bluuebunny/crossref_metadata_2025 huggingface dataset:
188 DIRECT-THAW TRANS-CERVICAL TRANSFER OF RED DEER FROZEN IN VITRO BLASTOCYSTS CAN RESULT IN PREGNANCIES
Authors: Beaumont S.E. , Berg M.C. , Strongman K. , Saywell D.P. , Berg D.K.
Published: December 2004

The seasonal demand for farmed venison in New Zealand has necessitated the concentration of red deer breeding into the first month of the four-month breeding season. Because of this constraint it is difficult to obtain enough in vitro-produced blastocysts for transfer. Successful cryopreservation would enable embryos produced and stored throughout the breeding season to be available for transfer the following year. In vitro red deer calves have been successfully produced after trans-cervical transfers in a limited number of red deer (Berg DK et al. 2004 Reprod. Fert. Dev. 16, 201 abst). We determined the viability of frozen blastocysts following trans-cervical transfer to recipient hinds using the direct-thaw method. In two replications, abattoir derived red deer COCs were selected and matured in vitro (Berg DK et al. 2002 Ani. Reprod. Sci. 70, 85–98). Oocytes were randomly divided into two groups and fertilized with either red deer sperm using IVF-Deer SOF (DSOF), or wapiti sperm using IVF-SOF. All presumptive zygotes were cultured for 6 days in DSOF (Beaumont SE et al. 2004 Reprod. Fert. Dev. 16, 268 abst). Cleavage was recorded on Day 4 and embryos were evaluated on Day 7. Grade 1 and 2 blastocysts were selected and equilibrated in 1.5 M ethylene glycol with 0.1 M sucrose, frozen from −5 to −38°C at a rate of 0.3°C per min and plunged into liquid nitrogen. Twenty synchronized farmed deer hinds (13 red deer to receive red deer blastocysts, and 7 F1 wapiti/red hybrids to receive F1 blastocysts) were prepared for transfer (Berg DK et al. 2003 Theriogenology 59, 189–205). Only Grade 1 blastocysts were selected for transfer. Straws were thawed for 5 s in air, immersed in a 30°C water bath for 20 s, directly diluted, and loaded into cattle transfer pistolettes. Each embryo was deposited in the uterine horn. A modified pistolette, fitted with a Mariensee tip (Minitüb, 84184 Tiefenbach, Germany) was used to dilate difficult cervices (n = 4). Pregnancies were confirmed by ultrasonography on Day 35. Results were evaluated using chi-square analysis. Embryo cleavage rates ranged from 74 to 85% and were not different between the two sires. Blastocyst development rates (from cleaved zygotes) were similar for both sires; wapiti 15% (43/279) and red deer 14% (34/246). A total of 24 wapiti/red hybrid and 17 red deer blastocysts were frozen. Eighteen of 20 hinds (90%) received embryos, 11/13 red deer receiving red deer embryos and 7/7 F1 wapiti/red hybrids receiving F1 wapiti/red hybrid embryos. The cervices of two red deer hinds were impenetrable. Pregnancy rates were not different between the 2 groups of recipients, with 29% (2/7) of the wapiti hybrids and 45% (5/11) of red deer confirmed pregnant. These preliminary results demonstrate, for the first time, that farmed deer pregnancies can be established from frozen in vitro-produced embryos after direct-thaw and trans-cervical transfer to synchronized hinds.
193 TESTIS TISSUE XENOGRAFTING TO PRESERVE GERM CELLS FROM A CLONED BANTENG CALF
Authors: Honaramooz A. , Zeng W. , Rathi R. , Koster J. , Ryder O. , Dobrinski I.
Published: December 2004

In April 2003, two banteng (Bos javonicus) calves were born after heterologous nuclear transfer of donor cells from a genetically valuable individual frozen in 1978. One of the cloned banteng calves died at one week of age. The calf was found to have one scrotal and one abdominally cryptorchid testis. In an attempt to preserve male germ cells from this valuable animal, parts of each testis were shipped on ice to the University of Pennsylvania for xenografting. Grafting of testis tissue from immature domestic animals and monkeys under the back skin of immunodeficient mice can result in complete spermatogenesis, albeit with different levels of efficiency in different species. The objective of this experiment was to investigate if grafting of immature banteng testis tissue would result in spermatogenesis in a mouse host. Small fragments of tissue (about 1 mm, 3 each) from both testes were grafted under the back skin (4 pieces of scrotal testis on the right side and 4 pieces of retained testis on the left side) of 6 castrated male immunodeficient mice. Histological examination of the testis xenografts was performed 3, 6, 9, 12, and 15 months after transplantation. Weight of the seminal vesicles in the host mouse was recorded as an indicator of bioactive testosterone produced by the xenografts. At the time of grafting, both testes contained seminiferous cords with immature Sertoli cells and gonocytes. At 3, 6, and 9 months after grafting, pachytene spermatocytes were present in the xenografts of the scrotal testis whereas no germ cell differentiation was observed in grafts from the retained testis. However, spermatogenesis in grafts of the scrotal testis did not proceed further through meiosis in grafts analyzed at 12 and 15 months after grafting, with pachytene spermatocytes still the most advanced germ cell type present in grafts recovered 15 months after grafting. The weight of the seminal vesicles in the castrated host mice was restored to pre-castration values showing that xenografts were releasing bioactive testosterone. These results indicate that banteng spermatogenesis was initiated in the mouse host but became arrested at meiosis as observed previously in xenografts of immature bovine or equine testis. Therefore, haploid germ cells could not be recovered. This represents the first example of trying to preserve fertility from a rare, valuable newborn animal by testis tissue xenografting. While xenografting presents a previously unavailable option for preservation of male germ cells from immature individuals, the efficiency of sperm production in testis xenografts appears to be variable and has to be determined empirically for different donor species.
This work was supported by USDA 03-35203-13486.
199 BASIC CHARACTERISTICS AND CRYOBANKING OF BARBARY SHEEP (AMMOTRAGUS LERVIA) SEMEN
Authors: Pérez-Garnelo S.S. , Borque C. , Madrid-Bury N. , Delclaux M. , Talavera C. , Martínez E. , Palasz A.T. , De La Fuente J.
Published: December 2004

Barbary sheep (Ammotragus lervia) are considered vulnerable species by the World Conservation Union (IUCN). The purpose of this study was to describe the basic characteristics of fresh semen, test the efficacy of commercial extender Triladyl, and collect necessary data that may help to create a frozen semen bank for the Barbary sheep in Spain. A total of 21 ejaculates were collected by rectal-probe electroejaculation from one dominant (D) and three minor (M) adult males housed in the Madrid Zoo. After ejaculation, semen volume, concentration, and mass motility were assessed. Remaining raw semen was diluted at 37°C with TRIS-based extender Triladyl (Minitüb, Tiefenbach, Germany) and 20% egg yolk to a final concentration of 200 × 106 sperm per mL. Diluted samples were kept at 5°C for 4 h and then loaded into 0.25-mL French straws, frozen at 5 cm above liquid nitrogen (LN2) for 10 min and then plunged into LN2. Samples were thawed in a water bath at 37°C for 30 s. Post-thaw semen survival was evaluated by sperm motility (%M), quality of movement (Q), normal acrosome status (%NAS), normal sperm morphology (%NOR), membrane integrity (hypo-osmotic test; %HOST), and sperm viability (eosin-nigrosin vital staining; %V), and were compared with the same parameters in the fresh semen. Data between D and M males were analyzed by one way ANOVA. Mean volume of ejaculates, total sperm concentration and mass motility of raw semen were respectively; 5.2 ± 1.56 mL, 2800.0 ± 1290.5 × 106 and 3.4 ± 0.4 for the D male, and 3.5 ± 3.2 mL, 251.2 ± 103.9 × 106, and 1.88 ± 1.4 for M males (P < 0.05). Remaining semen parameters evaluated in raw semen showed no differences between D and M males. However, post-thaw semen quality was significantly (P < 0.05) reduced in all analyzed parameters except %NAS and %NOR in M males groups as compared to the D male (Table 1). It can be concluded that Barbary sheep raw semen collected by electroejaculation is of sufficient quality to be used in an artificial insemination program and can be successfully frozen in commercially available Triladyl extender. However, the post-thaw viability of semen may considerably depend on the male reproductive status in the flock.

Table 1.
Characteristics of fresh and cryopreserved Barbary sheep semen

This work was supported by CAM 07B/007/1999 (Analysis of ejaculate traits and development of methods of semen preservation in wild ungulates from the Madrid Zoo).
211 TEMPORAL AND SPATIAL EXPRESSION PROFILE OF THE UTERINE MILK PROTEIN — A MEMBER OF THE SERINE PROTEASE INHIBITOR SUPERFAMILY — IN THE BOVINE ENDOMETRIUM
Authors: Bauersachs S. , Ulbrich S.E. , Gross K. , Schmidt S. , Wenigerkind H. , Meyer H.H.D. , Blum H. , Wolf E.
Published: December 2004

A combination of subtracted cDNA libraries and cDNA array hybridization was applied to identify differentially expressed genes in the bovine intercaruncular endometrium at estrus and diestrus. Six cyclic Simmental heifers were cycle-synchronized and slaughtered the morning after standing heat occurred (n = 3) or 12 days after estrus (n = 3), respectively. The uterus was divided into seven sections: corpus, plus the caudal, middle, and cranial parts of both uterine horns. Samples were collected from the epithelial layer of the lamina propria of the intercaruncular endometrium. Two subtracted libraries were prepared and analyzed by array hybridization with probes produced from the 42 tissue samples. More than 130 differentially expressed genes were identified. Among these genes, the mRNA for the uterine milk protein (UTMP) showed a much higher expression level at estrus versus diestrus. Furthermore, the expression level differed markedly between the uterus sections. Statistical significance was tested using ANOVA and Student Newman Keuls post-hoc test (GeneSpringÂ® version 6.1, Silicon Genetics, Redwood City, CA, USA). There was a pronounced expression gradient from both cranial uterine horns (highest) to the corpus (lowest) at estrus. UTMP mRNA level was highest in the ipsilateral cranial horn. A more accurate quantification was done by quantitative real-time RT-PCR using the same RNA samples as for array hybridization and, in addition, samples from further time points of the estrous cycle and early pregnancy. The results obtained by array hybridization were clearly confirmed. UTMPs, also known as uterine serpins (US), were previously shown to be the major products secreted by the uteri of cattle, sheep, and pigs during pregnancy. The US are glycoproteins belonging to the serpin (serine proteinase inhibitor) superfamily of proteins. However, no inhibitory activity to any serine proteinase has been found. The ovine uterine serpin (OvUS) has been shown to inhibit a wide variety of immune responses, including mixed lymphocyte reaction, T cell-dependent antibody production, and NK (natural killer)-like activity, indicating a major role in protecting the conceptus from maternal immune reactions. The four members of the US family form a clade distinct from other serpins, which have probably adopted important biological functions in the reproductive biology of Artiodactyla. Therefore, the identified expression gradient of the UTMP mRNA at estrus may be of particular importance, e.g. for sperm selection, and deserves further investigation at the protein level and in functional studies.
This work was supported by the Deutsche Forschungsgemeinschaft (Research Unit “Mechanisms of Embryo-Maternal Communication”; FOR 478/1).
217 PROMOTER-SPECIFIC EXPRESSION OF THE IMPRINTED Igf2 GENE IN CATTLE
Authors: Curchoe C. , Zhang S. , Bin Y. , Yang L. , Tian X.C.
Published: December 2004

Insulin-like growth factor II (Igf2) is an imprinted gene crucial to fetal development and maternal/fetal nutrient transfer. Studies in the pig have demonstrated that this quantitative trait locus controls muscle growth, fat deposition, and heart size. Additionally, studies have shown that four splice variants transcribed from promoters (P1–P4) regulate the complex temporal and spatial expression of Igf2 in mice, humans, pigs, and horses. Cattle have a cotyledonary placenta and therefore a distinct type of maternal/fetal interaction. Here we have studied for the first time the promoter-specific expression of Igf2 in cattle, an economically valuable livestock species. Five naturally reproduced animals obtained from an abattoir were used in this study (2 mid-gestation fetuses, 1 calf, and 2 adults) of which all major internal organs were tested. Here we used RT-PCR to show that, like that of the pig and the human, the bovine Igf2 is expressed from four different promoters in a temporal and spatial manner. However, unlike for pigs and humans, we have found that transcripts from promoter P1 were present in several bovine fetal and adult tissues including the liver, heart, kidney, lung, placenta, and spleen. Promoter P2 was expressed only in mid-gestation fetal tissues including the liver, bladder, lung, and kidney. Promoter P2 was not detected in the brain. Promoter P3 was expressed ubiquitously throughout fetal and adult life; however, expression appears to be lower in the heart. Promoter P4 was expressed in all mid-gestation fetal tissues. Transcription from P4 decreased with age until transcripts were detected only in the kidney, lung, heart, and spleen. Using single-stranded conformational polymorphism polyacrylamide gel electrophoresis and a single nucleotide polymorphism in exon 10 of Igf2, we have confirmed that a loss of imprinting occurs with age from all transcripts (P1–P4) and biallelic expression is observed in most adult tissues studied.
This work was funded by a grant from the USDA.
219 METHYLATION STATUS OF A DIFFERENTIALLY METHYLATED REGION (DMR) WITHIN THE BOVINE Igf2 GENE IN PREIMPLANTATION EMBRYOS
Authors: Gebert C. , Wrenzycki C. , Herrmann D. , Reinhardt R. , Gröger D. , Lucas-Hahn A. , Carnwath J.W. , Niemann H.
Published: December 2004

Specific DNA regions within imprinted genes become differentially methylated on the maternal and paternal chromosomes during germ cell development. These DMRs play a crucial role in the regulation of imprinted gene expression. The murine insulin-like growth factor2 gene (Igf2) is imprinted and contains an intragenic DMR within the last exon. Recently it became known that the bovine Igf2 gene is also imprinted (Dindot et al. 2004 Biol. Reprod. 71, 470–478) where we have now identified an intragenic DMR in the last exon with the paternal allele being methylated. Aberrant methylation patterns within the bovine Igf2 gene could result in deregulated gene expression and could therefore be involved in the development of fetal abnormalities such as the large offspring syndrome (LOS) in cattle. We have studied the methylation status of 27 CG dinucleotides within this DMR in bovine pre-implantation embryos of different origin by bisulfite sequencing. DNA was isolated from expanded blastocysts collected in vivo and generated by in vitro fertilization (IVF), somatic nuclear transfer (NT), and parthenogenesis (PA). Additionally, DNA was obtained from fibroblasts derived from a female and a male adult animal and used as donor cells for NT and from zygotes and 4-cell embryos both produced by IVF. After PCR amplification of the bisulfite-treated DNA, PCR products were cloned and sequenced. Methylation percentages were calculated for each individual clone by division of the 27 CpGs with the number of methylated CpGs per sample. The methylation levels (%) from each sample were then used to obtain the global methylation levels of the analyzed region. Methylation decreased during the transition from the zygote (28.4% ± 3.8 SEM) to the 4-cell embryo (6.3% ± 2.2 SEM) indicating that the DMR is demethylated after fertilization. An increased methylation level was observed in expanded blastocysts (in vivo: 10.2% ± 1.2 SEM; IVF: 10.1% ± 0.7 SEM; female NT: 12.4% ± 1.4 SEM). Thus, remethylation starts before the blastocyst stage. The higher methylation level of male NT blastocysts (22% ± 1.9 SEM) in comparison to their in vivo and IVF counterparts could be due to an insufficient reprogramming of the donor cells after nuclear transfer. Female and male donor cells were both heavily methylated (77% ± 2.2 SEM; 72% ± 2.9 SEM, respectively). Parthenogenetic expanded blastocysts were less methylated (2.3% ± 1 SEM), probably due to their diploid maternal genome. Results show for the first time that the methylation status at this DMR is associated with the origin of the embryo. Analysis of methylation patterns in pre-implantation embryos could provide a diagnostic tool to unravel mechanisms involved in fetal malformations often observed after the use of in vitro fertilization and/or nuclear transfer.
225 RECOMBINANT HUMAN ERYTHROPOIETIN PRODUCED IN THE MILK OF TRANSGENIC MICE: FUNCTIONAL CHARACTERIZATION AND PHARMACEUTICAL APPROACH
Authors: Kwon D.N. , Park J.Y. , Lee S.Y. , Kang S.J. , Kim J.H.
Published: December 2004

The proper post-transcriptional modification of recombinant human erythropoietin (rhEPO) is critical to retain its biological functions, either in vivo or in vitro. The major glycosyltransferases for the determinant of glycosylation patterns of rhEPO are N-acetylglycosaminyltransferase (GnT) and α-1-3/4 fucosyltransferase (Fut). GnT-III expression (388 ± 19.09) in the mouse mammary gland has been shown to be dramatically different from that in CHO cells, although FuT-VIII expression in CHO cells (1970 ± 255.9) is comparable to mouse mammary gland (272 ± 14.8), suggesting that the mammary gland may proceed with the proper glycosylation of rhEPO as shown in CHO cells. To identify this hypothesis and establish the rhEPO bioreactor system for mass production of protein in transgenic animals, we have generated two transgenic mouse lines that express rhEPO in milk. Both lines of transgenic mouse express only rhEPO in the lactating mammary gland, and the protein yield of rhEPO in lactating milk is comparable to that in CHO cells. After determining the protein expression in lactating milk, using three different methods – enzymatic release of oligosaccharide analysis, two-dimensional electrophoresis, and 2-aminobenzamide-labeled analysis – we report that the rhEPO produced by the animal bioreactor system has the proper glycosylation patterns as shown in CHO cell-derived Epoietin α, and has more tetra-acidic oligosaccharide structures than Epoietin α, which is the widely used rhEPO for therapeutic purposes. The in vitro biological property of transgenic mouse milk-derived rhEPO has been tested by measuring luciferase activity in MCF-7 cells, indicating that rhEPO from mammary gland up-regulates the EPO-receptor-mediated STAT5 gene expression in a dose-dependent manner the same as Epoietin α does. In addition, in vivo biological activity demonstrated that direct injection of rhEPO into a mouse vein increases blood components such as RBC and HCT. In light of these findings, we suggest that high levels of tetra-acidic structures observed in transgenic mouse milk-derived rhEPO may be related to the high level of expression of glycosiltransferases (GnT-III and FuT-VIII) in mammary gland; thus the bioreactor system using the mammary gland of a transgenic animal could be a good candidate for production of rhEPO for pharmaceutical purposes.
This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea. D.N. Kwon is the recipient of a scholarship from the BK21 program, granted by the Ministry of Education, Korea.
227 CONSTRUCTION OF STAGE-SPECIFIC cDNA MICROARRAY, AND ANALYSIS OF IN VITRO PRODUCED PRE-IMPLANATION STAGE BOVINE EMBRYOS FOR DEVELOPMENTAL COMPETENCE
Authors: Mamo S. , Sargent C.A. , Affara N.A. , Wimmers K. , Ponsuksili S. , Gilles M. , Schellander K.
Published: December 2004

Microarray technology currently has wide acceptance as a research tool in the study of gene expression profiling, mainly as a result of its use for monitoring the expression profiles of thousands of genes in a single experiment. However, its use in analyzing gene expression in the pre-implantation stage of bovine embryo development has been limited for reasons such as the large amount of RNA required and the lack of bovine specific cDNA clone collections (Smith L and Greenfield A 2003 Hum. Mol. Genet. 12, 1–8). In this study, with the objectives of producing pre-implantation-stage-specific bovine cDNA clones and examining the developmental competence, eighty-two selected target clones of pre-implantation-stage-specific genes were prepared and spotted on the glass slide. Embryos were produced in vitro and mRNAs were isolated from contrasting probes of good quality matured oocytes and blastocyst-stage embryos using a Dynabead mRNA isolation kit by following the manufacturer's instructions. First-strand cDNA syntheses were primed with T7 Oligo d(T)21 primer, followed by random primed second-strand syntheses using a DOP master kit (Roche Diagnostics, Mannheim, Germany) and global amplification using the same primers used for the first- and second-strand syntheses. In vitro transcription was performed to amplify the RNA by using the AmpliScribe T7 transcription kit (EPICENTRE Technologies, Oldendorf, Germany), and the amplified RNA (aRNA) was purified using a RNeasy Mini kit (Qiagen, Hilden, Germany). Finally, the results of different RNA amplifications (aRNA) were tested by hybridization on microarrays and also using real-time PCR techniques. With these analyses, the sufficiency of the yield and linearity of amplification procedures were confirmed. Three micrograms each of aRNA were labelled with Cy3 and Cy5 dyes and hybridized to the array. After overnight incubation at 42°C, the slides were sequentially washed and scanned using an ArrayWorx biochip reader (Applied Precision, Marlborough, UK), and quantifications as well as all analyses were carried out using different TIGR software modules (Saeed AI et al. 2003 Biotechniques 34(2), 374–378). Analyses of the results of repeated hybridizations showed that 35 genes (43%), which belong to different functional groups, were differentially expressed between the two stages. Further independent analyses using real-time PCR confirmed the results of 25 genes. Hence, it is possible to conclude that the established methods can be used for large scale gene expression analysis, and the identified genes can be potential candidates for characterizing developmental competence.
242 DEVELOPMENT OF BAC FISH PROBES FOR GENETIC ANALYSIS OF NON-HUMAN PRIMATE GAMETES AND EMBRYONIC STEM CELLS
Authors: Froenicke L. , Nichols S.M. , Kubisch H.M. , Lyons L.A. , Bavister B.D. , Brenner C.A.
Published: December 2004

Several factors relating to reproductive health and gamete competence must be considered when implementing programs for propagation of genetically valuable primates via natural breeding or assisted reproductive technologies. These relevant factors include ascertaining gamete chromosome normality of individuals. Our early attempts at characterizing aneuploidy levels of gametes within a model primate species, the rhesus macaque, utilizing Vysis centromeric fluorescent in situ hybridization (FISH) probes, failed to produce a signal. Commercially available probe sets for interphase cytogenetics rely partly on repetitive DNA probes. Therefore, we have employed either pools of human BACs or clones selected from a rhesus macaque BAC library for interphase cytogenetics. Bacterial artificial chromosome (BAC) FISH probes specific to regions of human DNA that are homologous to macaque DNA were produced, in collaboration with researchers at the University of California-Davis. The most common aneuploidies implicated in human birth defects or pregnancy failures are those involving the sex chromosomes and chromosomes HSA (human) 13, 16, 18, and 21 (Munne 2003 Placenta 24, 70–76). Hence, we have initially used human BAC FISH probes on the equivalent chromosome homologs in Macaca mulatta (MMU): X, Y, MMU 17 (HSA13), MMU 20 (HSA16), and MMU18 (HSA18). The homolog of HSA 21 is part of a larger chromosome in the macaque (MMU3) that also contains HSA 7 (based on nomenclature by Cambefort et al. 1976 Ann. Genet. 19, 5–9). Since aneuploidy has been linked to chromosome size and MMU3 is large, this macaque chromosome may be involved in aneuploidies. In addition, we determined the suitability of commercially available “chromosome paints” that target repetitive sequences and are widely used in determining karyotypes in human fertility clinics. Initial work was performed on cultured macaque blood cells exposed to Colcemid. Our preliminary data clearly demonstrate the feasibility of using probes that are commonly used in the analysis of human karyotypes to examine chromosomal anomalies in macaque gametes and embryos. Additional probes for other chromosomes are currently under investigation. The development of a large panel of available probes will provide a rapid and accurate method to assess aneuploidy/mosaicism levels in genetically valuable primate spermatozoa, in vitro-matured oocytes and blastomeres of IVP embryos. Furthermore, these probes will be useful to determine the karyotypic normalcy of cultured non-human primate embryonic stem cells.
This research has been supported by the University of New Orleans to CAB, Tulane National Primate Research Center base grant NIH 5P51 RR00164-41, and the Joe W. and Dorothy Dorsett Brown Foundation Cellular Imaging Facility.
250 DEHYDRATED COCONUT WATER FOR IN VITRO SPERM CAPACITATION IN SWINE
Authors: Nascimento A.B. , Oliveira V.P. , Marques M.G. , Toniolli R. , Gerger R.P.C. , Coutinho A.R.S. , Feitosa W.B. , Visintin J.A.
Published: December 2004

Recently, progress has been achieved in reproduction biotechnology with coconut water as semen extender for domestic animals and in bovine oocyte in vitro maturation and embryo culture. The aim of this study was to evaluate the dehydrated coconut water (ACP250) as semen extender and its effect on in vitro sperm capacitation in swine. Eighteen ejaculates were collected from three crossbred boars of Landrace and Duroc. Semen samples were extended in ACP250 solution or Beltsville Thawing Solution (BTS) and submitted to sperm capacitation after extension (EXT) or after cooling (COO) at 16°C for 24 h. The semen was centrifuged at 400g for 8 min. After centrifugation, the samples were standardized at 2 × 107 spermatozoa/mL, subjected to sperm capacitation in TALP medium with 5 mM of caffeine, and incubated at 38.5°C and 5% of CO2 in high humidity for three h. Four treatments were tested, T1 (ACP250 + EXT), T2 (ACP250 + COO), T3 (BTS + EXT), and T4 (BTS + COO). Sperm capacitation was evaluated by Coomassie Blue G stain, as follows: capacitated spermatozoa were stained in tenuous blue and not capacitated in intense blue. The Coomassie Blue G staining technique was described by Larson and Miller (1999 Mol. Reprod. Dev. 52, 445–449), who demonstrated that sperm from a variety of mammalian species can be stained by this technique, and an adequate observation of the acrosomal status was possible as well. This procedure was described as simple, fast, inexpensive and reliable, and it requires no fluorescence or DIC optics. For statistical analysis, SAS (System for Windows; SAS Institute, Inc., Cary, NC, USA) was utilized (P < 0.05). Comparing the sperm capacitation rates in relation to fresh vs. cooled semen, there were significant differences between T1 (22.3%) and T2 (43.7%) and between T3 (23.4%) and T4 (48.1%) (P < 0.05). In relation to BTS or ACP250 extenders, there were no significant differences between T1 and T3 and between T2 and T4 (P > 0.05). In conclusion, the ACP250 solution is an option for in vitro sperm capacitation in swine, specially with chilled extended semen.
This work was supported by FAPESP 01/11931-8 and ACP Biotechnology – CE.
252 COMPARISON OF TWO SPERM SEPARATION PRODUCTS FOR USE IN BOVINE IVF
Authors: Pryor J. , Romo S. , Varner D.D. , Hinrichs K. , Looney C.R.
Published: December 2004

In commercial bovine in vitro fertilization (IVF) companies, there is a continuous need to improve results. Efforts to maximize in vitro embryo production have included modifications in the use of sperm separation gradients. The development of commercially available sperm centrifugation gradients represents a new possibility of increasing the number of viable sperm that can be obtained from low concentration (fresh or frozen, sexed or unsexed) semen samples in order to improve the efficiency of the IVF system to make embryo production as efficient as possible. The objective of this study was to compare two different separation gradients, as follows: Group 1: Percoll (Sigma, St. Louis, MO, USA), in 45% and 90% gradients; Group 2: EquiPure (Nidacon, Gathenburg, Sweden), in top and bottom layers. Before and after separation, sperm were evaluated at 200× magnification for total motility, and then stained to assess viability at 400× with fast-green/eosin stain (Sigma). Sperm separation was performed using frozen/thawed semen from one bull. Semen was separated by centrifugation at 200g for 30 min in both density gradients. Results obtained from Groups 1 and 2 were compared by chi-square test. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 92 × 106, vs. 159 × 106 sperm/mL for EquiPure; P < 0.05) but resulted in higher motility (60% vs. 39%, respectively; P < 0.05) of separated sperm. Rates of live sperm cells were not significantly different between groups (69.5% vs. 70%, respectively; P > 0.1). These results indicate that the commercial separation medium EquiPure may be associated with higher sperm concentration levels but with lowered sperm motility when compared to Percoll for bovine sperm separation. However, Equipure provided similar percentages of live sperm when compared to Percoll, which is currently used in our laboratory.
284 KINETICS OF OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENT	OF PARTHENOGENETIC PORCINE EMBRYOS AFTER MEIOTIC INHIBITION WITH ROSCOVITINE
Authors: Kim D.H. , Kim S.W. , Im G.S. , Yang B.C. , Lee D.R. , Park H.S. , Hwang I.S. , S Seo J. , Yang B.S. , Chang W.K.
Published: December 2004

Maturation of mammalian oocytes is a very important process for subsequent embryo development after fertilization. Prolonged maturation time by meiotic inhibitors could be an effective method for improvement in the meiotic and developmental competence of mammalian oocytes. Roscovitine, a cyclin dependent kinase inhibitor, is known to specifically inhibit M-phase promoting factor (MPF) kinase activity and prevent the resumption of meiosis. The aim of this study was to examine the effect of roscovitine on the maturation and subsequent development of porcine oocytes. Ovaries were collected from slaughtered prepubertal gilts and COCs were aspirated from 2- to 5-mm antral follicles. In control, porcine cumulus oocyte complexes (COCs) were cultured in the maturation medium (TCM-199 supplemented with 0.3% BSA, 1 μg/mL FSH, 1 μg/mL LH, and 10 ng/mL EGF) for 44 h. In the experimental group, COCs were cultured in the inhibition medium (TCM-199 supplemented with 0.3% BSA and roscovitine) for 24 h, and then further cultured in the maturation medium for 44 h. Matured oocytes from both groups were activated by electrical pulse (1.2 kV/cm for 30 μs), and then cultured in PZM-3 medium for 6 days. Apoptotic cells in blastocysts were detected by TUNEL assay and total cell number was examined by propidium iodide (PI) counterstaining. Data were analyzed by chi-square and Student's t-test. The first experiment was conducted to determine the effect of roscovitine (0, 12.5, 25, 50, and 100 μM) on meiotic inhibition of GV oocytes. This effect was dose-dependent, and a concentration of 50 μM was sufficient to prevent meiotic resumption in 79.2% (76/96, 5 replicates) of the porcine oocytes after 24 h of culture when compared to 0 (15.4%, 15/97), 12.5 (32.1%, 36/112), 25 (57.4%, 54/94), and 100 μM (77.8%, 77/99). The second experiment was carried out to examine the kinetics of maturation of roscovitine-treated porcine oocytes. The concentration of roscovitine used was 50 μM. A total of 75.8% (50/66, 3 replicates) of roscovitine-treated oocytes reached metaphase II stage compared with 70.8% (46/65) of control. The third experiment was performed to compare embryo development between control and treated group after parthenogenetic activation. No differences (P > 0.05) were found between the control and the treated group in cleavage rate (77.2%, 132/171 vs. 68.0%, 115/169), blastocyst rate (26.9%, 46/171 vs. 17.8%, 30/169), and total (33.7 ± 12.4 vs. 35.1 ± 12.6) and apoptotic (2.2 ± 2.4 vs. 2.2 ± 1.2) cell number per blastocyst (4 replicates). The results suggest that roscovitine can be used to prolong maturation time of porcine oocytes without reducing meiotic maturation but also without significantly decreasing their subsequent developmental competence. Further studies are necessary to improve the developmental competence of porcine oocytes treated with roscovitine.
291 EFFECT OF DIFFERENT TRANSPORT TEMPERATURES (+4°C, +32°C) ON IN VITRO MATURATION OF OOCYTES COLLECTED FROM CATTLE AND SHEEP OVARIES
Authors: Ozdas O.B. , Tas M. , Cirit U. , Evecen M. , Demir K. , Bacinoglu S. , Ak K. , Ileri I.K.
Published: December 2004

At present, blastocyst rates in embryos obtained from in vitro maturation of oocytes, and their fertilization and culture, is still not at the desired level. One of the most important problems encountered in in vitro culture studies is seen in the maturation period of oocytes until they reach the fertilizable level. Transport time of the ovaries and, in particular, temperature of the transport medium used are among the factors affecting complete maturation. The aim of this study was to determine the effects of different transport temperatures (4°C, 32°C) of sheep and cattle ovaries on the in vitro maturation of oocytes. Two experimental groups were formed in the study. Sheep and cattle ovaries were put into saline solution at 32°C. The ovaries were transported at the same temperature (Group I) or at 4°C following a 10-min incubation at room temperature (Group II), in 2–4 h to the laboratory (n = 6). For each group, oocytes were collected from ovaries using the dissection method and selected oocytes were matured in their own group in 700 μL TCM-199 (supplemented with pyruvate, LH, FCS) for 23 h at a gas atmosphere of 5% CO2, 5% O2, 90% N2 and at 38.8°C. At the end of maturation, oocytes were cleansed from their cumulus oophorus cells and fixed in acetic acid-ethyl alcohol (1:3) for 48 h. The developmental stages until MII of oocytes stained with aceto-orcein were then examined under the phase contrast microscope. The chi-square test was used for statistical analysis (Table 1). While oocytes obtained from sheep ovaries transported at +32°C reached the MII stage at a faster rate compared to those at +4°C (P < 0.001), no statistically significant difference was observed between the maturation to the MII stage of oocytes obtained from cattle ovaries transported at +4°C and +32°C. As a result of this study, while it was established that cattle ovaries could be transported at both +4°C and +32°C and that there was no difference in oocyte maturation, a medium temperature of +4°C was determined to be unsuitable for transporting sheep ovaries.

Table 1.
Stages of development in sheep and cattle oocytes after 23 h of culture

This work was supported by Istanbul University.
314 FULL-TERM DEVELOPMENT OF RAT OOCYTES MICROINSEMINATED	WITH FREEZE-DRIED SPERMATOZOA
Authors: Hirabayashi M. , Kato M. , Hochi S.
Published: December 2004

Since freeze-dried spermatozoa can be stored at ambient or refrigerated temperature, the costs required for maintenance and shipping of spermatozoa can be reduced. To date, viable offspring in mice (Wakayama and Yanagimachi 1998 Nat. Biotech. 16, 639) and rabbits (Liu et al. 2004 Biol. Reprod. 70, 1776) have been produced by intracytoplasmic sperm injection (ICSI) using freeze-dried samples. The objectives of the present study were to examine whether freeze-dried rat spermatozoa can participate in full-term development by ICSI, and whether sonication prior to freeze-drying of the spermatozoa influences the offspring rate. Spermatozoa from cauda epididymides of Sprague-Dawley (SD) rats were collected in 10 mM TRIS/HCl buffer supplemented with 50 mM NaCl and 50 mM EGTA. A 2 × 3 factorial-designed experiment was conducted. The sperm suspensions were either sonicated for 10 s using a 10% power output from an ultrasonic cell disruptor or not sonicated. The sperm suspensions were then processed for freeze-thawing (100-μL sample in 1.0-mL cryotube was cooled in liquid nitrogen vapor, stored at -196°C for 48 h, and thawed in a 25°C water bath) and freeze-drying (100-μL sample in 1.5-mL polypropylene tube was frozen in liquid nitrogen for 20 s, lyophilized for 6 h by a freeze-drying apparatus, stored at 4°C for 48 h, and rehydrated with 100 μL ultra pure water), or were subjected to immediate use for ICSI. The sperm heads were microinjected into denuded SD oocytes using a piezo-driven micropipette 2–4 μm in diameter, as described previously (Hirabayashi et al. 2002 Transgenic Res. 11, 221). The presumptive zygotes were transferred into oviducts of pseudopregnant Wistar female rats. The in vivo developmental potential of rat oocytes microinseminated with fresh, freeze-thawed, and freeze-dried spermatozoa is shown in the table below. Viable rat offspring were produced in all six experimental groups, with the offspring rates at 2.5–35.0%. Sonication treatment of rat spermatozoa to induce membrane disruption and tail/midpiece dissociation from the heads was effective in increasing the offspring rate after ICSI. The positive effect of sperm sonication may be explained as facilitating decondensation of sperm heads by membrane disruption in the spontaneously activating rat oocytes. Thus, successful participation of freeze-dried rat spermatozoa into full-term development was demonstrated by applying the ICSI.

Table 1.
In vivo development of rat oocytes microinseminated with fresh, freeze-thawed, and freeze-dried spermatozoa
318 ENHANCEMENT OF FERTILIZATION BY DIGITONIN IN ROUND SPERMATID INJECTION
Authors: Kishigami S. , Mizutani E. , Wakayama S. , Wakayama T.
Published: December 2004

Reproductive technologies allow us to produce offspring using a variety of cells including sperm, spermatids, spermatocytes, somatic cells, and even parthenogenetic oocytes. In each of these technologies, failure of pronuclear formation after injection often prevents successful artificial reproduction. One of the possible causes is assumed to be that the breakage of the cytoplasmic membrane by simple pipetting is not enough to expose the nuclei to the ooplasm for pronuclear formation. To overcome this problem, we applied digitonin, a mild nonionic detergent, for the purpose of the permeabilization of cellular and nuclear membranes before injection. In this study, round spermatid cells in the mouse were used as a model because of their low pronuclear formation rate after injection. First, to examine the permeabilization of spermatids by digitonin, spermatid cells were incubated in CZB medium including 10 μg/mL of digitonin. Interestingly, the spermatids were lysed within 30 s after transfer but not other spermatogenic cells or somatic cells. Next, we conducted round spermatid injection (ROSI) using PVP including digitonin in a similar manner. Spermatids were picked up by injection pipette from spermatogenic cells suspended in a drop of PVP. These spermatids were transferred into another PVP drop including 1 μg/mL or 10 μg/mL of digitonin and left for 30 s. These digitonin-treated spermatids were then directly injected into previously activated oocytes. Six hours after injection, the fertilized oocytes were examined. Pronuclear formation rates were calculated as a proportion of oocytes with two pronuclei as well as one second polar body to total oocytes with one second polar body (Table 1). After digitonin treatment, fertilization rates significantly increased compared with ROSI without digitonin (Table 1). Further, these fertilized oocytes developed into blastocysts in vitro at comparable or higher rates. To further elucidate the effects of digitonin pretreatment on in vivo development, embryos were transferred into surrogate mothers 24 h after injection for offspring production. Although it is preliminary, we succeeded in the delivery of pups after ROSI with digitonin pretreatment (8 pups out of 14 transferred embryos). Thus, digitonin pretreatment is suggested to improve the success rate of ROSI.

Table 1.
Fertilization and in vitro development after ROSI with digitonin
339 ULTRASOUND GUIDED OVUM PICK UP (OPU) IN PREPUBERTAL SWAMP BUFFALO USING THREE DIFFERENT VACUUM PRESSURES
Authors: Techakumphu M. , Promdireg A. , Phutikanit N. , Singlor J. , Thongjan S. , Onwan N. , Na-Chiengmai A.
Published: December 2004

Our group successfully developed ovum pick-up in prepubertal swamp buffalo, however the quality of the oocytes that were collected was poor especially those without a cumulus mass (Techakumphu et al. 2003 Theriogenology 61, 1705–1711). The vacuum pressure used for oocyte collection was one of the factors influencing oocyte quality (Bols et al. 1997 Theriogenology 47, 1221–1236). The objective of this study was to compare the effect of three vacuum pressures on both the recovery rate and oocyte quality in prepubertal swamp buffaloes. Oocyte recovery and oocyte quality were using different groups of aspiration vacuum pressures. The maturation stages of the recovered oocytes were immediately assessed by fixation and rapid staining with basic carbol fuchsin. Twelve prepubertal calves, aged 1.5 yrs were a total of 180 mg FSH given, twice a day, in divided doses over 3 d (40/40, 30/30, 20/20). The animals were randomized into 3 groups, according to the different vacumm pressures, 100 (n = 8), 80 (n = 8) and 60 mmHg (n = 8). Two sets of treatments, carried out, with a 2 month interval between them. The oocyte recovery rates using 100 and 80 mmHg, were not different at 78.4% (29/37) and 83.6% (61/73). The 60 mmHg gave a lower rate, 65.7% (23/35) which was statistically different from the 80 mmHg group (P < 0.05). The oocytes recovered per donor showed no significant difference among the groups; 5.8 ± 4.9 for 100 mmHg, 7.6 ± 8.6 for 80 mmHg and 3.3 ± 2.1 for 60 mmHg respectively. The percentage of cumulus-oocyte complex (COC), single layered+partial cumulus oocytes (S + P) and expanded cumulus oocytes (EXP) showed no differences for any of the pressures, being 79.3, 65.5 and 82% of the total recovered. The experiment showed that follicles with a size of 2–6 mm were dominant after FSH treatment, being around 80% of the total number. Furthermore, the maturation stages of these oocytes were at prophase I and metaphase I. In conclusion, the vacuum pressure used for the oocyte retrieval technique influenced the recovery rate but not the oocyte quality.
This study was supported by Rajadapisek Sompoj Fund, Chulalongkorn University year 2002. AP was PhD candidate under Royal Golden Jubilee, Thailand Research Fund.
36 IMPROVING THE APPLICATION OF NUCLEAR TRANSFER FOR PRODUCING NON-DOMESTIC FELIDS
Authors: Gomez M.C. , Pope C.E. , Lyons L. , Cole A. , Lopez M. , Dumas C. , Dresser B.L.
Published: December 2004

One of the most remarkable aspects of somatic cell nuclear transfer (NT) is the possibility of avoiding extinction when there are few remaining animals of a specific felid population. Previously, we produced live male African Wildcat (AWC; Felis lybica) cloned kittens using inter-species nuclear transfer (Gomez et al. 2004 Cloning and Stem Cells 6, 217–228). The production of females is a primary objective of most breeding programs. Therefore, the purpose of the present study was to determine (1) if we could produce live female AWC cloned kittens at a proportion similar to that previously demonstrated with males, and (2) if our inter-species NT technique used to produce AWC is applicable to in vitro production of another non-domestic felid species. Specifically, we evaluated the in vivo developmental competence of NT embryos derived by fusion of Black footed cat (BFC, Felis nigripes) and AWC fibroblasts with domestic cat (DSH, Felis catus) cytoplasts, after transfer into domestic cat recipients. Fibroblast cell lines were established from skin biopsies of BFC (6-year-old), and AWC (12-year-old) adult females. After at least three passages, cells were serum-starved for 5 days and injected into the perivitelline space of enucleated domestic cat oocytes. Fusion of cell-cytoplast couplets was induced by applying a 3-s AC pre-pulse of 20 V, 1 MHz, followed by two 30-μs DC pulses of 240 V/mm. Fused couplets were activated 2 to 3 h after fusion by exposure to two 60 μsec DC pulses of 120 V/mm, followed by 4 h incubation with 10 μg/mL cycloheximide and 5 μg/mL cytochalasin B. Reconstructed BFC (n = 16) and AWC (n = 536) NT Day 1 embryos were transferred by laparoscopy into the oviducts of 1 and 12 gonadotrophin-treated DSH recipients, respectively, on Day 1 after induced ovulation. Pregnancy was assessed by ultrasonography on Day 22. One cat (100%) receiving BFC NT embryos and 5 (41.6%) cats receiving AWC NT embryos became pregnant. Twenty-three AWC cloned embryos implanted and 11 kittens were born. Three BFC NT embryos implanted and the pregnancy is currently ongoing. AWC cloned kittens were phenotypically and genetically identical to their somatic cell donor. Their clonal identity was assessed by multiplex PCR amplification of 20 microsatellite markers, including seven markers that are known to be on the X chromosome.
In summary, these results indicate that female AWC cloned kittens can be produced and BFC pregnancy can be established in domestic cat recipients. The embryo implantation rate and viability of AWC female cloned embryos was higher than that observed after the transfer of AWC male cloned embryos. The difference may be due to improvements in the NT procedure, rather than to differences in the sex of the cell lines.

Table 1.
Implantation rate and fetal survival to term of AWC and BFC NT embryos in pregnant domestic cat recipients