bluuebunny/crossref_metadata_2025 huggingface dataset:
213 POLARIZED LIGHT MICROSCOPY REVEALS AN ASSOCIATION BETWEEN ZONA BIREFRINGENCE INTENSITY AND EMBRYONIC DEVELOPMENT IN BOVINE OOCYTES AND ZYGOTES
Authors: Mohammadi-Sangcheshmeh A. , Koester M. , Schimming T. , Tesfaye D. , Montag M. , Schellander K. , Hoelker M.
Published: December 2008

To increase the efficiency of human IVP, noninvasive parameters to predict the developmental competence of oocytes and zygotes would be useful for selecting the right embryo to transfer. However, human oocytes and zygotes for experimental studies are rare. Therefore, using a bovine model with a precisely large sample size was the aim of the present study to investigate whether zona birefringence intensity (ZBI), measured by polarization light microscopy of metaphase II (MII) oocytes and zygote-stage embryos, was associated with subsequent development. In this study, ZBI was measured through 2 different parameters, the birefringence peak (PV, average signal strength of polarized light) and the birefringence peak combined with the surface of the signal (CV, average signal strength of polarized light multiplied by the surface of the signal), by using polarized light microscopy and OCTAX polarAIDE-software. Statistical analysis was done by the Tukey-Kramer test using SAS version 9.1. Cumulus–oocyte complexes (COC) were recovered from slaughterhouse ovaries by aspiration. After in vitro maturation, MII oocytes were denuded and activated parthenogenetically (4-min exposure to 5 μm Ionomycin, followed by 3.5 h of incubation in 2 mm 6-DMAP) or fertilized in vitro before denudation. Subsequently, ZBI of MII oocytes (directly after activation) and zygotes (24 h after the beginning of IVF) were measured. To allow tracking of subsequent development, embryos were cultured individually in a well-of-well (WoW) culture system until Day 7 in CR1aa medium (5% CO2, 38.7°C, humidified air). When parthenogenetically activated embryos were cultured (n = 365), 287 (78.6%) cleaved and 115 (31.5%) reached blastocyst stage. The ZBI of MII-activated oocytes did not indicate any differences between those that cleaved and those that did not. On the contrary, we observed significant differences (P < 0.05) for mean values of PV (41.4 v. 43.3) and CV (21.3 v. 22.6) for MII-activated oocytes that subsequently reached the blastocyst stage at Day 7 and those that did not. When IVF zygotes (n = 415, 80.5% cleavage rate and 26.0% blastocyst rate) were analyzed, significantly (P < 0.05) lower mean values of PV (47.1 v. 51.5) and CV (24.7 v. 27.1) were observed for zygotes that cleaved within 48 h compared with zygotes that did not cleave. In addition, mean values of PV (45.5 v. 47.9) and CV (24.3 v. 25.4) for the zygotes that developed to the blastocyst stage at Day 7 were significantly lower in comparison with zygotes that did not develop to that developmental stage. In conclusion, we observed a connection between birefringence intensity at the MII/zygote stage and further development. This may offer benefits for the identification of favorable MII oocytes that could have implications for bovine somatic cell nuclear transfer as well as for human-assisted reproduction technology.
214 EFFECT OF OOCYTE SOURCE ON THE DEVELOPMENTAL CAPACITY OF SOUTH AFRICAN IN VITRO-PRODUCED INDIGENOUS CATTLE EMBRYOS
Authors: Nedambale T. L. , Raito M. B. , Mphaphathi M. L.
Published: December 2008

The present study was undertaken to determine whether the source of oocytes (slaughterhouse ovaries from feedlot cows or naturally grazing indigenous cows) would affect in vitro bovine embryo production. Bovine oocytes (n = 1047), aspirated from slaughterhouse ovaries from feedlot cows and naturally grazing indigenous cows were randomly allocated to Sanyo, Forma, and Thermo 5% CO2 incubators. Oocytes were then in vitro matured in TCM-199 plus 10% fetal bovine serum, 1 μg mL–1 for both FSH and LH at 39°C for 24 h, and fertilized in Brackett and Oliphant (BO) medium per treatment group at 39°C. Presumptive zygotes were cultured in vitro per treatment group. Total cleavage and blastocyst rates were recorded postfertilization. Data were analyzed by ANOVA. Preliminary results demonstrated that there was no effect of incubator or source of oocytes on cleavage and 8-cell embryos. However, the cleavage and embryo developmental rate tended to be lower for the feedlot group, regardless of the incubator used (Table 1). In conclusion, this study suggests that slaughterhouse ovaries obtained from South African indigenous cows from a feedlot resulted in a lower blastocyst formation rate. Further studies are currently underway to count the cell numbers and to conduct embryo transfer.

Table 1.Comparison of three different incubators and source of oocytes on embryo development in vitro

This work was funded by the South African National Department of Agriculture, DST-PDP, and the National Research Foundation (NRF, Grant. Nos. RT21 and 24000).
217 KINETICS OF SPERM PENETRATION IS CORRELATED WITH IN VITRO FERTILITY OF BUFFALO (BUBALUS BUBALIS) BULLS
Authors: Rubessa M. , Di Fenza M. , Mariotti E. , Di Francesco S. , de Dilectis C. , Di Palo R. , Zicarelli L. , Gasparrini B.
Published: December 2008

It was previously demonstrated that the kinetics of early cleavage could be used to discriminate between bovine bulls with high and low field fertility (Ward F et al. 2001 Mol. Reprod. Dev. 60, 47–55). Marked differences exist in the kinetics of sperm penetration between bulls, and this may be a useful predictor of field fertility in cattle (Ward F et al. 2002 Theriogenology 57, 2105–2117). It is well known that the ability to fertilize oocytes in vitro and to sustain embryo development varies significantly among buffalo bulls. Therefore, the aim of this work was to evaluate whether the speed of oocyte penetration after IVF was correlated with the blastocyst rates obtainable with different bulls in buffalo species. In Experiment 1, in vitro-matured buffalo oocytes were co-incubated with MitoTracker-labeled spermatozoa (Ward F et al. 2002 Theriogenology 57, 2105–2117) from 6 different bulls, over 2 replicates. Oocytes were subsequently fixed every 3 h (up to 18 h) postinsemination (pi). At each time point, oocytes were denuded, dezoned, fixed in ethanol overnight, and stained with 4′,6-diamidino-2-phenylindole for nuclei examination under a fluorescence microscope. In Experiment 2, in vitro-matured oocytes were fertilized with sperm from the same 6 bulls and were cultured to the blastocyst stage, over 4 replicates. Bulls were tested, collectively, on each batch of ovaries in both experiments. Differences in the percentages of monospermic penetration among bulls were analyzed by chi-square test. Correlation and multiple regression analyses were also carried out between the speed of penetration and blastocyst yields. Marked differences in the kinetics of sperm penetration were found among buffalo bulls, as shown in Table 1. Interestingly, a correlation was found between the blastocyst rate and the percentage of oocytes penetrated at 6 h (r = 0.71; P < 0.01), at 9 h (r = 0.65; P < 0.05), at 12 h (r = 0.77; P < 0.01), and at 18 h pi (r = 0.59; P < 0.05). Regression analysis showed that the optimal time of penetration for predicting the blastocyst rate was 12 h pi (R2 = 0.6). In conclusion, the kinetics of sperm penetration may be a useful marker to predict the in vitro-fertilizing ability of buffalo bulls. The great variability in the speed of oocyte penetration suggests inserting this assessment in the preliminary screening of bulls before their utilization in IVF programs. This may be helpful in selecting high-fertility bulls and identifying the optimal gamete co-incubation times for each bull used.

Table 1.Percentage of oocytes penetrated at each time point (hpi, h postinsemination) by different bulls1
221 EFFECT OF ROCKING ON IN VITRO PRODUCTION OF BOVINE EMBRYOS
Authors: Serita Y. , Kubota C. , Kojima T.
Published: December 2008

This study tested whether embryo development yield using in vitro fertilization (IVF) could be improved by rocking cultures. Bovine ovaries were obtained at a slaughterhouse and transported to the laboratory within 6 h. Cumulus–oocyte complexes were collected and 20–25 were transferred in 100-μL drops of TCM-199 containing 10% fetal bovine serum and antibiotics under paraffin oil. Maturation was for 20–24 h at 38.5°C under 5% CO2 and 95% air in a humid atmosphere (IVM). In vitro fertilization was carried out for 6 h using frozen–thawed sperm from a single bull in modified Brackett and Oliphant (BO) medium. Presumptive zygotes were cultured in CR1aa supplemented with 10 mg mL–1 of BSA or 5% FBS for 9 d at 38.5°C under 5% CO2, 5% O2, and 90% N2 in a humid atmosphere (IVC). Rocking was performed to a height of 6 cm every 7 s using a Profile Rocker (New Brunswick Scientific Co., Edison, NJ, USA) in an incubator. Dishes were placed at a 15-cm distance from the fulcrum of the rocker. The conventional method (no rocking) served as a control, and every experiment was replicated 3 times. For Experiment 1, the effect of the period of rocking on developmental competence was examined when COC or zygotes were subjected to rocking for IVM, IVF, or IVC (IVM-move, IVF-move, and IVC-move). There were no significant differences in rates of oocyte maturation, cleavage, and development for IVM-move v. the control, or for rate of development between IVC-move and the control. However, the rate of fertilization for IVF-move was higher than that of the control (88.9 v. 67.5%; P < 0.01), and the rate of development was higher for IVF-move than for the control (39.0 v. 25.7%; P < 0.05). For Experiment 2, the effect of rocking frequency during IVF on development was determined. The IVF cultures were rocked every 7, 3.5, and 1.5 s (IVF-1move, IVF-2move, IVF-3move). The rates of cleavage on IVF-1move, IVF-2move, IVF-3move, and the control were 74.3, 69.8, 68.8, and 60.4%, and the rates of development were 39.0, 48.3, 26.2, and 25.7%, respectively. The rates of development on IVF-1move and IVF-2move were significantly different from the control and IVF-3move (P < 0.01). These results showed that rocking during IVF improved fertilization and embryo yield, and that frequency of rocking affected embryo development.
226 TYROSINE PHOSPHORYLATION PATTERN IN PORCINE EJACULATED AND EPIDIDYMAL CAPACITATED BOAR SPERM
Authors: Aviles-Lopez K. , Garcia-Vazquez F. A. , Matas C.
Published: December 2008

After ejaculation, mammalian sperm must undergo a preparation period know as capacitation to be competent to fertilize. Capacitation normally occurs in the female genital tract; however, it can also be achieved in vitro by removing seminal plasma and incubating sperm in capacitation media. In previous studies, we have demonstrated that ejaculated spermatozoa washed through a Percoll® gradient can penetrate the oocyte faster than epididymal sperm. It has been shown that capacitation is associated with tyrosine phosphorylation of sperm proteins, and many studies utilize protein phosphorylation as an indicator of sperm capacitation (Tardif S et al. 2001 Biol. Reprod 65, 784–792). The main objective of this experiment was to determine the protein tyrosine phosphorylation patterns of ejaculated and epididymal boar spermatozoa washed through a Percoll® gradient and incubated in a capacitating medium (TALP) without albumin. Ejaculated and epididymal spermatozoa were each divided into 3 groups: (1) spermatozoa without any treatment (control = C), (2) spermatozoa washed through Percoll® gradient (P), and (3) spermatozoa washed through Percoll® and incubated 3 h in TALP medium (P-3hr). The sperm plasma membranes were isolated (as described in Bravo M et al. 2005 Mol. Reprod. Dev. 71, 88–96), and sperm soluble proteins were separated by SDS-PAGE 12% acrylamide. Afterward, the proteins were transferred onto a nitrocellulose membrane and blocked with TBST-albumin 5% and were then incubated with an anti-phosphotyrosine antibody (4G10 Upstate Biotechnology, Lake Placid, NY, USA; 1:20000), then with an anti-mouse IgG (1:20000; Biorad, Hercules, CA, USA) and revealed with ECL + Plus (Amersham, São Paulo, Brazil). Five replicates were conducted for this experience. The results showed a band of tyrosine phosphoprotein of 19 kDa in all experimental groups except the ejaculated control group. A band with a range of 40–43 kDa appears strongly phosphorylated in the ejaculated spermatozoa, while in the epididymal spermatozoa was poorly labeled. In conclusion, the ejaculated and epididymal spermatozoa have different pattern of tyrosine phosphorylation with the same treatment. It is probably that the seminal plasma proteins could be regulated by the phosphorylation of the 19 kDa of protein. However, further studies are necessary to elicit what kind of proteins in seminal plasma change this stage of phosphorylation and their role in an IVF system.

Supported by MEC (AGL2006-03495).
227 AN ALTERATION IN GENE EXPRESSION PATTERNS INDUCED BY DI-(2 ETHYLHEXYL) PHTHALATE AND FLUTAMIDE IN THE TESTIS OF IMMATURE MALE RATS
Authors: Choi K.-C. , Vo T. T. B. , Jung E.-M. , Dang V. H. , Jeung E.-B.
Published: December 2008

In a previous study, we demonstrated that although endocrine disruptors (EDs) with androgenic and anti-androgenic effects may alter reproductive function, their effects on the developing male reproductive organs may be distinct. To continue this line of study, we treated immature rats to examine the adverse effects of di-(2 ethylhexyl) phthalate (DEHP) and flutamide (Flu) on the male reproductive system. Immature male SD rats were treated daily with DEHP and/or Flu at postnatal day (PND) 21 to 35 in a dose-dependent manner, and the changes evoked by these EDs were determined by differences in male reproductive tract and other organ weights, testicular histology, and serum LH and testosterone levels in combination with global microarray analysis. Interestingly, the testes, prostate, seminal vesicle weight, and anogenital distances were significantly decreased in response to the highest dose of DEHP and Flu. There were no differences in serum LH and testosterone concentration at PND 35 for immature male rats exposed to DEHP and/or Flu. However, treatment with DEHP and/or Flu caused histopathological changes in testes in which the degeneration and denseness of germ cells and/or dilatation of the tubular lumen were observed in response to the high dose [500 mg kg–1 of body weight (BW)] of DEHP and medium dose (10 mg kg–1 of BW) of Flu. Additionally, the results from cDNA microarray indicated that 1272 genes were up-regulated (more than 2-fold) and 1969 genes were down-regulated in response to DEPH and/or Flu. These genes were identified based on their roles in some physiological processes (i.e. lipid and cholesterol homeostasis, steroidogenesis, sex determination, and calcium signal transduction). The significant decreases were observed in the expressions of steroidogenic genes (i.e. Star, Cyp11a1, or Hsd3b). In addition, a common set of targeting genes, including CaBP1, Vav2, Plcd1, Lhx1, and Isoc1, were altered following EDs exposure, suggesting a potential set of biomarker genes for screening anti-androgenic and/or androgenicity of EDs. Taken together, we demonstrated that exposure to DEHP and/or Flu resulted in a temporal alteration in gene expression profile in the testes of immature male rats, and their toxicological effects on male reproductive system are distinct depending on their anti-androgenicity, suggesting new insight into molecular mechanism(s) underlying detrimental impacts of EDs with anti-androgenic activities in human and wildlife.
237 SHORT-TERM TREATMENT OF 6-DMAP AND DEMECOLCINE IMPROVE DEVELOPMENTAL COMPETENCE OF PORCINE PARTHENOGENETIC EMBRYOS
Authors: Park S. J. , Koo O. J. , Kwon D. K. , Park H. J. , Lee S. S. , Nam Y. H. , Lee B. C.
Published: December 2008

Artificial activation is one of the essential factors for successful development of embryos derived from parthenogenetic activation or somatic cell nuclear transfer (SCNT). This study was carried out to confirm whether the short-term treatment of 6-dimethylaminopurine (6-DMAP) or demecolcine would improve developmental competence of porcine parthenogenetic activated embryos and find out the optimal activation protocol. Ovaries were collected from gilts at a local slaughterhouse and cumulus–oocyte complexes were cultured for 44 h in the tissue culture medium-199 with 10% of porcine follicular fluid. Matured oocytes were chemically activated with 10 min treatment of 0.2 mm thimerosal (Thi) followed by 30 min treatment of 8 mm dithiothrethiol (DTT). Additional treatments of 6-DMAP (2 mm) and/or demecolcine (0.4 μg mL–1) for a short term (40 min) or a long term (3 h) were conducted. Activated embryos were cultured in PZM-3. Cleavage rate, blastocyst (BL) formation rate and total cell number of BL were assessed at Day 2, 7 of culture, respectively. All data were subjected to one-way ANOVA followed by Tukey’s test using Prism version 4.0 (Graphpad Software, San Diego, CA, USA) to determine differences among experimental groups. To begin with, we evaluated that effect of short-term treatment of 6-DMAP or demecolcine. Short-term treatment of 6-DMAP improved developmental competence of activated embryos compared to long-term treatment (47.0 ± 1.0% v. 50.5 ± 1.5% for cleavage rate, 5 ± 3% v. 3 ± 1% for BL formation rate, 70 ± 13 v. 37 ± 6 for total cell numbers). Moreover, short-tem treatment of demecolcine also inhibited second polar body extrusion effectively compared with nontreated control (14 ± 2.1 v. 8.3 ± 4.1%). We confirmed that short-term treatment of 6-DMAP or demecolcine is valid. To optimize activation procedure with short-term treatment, development of embryos derived from each treatment were compared among nontreated control (C), 6-DMAP only treatment (6O), demecolcine only (DO) treatment, and 6-DMAP + demecolcine treatment (6 + D) groups. Cleavage rates of 4 groups were similar (53.5 ± 3.0, 62.1 ± 2.9, 53.6 ± 3.0 and 57.0 ± 3.0%, respectively). However, 6 + D group shows significantly greater BL formation rate (18.0 ± 2.5%) compared with C, 6O, or DO groups (9.4 ± 1.9, 14.0 ± 2.3 and 4.3 ± 1.3%, respectively). Total cell number of BL also increased in 6 + D group (52.8 ± 13.0) compared with C, 6O, or DO groups (47 ± 21.5, 31.5 ± 4.22 and 45.2 ± 13.7, respectively). In conclusion, we elucidated that short-term treatment of 6-DMAP or demecolcine improves developmental competence of embryos compared with long-term treatment. We recommend a combination of Thi/DTT with short-term treatment of both 6-DMAP and demecolcine for optimal activation procedure of porcine embryos.

This study was supported by BK21, KOSEF (#M10625030005-07N250300510), HANWHA L&C Corp, and Gyeonggido Veterinary Service.
240 EFFECTS OF TIMING AND DIPLOIDIZATION ON ACTIVATION AND DEVELOPMENT OF RAT OOCYTES
Authors: Zhu J. , Amarnath D. , Campbell K.
Published: December 2008

The objectives of these studies were (1) to compare the effects of Cytochalasin B (CB) or 6-dimethyl amino purine (DMAP) in combination with SrCl2 on activation and development of rat oocytes and (2) to determine the optimal timing for collection and activation of rat oocytes following superovulation. In Experiment 1, rat oocytes collected at 16–18 h after hCG injection were immediately cultured in 10 mm SrCl2 alone, 10 mm SrCl2 + 2 mm DMAP for 4 h and 10 mm SrCl2 + 5 μg mL–1 CB for 5–6 h, respectively, they were then cultured in KSOM for 20 h and finally cultured in R1ECM for an additional 4 days, all at 37°C, 5% CO2 in air. There were no significant differences in pronuclear formation (SrCl2 alone: 51/53; DMAP: 139/140; CB: 139/140) and cleavage rate (SrCl2 alone: 52/53; DMAP: 129/140; CB: 130/140) observed among three groups (χ2-test, P > 0.05). On Day 5 the frequency of development to blastocyst was not statistically different (P > 0.05) between DMAP (55.71%, 78/140) and CB (54.23%, 76/140) treated groups; however, no blastocysts were produced in 10 mm SrCl2 alone. In Experiment 2, oocytes were collected at 14, 16, 18, 20 and 22 hours after hCG injections, respectively, and they were immediately cultured in 10 mm SrCl2 + 2 mm DMAP for 4 h and then cultured in the same conditions described for Experiment 1. Although development to blastocyst was slightly lower in the 14-h group (43.50%, 47/108), no significant differences in the frequencies of pronuclear formation, cleavage on Day 1 or development to blastocyst on Day 5 were observed between groups (χ2-test, P > 0.05) (Table 1). All experiments were repeated at least three times. In conclusion, our results showed that SrCl2 combined with either CB or DMAP could equally, effectively activate rat oocytes. In contrast rat haploid oocytes activated in the absence of CB or DMAP were not able to develop to the blastocyst stage. Furthermore, rat oocytes collected between 14 and 22 h after hCG injection had an equal potential to develop to the blastocyst stage after activation by 10 mm SrCl2 + 2 mm DMAP.

Table 1.Development of rat oocytes activated at different time points following
241 DIBUTYRYL CYCLIC AMP AND EGF-LIKE FACTORS SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM WITHOUT GONADOTROPINS
Authors: Akaki Y. , Yoshioka K. , Funahashi H.
Published: December 2008

Exposure of porcine oocyte–cumulus complexes (OCC) to gonadotropins induces meiotic resumption, but the details of this mechanism are still unknown. The present study was undertaken to examine combinational effects of EGF-like factors and dibutyryl cyclic AMP (dbcAMP) in a chemically defined medium on in vitro maturation (IVM) of porcine oocytes. The OCC were aspirated from 3- to 6-mm-diameter follicles of prepuberal ovaries and used in the current study. The basic culture medium was a chemically defined medium, Porcine Oocyte Medium (POM; Research Institute for the Functional Peptides, Yamagata, Japan). In the first experiment, various concentrations (0, 10, and 1000 ng mL–1) of EGF-like factors (EGF, amphiregulin, and betacellulin) were added to POM during an entire IVM period (44 h). In the second experiment, to determine the additive effect of EGF-like factors, each EGF-like factor with an effective concentration was combined with the others. In the last experiment, to examine the combined effect with dbcAMP, OCC were exposed to EGF (10 ng mL–1), amphiregulin (1000 ng mL–1), and dbcAMP (1 mm) during the first 20 h of IVM and then the culture was continued in the absence of EGF-like factors and dbcAMP. After culture, in all experiments, meiotic resumption and the progress of oocytes were examined after denuding, fixing, and staining. Statistical analyses was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In the first experiment, all treatments without supplementation with 10 ng mL–1 amphiregulin increased the incidence of oocytes maturing to the MII phase, as compared with controls (29.1 to 39.3% v. 11.1%, P < 0.05). In the second experiment, combinations with 2 kinds of EGF-like factor slightly (but not significantly) improved the percentage of oocytes at the MII stage (37.7 to 47.4%). In the last experiment, supplementation with 1 mm dbcAMP during the first 20 h of IVM, regardless of the presence of EGF-like factors, significantly increased the incidence of MII oocytes as compared with controls, whereas the incidence was the highest when 1 mm dbcAMP, 10 ng mL–1 EGF, and 1000 ng mL–1 amphiregulin were supplemented (75.5%). When those oocytes were cultured in a chemically defined medium after in vitro fertilization, the developmental competence of oocytes to the blastocyst stage (25.0%) was not different from oocytes matured in the presence of gonadotropins and dbcAMP during the first 20 h of IVM (17.3%). These observations indicate that supplementation of a chemically defined maturation medium with EGF-like factors and dbcAMP during the first 20 h of IVM can support the meiotic progress and developmental competence of porcine oocytes well. Currently, we are examining the developmental competence of those oocytes after embryo transfer. The results will be presented at the meeting.

This study was supported by MAFF AgriBio1605.
242 DETECTION OF MICROTUBULES BY POLARIZED LIGHT MICROSCOPY IN BOVINE OOCYTES
Authors: Caamaño J. N. , Díez C. , Muñoz M. , Martin D. , Morató R. , Mogas T. , Carrocera S. , Gómez E.
Published: December 2008

Polarized light microscopy (PLM) is being used as a tool in assisted reproductive technologies in humans and, more recently, in farm animals. Polarized light microscopy may allow for a quick localization or removal of nuclear structures, or both in the oocyte without fluorochrome staining and ultraviolet exposure, and may reduce nuclear damage when performing intra-cytoplasmic sperm injection. The aim of this study was to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured bovine oocytes. Cumulus–oocyte complexes from slaughterhouse ovaries were matured in vitro for 23 h in bicarbonate-buffered TCM-199, FSHp (1 μg mL–1), LH (5 μg mL–1), 17β-estradiol (1 μg mL–1), and 10% fetal calf serum at 38.7°C in 5% CO2 in air with high humidity. After in vitro maturation, oocytes (n = 98) were denuded with 0.5% hyaluronidase and were placed individually in 10-μL drops of TCM-199-HEPES-BSA in a glass Petri dish. Polarized light microscopy was used to detect the presence of polymerized protein that could be forming a meiotic spindle. To confirm the presence of the polymerized protein and the meiotic spindle, each individual oocyte was subjected to immunostaining and chromatin detection as described by Morató et al. 2008 Mol. Reprod. Dev. 75, 191–201. The experiment was replicated 6 times. There was an absolute positive correlation (r = 1; P < 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by immunostaining. A barrel-shaped spindle was observed in 40% of the individual samples, whereas disorganized microtubule structures were obtained in 55% of the samples. The biological implication of these findings needs to be explored. However, PLM seems to be an efficient system to detect polymerized protein in in vitro-matured bovine oocytes.
243 EFFECT OF OOCYTE RECOVERY TECHNIQUES ON IN VITRO MATURATION OF SWINE OOCYTES
Authors: de Barros F. R. O. , Marques M. G. , Goissis M. D. , Peres M. A. , Milazzotto M. P. , Maria F. S. , Assumpção M. E. O. A. , Visintin J. A.
Published: December 2008

The aim of this study was to compare 2 different techniques to obtain swine oocytes from abattoir ovaries. Ovaries were washed in saline at 35°C and submitted to slashing or aspiration, simultaneously. For the slashing group, ovaries were held with a hemostat inside a beaker containing 35 mL of HEPES-buffered Tyrode’s media (HbT) and follicles (2–6 mm) were incised with a scalpel. For every 5 slashed ovaries, HbT-containing follicular fluid was transferred to 50-mL centrifuge tubes. For the aspiration group, follicles (2–6 mm) were aspirated using an 18-gauge needle and a 5-mL syringe. The follicular fluid of each ovary was transferred to a 50-mL centrifuge tube. Tubes from both techniques were placed in a water bath at 35°C for 15 min to allow settling of the cumulus–oocyte complexes (COC). The supernatant was removed and the sediment was resuspended in HbT and placed in water bath at 35°C for an additional 15 min. The sediment was resuspended in 15 mL of HbT and COC were recovered under stereomicroscopy. Oocytes were in vitro matured for 44 h in TCM-199 added with 10% porcine follicular fluid (PFF) and hormones (LH and FSH) at 38.5°C, 5% CO2 and high humidity. The oocyte recovery rate of each technique was determined by the ratio between the number of COC and ovaries used. To verify nuclear maturation by epifluorescence microscopy (Zeiss), oocytes were fixed, permeabilized, and incubated in 10 μg mL–1 of RNAse for 30 min and in 10 μg mL–1 of propidium iodide for 10 min. Heat shock protein 70 (HSP70) content was assessed as described in Kawarsky and King (2001 Zygote 9(3), 39–50) to verify the metabolic stress. Data were analyzed by ANOVA and Tukey’s test using the software Statistica for Windows. A level of 5% was considered significant in all assessments. The oocyte recovery rate (COC/ovary) was higher for the slashing group (2.665 ± 0.38) compared with the aspiration group (1.762 ± 0.15). The percentage of oocytes that reached the germinative vesicle (GV) stage (h 0 of maturation) did not differ between groups (100 ± 0 and 86.66 ± 13.36, slashing and aspiration group, respectively). The same was observed for the percentage of oocytes that reached the metaphase II stage (MII, after 44 of maturation; 79.99 ± 9.74 and 96.00 ± 4.00, slashing and aspiration group, respectively). Moreover, no difference at pixel quantification of HSP70 was observed between groups (256.50 ± 42.42 and 238.61 ± 71.18, slashing and aspiration group, respectively). In conclusion, the slashing procedure provided a better oocyte recovery rate compared with the aspiration of ovaries. This technique does not affect nuclear maturation, because no differences were observed regarding the percentage of oocytes that reached the GV and MII stages. In addition, it does not affect HSP70 content, suggesting that the slashing of ovaries does not increase the basal stress of oocytes in an in vitro-maturation system.
261 FOALS PRODUCED AFTER ICSI USING FROZEN, SEX-SORTED, REFROZEN SPERM
Authors: Carnevale E. M. , Graham J. K. , Suh T. K. , Stokes J. E. , Squires E. L.
Published: December 2008

Obtaining adequate numbers of sex-selected sperm for uterine inseminations can be difficult, especially if fresh semen is not available or sperm numbers are limited. Intracytoplasmic sperm injection (ICSI) requires minimal sperm numbers and can be used for the assisted fertilization of oocytes. The objectives of this study were to: (1) thaw, sex-sort, and refreeze semen, and (2) determine if cleavage rates of oocytes were similar after ICSI using frozen v. frozen, thawed, sex-sorted, and refrozen sperm. The final aim was to produce foals by ICSI using sex-sorted sperm. Light-horse mares between 3 and 15 years were used as oocyte donors. When a follicle 30 to 35 mm and endometrial edema was imaged using ultrasound, deslorelin (1.5 mg, i.m., Franck’s Pharmacy, Ocala, FL, USA) was administered to induce follicular maturation. Between 20 and 24 h after deslorelin, oocytes from preovulatory follicles were collected by transvaginal, ultrasound-guided follicular aspirations. Oocytes were cultured for 16 to 18 h in TCM 199 with 10% FCS, 0.2 mm pyruvate at 38.5°C and in an atmosphere of 6% CO2. Semen from a single ejaculate of two stallions was frozen for the experiment. Some of the frozen semen (control) was thawed and used for ICSI. Sperm from other straws were thawed, sex-sorted by flow cytometry, treated with cholesterol-loaded cyclodextrin, and refrozen in a skim milk-egg yolk diluent containing 0.52 m dimethyl formamide before thawing for ICSI. Sperm were thawed by cutting a thin section of a straw under liquid nitrogen; the straw section was dropped directly into medium at 38.5°C. Sperm were incubated for 10 min, before 1 μL of supernatant was removed and placed into a 5 μL drop of medium with 5% polyvinylpyrrolidone. From the droplet, sperm with progressive motility and normal morphology by visual inspection were selected for ICSI. Injected oocytes were placed in DMEM/F12 with 10% FCS for 48 h (±2 h) before assessment of cleavage. Numbers of cleaved per injected oocytes were compared by Fisher’s Exact Test and were lower (P < 0.001) for sex-sorted, refrozen sperm than for frozen control sperm (6/20, 30% and 15/18, 83%, respectively). At the completion of the project, three additional oocytes were injected with X-bearing sperm. The injected oocytes cleaved and developed into embryos under culture conditions [DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) with 10% FCS at 38.5°C and an atmosphere of 5% CO2, 5% O2 and 90% N2]. The resulting morula, blastocyst, and expanded blastocyst were transferred nonsurgically into recipients’ uteri. Pregnancies were established in recipients receiving the morula and expanded blastocyst, and two fillies were born in July 2008. Both foals appeared normal at birth; however, one foal became septic and was euthanized before 2 weeks of age. In this study, frozen, thawed, sex-sorted, and refrozen sperm were successfully incorporated into an ICSI program to produce early-stage embryos and sex-selected foals.
263 LABELING BOVINE SPERM Y-CHROMOSOME SEQUENCE USING DNA MIMICS
Authors: Didion B. A. , Bleher R.
Published: December 2008

Flow cytometric separation of X- and Y-chromosome bearing bovine sperm is an accepted technology for use at the commercial level. Nevertheless it is important to continue researching the area of gender-preselected sperm for improved efficiencies. We used a synthetic DNA mimic conjugated to a fluorescent dye for in situ detection of Y chromosomes in metaphase preparations of bovine somatic cells and spermatozoa. Peptide nucleic acids (PNA) are a type of DNA mimic having a higher affinity and stability than conventional DNA probes and are used as hybridization probes to complementary DNA. Using male bovine somatic cells and the Y-chromosome as a template, we arranged for the synthesis of a CY3-conjugated PNA to bind 13 to 15 base pairs of unique, Y-chromosome sequence. By testing different labeling conditions, we found that brief incubation (~1 h) of metaphase chromosomes with the PNA produced a localized signal on the Y-chromosome. No signals were observed when chromosomes of female bovine somatic cells were incubated with the same PNA probe. Because chromosomes occupy non-random territories in all cell nuclei, including sperm, we proposed to find centrally-located signals in 50% of fixed bovine sperm when treated with the same PNA as used for the somatic cells. As expected, we found the PNA signals present in 50% sperm (23/43) existing as a single, centrally-located, round fluorescent dot in the sperm head. Validation studies were also conducted using bovine sperm previously flow sorted into X or Y populations, and we found the signals in accordance to an expected signal present using the PNA (146/165 or 88.5% with PNA signal in presorted Y sperm heads and 13/174 or 7.5% with PNA signal in presorted X sperm heads).
264 TESTOSTERONE IN THE OOCYTE CULTURE DOES NOT ALTER SEX-RATIO OF IN VITRO PRODUCED BOVINE EMBRYOS
Authors: Díez C. , Bermejo-Alvarez P. , Gutiérrez-Adan A. , Caamaño J. N. , Muñoz M. , Carrocera S. , Martín D. , Gómez E.
Published: December 2008

The production of sex-known offspring is a main objective in reproductive biotechnology. It has been reported that bovine ova developed in follicles with high concentrations of testosterone in vivo yielded significantly more male embryos in vitro (Grant V et al. 2008 Biol. Reprod. 78, 812–815). In this work we aimed to test the effects of testosterone on sex ratio of bovine embryos produced in fully in vitro conditions. Immature bovine cumulus–oocyte complexes (COCs; n = 750) from slaughterhouse ovaries were cultured in 199 HNaCO3 with polyvinyl alcohol (PVA) 0.1 mg mL–1 as a basic medium. Culture was made in two steps, a 24 h meiotic arrest (roscovitine 25 μm), and a subsequent in vitro maturation period with FSH-LH for 24 h. Testosterone (T-86500, Sigma-Aldrich, St. Louis, MO, USA) was added throughout the entire oocyte culture at 0, 30, 300, and 1500 nm. After in vitro fertilization (Day 0), zygotes were freed of cumulus cells by pipetting, and subsequently cultured in SOF + 6 g L–1 BSA up to Day 3. At this time, embryo development was recorded, and all embryos having 3 or more cells were treated with pronase to remove the zona pellucida. Zona-free embryos were washed in PBS containing PVA 0.1 mg mL–1 and individually frozen at –80°C until sex analysis by PCR (Bermejo-Alvarez P et al. 2008 Biol. Reprod. doi:10.1095/biolreprod.108.070169). A total of 252 embryos from 5 replicates were sexed. Data for development and sex-ratio are presented as % LSM ± SD. There were no interactions between testosterone treatment, embryonic sex, and embryonic stage analyzed. Testosterone did not affect development rates (P > 0.05) at any stage: cleavage (47.8 ± 6.8, 56.5 ± 6.8; 50.9 ± 6.8; 62.2 ± 6.8), 3 to 4 cells (40.6 ± 5.2, 45.8 ± 5.2; 37.8 ± 5.2; 47.7 ± 5.2) and >5 cells rates (24.5 ± 4; 27.3 ± 4; 21.3 ± 4; 25.3 ± 4) for 0, 30, 300, and 1500 nm testosterone, respectively. Cumulative percentages of male embryos were as follows: 53 ± 8 (n = 56), 42.6 ± 8 (n = 52), 53.6 ± 6 (n = 81) and 57.6 ± 8 (n = 63) for 0, 30, 300, and 1500 nm groups respectively (P > 0.05). These results show that the testosterone effects on oocyte ability to select Y-chromosome bearing spermatozoa are not reproducible in vitro under the present experimental conditions.

Grant support: MEC, project AGL2008-01530; RTA2008-0082; M. Muoz is supported by FICYT.
268 IDENTIFICATION OF X- AND Y-BEARING SPERMATOZOA IN SORTED BUFFALO (BUBALUS BUBALIS) SEMEN BY FLUORESCENCE IN SITU HYBRIDIZATION
Authors: Zhang M. , Zhuang X. J. , Lu Y. Q. , Hu C. H. , Lu S. S. , Lu K. H.
Published: December 2008

Flow cytometry sorting technology has been successfully used to sort the X- and Y-chromosome bearing sperm. Previous studies showed that fluorescence in situ hybridization (FISH) method was a simple and reliable procedure for assessing the effectiveness of separation of X- and Y-sperm in the swine (Kawarasaki T et al. 1998 Theriogenology 50, 625–635) and the bovine (Rens W et al. 2001 Reproduction 121, 541–546). Reports of sex-preselection by flow-cytometry sorting of the X- and Y-sperm were also seen in the buffalo (Presicce GA et al. 2005 Reprod. Dom. Anim. 40, 73–75; Lu YQ et al. 2006 Anim. Reprod. Sci. 100, 192–196). There was, however, no report to date for using the FISH method to assess the purity of the sorted buffalo sperm. The objective of the present study was to verify the purity of flow cytometrically-sorted buffalo X- and Y-sperm by FISH using bovine X- and Y- chromosome painting probes prepared by microdissection. The X- and Y- chromosomes of bovidea were microdissected respectively from the metaphase spreads of Holstein blood cells with a glass needle controlled by a micromanipulator and amplified by degenerate oligo-nucleotide primer-PCR (DOP-PCR) (Mariela N et al. 2005 Genet. Mol. Res. 4, 675–683). The DOP-PCR products of X- and Y- chromosome were labeled with CY3-dUTP and Biotin-11-dUTP, respectively. The buffalo X- or Y-sperm DNA from unsorted semen and sorted semen were hybridized to the labeled probes, respectively. The results showed that the hybridized signals were clearly visible in the metaphase karyotype of bovine and buffalo semen samples. About 47.7% (594/1246) and 48.9% (683/1396) of the unsorted buffalo sperm emitted strong fluorescent signals when assessed by Y- and X-chromosome painting probes, respectively, which was conformed to the sex ratio in normal buffalo sperm (50%:50%). About 86.1% (1529/1776) hybridization signals of the sperm in the sorted X-semen assessed by X-chromosome painting probes were detected, while 82.2% (2232/2716) of the Y-sorted buffalo sperm emitted strong fluorescent signals when assessed by Y-chromosome painting probe. The results of the flow cytometer re-analysis revealed that the proportions of X- and Y-bearing sperm in the sorted semen were 89.6% and 86.7%, respectively. There were no apparent differences between the two assessment methods of sperm separation by flow cytometry re-analysis and by FISH with the X-Y paint probe. In conclusion, bovine X- and Y-chromosome painting probes prepared using microisolation method could be used to verify the purity of the sorted sperm in the buffalo.

This study was supported by the Guangxi Department of Science and Technology (0626001-3-1) and National Key Technology R&D Program, The People’s Republic of China (2006BAD04A18). The authors (M. Zhang, X.J. Zhuang, and Y.Q. Lu) contributed equally to this work.
277 PLASMID MEDIATED EXPERIMENTAL TELOMERE EXTENSION IN BOVINE EMBRYOS BY ECTOPIC EXPRESSION OF HUMAN TERT
Authors: Iqbal K. , Kues W. A. , Niemann H.
Published: December 2008

Telomeres are composed of repetitive hexanucleotide sequences, (TTAGGG)n, encompassing several kilobase pairs, and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage and up to the morula stage, telomerase is not active or is found only at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) has 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from a local abattoir, followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. Two plasmid encoding CMV promoter-driven sequences of hTERT and green fluorescent protein (GFP) were coinjected in bovine zygotes, and GFP driven by a muscle specific promoter was injected for mock experiments. The hTERT and GFP were co-injected to allow live separation of embryos. A total of 768 bovine embryos were injected; 468 (61%) of the treated embryos showed specific GFP-fluorescence. Of a total of 132 blastocysts (17%), 45 showed GFP fluorescence (34%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic hTERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. The mRNA level of the ectopic hTERT began to increase from the 4- to the 8-cell stage and remained high up to the morula stage. Embryos at the morula and blastocyst stages were spread on slides and analyzed by quantitative fluorescence in situ hybridization (qFISH). A Cy3-labeled 18-mer peptide nucleic acid (PNA) probe was used to hybridize the telomeres. The resulting spot intensities were quantified by using TFL-Telo software and were statistically analyzed. A modest increase in telomere length was observed in hTERT injected [775 ± 69 fluorescence unit (fu)] group compared to uninjected control (679 ± 75 fu) group at blastocyst stage. In conclusion, this study demonstrates that the ectopic expression of hTERT in embryos results in telomere elongation; overexpression of TERT may facilitate the derivation of bovine embryonic stem cells.

Supported by DFG and Goyaike SAACIYF.
285 ISOLATION AND CHARACTERIZATION OF BOVINE ADIPOSE-DERIVED SOMATIC STEM CELLS
Authors: Picou A. A. , MacLean R. A. , Dresser B. , Godke R. A. , Bondioli K. R.
Published: December 2008

Adipose tissue has proven to serve as an abundant and rich source of adult stem cells with pluripotent properties. It has been suggested that pluri potent cell could serve as en efficient source of donor cells for nuclear transfer. The objective of this study was to develop an isolation and culture protocol for bovine adipose tissue-derived stem cells Tissue was collected post-mortem from the brisket of cows at a local abattoir. For cell isolation, the tissue was finely minced and weighed. The tissue was washed two times with PBS plus 2% penicillin-streptomycin (P/S) and enzymatically digested in a collagenase type 1 solution. Isolation conditions were determined by using a 3 × 3 factorial design. The tissue was digested in PBS with 0.1%, 0.25%, or 0.50% collagenase type 1 and 1% BSA, 2% P/S, and 2% Fungazone. Each enzyme concentration was incubated for 1, 2, or 3 hours in a shaking incubator at 39°C. The cellular mixtures were centrifuged at 300g for 5 minutes, floating adipocytes removed, and the remaining cell suspension passed through filter apparatus consisting of 180 and 20 μm nylon meshes. Cells were resuspended in PBS plus 1% BSA and centrifuged again for 5 minutes at 300g. Pelleted stromal cells were washed and nucleated cells were counted using Hoechst stain at a concentration of 2 μg mL–1. Primary cell cultures were plated in a 12.5 cm2 culture flask in DMEM (high glucose) supplemented with 10%FBS and 1% P/S. Cells were cultured for 48 h in a 38°C incubator in a humidified atmosphere of 5% CO2 in air. Nonadherent cells were removed by washing with PBS. For some cultures adherent cells were removed by trypsinization to determine the proportion of cell adhering. On average 23.38% of cells remained adherent, and the average cell cycle length was 1.85 days. Table 1 displays the average number of nucleated cells that were released per gram of fat for the various collagenase concentrations and exposure times. Analysis of data by one-way ANOVA show no differences. Growth factor supplementation is currently being evaluated on the effects of culture duration and passage number. Isolation conditions of 0.25% collagenase for 2 hours incubation has been consistently successful at obtaining viable ADAS cultures from cattle and common eland (Taurotragus oryx).

Table 1.Average number of cells released and viability after collagenase treatments

Funding provided by LSU/ACRES collaborative research program.
292 EFFECT OF A SINGLE INJECTION OF FSH DISSOLVED IN POLYETHYLENE GLYCOL ON SUPEROVULATION RESPONSE IN KOREAN NATIVE COWS
Authors: Choi S. H. , Cho K. H. , Park Y. S.
Published: December 2008

Superovulation in bovine is usually achieved after several FSH injection in a time-consuming protocol, and the half-life of FSH is very short. This study was carried out to assess the efficiency of embryo production after induction of superovulation with a single injection of FSH (Folltropin-V, Vetrepharm, Canada) dissolved in polyethylene glycol (PEG, 8000 MW, Sigma-Aldrich Corp., St. Louis, MO, USA) in Korean Native cows. Eighty-eight cows were randomly assigned to one of four groups. In Group 1 (control), cows were intramuscularly treated with twice-daily administration of 50 mg FSH for 4 days. In Group 2 and 3, cows received a single subcutaneous injection of 400 mg and 200 mg FSH dissolved in 30% PEG, respectively. In Group 4, cows received a single subcutaneous injection of 200 mg FSH dissolved in 30% PEG given seven days after CIDR insertion. On days 10 to 12 after observed estrus, superovulation was started. Luteolysis was induced by administration of 25 mg of PGF2α (Pharmacia, Puurs, Belgium) IM 2 days after the start of the FSH injection. Donors were timed-inseminated with frozen–thawed semen at 48 and 60 h after the administration of PGF2α. Embryos were collected by non-surgical methods on 7 days after the second AI. The numbers of corpus lutea (CL), total ova, transferable embryos, and progesterone concentration were analyzed by General Linear Models Procedure with the Statistical Analysis System (SAS; SAS Institute, Inc., Cary, NC, USA) and means were compared with the Duncan’s multiple range test. Data on the pregnancy rate was analyzed by chi-square test. P values <0.05 were considered to be significantly different. The number of CL in Group 2 was significantly (P < 0.05) higher compared to Groups 1, 3 and 4 (18.5 ± 7.5 v. 11.2 ± 5.1, 13.1 ± 5.1 and 13.9 ± 6.4, respectively). However, the number of total ova (7.9 ± 6.0 to 10.4 ± 7.2) and transferable embryos (3.7 ± 3.3 to 4.7 ± 3.9) did not differ among treatment groups. In addition, blood progesterone concentrations at embryo recovery did not differ among all groups (10.0 ± 3.5 ng mL–1 to 15.7 ± 3.2 ng mL–1). No difference was observed among groups in pregnancy rate after transferring the recovered embryos (36.0 to 50.0%). In conclusion, our results show that a single injection of FSH dissolved in polyethylene glycol is an efficient and simple procedure for inducing superovulation in Korean native cows.