bluuebunny/crossref_metadata_2025 huggingface dataset:
33 USE OF ADULT MESENCHYMAL STEM CELLS FOR CLONING PIGS
Authors: Faast R. , Beebe L. F. S. , Harrison S. J. , McIlfatrick S. M. , Ashman R. J. , Nottle M. B.
Published: December 2006

Current cloning efficiencies are relatively low and there is evidence to suggest that a less differentiated cell type can increase these (Jaenisch et al. 2002 Cloning and Stem Cells 4, 389-396). As part of an ongoing study that aims to develop cloning as a breeding tool for the pig industry, we examined whether mesenchymal stem cells (MSCs) isolated from bone marrow (Experiment 1) and blood (Experiment 2) could increase cloning efficiencies compared with fibroblasts isolated from ear tissue of live animals. MSCs were isolated from the femurs and blood of two pigs over a Histopaque-1077 density gradient. Cells were seeded and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics/antimycotics. Blood MSCs were plated onto fibronectin-coated dishes (Faast and Nottle, Proc. Australian Health Med. Res. Congress 2004, abst. 1272). Cells from passages 2-4 were used for nuclear transfer by means of a fusion before being subjected to the activation protocol described previously (Harrison et al. 2004 Cloning and Stem Cells 6, 327-331). Embryos were cultured in a modified NCSU23 for 7 days. In Experiment 1 there was no difference in the number of fused embryos that cleaved in the bone marrow MSC and fibroblast groups (374/447; 84% vs. 370/446; 83%, respectively; 9 replicates). The number of embryos that developed to the blastocyst stage by day 7 was significantly higher in the bone marrow MSC group compared with the fibroblast group (136/447; 30% vs. 79/446; 18%, respectively, Chi square test; P < 0.01). In Experiment 2 there was no difference in the percentage of embryos that cleaved in the blood MSC and fibroblast groups (287/375; 77% vs. 275/347; 79%, respectively; 8 replicates). The number of embryos that developed to the blastocyst stage by day 7 was also similar between the blood MSC and fibroblast groups (67/375; 18% vs. 63/347; 18%, respectively). In conclusion, our results suggest that bone marrow MSCs may be a more efficient cell type, compared with fibroblasts, for the cloning of live animals.
330 THE PROTEIN EXPRESSION PROFILE (SECRETOME) OF INDIVIDUAL CUMULUS - OOCYTE COMPLEXES DURING IVM IN THE MOUSE IS AFFECTED BY FSH
Authors: Katz-Jaffe M. G. , Sheehan C. , Schoolcraft W. B. , Gardner D. K.
Published: December 2006

Studies of the protein expression profile into the surrounding medium (secretome) of in vitro-matured cumulus-oocyte complexes (COCs) have the potential to elucidate biochemical pathways involved in oogenesis, including the complex dialogue between the oocyte and its supporting cells. The understanding of these processes should assist in improving IVM success and fertility outcome, as early embryo development reflects the quality of the oocyte and its cumulus cells. Through the analysis of the individual COC secretome, we have investigated the effects of adding follicle stimulating hormone (FSH) to a defined maturation medium during IVM. COCs were collected from 3-week-old female mice (C57BL/6 � CBA) 48 h post-pregnant mare serum gonadotropin (PMSG) (5/iu) injection. Individual COCs were cultured in 5-�L drops of a defined maturation medium (0.25 mg/mL recombinant albumin) with the addition of 0, 2, 20, or 200 ng/mL FSH, under oil for 17 h. Oocytes were denuded and maturity recorded. Each microdrop of media (n = 8 oocytes per group) was collected, processed through an optimized series of buffers and washes prior to analysis by time-of-flight mass spectrometry (TOF-MS). Differential protein expression profiles were obtained from the secretome of individual COCs producing MII oocytes after maturation in differing doses of FSH. Statistical analysis revealed significant differences observed across 10 proteins/biomarkers with mass-to-charge (m/z) ratios ranging from 2.7 to 6 kDa (Mann-Whitney non-parametric test; P < 0.05). In addition, hierarchical and horizontal clustering analysis identified unique clusters of both up-regulated and down-regulated proteins/biomarkers within the m/z range of 2 to 18 kDa in the 2 ng/mL FSH group. Several of the individual COCs from the 20 ng/mL FSH group were also clustered alongside the 2 ng/mL group with similar protein expression profiles. In contrast, COCs cultured in the presence of 0 ng/mL and 200 ng/mL FSH were observed to cluster as a separate branch with distinctly different protein expression profiles. This study has determined for the first time the secretome profiles of individual COCs after IVM. These data have shown that the FSH dose in a defined maturation medium affects the secretome of an individual COC. Further investigation is currently underway to characterize these protein differences. The development of this proteomics approach will assist in revealing the intricate cellular function of an individual COC and elucidate critical pathways involved in mammalian oocyte maturation.
335 DEVELOPMENTAL ABILITY OF PIG OOCYTES TREATED WITH EGF DURING IN VITRO MATURATION
Authors: Lee H. T. , Uhm S. J. , Han D. W. , Lee S.-H. , Kim A. R. , Park S. J. , Gupta M. K. , Park C. K. , Chung H. M. , Kim Y. B. , Chung K. S.
Published: December 2006

Epidermal growth factor (EGF) is a major follicular factor affecting maturation of oocyte in many species. The insulin-like growth factor II (IGF2) gene is an imprinted gene in embryonic development that functions primarily as a regulator of cell growth and differentiation. Thus, this study examined the maturation and developmental ability of in vitro-fertilized (IVF) pig immature oocytes cultured in maturation medium supplemented with EGF. The blastocysts derived from these oocytes were further examined for the expression level of IGF2 as a cell survival activity. Pig immature oocytes were cultured in TCM-199 medium (with no supplement) with or without 10 ng/mL EGF for 42–14 h, and then matured oocytes were co-incubated with 5 × 105 sperm/mL in modified Tris-buffered medium containing 1 mM caffeine sodium benzonate and 0.1% bovine serum albumin (BSA) for 6 h for IVF. Subsequently, embryos were cultured in 50 μL of NCSU-13 containing 0.4% BSA for 7 days at 39°C in a humidified atmosphere of 5% CO2 in air. Total cell numbers in blastocysts were examined by fluorescence staining with Hoescht 33342, and the expression level of IGF2 was analyzed with a fluorescence-monitored quantitative real-time reverse transcriptase-polymerase chain reaction method. We found that pig oocytes matured with EGF showed significant improvement of their development ability (Table 1). Presence of EGF in TCM-199 medium significantly increased (P < 0.05) the rates of maturation, sperm penetration, male pronucleus (MPN) formation, cleavage, and blastocyst formation. Furthermore, blastocysts derived from oocytes cultured with EGF were 24-fold higher in the relative expression level of IGF2 than those without EGF. Therefore, these data suggest that pig oocytes matured in medium supplemented with EGF increases the developmental ability and cell viability during cell divisions following IVF. In conclusion, EGF may increase cytoplasmic as well as nuclear maturation of pig immature oocytes.

Table 1.
Improvement of developmental ability of pig oocytes matured with EGF

This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea.
336 SHEEP OOCYTES MATURED IN A SIMPLEX MEDIUM (HECM-1), AN ANSWER TO IN VITRO FERTILIZATION
Authors: Navarro-Maldonado C. , Ducolomb-Ramirez Y. , Galindo-Rodriguez A. , Rosado-Garcia A.
Published: December 2006

In vitro maturation and in vitro fertilization (IVM and IVF) of mammalian oocytes still show unsatisfactory results when applied to the study of embryo development. This is probably due to inadequate information about the use of media components and supplements for oocyte maturation and to a discrepancy between results obtained by focusing strictly on oocyte maturation and those that are focused on IVF. A conventional medium that provides adequate results in studies of oocyte maturation (TCM-199) contains hypoxanthine, phosphate ions, and glucose, all known to inhibit embryo development in vitro in some species. In contrast, it has been shown that a simpler medium (HECM-9) increases embryo development in bovine although its use for oocyte maturation has not been defined. This medium contains taurine (an amino acid that reduces intracellular peroxide content) and is supplemented with polyvinyl alcohol (PVA) instead of using protein components, making it a simple defined medium that reduces variability in embryo development. Adding sodium panthothenate to media also confers cell protection against reactive oxygen species. Finally, supplements such as epidermal growth factor (EGF) increase the number of oocytes that complete maturation (Metaphase II, MII) and facilitate embryo development. An adequate combination of our knowledge about in vitro maturation and fertilization of oocytes, together with the requirements for embryo development, is important for the preparation of culture media to study regulatory mechanisms for embryo development and to increase the number of viable and normal term individuals. In this study we compared the effects of HECM-9 (containing panthothenate) vs. TCM-199 (both media supplemented with PVA, EGF, and FSH/LH) on the integrated processes involving IVM and IVF. No significant differences were found between the results obtained with these media in relation to oocyte maturation (65% MII for HECM-9 vs. 71% for TCM-199); however, those oocytes matured in HECM-9 showed a highly significant difference in in vitro fertilization using a conventional IVF medium (SOFm) (25% in HECM-9 vs. 6% in TCM-199). Maturation results were relatively low but in accordance with those reported by other groups, whereas IVF results are lower than those reported in the literature, perhaps because we have been using frozen and thawed samples and do not have complete control over the sperm quality. At present, we are extending our investigation using fresh semen samples.
339 DIFFERENTIAL MECHANISM OF LEPTIN ACTION IMPROVING NUCLEAR MATURATION AND DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES
Authors: Paula-Lopes F. F. , Boelhauve M. , Habermann F. , Sinowatz F. , Wolf E.
Published: December 2006

Leptin is a pleiotrophic peptide that has been implicated in the events associated with oocyte maturation and acquisition of developmental competence. Previous studies indicated that leptin supplementation during oocyte maturation has long-term effects, increasing blastocyst development and reducing the proportion of TUNEL-positive cells per blastocyst. Moreover, blastocysts derived from leptin-treated oocytes showed increased BIRC4 and reduced BAX expression (Boelhauve et al. 2005 Biol. Reprod. 73, 737-744). The objective of the current study was to determine the mechanism of leptin action during bovine oocyte maturation. In the first series of experiments cumulus-oocyte complexes (COCs) were matured in serum-free medium containing 0, 1, or 10 ng/mL leptin or 10% estrous cow serum (ECS) as positive control. Leptin reduced the proportion of cumulus cells undergoing cell death through apoptosis as determined by TUNEL staining (6.8 � 0.4, 1.8 � 0.4, 1.5 � 0.4, and 6.3 � 0.5% for 0, 1, or 10 ng/mL leptin or 10% ECS, respectively; P < 0.0001), but had no effect on the frequency of apoptotic oocytes. Nuclear maturation was also enhanced by leptin. The proportion of oocytes with extruded polar bodies (64.0 � 2.9, 75.7 � 2.9, 73.8 � 2.9, and 63.3 � 2.9% for 0, 1, or 10 ng/mL leptin or 10% ECS; P < 0.05) and the proportion of DAPI-stained metaphase II oocytes (72.9 � 2.9, 90.5 � 2.9, 85.8 � 2.9, and 82.0 � 2.9% for 0, 1, or 10 ng/mL leptin or 10% ECS; P < 0.05) were increased by 1 and 10 ng/mL leptin. There was no effect of ECS in any of the parameters examined. A second series of experiments tested whether the maturation-promoting activity of leptin was mediated by cumulus cells. Denuded oocytes (DO) and COCs were matured in serum-free medium containing 0 or 10 ng/mL leptin. The percentage of oocytes with extruded polar bodies (COCs: 59.1 � 3.7% vs. 75.7 � 3.7%, and DO: 45.5 � 3.7% vs. 54.8 � 3.7% for 0 or 10 ng/mL leptin; P < 0.01) was increased by leptin regardless of cumulus cells. Even though leptin did not affect cleavage rate, it increased blastocyst development. The proportion of COCs that developed to the blastocyst stage increased from 22.3 � 4.6% in the control group to 35.2 � 4.1% in the leptin-treated group. On the other hand, DO matured without cumulus cells did not acquire developmental competency, and this was not reversed by leptin supplementation (1.0 � 5.0% vs. 1.0 � 4.7% for 0 or 10 ng/mL leptin; P < 0.05; leptin � oocyte interaction P < 0.05). In conclusion, leptin enhanced oocyte maturation by acting directly in the oocyte and indirectly through cumulus cells. This suggests that leptin modulates the release of cumulus-derived factors secreted in the oocyte through gap junction coupling and in the extracellular environment.
34 DEVELOPMENTAL ABILITY OF ZONA-FREE PORCINE CLONED EMBRYOS RECONSTRUCTED BY SOMATIC CELLS AND CENTRIFUGED CYTOPLASTS
Authors: Fahrudin M. , Kikuchi K. , Karja N. W. K. , Ozawa M. , Somfai T. , Noguchi J. , Kaneko H. , Nagai T.
Published: December 2006

The combination of bulk enucleation and zona-free cloning will offer simplification of the conventional nuclear transfer technique. A bulk enucleation method such as enucleation by centrifugation could reduce the time of manipulation that is necessary for removing genetic materials from the oocytes. The present study was conducted to examine the ability of cytoplasts obtained by centrifugation of zona-free in vitro maturation (IVM) porcine oocytes to support remodeling of the somatic cell nucleus and the subsequent development in vitro of somatic cell nuclear transferred (SCNT) embryos. A primary culture of cumulus cells was used as the source of donor cells, and recipient cytoplasts were derived from IVM oocytes that were cultured for 48 h, denuded of zonae pellucidae, and subjected to gradient centrifugation in Percoll solution to separate the ooplasm into fragments. Fragments were stained with Hoechst-33342 and cytoplasts were selected under an epifluorescence microscope. Then two or three cytoplasts were aggregated with a single somatic cell in phytohemagglutinin solution (500 �g/mL). Fusion between somatic cell and cytoplasts was induced by two DC pulses of 1.5 kV/cm for 20 �s, and activation was accomplished by two DC pulses of 0.8 kV/cm for 30 �s at 1 h after fusion in 0.28 M mannitol solution supplemented with 0.05 mM CaCl2 and 0.1 mM MgSO4. The resultant embryos were transferred to a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) and cultured in glucose-free NCSU-37 containing 4 mg/mL BSA supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2; from Days 2 to 7 they were cultured in NCSU-37 supplemented with 5.55 mM {D}-glucose and 5% FCS. Some of the reconstructed embryos were fixed at 1, 10, and 24 h after activation and stained with 1% (w/v) orcein to display the morphology of the transferred somatic nuclei. The results showed that 53.6% (30/56) of the SCNT embryos underwent premature chromosome condensation at 1 h, 90.9% (50/55) formed pseudo-pronuclei at 10 h, and 21% (19/90) of them cleaved to the two-cell stage at 24 h after the activation. The development to the blastocyst stage of the embryos that were reconstructed by quartet cells (three cytoplasts and one somatic cell; 8.9%, 10/112) was significantly higher (P < 0.05) than that of the triplet ones (2.2%, 3/139). However, these blastocyst rates were significantly lower (P < 0.05) than the blastocyst development rate of parthenogenetic embryos with the intact zonae pellucidae (28.3%, 17/60). These results suggest that (1) cytoplasts obtained by gradient centrifugation could support reprogramming of somatic cells and in vitro development of SCNT embryos to the blastocyst stage, and (2) the volume of cytoplasts apparently affects their in vitro development in pigs.
342 DEVELOPMENT OF PIG OOCYTES MATURED AND FERTILIZED IN CHEMICALLY DEFINED MEDIUM CONTAINING PVA, PVP, AND PFF
Authors: Seok H.-B. , Kim I.-D.
Published: December 2006

This study was conducted to develop a serum-free, defined medium for IVM of pig oocytes. TCM-199 with supplemented with polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and porcine follicular fluid (pFF) were used as basal medium. The effects of the these additives on the rates of maturity and development during in vitro fertilization and in vitro culture were examined and subsequently considered as possibile substitutes for bovine serum albumin (BSA). In an experiment that contained nine replicates, pig oocytes from abattoir-derived ovaries were matured in TCM-199 containing pyruvic acid, gentamycin, L-cysteine, �-estradiol, and FSH, and supplemented with 0.1% PVA, 0.1% PVP, 10% pFF, or 0.1% BSA (control) for 44 h at 39�C, 5% CO2. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate, and the same indicated supplements for 2 days. Maturation, fertilization, and morula and blastocyst formation were examined at 44 h after IVM and at 12, 48, and 120 h after IVF, respectively. Morphology of oocytes and cleaved cells was desplayed by staining with Hoechst 33342. Maturation rates of pig oocytes in IVM media containing PVA (82.4%), pFF (89.4%), or BSA (90.0%) were significantly higher (P < 0.05) than that with PVP (78.6%). Cleavage rates after IVF with PVP (64%) was significantly lower (P < 0.05) than those in PVA (73%), pFF (77%), or BSA (73%) supplements. In vitro development rates to morulae and blastocysts with PVP (54%) were also significantly lower (P < 0.05) than those with the supplements of PVA (63%), pFF (69%), or BSA (65%). In comparison of maturation and fertilization rates of pig oocytes in each of the supplements, the maturity rates with PVA (82.4%), pFF (89.4%), or BSA (90.0%) were significantly lower (P < 0.05) than that with PVP (72.4%) and the fertilization rates with pFF (87.1%) or BSA (89.1%) were significantly higher (P < 0.05) than those with PVA (78.0%) or PVP (70.6%). It may be concluded that PVA and pFF can be substituted for BSA in medium for culturing pig oocytes, and that PVP would not be an acceptable substitute for BSA.
344 PENTOSE PHOSPHATE PATHWAY ACTIVITY CONTROLS NUCLEAR MATURATION OF PORCINE OOCYTES
Authors: Tubman L. , Peter A. , Krisher R.
Published: December 2006

Diphenyleneiodonium (DPI), an inhibitor of the pentose phosphate pathway (PPP), arrests nuclear maturation of porcine oocytes. This inhibition is reversed using products or cofactors of PPP such as nicotinamide adenine dinucleotide phosphate (NADP), phosphoribose diphosphate (PRPP), and ribose-5-phosphate (R5P). The objective of this study was to determine the relationship between DPI-mediated meiotic inhibition, reversal of this inhibition, and metabolism of in vitro-matured (IVM) porcine oocytes. Oocytes were aspirated, searched, and selected in the presence of DPI, with the exception of control oocytes. Oocytes were then matured in one of five treatments for 40 h in 7% CO2 in air at 39°C in defined Purdue Porcine Medium for maturation (PPMmat). Treatments included control, 50 nM DPI (DPI), DPI + 5 mM NADP (NADP), DPI + 12.5 mM PRPP (PRPP), and DPI + 10 mM R5P (R5P). Following IVM, oocytes were denuded by vortexing. Glycolysis and PPP activities were measured in 4 μL hanging drops containing labeled glucose (0.0125 mM 5-3H glucose and 0.482 mM 1-14C glucose, respectively) for 3 h in 6% CO2. Oocytes were then individually fixed in a 3:2:1 solution of ethanol:acetic acid:chloroform and stained with aceto-orcein for determination of meiotic stage (germinal vesicle = 1 through metaphase II = 7). Data were analyzed using one-way ANOVA. The use of DPI inhibited PPP and nuclear maturation; additionally glycolysis was decreased by DPI compared to control. Addition of NADP and PRPP increased both metabolic pathways and nuclear maturation compared to DPI. R5P restored glycolysis and nuclear maturation to control levels, and PPP to above the control level. There were no significant differences among meiotic stages relative to glycolytic activity. PPP activity was significantly different (values with different superscripts; P < 0.05) among oocytes of different meiotic stages (germinal vesicle = 0.24 ± 0.03ad, germinal vesicle breakdown = 0.40 ± 0.05bcde, condensed chromatin = 0.44 ± 0.05bcd, metaphase I = 0.45 ± 0.12abcd, anaphase = 0.76 ± 0.50abcde, telophase = 0.92 ± 0.17be, metaphase II = 0.74 ± 0.08be). Percentages of oocytes reaching MII were 43.48 (control), 2.08 (DPI), 28.30 (NADP), 18.18 (PRPP), and 46.94 (R5P). These results demonstrate that the PPP is a critical control mechanism for nuclear maturation of porcine oocytes, as inhibition of this metabolic pathway resulted in arrest of nuclear maturation. Addition of PPP cofactors or end products to the arresting medium led to reversal of inhibition as demonstrated by restoration of PPP activity resulting in nuclear maturation.

Table 1.
Meiotic stage, glycolysis, and pentose phosphate pathway activity after in vitro maturation of porcine oocytes
345 INFLUENCE OF DIFFERENT MATURATION MEDIA ON IN VITRO SWINE EMBRYO DEVELOPMENT
Authors: Visintin J. A. , Lima A. S. , Nascimento A. B. , Nicacio A. C. , Marques M. G. , Assumpção M. E. O. A.
Published: December 2006

The aim of this work was to study the influence of maturation media NCSU23, Whitten, and TCM199 on in vitro development of swine embryos. Oocytes from slaughtered gilt ovaries were in vitro-matured (IVM) with NCSU23 (n = 148), Whitten (n = 151), and TCM199 (n = 170) media for 44 h. After IVM, all oocytes were fertilized in mTBM medium for 6 h and cultured in NCSU23 with 0.4% BSA for 5 days. After Day 5, all embryos were cultured in NCSU23 supplemented with 20% FCS for 4 days. Cleavage, blastocyst, and hatching rates were evaluated. Cleavage rates were 29% for NCSU23, 35.8% for Whitten, and 42.3% for TCM199 maturation media, with no significant difference among media (P > 0.05). Blastocyst rates were similar (P > 0.05), 18.9%, 21.8%, and 20%, respectively, for NCSU23, Whitten and TCM199 maturation media. Hatching rates were 16.1%, 34.3%, and 32.3%, respectively, for NCSU23, Whitten, and TCM199 maturation media, with no significant differences (P > 0.05). In conclusion, all three maturation media supported IVF, allowing embryo development from cleavage until blastocyst hatching.
349 IMPROVING THE FUNCTIONALITY OF STORED FLOW CYTOMETRICALLY SEX-SORTED BOAR SPERMATOZOA: SEDIMENTATION VS. CENTRIFUGATION
Authors: Parrilla I. , Vazquez J. M. , Gil M. A. , Garcia E. M. , Caballero I. , Roca J. , Martinez E. A.
Published: December 2006

Centrifugation has been the routine procedure used to concentrate the highly diluted spermatozoa obtained after sorting. However, this harmful procedure has a detrimental effect on the lifespan of the spermatozoa, particularly when a short-term storage period is required to perform the inseminations in the farms. The aim of the study was to evaluate the procedure of concentration by sedimentation of the flow cytometrically sex-sorted boar spermatozoa after a 24-h storage period, in terms of sperm functionality and recovery efficiency. Spermatozoa were sex-sorted by flow cytometry (Johnson and Welch 1999 Theriogenology 52, 1323-1342) and collected in 50-mL tubes containing 5 mL of Beltsville thawing solution (BTS) with 10% seminal plasma (SP). The collected samples were split into two aliquots. The first aliquot was concentrated by centrifugation (800g/5 min) just after sorting and stored at 17�C for 24 h at a concentration of 6 � 106 sperm/mL. The second aliquot was directly stored (0.5 � 106 sperm/mL) at 17�C for 24 h. In this case, after storage the supernatant was discarded and the pellet obtained by sedimentation was adjusted to 6 � 106 sperm/mL. For assessment of the sperm functionality after the storage, motility and viability of the spermatozoa were analyzed using a CASA system (Sperm Class Analyzer; Microptic, Barcelona, Spain) and a EPICS XL flow cytometer (Coulter Corporation, Inc., Miami, FL, USA) after the sperm were stained with propidium iodide and SYBR-14, respectively. For assessment of the recovery efficiency, the total number of spermatozoa in each sample was counted. The results are expressed as percentages of five replicates. Statistical analysis was performed using a �2 test. Percentages of motile and viable spermatozoa just after sorting were 80% and 83.3%, respectively. After storage at 17�C for 24 h, the percentage of viable spermatozoa was significantly higher (P < 0.05) in settled spermatozoa (82.4%) compared to centrifuged spermatozoa (63.3%). Similarly, the percentage of motile spermatozoa was significantly higher (P < 0.05) in settled spermatozoa (78.3%) compared to centrifuged spermatozoa (58.3%), as was the percentage of progressive motility (63.6% vs. 35.5%, respectively; P < 0.05). Interestingly, the percentage of cell recovery was significantly higher (P < 0.05) after sedimentation (80% of the sorted population) than after centrifugation (60.3% of the sorted population). The results show that sedimentation is more beneficial than centrifugation when short-term storage of sex-sorted boar sperm is required.
This work was supported by CDTI and Fundaci�n Seneca.
353 IN VITRO FERTILIZATION WITH FLOW-SORTED BUFFALO SPERM
Authors: Zhang M. , Liang X. W. , Lu Y. Q. , Lu K. H.
Published: December 2006

Flow cytometrically sorted X and Y sperm have been successfully used for IVF and the production of offspring in cattle and sheep (Maxwell et al. 2004 Anim. Reprod. Sci. 82–83, 79–95).The objective of this study was to test the feasibility of flow sorted buffalo sperm used in IVF systems and to establish a suitable IVF system for sorted buffalo sperm. Oocytes were aspirated from 2–6 mm follicles on the buffalo ovaries from a slaughterhouse and matured for 22–24 h in a 1-mL dish containing TCM199 + 10% OCS + 3% BFF (bovine folliciular fluid) + hormones at 38.5°C, 5% CO2 in air with maximum humidity. Semen was sorted by a flow-sorter (Clontech, Mountain View, CA, USA) into X- and Y-chromosome bearing sperm at 90% accuracy and stored at 4°C for later use with IVF. Sorted sperm were prepared for IVF by centrifugation (700g, 20 min) through a Percoll gradient (90%:45%), and washed (centrifugation at 700g, 5 min) in mTALP-BSA. For IVF, groups of 10–15 oocytes were transferred to 40-μL drops of mTALP-BSA and incubated with motile sperm at a concentration of 2 × 106 sperm mL−1 in each fertilization drop for 8–10 h under mineral oil. Presumptive zygotes were cultured until Day 8 in 25-µL drops of TCM–199 supplemented with 0.33 mM pyruvate and 10% NCS with granulosa cells at 38.5°C under 5% CO2 in air. Cleavage and blastocyst rates per oocyte insemination were recorded on Day 2 and Days 6–8 after insemination, respectively. Data were analyzed by ANOVA procedures with replicates and treatments in the model. There were significant differences in cleavage rate (42.23% vs. 52.28%) and blastocyst rate (20.62% vs. 27.67%) between sorted and unsorted sperm, respectively (Table 1). There were no significant differences in the proportions of blastocyst development rates on Days 6, 7, or 8 after insemination with sorted and unsorted sperm. The results indicate that sorted buffalo sperm from two bulls have been successfully used in an IVF system to produce sex-controlled embryos.

Table 1.
Cleavage and blastocyst rates with different sperm types

This research was supported by grants from the National Natural Science Foundation of China (30360073) and the Guangxi Department of Science and Technology (0330004–13). The authors (M. Z. and X.W. L.) contributed equally to this work.
355 IMPROVEMENT OF CUMULUS-FREE IN VITRO MATURATION AND ITS APPLICATION TO MICROINSEMINATION WITH PRIMARY SPERMATOCYTES IN MICE
Authors: Miki H. , Ogonuki N. , Inoue K. , Baba T. , Ogura A.
Published: December 2006

Manipulation of immature oocytes requires removal of cumulus cells, which may compromise ooplasmic maturation. This problem can be overcome by cytoplasm exchange at the MII stage, but this requires a second micromanipulative step. This study was undertaken to determine whether cumulus-free IVM can be improved by modifying IVM medium. We assessed the quality of oocytes by several cytochemical parameters, and by the subsequent development of embryos constructed by IVF, parthenogenetic activation, or microinsemination using primary spermatocytes. GV-stage oocytes were collected from superovulated B6D2F1 females. After freeing the oocytes from cumulus cells, they were subjected to IVM in either �MEM or TYH media, or a 1:1 mixture of the two (termed TaM). IVM was performed under chemically semidefined conditions by using BSA as the only protein source. Oocytes that reached the MII stage were analyzed for distribution of mitochondria, activity of the catalytic p34cdc2 kinase subunit of the maturation promoting factor (MPF), and chromosomal alignment within the MII spindle. Microinsemination with primary spermatocytes was performed in oocytes at the MI stage as described previously (Ogura et al. 1998 PNAS 95, 5611-5615), but without MII chromosome exchange. TYH and �MEM media had opposing effects on several parameters. TYH resulted in a better maturation rate (181/196, 92.3%) than �MEM (184/257, 71.6%). In contrast, �MEM supported better embryo development to the morula/blastocyst stage than TYH following IVF (93.3% vs. 76.5%) or parthenogenetic activation (82.4% vs. 60.4%). Mitochondrial distribution in MII oocytes was diffuse in �MEM, but aggregated in TYH. MPF activity in MII oocytes was significantly higher in TYH than in �MEM (P < 0.05). Oocytes derived from TaM had intermediate characteristics between TYH- and �MEM-oocytes and essentially resembled in vivo-matured oocytes, with the mitochondrial distribution pattern being most typically intermediate. By using TaM, 23.8% of GV oocytes developed into full-term fetuses following IVF and embryo transfer (compared to TYH: 6.3%, and �MEM: 18.2%). This IVM system was then applied to oocytes to be injected with spermatocytes when in the MI stage. Approximately 60% of the oocytes survived injection. After 127 two-cell embryos were transfered, two normal-looking offspring were obtained. This is the first successful birth of pups derived from fertilization with spermatocytes by single injection, without serial nuclear transfer. Chromosomal analysis at MII revealed that the low developmental efficiency after embryo transfer could be attributed mainly to premature sister chromatid separation, as reported previously (Ogura et al. 1998). These results demonstrate that optimization of IVM media can improve the quality of cumulus-free IVM oocytes in mice, although it cannot overcome defects in the kinetics of injected spermatocyte chromosomes.
358 MICROINSEMINATION USING MALE GERM CELLS FROM EPIDIDYMIDES AND TESTES STORED IN FREEZERS WITHOUT CRYOPROTECTANT
Authors: Ogonuki N. , Mochida K. , Shinmen A. , Ohkawa M. , Miki H. , Inoue K. , Fray M. , Moriwaki K. , Obata Y. , Ogura A.
Published: December 2006

Cryopreservation of male germ cells is a strategy for the conservation of species and strains valuable to biomedical researchers. However, to minimize damage that may occur during freezing and thawing, complex cryopreservation protocols that have been optimized for the stage and species of the male germ cell are usually employed. This study was undertaken to see whether mouse male germ cells could be safely cryopreserved for later use by freezing the whole epididymides and testes without cryoprotectant. Furthermore, we examined whether frozen male germ cells maintained their fertilization ability after international transportation on dry ice. Epididymides and testes were collected from sexually mature male ICR and C57BL/6Cr mice and placed in polypropylene cryotubes. The cryotubes were frozen at -80�C with or without a freezing container, or were plunged directly into liquid nitrogen (LN2). They were stored at -80�C or in LN2 from between one week and one year. Epididymides and testes were thawed by placing the cryotubes in a water bath at room temperature. B6D2F1 and C57BL/6Cr oocytes were microinseminated with either epididymal and testicular spermatozoa or round spermatids. After embryo transfer into pseudopregnant females, normal pups were obtained irrespective of the method of cryopreservation and cell type used. However, their birth rates (2-33%) were lower than those of our conventional microinsemination using fresh sperm or spermatids (20-60%). For transportation experiments, testes were collected from C57BL/6J mice and placed in a cryotube. The cryotubes were frozen at -80�C in a freezing container. On the day of transportation, the cryotubes were placed in a polystyrene foam case filled with dry ice and were transported from Harwell (UK) to Tsukuba (Japan) by air and land. After three days, the samples were delivered to the recipient facility and were stored at -80�C until use (about 1 month). After thawing and collection of spermatogenic cells, C57BL/6J oocytes were microinseminated with either testicular spermatozoa or elongated spermatids. After embryo transfer, 24 (34% per transfer) and 8 (16%) offspring, respectively, were obtained from the two groups. These results indicate that mouse male germ cells retain their nuclear integrity even after freezing epididymides or testes in freezers without cryoprotectant. Since this cryopreservation technique is very simple and allows storage at -80�C for at least several months, it may enable transportation of mouse male germ cells internationally on dry ice, even when the senders are not specialized in cryopreservation.
36 DEVELOPMENTAL COMPETENCE OF HUMAN AGED OOCYTES AS HOST CELLS FOR NUCLEAR TRANSFER
Authors: Hall V. , Compton D. , Stojkovic P. , Nesbitt M. , Herbert M. , Murdoch A. , Stojkovic M.
Published: December 2006

The use of aged metaphase II oocytes (cultured in vitro for more than 14 h) for somatic cell nuclear transfer (SCNT) in varying species has resulted in lower developmental outcomes compared with non-aged in vitro- or in vivo-matured oocytes. However, due to limited resources of fresh oocytes for the derivation of nuclear transfer stem cell lines, further investigation in using spare oocytes is required. Aged spare oocytes (48 h post oocyte retrieval) were consigned for research (under HFEA and local ethics approval) by couples undergoing either in vitro fertilization (failed IVF oocytes, f-IVF) or intracytoplasmic sperm injection (failed-ICSI oocytes, f-ICSI) treatments. Aged oocytes were randomly assigned for double-labeling immunocytochemical analysis (f-IVF, n = 10; f-ICSI, n = 7) for the microtubule markers, NuMA and �-tubulin, or parthenogenetic activation. Immunocytochemical analysis was performed as previously described (Chatzimeletiou et al. 2005 Hum. Reprod. 20, 672-682) using primary anti-rabbit NuMA (gift from D. Compton, Dartmouth Medical School, Hanover, NH, USA) and anti-mouse DM1-�. Secondary antibodies were donkey anti-rabbit and anti-mouse immunoglobulins. Oocytes were counterstained with Hoechst 33342. Negative controls were performed as above with blocking solution substituting for primary antibodies. Parthenogenetic activation was performed for 4 h using 10 �M calcium ionophore (5 min) and 2 mM 6-dimethylaminopurine (Ca-I/DMAP) for f-IVF (n = 10) and f-ICSI oocytes (n = 11) or 10 �g/mL puromycin (Ca-I/Pur) for f-IVF (n = 12) and f-ICSI oocytes (n = 10) (4 h). Activated oocytes were cultured in a biphasic system, G1.3" and G2.3" (Vitrolife UK, Ltd., Ediburgh, Lothian, UK) for 5 days at 37 �C in 5% CO2 in humidified air. NuMA was localized to the metaphase spindle in 6/10 (60%) and 7/7 (100%) oocytes for f-IVF and f-ICSI, respectively, and/or in cytoplasmic cytasters. One f-IVF oocyte and four f-ICSI oocytes had visible tetrapolar spindles. Unusual patterns of diffuse NuMA staining containing dense foci within these regions, but not associated with the cytasters or metaphase spindle, were also observed in two f-IVF oocytes. The majority of oocytes displayed ring-like staining of DM1-�, which was aberrant in two f-ICSI oocytes. Parthenogenetic development was poor for both treatments. Cleavage rates were 17% and 20% for f-IVF using Ca-I/PUR and Ca-I/DMAP, respectively, and 40% and 45% for f-ICSI using Ca-I/PUR and Ca-I/DMAP, respectively. Fragmentation rates were high across all treatments. No parthenogenetic embryos developed beyond the 6-cell stage. Thus, the use of aged human oocytes for SCNT may be difficult due to their incapacity to artificially activate using current activation protocols and, in addition, due to the microtubule abnormalities observed in many of these aged oocytes.
360 THE EFFECTS OF PROGESTERONE AND PRESENCE OF CORPUS LUTEUM ON SUPEROVULATION IN BEEF CATTLE (BOS TAURUS)
Authors: Burns M. , Lapin D. , Brown A. , Gibbons J.
Published: December 2006

Bovine embryo transfer is a multi-million dollar industry that relies on hyperstimulation of cattle ovaries with exogenous follicle stimulating hormone (FSH). This process is variable among donors, breeds, and embryo transfer organizations. Historically, donor cattle are administered FSH (IM; <15 to 50 mg) for 4 days and a luteolytic dose of prostaglandin F2� (PGF2�; IM; 25 mg twice daily) on Day 3. The presence of a functional corpus luteum (CL) is crucial for this protocol; however, there are embryo donor cattle that do not exhibit progesterone indicative of a functional CL. Experiment 1 was performed to determine whether the number of embryos and ovulations that could be generated from donors without a functional CL might be increased by progesterone supplementation via a vaginal insert (CIDR). Experiment 2 was performed to determine an optimal FSH (Sioux Biochemical, Inc., Sioux Center, IA, USA) dosage (high dose = 30 units, n = 9; low dose = 20 units, n = 7) for cattle without a functional CL. Embryos were collected nonsurgically by uterine lavage and were evaluated morphologically according to the IETS system. Progesterone concentrations were determined using an RIA on venous blood samples (10 mL). Ovulations (as evident by resulting CL) were counted using transrectal ultrasonography. Embryo, ovulation, and progesterone data were analyzed using ANOVA with SAS (SAS Institute, Inc., Cary, NC, USA), to evaluate the main effects of group and time (progesterone concentrations only). In Experiment 1, there was no difference in the number of high-quality embryos that were recovered from hyperstimulated (IM; 35 units) Angus-cross cattle that either had a functional CL (n = 12) or a CIDR insert (n = 12) following PGF2� injection. Progesterone concentrations were similar until Day 3 (Day 0 = injection of PGF2� and CIDR insertion). On Day 3, the CL group had higher (P < 0.05) progesterone concentrations (3.5 � 0.4 ng/mL) than the CIDR group (1.5 � 0.1 ng/mL). Further, there was no difference between the average number of ovulations for the cows with a CL (22.5 � 2.4) or cows without a CL (22.8 � 1.5). In Experiment 2, there was no difference in the number of high-quality embryos recovered from cattle in the high-dose group (8.1 � 0.4) compared to cattle in the low dose group (5.7 � 1.3). There were no differences during the treatment period in progesterone concentrations between high- and low-dose groups. Although there was no statistical difference in the number of ovulations between cattle in the high dose group (20.2 � 2.5) compared to cattle in the low dose group (15.6 � 0.9), the higher FSH dose did yield over 30% more ovulations. Embryo collection from cattle that do not show signs of a CL can increase the number of offspring and embryos, and the genetic progress of otherwise unavailable cattle, thus benefiting the beef and beef cattle industries.
368 OOCYTE ASPIRATION FROM TRANSITIONAL AND CYCLING MARES TREATED WITH eFSH
Authors: Purcell S. H. , Suh T. K. , Seidel Jr G. E.
Published: December 2006

A major obstacle in studying equine oocyte maturation is a source of oocytes. The aim of this study was to determine if oocyte recovery following transvaginal ultrasound-guided aspirations could be improved by using eFSH. Fourteen mares ranging from 3 to 16 years of age were used as oocyte donors during the transitional (March to April) and cycling (May to June) seasons. Each follicle was aspirated with a 12-gauge double-lumen needle using a pressure of 150 mmHg and then flushed eight times using PBS supplemented with 10 U/mL heparin and 0.05% BSA. Large follicles (>20 mm) were aspirated first, followed by small follicles (10–20 mm). The first aspiration in each season was used to deplete the follicular pool of all follicles >10 mm; these data were not considered. Following this initial aspiration, mares were subjected to four rounds of aspirations every 10 to 11 days. In each round during the transitional season, half the mares received 12.5 mg eFSH (Bioniche Animal Health, Belleville, Ontario, Canada) once daily for four days prior to aspiration. For each round during the cycling season, half the mares received 12.5 mg eFSH twice daily for three days prior to aspiration. In each replicate, mares were alternated from treated to control groups during both seasons. Oocyte recovery and superstimulatory responses were analyzed using the GLM procedure of SAS (SAS Institute, Inc., Cary, NC, USA). During both the transitional and the cycling season, treatment with eFSH did not have a significant effect (P > 0.1) on the number of small or large follicles present at the time of aspiration (Table 1). For both seasons and size classes, the number of follicles varied by mare (P < 0.02). Oocyte recovery from small follicles during the transitional season improved during the later two rounds of aspiration (0.58 vs. 1.49; P < 0.01) and was also affected by mare (P = 0.06). During the cycling season, recovery of oocytes from both small and large follicles varied by mare (P < 0.05). Treatment with eFSH increased the number of oocytes recovered from large follicles in cycling mares (P < 0.05). This study indicates that responses to short-term eFSH treatment for the purpose of oocyte recovery vary greatly by mare. While treatment with eFSH did not increase the number of follicles available for aspiration (P > 0.1), it did increase the number of oocytes recovered from large follicles during the cycling season.

Table 1.
Oocyte recovery and superstimulatory response of mares treated with eFSH
37 CELL CYCLE ANALYSIS IN IN VITRO-CULTURED ADULT SKIN FIBROBLASTS OF THE GORAL (NAEMORHEDUS CAUDATUS)
Authors: Hashem M. A. , Dilip P. B. , Kang S. K. , Lee B. C. , Hwang W. S.
Published: December 2006

The present study was undertaken to examine the cell cycle characteristics of goral adult skin fibroblasts under a variety of cell cycle-arresting treatments. Gorals are listed as an endangered animal in CITES Appendix I. Experimental findings suggest that G0/G1 stages of the cell cycle give better results in somatic cell nuclear transfer (SCNT) in terms of normal ploidy and development of reconstructed embryos. In this regard, synchronization of the cell cycle stages in G0/G1 phase is suggested to be one of the key factors for determining the success of nuclear transplantation. Serum deprivation, contact inhibition, chemical inhibitors such as protease, and antioxidant inhibitors are widely used for cell cycle synchronization and inhibition of apoptosis. Four experiments were performed, and each with one-way completely randomized design involving three replicates of all treatments using the general linear models procedure in the statistical analysis system (SAS Institute, Inc., Cary, SC, USA). Least significant difference was used to determine statistical significance (P < 0.05) among treatment groups. In Exp. 1, effects of cyclic confluency, serum starvation, or full confluency on goral cells were studied. In Exp. 2, treatments with different antioxidants such as 2 mM beta-mercaptoethanol (B-ME), cysteine, or glutathione for 24 h were examined in fully confluent cells without serum starvation. In Exp. 3, three protein inhibitors, 2 mM 6-dimethylaminopurine (6-DMAP), 7.5 �g/mL cycloheximide, or 7.5 �g/mL cytochalasin B, were treated as in Exp. 2. In Exp. 4, different concentrations (0, 0.5, 1.0, or 2.5%) of dimethyl sulfoxide (DMSO) were treated as in Exp. 2. In all experiments, cell cycle stages were analyzed by fluorescence-activated cell sorting (FACS) analysis. In Exp. 1, more cells (70.4%) were found in the G0/G1 stage in the serum starved medium compared to cells in the cyclic (66.1%) and fully confluent growth phase (65.5%). In Exp. 2, supplementing with B-ME (61.8%) or cysteine (62.0%) in the culture medium synchronized cell cycle to the G0/G1 stage better than glutathione (53.1%). In Exp. 3, more cells were synchronized to the G0/G1 stage in media supplemented with cyclohexamide (62.3%) than with 6-DMAP (5.7%) or cytochalasin B (13.3%). Incidence of apoptosis was higher in media containing 6-DMAP (91.8%) or cytochalasin B (76.6%). In Exp. 4, supplementing the medium with 0.5% (76.0%) or 1.0% (75.9%) DMSO synchronized the cells effectively to the G0/G1 stage compared to the untreated control medium (69.3%) or medium supplemented with 2.5% of DMSO (8.0%). In conclusion, cyclohexamide, B-ME, cysteine, or DMSO at the optimum concentration can synchronize the cell cycle effectively, which may have a positive impact on the outcome of SCNT in the goral.
This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-04-00).