that this developmental step is impaired.

Table 1.
Interspecies NT embryo development

This work was funded by grant ISS CS 11 and ESF.
65 ASSESSMENT OF LIVE OFFSPRING SIRED BY A SOMATIC CELL NUCLEAR TRANSFER BULL, STARBUCK II
Authors: Ortegon H. , Lin L. , Coppola G. , Perrault S. , Kroetsch T. , Betts D. H. , King W. A. , Bousquet D. , Blondin P.
Published: December 2006

It appears that overt phenotypic abnormalities observed in some domestic animal clones are not transmitted to their progeny, suggesting that epigenetic and genetic alterations are reset in the germline of clones. Still, little is known about the normalcy and performance of the offspring from cloned animals. The current study monitored Holstein heifers sired by a cloned bull, Starbuck II, from weaning to puberty. Genomic stability was assessed by telomere length status and chromosomal assessments. Growth parameters, blood profiles and physical exams were performed and the results were compared to those for a group of control heifers with matched breed and ages born from normal cows located in the Ponsonby Research Station (Guelph, Ontario, Canada). Once circulating progesterone concentrations were determined to be >1 ng/mL, the heifers were monitored by ultrasound to investigate follicular dynamics. Statistical analysis was performed using descriptive statistics, paired t-test, and a Tukey-Kramer post-hoc test for multiple comparisons. Compared to controls during the observation period, progeny sired by the cloned bull did not show any significant (P > 0.05) difference as in weight (mean � SEM) (231.5 � 29.4 vs. 237.6 � 31.0 kg), length (160.6 � 13.0 vs. 172.4 � 2.7 cm), height (105.3 � 3.72 vs. 95.5 � 11.0 cm) and temperature (39.06 � 0.01 vs. 39.16 � 0.0�C). However, the cases displayed a relaxed behavior and were easier to handle than controls. This was reflected by significant differences in heart rates (83.3 � 3.3 vs. 101.3 � 2.6 bpm) (P = 0.005) and respiratory rates (35.4 � 2.7 vs. 46.7 � 3.3 rpm) (P = 0.04) for progeny and controls, respectively. Blood profiles were within normal ranges for both groups. External and internal genitalia were normal at palpation and ultrasound exam. Both groups began puberty at the expected age (10-12 months) and weight (318 to 365 kg). Follicular dynamics and progesterone concentrations corresponded to normal adult cows and were similar between groups after puberty. Telomere lengths of spermatozoa and blood samples from Starbuck II (22.23 kb and 14.19 kb, respectively) were not statistically different from normal telomere length profiles for non-cloned cattle. Telomere lengths of Starbuck II's progeny (22.52 � 0.38 kb, n = 32) were no different compared to age-matched controls (22.20 � 0.56 kb, n = 20). In addition, the progeny exhibited a normal karyotype in their peripheral blood leukocytes (83.14 � 5.22% normal diploid cells). In summary, heifers sired by a cloned bull have normal chromosomal stability, growth, physical, hematological and reproductive parameters compared to normal heifers. Also, these heifers had a better response with lower stress to regular handling and restraining procedures.
This work was funded by OCAG, OMAF, and CRC.
70 NON-PLATED GRANULOSA AND CUMULUS CELLS AND FIRST PASSAGE FIBROBLASTS AS NUCLEUS DONOR FOR GOAT CLONING
Authors: Salamone D. , Catala M. , Gibbons A. , Pereyra Bonnet F. , Cueto M.
Published: December 2006

Different types of somatic cells have been used as nucleus donors for cloning. Most of them were previously cultured in vitro as a monolayer through several plate passages. The experiment reported here was conducted to study the potential usages of granulosa and cumulus cells for cloning without previous culture as a monolayer. A first-plate-passage fibroblast was also used. Oocytes were aspirated by laparoscopy from Criolla goats and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL HEPES with 1 mg/mL bovine testis hyaluronidase. Metaphases were assessed and oocytes were enucleated by visualization with Hoechst 33342 (5 μg/mL) under UV light (<6 s). Granulosa and cumulus cells were also recovered by laparoscopy and maintained in maturation medium in cryotube for 20 h at room temperature or 39°C, respectively. Goat adult ear fibroblasts were cultured for 1 or 2 weeks and used 2 days after confluence. All types of donor cells were transferred to the perivitlline space of enucleated oocytes and fused by an electrical pulse. After 2 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium and atmosphere of 5% CO2 + 5% O2 + 90% N2. Cleavage (Day 2) and development to blastocysts (Day 6) were recorded and analyzed by chi-square test. The cleavage rate for non-plated granulosa cells was higher than for the other treatment goups; cumulus cells had a lower rate of development to blastocysts (Table 1). These results suggest that granulosa cells collected and maintained for 24 h at room temperature could be used to produce cloned blastocysts.

Table 1.
Effect of non-plated granulosa and cumulus cells and first passage fibroblasts as donor nucleus oocytes in goat cloning