bluuebunny/crossref_metadata_2025 huggingface dataset:
112EFFECT OF THE IVM PROTOCOL OF BOVINE OOCYTES ON SURVIVAL RATES AFTER
VITRIFICATION BY OPEN PULLED STRAW METHOD
Authors: Moran E. , Gomez E. , Rodriguez A. , Hidalgo C.O. , Facal N. , Diez C.
Published: February 2004

The meiotic stage and the cryopreservation protocol influence the ability of the oocytes to survive cryopreservation. The in vitro maturation (IVM) methods affect nuclear and cytoplasmic maturation and, consequently, the developmental competence of the oocytes. On the other hand, the cytoplasm of the bovine oocyte contains large amounts of lipids which, as demonstrated in the bovine embryo (Díez et al., 2001 Theriogenology 55; 923–936), can negatively affect post-thaw survival. The aim of this work was to analyze the effect of fetal calf serum (FCS) during IVM on the freezability of the bovine metaphase II oocyte. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries. Oocytes with compact cumulus cells and evenly granulated cytoplasm were matured for 22h in TCM199, NaHCO3, FSH, LH and 17βestradiol. Approximately half of the oocytes were allowed to mature in 10% FCS, and the remainder were matured in polyvinyl-alcohol (PVA; 0.3gL−1). For vitrification, oocytes were matured for 22h, partially denuded of cumulus cells, and then vitrified (v-FCS and v-PVA) by the OPS system (Vajta et al. 1988 Mol. Reprod. Dev. 51; 53–58). Fresh untreated controls (c-FCS and c-PVA) were allowed to mature for 24h and immediately fertilized in modified TALP medium with swim-up separated sperm, and cultured. After warming and dilution, vitrified oocytes were cultured in IVM medium for 2h and then fertilized (Day 0). Presumptive zygotes with normal morphology were cultured in SOFaa+amino-acids+myo-inositol+5% FCS (Day 3), and oocytes with a degenerated appearance were counted and discarded. Data were analyzed by ANOVA and Duncan’s test. Results are shown in the Table 1. After warming, we observed severe cryodamage in both v-FCS and v-PVA groups. Rates of degenerated oocytes were 17.8±9.6 and 12.0±9.6 for v-FCS and v-PVA groups, respectively (P>0.05). The presence of PVA instead of FCS did not improve the blastocyst rates obtained from vitrified/warmed oocytes. The use of PVA during IVM (c-PVA) yielded lower (P<0.05) blastocyst rates compared to the FCS control (c-FCS). Ultrastructural studies are in progress to analyze alterations in meiotic spindle, cytoplasmic organelles and cortical granules as possible causes of reduced oocyte competence after vitrification. Supported by CICYT, AGL2001-379.

Table 1
114EFFECT OF CYTOCHALASIN B TREATMENT ON VITRIFICATION OF CAPRINE
PARTHENOGENIC BLASTOCYSTS
Authors: Nims S. , Melican D. , Jellerette T. , Butler R. , Gavin W.
Published: February 2004

Production of human recombinant proteins in the milk of transgenic animals has been shown to be a viable production system. Protection of the animal genetics involved is paramount. Vitrification of embryos is a simple, time-efficient way of preserving an animal’s genetics without the formation of damaging ice crystals during the freezing process. Cytochalasin B has been shown to increase the viability of porcine blastocysts by reducing damage to microfilaments and other cytoskeletal components. These experiments utilized caprine parthenogenic blastocysts as a model to compare the viability of parthenotes treated with or without cytochalasin B prior to and during vitrification. Abattoir oocytes were in vitro-matured in M199 with 10% goat serum containing FSH, LH and gentamycin for 18 to 21h. Parthenogenic blastocysts were produced by treating in vitro matured abattoir oocytes with ionomycin for 5min (5μM) and with 6-dMAP (3mM) for 3h followed by culturing in SOF+0.8% BSA for 7 to 8 days at 38°C with 6% O2, 5% CO2, and 89% N2 in a modular incubator chamber. The experimental group was treated with cytochalasin B (5μg/mL)in the culture media for 30 to 45min prior to and thereafter throughout the vitrification process. All blastocysts (both the experimental group and the control group) were washed through two ovum culture media (OCM) droplets for 5min each. The blastocysts were incubated in vitrification solutions 1 and 2 (10% glycerol in OCM and 10% glycerol+20% ethylene glycol in OCM, respectively) for 5min each, followed by vitrification solution 3 (25% glycerol+25% ethylene glycol in OCM). They were then aspirated immediately into a 0.25cc cryopreservation straw, followed by an air bubble, and then a 0.25M sucrose solution in OCM. The straws were immediately plunged into liquid nitrogen and stored at −196°C. One to four days later, straws were thawed in air for 5s at room temperature, then in 22°C water for 15s. After thawing, the contents of the straw were expelled, mixed, held for 5min, and finally placed in OCM for 5min. Recovered embryos were placed in SOF+20% FBS and incubated at 38°C with 5% CO2 in air overnight. Viability was determined by re-expanding and subsequent hatching of the blastocyst. As shown in Table 1, there were no significant differences between re-expansion and hatching of blastocysts with cytochalasin B treatment compared to blastocysts not treated with cytochalasin B. These results suggest that, unlike porcine embryos (Dobrinsky et al., 2000 Biol Reprod 62, 564–570), cytochalasin B treatment does not improve the post-thaw viability of vitrified caprine parthenogenic blastocysts.

Table 1
119CRYOPRESERVATION OF BIOPSIED BOVINE EMBRYOS PRODUCED IN VITRO USING
ARABINOGALACTAN AND 1.5M ETHYLENE GLYCOL
Authors: Shangguan B. , Yang N. , Vanderwal R. , Darrow M.D.
Published: February 2004

Arabinogalactan (AG) in combination with 1.5M ethylene glycol (EG) has been used successfully in cryopreserving biopsied in vivo bovine embryos (Darrow, 2002 Theriogenology 57(1), 531). This study was undertaken to investigate the efficiency of AG addition in a freezing medium (FM) to cryopreserve biopsied bovine embryos produced in vitro (IVP). Blastocysts of grade 1 were collected at Days 7 and 8 post-insemination. After biopsy with a small blade, embryos were transferred to CR1aa medium and cultured for 2 hours (h) before being frozen. In experiment 1, a group of unbiopsied embryos were handled in a manner similar to that used for the biopsied embryos. Embryos were frozen using either 1.5M EG+0.1M sucrose (EG+) (AB Technology, Pullman, WA, USA) or a FM containing 1.5M EG and different concentrations of AG (AG1, 2 and 3, courtesy of AB Technology). Embryos remained in FM for 10 (exp.1), 5 (exp.2), 5 and 10 (exp.3) or 5, 10, and 20 (exp.4) minutes before being loaded into a freezer and cooled down to −35°C at 0.3°C/min. Frozen embryos were thawed (35°C, 20 seconds) and cultured in CR1aa at 38.5°C for 3 days. Embryo survival rates (S%) were recorded at 24, 48 and 72h post-thawing. Data were compared with t-test or ANOVA procedures using SigmaStat 3.0. Results from exp.1 (Table) indicate that biopsied and unbiopsied embryos survived well in EG+ or AG2. While the biopsy procedure did not affect the post-thaw S% of embryos in either FM, no significant differences were observed between embryos frozen with EG+ and AG2 (P=0.055). Reducing or increasing AG concentration in FM by 2-fold (AG1 and 3, respectively) did not significantly affect the post-thaw S% at 24h (EG+, 80.0%, n=133; AG1, 83.3%, n=135; AG2, 71.4%, n=137 and AG3, 75.0%, n=135; P=0.217, exp.2). However, shortened exposure from 10 to 5 minutes to AG2 resulted in an improvement in S% at 24h, from 35.7% (n=80) to 61.4% (n=82, P<0.05; exp.3). When AG1 (=0.5×AG2) was used in the FM the S% at 24h after different exposure times was not significant (5 minutes, 77.8%, n=179; 10 and 20 minutes, 66.7%, n=179 and 183; P=0.472, exp.4). This study demonstrates that addition of AG to the FM effectively sustains the viability of biopsied IVP embryos during freezing and any potential harmful impact of AG on embryo survival can be minimized by reducing AG concentration or the time of embryo exposure to AG prior to freezing. Further studies are needed to determine optimal AG concentration. Currently, field trials are underway to evaluate the ability of AG medium to promote pregnancies from frozen, biopsied IVP embryos.

Table 1
Post-thaw survival rates of biopsied IVP embryos frozen in ethylene glycol with sucrose (EG+) and a FM containing arabinogalactan (AG2). Data are means±SEM
122COMPARISON OF TWO CRYOPROTECTANT DILUTION TREATMENTS FOR QUICK FROZEN
IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Visintin J.A. , Nicácio A.C. , Yamada C. , Amaral H.V.C. , Simões R. , Milazzotto M. , Marques M.G. , Mendes C.M.
Published: February 2004

The aim of this study was to compare the viability of in vitro-produced bovine embryos following quick freezing in ethylene glycol (EG) and subsequent dilution of EG by either a two- or a three-step procedure. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries. COCs were matured in TCM199 containing 10% bovine fetal serum, LH, FSH and E2, and fertilized. Presumptive zygotes were co-cultured in TCM199 with a granulosa cell monolayer, at 39°C in humidified atmosphere of 5% CO2 in air. Grade 1, expanded blastocysts (n=544) were selected 7 and 9 days after insemination and randomly distributed to one of three EG equilibration treatment groups. Embryos were exposed to 10% EG for 10min, and then to 17%, 22% or 28% EG for 30s (respectively referred to as EG 17, EG 22 and EG 28). In all treatment groups, EG solutions were prepared in PBS+0.2% BSA, and embryos were exposed to EG solutions at 22°C. Embryos were loaded into 0.25-mL straws which were then heat-sealed. Straws were cooled in liquid nitrogen vapor for 2min, and then plunged and stored in liquid nitrogen. Straws were thawed in room temperature air for 10s, and then in 25°C water for 20s. The thawed embryos of the EG 17, EG 22 and EG 28 groups were randomly assigned to one of two EG dilution procedures. Two-step dilution consisted of transfer of embryos into PBS+0.2% BSA+0.3M sucrose solution for 3min, and then PBS+0.2% BSA for 3min. Three-step dilution consisted of transfer of embryos into PBS+10% EG+0.2% BSA+0.3M sucrose for 3min, PBS+0.2% BSA+0.3M sucrose for 3min, and then PBS+0.2% BSA for 3min. Embryos were co-cultured on a granulosa cell monolayer in TCM199 and evaluated after 24h for blastocyst re-expansion (EXP), and again at 48, 72 and 96h for hatching (HAT). A total of 724 in vitro-produced bovine blastocysts were used as controls to determine hatching rates. The results are presented in the Table. No significant differences were found between the two- and three-step dilution procedures (P>0.05) for in vitro-produced bovine embryos cryopreserved by quick freezing. This project was supported by FAPESP (01/11266-4).

Table 1
In vitro re-expansion and hatching rates (%) of rapidly frozen embryos after two- or three-step dilution
124CRYOPRESERVATION OF WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) SEMEN
Authors: Williams B. , Flores-Foxworth G. , Chapman S. , Romano J. , Kidd B. , Fuchs G. , Westhusin M. , Kraemer D. , Frels D.
Published: February 2004

The methods for collecting and freezing deer semen have been, for the most part, limited to two species; red deer ( Cervus elaphus) and fallow deer ( Dama dama) (Asher et al., 2000 Anim. Reprod. Sci. 62, 195–211). The object of this study was to evaluate the progressive motility and effects of a thermal stress test on white-tailed deer (WTD) semen frozen in Biladyl extender (Mini Tube, Verona, WI, USA). Semen was collected by electroejaculation from WTD bucks (n=7, ages 1.5–2.5 years) during the breeding season. This trial was the second collection for one buck (#0025) and the third collection for the other 6 bucks. The bucks were immobilized with a xylazine/ketamine mixture i.m. (2mgkg−1 Vedco, Inc., St. Joseph, MO, 2.2mgkg−1 ketamine HCl, Fort Dodge Animal Health, Fort Dodge, IA, USA) and electroejaculated with a Pulsator IV unit (Lane Manufacturing, Denver, Co). Semen was extended 1:1 with Biladyl A, and then slowly cooled to 4°C. Once cooled, semen was extended with equal amounts of Biladyl part A, then part B, to a final concentration of 160×106cells/mL. The extended semen was then loaded into 0.25-cc straws, placed over liquid nitrogen (LN2) in vapors (−80°C) for 10min, and then plunged into LN2. Straws were stored in a LN2 tank for 3 months. Semen was thawed in a 38.5°C water bath for 45s, then placed in a warm test tube and incubated at 38.5°C for 5min before progressive motility was evaluated using a computer program (Sperm Vision, Mini Tube). A thermal stress test was performed by incubating thawed samples at 38.5°C for 1h. Results of the stress test were graded as either passed (progressive motility ≥50%) or failed (progressive motility<50%). Results are shown in the table below. Our results show that the protocol described above is suitable for the cryopreservation of white-tailed deer semen. These data suggest that the initial post-thaw progressive motility may not accurately represent the potential progressive motility of the spermatozoa (e.g. WTD s 0038 & 0103).

Table 1
Volume collected and post thaw evaluation of white-tailed deer semen
127APOPTOSIS IN BOVINE BLASTOCYSTS FROM FIVE DIFFERENT PRODUCTION SYSTEMS
Authors: Gjørret J.O. , Maddox-Hyttel P.
Published: February 2004

Regulation of apoptosis may be affected by factors during preimplantation development, and this is possibly related to embryo developmental potential. Here we investigate differences in the incidence of apoptotic nuclei in Day 7 bovine blastocysts produced by two different in vivo and three different in vitro methods. In vivo embryos were produced either by a regular superovulation procedure (reg group; n=29; Laurincik et al., 2003, Mol. Reprod. Dev. 65, 73–85), or by postponement of the LH surge (pp group; n=35; van de Leemput et al., 2001, Therio. 55, 573–592). In vitro embryos were derived from systems using either co-culture (cc group; n=30, Avery and Greve 2000, Mol. Reprod. Dev. 55, 438–445), or culture in synthetic oviduct fluid (SOF) with (S+group; n=35) or without serum (S− group; n=38; Holm et al., 1999, Theriogenology, 52, 683–700). Embryos were collected at approx. 168h post ovulation/insemination and subjected to chromatin staining and detection of DNA degradation by TUNEL reaction. The total number of nuclei, number of nuclei displaying apoptotic morphology (+M), number of nuclei displaying TUNEL reaction (+T), and number of nuclei displaying both markers simultaneously (M&T) were scored according to J.O. Gjørret et al. (2003 Biol. Reprod. 69. in press). Only M&T nuclei were regarded as apoptotic, and +M, +T, and apoptotic (M&T) indices (%) were calculated for the trophoblast (tb), inner cell mass (i) and the total blastocysts (t) in each group. Significant differences were observed for all parameters when all groups were compared (ANOVA, P ranging from 0.024 to<0.0001). Highest number of total nuclei were observed in the S+ group, whereas the lowest indices were observed in the pp group, which had significant lower indices in the i and t than in the reg., S+ and S− groups P<0.05; Tukey’s post test for ANOVA). Highest indices were generally observed in the S− group. The results demonstrate that not only embryo cell numbers but also incidences of apoptotic markers are affected by the mode of production. However, in Day 7 bovine blastocysts high cell number is not consistent with a low incidence of apoptosis. Even though cell numbers appeared comparable in the two in vivo groups, their incidences of apoptosis were different, and the reg group displayed indices comparable to the in vitro groups, highlighting the importance of ovulation protocols when in vivo embryos are used as reference material in general.

Table 1
135DNA METHYLATION PROFILES IN THE PREIMPLANTATION PORCINE EMBRYOS
Authors: Yeo S. , Kang Y.-K. , Koo D.-B , Han J.-S , Chang W.-K , Park J.-K. , Han Y.-M. , Lee K.-K.
Published: February 2004

DNA methylation at CpG dinucleotides is an important epigenetic regulation process, which is associated with gene expression without any change in DNA sequence. During early development of the mouse embryo, dynamic changes in DNA methylation of the genome occur. After fertilization, active demethylation occurs on the paternal genome followed by passive demethylation until morula stage and then de novo methylation at the blastocyst stage. This study was designed to investigate changes in DNA methylation of in vivo- and in vitro-fertilized (IVF) porcine embryos. DNA methylation states were observed in preimplantation porcine embryos by using an immunofluorescence method after staining with an antibody against 5-methylcytosine. In contrast to the data from mouse embryos, active demethylation of the genome from the paternal pronucleus was not observed in the porcine embryos. Also, no passive demethylation was detected in in vivo- and IVF-derived embryos until the morula stage. Moreover, differential de novo methylation was not shown on the genome of the inner cell mass. Whole genomes of inner cell mass and trophectoderm cells were fully methylated. Our results demonstrate that DNA methylation of porcine embryos is different from that of mouse embryos during preimplantation development, suggesting that the machinery to regulate DNA methylation may be species-specific in mammals.
139PROSTAGLANDIN F2± COMPROMISES DEVELOPMENT
OF PRE-IMPLANTATION BOVINE EMBRYOS DURING COMPACTION
Authors: Scenna F.N. , Edwards J.L. , Schrick F.N.
Published: February 2004

Several studies have implicated prostaglandin F2α (PGF) as a major embryotoxic factor during early embryonic development in cattle. Elevated uterine concentrations of PGF were negatively associated with embryo development, quality and pregnancy rates (Schrick FN et al. 1993 Biol. Reprod. 49, 617–621; Hockett ME et al. 1998 J. Anim. Sci. 76 (Suppl 1), 241 abst; Seals RC et al. 1998 Prostaglandins 56, 377–389). Moreover, addition of PGF to culture medium decreased hatching rates of compacted morulae (Scenna FN et al. 2002 Theriogenology 53, 512 abst) and decreased development of pre-compacted (16–32 cell) bovine embryos to blastocyst stage (Scenna FN et al. 2003 Theriogenology 59, 335 abst). Furthermore, administration of an inhibitor of PGF synthesis at the time of embryo transfer improved pregnancy rates in cattle (Schrick FN et al. 2001 Theriogenology 55, 370 abst). The objective of the current study was to identify the period of time during early embryonic development that is most susceptible to the deleterious effects of PGF. After in vitro maturation and fertilization of bovine oocytes, putative zygotes were cultured in KSOMaa plus 0.3% BSA. On Day 4 post-insemination, pre-compacted (16–32 cell) embryos were removed from culture, evaluated for quality, and randomly assigned to one of the following treatments: 1) Control (KSOMaa plus 0.3% polyvinyl alcohol (KSOM-PVA; n=470) or 2) PGF-1 (1ngmL−1 PGF in KSOM-PVA; n=473; Scenna FN et al. 2003 Theriogenology 59, 335 abst). After 48h of incubation in assigned treatments, assessment of development to compacted morula stage was determined. Thereafter, embryos were kept separate according to treatments, sorted by stage of development and quality, and randomly assigned to receive either Control (CON) or PGF-1 supplemented medium until assessment of blastocyst development on Day 9. This random sorting resulted in the formation of four treatment groups comprising the initial treatments and assigned treatments during Days 6–9 (CON-CON, n=366; PGF-CON, n=226; CON-PGF, n=149; PGF-PGF, n=287). Analyses were performed incorporating a randomized incomplete block design using mixed models of SAS (2000) to determine effects of PGF on Days 4–6, 6–9 and 4–9 of development. Data were also analyzed using chi-square. Addition of 1ngmL−1 of PGF to culture medium on Days 4–9 decreased the percentage of pre-compacted embryos reaching blastocyst stage (CON-CON, 47.8%; PGF-PGF, 36%; P<0.05). Moreover, addition of 1ngmL−1 of PGF to the culture medium of pre-compacted bovine embryos on Days 4–6 of development decreased the percentage of compacted morulae on Day 6 (Control, 68.1%; PGF-1, 60.5%; P=0.01). However, the percentage of embryos developing to blastocyst was not decreased following addition of 1ngmL−1 of PGF on Days 6–9 of development (CON-CON, 47.8%; CON-PGF, 42.6%; P>0.05). Results suggest that morula stage embryos during compaction are most susceptible to deleterious effects of PGF.
142DETECTION OF LATE EMBRYONIC MORTALITY BY MEANS OF OVINE PREGNANCY ASSOCIATED GLYCOPROTEINS
Authors: Verberckmoes S. , Vandaele L. , De Cat S. , El Amiri B. , Sulon J. , Duchateau L. , Van Soom A. , Beckers J.-F.
Published: February 2004

In sheep, conception rates of 80% are obtained after first mating. Some of the ewes that were not pregnant may have conceived, but underwent embryonic mortality. Embryonic mortality can be distinguished as early (EEM) or late (LEM) embryonic mortality. When EEM occurs, sheep return to estrus 17±2 days after mating, which makes the detection of EEM very difficult. Because determination of ovine pregnancy-associated glycoprotein (ovPAG) concentration can be used for pregnancy diagnosis from Day 25 of conception, with a specificity and sensitivity of 100% and 99%, respectively, it possibly could be used for detection of LEM (Verberckmoes et al., 2003. Pregnancy diagnosis in sheep by means of ultrasound and ovine pregnancy associated glycoproteins (ovPAG), 10th International Symposium on Veterinary Epidemiology and Economics (ISVEE 10), 17–21 November, 2003.). Therefore, in this study the prevalence of LEM and the usefulness of ovPAG concentration for the detection of LEM were examined. In a field trial, serum samples of 160 Texel and 127 Suffolk ewes were collected at the moment of estrus synchronization (T0), and at 25 (T25), 35 (T35), and 45 days (T45) after mating. Concentrations of ovPAG were determined by means of homologous RIA using ovine PAG as standard and tracer, and rabbit antibodies against ovPAG as antiserum. Ewes were considered as being pregnant when ovPAG concentrations were higher than 2ngmL−1. The F-test in a mixed model with ewe as random effect was used to compare the mean PAG concentrations. Of the 287 ewes examined, 5 Suffolk and 2 Texel ewes showed an abnormal PAG profile which was indicative of LEM. The average ovPAG concentration of the 7 former ewes at T25 was 5.6±2.0ngmL−1, which indicates that they were pregnant. However, in contrast to the normal pregnant ewes, they had significantly lower ovPAG concentrations at T35 (2.8±1.5ngmL−1 v. 9.5±2.3ngmL−1) and T45 (0.9±0.3ngmL−1 v. 9.0±1.9ngmL−1) (P<0.01), and they did not lamb at 145±5 days after mating. In conclusion, late embryonic mortality occurred in 2% of the ewes. Determination of the ovPAG concentration by means of homologous RIA is a useful tool for the detection of LEM in sheep.
145COMBINED GLUCOSE AND FRUCTOSE SUPPLEMENTATION IN PROTEIN-FREE KSOM
IMPROVES PREIMPLANTATION DEVELOPMENT OF BOVINE TRANSGENIC CLONED EMBRYOS
Authors: Bhuiyan M.M.U. , Jang G. , Park E.-S. , Ko K.-H. , Jeon H.-Y. , Kang S.-K. , Lee B.-C. , Hwang W.-S.
Published: February 2004

The present study investigated the effect of fructose supplementation in protein-free potassium simplex optimization medium (KSOM) on the preimplantation development of bovine transgenic cloned embryos. An expression plasmid containing bovine mutant PrP gene and enhanced green fluorescent protein (eGFP) as a marker gene was constructed and transfected into bovine fetal fibroblasts using FuGene6 (Roche, Indianapolis, IN, USA) as a lipid carrier. Transfected cells were cultured for 5 to 6 days to achieve chromosomal integration of the gene and then used for nuclear transfer. The somatic cell nuclear transfer was carried out by transferring a GFP-expressing donor cell into the perivitelline space of an enucleated oocyte. After electrical fusion and chemical activation, 525 (11 replicates) fused embryos were cultured in KSOM supplemented with 0.01% (w/v) PVA for 192 h at 39°C under 5% CO2, 5% O2 and 90% N2 gas atmosphere. The embryos were randomly allocated for 4 culture groups; KSOM supplemented with 1) 0.2mM glucose, 2) 1.5mM fructose, 3) 0.2mM glucose+1.5mM fructose and 4) vehicle (without glucose and fructose). We used 0.2mM glucose as per formulation of KSOM (Biggers JD et al. 2000 Biol. Reprod. 63, 281–293) and 1.5mM fructose due to its benefical effect on embryo development (Kwun J et al. 2003 Mol. Reprod. Dev. 65, 167–174). As a control experiment, 1043 (17 replicates) in vitro fertilized (IVF) embryos were cultured in the same culture system. The data were analyzed by PROC-GLM using SAS program. In IVF embryos, no significant differences in rates of cleavage (71.7 to 75.5%), morulae (34.1 to 37.1%) and blastocysts formation (21.0 to 24.5%) among the culture groups were observed. In contrast, significantly (P<0.05) higher rate in blastocysts formation (19.2%) was obtained when transgenic cloned embryos were cultured in KSOM supplemented with 0.2mM glucose+1.5mM fructose than with 0.2mM glucose (10.0%). However, the differences in blastocysts formation rates among other culture groups (13.2% for 1.5mM fructose and 11.9% for vehicle) were not significant. Moreover, the rates of cleavage (76.2 to 79.1%), morulae (19.3 to 23.8%) and GFP expression in blastocysts (68.0 to 78.6%) did not differ significantly among the culture groups for transgenic cloned embryos. This study demonstrated that the requirement of energy substrates in culture medium for bovine transgenic cloned and IVF embryos might be different. Now, we are conducting experiments to confirm whether the beneficial effect on transgenic cloned embryo development is due to combined effect of glucose and fructose or due to total concentration of available energy. This study was supported by Biogreen 21-1000520030100000.
148EPIDERMAL GROWTH FACTOR INDUCES BCL-XL GENE EXPRESSION AND REDUCES APOPTOSIS IN PORCINE PARTHENOTES DEVELOPING IN VITRO
Authors: Cui X.S. , Jeong Y.J. , Cheon S.H. , Kim N.-H.
Published: February 2004

Epidermal growth factor (EGF) induces well-documented mitogenic and differentiating effects on murine and bovine preimplantation embryos. However, the effects of EGF on apoptosis and apoptosis-related gene expression in porcine embryos developing in vitro have not been evaluated. The objective of this study was to determine the effects of exogenous EGF in the presence and absence of BSA on the preimplantation development of porcine diploid parthenotes. In addition, we measured cell number, apoptosis, and expression of apoptotic-related genes of the blastocysts that developed in these culture conditions. In vitro-matured oocytes were parthenogenetically activated by square electrical direct current pulses. After 3h of culture in North Carolina State University (NCSU) 23 medium with 0.4% BSA containing 7.5mgmL−1 cytochalasin B, embryos were washed and cultured in NCSU 23 medium with 0.4% BSA for 48h. The general linear models (GLM) procedure in the Statistical Analysis System was used to analyze developmental rates. A paired Student’s t-test was used to compare relative gene expression. Presumptive diploid 4-cell parthenote embryos were randomly cultured in the same medium containing 0 or 10ngmL−1 EGF in the presence and absence of 0.4% BSA. More 4-cell embryos developed into blastocysts at Day 7 when BSA was present at 0.1% (54.8%±4.9) and 0.4% (55.1%±3.1) than when BSA was absent (43.0%±3.5, P<0.05). The addition of 10ngmL−1 EGF into the medium did not significantly increase the developmental rate (40.2±2.2 v. 35.1±2.1), but EGF in the presence of 0.1 and 0.4% BSA significantly increased the cell numbers per blastocyst (51.3±2.8 v. 37.8±2.5 in 0.1% BSA; 51.8±2.0 v. 42.7±2.3 in 0.4% BSA, P<0.01 for both comparisons). Furthermore, EGF treatment in the absence of BSA did not inhibit apoptosis in the blastocysts (6.8%±0.9 v. 8.8%±1.0), while addition of EGF in the presence of 0.4% BSA significantly reduced the degree of apoptosis in the blastocysts (4.0%±0.9 v. 9.3%±1.0, P<0.01). To investigate whether EGF modulates mRNA expression of apoptosis-related genes, mRNA was prepared from single blastocysts and each preparation was subjected to RT-PCR for Bcl-xL and Bak transcripts. EGF enhanced the relative abundance of Bcl-xL expression in the presence of BSA (P<0.01). The relative abundance of Bak expression was not altered by EGF treatment in either the presence or the absence of BSA. These results suggest that EGF and BSA synergistically enhance Bcl-xL gene expression, which may result in a net increase in cell number in porcine presumptive diploid parthenotes developing in vitro.
15EFFECT OF DOSE OF ESTRADIOL VALERATE ON OVARIAN FOLLICULAR DYNAMICS IN CIDR-TREATED BEEF COWS
Authors: Mapletoft R.J. , Colazo M.G. , Small J.A. , Ward D.R. , Kastelic J.P.
Published: February 2004

The objective was to investigate the effect of dose of estradiol valerate (EV) on ovarian follicular growth profiles, intervals to follicular wave emergence and, following CIDR removal, estrus and ovulation in beef cows. On Day 0, 43 non-lactating, crossbred beef cows, 3 to 9 yr of age and at random stages of the estrous cycle, received a CIDR (Bioniche Animal Health; Belleville, Ontario, Canada) and were randomly allocated to one of four groups to receive no further treatment (Control; n=10), or an injection of 1mg (n=11), 2mg (n=10), or 5mg (n=12) i.m. of EV (Sigma Chemical Co, St. Louis, MO, USA) in 2mL canola oil. On Day 7, CIDR were removed and cows received 500μg i.m. of cloprostenol (Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada). Ovaries were examined by transrectal ultrasonography once daily until 48h after CIDR removal to detect ovarian follicle growth profiles, and twice daily thereafter to detect ovulation. Data were analyzed by ANOVA (LSD and Bartlett’s tests) and chi-square procedures. One cow (5mg EV group) lost the CIDR and was removed from all analyses. There was an effect of day (P<0.0001) on CL diameter, but the effects of treatment (P=0.3), and the treatment-by-day interaction (P=0.1), were not significant. Follicular wave emergence occurred within 7d in 7/10 (70%) Control cows and 31/32 (97%; P<0.04) EV-treated cows (one cow in late diestrus at the time of treatment did not respond to 1mg EV). Mean (±SD) interval from treatment to wave emergence was longer (P<0.03) in cows treated with 5mg EV (4.8±1.2d) than in those treated with 1mg (3.2±0.9 days) or 2mg EV (3.4±0.8 days), while Control cows were intermediate (3.8±2.0 days). Although follicular wave emergence tended (P<0.09) to be more synchronous in cows receiving EV, intervals from CIDR removal to estrus (P=0.7) and ovulation (P=0.8) did not differ among groups. Diameter of the dominant follicle was smaller (P<0.04) at CIDR removal and tended to be smaller (P<0.08) just prior to ovulation in the 5mg EV group (8.5±2.2 and 13.2±0.6mm, respectively) than in the Control (11.8±4.6 and 15.5±2.9mm, respectively) or 1mg EV (11.7±2.5 and 15.1±2.2mm, respectively) groups, with the 2mg EV group (10.7±1.5 and 14.3±1.7mm, respectively) intermediate. Diameter of the dominant follicle at CIDR removal was less variable (P<0.01) in the 2 and 5mg EV groups than in the Control group and intermediate in the 1mg EV group. In summary, dose of EV affected follicular dynamics, interval to and synchrony of follicular wave emergence, and dominant follicle diameter at CIDR removal and just prior to ovulation in CIDR-treated cows. However, interval from CIDR removal to estrus and ovulation was not affected by treatment. Results suggest that a dose of 2mg EV may be most efficacious in synchronizing follicular wave emergence in CIDR-treated cows.
150PREGNANCY AND PARTURITION FOLLOWING TRANSFER OF BOVINE EMBRYOS
CULTURED IN TWO MEDIA UNDER TWO OXYGEN CONCENTRATIONS
Authors: Fischer-Brown A. , Monson R. , Northey D. , Kuhlka T. , Rutledge J.
Published: February 2004

Developmental aberrations following transfer of in vitro-produced bovine embryos can result in early gestational losses and offspring abnormalities. An ongoing study tests the hypothesis that such aberrations occur with equal frequency among commonly employed culture systems. In year 1, embryos were produced using oocytes from abattoir-derived ovaries (breed unspecified) and a proven Angus bull selected for low birth weight. IVC treatments were 2×2 factorial for medium (KSOMaa or SOFaa) and oxygen concentration (5% or 20%). Angus recipients (n=61; 32 cows, 29 heifers) were randomly allotted to treatments for Day 7 transfers. Pregnancy was diagnosed with ultrasound several times during gestation (Table 1). At parturition calf weight, shoulder height, chest circumference, crown-rump length, and humeral and femoral length data were collected. Statistical analyses (Statistical Analysis System, Cary, NC) were logistic regression with a binomial distribution for pregnancy rate, and the general linear models procedure for calf measurements; included were fixed effects of medium, oxygen, and their interaction, with additional fixed effects of dam parity and calf sex where appropriate. No significant effects of medium or oxygen were found for pregnancy rate or calf measurements other than birth weight. Mean birth weight was higher in the KSOM, 20% oxygen treatment (Table 1), and medium-oxygen interaction for calf weight was also significant (P<0.01). In year 2 embryos were produced using the same Angus bull and Angus oocytes. Angus recipients (n=38; 32 cows, 6 heifers) were randomly allotted to treatments. Fetal crown-rump lengths were measured by ultrasound weekly from Days 33 to 54 and were analyzed as repeated measures using the mixed procedure. Pregnancy outcome and LS means for crown-rump lengths are included in Table 1. Though insufficient recipient numbers preclude determination of statistical significance, of interest is the relatively small fetal size in early gestation and large birth weights in the KSOM, 20% oxygen treatment. This treatment also contained a Day 33 pregnancy, subsequently lost by Day 40, in which the fetus was too small to obtain an accurate measurement. Fetal growth will continue to be monitored throughout gestation. Data will be collected at parturition as in year 1, and pooled analyses will be done.

Table 1
154THE EFFECT OF FIBROBLAST GROWTH FACTOR-4 ON THE DEVELOPMENT OF
NUCLEAR TRANSFER BOVINE EMBRYOS
Authors: Kim D.H. , Yang B.C. , Lee S.K. , Park H.S. , Park S.B. , Chang W.K.
Published: February 2004

Fibroblast growth factor-4 (FGF-4) has been shown to be preferentially produced in the inner cell mass (ICM) of mouse blastocysts, to stimulate the proliferation of mouse trophectoderm (TE) cells and to repress their transformation and differentiation into giant trophoblasts. Recent studies have shown that nuclear transfer (NT) bovine embryos have aberrant allocations of ICM and TE cells (Koo et al., 2002 Biol. Report. 67, 487–492) and aberrant expression of FGF-4 gene (Daniels et al., 2000 Biol. Reprod.63, 1034–1040). In this study, we examined whether recombinant human FGF-4 (rhFGF-4) stimulates development of nuclear transfer bovine embryos. As donor cells for NT, bovine ear skin fibroblast cells of passage 5 to 8 were used. Oocytes were enucleated after in vitro maturation in TCM 199 supplemented with 10% FBS, 1μgmL−1 FSH and 1μgmL−1 estradiol-17β for 20h. Enucleated oocytes were fused with donor cells by a DC pulse of 25V/150μm for 10μs in Zimmerman cell fusion medium. For activation, fused oocytes were exposed to 10μM Ca-Ionophore for 5min, followed by 2mM 6-dimethylaminopurine for 3h. NT embryos were subsequently cultured in CR2 medium (containing 0.5% BSA) without or with rhFGF-4 (1, 10 and 100ngmL−1) at 39.0°C in 5% O2, 5% CO2 and 90% N2. After 7 days of culture, blastocyst formation was observed. Apoptotic cells in blastocysts were detected by a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay and total cell number was examined by propidium iodide (PI) counterstaining. Data were analyzed by chi-square test and Student’s t-test. Supplementation of serum-free medium with rhFGF-4 increased the proportion of embryos developing to the blastocyst stage (18.4, 29.4 and 23.5% for 0, 1 and 10ngmL−1 FGF-4, respectively) and total cell number of blastocysts (66.3±11.4, 75.9±25.5 and 74.4±22.4 for 0, 1 and 10ngmL−1 FGF-4). Particularly, 100ngmL−1 FGF-4 significantly (P<0.05) increased the proportion of blastocysts (40.4%) and total cell number of blastocysts (86.7±26.5) when compared with TCM 199 medium alone. FGF-4 also decreased the mean proportion of apoptotic cells in blastocysts (10.6±7.8, 7.4±5.3, 8.6±5.3 and 7.5±4.1% for 0, 1, 10 and 100ngmL−1 FGF-4). Our results suggest that FGF-4 may play a role in the early development of NT bovine embryos and might be a useful molecule for increasing development of NT bovine embryos in serum-free culture systems.
161EFFECT OF BOVINE PITUITARY EXTRACT ON HATCHING AND POST-HATCHING
DEVELOPMENT OF IVP BOVINE EMBRYOS
Authors: Talbot N.C. , Powell A.M.
Published: February 2004

The effect of bovine pituitary extract (BPE; BD Biosciences, Inc., San Jose, CA, USA) on development of in vitro-produced (IVP) bovine blastocysts after Day 7 was studied. IVP embryos were produced from in vitro-matured COCs processed from local slaughterhouse ovaries or obtained from Bomed, Inc., Madison, WI, USA. The first 7 days of embryo culture following in vitro fertilization were in G1/G2 medium in 5%O2+5%CO2+90%N2. Embryos that had reached the compacted morula or early blastocyst stage on Day 7 (29% of total) were identified and randomly assigned to four media treatments (n=75/treatment, r=7). DMEM/199 medium (D2) or G2, each supplemented by the addition of BSA, insulin, transferrin, and selenium (ITS), were used with or without BPE at 30μgmL−1. Extended embryo culture was continued until Day 11. Further development was assessed by counting hatched blastocysts and measuring the size (cross-sectional index) and the number of cells per blastocyst. Size diameters of blastocysts ranged from 200μm to 400μm across treatments. Cell counts were performed by staining the fixed blastocysts with Hoechst 33342 and counting the nuclei of squashed blastocysts with a fluorescent microscope. The percentage of hatched blastocysts was significantly greater (P<0.05) in G2 with BPE (63.7±6.8%) than in G2 alone (29.6±7.3%). D2 with BPE (59.3±6.8%) was not significantly different from D2 alone (56.8±7.9%). Area index gave similar results; G2+BPE (267±168) was significantly higher (P<0.05) than G2 alone (76±28), and D2+BPE (192±139) and D2 alone (155±106) were not different. Cell number gave similar results (P<0.05); G2+BPE (303±164), G2 alone (123±47), D2+BPE (254±166) and D2 alone (233±120). A second experiment randomly assigned 7 day embryos (33% of total) to extended culture in G2 or D2 medium with or without irradiated STO feeder cells and under either 5% O2 or 20% O2 (n=49/treatment, r=5). All treatments contained BPE and a layer of agarose. Hatched blastocysts were counted on Day 11 and percentages were not different (P<0.05) for both media without STO in low O2 (G2=75±12 %; D2=67±8%) and in air (G2=82±19%; D2=83±11%). Hatching was not different (P<0.05) with STO in both media in air (G2=73±10%; D2=68±15%), but was significantly reduced (P<0.05) when STO was combined with low oxygen (G2=28±14%; D2=4±6%). Further culture to 18 days was completed, but many of the embryos collapsed by Day 12 and thereafter grew as lobed bodies and occasionally attached to each other. By 18 days a few embryos continued uncomplicated growth, however, and were composed of as many as 6900 cells. These results indicate that promotion of in vitro bovine blastocyst development in a minimal medium such as G2 requires BPE while a more complex medium, D2, supports further development without BPE. STO feeder cells in combination with low oxygen culture was inhibitory to blastocyst hatching and survival.
167THE USE OF BULL ROUND SPERMATIDS FOR PRODUCING RECONSTRUCTED EMBRYOS
Authors: Ock S.A. , Kwack D.O. , Choe S.Y. , Rho G.J.
Published: February 2004

Recently, sperm has been used as a vector to carry exogenous genes for the production of transgenic animals. However, the success in cattle is low, due to deficiencies in oocyte activation and sperm decondensation caused by high disulphide bond (S=S) content in mature sperm. This study was carried out to develop an effective method for producing transgenic animals with round spermatids (RS). Two methods of embryo production—electric fusion (EC) or intracytoplasmic injection (IC)—and three activation treatments were compared. RS were isolated from bull testes by Percoll density gradients (20, 35, 40, 45 and 90%). Fusion between ooplast and RS was performed with a single DC electric pulse delivered by BTX 200 (1.0KVcm−1, 45μs) in 0.28M mannitol solution supplemented with 100μM CaCl2 and 100μMMgCl2. MII oocytes were divided into three activation groups for EC and IC methods, respectively. In Group 1, oocytes were activated with ionomycin (5μM, 5min) prior to EC/IC. In Group 2, oocytes were activated with ionomycin (5μM, 5min) after EC/IC. In Group 3, oocytes were activated twice with ionomycin, before and after EC/IC. All eggs were then incubated in cycloheximide (10μgmL−1) for 5h and cultured in CR1aa medium to Day 8. The rates of cleavage and blastocyst development were evaluated on Days 2 and 8, respectively. All experiments were performed as 5 replicates, and statistical differences were analyzed with one-way ANOVA (P<0.05). In IC, cleavage rates were significantly higher (P<0.05) in Group 3 (99/123, 80.5%) compared to Groups 1 and 2 (31/68, 45.6% and 45/70, 64.3%, respectively). Blastocyst development rates were also significantly higher (P<0.05) in Group 3 (15/123, 12.2%) compared to Group 2 (1/70, 1.4%), but did not differ compared to Group 1 (4/68, 5.9%). In EC, the cleavage rates in all three activation treatment groups remained low (43–45%), and no blastocysts developed. These results suggest that intracytoplasmic RS injection combined with repeated ionomycin activation is more efficient than electric fusion for producing developmentally competent embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010 and Cho-a Pharm. Co. LTD.]
169PREGNANCY RATES OF THREE DIFFERENT RECIPIENT POOLS IN A COMMERCIAL
BOVINE OPU/IVP PILOT TRIAL-NEW ZEALAND
Authors: Berg M.C. , Peterson A.J.
Published: February 2004

Commercial bovine IVP is being extended into new markets and regions worldwide. This study presents pregnancy results from 457 OPU (n=2860 oocytes) sessions performed on a range of commercial donors (n=43) collected twice weekly during a 6-month period in 2002 at a central donor facility. A SOF-culture system was utilized to produce blastocysts (n=750, 1.6/session) for either fresh or frozen-thaw transfer into a wide range of recipient cattle on three sites. Cattle were synchronised with prostaglandin, were observed for behavioural estrus and were eligible to receive an embryo 7 days later. All OPU and embryo transfer were done by a single technician, and pregnancies were confirmed by ultrasound examination at 35 days of gestation. The following table summarizes early pregnancy results from 556 transferred embryos. Recipient selection remains a critical variable as shown in this pilot trial data set. New Zealand’s seasonal calving meant the lactating beef cattle were approximately 60 days postpartum at the start of the embryo transfer programs and the lactating dairy cows were 80 days postpartum. Postpartum interval and body condition score could be interesting variables to elucidate. Bulls were run with the beef cows shortly after embryo transfer resulting in 68% pregnancy rate of those cows whose embryos failed and then were naturally mated. Despite the disappointing results with the lactating beef cows, the overall results have led to OPU/IVP being commercially offered in this new market. The authors would like to acknowledge the ArTech IVP team and the enthusiastic cattle breeders of New Zealand.
171IMPROVEMENT IN EMBRYO RECOVERY USING UTERINE DOUBLE FLUSHING
Authors: Castro Neto A. , Sanches B.V. , Perri S.H.V. , Seneda M.M. , Garcia J.F.
Published: February 2004

The aim of the present study was to evaluate the effects of uterine double flushing on embryo recovery rates (total structures and viable embryos), after a resting period with the uterus filled with PBS. There were 210 embryo recovery procedures conducted using the uterine double flushing method, and the results were compaired with 432 conventional single flushing procedures. All procedures were conducted with Limousin (n=403) and Guzerá (n=239) cows, following the same superovulation protocol. Cyclic donors received a progestagen implant (CIDR) and 1mL of oestradiol benzoate (Estrogin®) at Day 0. Between Day 5 and Day 9 animals received doses ranging from 200 to 300UI (zebu cows) and 300 to 500UI (taurine cows) of FSH (Pluset® – Serono) in decreasing doses. Between Day 6 and Day 8 PGF2a (Ciosin®) was administered, followed by withdrawal of the progestagen after 24h. Artificial insemination was performed between 14 and 26h after the beginning of treatment. For the double flushing procedure, after the first uterine flushing of both horns with 1L DPBS (Nutricell), a Foley catheter was positioned in the uterine body in order to fill the uterus with the same solution (80 to 150mL). After this procedure the catheter was closed with a disposable 5-mL syringe gasket, and the animals were allowed to rest in the surroundings of the work place for 30min. After this period, a second flush was performed in order to recover the remaning liquid used during manipulation of the uterus. Animals from the control group (group A) were subjected to a single uterine flushing procedure. From 210 double flushing procedures (group B – test), 1409 viable embryos were produced, classified as grades I, II, III and IV (IETS), (average of 6.7 embryos per procedure), whereas, in the 432 single flushing procedure (group A – control), 1993 embryos were produced (average of 4.6). Statistical analysis showed the increase of viable embryo recovery rate. When consecutive double flushing was performed, the average of recovered embryos increased from 8.3 to 12.7 (P<0.05) in Limousin cows, and from 7.9 to 11.5 (P<0.05) in Guzerá cows. Comparing recovery after single flushing with that after double flushing, the mean number of viable embryos increased from 4.7 to 6.9 (P<0.05) in Limousin cows and from 4.5 to 6.4 (P<0.05) in Guzerá cows. In order to assure the nonexistence of negligence effects or operator influence on results, the mean values of total embryo recovery rate after single flushing (control group) was compared to the mean values of the same rate after the first uterine flushing on test group. Results indicated no difference between recovery rates. The present work showed the viability of using the uterine double flushing procedure for improvement of embryo recovery rates in cattle.