bluuebunny/crossref_metadata_2025 huggingface dataset:
258 MESSENGER RNA EXPRESSION PATTERNS OF HISTONE MODIFICATION GENES IN BOVINE EMBRYOS DERIVED FROM DIFFERENT ORIGINS
Authors: Nowak-Imialek M. , Wrenzycki C. , Herrmann D. , Lagutina I. , Lucas-Hahn A. , Lemme E. , Lazzari G. , Galli C. , Hadeler K.-G. , Niemann H.
Published: December 2006

Epigenetic modifications of the genome, such as covalent modifications of histones, are crucial for transcriptional regulation during development. The N-terminal tails of the histones are subject to post-translational modifications, including acetylation, deacetylation and methylation. histone acetylation loosens chromatin packing and correlates with transcriptional activation. The enzymes Histone acetyltransferases (HATs) transfer acetyl moieties to the lysine residues of histones H2A, H2B, H3, and H4. Histone acetylation is a reversible process which is catalyzed by the histone deacetylase (HDAC) and results in transcriptional repression. Histone methyltransferase (HMT) is responsible for the methylation of arginine in histones 3 and 4, playing an important role in transcriptional activation of genes. In contrast, the H3 Lys 9 methylation is associated with a transcriptionally repressive heterochromatin. The objective of the present study was to determine the effects of different origins of embryos on the relative abundance of transcripts for the histone acetyltransferase 1 (HAT1), histone deacetylase 2 (HDAC2), histone metyltransferases (SUV39H1 and G9A), and heterochromatin protein 1 (HP1). Messenger RNA expression profiles of these genes were investigated in bovine oocytes and pre-implantation embryos up to the blastocyt stage produced either in vitro by two different culture systems, i.e. SOF+BSA or TCM+SERUM, by somatic cloning using adult male and female fibroblasts, parthenogenetic activation, and androgenetic construction, or in vivo, employing semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Significant differences are described below. HAT1, SUV39H1, G9A, and HP1 mRNA transcripts decreased in enucleated oocytes, compared with immature oocytes. The relative abundance of HAT1 and SUV39H1 transcripts was significantly increased in NT-derived zygotes produced from adult female fibroblasts, compared to their in vitro fertilized and parthenogenetic counterparts. No differences were found in the relative abundances of gene transcripts at the 8-16-cell stage, except for parthenogenetic embryos in which SUV39H1 transcripts were significantly higher than in all other 8-16 cell groups. The relative abundance of SUV39H1, G9A, and HP1 transcripts were significantly higher in NT-derived blastocysts derived from adult male fibroblasts than in their in vivo-generated counterparts. HP1 and G9A transcript levels were significantly increased in NT-derived blastocysts derived from male fibroblasts compared to NT-derived embryos produced from female fibroblasts. The results show that the in vitro environment and the nuclear transfer protocol affect mRNA expression patterns of histone modification genes in pre-implantation bovine embryos.
259 LOCALIZATION OF LEUKEMIA INHIBITORY FACTOR (LIF) GENE IN BOVINE PLACENTA
Authors: Oshima K. , Watanabe H. , Kojima T. , Komatsu M. , Yamamoto N.
Published: December 2006

Leukemia inhibitory factor (LIF), a member of the group of hemopoietic cytokines, plays a primary role in the control of embryo development and implantation and in the growth of the placenta in humans and mice. The objective of this study was to investigate the localization of LIF gene in bovine placenta tissues during pregnancy using in situ hybridization (ISH). Eleven Japanese Black cows aged between 1.8 and 14.5 years, with normal estrous cycles, were used in this study. They were observed daily for estrous behavior, and the day of estrus was considered as Day 0. They were artificially inseminated, and their uteri were collected on Days 61 to 63 (n = 3), 127 to 142 (n = 4), and 225 to 232 (n = 4) of pregnancy. Pregnancy was confirmed by the presence of a conceptus in the uterus, and the uterus was isolated by dissection, avoiding damage of the uterine artery. The uterus was perfused with 4% paraformaldehyde (PFA) solution using catheters inserted into the uterine artery, and placental tissues were isolated by dissection. Each tissue was cut into small pieces (5 mm thick) and fixed in 4% PFA solution for 20-24 h, after which they were embedded in paraffin using routine procedures. Several tissue pieces were collected from each individual cow. Six micrometer-thick sections were cut and placed on MAS coated glass slides (Matsunami Glass, Kishiwada, Japan). The sections were dried in an oven for three days at 40�C. Anti-sense and sense biotin-labeled oligonucleotide probes for bovine LIF were designed from the sequence information (GenBank accession number D50337). The sense probe was used as the negative control. ISH was carried out using GenPoint System (DakoCytomation, Glostrup, Denmark) according to the manufacturer's instructions. The sections were observed under an Eclipse 800 microscope (Nikon, Tokyo, Japan) to detect the positive signal for LIF gene. Density analysis was performed with Scion Image (Scion Corporation, Frederick, MD, USA). Data were analyzed by one-way ANOVA and Tukey-Kramer's HSD, using 0.05 as a significant level. Leukemia inhibitory factor gene was expressed in stromal cells of the fetal cotyledonary villus and caruncular crypt and in mono- and multi-nuclear epithelium cells of the fetal cotyledonary villus and caruncular crypt. Intensities of LIF gene positive signals in all positive cells at Days 127 to 142 tended to be weak compared with those at other periods. Furthermore, intensities of LIF gene positive signals of multinuclear cells of the villus and crypt demonstrated a tendency to be strong compared with those of other cells. These results suggest that LIF is produced in several cell types in the placenta and may play important roles in pregnancy.
260 COMPREHENSIVE IDENTIFICATION OF RELATIVE GENES CAUSING ABNORMAL DEVELOPMENT OF BOVINE EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Park J. , Minami N. , Imai H.
Published: December 2006

Developmental failure of a cloned animal using somatic cell nuclear transfer (SCNT) procedures is considered to be the result of abnormal expression of developmentally important genes caused by incomplete reprogramming of the donor cell nuclei. However, there are few reports about stage-specific gene expression during cleavage progression of cloned embryos. The aim of this study was to identify using fluorescein differential display method, the differentially expressed genes in cloned embryos at early developmental stages compared with those produced by in vitro fertilization. Bovine cumulus-oocytes complexes (COCs) were aspirated from follicles (2-8 mm in diameter) of slaughterhouse ovaries and cultured in TCM-199 supplemented with 10% fetal calf serum (FCS) for 18 h for somatic cell nuclear transfer (NT) or 24 h for in vitro fertilization (IVF) at 39�C. Removal of oocyte nuclei for NT was performed by squeezing out a small amount of the cytoplasm laying beneath the first polar body by means of a glass needle. Donor cells for NT were obtained from skin cells of an adult cow and cultured in DMEM supplemented with 10% FCS. After the transfer of somatic cell into enucleated oocytes, DC electric pulses at 200 V/mm for 2 � 10 �s were used for fusion, and the reconstructed embryos were treated with 10 �g/mL cycloheximide for 6 h. The embryos were then cultured for 120 h (morula stage) or 168 h (blastocyst stage) in modified SOF medium under 5% CO2, 5% O2 and 90% N2 at 39�C. Total RNA obtained from NT and IVF embryos were analyzed by differential display RT-PCR (DDRT-PCR) as previously described (Minami et al. 2001 Biol. Reprod. 64, 30-35). We obtained several differences in gene expression patterns between NT and IVF embryos at the morula and blastocyst stage. A total of 52 cDNA fragments were isolated and analyzed. Semiquantitative analysis revealed that some genes (NADH dehydrogenase subunit 1, SR rich protein, KIAA0107, ribosomal protein L19) were highly expressed in IVF embryos compared with NT embryos, whereas other genes (CASK) were highly expressed in NT embryos compared with IVF embryos. These results indicate that the differentially expressed genes observed in NT embryos may be representative of marker genes for the production of normal NT offspring and DDRT-PCR procedure is quite useful for identification of several genes that are differentially expressed between NT and IVF embryos.Although the detailed function of the genes and their products remains to be determined, it is likely that the reprogramming mechanisms can be elucidated genetically by the analysis of differentially expressed genes in the future.
268 BOVINE AND EQUINE RECOMBINANT FOLLICLE-STIMULATING HORMONE PRODUCED IN TWO HETEROLOGOUS HOSTS: ESCHERICHIA COLI AND TRYPANOSOMA BRUCEI
Authors: Wilson M. , Morris J. , Gibbons J.
Published: December 2006

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone consisting of � and � subunits produced by different genes. Following release from the anterior pituitary, FSH is responsible for development of ovarian follicles from the time of emergence of a follicular wave until divergence of the dominant follicle. Treatment of cattle and horses with recombinant FSH would be valuable for induction of superovulation in an effort to harvest multiple embryos for use in embryo transfer. Glycosylation of the subunits may be essential for proper function and receptor recognition, thereby complicating the production of recombinant FSH. Therefore, a eukaryotic, rather than a prokaryotic, host would be most beneficial for generation of recombinant protein. To explore recombinant protein synthesis, we utilized the ancient protozoan Trypanosoma brucei. Perhaps best known for its parasitic life style, T. brucei has been the focus of extensive biochemical and genetic research, with the nonpathogenic insect stage of the parasite serving as a model eukaryote for the study of many fundamental eukaryotic pathways, including glycosyl phosphatidylinositol (GPI) anchor biosynthesis and RNA editing. The ease of molecular manipulation of this protozoan, and the organism's robust glycosylation machinery, has led us to explore the possibility of using the non-pathogenic life stage for recombinant protein production. The objective of this study was to produce recombinant bovine and equine FSH in both prokaryotic and eukaryotic expression systems. Protein has been produced in Escherichia coli using the pQE30 expression system. Subunit genes were ligated into pQE30 and expressed in M15 E. coli cells. These cells were found to produce detectable amounts of the individual subunit protein after a three hour induction. To express recombinant protein in T. brucei, subunit genes were cloned into pLEW2T7(6His), a stably integrated vector designed to allow inducible expression of proteins fused to a C-terminal six-histidine tag. Clones were subsequently transformed into T. brucei and are awaiting protein expression. All protein purification will be performed via nickel affinity chromatography and protein concentration will be measured using radioimmunoassay. Bioactivity will be analyzed using a rat ovarian weight assay prior to use in cattle and horses. The purified protein will be administered as intramuscular injections twice daily for four days beginning on the day of follicular wave emergence. Producing recombinant FSH will be useful for the embryo transfer and food safety industries by increasing consistency of biologic activity, decreasing production costs, and, perhaps most importantly, decreasing the opportunity for zoonotic transmission of prions such as bovine spongiform encephalopathy.
270 APOPTOSIS AND ONCOSIS ASSESSMENT IN CRYOPRESERVED MOUSE EMBRYOS
Authors: Assumpção M. E. O. A. , Coutinho A. R. S. , Feitosa W. B. , Mendes C. M. , Simões R. , Visintin J. A.
Published: December 2006

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnology. Recent evidence indicates that apoptosis of cryopreserved embryos may be a negative factor for their viability. The aim of this study was to detect apoptosis and to characterize and quantify the embryonic cell death caused by cryopreservation. Mouse morulae were separated to be subjected to two cryopreservation protocols (slow freezing and vitrification) and a control group (fresh). In the slow-freezing procedure, embryos were exposed to 10% ethylene glycol (EG) for 10 min. Straws were placed in a methanol bath at -7�C until it reached -31�C and then plunged and stored in liquid nitrogen. The embryos were thawed in air for 10 s and in a 25�C water bath for 20 s. In the vitrification method, embryos were exposed to 10% and 20% EG for 5 min, followed by 40% EG + 18% Ficoll + 10% sucrose (EFS) for 30 s and then plunged and stored in liquid nitrogen. These embryos were thawed in a 25�C water bath for 20 s. For the cell death evaluation, cell membrane integrity from the fresh and cryopreserved embryos was assessed by Hoechst and propidium iodide (H/PI staining). Morphology and apoptosis were assessed by means of the haematoxylin-eosin staining (HE) and by electron microscopy (MET). To confirm apoptosis, 64 cryopreserved mouse morulae (34 submitted to slow freezing and 30 to vitrification) were used to evaluate Caspase-3 activity. The cryopreserved embryos were divided into experimental and control groups and incubated with Caspase-3 and buffer solution, respectively. Afterward, the embryos were incubated with rhodamine and the Caspase activity was determined under a fluorescence microscope. H/PI staining detected more membrane permeability in the vitrification (69.7%) than in the slow-freezing (48.4%) or fresh (13.8%) groups (P < 0.05; Wilcoxon's test). Nuclear evaluation by HE revealed that vitrification and slow freezing induced pyknosis and chromatin condensation. HE staining revealed weakly staining cytoplasm and degenerated cells in the vitrification group (indicating oncosis), whereas in the slow-freezing the presence of cytoplasmic condensation and eosinophilic structures indicating apoptosis were observed. MET examination of the ultrastructure confirmed the HE results. The Caspase-3 activity showed a fluorescence increase in both experimental groups compared with the control group. In conclusion, staining with HE allows detection of oncosis and apoptosis in cryopreserved embryos. Regarding the cryopreservation techniques, both slow freezing and vitrification showed oncosis and apoptosis injuries. However, in this experiment vitrification caused more cellular injuries, with less embryo viability, than slow freezing.
This work was supported by FAPESP 04/01252-4 and CAPES.
272 DEVELOPMENTAL COMPETENCE OF OOCYTES OBTAINED FROM BOS TAURUS AND BOS INDICUS DAIRY COWS RAISED IN A TROPICAL CLIMATE
Authors: Camargo L. S. A. , Viana J. H. M. , Sa W. F. , Ferreira A. M. , Ramos A. A. , Freitas C. , Vale Filho V. R.
Published: December 2006

The effects of heat stress on Bos taurus reproductive performance in tropical and subtropical regions are well known, and have been associated with lower oocyte developmental capacity. The aim of this study was to evaluate the developmental competence of oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) dairy cows raised in a Brazilian tropical region, located at 21°35′S latitude, 43°51′W longitude, and 435 meters altitude. Cumulus–oocyte complexes (COCs) were recovered by oocyte pickup (OPU) from mature non-lactating Holstein (n = 9) and Gyr (n = 13) donor cows between the end of spring and the beginning of autumn, with at least two OPU sessions/cow. COCs were in vitro-maturated in TCM-199 (GIBCO, Grand Island, NY, USA) with 10% inactivated estrus cow serum for 24 h under 5% CO2 at 38.5°C in air. Bos taurus and Bos indicus semen with similar cleavage rates, previously evaluated by in vitro fertilization with oocytes obtained from slaughterhouse ovaries, were used to reduce bull effect. Holstein and Gyr spermatozoa were obtained through swim-up method and co-incubated with Holstein (n = 390) and Gyr (n = 505) oocytes, respectively, in Fert-TALP medium (Parrish et al. 1988 Biol. Reprod. 38, 1171–1180) supplemented with 10 μg/mL heparin (Sigma-Aldrich, Sao Paulo, Brazil) and 6 mg/mL fatty acid-free bovine albumin (Sigma) for 18 h in 5% CO2 at 38.5°C in air. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa medium (Wilkinson et al. 1996 Theriogenology 45, 41–49) supplemented with 10% fetal calf serum in humid atmosphere of 5% CO2 at 38.5°C in air. On Day 7 to 8 of co-culture, Gyr and Holstein blastocysts were assessed and those classified as grade 1 (IETS Manual) were transferred to synchronized Bos indicus × Bos taurus crossbred recipients managed under the same nutritional and environmental conditions. Pregnancy diagnosis was performed between 35 and 50 days after estrus. Cleavage, blastocyst, and pregnancy rates were analyzed by chi-square test. Cleavage and blastocyst rates were greater (P < 0.05) in Gyr than in Holstein (66.7% vs. 53.1% for cleavage and 19.6% vs. 10.8% for blastocyst, respectively), but the pregnancy rate was similar (P > 0.05; 44.5% vs. 60% for Gyr and Holstein, respectively). These results show that Gyr oocytes obtained in a tropical region have greater developmental capacity than Holstein oocytes, suggesting an interaction between genotype and environment that influences the success of an in vitro embryo production program; nevertheless, the blastocyst viability after transferring to recipients is similar for both breeds.
274 LAPAROSCOPIC OVUM PICK-UP IN SHEEP AND GOATS: EFFECTS OF REPEATED RECOVERIES AND FOLLICULAR DIAMETER
Authors: Gibbons A. , Pereyra Bonnet F. , Cueto M. , Catala M. , Salamone D.
Published: December 2006

Laparoscopic ovum pickup (LOPU) is a modern technique that may be used in programs of sheep and goat in vitro embryo production. The effects of repeated recoveries and follicular diameter on the efficiency of LOPU were evaluated in these species. In six Merino donor sheep and five Criolla goats, ovarian stimulation for follicular development was performed by administering a single dose of 60 mg NIH-FSH-P1 (Folltropin®; Bioniche, Canada) and 300 UI of eCG (Novoromon 5000®; Syntex, Argentina). Every four days, a total of 4 LOPU in sheep and 3 in goats were performed 24 h after each ovarian stimulation treatment. The intravaginal sponge with 60 mg of medroxyprogesterone (Esponjas Vaginales Syntex®; Syntex, Argentina) were removed after the last LOPU. Follicular fluid was aspirated under laparoscopic observation according to the follicle diameter with a cannula for intrauterine artificial insemination without a vacuum pump. The number and size of follicles, oocyte quantity and quality and recovery rate were evaluated. A total of 566 follicles were aspirated (14.4 ± 2.4 follicles/sheep and 14.6 ± 7.5 follicles/goat), yielding a total of 263 oocytes (6.8 ± 1.5 oocytes/sheep and 6.6 ± 2.9 oocytes/goat) and a recovery rate of 47% in sheep and 46% in goats (Table 1). In both species, there were no significant differences in the number of aspirated follicles, recovered oocytes, overall recovery rate, and the recovery rate of good quality oocytes in the repeated LOPU. The oocyte recovery rates in sheep differed significantly according to the follicular size (55% in large follicles, 36% in small follicles; P < 0.05), whereas in goats recovery rates of oocytes related to follicular size showed no significant differences (42 and 54% for large and small follicles, respectively). Follicular sizes from which oocytes were obtained was not a determining factor in the recovery rate of good quality oocytes. The technique used would allow effective and repeated oocyte recoveries for in vitro embryo production in sheep and goats.

Table 1.
Repeated recoveries by laparoscopic ovum pickup (LOPU) in sheep and goats
276 IN VITRO FERTILIZATION PROCEDURES AFFECT THE HEPATIC GENOMIC CYTOSINE METHYLATION LEVEL AND THE PHENOTYPE OF BOVINE FETUSES
Authors: Hiendleder S. , Mund C. , Klempt M. , Reichenbach H.-D. , Stojkovic M. , Weppert M. , Wenigerkind H. , Elmlinger M. , Lyko F. , Wolf E.
Published: December 2006

Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable bovine Day 80 fetuses to assess the effects of different in vitro fertilization (IVF) protocols on hepatic genomic cytosine methylation levels and on fetal phenotype. Two groups of IVF-fetuses (IVF1, n = 5 and IVF2, n = 10) were compared with control fetuses generated by artificial insemination (AI, n = 14). Both IVF protocols were previously employed to generate live offspring but differed with respect to gonadotropins in the oocyte maturation medium (0.01 units/mL b-FSH and b-LH for IVF1 versus 0.2 units/mL o-FSH for IVF2) and serum concentrations in the embryo culture media (5% estrous cow serum in IVF1 versus 10% estrous cow serum in IVF2). Analysis of variance (General Linear Model Procedure, SPSS for Windows version 12.0; SPSS GmbH Software, Munich, Germany) showed that fetus group significantly affected fetus weight and length (P < 0.01), heart (P < 0.05) and liver (P < 0.01) weight, and 5-methylcytosine (5 mC) content of liver DNA (P < 0.001). Comparison of group means (t-test) showed that methylation levels in both groups of IVF fetuses differed significantly from AI controls. We observed hepatic DNA hypomethylation (-15.4% vs. AI control, P < 0.01) in IVF1 fetuses, and hypermethylation (+11.6% vs. AI control, P < 0.001) in IVF2 fetuses, but only IVF2 fetuses showed phenotypic abnormalities. The IVF2 fetuses were significantly heavier (18.6%, P < 0.01) and longer (4.3%, P < 0.05) than AI fetuses, with increased heart (21.8%, P < 0.05) and liver (25%, P < 0.001) weights, and thus displayed an overgrowth phenotype. A clinical-chemical screen of 18 plasma parameters failed to detect abnormalities in IVF1 fetuses but revealed significantly increased levels of insulin-like growth factor 1 (40.8%, P < 0.001) and creatinine (28.5%, P < 0.05) in IVF2 fetuses. Our data indicate that bovine IVF-procedures can induce protocol-specific and persistent changes in hepatic cytosine methylation level with or without obvious concomitant changes in fetal phenotype. This suggests that epigenetic change after bovine IVF could be more widespread than previously thought and highlights the value of epigenetic diagnostic screening.
277 FOLLICULAR GROWTH, SUBSEQUENT OVUM PICKUP, AND DOMINANT FOLLICLE REMOVAL IN COWS
Authors: Imai K. , Tagawa M. , Matoba S. , Narita M. , Kanayama K.
Published: December 2006

The present study was designed to assess the recruitment of follicles after ovum pickup (OPU) and dominant follicle (DF) removal on the follicular wave after OPU in Holstein dry cows. Cows were reared under the same feeding and environmental conditions. In Experiment 1, follicle aspiration (>2 mm in diameter) by OPU using a 7.5-MHz linear transducer with needle (COVA needle; Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200; ALOKA, Tokyo, Japan) was performed in four cows. Then, ovaries were observed after OPU from Day 1 (Day 0 = the day of OPU) to Day 11 to assess the number of follicles developed. In Experiment 2, two sessions of OPU were performed with a 7 day interval between sessions, with or without dominant follicle removal, to assess the quality of developing follicles and oocytes. In the DF removal group, >8-mm follicles were aspirated at Day 5 after the first OPU session, and the same cows without DF removal were designated as a control (n = 4, crossover trial). Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the developmental competence of oocytes, Grades 1 and 2 cumulus-oocyte complexes (COCs) were collected, matured, fertilized, and cultured as described by Imai et al. (2002 J. Vet. Med. Sci. 64(10), 887-891). Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst formation rate on Days 7 to 9 (the day of insemination = Day 0). Data were analyzed by ANOVA or Student t-test. In Experiment 1, a dominant follicle (>8 mm in diameter) was developed during Days 3 to 5 after OPU in each donor. The mean number of developing follicles (>2 mm in diameter) were increased from Day 1 to Day 9 (Day 1: 7.5 � 2.1, Day 3: 19.0 � 1.2, Day 5: 23.3 � 9.0, Day 7: 30.3 � 11.0, Day 9: 42.0 � 15.8 and Day 11: 41.0 � 16.7 (mean � SD), P < 0.05). In Experiment 2, there was no difference in the mean number of developing follicles on the day of OPU and collected oocytes between DF removal and control groups (follicles: 47.8 � 23.0 and 39.3 � 6.2; oocytes: 27.0 � 11.6 and 26.5 � 5.4, respectively). The number of Grades 1 and 2 oocytes for the DF removal group was significantly higher (P < 0.05) than that for the control (83.6 � 1.5 and 63.2 � 14.2, respectively), and no significant difference was found within cleavage (60.0 � 37.2, 53.6 � 23.2) and blastocyst rates (34.1 � 33.9, 34.4 � 16.8). These results indicate that populations of follicles were increased till Day 9 after OPU, and the DF removal was effective at increasing oocyte quality in the developing follicles.
279 LIVE RAT OFFSPRING FROM CRYOPRESERVED EJACULATED SPERMATOZOA THROUGH IN VITRO FERTILIZATION
Authors: Kashiwazaki N. , Seita Y. , Shino M. , Hisamatsu S. , Inomata T.
Published: December 2006

We have previously reported successful cryopreservation of epididymal rat spermatozoa (Nakatsukasa et al. 2001 Reproduction 122, 463). However, the procedure for cryopreservation of rat spermatozoa has a disadvantage; a male has to be euthanized for collection of spermatozoa from its epididymides. Obtaining ejaculated spermatozoa repeatedly from the same male could be useful for cryopreservation of invaluable spermatozoa which carry mutations including transgenes. The objective of the present study was to develop a reliable system for cryopreservation of ejaculated rat spermatozoa and efficient production of offspring from the cryopreserved spermatozoa. Matured Wistar females were mated with three males of the same strain, and killed by cervical dislocation after formation of vaginal plugs. The uteri of mated females were excised and flushed with freezing medium containing 23.0% egg yolk, 8.0% lactose, and 0.7% Equex STM to recover ejaculated spermatozoa. The semen samples were loaded into 0.25-mL straws. The straws were cooled to 5.0�C at 0.5�C/min in a programmable freezer and then exposed to liquid nitrogen (LN) vapor at 4 cm (-150�C) above the LN level for 15 min. The straws were then plunged into LN and stored for at least a week. The straws were thawed in a 37.0�C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 � 106 sperm/mL into a 200-�L droplet of R1ECM and then pre-incubated for 5 h. Ovulated oocytes collected from superovulated females were introduced into the droplet and co-cultured for 10 h for in vitro fertilization (IVF). The oocytes were denuded and examined for the presence of two pronuclei (2PN) microscopically. The denuded oocytes with 2PN were transferred into the oviducts of pseudo-pregnant females. The rates of sperm motility at recovery, post-thaw, and the initiation of IVF (after pre-incubation) were 57 � 6%, 24 � 5%, and 18 � 3%, respectively. After co-culture, 46 (14%) of the total 329 co-cultured oocytes were confirmed to contain 2PN. A total of the 44 putative zygotes were transferred to five recipients, and a total of 21 live young (48%) were born from all of the transferred recipients. We were able to produce zygotes and offspring derived from cryopreserved ejaculated spermatozoa of all three males used in the present study. In conclusion, the cryopreservation system for ejaculated rat spermatozoa used in the present study is a workable protocol for banking of valuable genetic resources of laboratory rats. Further studies on the IVF procedure with cryopreserved ejaculated spermatozoa in the rat are needed to improve the fertilization rate.
28 EFFECT OF TREATMENT OF OVINE OOCYTES WITH CAFFEINE ON GENE EXPRESSION IN NUCLEAR TRANSFER EMBRYOS
Authors: Choi I. , Lee J.-H. , Campbell K.
Published: December 2006

The efficiency of animal production by somatic cell nuclear transfer (SCNT) remains low and this has been linked to incomplete epigenetic reprogramming of the donor somatic cell nucleus. Previous studies have reported that embryos produced by SCNT exhibit abnormal expression patterns for a number of genes, including IL6, FGF4, FGFr2, Hsp, IF-tau, DNMT, and Mash2 (Daniels et al. 2000 Biol. Reprod. 63, 1034-1040; Wrenzycki et al. 2001 Biol. Reprod. 65, 309-317). Recently, we demonstrated that treatment of telophase I or metaphase II ovine oocytes with 10 mM caffeine for 6 h increased the activities of both MPF and MAPK kinases. In NT embryos produced using caffeine treated oocytes as recipient cytoplasts, no increase in the frequency of development to the blastocyst stage was observed; however, blastocyst stage embryos had an increased cell number (Lee and Campbell 2005 Reprod. Fertil. Dev. 16, 125). The objective of the present study was to examine the effects of caffeine treatment on the expression levels of a number of genes involved in early development. Target genes were categorized into seven groups based on their function: (1) transcription factors (Oct-4, Sox-2), (2) growth factors (IGF-1, IGF-1r, IGF-2r, FGF-2, and FGF-4), (3) stress adaptation (Hsp70.1 and Hsp27), (4) metabolism (Glut-1, Glut-3, and Glut-4), (5) compaction/cavitation (DcII), (6) trophoblastic function (IF-tau), and (7) nuclear reprogramming factors (Hist4h4, H2A.Z, and Lmna). To determine the transcript levels semiquantitatively, different PCR cycle parameters were used (35 and 40 cycles). Expression levels were compared in blastocyst-stage embryos obtained from five groups produced as previously described (Lee and Campbell 2005; Maalouf et al. 2005 Reproduction 32, 49): (I) IVF embryos, (II) caffeine-treated IVF embryos, (III) parthenotes, (IV) SCNT embryos, and (V) caffeine treated SCNT embryos. No differences in overall expression patterns were observed among groups I, II, and III. In group IV non-treated SCNT-derived embryos, an aberrant gene expression pattern was found with respect to Oct-4 and genes regulated by Oct-4: H2A.Z, IF-tau, and FGF-4, Oct-4, H2A.Z, and FGF-4 were down-regulated and IF-tau was up-regulated. In contrast, the expression patterns of group V caffeine-treated SCNT embryos resembled those of control groups I, II, and III. In comparison to gene expression in group IV embryos, Oct-4, FGF-4, and H2A.Z were up-regulated but IF-tau was down-regulated. Previous studies have demonstrated that FGF-4 and H2A.Z play an important role in early development and implantation, whereas expression of IF-tau is related to embryo quality (Feldman et al. 1995 Science 267, 246-249; Fasst et al. 2001 Curr. Biol. 11, 1183-1187; Wrenzycki et al. 2001). Our results demonstrate that treatment of oocytes with caffeine prior to embryo reconstruction can alter the expression patterns of developmentally regulated genes in SCNT embryos to more closely resemble those of IVF controls.
280 PORCINE BLASTOCYSTS DERIVED FROM IN VITRO-MATURED OOCYTES INJECTED INTRACYTOPLASMICALLY WITH SPERM FROM TESTICULAR TISSUE XENOGRAFTED INTO NUDE MICE
Authors: Kikuchi K. , Nakai M. , Kashiwazaki N. , Ozawa M. , Maedomari N. , Noguchi J. , Ohnuma K. , Kaneko H.
Published: December 2006

The utilization of spermatogonia from testicular tissue after xenografting into immuno-deficient mice should lead to new insights for the conservation of male gametes. However, successful embryo production using sperm cells from xenografted testicular tissues has been limited to rhesus monkeys (Honaramooz et al. 2004 Biol. Reprod. 70, 1500-1503). In the present study, the objective was to establish this new technology for pig conservation in combination with intracytoplasmic sperm injection. Testes were obtained from male piglets 6 to 15 days old, in which most of the germ cells were gonocytes; these were minced into pieces of approximately 1.5 � 1.5 � 1.5 mm. Approximately 20 fragments were transplanted under the back skin of castrated nude mice 5 to 8 weeks old. The testicular grafts were recovered between 125 and 192 days after xenografting, minced in Dulbecco's phosphate-buffered saline, and centrifuged several times, to serve as a sperm suspension. In vitro maturation of the recipient oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041) and injection with an intact spermatozoon, followed by electrical stimulation at 1 h post-injection (Nakai et al. 2003 Biol. Reprod. 68, 1003-1008), were carried out. The putative zygotes were cultured in vitro for 6 days (Kikuchi et al. 2002), and were then fixed, stained, and assessed for embryonic development and quality. From a total of 27 mice that were xenografted with testicular tissues, spermatids and spermatozoa were obtained in 19 of the mice (70.4%). Most of the spermatozoa were matured morphologically, showing faint motility after release into the collection medium. From a total of 253 oocytes (four replications) that were injected with sperm, 63 (24.9 � 7.1%) oocytes developed to the blastocyst stage. The average total cell number was 41.9 � 3.9. These values are comparable to those in in vitro fertilization by frozen-thawed spermatozoa, resulting in developmental ability to piglets after embryo transfer (25.3% and 48.7 cells; Kikuchi et al. 2002). These results suggest the possibility of embryo production using porcine spermatozoa that are differentiated from gonocytes within the xenografts.
281 ALGINATE-ENCAPSULATED BOVINE EMBRYOS SUPPORT IN VITRO DEVELOPMENT OF A SMALL NUMBER OF EMBRYOS
Authors: Kobayashi S. , Sakatani M. , Kobayashi S. , Takahashi M.
Published: December 2006

Ova are genetic resources that can be obtained from slaughterhouse ovaries or live cows by ovum pickup (OPU). However, the number of oocytes recovered by OPU is low. Previous studies show that embryos cultured in large numbers have better developmental competence than those in small numbers in mice, sheep, and cattle. Therefore, to improve development of small numbers of embryos, co-culture with other types of embryos is an efficient way. However, it is necessary to distinguish the desired embryos from the co-cultured embryos. Recently, encapsulation of embryos using calcium-alginate was reported to be useful for handling and in vivo culture of porcine embryos (Iwamoto et al. 2003 Theriogenology 59, 261). In the present study, we investigated the effect of co-culture of embryos encapsulated with calcium-alginate on development of small numbers of embryos. In vitro-matured and fertilized zygotes from slaughterhouse-derived ovaries were used for the experiment, and data were analyzed by Student t-test. Encapsulation was carried out by putting the 1% sodium alginate solution containing zygotes slowly into 0.1% calcium chloride solution (microcapsule). We used the microcapsule for the following experiments. In Experiment 1, twenty zygotes were cultured in CR1aa containing 5% FCS with a capsule containing 20 zygotes or without (control) a microcapsule. The rate of cleavage (capsule: 80.0% vs. control: 72.1%) and development to blastocyst stage (capsule: 31.7% vs. control: 33.7%) were not significantly different. This result indicates that the microcapsule is not toxic to embryo development. In Experiment 2, five zygotes were co-cultured with 15 zygotes (microcapsule), and culture of five zygotes without capsules served as a control. The rate of cleavage (co-culture: 81.4% vs. control: 80.0%) was not significantly different, but the rate of development to the blastocyst stage was significantly higher (P < 0.05) in the co-culture (47.1%) than in the control (30.6%). This result indicates that co-culture with a microcapsule including zygotes enhances the development of small numbers of embryos. In Experiment 3, five zygotes derived from a single cow were encapsulated, and four microcapsules from different cows were cultured in the same droplet. The microcapsules could be distinguished by the inclusion of different numbers of glass beads with the zygotes. Culture of five zygotes without capsules was assigned as a control. The rate of cleavage (co-culture: 75.6% vs. control: 69.6%) was not significantly different, but the rate of development to the blastocyst stage was significantly higher (P < 0.05) for the co-culture (30.6%) than for the control (17.8%). These results indicate that co-culture with bovine embryos encapsulated with calcium-alginate may improve development of small numbers of embryos.
283 EFFECTS OF CUMULUS CELL REMOVAL ON IN VITRO OOCYTE MATURATION, FERTILIZATION, AND EMBRYONIC DEVELOPMENT IN PIGS
Authors: Maedomari N. , Kashiwazaki N. , Ozawa M. , Takizawa A. , Noguchi J. , Kankeo H. , Shino M. , Kikuchi K.
Published: December 2006

It is generally accepted that cumulus cells (CCs) support the nuclear maturation of immature oocytes in mammals. However, the precise mechanism of interaction between cumulus cells and oocytes has not been clarified. Furthermore, the role of cumulus cells in embryonic development has not been reported. In the present study, the effect of denuding cumulus cells from porcine oocytes on oocyte maturation, ertilization, and their subsequent development to the blastocyst stage was examined in vitro. In vitro maturation, fertilization, and culture were carried out as previously reported (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Porcine cumulus-oocyte complexes (COCs) were collected; some of them were completely denuded of cumulus cells immediately after the collection (DO-0 group). The remaining intact COCs and the DO-0 oocytes were cultured for 24 h in the presence of dbcAMP and hormones. After the initial culture, some of the intact COCs were denuded either completely (DO-24 group) or partially (H-DO-24 group). Additionally, some of DO-24 oocytes were co-cultured with the cumulus cells removed at 0 h and pre-cultured for 24 h
(DO-24 + CCs group). The denuded oocytes in each experimental group and intact COCs (control) were further cultured for total 46 h. The remaining oocytes with a first polar body were either examined for the levels of intracellular glutathione (GSH) or fertilized in vitro with frozen-thawed boar
spermatozoa. The inseminated oocytes were cultured and examined for their fertilization status after 10 h and for their developmental competence after 6 days. Data were analyzed by ANOVA, followed by the Duncan's multiple range tests. The maturation rates of all denuded groups were
significantly lower (P < 0.05; 34.3 to 45.0%) than that of the control group (64.5%). Intracellular GSH concentrations of all denuded groups were
also significantly lower (P < 0.05; 4.03 to 7.00 pmol/oocyte) than that of the control group (9.60 pmol/oocyte); however, the GSH level of H-DO-24
oocytes was significantly higher (P < 0.05) than the GSH levels in the other denuded groups. Male pronuclear formation rates of completely denuded
oocytes (DO-0, DO-24, and DO-24 + CCs groups) were significantly lower (P < 0.05; 41.4 to 59.3%) than those of the control (89.4%) and the H-DO-24 (80.0%) groups. The blastocyst rate of the control group was significantly higher (P < 0.05; 19.9%) than that of H-DO-24 group (11.6%), and these rates were significantly higher (P < 0.05) than those of the completely denuded groups (3.0 to 4.5%). The results suggest that the presence of cumulus cells during maturation culture improves nuclear maturation of oocytes and plays an important role in embryonic development to the blastocyst stage in vitro.
285 VARIATION FACTORS INFLUENCING CLEAVAGE AND EMBRYONIC DEVELOPMENT RATES OF BOVINE OOCYTES COLLECTED ON A FARM IN A COMMERCIAL OPU-IVP SYSTEM
Authors: Marquant-Le Guienne B. , Aymar F. , Ponsart C. , Guyader-Joly C. , Ponchon S. , Humblot P.
Published: December 2006

The aim of this work was to identify factors influencing the success rate of each production step through a retrospective study conducted for 362 commercial OPU-IVP sessions performed on a farm. Donor females were stimulated with FSH in five decreasing doses (400 μg for cows and 250 μg for heifers). Collected oocytes were matured for 24 h in M199 plus fetal calf serum, FSH, epidermal growth factor (EGF), and estradiol. They were then fertilized in TALP with frozen-thawed semen; zygotes were cultured for 6 days on Vero cell monolayers in B2 medium. Embryos were transferred as fresh to recipients on Day 7. The effects of donor characteristics, OPU and IVP conditions on cleavage, development rates, and number of transferred embryos (TE) were analyzed by ANOVA (GLM program in SAS; SAS Institute, Inc., Cary, NC, USA). Effects are mentioned when significant at P < 0.05. The mean number of collected oocytes per session was 14; 11.6 were selected for IVM, 8.5 cleaved, and 3.9 developed to the blastocyst stage (40.4% of the embryos were Grade 1, 35.3% G2, and 20.5% G3). On average, 2.9 embryos were transferred into recipients. The number of transferred embryos was higher when the dominant follicle (DF) was punctureed prior to OPU (see Table 1). This resulted in a better cleavage rate in punctured DF donor females. Higher cleavage rates were observed in infertile females as well as numbers of TE per session. Pregnancy and body condition score (BCS) recorded at OPU only influenced cleavage rates. Embryonic development rates were mainly influenced by donor breed and parity. In the Abundance breed, 87.5% of the sessions resulted in at least one embryo being transferred compared to 58.6% in Holsteins. Higher numbers of embryos were transferred per session when donor females were cows rather than heifers. Fertilization conditions (heparin and sperm concentrations) had no effect on cleavage and embryonic development rates. To conclude, cleavage and development rates were mainly influenced by donor characteristics (breed, parity, physiological status). Improvement of results may be achieved by systematic puncture of DF prior to OPU. Cleavage rates were dependent on BCS. To improve management of body condition, BCS variations before OPU could be a new parameter to be followed in donor females.

Table 1.
Factors affecting steps of the OPU-IVP procedure
287 EFFECT OF CYSTEAMINE DURING IN VITRO MATURATION OF OPU DERIVED BOVINE OOCYTES ON FURTHER IN VITRO EMBRYONIC DEVELOPMENT AND PREGNANCY RATE
Authors: Merton J. S. , Landman B. , Mullaart E.
Published: December 2006

Glutathione (GSH) plays an important protective role in relation to reactive oxygen species generated by normal oxidative metabolism in the cell. The presence of cysteamine during in vitro maturation may facilitate the synthesis of GSH by immature oocytes. In a previous study we showed a positive effect of the presence of cysteamine during in vitro maturation of slaughterhouse derived bovine oocytes on subsequent in vitro embryonic development (Merton et al. 2004 Rep. Fert. Dev. 16, 279 abstract). This report shows the results of a field trial with ultrasound guided transvaginal oocyte collection (OPU) derived oocytes, in order to confirm our previous results obtained with slaughterhouse derived oocytes. Immature cumulus–oocyte complexes (COCs) were recovered twice weekly by ovum pick-up (OPU) at two collection centres from 11 cows and 147 pregnant heifers. COCs were matured in vitro in TCM199/FCS/LH/FSH supplemented either with or without cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. At Day 7, Morula grade 1 (IETS) were transferred fresh and early-, mid- and exp-Blast grade 1 and 2 were transferred either fresh or frozen/thawed. The experimental design was a 2 × 2 factorial. Results were analysed by Chi-square analyses. The results show that the presence of cysteamine during in vitro maturation significantly affected embryo production from OPU derived COCs (23.4% and 34.4% Morula + Blastocyst rate at Day 7 for control and cysteamine, respectively; Table 1). This higher embryo production rate was mainly due to an increased number of Blastocysts. Also the proportion of grade 3 embryos was significantly reduced in the cysteamine group (P < 0.01). The number of transferable embryos per session was 1.06 and 1.73 for control and cysteamine, respectively. Pregnancy rate was not significantly affected by the presence of cysteamine during in vitro maturation for both fresh and frozen/thawed embryos (fresh: 40.5% and 44.8%, frozen/thawed: 44.4% and 47.2% for control and cysteamine, respectively). These results show that the presence of cysteamine during in vitro maturation affects further in vitro embryonic development, resulting in a higher embryo production rate. This suggest that an apparently ‘simple’ extra protection of the oocyte, due to the free radical scavenging potency of GSH, can have an enormous effect (63.2% relative increase in transferable embryos) on its in vitro developmental potency. The intrinsic quality of the ‘extra’ produced transferable embryos seems not to be different, since pregnancy rate was not affected.

Table 1.
Effect of cysteamine during in vitro maturation of OPU-derived bovine oocytes on subsequent in vitro embryonic development
288 EFFECT OF IN VITRO MATURATION PERIOD AND OOCYTE ACTIVATION BY CALCIUM IONOPHORE ON MONOSPERMIC PORCINE EMBRYO RATES
Authors: Oliveira V. P. , Marques M. G. , Nascimento A. B. , Caetano H. V. A. , Assumpção M. E. O. A. , Visintin J. A.
Published: December 2006

It has already been reported that polyspermy in prepubertal pig oocytes is higher than in mature oocytes. Oocyte activation by calcium ionophore may help cortical granule exocytose. Normally, pig zygotes stay in maturation medium for 44 h, but at 36 h oocytes are already at metaphase II stage (MII). Arrest at MII for a longer period may cause deterioration of various cytoskeletal components. The aim of this study was to evaluate the effect of maturation period (36 or 44 h) and 0 or 50 �M calcium ionophore on monospermic pig embryo rates. Prepubertal pig oocytes were matured in TCM-199 with 10 IU eCG + 10 IU hCG + 50 IU epidermal growth factor (eGF) + 90 �L porcine follicular fluid (pFF) for 20 h and in the same medium without hormones for the last 16 or 24 h. In vitro fertilization was performed in mTBM medium with 1 � 105 spermatozoa/mL in three different periods (2, 4, and 6 h). For nuclear evaluation, the zigotes were stained by Hoechst 33342 18 h after fertilization. Results were analyzed by PRC GLM of SAS (ANOVA and Tukey test) at a 5% level. Data were expressed as mean � standard error of the mean non transformed. Independent variables were maturation period, effect of calcium ionophore, interaction among calcium*maturation period, and manipulations. In non-fertilized oocytes, there was no effect of maturation period, manipulation, or interaction calcium*maturation, but there was effect of calcium ionophore, having 43.5 � 2.5 more non-fertilized oocytes at 36 h and 43.1 � 5.1 more at 44 h of maturation (P < 0.01). At 36 h oocytes not activated by calcium ionophore showed 29.7 � 4.9 more monospermic embryos than activated oocytes (P < 0.01). However, at 44 h of maturation, activation with calcium ionophore produced 47.4 � 7.2 more monospermic embryos than non activated oocytes. From this study we conclude that monospermic embryo rates were influenced by maturation period, with the best results at 36 h in the absence and at 44 h in the presence of 50 �M of calcium ionophore.
This work was supported by CAPES.