bluuebunny/crossref_metadata_2025 huggingface dataset:
339 EFFECTS OF 17-β ESTRADIOL AND CYSTEAMINE ON IN VITRO MATURATION OF BEEF CATTLE OOCYTES
Authors: Guo L. , Tang B. , Ma X. , Gao F. , Zhang J. B. , Li Z. Y.
Published: August 2010

IVM is a critical step in in vitro bovine embryo production. Some materials supplemented in the IVM medium could improve the maturation rate. It was reported that 17-β estradiol (E2) stimulated the FSH-induced follicular growth and expression of the LH receptor in mural granulosa cells, and cysteamine could enhance glutathione synthesis, protect cells against harmful effects caused by oxidative injuries. In order to optimize IVM system of beef cattle oocytes, effects of different supplements (E2 and cysteamine), and dose of supplements on IVM of beef cattle oocytes were investi- gated in this study. The COCs were collected and cultured in the basic IVM media (TCM-199 + 10% FCS + 10 μg mL-1 FSH + 30 μg mL-1 LH) supplemented with hormonesof 0 μg mL-1 (control), 1 μg mL-1, 2 μg mL-1, and 4 μg mL-1 E2, respectively. Statistical analyzes were performed using SPSS (version 9.0) one-way ANOVA. Oocyte maturation rates (mean ± SEM) of four supplement groups were 60.52 ± 1.4%, 74.91 ± 0.25%, 77.25 ± 2.08%, and 62.20 ± 1.87%, respectively. No statistical differences were observed (P > 0.05) between 1 μg mL-1 and 2 μg mL-1 E2 groups; a similar trend was seen between 0 μg mL-1 and 4 μg mL-1 E2 groups. However, oocyte maturation rates (74.91-77.25%) of groups of 1 μg mL-1 and 2 μg mL-1 E2 were significantly higher (P < 0.05) than those (60.52-62.20%) of the other two groups. When basic IVM medium was sup- plemented with 0 μg mL-1 (control), 50 μg mL-1, and 100 μg mL-1 cysteamine, oocyte maturation rates were 75.25 ± 0.25%, 80.56 ± 0.57%, and 76.83 ± 1.82%, respectively. No significant difference (P > 0.05) was observed among them. However, there was approximately five percent higher maturation rate in the supplement group with 50 μg mL-1 cysteamine than those of groups supplemented with 0 μg mL-1 and 100 μg mL-1 cysteamine. Our results indicated: i) the appropriate supplement of E2 (1 μg mL-1 to 2 μg mL-1) could improve in vitro maturation of beef cattle oocytes. Also, the excessive supplement of E2 (>4 μg mL-1) could inhibit the maturation; ii) appropriate supplement of cysteamine (50 μg mL-1) was benefit to the IVM of beef cattle oocytes.
This work was supported by the grant from national support plan, China, No. 2007BAD55B03; Corresponding author: Z.Y. Li.
340 EPIGENETIC ANALYSIS OF DEVELOPMENTALLY IMPORTANT GENES IN BOVINE OOCYTES OF DIFFERENT ORIGINS
Authors: Heinzmann J. , Hansmann T. , Wrenzycki C. , Zechner U. , Haaf T. , Niemann H.
Published: August 2010

A critical step in assisted reproductive technologies (ART) is the IVM of oocytes. The quality of the oocyte is crucial for successful fertilization and subsequent embryo development. Studies in bovine ART, and epidemiological studies in children from ART, reveal a degree of abnormal development thought to be primarily caused by aberrant DNA methylation patterns in imprinted and non-imprinted genes. Due to the inherent similarities in bovine and human preimplantation embryonic development, bovine oocyte and embryo development is increasingly being used as a model for human development. The goal of this project is to investigate the effects of specific IVM conditions on the DNA methylation profile of bovine oocytes and early embryos and to determine the relationship between DNA methylation and mRNA expression for selected developmentally important genes (PEG3, IGF2R, SNRPN, GDF9, Glut8, PRDX1, Dnmt1A and Dnmt3a/b). Two different IVM systems have been established, the first is the routinely used TCM-199 medium at 39°C, 5% CO2 in air, the second uses synthetic oviduct fluid medium (SOF) as the basic medium with minor modifications (10 mM glucose, 1 mM glutamine) at 39°C, 5% CO2 and reduced oxygen tension (5%). Oocytes for IVM were retrieved by slicing of ovaries collected from local slaughterhouses. Successful maturation was confirmed by extrusion of the first polar body for 78% (TCM) and 72% (SOF) of the oocytes, respectively. Results of quantitative real-time PCR experiments indicate that transcripts of the maternally imprinted gene PEG3 and the paternally imprinted gene IGF2R as well as transcripts of other developmentally important genes (GDF9, PRDX1 and Glut8) are present in immature and IVM oocytes and are applicable targets for further expression and methylation studies. Relative quantification of mRNA transcripts normalised to an external standard revealed that, while expression of the maternally imprinted PEG3 gene is lower in all groups of oocytes (immature: 9.8, matured in TCM: 3.3, and in SOF: 6.4), compared to the paternally imprinted IGF2R (immature: 29.3, TCM: 14.2, SOF: 19.2), no significant difference between the both groups of IVM oocytes could be detected. However, determination of specific effects of IVM will require more replicates and the comparison with IVM oocytes. This is currently underway and together with the ongoing methylation analysis based on the novel identification of differentially methylated regions in bovine imprinted genes, this study will provide deeper insight into the epigenetic profile of bovine oocytes as a potential causative mechanism implicated in the increased incidence of developmental abnormalities in human and bovine ART.
Funded by DFG Research Unit Germ Cell Potential.
349 EFFECT OF CUMULUS CELLS AND MATURATION CULTURE PERIOD OF OOCYTES ON THE SEX RATIO OF IN VITRO-FERTILIZED BOVINE EMBRYOS
Authors: Mingoti G. Z. , Leão B. C. S. , Gottardi F. P. , Alonso R.-V.
Published: August 2010

Studies have reported a high proportion of males from IVP of embryos systems. Factors such as the stage of oocyte maturation at the time of insemination and conditions of the IVC may influence the proportion of sex. Although cumulus cells (CC) improve the fertilization ability of sperm cells, there are some evidence that exposure of sperm to CC increases the proportion of male embryos produced in vitro. The objective of this study was to investigate the effects and interaction between CC and IVM culture period of bovine oocytes on the sex ratio of in vitro-produced embryos. COCs (n = 781) were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (18, 20, 22, and 24 h). Maturation medium consisted of TCM-199 with 0.2 mM pyruvate, 25 mM sodium bicarbonate, 75 μg mL-1 gentamicin, 0.5 μg mL-1 FSH, 100 IU mL-1 hCG, and 10% FBS. After maturation culture, oocytes were inseminated with frozen-thawed spermatozoa. Oocytes were inseminated included in CC (COC) or denuded of such cells (denuded oocytes, DO). All zygotes were then co-cultured in vitro with CC in SOF medium with 0.5% BSA and 2.5% FBS. Cultures were carried out at 38.5°C and 5% CO2 in air. Cleavage and blastocyst development rates were observed, respectively, at 48 (Day 2) and 168 (Day 7) h post-insemination (hpi). Blastocysts were harvested on Day 7, and the sex of the embryos was examined using the pair of primers S4B (Kageyama S et al. 2004 Theriogenology 66, 509-514). The data (mean ± SEM) were analyzed by ANOVA, in which the effects of the existence of CC (COC v. DO) and IVM culture periods (18 h, 20 h, 22 h v. 24 h), and interaction between these variables were evaluated. Multiple comparisons of means were followed using Tukey’s test (P < 0.05). The chi-square test was utilized to test the difference in the proportion of sex of embryos. Independently of the presence or absence of CC during IVF, there was no effect of IVM culture duration (P > 0.05) on cleavage (81.5 to 94.8% for COC; and 71.7 to 75.8% for DO) and blastocyst rates (20.0 to 35.9% for COC; and 10.0 to 15.5% for DO). But independently of IVM culture duration, the comparison between COC and DO groups revealed that absence of CC adversely affected (P < 0.05) the cleavage (87.0 and 73.3%, respectively; averaged results) and blastocyst rates (24.5 and 13.5%, respectively; averaged results). In COC, the proportion of male blastocysts increased with the progression in maturation culture period from 18 up to 22 h (54.2, 65.9, and 83.3%, respectively). But for DO, the proportion of females was higher, with exception of 22 h IVM group (65.4, 32.3, 53.3, and 60.0%, respectively from 18 to 24 h of IVM). These results indicate that the sex ratio of in vitro-fertilized embryos is apparently influenced by the maturation culture period of the oocytes and by the presence of CC.
FAPESP.
351 POLARIZED LIGHT MICROSCOPY: DETECTION OF MICROTUBULES AND ITS EFFECTS ON THE VIABILITY OF IN VITRO-MATURED PORCINE OOCYTES
Authors: Molina I. , Muñoz M. , Díez C. , Gómez E. , Martínez E. A. , Martín D. , Trigal B. , Carrocera S. , Gil M. A. , Sánchez-Osorio J. , Caamaño J. N.
Published: August 2010

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) is used as a tool in human and, recently, in farm animals assisted reproductive technologies. PLM could be useful for a non-invasive evaluation of the meiotic spindle. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured porcine oocytes and to examine the effects of PLM on the oocyte developmental competence. Cumulus-oocyte complexes from slaughterhouse ovaries were matured in vitro for 42 h as described by Gil et al. (2004 Theriogenology 62, 544-552). In the first experiment, a total of 97 oocytes from 6 replicates were placed individually in 10-μL drops of TCM-199-Hepes-FCS in a glass Petri dish. PLM was used to detect the presence of polymerized protein which could be forming a meiotic spindle. The presence of polymerized protein and a meiotic spindle was confirmed in individual oocytes by inmunostaining and chromatin detection as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191-201). In the second experiment, a total of 160 oocytes from 4 replicates were exposed or not (controls) to PLM for 10 minutes. Thereafter, the oocytes were parthenogenetically activated and cultured in vitro. Cleavage rate, total blastocyst rate, expanded blastocyst rate on Day 7 and total cell numbers in expanded blastocysts were assessed. Data were analyzed by GLM procedure of SAS. There was a positive correlation (r = 1; P < 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by inmunostaining. A positive PLM signal was detected in 98.9% of the oocytes. A barrel-shape spindle was observed in 94.8% of the individual samples by inmunostaining and all of these oocytes were positive to PLM. Moreover, oocytes exposed to PLM did not differ significantly from controls on cleavage rate (83.7 ± 1.5 v. 84.4 ± 1.5), total blastocyst rate (36.9 ± 3.6 v. 41.2 ± 3.6) and expanded blastocyst rate on Day 7 (21.9 ± 1.7 v. 26.2 ± 1.7), respectively. There were also no differences in total cell numbers counted in expanded blastocysts (32.8 ± 2.6 v. 35.6 ± 2.5). These results indicate that polarized light microscopy did not exert detrimental effects on porcine oocyte developmental competence and it seems an efficient system to detect polymerized protein in in vitro-matured porcine oocytes.

Grant support: INIA: RZ2007-00013-00-00. I. Molina, M. Muñoz, B. Trigal and D. Martín are sponsored by INIA, RYC08-03454, Cajastur and PTA2007-0268-I, respectively.
352 DISSOCIATION OF NUCLEAR AND CYTOPLASMIC MATURATION OF BOVINE OOCYTES IN DEFINED CULTURE SYSTEM
Authors: Oliveira e Silva I. , Vasconcelos R. B. , Caetano J. V. O. , Gulart L. V. M. , Camargo L. S. A. , Báo S. N. , Rosa e Silva A. A. M.
Published: August 2010

Insufficient cytoplasmic maturation of MII oocytes may be one reason of the low rate of embryo production in vitro. To solve this problem, a two-step procedure of IVM is tested. In this case, there is a prematuration period (PMP) in which meiosis is arrested, followed by a resumption period (RP) during which the oocytes are liberated from the inhibition of meiosis. Preliminary studies have shown that a defined medium (DM) inhibits nuclear oocyte maturation (NM) (Unpublished data). Thus, our purpose was to verify if meiosis arrest is followed by an inhibition of cytoplasmic maturation (CM). Herein we investigated the effects of DM on NM and CM using the two-step procedure for IVM. COCs were selected from bovine ovaries and cultured in non-defined medium (NDM), composed of TCM-199 suplemented with 10% fetal calf serum and 200 ηg mL-1 FSH or in a defined medium (DM), MEMα supplemented with 0.1% Polyvinyl Alcohol. Both media are from Invitrogen-Gibco/BRL, Grand Island, NY, USA. In the PMP (experiment 1), COCs (n = 232) were distributed in G1 (NDM) or G2 (DM) and cultured for 24 h as usual. After this, NM and CM were analyzed. As inhibition of meiosis was observed in G2, a second step of culture (experiment 2) was performed to evaluate the ability of the COCs to reverse meiosis arrest. Thus, COCs (n =202) were distributed in G1 (NDM) or G3 (DM +NDM). COCs from G1 were cultivated in NDM for 24 h, and that ones from G3 in DM for 24 h followed by cultivation in NDM for an additional 24 h (RP). After this, NM and CM were analyzed. The CM evaluation (distribution of organelles) was performed in fresh oocytes (0 h) and in G1, G2, and G3 after usual preparation for transmission electron microscopy (TEM) analysis. The NM data were analyzed by Chi-square test. After the PMP period, G1 COCs presented 83.17% (84/101) of complete nuclear maturation whereas G2 presented 74.05% (97/131) of NM inhibition (oocytes in GV or GVBD) (P < 0.05). This meiosis arrest observed in DM was reversed after 24 h of cultivation in NDM, 79.17% (90/110) of G3 COCs were at MII. The analysis of CM has shown fresh COCs with ultrastructural characteristics of immaturity, as presence of mitochondria in the periphery of the ooplasm, and cortical granule clusters in the ooplasm; G1 COCs with mature cytoplasmic characteristics as mitochondria spread throughout the ooplasm and cortical granules located close to the oolema and G2 COCs with both immature and mature characteristics as presence of ooplasm granule clusters and presence of mitochondria spread throughout the ooplasm. G3 have shown mature cytoplasmic characteristics in the end of RP. In conclusion, a two-step procedure of IVM using a DM was able to maintain meiosis arrest while the cytoplasm undergoes maturation (G2). This could be a strategy to improve the rate of viable embryos obtained at in vitro embryo production.

FAP-DF, Finatec, CNPq, Capes.
360 EMBRYONIC DEVELOPMENT AND STEROIDOGENESIS OF BOVINE CUMULUS - OOCYTE COMPLEXES MATURED IN α-MEM MEDIUM SUPPLEMENTED WITH PVA OR PVP-40
Authors: Vireque A. A. , Camargo L. S. A. , Ferreira E. M. , Rosa e Silva A. A. M. , Watanabe Y. F. , Bos-Mikich A. , Martins W. P. , Campos J. R. , Navarro P. A. A. S. , Ferriani R. A.
Published: August 2010

The influence of the culture medium and its supplements on IVM and steroid secretion by bovine COCs was examined. Immature COCs were matured for 24 h in a-MEM supplemented with IGF-1 (10 ng mL-1), insulin (100 ng mL-1) and 0.1% polyvinyl alcohol (PVA; Sigma Chemical Co., St. Louis, MO, USA) or 0.1% polyvinylpyrrolidone-40 (PVP; molecular weight of 40 000; Sigma). Neither FSH nor LH was used in either treatment. The control group consisted of COCs matured in TCM supplemented with FSH (20 μg mL-1) and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Steroid production was measured in culture medium after IVM and cumulus cells (CC) aromatase activity was estimated by estradiol (E2) production and by the determination of the ratio of E2 to testosterone (T). Cleavage, blastocyst, and hatching rates were evaluated 168-224 h post-insemination. Hormone determination data were analyzed using the GraphPad 5.0 for Windows software (GraphPad Software, San Diego, CA, USA). The means and standard deviations were first calculated for all variables, followed by the Kolmogorov-Smirnov normality test. Since the values were not normally distributed, the treatments were compared by the Kruskal-Wallis test. Blastocyst formation, cleavage, and hatching rates were analyzed by the chi-square test. The level of significance was set at P < 0.05 in all analyses. High aromatase activity (E2:T ratio > 1.0) was detected in the culture medium of both chemically defined IVM systems. E2 concentrations were 22.86 ng mL-1 and 22.46 ng mL-1 for PVA and PVP-40, respectively, and 0.27 ng mL-1 (P < 0.001) for the control group. Progesterone secretion was lower in a-MEM + PVP-40 medium. Testosterone was not secreted by COCs matured in control medium. There was a significantly higher cleavage rate in the control group, but no differences (P > 0.05) in blastocyst (48.92%, 49.56%, and 44.21%) or hatching (38.46%, 41.96%, and 40.78%) rates between the PVA, PVP, and control groups, respectively. Our results show that CC of COCs cultured in serum-supplemented medium show decreased aromatase activity. Also, the addition of IGF-I/insulin and PVA or PVP-40 to IVM medium had no significant effect on the rates of oocyte maturation and embryonic development when compared with results obtained in medium supplemented with estrous cow serum and FSH.

Supported by FAPESP/FAEPA-HCFMRP/USP.
361 EFFECT OF HORMONES, EGF AND β-MERCAPTOETHANOL ON IN VITRO MATURATION OF CAPRINE OOCYTES
Authors: Yadav P. , Kharche S. D. , Goel A. K. , Jindal S. K. , Sharma M. C.
Published: August 2010

In vitro maturation of oocytes is an integral part of in vitro culture system. In almost all studies of mammalian in vitro maturation, the basic medium is supplemented with serum and hormones. The maturation medium and selection of protein supplements, growth factors, antioxidants and hormones for IVM play an important role in subsequent in vitro fertilization and in vitro embryo development. The objective of the present experiment was to study the effect of exogenous addition of hormones, epidermal growth factor, insulin and β-mercaptoethanol to the maturation medium for in vitro maturation of caprine oocytes. A total of 1540 oocytes were collected from slaughtered goat of 1.5 to 2.5 year of age and randomly divided in to treatment groups. Group 1; COCs were matured in TCM-199 medium containing 10% calf serum (NCS) and 3 mg mL-1 BSA (used as a base medium) for control, Group 2; COCs were matured in a base medium supplemented with hormones (5 μg mL-1 FSH, 5 μg mL-1 LH and 1 μg mL-1 estradiol-17β), Group 3β COCs were matured in a base medium supplemented with 50 ng mL-1 insulin, Group 4β COCs were matured in a base medium supplemented with hormones and 10 ng mL-1 EGF, Group 5; COCs were matured in a base medium supplemented with hormones and 50 mM β-mercaptoethanol and Group 6; COCs were matured in a base medium supplemented with hormones and 50 ng mL-1 insulin. These COCs were matured at 38.5°C in 5% CO2 in air for 27 h. After the maturation, oocytes were separated from cumulus and corona cells by treatment with 0.1% hyaluronidase and by passing through a fine pipette. They are them fixed in 2.5% glutaraldehyde, stained with DAPI and observed under fluorescent microscope for evidence of nuclear maturation. The maturation rates in groups 1 to 6 were 33.6%, 38.0%, 39.7%, 60.0%, 37.4%, and 44%, respectively. Statistical analysis (ANOVA) after arcsin transformation revealed that the maturation rate in group 4 was statistically significant (P < 0.05) as compared to those in groups 1, 2, 3, 5, and 6. The results suggest that the supplementation of EGF in maturation medium significantly enhances the in vitro maturation rate of caprine oocytes.
367 INCIDENCE OF CHROMOSOMAL ABNORMALITIES IN MALE AND FEMALE BOVINE EMBRYOS DERIVED FROM SEX-SORTED SPERM
Authors: Garcia-Herreros M. , Carter T. F. , Villagómez D. A. F. , King W. A. , Lonergan P.
Published: August 2010

Previous studies from our laboratories have shown that developmental rates following IVF with frozen-thawed sex-sorted sperm are lower than with non-sorted sperm and that this may be related to a difference in the kinetics of development. In addition, it is known that the incidence of chromosomal abnormalities in in vitro produced bovine blastocysts is significantly greater than that observed in blastocysts derived in vivo (Viuff D et al. 1999 Biol. Reprod. 60 123-128). In this study, FISH with X- and Y-chromosome-specific probes was used to assess the extent of chromosome abnormalities in bovine blastocysts derived from IVF with X-sorted, Y-sorted, or unsorted semen from the same bull (n = 15 blastocysts per group). Individual blastocysts were transferred to a small drop of spreading solution (0.01 N HCl, 0.1% Tween 20) on a glass slide. Following digestion of the zona pellucida and cytoplasm, the isolated nuclei were fixed in 3:1 methanol:glacial acetic acid at 4°C for 24 h. Slides were then air dried, incubated at 60°C overnight and stored at -80°C until FISH analysis. The StarFISH X-Y FISH bovine sex test kit was used following minor modification and optimization of manufacturer protocols for use with blastocyst preparations. Slides were examined with standard fluorescence microscopy. Overall, 80% (36/45) of blastocysts were considered to be mixoploid i.e. were a mixture of normal diploid and aneuploid and/or non-diploid cells (10/15 for X-sorted, 14/15 for Y-sorted, and 12/15 for unsorted; P > 0.05). The severity of the chromosomal aberration was relatively low in all groups, however; in other words, only a small fraction of the total number of cells per embryo were chromosomally abnormal (1.64%, 5.62%, and 7.93% for X-sorted, Y-sorted, and unsorted, respectively). The most common chromosome abnormalities observed were diploid-triploid-tetraploid mixoploidies. Interestingly, 20% (3/15) of blastocysts derived from unsorted sperm were chimeric (XX/XY). If such chimeric embryos had been sexed by PCR they would have been ascribed as males. In addition, FISH with probes for non-sex chromosomes would not have revealed such chimerism. In conclusion, the incidence of chromosomal abnormalities was not different between blastocysts derived from sorted and unsorted sperm. The occurrence of mixed sex chimeras amongst blastocysts derived from unsorted sperm is interesting and requires further study. Whether it represents a previously undetected abnormality of IVF is as yet unknown.

MGHwas supported by a Posdoctoral Research Grant (2008-0198) from the Spanish Government.
370 ASSESSMENT OF SWIM-UP AND Percoll DENSITY GRADIENT FOR SPERM SEX PRESELECTION
Authors: Lucio A. C. , Resende M. V. , Dernowsek-Meirelles J. A. , Perini A. P. , Diaz I. P. S. , Oliveira L. Z. , Carmo A. S. , Miguel M. C. V. , Moreira-Filho C. A. , Alves B. C. A. , Albuquerque L. G. , Hossepian de Lima V. F. M.
Published: August 2010

The low cost of sperm sexing methods combined with in vitro embryo production in genetic improvement programs can increase the profitability of cattle production, in particular when it does not decrease reproductive efficiency. The aim of this work was to evaluate the sex ratio deviation of thawed bovine semen processed by density gradient centrifugation and swim-up. Semen doses were collected from ten bulls of different breeds, and each experimental group was replicated ten times.A Percoll™ gradient was prepared by mixing Dulbecco’s Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) isotonic solutions with Percoll™ (GE Healthcare Bio-Science AB, Uppsala, Sweden) stock solution with 0.3% BSA, resulting in densities ranging from 1.110 to 1.123 g mL-1. The layers of discontinuous density gradient were disposed from the larger density (bottom of the tube) to the smaller into 15-mL conical centrifuge tubes. About 40 million sperm were overlaid on Percoll™ gradient and were centrifuged at 500 g for 15 min, at 22°C.The thawed semen samples were deposited in 15-mL conical centrifuge tubes containing 5 mLof DMEM and centrifuged twice at 300 g for 5 min for extender removal. After the second centrifugation, the supernatant was discarded and the sediment diluted in 1.0 mL of DMEM supplemented with 0.3% BSA. The tube was maintained in an incubator at 38.5°C and 5% CO2 for 1 h. One milliliter of supernatant was recovered and evaluated for the sperm motility and vigor. Eighty million sperm were submitted to the swim-up method. For the association of density gradient and swim-up, the swim-up supernatant was recovered and overlaid on Percoll™ gradient. The gradients were centrifuged and the sperm pellet recovered. The recovered sperm were submitted to DNA extraction with phenol-chloroform. Quantitative Real Time PCR (Parati et al. 2006 Theriogenology 66, 2202-2209) was used for the determination of the proportion of X-chromosome-bearing sperm, after centrifugation through density gradient and after swim-up. The results of amplification were analyzed in 7500 Sequence Detection System Software (Applied Biosystems, Foster City, CA, USA), using Relative Quantification (Ct) Study assay. The results of X-sorted sperm samples analyzed by multiple pairwise comparisons (Tukey) were not different (P > 0.05). The percentage of X-chromosome-bearing sperm after the density gradient comprised 50.3% of the sample, after the swim-up was 49.9% and after swim-up combined with Percoll™ gradient centrifugation was 56.6%. Therefore, sperm sex selection using Percoll™ gradient centrifugation and swim-up was not effective.
375 PERFORMANCE OF DIFFERENT BOVINE BREEDS ON IN VITRO COMMERCIAL EMBRYO PRODUCTION USING SEX-SORTED SEMEN
Authors: Yamazaki L. T. S. , Benzi E. C. D. , Corneglian D. P. , Romano M. , Landim Junior L. P. , Castro Netto A. , Guidorizzi F. , Santos G. L. , Yamazaki W.
Published: August 2010

Commercial bovine in vitro production (IVP) systems using sex-sorted semen, instead of the invasive technique involving embryo biopsy followed by PCR, have a superior influence on embryo production rates. However, due to damage from flow cytometric procedures and cryopreservation, blastocyst rates using sex-sorted semen remain lower than from conventional non-sorted semen. The aim of this study was to describe embryo production rates using sex-sorted semen during the past year in different bovine breeds. Cumulus oocyte complexes obtained by ovum pickup (OPU) from high genetic merit donor cows were matured (TCM-199, supplemented with FCS, LH, FSH, estradiol, pyruvate, and antibiotics) for 24 h and fertilized (Fert-TALP supplemented with BSA, phenylalanine, and heparin) for 18-22 h (Day 0) in vitro. Frozen sex-sorted semen samples from different bulls were thawed at 35°C for 30 s in a water bath and then selected by centrifugation at 800 g on discontinuous Percoll™ gradients (45 : 90%). Presumptive zygotes were transferred to culture medium (SOFaa supplemented with BSA and FCS) on Day 1 and blastocyst rate was evaluated on Day 7. During the past year, sex-sorted semen was used more frequently on Bos taurus (Holstein), Bos indicus (Gyr), and crossbred (Gyrolando) milk cattle than in meat breeds (Nelore, Guzera, and Brahman). The large numbers of oocytes routinely recovered in Nelore cows could be attributed to the female valorization in the market due to high potential in new generations and repositioning of genetic value. The effect of subspecies (Bos taurus taurus and Bos taurus indicus) could be also observed in blastocyst rate, with better results than Holstein, showing the same tendency when conventional (non-sexed) semen is used. Embryo production in Brahman and Nelore with sex-sorted semen was lower than with conventional (non-sexed) semen, probably due to damage to the sperm during the sorting process, thereby changing patterns of fertilization, involving the acrosome reaction, for example. This points to the need for more studies on cellular integrity during the sorting process as well improvement of IVP procedures with sex-sorted semen.

Table 1.Embryo production rates according to different bovine breeds
376 FAILURE OF SPERM DECONDENSATION AFTER INTRACYTOPLASMIC SPERM INJECTION IN SWAMP BUFFALO (BUBALUS BUBALIS)
Authors: Chankitisakul V. , Tharasanit A. , Thaseephoo K. , Techakumphu M.
Published: August 2010

Intracytoplasmic sperm injection (ICSI) has been intensively used to examine the early events of gamete activation, but few studies have been reported for swamp buffalo. The first objective (Exp. 1) was to compare the developmental competence of oocytes after ICSI using either live or dead frozen-thawed spermatozoa. Matured oocytes were fertilized by ICSI using live (n = 148) or dead (n = 151) spermatozoa, followed by chemical activation using calcium ionophore (A23187) and cyclohexamide (CHX) in SOF medium. In vitro fertilization (n = 149) served as thecontrol. Cleavage rate was recorded on Day 2 and blastocyst formation rate was evaluated on Day 7. The second objective (Exp. 2) was to examine the effects of ICSI and activation regime on the decondensation of buffalo spermatozoa. A total of 148 matured oocytes were subjected to ICSI. The sperm-injected oocytes (n = 87) were then activated using the activation protocol as described in Exp. 1. The ICSI oocytes without activation (n = 61) and sham-injected oocytes with activation (n = 35) were used as controls. Nuclear changes of presumptive zygotes were mor- phologically evaluated for pronuclear formation using 4′,6-diamidino-2-phenylindole procedure and epifluorescent microscopy at 18 h post-ICSI. Statistical differences were determined among the groups using chi-square test. In Exp. 1, the results showed that the percentages of cleavage and blastocyst formation rate were 79.7, 77.0, and 41.6% and 33.8, 30.5, and 14.8% in the live sperm, dead sperm, and IVF groups, respectively. Embryo development rates did not significantly differ between ICSI groups; however, these rates were significantly higher than in the IVF group (P < 0.05). In Exp. 2, the pronuclear formation rate was significantly higher in the ICSI with chemical (70.1%) and sham injection with chemical (60.6%) groups than in the ICSI without chemical group (3.2%; P < 0.01). However, most of the presumptive zygotes with pronuclear formation from the ICSI with chemical activation group showed only intact sperm heads instead of the full male pronuclear formation. Our study suggests that the chemical activation directly affected the female pronuclear formation and embryo development but that it was not associated with the male pronuclear formation. It is postulated that ICSI oocytes that developed to cleavage and blastocyst stages underwent parthenogenesis after chemical activation.

This work was supported by TRF-MAG (MRG-WII515S056) and CHE-TRF Senior Research Fund (RTA5080010).
377 EMBRYO PRODUCTION BY OVUM PICKUP-INTRACYTOPLASMIC SPERM INJECTION-IVC IN AN EQUINE OVUM PICKUP PROGRAM USING SEMEN FROM FERTILE AND INFERTILE STALLIONS
Authors: Colleoni S. , Duchi R. , Lazzari G. , Galli C.
Published: August 2010

The introduction in equine reproduction of ovum pickup (OPU) combined with intracytoplasmic sperm injection (ICSI), IVC, and embryo transfer, has allowed for the production of offspring from donors and stallions that could not reproduce by conventional techniques. For this reason, we used in our OPU-ICSI-IVC program both fertile stallions and stallions with field records of low or no fertility. Overall, 805 and 584 OPU oocytes were fertilized with sperm from fertile and infertile stallions, respectively. Cleavage rate was statistically lower in the latter group (65.94 v. 59.24%, chi square test; P < 0.05) but embryo development was similar (11.67 v. 8.20% blastocysts/injected oocytes, chi-square test). In order to further investigate the stallion effect on embryo development, we selected 3 stallions with low (A) or no (B, C) fertility in the field and we compared the results of the OPU program with embryo development obtained using oocytes recovered from abattoir ovaries and matured, fertilized, and cultured in vitro as the OPU oocytes. Part of the abattoir oocytes was fertilized with a stallion with known high fertility both in vivo and in vitro (abattoir fertile). Overall, the results (shown in the table) suggest a reduction in the efficiency of stallions A, B, and C compared with to the fertile stallion used as control (10.79, 7.69, and 5.0% v. 17.35%, respectively). For stallions A and B, the efficiency was further reduced in the OPU setting, indicating that the female component can play a role in the overall efficiency of the procedure. In particular, 4 mares out of 8 had a history of no pregnancy and all mares had some rate of inbreeding with the respective stallion used for the ICSI. Instead, the oocytes from the abattoir ovaries were collected in large pools from several mares, representing an average oocyte quality, and the mares were of different breed than the stallions. All data were analyzed by chi-square test and significance was set at P < 0.05. In conclusion, we demonstrated that, for those stallions in which fertility in the field is low or absent, OPU-ICSI-IVP is a suitable choice to obtain embryos, although the efficiency is variable depending not only on the stallion but also on the origin of the oocytes.

Table 1.Stallion effect on embryo development of ovum pickup (OPU) and abattoir oocytes

This work was supported by Fondazione Cariplo and Regione Lombardia.
38 APPLICATION OF A FIVE-DAY PROGESTERONE-BASED SYNCHRONIZATION PROTOCOL FOR TIMED AI WITH SEXED SEMEN IN DAIRY HEIFERS
Authors: Rabaglino M. B. , Risco C. A. , Thatcher M. J. , Santos J. E. P. , Thatcher W. W.
Published: August 2010

It is expected that pregnancy per AI with sexed semen would be 70 to 75% of that with conventional semen, and the use of sexed semen with timed AI (TAI) protocols is discouraged (DeJarnette et al. 2009 Theriogenology 71, 49-58). It was hypothesized that the 5-day Co-Synch + CIDR (controlled internal drug-release) protocol with 1 injection of PGF2α would be an acceptable reproductive management program for TAI of dairy heifers with sexed semen. The objectives were to compare pregnancy per TAI (P/TAI) using conventional or sexed semen for the first TAI (experiment 1), and to evaluate P/TAI of a reproductive management program with sexed semen for the first TAI and sexed or conventional semen for the second TAI (experiment 2). A total of 1,000 Holstein heifers between 13 to 14 months of age were synchronized with the 5-day Co-Synch + CIDR protocol, that consisted of an administration of GnRH (100 μg i.m. Cystorelin®, Merial Ltd., Iselin, NJ, USA) and a CIDR insert containing 1.38 g of progesterone inserted at Day 0; on Day 5 the CIDR was removed and one injection of PGF2α (25 mg, i.m. Lutalyse®, Pfizer Animal Health, New York, NY, USA) administered; on Day 8 a second injection of GnRH was administered concurrent with TAI. In experiment 1, 198 heifers were assigned randomly to be TAI with conventional (n = 98) or sexed (n = 100) semen for the first TAI. Commercial straws with sexed or conventional semen were obtained from 2 sires. In experiment 2, a total of 802 heifers received TAI to the first service with sexed semen. For the resynchronized TAI, nonpregnant heifers at 32 days after the first TAI were resynchronized with the 5-day Co-Synch + CIDR and were TAI with either sexed semen (n = 114) or with conventional semen (n = 373). Pregnancy per TAI to first and second TAI was analyzed by logistic regression using the GLIMMIX procedure of SAS (SAS Version 9.1 for Windows, SAS Institute, Cary, NC, USA) with heifer treated as a random effect. In experiment 1, P/TAI was 51.0% and 42.0% at 45 days for conventional and sexed semen, respectively (P = 0.1). Pregnancy per TAI with sexed semen was 82.3% of conventional semen. In experiment 2, P/TAI at first service with sexed semen was 35.9% at 45 days after TAI. For the second TAI, P/TAI was 40.4% with sexed semen at 45 days and 59.2% with conventional semen at 60 days (P < 0.001). As expected, P/TAI was lower with sexed compared to conventional semen. Nevertheless, the application of the 5-day Co-Synch + CIDR protocol with one injection of PGF2α, as a reproductive management program for TAI of dairy heifers, achieved an acceptable P/TAI with sexed semen. We conclude that sexed semen can be used with TAI to effectively manage reproduction in dairy heifers by removing the challenges of detection of estrus and increase the number of females born.
387 DERIVATION OF PUTATIVE EMBRYONIC STEM CELLS FROM PORCINE PARTHENOGENETIC BLASTOCYSTS AND THE LOSS OF PARENTAL-SPECIFIC EXPRESSION PATTERNS IN Igf2/H19 GENES
Authors: Lee C. K. , Uh K. J. , Park J. K. , Kim H. S. , Kim H. M. , Hwang J. Y. , Ka H.
Published: August 2010

Porcine embryonic stem cells (ESC) can be a useful tool for the production of a transgenic animal and the study of developmental gene regulation. The study of porcine parthenogenetic ESC might also provide advantages in the understanding of changes in human parthenogenetic embryonic stem cells in the culture environment. Because human embryonic stem cells must be maintained stably for therapeutic uses, parthenogenetic porcine embryonic stem cells can give us precious information to help understand human parthenogenetic embryonic stem cells. Three putative porcine embryonic stem cell lines were derived from 99 parthenogenetic embryos. Cumulus-oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro. Diploid parthenogenetic zygotes were produced by electrical activation followed by cytochalasin B treatment to suppress second polar body extrusion. Embryos were cultured to the blastocyst stage. Hatched blastocysts were directly cultured on mitomycin C-inactivated murine embryonic fibroblasts as feeder layers. Primary colonies were formed after 7 days of culture, and the colonies were transferred to new culture dishes 7 days after. They were passsaged every 5 days by physical dissociation, with one colony divided into small clumps and maintained for over 30 passages. These cells morphologically resembled human embryonic stem cells and consistently expressed the markers of pluripotent cells such as alkaline phosphatase, NANOG, OCT-4, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. They could be maintained holding the previous characteristics after cryopreservation. Furthermore, we conducted experiments to confirm the expression patterns of the imprinted genes Igf2 and H19 in these ESC and IVF/parthenogenetic blastocysts using quantitative real-time PCR. At the blastocyst stage, the 2 genes were expressed in a parental-specific manner according to their origins in normal fertilized embryos and uniparental embryos. The putative parthenogenetic ESC, on the other hand, showed a high expression of Igf2, the paternally expressed gene, when compared with their blastocyst counterparts. Current work aims to confirm the authenticity of these ESC via teratoma formation in severe combined immunodeficiency mice following injection with these putative parthenogenetic ESC.

This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).
390 ISOLATION AND ADIPOGENESIS OF GRIZZLY BEAR MESENCHYMAL STEM CELLS
Authors: Maki A. J. , Omelogu I. , Monaco E. , McGee-Lawrence M. E. , Bradford R. M. , Nelson O. L. , Robbins C. T. , Donahue S. W. , Wheeler M. B.
Published: August 2010

During winter hibernation, grizzly bears (Ursus arctos horribilis) do not eat but instead rely on internal fat stores as a primary source of metabolic energy. The resulting seasonal fluctuations in appetite and body mass make the grizzly bear a naturally occurring animal model for human conditions such as obesity and anorexia. An in vitro model of hibernating bear stem cells might enhance our understanding of processes such as stem cell proliferation and differentiation. Mesenchymal stem cells, derived from bone marrow and adipose tissue among others, differentiate into adipocytes and might play important roles in energy metabolism. In the current study, we examined the in vitro viability and morphology of mesenchymal stem cells isolated from grizzly bear adipose tissue (ADSC) and bone marrow (BMSC); these ADSC and BMSCs underwent adipogenic differentiation for 0, 7, 14, 21, and 28 days. Bone marrow stem cells and ADSC were isolated using mechanical disaggregation, collagenase digestion, centrifugation, and plating onto tissue culture polystyrene. Cell viability and proliferation was quantified using the colony forming unit assay and a hemocytometer. Both stem cell types were differentiated into adipocytes using 10 μM insulin, 1 μM dexamethasone, and 0.5 mM isobutylmethylxanthine (all Sigma- Aldrich, St. Louis, MO, USA) with the addition of 10% fetal bovine (FBS) or bear serum from the active feeding period. Adipogenic differentiation was confirmed using Oil Red O and quantified using ImageJ. Statistical analysis was performed using an unpaired t-test between treatments of the same time point. All cells were isolated within 28 h of tissue harvest. Adipose-derived stem cells formed an average of 11 colonies (0.011%), whereas BMSC formed 1.5 colonies (0.0015%) per 100 000 cells. Doubling time forADSC was approximately 54 h in 10% FBS. BothADSC and BMSC had an initial spindle-shaped morphology, which gradually became more rounded during adipogenic differentiation. For bear serum at Day 28, ADSC had a significantly (P < 0.01) greater stained area per cell than did BMSC. In summary, both types of mesenchymal stem cells successfully differentiated into adipocytes and maintained viability. In conclusion, grizzly bear mesenchymal stem cells canbesuccessfully isolated, expanded, and differentiated in culture. These results allow for future studies using the bear as an in vitro model for fat metabolism during hibernation and active periods.

This work was partially supported by the Carle Foundation Hospital, the Intel Scholar’s Research Program, USDA Multi-State Research Project W1171, and the Illinois Regenerative Medicine Institute (IDPH # 63080017). In addition, the authors would like to thank Agatha Luszpak for support with the analysis.
394 CHARACTERIZATION OF BOVINE ADULT-DERIVED ADIPOSE STEM CELLS FOR USE IN NUCLEAR TRANSFER
Authors: Picou A. A. , Wilson J. , Dresser B. , Gentry G. T. , Godke R. A. , Bondioli K. R.
Published: August 2010

Adipose tissue is an abundant source of adult-derived cells that have displayed multipotent properties in vitro. The goal of this research was to study the characteristics of bovine adipose-derived adult stem (ADAS) cells to determine the feasibility for use in NT. Adipose tissue was isolated from the brisket of adult cattle postmortem. Cells were isolated by incubation for 2 h with 0.25% collagenase solution, separation of stromal cells by centrifugation, and selection by adherence to plastic. The lifespan and growth characteristics for culture conditions were determined by a 2 × 2 factorial with DMEM or DMEM:F12 and with or without growth factor (GF) supplementation.A two-way ANOVA, followed by multiple pair-wise comparisons using Tukey’s test when applicable, was used to detect differences in population doublings (PD) until senescence for media treatments and GF supplementation. Dulbecco’s modified Eagle’s medium with GF supported significantly less (PD) (P > 0.05) than DMEM : F12. The average lifespan was approximately 30 PD, with a cell length of 48 h until passage 8 (P8). As cells approached replicative senescence, the cell cycle length was inconsistent. Two ADAS and one adult-derived skin fibroblast cell lines from different animals were subjected to differentiation conditions for adipocytes, chondrocytes, and osteoblasts at P2, P6, and P11. Differentiation was confirmed by histological staining. Passage 2 ADAS cells differentiated more efficiently than did P6, P11, or skin fibroblasts. Global levels of DNA methylation and histone acetylation were analyzed from P1 to P6 in 3 sets of cell lines consisting of ADAS and skin cells from the same animals by immune staining and flow cytometry. There was no significant difference (P > 0.05) between cell types by one-way ANOVA. Nuclear transfer was performed using ADAS cells as donor cells and commercially supplied oocytes. Mature, enucleated oocytes were reconstructed with either adult skin fibroblasts or ADAS cells. The percentage of cleaved and blastocysts from ADAS cells (62% and 8%, n = 163) and skin fibroblasts cells (42% and 8%, n = 170) were not different (P > 0.05) by chi-square analysis. Interspecies NT was attempted with eland (Taurotragus oryx) ADAS cells and enucleated bovine oocytes. Two groups of enucleated oocytes were reconstructed with bovine (n = 234) and eland (n = 290) ADAS cells. There was no significant difference between the number of cleaved embryos (38% and 39%) or blastocysts formed by chi-square analysis. A total of 3 interspecies embryos (1%) and 5 bovine embryos (14%) developed to blastocysts. Bovine ADAS cells are not more efficient than bovine adult-derived skin fibroblasts as donor cells, but they do represent a viable option for use in NT because of their higher in vitro development. Eland ADAS cells resulted in development to the blastocyst stage after interspecies NT.
406 EFFECT OF DIFFERENT FOLLICLE-STIMULATING HORMONE DOSES ON SUPEROVULATORY RESPONSE OF Red Sindhi COWS
Authors: Carvalho B. P. , Mello M. R. B. , Baldrighi J. M. , Campanati J. S. , Mello R. R. C. , Dias A. J. B.
Published: August 2010

The use of bovine embryo transfer to increase the reproductive potential of animals with high genetic merit is unquestionable. However, studies that evaluate the response to exogenous FSH stimulation in Red Sindhi cows are rare. Therefore, the objective of this study was to compare the superovulatory response of Red Sindhi cows treated with 3 different doses of FSH (Folltropin®-V, Bioniche Animal Health, Belleville, Ontario, Canada). The animals used had body condition scores ranging from 3.5 to 4.0 (1-5 scale), were between 6 and 10 years of age, and were randomly assigned to 1 of 3 treatment groups. Three doses of FSH were administered: 100 mg (n = 16); 133 mg (n = 13), and 200 mg (n = 14). The treatments were initiated on random days of the estrous cycle. On Day 0, animals received 2 mg of estradiol benzoate i.m. (Estrogin®, Farmavet, Jaboticabal, Brazil) and an intravaginal device (CIDR; InterAg, Hamilton, New Zealand) with 1.9 g of progesterone. On Day 4, donors received FSH in 8 i.m. injections that decreased in dose during 4 consecutive days. A dose of 265 μg of cloprostenol (Ciosin®, Schering-Plough, Cotia, Brazil) was given i.m. concomitantly with the fifth FSH injection. Twelve hours after the last FSH injection, donors received 25 μg of lecirelin i.m. (GnRH; Gestran Plus®, ARSA SRL, Buenos Aires, Argentina). TheAI were performed 12 and 24 h after GnRH. Seven days after the first AI, embryos were recovered and classified as described in the IETS manual. Data were evaluated using KruskalWallis test at a 5% level of significance. Ovarian ultrasonography was also performed to count the number of CL present at the time of ova/embryo collection. Results are summarized in Table 1. The number of CL and total ova/embryos was higher in donors receiving 200 mg of FSH than in those in the other groups. However, the number of transferable or cryopreserved embryos did not differ among groups. In conclusion, the dosage of 200 mg of Folltropin®-V seemed to be the more adequate dosage for this breed of cattle.

Table 1.Effect of different follicle-stimulating hormone (FSH) doses on CL number, total structures, transferable embryos, and cryopreserved embryos in Red Sindhi cows (mean ± SEM)

Financial support provided by FAPERJ (E26-170.529/2007).