bluuebunny/crossref_metadata_2025 huggingface dataset:
245 OOCYTE RECOVERY BY OVUM PICK UP AND EMBRYO PRODUCTION IN MURRAH AND NILI-RAVI BUFFALOES (BUBALUS BUBALIS) IMPORTED IN CHINA
Authors: Huang Y. , Zhang X. , Gasparrini B. , Presicce G.A.
Published: December 2004

In this preliminary study, in vitro embryo production and cryopreservation in two river type buffaloes (Murrah and Nili-Ravi) imported into China have been carried out. The objective of the study was enhancement of the genetic merit and productive performances of imported river buffaloes in conjunction with the utilization of local swamp buffaloes. In order to improve milk and meat production in China local swamp buffaloes (2n = 48), which are the predominant subspecies, have been crossbred with imported river buffaloes (Murrah and Nili-Ravi: 2n = 50). At present, several hundred thousand crossbred heads have been produced, and although both males and females can reproduce with 2n = 49 crossbred buffaloes, their reproductive performances are significantly reduced when compared to 2n = 50 buffaloes. As an alternative approach, a program of embryo production in river buffaloes and transfer into both river and swamp buffaloes has been implemented at the Guangxi Buffalo Research Institute, in Nanning, P.R. China. Some preliminary results are presented: from a start-up experiment, a total of 46 river buffaloes were subjected to 2 to 3 ovum pickup sessions at 4-day intervals. A total of 750 antral follicles were punctured and 495 (66%) cumulus-oocyte complexes (COCs) were retrieved. Only COCs characterized by at least one layer of granulosa cells (n = 451; 91.1%) were considered for in vitro maturation (IVM). COCs were matured in TCM 199 + 10% FCS, 0.5 μg/mL FSH, 5 μg/mL LH, and 1 μg/mL estradiol in the presence of cysteamine (50 μM) at 39°C under 5% CO2 in humidified air for 24 h. Of the initial 451 COCs matured, only 277 could be considered for in vitro fertilization (IVF). IVF was performed at 39°C under CO2 in humidified air in TALP medium supplemented with 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin. Frozen/thawed sperm from a tested bull was treated by swim-up procedure and used at a final concentration of 20 million/mL. Following 20 to 22 h of co-incubation, presumptive zygotes were cultured in SOF medium, supplemented with essential and non-essential amino acids and 8 mg/mL BSA, in a gas atmosphere of 5% CO2, 7% O2, and 88% N2. A total of 41 (14.8%) blastocysts were produced, of which 33 were vitrified and 8 transferred immediately into available swamp and river buffalo recipients. Two calves were born (25%) from the transfer of fresh embryos into one river and one swamp buffalo. In vitro embryo production in the buffalo species is still characterized by a high degree of variable results. However, these preliminary results reinforce the need to implement newly developed reproductive technologies not only for speeding up genetic gain of already productive species, but also for the utilization of local breeds characterized by reduced productive performance.
248 EMBRYO DEVELOPMENT IN MICRODROPS AND MICROCHANNELS: COMPARISON OF NCSU23 SEQUENTIAL AND NON-SEQUENTIAL CULTURE MEDIUM
Authors: Lima A.S. , Ferguson C.E. , Wheeler M.B.
Published: December 2004

The in vitro culture systems used to produce pig embryos generally result in few embryos developing to the blastocyst stage. The use of pyruvate (pyr) and lactate (lac) during the culture of zygotes to the 8-cell stage followed by glucose (glu) supplementation replacing pyr and lac appears to be beneficial for embryo development in the pig. The aim of this study was to compare the embryo development rates from pig oocytes fertilized with and without cumulus cells in 100-μL microdrops (MD) and cultured in 100-μL MD or microchannels (MC), using NCSU23 containing 8 mg/mL of BSA and supplemented with (1) glu or (2) pyr/lac or (3) pyr/lac for the first three days and then with just glu for the remainder of culture period (pyr/lac-glu). Sow oocytes were matured in TCM199 supplemented with gonadotropins for the first 22 h, and for an additional 22 h without hormones. After 44 h of maturation, oocytes were placed in MD of modified tris-buffered medium to be fertilized using 3 × 105 sperm/mL. Oocytes were divided into two groups for fertilization: with and without cumulus cells. Following 6 h of fertilization, all inseminated oocytes were washed, divided into groups of 15, allotted to the three culture media treatment groups as described above, and incubated in either MD or MC. With the exception of one treatment there were no significant differences in development rates among embryos cultured in MD or MC, hence data were pooled from these two culture devices. Only oocytes fertilized without cumulus cells and cultured in pyr/lac in MC appeared to have lower rates of blastocyst formation (11.67%) than those cultured in MD (26.67%) in the same culture medium. When the six treatments were compared, oocytes fertilized with cumulus cells and cultured in glu had significantly higher (P < 0.05) blastocyst rates and hatching rates compared with the other treatments, with the exception of those fertilized without cumulus cells and cultured in pyr/lac-glu. There were no significant differences among other treatments in Day 7 blastocyst or in Day 9 hatching rates. In conclusion, both culture devices can be used to reach similar blastocyst rates with different treatments. In this experiment, the removal of cumulus cells before fertilization appeared to enhance embryo development in vitro when sequential media are used. On the other hand, the presence of cumulus cells before fertilization seems to enhance embryo development when non-sequential glu medium is used.

Table 1.
Embryo development rates on Day 9 for three different culture treatments
258 APOPTOSIS IN FAST- AND SLOW-CLEAVING BOVINE EMBRYOS IN VITRO
Authors: Vandaele L. , Mateusen B. , Maes D. , Van Soom A.
Published: December 2004

Fast-cleaving embryos have significantly higher chances of reaching advanced developmental stages, and hatched blastocysts developed from fast-cleaving embryos have higher total cell and inner cell mass cell numbers. Few data are available on the prevalence of apoptosis in blastocysts resulting from fast-cleaving bovine embryos. Therefore, it was the aim of this study to search for a correlation between this early quality marker (timing of first cleavage) and a later quality marker (apoptosis in blastocyst stage). A total of 836 immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes were denuded 24 h after fertilization and cultured in 50 μL droplets of modified SOF medium without serum at 39.0°C in 5% CO2, 5% O2, and 90% N2. At 30 h, 36 h and 48 h post-fertilization (PF), zygotes that had developed to the 2-cell stage or beyond were placed in new droplets and cultured in separate groups. In each of the 3 replicates a control group of 100 presumed zygotes was cultured in SOF medium without manipulation. After 24 h of culture, the SOF medium was supplemented with fetal calf serum (FCS) up to 10%. Blastocysts were evaluated at Days 7 and 8, and fixed in 4% paraformaldehyde. After TUNEL staining, total cell number and TUNEL-positive cells were counted for each group. An univariate analysis of variance was used with % apoptotic cells as the dependent variable, timing of first cleavage as the fixed factor, and replicate as the random factor (mixed model ANOVA). The mean cumulative blastocyst yields in the control group were 20.9% and 27.0% at Days 7 and 8, respectively, which were not different from the total yields from manipulated embryos (Table 1). The cumulative cleavage rates for manipulated embryos were 44.9, 65.1, and 85.7% at 30, 36, and 48 h PF, respectively. The blastocyst yields at Days 7 and 8 PF were declining for embryos which cleaved later, with a distinct drop (P < 0.05) between the 36-h and 48-h groups. The percentage of apoptotic cells in Day 7 blastocysts was significantly lower in the 30-h compared with the 36-h and 48-h groups (P < 0.05). Within the 30-h and 36-h groups, Day 8 blastocysts had significantly more apoptotic cells than Day 7 blastocysts (P < 0.05), but no differences could be detected between groups at day 8. In conclusion, this experiment has confirmed the increased chances of early-cleaving embryos to reach the blastocyst stage. The higher percentages of apoptosis in late cleaving embryos at Day 7 and in all blastocysts at day 8 as determined by TUNEL staining could be an indication of lower blastocyst quality.

Table 1.
Blastocyst yield (number and percentage) and percentage of apoptotic cells (APC) at Days 7 and 8 PF
26 ASSESSMENT OF CHROMOSOME ABNORMALITIES IN SHEEP PARTHENOGENETIC AND NUCLEAR TRANSFER EMBRYOS: EFFECT OF 6-DMAP AND CYCLOHEXIMIDE ON PLOIDY
Authors: Alexander B. , Coppola G. , Di Berardino D. , Betts D.H. , King W.A.
Published: December 2004

In current somatic cell nuclear transfer (NT) protocols, the reconstructed embryos are activated by incorporation of secondary oocyte activation compounds such as 6-DMAP or cycloheximide (CHX). The effects of these compounds on the chromosome complement of sheep NT embryos have not been studied in detail. Therefore, the aim of this study was to assess the chromosome abnormalities using sex chromosome specific probes of Day 6 blastocyst-stage sheep embryos produced from parthenogenetic activation and NT. Following 20–22 h of IVM, the oocytes were activated by electric pulsing followed by 30-min culture in cytochelasin B. They were reactivated using ionomycin (5 min) followed by 2-h culture in 6-DMAP or CHX. In contrast, NT embryos were produced using standard NT procedures using male sheep fetal fibroblasts. Reconstructed embryos were activated using the same methods described earlier. The embryos (compact morulae and blastocysts) were fixed and subjected to FISH analysis using cattle X and Y chromosome painting probes. The data were analyzed using Fisher's exact test. Of the parthenogenetic embryos (6-DMAP, n = 28; CHX, n = 32) analyzed, none of the embryos was totally haploid (X) or totally polyploid. When all of the nuclei per embryo were considered, normal (XX) genotype embryos were 6.2% and 0.0% in CHX and 6-DMAP groups, respectively. The rest of the embryos were abnormal due to mixoploidy (100% vs. 93.8%, P < 0.05) in 6-DMAP and CHX treatment groups, respectively. The abnormal nuclei per embryo ranged from 7.3% to 72.2%. The mean total cell number of parthenogenetic blastocysts was 91.2 ± 4.3 and 81.8 ± 6.2 (mean ± SE) in 6-DMAP and CHX, respectively. Among NT embryos analyzed, (6-DMAP, n = 30; CHX, n = 32) only 40.0% and 43.8% of embryos were completely normal for XY chromosomes in 6-DMAP- and CHX-treated groups, respectively. The rest of the embryos were abnormal due to mixoploidy (60.0% vs. 56.2%, P > 0.05) in 6-DMAP and CHX groups, respectively. Monosomy (XO or OY), trisomy (XXY), and tetrasomy (XXYY) were the common abnormalities detected in mixoploid embryos. The abnormal cells per embryo ranged from 3.8% to 41.8% in both treatment groups. The mean total cell number of NT blastocysts was 71.2 ± 9.8 and 63.8 ± 8.4, in 6-DMAP and CHX treatment groups, respectively. In conclusion, the 6-DMAP-treated embryos derived from parthenogenetic activation had significantly higher chromosomal abnormalities than CHX-treated embryo groups (P < 0.05). In contrast, the NT embryos derived from either 6-DMAP or CHX treatment did not show any significant difference in producing chromosomally abnormal embryos at the blastocyst stage. This study also highlights the feasibility of using bovine chromosome painting probes on ovine embryo spreads.
This work was supported by NSERC, OMAFRA, and ICCS.
270 COMBINED ELECTRICAL AND CHEMICAL ACTIVATION OF ZONA-FREE PORCINE OOCYTES
Authors: Kragh P.M. , Mtango N.R. , Corydon T.J. , Bolund L. , Callesen H. , Vajta G.
Published: December 2004

Activation is a crucial step in mammalian somatic cell nuclear transfer (SCNT). Recently we described the application of the handmade cloning technique for porcine SCNT that uses oocytes without zonaa pellucidae (zona-free) in a micromanipulation-independent procedure (Kragh et al. 2004 Reprod. Fertil. Dev. 16, 315–18). The purpose of the present study was to investigate the effect of a combined electrical and chemical activation of zona-free porcine oocytes. Cumulus-oocyte complexes were aspirated from ovaries of sows and matured for 41 h. Subsequently, the cumulus cells were removed by the addition of 1 mg/mL hyaluronidase in HEPES-buffered TCM-199. For zonae pellucidae removal, oocytes were incubated in 8 mg/mL pronase in HEPES-buffered TCM-199 supplemented with 20% cattle serum for 10 s. Three independent experiments with four treatments were conducted, and oocytes were activated by a combined electrical and chemical activation. Oocytes were washed once in activation medium (0.3 M mannitol, 0.1 mM MgSO4, 0.1 mM CaCl2, and 0.01% polyvinyl alcohol) and transferred to a chamber with two wires (0.5-mm separation) covered with activation medium. After the electrical pulse, oocytes were incubated in culture medium (NCSU-37 containing 4 mg/mL BSA) supplemented with 5 μg/mL cytochalasin B and 10 μg/mL cycloheximide for 6 h. Activated oocytes were cultured in culture medium using the wells of wells system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–64) in the submarine incubation system (Vajta et al. 1997 Theriogenology 48, 1379–85). The rate of development into blastocysts was checked on Day 7 of culture. In treatment 1, zona pellucida-intact oocytes were first activated by a single DC pulse of 1.25 kV/cm for 80 μs, and subsequently cultured in cytochalasin B and cycloheximide for 6 h. In treatments 2 and 3, oocytes without zonae pellucidae were activated by a single DC pulse of 1.25 and 0.85 kV/cm for 80 μs, respectively, and subsequently cultured in cytochalasin B and cycloheximide for 6 h. In treatment 4, oocytes without zonae pellucidae were bisected by hand under a stereomicroscope using a microblade in 5 μg/mL cytochalasin B in TCM-199 supplemented with 15 mg/mL BSA, re-fused/activated by a single DC pulse of 1.25 kV/cm for 80 μs in activation medium, and cultured in cytochalasin B and cycloheximide for 6 h. The rates of blastocyst formation from activated oocytes (mean ± SEM) in treatments 1, 2, 3, and 4 were 55 ± 4%, 40 ± 2%, 49 ± 1%, and 41 ± 8%, respectively. In conclusion, the combined electrical and chemical activation efficiently induced parthenogenetic development of zona-free oocytes. Also, a more authentic control model for activation during SCNT was established by activating and producing blasctocysts from re-fused bisected oocytes.
28 CLONED EMBRYOS CAN BE PRODUCED USING DONOR CELLS OBTAINED FROM A 72-HOUR COOLED CARCASS
Authors: Arat S. , Bagis H. , Odaman Mercan H. , Dinnyes A.
Published: December 2004

There are few reports on the use of cells from a dead mammal for nuclear transfer (NT). So far, most calves have been cloned from live adult cows or fresh fetal samples. The ability to produce cloned animals using postmortem tissue can provide an additional application to the field of NT. This study was conducted to investigate whether viable cells could be obtained from tissues chilled for 72 h and whether these cells could be used for NT. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal calf serum (FBS), 50 μg/mL sodium pyruvate, 1% v:v penicillin-streptomycin (10,000 U/mL penicillin G, 10,000 μg/mL streptomycin), 10 ng/mL EGF, 0.5 μg/mL FSH, and 5 μg/mL LH. A cell line (MC) was established from leg muscle of a cow carcass stored at 0°C for 72 h. Tissues from muscle were cut into small pieces. Tissue explants were cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Bovine granulosa cells (GC) were isolated from ovarian follicles and used for NT as control cells. Prior to NT, all somatic cells were allowed to grow to confluency (G1/G0) in DMEM-F12 medium supplemented with 10% FBS. Cumulus cells were removed by vortexing with hyaluronidase at 18 h after the start of maturation. Matured oocytes labeled with DNA fluorochrome Hoechst 33342 were enucleated under UV to ensure full removal of the chromatin. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused by a DC pulse of 133V/500 μm for 25 μs. After fusion, NT units were activated using a combination of calcium ionophore (5 μM), cytochalasin D (2.5 μg/mL) and cycloheximide (10 μg/mL) and cultured for 7 days in BARC or G1.3-G2.3 medium. Differences (developmental potential and cell numbers) among groups were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. The results suggest that viable cells can be obtained from muscle of a cow carcass stored at cold temperature for 72 h and that these cells have ability to generate NT blastocysts at rates similar to those obtained with fresh GCs. In addition, G1.3 and G2.3 culture medium supported embryo development better than BARC medium.

Table 1.
In vitro development of NT embryos

This study was supported by a grant from TUBITAK, Turkey (VHAG-1908 and Turkey-Hungary bilateral project VHAG-2022).
282 EFFECT OF THE MATURATION TIME ON THE OUTPUT OF THE INTRACYTOPLASMIC SPERM INJECTION (ICSI) OF PIG OOCYTES PRECULTURED WITH ROSCOVITINE
Authors: García-Roselló E. , Matás C. , García-Vázquez F. , Cánovas S. , Coy P.
Published: December 2004

This study was conducted to evaluate the effect of different maturation times on the output of intracytoplasmic sperm injection (ICSI) with pig oocytes precultured with roscovitine, a specific inhibitor of MPF kinase activity (Meijer and Raymond 2003 Acc. Chem. Res. 36, 417–425). Cumulus-oocyte complexes (COCs) were collected from nonatretic follicles from slaughtered gilts. Just after recovery, oocytes (n = 456) were cultured in the presence of 50 μÂ­M roscovitine for 28–30 h, which maintains them at the GV stage until use without affecting further development (Coy et al. 2004 Human Reprod. 19, 41–42). Then, oocytes were matured in a conventional maturation system (Coy et al. 2002 Reproduction 124, 279–288) for 36, 40, or 44 h. Matured oocytes were fertilized by ICSI. Injection was performed as described by Martin (2000 Biol. Reprod. 63, 109–112) but in D-PBS medium with 10% FCS. Injected oocytes were transferred to TALP medium (Rath et al. 1999 J. Anim. Sci. 77, 3346–3352) for 24 h, after which they were fixed and Hoechst-stained to check for fertilization. Results (data were analyzed by ANOVA and Tukey test) showed similar activation rates (oocytes with female pronucleus) for 36, 40, and 44 h (83.6%, 81.7%, and 90.7%, respectively). Differences were observed in male pronuclear formation (80.2%, 67.6%, and 57.8%) and in putative embryos (zygotes showing two pronuclei or two cell stage 24 hpi; 74.3%, 62.9%, and 55.1%) for 36, 40, and 44 h, respectively (P < 0.05), where the 36-h group showed the best result. Data show that pre-treatment of pig oocytes with roscovitine and further maturation for 36 h can improve the output of ICSI.
This work was supported by Ministerio de Ciencia y Tecnología (AGL2003-03144).
285 SUPPRESSION OF MPF AND MAPK ACTIVITIES BY DIBUTYRYL CAMP DURING FIRST MEIOTIC MATURATION IMPROVES SUBSEQUENT DEVELOPMENT OF PORCINE OOCYTES
Authors: Kim J.-S. , Koo D.-B.n , Chae J.-I. , Choo Y.-K. , Lee K.-K. , Han Y.-M.
Published: December 2004

Maintaining the germinal vesicle (GV) stage in growing oocytes is essential for developmental competence of the eggs. In pig oocytes, MPF and MAPK activities are low during the GV stage, and their activities increase with progression of meiosis I. In general, cAMP that exists at high levels in GV oocytes inhibits germinal vesicle break down (GVBD). After gonadotrophin stimulation, the amount of cAMP in oocyte cytoplasm is decreased gradually for meiotic resumption. This study was conducted to examine the effect of dibutyryl cAMP (dbcAMP) on nuclear maturation, fertilization, and early embryo development of porcine oocytes. Oocytes were cultured in NCSU23 medium with or without dbcAMP for 22 h, and then cultured in fresh maturation medium for an additional 22 h prior to fertilization. The activities of MPF and MAPK were evaluated by Western blot analysis by using specific antibodies such as anti-cdc2 and anti-ERK1/2 in maturing pig oocytes at 22 h and 44 h. In vitro fertilization was performed with fresh ejaculated spermatozoa in the modified TRIS-buffered medium, and fertilized embryos were cultured in NCSU23 medium. When treated with dbcAMP for 22 h, most oocytes (204/224, 91.9%) were arrested in GV stage by blocking meiotic resumption. The activities of constituent proteins (cdc2 and ERK1/2) of MPF and MAPK were also suppressed in dbcAMP-treated oocytes. After completion of IVM, dbcAMP-treated oocytes showed a higher proportion of the metaphase II stage than non-treated ones (156/171, 91.3% vs. 121/167, 72.8%; P < 0.05). Furthermore, incubation of 44 h matured oocytes with dbcAMP for 22 h increased the MPF and MAPK activities. In the dbcAMP-treated group, penetration rate was increased (126/145, 80.0%) and polyspermy rate was reduced (26/126, 22.4%) as compared to the nontreated group (97/140, 69.3% and 46/97, 47.4%, respectively; P < 0.05). Furthermore, blastocyst formation of dbcAMP-treated eggs was also improved compared to the nontreated group (47/126, 37.0% vs. 28/146, 17.2%; P < 0.05). Our results suggest that the synchronization of meiotic resumption by dbcAMP may support meiotic maturation and in vitro development of pig eggs.
292 MATURATION IN A STRAW IS EFFECTIVE ON THE DEVELOPMENT	OF BOVINE OOCYTES IN VITRO
Authors: Park Y.M. , Kim S.S. , Lee J.H. , Park Y.S. , Park H.D.
Published: December 2004

In vitro embryo development is strongly influenced by oocyte maturation environments. Maturation of bovine oocytes is processed in a culture dish. However, the development rate to the transferable blastocyst stage was 10 to 30%. This experiment was to examine the effect of the size of straw and the medium exchange on the development of Korean Native Cow (KNC) oocytes. Ovaries of KNC were obtained from a local slaughterhouse and cumulus oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. Groups of 15 COCs were matured in TCM-199 supplemented with 10% fetal calf serum (FCS), 1 μg/mL FSH, 10 μg/mL LH, and 1 μg/mL estradiol-17β for 18 h. In vitro-matured oocytes were fertilized using frozen-thawed percoll-separated spermatozoa (Day 0) in fer-TALP medium for 20 h and cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (after Day 3). All cultures were maintained in an incubator at 39°C, 5% CO2 in air with maximum humidity. Data from three replicates were analyzed by chi-square test. In Experiment 1, we examined the effect of the instrument of maturation (dish or 0.25-mL and 0.5-mL straws) on embryo development. There were no difference in the cleavage (2-cell) among treatment groups. However, the development rate to the 8-cell and blastocyst stage was significantly higher in the 0.5-mL straw (38.5 and 17.0%) than in the 0.25 mL-straw (26.6 and 7.4%, all respectively). In Experiment 2, the KNC oocytes were matured in 0.5-mL straws based on the results of Experiment 1, and we examined the effect of the conditions such as circulation and exchange of maturation medium at 9 h after the start of IVM on embryo development. The development rates to the 2-cell, 8-cell, and blastocyst stage were significantly higher in the circulation group (83.3, 58.0 and 31.3%) than in the control (72.0, 44.7 and 19.3%) and exchange groups (71.3, 40.0, and 18.0%, all respectively). The results of this study suggest that the maturation of KNC oocytes in 0.5-mL straws accompanied by circulation of medium at 9 h is effective in the development to the blastocyst stage.
306 EMBRYO ANALYSIS IN FIELD MOET: HIGH SUCCESS RATE AFTER OVERNIGHT CULTURE OF MICROBLADE-BIOPSIED CATTLE EMBRYOS
Authors: Kananen-Anttila K. , Vartia K. , Hyvönen A. , Virta J. , Peippo J. , Halmekytö M.
Published: December 2004

Embryo storage before embryo transfer is a necessity in field MOET to enable complex embryo analysis. Cryopreservation of biopsied embryos may reduce pregnancy rates to unacceptable levels, below 40% (Shea BF 1999 Theriogenology 51, 841–854). Our primary objective was to investigate the effect of short-term storage in overnight culture on the pregnancy rate of microblade-biopsied and sexed cattle embryos. A specific aim was to apply embryo storage in culture to marker-assisted selection of MOET embryos.
Day 6.5 embryos were produced using standard superovulatory, AI, and embryo flushing procedures. Embryo donors were lactating cows of top genetic merit. Embryos were transported in straws in Holding medium (ICPbio, Auckland, New Zealand) to the laboratory and individually biopsied by a microblade. Biopsied embryos were cultured overnight in individual oil-overlaid 20-μL drops of Medium 199 with glutamax-1 (GIBCOTM, Baisley, UK) containing 0.25 mM sodium pyruvate, antibiotics, and 4 mg mL−1 fatty acid-free albumin. The biopsies were lysed in proteinase K and analyzed for sex with the BOV-Y/kappa-casein PCR method (Peura et al. 1991 Theriogenology 35, 547–555). Overnight-cultured grade I–II female embryos were transferred into the uteri of heat-synchronized recipients (Day 7.5). Pregnancy was confirmed by rectal palpation at 2–3 months after ET. Grade I-II male embryos as well as embryos of unknown sex were frozen in ethylene glycol, stored in liquid nitrogen, thawed, and cultured overnight for estimation of re-expansion rate and cell counts.
In total, 74 embryos of eight donors were overnight-cultured and sexed. The success rate of the sexing method was 95%. Of the successfully analyzed embryos, 41% (29 of 70) were females and 59% (59 of 70) males. The survival rate of microblade-biopsied overnight-cultured embryos was 99% (73 of 74); 73% of the surviving embryos were of grade I, 23% of grade II, and 4% of grade III. Sixteen of the 27 (59%) grade I–II female embryos transferred resulted in pregnancy. Forty-two grade I–II embryos were frozen and 36 (86%) re-expanded after thawing and overnight culture. Twenty-four of the re-expanded embryos (67%) were of grade I. The re-expanded embryos had on average 73 cells (range 43–114).
In conclusion, overnight culture of microblade-biopsied cattle embryos does not compromise embryo viability, resulting in high pregnancy rate and post-thaw re-expansion rate. The method can be utilized as a short-term embryo storage in the field MOET scheme and it will be applied in marker-assisted selection of MOET embryos for genotypes associated with milk production.
The study was supported by the HAKA-Top breeding animals from North-Savo-project.
311 CATTLE SEX REGULATION BY SEPARATION OF X AND Y SPERMATOZOA PRELIMINARY RESULTS OF FIELD EXPERIMENT IN POLAND
Authors: Smorag Z. , Bochenek M. , Pilch J.
Published: December 2004

The only reliable and relatively fast method of sex regulation in cattle is X and Y sperm high-speed sorting by flow cytometry. In October 2003 a field experiment started in Poland. The aim of the experiment was to examine the efficiency of sperm sexing and the fertility rate in field conditions. The semen of three Holstein bulls was used for the experiment. The semen was sexed and frozen according to modified XY, Inc. (Fort Collins, CO, USA) method. Only the X-fraction of spermatozoa was sorted and frozen. Sexing was performed at a speed of 15–20 × 106 spermatozoa/h and frozen in doses of 2.5 × 106 spermatozoa/straw. Progressive motility of spermatozoa was 90–95% immediately after sorting and 50–70% after freezeing/thawing for all three bulls. The X-fraction sorting purity was checked by re-analysis and it ranged from 90% to 96%. In total, 316 inseminations at 10 farms were performed up to June 2004. Fertility data of 178 inseminations were collected by ultrasonographic (USG) examination during the same time. Average fertility rate was 37.08%. However, significant variations of fertility rates were observed between farms: it ranged from 22.22% to 84.21%. Four calves, all females, were born after insemination with the X-fraction until June 2004. The experiment will continue.
337 OVUM PICK UP IN NON-PREGNANT AND POSTPARTUM SWAMP BUFFALOES (BUBALUS BUBALIS) AFTER FSH PRETREATMENT
Authors: Promdireg A. , Techakumphu M. , Adulyanubap W. , Na-Chiengmai A.
Published: December 2004

The feasibility of OPU has already been reported in prepubertal swamp buffaloes and heifers (Promdireg et al. 2000 Thai J. Vet. Med. 30(1), 41–50; Techakumphu et al. 2003 Theriogenology 61, 1705–1711) and it has to be emphasized that the reproductive potential of females can be maximized, if OPU and IVEP are applied in the course of non productive periods such as anestrus and postpartum. The objective of this study was to evaluate the efficiency of Ovum Pick Up (OPU) in non lactating, pluriparous non-pregnant (n = 5) and lactating, postpartum swamp buffaloes, started 3 mts after calving (n = 6) with gonadotropin stimulation. OPU was performed every two weeks in the two groups of animals for a total of 6 sessions. To buffaloes receiving hormonal stimulation, a total of 400 mg of FSH was administered for three days in decreasing dose together with 100 μg of GnRH 24 h after the last FSH injection. The number of aspirated follicles between non-pregnant and postpartum buffaloes was not significantly different, 7.2 ± 3.7 (217) and 9.0 ± 3.2 (285), respectively (P > 0.05). Recovered oocytes between the two groups of hormonally stimulated animals was also not statistically different: 3.7 ± 2.7 (112) in the non pregnant and 5.9 ± 3.5 (198) in the postpartum group, respectively (P > 0.05). Among stimulated buffaloes, most aspirated follicles were of the small size (<5 mm). Overall oocyte recovery rate in both the groups was 61.8% (41–77.4%). Collectively the majority of recovered oocytes were single- and multi-layered, at a higher rate in non-pregnant than postpartum buffaloes. In conclusion, as already reported in cattle, this study confirms the possibility of retrieving oocytes by OPU from non-pregnant and postpartum buffaloes. The number and quality of recovered oocytes was similar in both groups of buffaloes.
This study was supported by National Research Council, Thailand (year 2002). AP is PhD candidate under Royal Golden Jubilee program, Thailand Research Fund.
338 SYNCHRONISATION OF OVULATION IN MERINO EWES WITH GnRH	IN THE BREEDING AND NON-BREEDING SEASON
Authors: Reyna J. , Thomson P. , Evans G. , Maxwell C.
Published: December 2004

The objective was to determine the effect of GnRH as an aid to synchronise the time of ovulation in Merino ewes during the non-breeding and breeding seasons as determined by transrectal ultrasound. Oestrus was synchronized in 20 nulliparous Merino ewes (11–12 months old; 2 replicates of 10 animals) during spring 2003 and autumn 2004 at Camden, NSW, Australia, using FGA sponges for 12 days (30 mg Ovagest, Bioniche Pty. Ltd., Armidale, NSW) and an i.m. injection of 400 IU of PMSG (Pregnecol, Bioniche Pty. Ltd., Armidale, NSW). Ultrasound evaluations of ovaries were recorded on VHS tapes every 12 h for 36 h starting at sponge removal (SR), then half of the animals received an i.m. injection of 40 μg synthetic GnRH (Fertagyl, Intervet Australia Pty. Ltd, Bendigo, VIC) and ultrasound evaluations were conducted every 6 h until 60 h. The positions of the largest follicles were recorded on ovarian maps and their growth was monitored. Time of ovulation was defined as the time of disappearance of the largest follicle from the ovary. Ten days after ovulation, the position and diameter of the CL was confirmed by ultrasound. Comparisons were made between treated and control animals, and between breeding and non-breeding seasons, using t-tests. During the non-breeding season ovulation took place from 42 to 54 h (mean 48 ± 2.83 h) vs. 42 to 60 h (mean 52.2 ± 5.69 h) after SR in GnRH-treated vs. control animals (P < 0.05), respectively. Ovulation was delayed in the breeding compared with the non-breeding season (P < 0.05), starting from 48 to 60 h after SR for treated (52.8 ± 3.79 h) and control animals (57.0 ± 4.24 h; P < 0.05). These results suggest that GnRH synchronized the time of ovulation compared with the controls but the time of ovulation was later in the breeding than in the non-breeding season.
34 LIFESPAN AND CHROMOSOMAL STABILITY OF BOVINE AND PORCINE FETAL FIBROBLAST CELLS CULTURED IN VITRO
Authors: Giraldo A.M. , Lynn J.W. , Pope C.E. , Godke R.A. , Bondioli K.R.
Published: December 2004

The low efficiency of nuclear transfer (NT) has been related to factors such as mitochondria heteroplasmy, failure of genomic activation, and asynchrony between the donor karyoplast and recipient cytoplast. Few studies have characterized donor cell lines in terms of proliferative capacity and chromosomal stability. It is known that suboptimal culture conditions can induce chromosomal abnormalities, and the use of aneuploid donor cells during NT can lead to a high incidence of abnormal cloned embryos (Giraldo et al. 2004 Reprod. Fertil. Dev. 16, 124 abst). The purpose of this study was to determine the lifespan and chromosomal stability of bovine and porcine fetal cells. Four bovine and four porcine fibroblast cells lines were established from 50-day and 40-day fetuses, respectively. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C in 5% CO2. Each cell line was passaged to senescence. Total population doublings (PDs) and cell cycle duration were calculated. To determine the chromosome numbers at different PDs, cells were synchronized in metaphase, fixed, and stained. ANOVA and chi-square tests were used to analyze differences in PDs and proportion of aneuploid cells between cell lines, respectively (P < 0.05). The results show that proliferative capacity was not different between cell lines derived from the same species. Cell lines derived from bovine and porcine fetuses had different in vitro lifespans (33 PDs vs. 42 PDs, respectively; P < 0.05). The mean length of the cell cycles for both bovine and porcine fetal fibroblasts was ∼28 h. The percentage of aneupliod cells in both bovine and porcine fetal cell lines increased progressively with duration of culture (see Table) and was high throughout the study. The proliferative capacity of cultured cells was similar within individuals of the same species, but growth characteristics differed between fetal bovine and porcine cell lines. The progressive increase of aneuploid cells could be due to suboptimal culture conditions or unusual chromosome instability in the particular fetuses used. These data demonstrate the importance of determining chromosome content and the use of cells at early passages to decrease the percentage of aneuploid reconstructed embryos and increase the efficiency of NT.
39 CHROMOSOME ABNORMALITIES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED BY 'HANDMADE CLONING'
Authors: Jakobsen A.S. , Vajta G. , Thomsen P.D. , Callesen H. , Greve T.
Published: December 2004

Embryos produced by nuclear transfer using somatic cells as donors exhibit a lower developmental competence compared to in vivo developed and IVM/IVF/IVC embryos. The so-called handmade somatic cell nuclear transfer (HSCNT or handmade cloning) that was recently presented (Vajta et al. 2003 Biol. Reprod. 68, 571–578) has, however, improved the reconstructed embryo and subsequent blastocyst rate. Further, the high total cell number (n = 216) and the high ratio of cells allocated to the inner cell mass (35%) as well as the initial pregnancy rate of 48% on Day 28 following nonsurgical transfer of HSCNT embryos indicated a high average quality. But still the pregnancy loss of HSCNT embryos was high and resulted in a birth rate of 8%. The aim of this study was to estimate the chromosomal variation in HSCNT embryos using fluorescent in situ hybridization (FISH) and to evaluate this as an additional parameter of embryo quality. Nuclei from 49 Day 7 HSCNT embryos from five independent trials were isolated by hypotonic treatment and fixed. Then the nuclei were hybridized with differentially labelled cJAB8 and p33E39 probes that hybridize specifically to the centromeric region of chromosome 6 and 7 (Viuff et al. 2000 Biol. Reprod. 63, 1143–1148). A total of 6715 nuclei were analyzed for chromosomal abnormalities, and the percentage of nuclei with false negative scores represented 1.8%. Only 4.1% of the embryos (2 of 49) had a completely normal diploid composition, while the remaining 47 embryos exhibited different types of mixoploidy. Of the 47 mixoploid embryos, 87% contained more than one type of chromosomal variation with diploid/triploid/tetraploid being the most frequent constitution (43% of the 47 mixoploid embryos). No pure polyploid embryos were observed. The percentages of HSCNT embryos in the groups of 0%, 1–25%, and 26–100% polyploid cells were 4.1%, 67.5%, and 28.6%, respectively. This is significantly higher than the corresponding figures produced and analyzed by comparable methodology of bovine IVM/IVF/IVC embryos (Viuff et al. 1999 Biol. Reprod. 60, 1273–1278). However, 71.4% of the HSCNT embryos contained less than 25% polyploid cells; this low level may not have compromised the developmental competence of these embryos.
40 CHARACTERIZATION OF EARLY G1 CELLS AS NUCLEAR DONORS FOR SOMATIC CELL CLONING IN CATTLE
Authors: Kasamatsu A. , Saeki K. , Tamari T. , Shirouzu K. , Taniguchi S. , Mitani T. , Aoyagi Y. , Urakawa M. , Ideta A. , Matsumoto K. , Hosoi Y. , Iritani A.
Published: December 2004

In somatic cell cloning, the cell cycle phase of the donor cells has critical impact on nuclear reprogramming and chromosomal normality of the reconstructed embryos. Recently, enhanced development to full term was obtained with embryos reconstructed with bovine fibroblasts soon after cell division (early G1 cells, Kasinathan P et al. 2001 Nat. Biotech. 19, 1176–1178; Urakawa M et al. 2004 Theriogenology 62, 714–728). In this study, to investigate the detailed cell cycle characteristics and gene expression of the early G1 cells as nuclear donors, we examined the cell proliferating and nuclear activity by detecting PCNA and Ki-67 in the cells, and the gene expression in the cells transfected with the luciferase gene. Bovine fibroblasts were transfected with chicken β-actin/firefly luciferase fusion gene (β-act/luc+), and stably transfected; cloned cells were used for cell analysis. We compared cell cycle characteristics for quiescent cells (0.4% serum for 7 days), cell doublets (early G1 cells) prepared by the “shake-off” method, and proliferating (30 to 40% confluency) cells. The presence of PCNA and Ki-67 and the incorporation of BrdU in the cells were determined by immunohistochemical analysis. The LUC+ signal (luminescence) in the cells was detected with an imaging photon counter for 10 consecutive min. Embryos reconstructed with these cells were cultured for 168 h for examination of blastocyst development. Experiments were repeated three times, and the data were analyzed with Fisher's PLSD test following ANOVA. Incorporation of BrdU was observed only in proliferating cells (24% of the cells). Neither PCNA nor Ki-67 signals were detected in the quiescent cells. PCNA was detected but Ki-67 was not detected in early G1 cells. Both PCNA and Ki-67 were detected in the proliferating cells. A strong LUC+ signal (6354 ± 673 pixels/cell) was detected in the proliferating cells, and weak signals were detected in the early G1 (2044 ± 303 pixels/cell, P < 0.05) and quiescent cells (617 ± 59 pixels/cell, P < 0.05). The rate of blastocyst development with early G1 cells was higher (45/133, 32%) than that with starved and proliferating cells (47/233, 21%, and 41/258, 14%, respectively, P < 0.05). These results indicate that early G1 cells were actively proliferating cells because of the positive PCNA signals, but their nuclei were silent because of the absence of Ki-67 signals and the weak LUC+ signals. These characteristics of the early G1 cells might enhance the development of the reconstructed embryos.
This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.
5 NEUTRAL SEGREGATION OF DONOR CELL MITOCHONDRIA IN FETAL AND ADULT TISSUES OF SOMATIC CELL CLONES IN CATTLE
Authors: Viramontes F. , Filion F. , Smith L.C.
Published: December 2004

Until now, animal cloning has been extremely inefficient: only 1–2% of nuclear transfer (NT) clones survive to birth. Some of these anomalies may be related to an incompatibility between nuclear and mitochondrial genes (Cummins JM 2001 Hum. Reprod. Update 7, 217–228). Controversy exists as to the levels of donor cell mitochondrial DNA (mtDNA) inheritance in somatic clones (heteroplasmy). Whereas some researchers found very low quantities (0.1–4%) (Steinborn R et al. 2000 Nat. Genet. 25, 255–257), others found levels of heteroplasmy ranging from 6 to 40% (Takeda et al. Mol. Reprod. Dev. 64, 429–437). Since it remains unclear whether mtDNA segregation is neutral or selective, the purpose of this study was to analyze the transmission of the mtDNA from donor somatic cells in fetal and adult clones using a particular mtDNA marker (mtDNA Bos taurus with one mutation in the D-loop of 40 base pairs plus than the wild type). Fibroblasts from a fetus of 60 days were used as donor cells. The fetus was produced by artificial insemination of a Holstein (Bos taurus) heifer carrying an mtDNA mutation with semen from a Zebu (Bos indicus) bull. Oocytes derived from slaughterhouse ovaries of Holstein cows carrying wild-type mtDNA were used as recipient cells. The presence of the mutated mtDNA from the donor cell (heteroplasmy) was analyzed in a male cloned fetus of 60 days and in three adult male clones at 18 months of age. Heteroplasmy was detected in 7 tissues in the foetus: muscle, skin, stomach, testicle, thymus, tongue, and umbilical cord. Three tissues were analyzed from the adult clones: semen, skin, and white blood cells. Heteroplasmy was detected in all the tissues by nested PCR amplification of the D-loop and analyzed by ANOVA and Tukey-Kramer multiple comparison test. The mean (%) of the mutated mtDNA of the donor cell in the seven tissues of the60-day-old fetus was 1.14 ± 0.34 (SEM). There was no differences in the means of heteroplasmy (%) between the tissues of the fetus (P > 0.05). The mean level of heteroplasmy in the three adult clones analyzed (clones A, B, and C) was 1.41 ± 0.18 (SEM). Analysis of heteroplasmy between the tissues of each clone showed no differences (P > 0.05) with the exception of clone B, where semen was different (P < 0.05) from white blood cells. There were significant differences (P < 0.05) between some clones (taking together all the results of all tissues of each clone). The heteroplasmy in clone B (%) (2.59 ± 0.18 SEM) was different (P < 0.05) from that of both clone A (1.04 ± 0.18) and clone C (1.46 ± 0.18). There was no difference between the heteroplasmy (%) of clone A and that of clone C (P > 0.05). These results show that the tissues of the fetus and the adult clones were heteroplasmic at similar levels, suggesting neutral segregation of the donor cell mtDNA during development and tissue differentiation.
73 ABNORMAL REPROGRAMMING OF HISTONE ACETYLATION IN CLONED BOVINE EMBRYOS
Authors: Wee G. , Koo D.-B. , Kang M.-J. , Moon S.J. , Lee K.-K. , Han Y.-M.
Published: December 2004

Histone acetylation plays an important role in the chromatin structure prior to zygotic gene expression during early embryonic development. Successful animal clones indicate that differentiated somatic nuclei must be reprogrammed to some extent during pre-implantation development. However, the molecular mechanisms regarding epigenetic reprogramming of somatic nuclei in the early-stage embryos are poorly understood. To test this, the patterns of hyperacetylated histone H4 lysine 5 (AcH4K5) in the nuclear-transferred (NT) embryos were monitored, comparing in vitro fertilized (IVF) embryos and Trichostatin A (TSA)-NT embryos with TSA-treated cells. The intensity signals of AcH4K5 were observed in early-stage embryos and somatic cells (bovine ear skin fibroblasts composed of about 80% at G0/G1 stage) by immunofluorescence analysis with anti-AcH4K5 using image the analyzer system, SigmaScan-pro V5.01 (SPSS, Inc., Chicago, IL, USA). Our data were analyzed by analysis of variance (ANOVA) using an SAS package (SAS Institute, Inc., Cary, NC, USA). Somatic cells were exposed to TSA (1 μM for 60 h), a specific inhibitor of histone deacetylase (HDAC), to induce hyperacetylation prior to somatic cell nuclear transfer. Signal intensity for AcH4K5 in TSA-treated cells (n = 80) was significantly increased (P < 0.05), which was approximately double compared to that of normal cells (n = 80). In normal cells, histone H4 acetylation was profoundly reduced from the pro-metaphase to the early telophase and then reappeared at the late telophase. Acetylation signals of TSA-treated cells gradually increased to the early anaphase, abruptly decreased at the late anaphase and the early telophase, and recovered during late telophase. During early embryonic development (1 cell to 8 cell stage), NT embryos (n = 8) were hypoacetylated at the metaphase, whereas IVF (n = 10) and TSA-NT embryos (n = 8) were hyperacetylated. Our findings demonstrate that aberrant epigenetic reprogramming of histone modification occurs as early as the pronuclear stage in cloned embryos.
74 ANATOMICAL ABNORMALITIES IN CALVES PRODUCED BY NUCLEAR TRANSFER
Authors: Williamson M. , Tecirlioglu R. , French A. , Holland M.
Published: December 2004

Two limitations currently restrict the acceptability and adoption of bovine cloning as a commercial reality. The first of these is its low rate of efficiency and the second is that some of the cloned calves are not healthy. Abnormalities in the placenta are thought to contribute to many of the losses in early gestation; however, less is known of the pathology of clone deaths in the perinatal period. To date, the majority of perinatal deaths have been attributed to the “large offspring syndrome” characterized by increased birth weight and a range of morphological abnormalities thought to be associated with in vitro culture and manipulation. This report describes multi-systemic abnormalities in aborted, stillborn, and neonatal genetically modified and unmodified cloned calves weighing less than 60 kg at birth and aged between 6 months gestation and 3 weeks postnatal, generated in various experiments. Three of 14 genetically modified cloned calves had cystic renal dysplasia and osteopetrosis. All three and a fourth had irregular nodular, fibrotic livers with biliary abnormalities. Another two had marked flexion of the fetlock joints. Eleven calves derived from an unmodified cloned cell line by nuclear transfer had nodular, fibrotic livers with biliary anomalies, 9 of 11 had cystic renal dysplasia and cardiomegaly, two had osteopetrosis, and two had contracted tendons. In addition, three calves had polymicrocerebral gyri, two had retinal dysplasia, and one had an aortic aneurysm. Only one calf from a second unmodified cloned cell line produced by nuclear transfer had no significant congenital abnormalities. All calves were negative for bovine virus diarrhea virus (BVDV) by competitive-antigen ELISA, and by virus isolation and no BVDV antibodies were detected by AGID assay. Furthermore, all cell lines and media used were negative for BVDV by virus isolation. Two calves were tested and found to be negative for Akabane virus and Aino virus. There are very few reports of the pathological abnormalities of cloned animals. Similar multi-systemic abnormalities have not been found in non-cloned calves, but several analogous conditions occur in humans, including Simpson-Golabi-Behmel and Zellwegers syndromes. Further ultrastructure studies and genetic analysis are needed to investigate the mechanisms of these multi-systemic disorders, which may ultimately elucidate mechanisms for improved reprogramming and increase the efficiency of generating cloned animals with somatic cells by nuclear transfer.