bluuebunny/crossref_metadata_2025 huggingface dataset:
72 HISTONE METHYLATION AND GENE EXPRESSION PATTERNS IN PRE-IMPLANTATION EMBRYOS DERIVED FROM INTRA- AND INTER-SPECIES NUCLEAR TRANSFER
Authors: Shi W. , Yang F. , Wolf E. , Zakharchenko V.
Published: December 2006

The differential epigenetic changes in embryos from different species provide a model to study how the nucleus from one species interacts with cytoplasm from another species. In this study we examined histone methylation at lysine 9 of histone 3 (K9H3) and lysine 20 of histone H4 (K20H4) and the expression levels of three early development-related genes (Oct-4, Hsp 70.1 and Hprt) in individual intra- and inter-species cloned and control embryos at the 1-, 2-, 4- and 8-cell stages. Mouse fetal fibroblast (MFF) nuclei were transferred into mouse, bovine, or rabbit oocytes. As control, we used in vivo derived (mouse and rabbit) or in vitro-produced (bovine) embryos. Histone methylation was detected by anti-MeK9H3 and anti-MeK20H4 antibodies. Gene expression analysis was performed using a quantitative RT-PCR technique (Daniels et al. 2000 Biol. Reprod. 63, 1034-1040). Data were analyzed by Student's t-test. No embryos from inter-species cloning (MFF-bovine and MFF-rabbit) survived beyond the 8-12 cell stage. MFF-mouse and MFF-bovine embryos exhibited demethylation of K9H3 and K20H4 at the 2-cell stage and the methylation level was increased at the 4-cell stage, but no demethylation was observed at the 2-cell stage of MFF-rabbit embryos and the methylation level in these embryos was significantly higher than that of in vivo rabbit embryos. The level of Oct-4 mRNA was low at the 1- and 2-cell stages of in vivo mouse embryos and increased at the 8-cell stage. No significant increase in Oct-4 transcript was detected at the 8-cell stage of inter-species cloned embryos. The expression of Hsp 70.1 in in vivo mouse embryos was increased at the 2-cell stage and decreased to a level similar to that in the zygote at the 8-cell stage. In cloned embryos, Hsp 70.1 transcripts were also increased at the 2-cell stage, but there was no significant decrease of Hsp70.1 mRNA abundance at the 8-cell stage of inter-species embryos as compared to the corresponding 2-cell stage. For MFF-mouse embryos, Hsp 70.1 expression was increased at the 2-cell stage, but at the 8-cell stage the transcript level was at the level similar to that in inter-species clones. Hprt expression was increased at the 8-cell stage of in vivo mouse embryos. The dynamic change of Hprt transcript in MFF-mouse embryos was not significantly different from that of in vivo mouse embryos, but no significant change of Hprt expression occurred in the development of MFF-bovine and MFF-rabbit embryos. Differential epigenetic characteristics of mouse somatic nucleus after transfer into oocytes from different species suggest the existence of incompatibilities of nuclear-cytoplasm interaction between distantly related species. This abnormal interaction at the time of genome activation may affect normal development.
This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.
80 EFFECT OF DNA METHYLATION ON SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT IN RHESUS MONKEY
Authors: Yang J. F. , Yang S. H. , Niu Y. Y. , Zhou Q. , Ji W. Z.
Published: December 2006

Up to now, no primate animals have been successfully cloned with somatic cell nuclear transfer (SCNT) and little is known about molecular events occurring in SCNT embryos. DNA methylation reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals. Epigenetic characteristics of donor cell nuclei and their epigenetic reprogramming in oocyte cytoplasm have been supposed as major factors influencing the development of SCNT embryos. In Experiment 1, on donor cells used in a previous SCNT at our laboratory, global DNA methylation and histone 3 lysine 9 acetylation (H3K9ac) of three cell lines (S11, S1-04, and S1-03) derived from ear skin were examined after serum starvation by immunofluorescence with monoclonal antibody to 5-methyl cytosine (Oncogene, Science, Inc., Cambridge, MA, USA) and anti-acetyl-Histone H3 (Lys 9) (Upstate Jingmei Biotech, Ltd., Shenzhen, China). In the results, two cells lines, S11 and S1-04, supporting higher blastocyst development (about 20%) than that (7.8%) of S1-03, showed a higher level of H3K9ac than the S1-03 cell line. Global DNA methylation levels in the three cell lines were decreased after serum starvation, but no obvious correlation between the level and SCNT embryo developmental potential was found among the three cell lines. In Experiment 2, on SCNT and IVF embryos, global DNA methylation reprogramming during pre-implantation development was investigated with immunofluorescence and laser scanning microscopy techniques. In IVF embryos, active demethylation of paternal genome occurred soon after fertilization; subsequently, passive demethylation resulted in remarkably reduced global methylation level at the 8-cell stage and the morula stage. Thereafter, genomewide remethylation started at the late morula stage and an asymmetric methylation pattern was formed in blastocysts, with higher methylated trophectoderm than inner cell mass (ICM). Compared with IVF embryos, most SCNT 2-cell embryos and ICM in blastocysts showed higher methylation levels, and the asymmetric methylation pattern was not as evident as that in IVF blastocysts. Some SCNT 8-cell embryos showed higher methylation, but others were slightly stained, even lower than IVF embryos. In conclusion, the higher global H3K9 acetylation level of donor cells may benefit chromatin remolding and development of SCNT embryos. Abnormal methylation reprogramming in most SCNT embryos, especially in ICM of blastocysts, may be one main obstacle for primate cloning, although relatively high blastocyst development rate was obtained. DNA methylation reprogramming in rhesus monkey pre-implantation embryos, on the whole, was as conservative as that reported in other mammals.
84 EVALUATION OF BULL SEMEN AT 1 H VS. 48 H OF POST-FROZEN STABILIZATION TIME
Authors: Brogliatti G. M. , Larraburu G. , Cavia R. , Carini M. E.
Published: December 2006

The process of cryopreservation of bull semen in liquid nitrogen at −196°C is usually carried out after 3 to 6 h of refrigeration at 4°C post-collection. To guarantee the quality of the final product, the frozen straws are evaluated after cryopreservation. The seminal samples are usually stabilized during 48 h before being analyzed (Hafez, Reproduction and Artificial Insemination in Animals, 1989); this would retard the possible commercialization. The objective of the present study was to determine motility parameters and viability of semen doses stabilized by 1 h or more than 48 h in liquid nitrogen at −196°C. A total of 122 ejaculated from 23 different adult bulls (Angus, Brangus, Braford, and Hereford) were evaluated in an artificial insemination center between January and April 2005. The semen was diluted in a semi-defined semen extender (Andromed, Minitub, Germany) and frozen in an automatic freezer (Digicool, IMV, France). Parameters of velocity average path (VAP, μm/s), velocity straight line (VSL, µm/s), amplitude lateral head (ALH, µm), linearity (LIN, %), percentage of rapid cells (RAPID, %), and viability (VIA, %) were determined by Computer Assisted Semen Analysis (CASA, HTM-ceros 12.1, Berkeley, CA, USA). The obtained results were analyzed statistically with T Student and are summarized in Table 1. The results indicate that there is no difference in the velocity of the spermatozoa evaluated 1 h or 48 h post-frozen. There is no difference in VAP, VSL, movement of amplitude lateral head (ALH), or linearity (LIN). The percentage of viable spermatozoa was not affected in either group. Statistical analysis indicates that there is no difference (P > 0.05) in any of the evaluated parameters. The results demonstrate that spermatic motility and viability of frozen bull semen could be evaluated before 48 h post-frozen. This allows reduction of the time between freezing and evaluation and immediate availability of the bull straws.

Table 1.
Parameters of motility and viability at 1 h vs. 48 h of post-frozen stabilization time

This research was supported by Centro Genético Bovino EOLIA S.A.
85 VITRIFICATION IN VIVO OF LARGE EQUINE EMBRYOS AFTER VITRIFICATION OR CULTURE
Authors: Campos-Chillon L. F. , Cox T. J. , Seidel Jr G. E. , Carnevale E. M.
Published: December 2006

Cryopreservation of large (>300 μm) equine embryos has been unsatisfactory using slow-cooling or vitrification techniques. The objective of the present experiments was to compare three methods for vitrification of large embryos using modified vitrification protocols for equine (Eldridge-Panuska et al. 2005 Theriogenology 63, 1308–1319) and bovine (2003 J. Anim. Sci. 81 Suppl. 1, 143) embryos. For Method 1, embryos (n = 14) 350–550 or 550–750 µm were exposed, respectively to, VS1 [1.4 M glycerol (G) in mPBS] for 5 min or 7 min, moved to VS2 [1.4 M G, 3.6 M ethylene glycol (EG) in mPBS] for 5 min or 7 min, and then transferred to VS3 (3.4 M G, 4.6 M EG in mPBS) for 40 or 60 s. In Method 2, embryos (n = 13) 350–550 or 550–750 μm were exposed, respectively, to VS1 for 10 or 14 min, VS2 for 10 or 14 min and VS3 for 60 or 90 s. Straws (0.25 mL) for Methods 1 and 2 were loaded with two columns of DS (0.5 M galactose in mPBS) and a small column of VS3 containing the embryo. Straws were heat-sealed and deposited in a goblet suspended in liquid N2 and containing vapor for 1 min and then plunged. Straws were warmed in air (24°C) for 10 sec and then in water at 20°C for 10 sec. Straws were shaken to mix the columns; after 3 min, embryos were expelled and re-hydrated in two additional solutions containing 0.3 and 0.15 M galactose for 3 min each. For Method 3, embryos (n = 17) 300–750 μm were placed in 1.5 M EG in mPBS for 5 min; 3 M EG in mPBS for 10 min; 5 M EG in mPBS for 5 min and 7 M EG, 0.5 M galactose, 18% w/v Ficoll 70 in mPBS for less than a minute. The droplet containing an embryo was loaded into a 0.25 mL straw that was preloaded with two columns of DS and followed by a small column of DS. Straws were heat-sealed and plunged vertically, sealed end fist, into liquid nitrogen covering the embryo, then the remainder of the straw was slowly immersed. Straws were warmed in air (24°C) for 10 s and then in water at 37°C for 10 s. Straws were shaken to mix the columns; after 3 min at 37°C, embryos were rehydrated as in Methods 1 and 2. Embryos were transferred nonsurgically to recipients 5 d after ovulation, and pregnancy diagnoses were performed 5 to 9 d after transfers. No embryonic vesicles were observed for embryos vitrified with Methods 1 and 2. The pregnancy rate for embryos vitrified with Method 3 was 35% (6/17) overall, and 55% (6/11) for embryos between 300–400 μm. No pregnancies resulted from embryos >400 μm. More studies are needed to optimize methodologies for dehydration, equilibration, and warming of large equine embryos.
91 PRELIMINARY RESULTS OF SURGICAL EQUINE EMBRYO TRANSFER AFTER OPEN PULLED STRAW VITRIFICATION
Authors: Duchamp G. , Guignot F. , Grizelj J. , Vidament M. , Mermillod P.
Published: December 2006

In equine species, embryo cryopreservation is not as widely developed as in some other species. Slow freezing has been applied to equine embryos but with relatively low success rates. This higher sensitivity to conventional freezing procedures may be explained by the presence of a capsule surrounding the equine embryo that may impair cryoprotectant penetration. Recently, good in vitro embryo survival rate was obtained after open pulled straw (OPS) vitrification (Moussa et al. 2005 Theriogenology 64, 1619–1632). The aim of the present study was to evaluate in vivo survival of vitrified embryos five days after surgical transfer into Welsh pony mares. Morulae (M), early blastocysts (EB), and blastocysts (B) ranging from 140 to 320 μm in diameter were collected (n = 20) in a Ringer lactate solution on Day 6.75 after ovulation. Before vitrification, embryos were assessed morphologically and their size was measured (McKinnon and Squires 1988 J. Am. Vet. Med. Assoc. 192, 401–406). Then, embryos were vitrified using the OPS method described by Berthelot et al. (2001 Reprod. Nutr. Dev. 41, 267–272). Briefly, embryos were washed twice in HEMES-TCM-199 + 20% newborn calf serum (NBCS) for 1 min, equilibrated in HEPES-TCM-199 + 20% NBCS with 7.5% dimethyl sulfoxide (DMSO) + 7.5% ethylene glycol (EG) for 3 min, and then with 18% DMSO + 18% EG + 0.4 M sucrose for 45 s. One embryo was then loaded per straw. For transfer, four straws were quickly thawed (5 s in air) and the narrow end of the straw containing the embryo was immersed in HEPES-TCM-199 + 20% NBCS + PBS + 0.2 M sucrose. Five to 8 min after thawing, four embryos were surgically transferred into the cranial portion of the uterine horn in each of five pony mare recipients. Five days after transfer, embryos recovered by transcervical flushing of the uterus were classified as viable if morphology was normal, no dark inner cells were present, the capsule was intact, and the diameter was at least 1000 μm. The results are shown in the table. One recipient of vitrified embryos had an endometritis and no embryo was recovered. From the four other recipients, nine embryos were recovered out of 16 (56%) transferred, seven of which were viable (44%). The results of the present preliminary study demonstrating survival of equine embryos transferred after OPS vitrification is very encouraging. However, the results should be confirmed by birth of foals after transfer of OPS-vitrified embryos to recipients.

Table
95 EFFECT OF CRYOPROTECTANT ADDITION AND REMOVAL AT 4°C ON MOTILITY AND PLASMA MEMBRANE INTEGRITY OF BOVINE CAUDAL EPDIDYMAL SPERMATOZOA
Authors: Guerrero C. , Leibo S. , Paccamonti D. , Eilts B. , Bondioli K. , Godke R.
Published: December 2006

Cryopreservation of spermatozoa harvested from the epididymides would be a means of salvaging germplasm from genetically valuable males that die unexpectedly from injury, disease, or poaching. It is well known that the addition of cryoprotective agents (CPAs) is essential for sperm survival following the freezing process. However, CPAs can cause loss in sperm viability due to osmotic damage or chemical toxicity. The objective of this study was to determine the effects of single-step addition and/or removal of glycerol (GLY) or ethylene glycol (EG) on motility and plasma membrane integrity of bovine epididymal spermatozoa. Paired testes were obtained from mature bulls (n = 10) at a local abattoir and transported to the laboratory at 25–28°C within 4–6 h post-mortem. Epididymal spermatozoa were harvested by multiple incisions from the caudae epididymides of each bull, pooled, and washed in Brackett-Oliphant medium by centrifugation for 5 min at 500g. Pellets were resuspended in egg yolk-Tris-glucose-citric acid monohydrate medium (EYT-GC) at a concentration of 120 × 106 cells/mL and cooled to 4°C at a rate of 0.1°C/min. Specimens were allocated to each of five treatment groups: control (no CPA), 7% GLY, and 14% GLY, 7% EG, 14% EG. Then, replicate samples were diluted 1:1 in EYT-GC medium containing twice the final desired concentration of CPA. After being exposed for 10 min, each sample was diluted directly into EYT-GC at 4°C. Motility was assessed by means of a computer assisted semen analysis system and plasma membrane integrity was determined by SYBR 14 and propidium iodide staining followed by fluorescence microscopy. Differences among treatments were analyzed using one way ANOVA (P < 0.05). The results (Table 1) show that maximum survival, as assessed by measurements of motility and membrane integrity, was achieved with spermatozoa exposed to 7% EG. Almost identical intermediate levels of survival were observed with spermatozoa exposed to 7% GLY or 14% EG. The lowest survival was observed for spermatozoa exposed to 14% GLY. The results indicate that the use of EG as a cryoprotectant may minimize toxicity and osmotic damage to fresh bovine epididymal spermatozoa. Its efficacy as a CPA is currently being determined.

Table 1.
Sperm motility and membrane integrity (mean ± SEM) after addition of CPA to epididymal sperm
96 VITRIFICATION OF PORCINE OOCYTES — A NEW APPROACH TO GAMETE CRYOPRESERVATION
Authors: Gupta M. K. , Lee H. Y. , Uhm S. J. , Lee H. T.
Published: December 2006

Cryopreservation of porcine oocytes has a special importance in reproductive biotechnologies owing to the high lipid content of the oocytes. The objective of this study was to investigate the efficiency of a new and simple method of oocyte vitrification for cryopreservation of porcine oocytes. Abattoir ovary-derived prepubertal porcine oocytes at germinal vesicle (GV) or metaphase II (MII) stage were exposed for 10-15 min to vitrification solution I that contained 4% (v/v) ethylene glycol (EG) in either TCM-199 supplemented with 20% FCS or phosphate-buffered NCSU23 medium supplemented with 0.4% BSA. Oocytes were then washed thrice in EG (35%; v/v)-based vitrification solution II containing sucrose (13.7%; w/v) and polyvinyl pyrrolidone (5%; w/v) and placed in groups of 25-50 oocytes per 1-2 �L droplet onto aluminum foil kept directly over the liquid nitrogen (LN2). After visually observed vitreous formation, they were transferred to cryovials and directly plunged into LN2 for storage. Thawing was performed in sucrose- or EG-based media. After warming, the viability was assessed by fluorescein diacetate (FDA) staining and in vitro maturation (IVM) of GV-stage oocytes was determined based on cumulus expansion and polar body extrusion. In addition, the developmental capacity of oocytes was assessed by in vitro fertilization (IVF). Effects of pretreatment with cytochalasin B (CB) on the post-thaw viability, nuclear maturation, and developmental capacity were also investigated. The results demonstrated that the rate of oocytes having normal morphology was more than 90% in all groups. Viability based on esterase enzyme activity and oolemma integrity, as determined by FDA staining, ranged from 80% (without CB) to 86% (with CB) for MII-stage oocytes and 79% (without CB) to 90% (with CB) for GV-stage oocytes. The IVM rate of pelleted GV-stage oocytes reached 70% based on cumulus expansion and 55% based on polar body extrusion. Sucrose-based thawing media was found to be superior to EG-based media. Although the vitrification method resulted in a high survival rate based on the morphological appearance, FDA staining, and IVM rate, developmental rates of pelleted oocytes were found to be compromised. However, we believe that there is a scope for improving the developmental rates with further modifications in the method.
109 EFFECTS OF VITRIFICATION PROCEDURES ON SUBSEQUENT DEVELOPMENT AND ULTRASTRUCTURE OF IN VITRO-MATURED SWAMP BUFFALO (BUBALUS BUBALIS) OOCYTES
Authors: Boonkusol D. , Faisaikarm T. , Dinnyes A. , Kitiyanant Y.
Published: December 2006

The purpose of this study was to investigate the effects of 2 vitrification procedures on the developmental capacity and ultrastructural changes of matured swamp buffalo (Bubalus bubalis) oocytes. In vitro-matured (IVM) oocytes were vitrified by using 35% and 40% ethylene glycol (EG) as vitrification solution (VS) for solid surface vitrification (SSV) and in-straw vitrification (ISV), respectively. Survival rate of vitrified–warmed oocytes was evaluated on the basis of homogeneous cytoplasm, membrane integrity, and complete zona pellucida. All developmental data were analyzed using chi-square analysis. P < 0.05 was considered significant. The blastocyst rates of parthenogenetic vitrified–warmed oocytes were significantly higher with SSV (89.3% and 13.6%, respectively) than with ISV (81.8% and 5.5%, respectively). However, they were still significantly lower than those of control (100% and 34.2%, respectively). For examining the ultrastructural changes, fresh VS-exposed (ISV and SSV), and vitrified–warmed oocytes were processed for transmission electron microscopy. In VS-exposed oocytes, reduction of microvilli abundance and damage of mitochondrial membrane were found only in the ISV group. In vitrified–warmed oocytes, however, it was clear that both methods of vitrification induced profound ultrastructural modifications to microvilli, mitochondria, oolemma, and cortical granules as well as to the size and position of vesicles. Damaged mitochondria were, however, more abundant in ISV vitrified oocytes than in SSV vitrified oocytes, which correlated with the developmental data, showing the superiority of the SSV method. This study demonstrated for the first time the feasibility of vitrification of IVM swamp buffalo oocytes.
112 VIABILITY AND APOPTOSIS OF BOVINE FIBROBLASTS FROZEN IN A CONVENTIONAL ( - 20°C) OR LOW-TEMPERATURE ( - 80°C) FREEZER
Authors: Chacón L. , Gómez M. C. , Jenkins J. , Leibo S. P. , Wirtu G. , Dresser B. L. , Pope C. E.
Published: December 2006

Fibroblasts are commonly frozen in dimethyl sulfoxide (DMSO, 10% v/v) at a cooling rate of 1°C min−1 in cryo-containers placed in a low-temperature (−80°C) freezer (LTF) overnight before storage in liquid nitrogen (LN2). LTFs are not always available and cell viability may be altered by freezing at different cooling rates. Therefore, the purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen at different cooling rates in a −80°C freezer or conventional −20°C freezer. Fibroblasts were generated from skin tissue collected from an adult female cow. Tissue was cut and cultured in DMEM for 7 to 10 days and passaged 2 or 3 times before being cryopreserved. Cells were frozen by one of 6 procedures; cells that were not frozen served as a control. To be frozen, cells were resuspended in DMEM + 10% FBS and 10% DMSO in 2-mL cryo-vials before being placed into freezing containers (Mr. Frosty, Nalgene). For groups 1 and 2, the container was placed in a −20°C freezer and cooled at ≈0.6°C min−1 to −7°C; ice formation was induced by seeding, after which cooling was continued at ≈0.6°C min−1 to −20°C. Vials in groups 1 and 2 were placed in LN2 after 1 h and 16 to 18 h, respectively. Group 3 and 4 vials were also cooled to −20°C at 0.6°C min, except that these samples were not seeded. After being held at −20°C for 16 to 18 h, group 3 vials were thawed; group 4 vials were stored in LN2. Vials in groups 5 and 6 were placed into a Mr. Frosty container and cooled at ≈1°C min−1 in a −80°C freezer. After being held at −80°C for 16 to 18 h, group 5 vials were thawed, and group 6 vials were placed into LN2. Group 1, 2, 3, and 4 vials were thawed in a 37°C water bath; group 5 and 6 vials were thawed in a 40°C water bath. Cell viabilities were quantified by using TB exclusion (TB) with microscopy. At least 20 000 cells from each group were thawed, and caspase 3, a key enzyme involved in apoptotic processes, was detected using a Caspase 3-FLICA apoptosis detection kit; propidium iodide-stained nuclei were assessed with cytometry (FCM) to determine viability (Table 1). Data were analyzed by ANOVA (P < 0.05). Although there were viability differences among groups, freezer type, seeding, holding time, and LN2 had no significant impact, as assessed by TB or FCM; however, no LN2 significantly increased the percentage of viable cells displaying apoptosis. In summary, bovine fibroblasts were successfully frozen in a conventional (−20°C) freezer before storage in LN2.

Table 1.
Viability and apoptosis of bovine fibroblasts frozen at −20°C or −80°C
114 CRYOTOP VITRIFICATION FOR IN VITRO-PRODUCED BUFFALO (BUBALUS BUBALIS) EMBRYOS
Authors: De Rosa A. , Attanasio L. , Boccia L. , Pellerano G. , Campanile G. , Gasparrini B.
Published: December 2006

The aim of this study was to compare the efficiency of different combinations of cryoprotectants for vitrification of IVP buffalo (Bubalus bubalis) embryos by the cryotop method (Kuwayama et al. 2005 RBM Online 11, 300–308). In group A, we evaluated the vitrification and warming solutions previously used to vitrify buffalo embryos in French straws (Gasparrini et al. 2001 Theriogenology 55, 307). Embryos were equilibrated in 1.4 M glycerol for 5 min and in 1.4 M glycerol and 3.6 M ethylene glycol (EG) for an additional 5 min. After being transferred into 3.4 M glycerol and 4.6 M EG for 25 s, individual embryos were picked up in an extremely small volume (<0.1 �L) of vitrification solution and placed on the top of a very fine polypropylene strip (0.4 mm wide � 20 mm long � 0.1 mm thick) attached to a hard plastic handle, kindly provided by M. Kuwayama. Each embryo was placed onto the thin strip of the Cryotop and immediately submerged into liquid nitrogen. For warming, the strip of the Cryotop was immersed directly into a 0.5 M sucrose solution; embryos were retrieved and transferred into 0.25 M sucrose for 5 min before culture in SOF medium. In group B, we examined the vitrification and warming solutions previously used for OPS vitrification of buffalo embryos (De Rosa et al. 2006 Reprod. Fertil. Dev. 18, 153). Embryos were equilibrated in 7.5% EG + 7.5% dimethyl sulfoxide (DMSO) for 3 min before transfer into 16.5% EG + 16.5% DMSO + 0.5 M sucrose. After 25 s, they were placed on the cryotop, as previously described, and submerged into liquid nitrogen. For warming, embryos were recovered into a 0.25 M sucrose solution for 1 min, transferred into 0.15 M sucrose for 5 min, and cultured in SOF. IVP buffalo embryos of excellent quality that, by Day 7 of culture (Day 0 = in vitro fertilization), had reached the blastocyst stage (n = 44 and 53 for groups A and B, respectively), over 6 replicates, were vitrified. Embryo survival rate was determined as the percentage of vitrified-warmed embryos undergoing further development during a 24-h in vitro culture period. Differences between methods were analyzed by chi-square test. A significantly higher embryo survival rate was recorded in Group B compared to Group A (67.9 vs. 43.2% respectively; P < 0.05). In conclusion, it was demonstrated that cryotop vitrification, with the combination of cryoprotectants used in group B, is a valid tool to cryopreserve IVP buffalo blastocysts.
12 GENERATION OF RAT OFFSPRING DERIVED FROM CRYOPRESERVED SPERMATOZOA IN JAPANESE NATIONAL BIORESOURCES
Authors: Kashiwazaki N. , Seita Y. , Naoi K. , Takizawa A. , Kuramoto T. , Serikawa T.
Published: December 2006

The purpose of the National BioResource project is to facilitate the availability of genetically and phenotypically standardized rat strains for life sciences. The BioResource is available to scientists worldwide. To bank genetic resources efficiently in the rat, cryopreservation of both sperm and embryos is a very important technology. The objective of the present study was to confirm the ability of banked and transported rat spermatozoa to fertilize oocytes through intrauterine insemination and for the embryos to develop to term, with the ultimate aim of developing a system for banking rat genetic resources. The epidydimal spermatozoa from the KLM rat, whose body size is small because the Prkg2 gene is partially defective, were frozen with egg yolk medium supplemented with 0.7% Equex Stm (Nakatsukasa et al. 2001 Reproduction 122, 463–467) and banked in the Institute of Laboratory Animals, Kyoto University. The cryopreserved sperm in 0.25-mL straws were transported to the laboratory at Azabu University, Kanagawa. Two straws from different males were thawed in a 37�C water bath for 15 s. Thawed semen was diluted with 1.0 mL of mR1ECM (Miyoshi et al. 1997 Biol. Reprod. 56, 180–185) with 0.4% (w/v) bovine serum albumin (BSA, fraction V; Sigma-Aldrich Japan K.K., Tokyo, Japan) at 37�C and then incubated at 37�C in 5% CO2 in humidified air until insemination. The percentage of motile spermatozoa was assessed visibly and determined by direct observation at 37�C under a light microscope at 100�. The thawed semen (50 �L, 3–4 � 105 sperm cells) was then inseminated into the top of both uterine horns of recipient females that were mated with a vasectomized male. The post-thaw motility of frozen spermatozoa was 10%. Seven of 15 inseminated females became pregnant and 13 live pups were born. It is thought that the low number of pups born in spite of the relatively high pregnancy rate was caused by sperm damage during the freezing and thawing procedure. The results of the present study show that rat spermatozoa cryopreserved in the BioResource have the ability to revive genetic resources through intrauterine insemination.
129 IMPROVEMENT OF BOAR SPERM CRYOSURVIVAL BY THE OPTIMIZATION OF CRYOPRESERVATION CONDITIONS
Authors: Roca J. , Hernandez M. , Cremades T. , Vazquez J. M. , Martinez E. A.
Published: December 2006

One of the most important limiting factors for the efficient commercial application of frozen–thawed semen on pig artificial insemination programs is the significant and consistent inter-boar variability in sperm cryosurvival. The objective of the present study was to evaluate the effectiveness of different cryopreservation conditions (CCs) for freezing and thawing boar spermatozoa, and to determine their suitability for individual boars, with particular reference to those that showed an intrinsic poor sperm cryosurvival. Using a split-ejaculate technique, single ejaculates from 53 boars were suspended in lactose-egg yolk extender containing 2 or 3% final glycerol concentration, packaged in 0.5-mL straws, cooled at rates of 10, 40, or 60�C min-1 using a programmable cell freezer, and stored in liquid nitrogen; the frozen samples were warmed at ≈1200�C min-1 (37�C water bath for 20 s) or ≈1800�C min-1 (70�C for 8 s). The cryopreservation condition including 2% glycerol, 40�C min-1 of cooling, and ≈1200�C min-1 of warming was considered as the control. Frozen–thawed sperm were evaluated at 30 and 150 min post-thawing for sperm motility (CASA system), plasma membrane integrity (SYBR-14 and propidium iodide), and acrosome membrane integrity (FITC-peanut agglutinin and propidium iodide). Data were analyzed using 2 different ANOVA mixed models. Whereas cooling rate had no influence (P ≥ 0.05), glycerol concentration and warming rate, both independently, affected (P ≤ 0.05) all post-thawing sperm assessments. No interaction (P ≥ 0.05) among effects was detected for any of the sperm parameters assessed. Evaluating the combined effect of glycerol concentration and warming rate, the highest post-thaw sperm quality was achieved from the semen samples frozen with 3% of glycerol and thawed at ≈1800�C min-1. Significant differences (P ≤ 0.05) among ejaculates (boars) to support the different CCs were shown in all post-thaw sperm assessments. Three different (P ≤ 0.05) ejaculate (boar) populations, defined by PATN analysis (PATN software package, CSIRO, Canberra, Australia), were identified according to post-thaw sperm assessments in semen samples frozen and thawed using control CC (populations so-called 'good', 'moderate', and 'bad' sperm freezers). Different (P ≤ 0.05) susceptibility in the tolerance of spermatozoa to support the different CCs was found among the ejaculate populations. Whereas spermatozoa from ejaculates considered as 'good' freezers are relatively unaffected (P ≥ 0.05), those from 'moderate' and, mainly, 'bad' freezers are very sensitive (P ≤ 0.05) to the modifications in the CCs. In conclusion, slight modifications in the CCs — glycerol concentration and warming rate for thawing — can improve the sperm cryosurvival of some ejaculates (boars), the improvement being particularly larger in those ejaculates (boars) classified as ' bad' sperm freezers.
This work was supported by CICYT (AGF2005-00760), Madrid, Spain.
133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES
Authors: Somfai T. , Ozawa M. , Noguchi J. , Kaneko H. , Ohnuma K. , Karja N. W. K. , Farhudin M. , Maedomari N. , Dinnyes A. , Nagai T. , Kikuchi K.
Published: December 2006

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P < 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P < 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P < 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P < 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P < 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes.
This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).
134 CONCEPTION RATES OF IN VITRO-PRODUCED BOVINE EMBRYOS CRYOPRESERVED IN 6% GLYCEROL AND TRANSFERRED BY THE DIRECT METHOD
Authors: Takada N. , Hayasaka S. , Chiba K.
Published: December 2006

Ethylene glycol has been used as the standard cryoprotectant for direct transfer of bovine embryos due to its high permeability. But Merton et al. reported that cryoprotectivity of glycerol for bovine embryos was superior to that of ethylene glycol (2001 Theriogenology 55, 312 abst). We previously reported that nonsurgical transfer of in vivo-derived bovine embryos cryopreserved in a lower concentration (5%) of glycerol and thawed by stepwise method resulted in a 55.4% conception rate, whereas direct transfer without removal of cryoprotectant showed only a 45.1% conception rate (Takada et al. 2005 Jpn. J. Embryo Transfer 27, 59–64). In this experiment, survival and conception rates of in vitro-produced (IVP) bovine embryos cryopreserved in 6% glycerol solution (GLY) were compared to those of embryos cryopreserved in 10% ethylene glycol plus 0.1 M sucrose solution (EG). Cumulus–oocyte complexes were matured and fertilized according to Numabe et al. (2000 Theriogenology 54, 1409–1420). Presumed zygotes were cultured in mSOF supplemented with 5% calf serum (CS) and 0.25% linoleic acid albumin at 38.5�C under 5% CO2, 5% O2, 90% N2 for 7 days. At the expanded blastocyst stage, embryos were placed in GLY or EG in PBS supplemented with 20% CS for 15 min at room temperature and loaded into 0.25-mL straws. Straws were placed directly into an alcohol freezer. When the cryoprotectant was GLY, straws were seeded at -4.0�C, held for 10 min, cooled at 0.5�C min to -30.5�C, and then plunged into liquid nitrogen. When the cryoprotectant was EG, the seeding point was -7.5�C, and the plunging point was -34.0�C, but the rest of the protocol was the same as for GLY. In Exp. 1, thawing in both groups was done in a 30�C water bath, and the contents were directly rehydrated in PBS with 20% CS. Thawed embryos were cultured in mSOF with 5% CS for 24 h to assess embryo survival rate, based on the re-expansion of the blastcoele and on their hatching ability. In Exp. 2, embryos in both groups were thawed and transferred to synchronous recipients without removing the cryoprotectant. Data were analyzed using chi-square analysis. In Exp. 1, the developmental rates of post-thaw embryos were similar in GLY (46/52, 88.5%) and EG (45/52, 86.5%); however, the hatching rate was significantly higher (P < 0.05) in embryos cryopreserved in EG (26/52, 50.0%) than in GLY (15/52, 28.8%). In Exp. 2, the conception rates of embryos were similar in both groups, GLY (7/15, 46.7%) and EG (6/15, 40.0%). In conclusion, after direct rehydration of embryos, the developmental ability of IVP bovine embryos cryopreserved in EG was superior to that of embryos cryopreserved in GLY in vitro. However, conception rates in vivo were similar in both groups. These results suggest that a lower concentration of glycerol might be still useful as a cryoprotectant for direct transfer of IVP bovine embryos.
138 EFFECT OF SEMINAL PLASMA ON CHILLING AND FREEZING OF CANINE SPERMATOZOA
Authors: Yu I. , Kim Y. J. , Kim I. S. , Leibo S. P. , Songsasen N.
Published: December 2006

Seminal plasma (SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing (Barrios et al. 2000 Biol. Reprod. 63, 1531–1537; Moore et al. 2005 Theriogenology 63, 2372–2381; Vadnais et al. 2005 Anim. Reprod. Sci. 87, 121–132). The purpose of this study was to determine the effect on sperm survival of adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from 4 healthy dogs (3–4 years old) of various breeds were pooled and centrifuged at 300g for 10 min at 25�C; the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris (EYT) buffer. The study comprised 2 experiments: Exp. 1: Sperm were suspended in EYT extender containing 0%, 20%, 50%, 80%, or 100% SP, and were slowly cooled to 4�C for 2 h or held at 25�C as controls. Exp. 2: Sperm samples, each of which contained 1 � 108 sperm mL-1, were assigned to 5 groups to be frozen. In group 1, sperm in EYT + 20% SP were cooled to 4�C, diluted to contain final concentrations of 5% glycerol + 10% SP in EYT, and then frozen. In the 4 other groups, sperm in EYT alone were first cooled slowly to 4�C, then diluted to contain 5% glycerol plus 0%, 20%, 40%, or 50% SP in EYT, and then frozen. Spermatozoa were frozen at 25�C min in plastic straws that were suspended above liquid nitrogen and thawed in water at 38�C for 30 s. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at 400� magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that 20%, 50%, 80%, or 100% SP did not improve motility, membrane integrity, or acrosome integrity of spermatozoa chilled to 4�C compared to those chilled without SP (P > 0.05). Survival of spermatozoa suspended in EYT + 20% SP and maintained at 25�C was significantly higher than for those that were chilled (P < 0.05). The results of the second experiment showed that spermatozoa suspended in EYT + 20% SP and then diluted at 4�C to contain 5% glycerol + 10% SP exhibited the highest progressive motility and membrane integrity after being frozen and thawed (P < 0.05). In summary, although seminal plasma did not affect spermatozoa that were only chilled, addition of seminal plasma did significantly improve survival of canine spermatozoa that were frozen and thawed.
141 INDICATIONS FOR DNA DOUBLE-STRAND BREAK REPAIR IN EARLY BOVINE EMBRYOS
Authors: Brero A. , Koehler D. , Cremer T. , Wolf E. , Zakhartchenko V.
Published: December 2006

DNA double-strand breaks (DSBs) are considered the most severe type of DNA lesions, because such lesions, if unrepaired, lead to a loss of genome integrity. Soon after induction of DSBs, chromatin surrounding the damage is modified by phosphorylation of the histone variant H2AX, generating so-called γH2AX, which is a hallmark of DSBs (Takahashi et al. 2005 Cancer Lett. 229, 171–179). γH2AX appears to be a signal for the recruitment of proteins constituting the DNA repair machinery. Depending on the type of damage and the cell cycle stage of the affected cell, DSBs are repaired either by nonhomologous end joining or by homologous recombination using the sister chromatid DNA as template (Hoeijmakers 2001 Nature 411, 366–374). We used immunofluorescence to analyze chromatin composition during bovine development and found γH2AX foci in both male and female pronuclei of IVF embryos. The number and size of foci varied considerably between embryos and between the male and female pronuclei. To test whether the observed γH2AX foci represented sites of active DNA repair, we co-stained IVF zygotes for γH2AX and 3 different proteins involved in homologous recombination repair of DSBs: NBS1 (phosphorylated at amino acid serine 343), 53BP1, and Rad51. We found co-localization of γH2AX foci with phosphorylated NBS1 as well as with Rad51 but did not observe the presence of 53BP1 at γH2AX foci in IVF zygotes. Our finding shows the presence of DSBs in IVF zygotes and suggests the capability of homologous recombination repair. The lack of 53BP1, a component of homologous recombination repair, which usually co-localizes with γH2AX foci at exogenously induced DSBs (Schultz et al. 2000 J. Cell. Biol. 151, 1381–1390) poses the possibility that the mechanism present in early embryos differs substantially from that involved in DNA repair of DSBs in somatic cells.
142 RELATIONSHIP BETWEEN APOPTOSIS IN BOAR SEMEN AND DNA FRAGMENTATION IN PORCINE EMBRYOS
Authors: Bryla M. , Trzcinska M. , Smorag Z.
Published: December 2006

Although apoptosis in somatic cells and in spermatocytes and spermatids in vivo is well established, the presence and significance of apoptosis in ejaculated animal sperm and its correlation with developmental competence of preimplantation embryos is still unresolved. The aim of this experiment was to study the relationship between apoptosis in boar semen and DNA fragmentation in porcine embryos. Two ejaculates from the same boar were used in the experiment. Both fresh ejaculates were analyzed using Vybrant Apoptosis Assay Kit #4 (Molecular Probes, Inc., Eugene, OR, USA). Then one of them was diluted in Biosolwens plus extender, stored for 5 days at 15°C and analyzed using YO-PRO-1/PI assay, which detect changes in the membrane of boar spermatozoa, based on the slight increase of membrane permeability. Both fresh and stored semen were used for insemination, of superovulated gilts (8 per group). After 5.5 days of insemination embryos were flushed out of the uterus and DNA integrity of obtained embryos were analyzed. DNA fragmentation and caspase-3 activity were detected in embryos using kits, Roche Diagnostics (Mannheim, Germany) and PhiPhiLux G2D2 (Calbiochem, San Diego, CA, USA), respectively. For both the fresh and stored semen, 3 groups of sperm were observed under a fluorescence microscope. In the fresh semen, 3 and 2% of apoptotic sperm, 13 and 9% of necrotic sperm, 84 and 89% of live sperm in first and second ejaculated, respectively, were observed. In stored semen, 14% of apoptotic sperm, 27% of necrotic, and 59% of live sperm were noted. In total, 141 expanded blastocysts for DNA fragmentation were analyzed. The results are summarized in Table 1. In conclusion, apoptosis in fresh boar semen was lower than in stored semen and was correlated with the TUNEL nucleus index in blastocysts. The expression of caspase-3 was positively correlated with cells positive for TUNEL.
Table 1.
Relationship between apoptosis in boar semen and DNA fragmentation in porcine embryos

This study was supported by Polish Research Committee, grant no. 2 P06D 024 30.
143 DEVELOPMENTAL EFFICIENCY IMPROVEMENTS OF NUCLEAR TRANSFER EMBRYOS BY REPROGRAMMING SOMATIC CELLS USING TESTIS-DERIVED CELL EXTRACTS
Authors: Choi H.-Y. , Won C. , Kim B.-W. , Kang Y.-J. , Kang G.-H. , Oh Y. , Lee E.-R. , Roh S. , Cho S.-G.
Published: December 2006

Somatic cell cloning has promise for medical treatment using embryonic stem cells derived from cloned embryos. However, porcine cloning by somatic cell nuclear transfer has been inefficient and, even after birth, cloned pigs are found to carry a variety of abnormalities. Moreover, recent molecular analyses of cloned embryos have revealed abnormal epigenetic modifications. Therefore, the prevention of epigenetic errors is expected to lead to the improvement of the success rate in animal cloning. Reports of recent studies indicate that the direct transformation of one differentiated somatic cell type into another is possible and would be advantageous for producing isogenic replacement cells. Therefore, in this study, we modulated the cell fate of somatic donor cells by introducing cell extracts derived from porcine testis. Several porcine somatic cells, including primary and stabilized porcine fibroblasts or epithelial kidney cells, were treated with streptolysin O (SLO; 230 ng mL-1), which reversibly permeablizes plasma membrane, and incubated for 30 min with testis cell-derived cell extracts (4 mg mL-1). To reseal plasma membranes, cells were placed in DMEM containing 30% FBS and 2 mM CaCl2 for 30 min. After resealing the cell membranes, we incubated the cells for 3 weeks and analyzed the expression of testis-specific genes such as protamine 1, protamine 2, SOX 9, mullerian inhibitory substance (MIS), preproacrosine (ACR), phosphoglycerate kinase 2 (PGK-2), protein C, and c-kit ligand. In the reprogrammed primary porcine fibroblasts or epithelial kidney cells, the porcine testis extracts were able to activate the expression of the porcine testis sertoli cell-specific genes. The male germ cell functions were sustained for more than 10 days after the reprogramming process. Then, in vitro-matured oocytes were enucleated and a single cell (either reprogrammed or intact) was injected directly into cytoplasm of the oocytes. The reconstructed embryos were activated electrically and cultured in vitro for 7 days. The rate of blastocyst formation was significantly higher (P < 0.05; chi-square test) in the reprogrammed nuclear donor cells (27/119; 22.7 � 5.0%) than in the control (intact) cells (11/83; 13.3 � 3.2%). Taken together, our results suggest that testis-derived cell extracts can be successfully used to reprogram fibroblasts to express male germ cell function, thus improving the developmental efficiency of the nuclear transfer embryos.