bluuebunny/crossref_metadata_2025 huggingface dataset:
204 RELATIVE MESSENGER RNA ABUNDANCE OF HYALURONAN RECEPTORS AND SYNTHASES ON IN VITRO- AND IN VIVO-DERIVED DAY 7 AND DAY 13 BOVINE EMBRYOS
Authors: Clemente M. , Palasz A. T. , de La Fuente J. , Lonergan P. , Gutierrez-Adan A. , Rizos D.
Published: December 2008

Hyaluronan (HA), which progressively increases during embryogenesis, is a glycosaminoglycan that plays a major role in oocyte/embryo development (Fenderson et al. 1993 Differentiation 54, 85–95). One of the main functions of HA is to participate in the cell proliferation and migration that are controlled by HA receptors, RHAMM and C44, and by the presence of different HA synthases, Has1, Has2, and Has3. All have very distinctive features and functions at different stages of embryo development. The objective of this study was to determine the relative mRNA abundance of HA receptors and synthases in Day 7 and 13 bovine embryos derived in vitro or in vivo. In vitro embryos were produced by standard oocyte maturation and fertilization procedures. Presumptive zygotes were cultured in groups of 25 in 25-μL droplets of synthetic oviduct fluid supplemented with 5% FCS at 39°C, 5% CO2, and 5%O2 with maximum humidity. In vivo blastocysts were collected from superovulated heifers on Day 7 (estrus = Day 0) by uterine flushing and on Day 13 immediately after slaughter by flushing the dissected reproductive tracts. All embryos were frozen in LN2 and stored at –80°C for mRNA extraction. Quantification of transcripts for RHAMM and CD44 receptors and Has2 and Has3 synthases was performed on groups of ten Day 7 blastocysts (3 groups for in vivo or in vitro) and individual Day 13 embryos (7 embryos in vivo or in vitro) by real-time quantitative RT-PCR. Data on differences in transcript abundance were analyzed by ANOVA. The relative abundance of the Has2 and Has3 synthases was similar between in vivo and in vitro embryos, irrespective of their developmental stage. The quantity of CD44 was significantly higher in in vitro compared with in vivo embryos only on Day 7. However, the quantity of RHAMM receptor was higher on Day 13 in in vitro compared with in vivo embryos. When the comparison was done between developmental stages (Day 7 v. Day 13) for in vivo and in vitro embryos, we found that in vivo-produced Day 7 blastocysts expressed significantly more RHAMM receptor than embryos on Day 13. The reverse pattern of expression was shown for CD44 receptor. For in vitro embryos, the only difference observed was for Has3, which was up-regulated on Day 13 compared with Day 7 embryos. In conclusion, the present study demonstrates, for the first time, developmental changes in the abundance of RHAMM and CD44 receptor mRNA and Has2 and Has3 synthase mRNA in in vivo and in vitro bovine-derived embryos on Day 7 and 13. We believe that our results will provide new insight into the potential role of this intriguing multifunctional molecule in bovine early embryo development.
206 ALPHA6, BETA1, AND BETA3 INTEGRINS EXPRESSED BY SPERM MAY BE INVOLVED IN CATTLE FERTILIZATION
Authors: Gonçalves R. F. , Bertolla R. P. , Mortara R. A. , Barnabe V. H.
Published: December 2008

Sperm-egg interaction is a complex molecular process leading to gamete fusion mediated by a series of molecular interactions. Some integrin subunits, which are adhesion molecules, are expressed on human and mouse sperm, but major questions about the roles of integrins in sperm-oocyte fusion remain unsolved. This study was conducted to determine the presence of integrin subunits on cattle (Bos indicus and Bos taurus) sperm, and whether fertilization might be affected by treating sperm with antibodies to integrin subunits. Frozen–thawed sperm, donated by ABS Pecplan, were centrifuged at 700g for 10 min and washed once in warm PBS (Nutricell®, Campinas, São Paulo, Brazil). Sperm were resuspended in PBS and treated with an equal volume of cold 4% paraformaldehyde (prepared fresh from formaldehyde) for 15 min on ice. All subsequent steps were conducted at RT. Fixed sperm were washed twice with PBS (10 000 rpm, 5 min) in an IEC Micromax (Needham, MA), and the pellet was resuspended in PBS containing 5% BSA. Samples of 1 mL, each containing 5 × 106 spermatozoa, were incubated overnight at 4°C with a monoclonal anti-integrin α6 immunoglobulin G (IgG), monoclonal anti-integrin β1 IgG, and monoclonal anti-integrin β3 IgG (Chemicon®). The next day, sperm were resuspended in Alexa Fluor® 488 goat anti-mouse IgG (Invitrogen). After being washed 3 times in PBS-BSA as before, a drop of sperm suspension was smeared on a slide, air-dried, mounted with antifade reagent with 4′,6-diamidino-2-phenylindole (Invitrogen), and covered with a coverslip for analysis. Frozen–thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1 h in fertilization medium (FM; 1), FM with a monoclonal anti-integrin α6 IgG (2), FM with monoclonal anti-integrin β1 IgG (3), and FM with monoclonal anti-integrin β3 (4). In vitro-matured cattle oocytes were incubated (39°C, 5% CO2 in air) with 10 × 104 washed pretreated spermatozoa per 25 oocytes for 18 h. The oocytes were fixed in acid alcohol, stained with 1% acetate-orcein, and observed to determine the presence of pronuclei. Immunoreactive α6, β1, and β3 were concentrated at the apical segment of the acrosome in all bulls tested. There was no detectable fluorescence in any of the controls, and not all the sperm in an ejaculate were immunoreactive. Addition of anti-integrin α6, anti-integrin β1, and anti-integrin β3 decreased fertilization (P < 0.05) compared with the control: (1) 91.2 ± 2.0%; (2) 17.4 ± 2.0%; (3) 14.3 ± 2.0%; (4) 24.3 ± 2.0%. These findings show that α6, β1, and β3 integrins are expressed by cattle spermatozoa and may be involved in sperm-oocyte fusion and fertilization.

This study was supported by FAPESP grants (2007/00363-5 and 2006/06008-0, Brazil). We acknowledge Nutricell and ABS Pecplan for their generous contribution.
212 EFFECTS OF VASCULAR ENDOTHELIAL GROWTH FACTOR ON THE EARLY DEVELOPMENT AND POLYSPERMY RATE OF OVINE EMBRYOS PRODUCED IN VITRO
Authors: Luo H. , Cao X. , Zhao Y. , Zhou P. , Shi G.
Published: December 2008

To investigate the effect of vascular endothelial growth factor (VEGF) on the early development and polyspermy rate of ovine embryos in vitro, 2 experiments were conducted with human recombinant VEGF165 supplemented to the media during maturation, fertilization, and culture in vitro, respectively. Ovaries were collected from ewes at a local slaughterhouse. All oocytes surrounded by a multilayer of cumulus cells were collected and rinsed 3 times in maturation medium (control medium and treatment medium, respectively). A total of 100 oocytes in each group were cultured in 4-well plates (Nunc) containing 800 μL of maturation medium at 38.5°C in an atmosphere of 5% CO2 with saturated humidity. Four replicates of each experiment were conducted. Statistical analyses were conducted by ANOVA with SPSS 12.0 software (SPSS Inc., Chicago, IL, USA). Data are expressed as means, and P < 0.05 was considered significant. In Experiment 1, to investigate the effect of VEGF on the early development of ovine embryos in vitro, VEGF was used at 5 ng mL–1 (treatment group A) and 10 ng mL–1 (treatment group B) in maturation medium (TCM-199 + BSA), HSOF fertilization medium, and SOF culture medium. The results showed that the maturation rate was increased significantly (P < 0.01), from 75.76% in the control treatment to 83.98 and 80.23% in treatment group A and treatment group B, respectively. The cleavage rate was increased from 75.85% in the control group to 79.39% in treatment group A (P > 0.05). The development rates of morulae (45.03%) and blastocysts (23.54%) in treatment group A were significantly higher (P < 0.01) than those in the control group (38.94 and 18.09%, respectively). In addition, the development rates of blastocysts in treatment group B (21.05%) were lower than those in treatment group A (P > 0.05) and higher than those in the control group (P > 0.05). In Experiment 2, to investigate the effect of VEGF on the polyspermy rate of ovine embryos in vitro, 5 ng mL–1 of VEGF was used in TCM-199 + BSA maturation medium in this experiment. The results showed that the fertilization rate after 18 h of IVF was increased significantly (P < 0.01), from 75.75% in the control group to 83.86% in the treatment group, and that the polyspermy rate was decreased significantly (P < 0.01), from 12.64% in the control group to 7.68% in the treatment group. These results indicate that VEGF significantly improved the maturation and fertilization rates of ovine oocytes and, consequently, the rate of embryo development in vitro, especially when the medium was supplemented with 5 ng mL–1 of VEGF. The VEGF obviously decreased the polyspermy rate and bated the phenomenon of polyspermy in the process of ovine oocyte IVF.

The present study was supported by the National Natural Science Foundation of China (No. 30371035).
233 DEHIDROLEUCODINE INDUCES PARTHENOGENETIC ACTIVATION OF BOVINE OOCYTES
Authors: Canel N. , Salamone D.
Published: December 2008

Dehydroleucodine (DhL) is a sesquiterpene lactone that inhibits germinal vesicle breakdown in Bufo arenarum oocytes. Its action takes place over early stages of the cdc25 activation cascade (Bühler MI et al. 2007 Zygote 15, 183–187). The aim of this study was to evaluate the potential of DhL to induce parthenogenetic activation by observing nuclear dynamics and second polar body (2PB) extrusion of bovine oocytes, in the presence or absence of Cytochalasin B (CB), comparing these treatments with 6-Dimethylaminopurine (DMAP), an activation agent widely used. Cumulus–oocyte complexes were collected from cow ovaries obtained from a slaughterhouse. They were matured in TCM 199, supplemented with 5% FCS, 10 UI mL–1 penicillin, 10 μg mL–1 FSH, 100 μM cysteamine, 0.3 mm sodium pyruvate and 2 mm glutamine, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were selected and treated with 5 μm ionomycin (Io) for 4 min. Afterwards, oocytes were randomly allocated into one of the following treatments: a) incubation with 2 mm DMAP for 3 h (DMAP); b) incubation with 5 μm DhL for 3 h (DhL); and c) incubation with 5 μm DhL and 5 μg mL–1 CB, for 3 h (DhL-CB). A control group was only treated with Io. Activated oocytes were cultured in the maturation medium during 4, 11 or 17 h (Io exposure = 0 h), stained with Hoechst 33342 and analyzed under fluorescence microscope to evaluate nuclear stage and 2PB extrusion. Activation data are presented in Table 1. Oocytes with two extruded polar bodies and a metaphase plate were considered as partially activated (PA) and those exhibiting one pronucleus (PN) or already cleaved, as fully activated (FA). Oocytes that remained arrested at MII were not included in the table. Rates of 2PB emission were 98.3, 4.9, 83.6 and 61.5% for Io, DMAP, DhL and DhL-CB, respectively. These percentages were determined over total number of activated oocytes (PA and FA) within each group, including results from all evaluation times because no differences were found between them. Nuclear evaluation suggests that DhL is as effective as DMAP to induce full activation when combined with CB, and its use does not induce the early PN formation observed with DMAP at 4 h post Io. Most of the oocytes activated with DhL extruded a 2PB; these results were statistically different from those observed for other groups. These results indicate that DhL might be a useful agent to induce parthenogenesis, allowing 2PB extrusion and avoiding early PN formation in bovine oocytes.

Table 1.Partial and full activation of bovine oocytes at 4, 11 and 17 h post treatments
235 A COMPARISON OF PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES SYNCHRONISED DURING MATURATION BY CYCLOHEXIMIDE OR cAMP
Authors: Chen W. C. , Zhu J. , Fisher P. , Amarnath D. , Campbell K. H. S.
Published: December 2008

In vitro maturation of porcine oocytes is characterized by a high level of asynchrony between oocytes. Previous studies reported that cycloheximide (CHX) and 3′, 5′-cyclic AMP (cAMP) synchronize porcine oocytes and improve development to blastocyst stage following IVF or have been used for somatic cell nuclear transfer (SCNT) (Ye et al. 2005 Biol. Reprod. 72(2), 399–406; Betthauser et al. 2000 Nat. Biotechnol. 18(10), 1055–1059). We previously reported that cAMP was more effective than CHX in synchronizing porcine oocyte maturation, producing MII oocytes in a shorter time window and providing a more homogenous population for future SCNT studies (Chen et al. 2008 SRF conference, 2008 abst, p34). Here we compared parthenogenetic development of porcine oocytes synchronized by these two treatments. Selected cumulus–oocyte complexes (COC) obtained from slaughtered gilts were randomly divided into three groups and cultured at 39°C, 5% CO2 in air in modified NCSU-23 medium (with 1 μm glutathione, 1 mm cysteine, 5 mg L–1 insulin, 10 ng mL–1 epidermal growth factor, 10% (v/v) porcine follicular fluid, 1% essential and 0.5% nonessential amino acids) ± hormones (10 IU mL–1 PMSG and 10 IU mL–1 hCG): (1) with hormones for the first 22 h and then without hormones until 44 h; (2) with hormones and 5 μg mL–1 CHX for 12 h, and then with hormones but no CHX until 44 h; (3) with hormones and 1 mm cAMP for 22 h, and then without hormones and cAMP until 44 h. Parthenogenetic development of cycloheximide and cAMP treated oocytes was compared by cleavage rate at 48 h postactivation (hpa) and blastocyst formation at 168 hpa. No significant differences were observed in the frequency of cleavage (96.7 ± 2.1% v. 81.4 ± 11.6% v. 84.5 ± 5.7%), development to blastocyst (28.3 ± 11.4% v. 27.1 ± 5.7% v. 32.8 ± 5.3%) between control, CHX or cAMP treated oocytes, respectively (chi-square test, P > 0.05). However, total cell number was significantly higher in the CHX group than cAMP group (42.7 ± 4.1 v. 31.8 ± 2.0, respectively; t-test, P < 0.05). The results demonstrate that synchronization of porcine oocytes by treatment with CHX or cAMP does not affect subsequent parthenogenetic development if judged by the blastocyst formation, although the meaning of the difference of total cell numbers between CHX and cAMP treatments is still unclear.
24 EFFECTS OF HISTONE METHYLATION RELATED GENES ON EPIGENETIC REPROGRAMMING AND ZYGOTIC GENE ACTIVATION IN OVINE SOMATIC CELL NUCLEAR TRANSFER (SCNT) EMBRYOS
Authors: Choi I. , Campbell K. H. S.
Published: December 2008

After fertilization, early embryo development is dependent upon maternally inherited proteins and protein synthesised from maternal mRNA until zygotic gene activation (ZGA) occurs. The transition of transcriptional activity from maternal to embryonic control occurs with the activation of rRNA genes and the formation of the nucleolus at the 8- to 16-cell stage that coincides with a prolonged fourth cell cycle in bovine and ovine embryos. However, previous studies have reported a shift in the longest cell cycle (fifth cell cycle) in bovine somatic cell nuclear transfer (SCNT) embryos, suggesting that the major genome activation is delayed, possibly due to incomplete changes in chromatin structure such as hypermethylation and hypoacetylation of histone (Memili and First 2000 Zygote 8, 87–96; Holm et al. 2003 Cloning Stem Cells 5, 133–142). Although global gene expression profile studies have been carried out in somatic cell nuclear transfer embryos, little is known about the expression of genes which can alter chromatin structure in early embryo development and possibly effect ZGA. To determine whether epigenetic reprogramming of donor nuclei affected ZGA and expression profiles in SCNT embryos, ZBTB33 (zinc finger and BTB domain containing 33, also known as kaiso, a methy-CpG specific repressor), BRG1(brahma-related gene 1, SWI/SNF family of the ATP-dependent chromatin remodeling complexes), JMJD1A (jumonji domain containing 1A, H3K9me2/1-specific demethylase), JMJD1C (putative H3K9-specific demethylase), and JMJD2C (H3K9me3-specific demethylase) were examined by RT-PCR at different developmental stages [germinal vesicle (GV), metaphase II (MII), 8- to 16-cell, 16- to 32-cell, and blastocyst in both parthenogenetic and SCNT embryos]. All genes were detected in parthenogenetic and SCNT blastocyts, and ZBTB33 was also expressed in all embryos at all stages tested. However, the onset of expression of JMJD1C, containing POU5F1 binding site at 5′-promoter region and BRG1 required for ZGA are delayed in SCNT embryos as compared to parthenotes (16- v. 8-cell, and blastoocyst v. 16-cell stage). Furthermore, JMJD2C containing NANOG binding sites at the 3′-flanking region was expressed in GV and MII oocytes and parthenogenetic blastocysts, whereas in SCNT embryos, JMJD2C was only observed from the 16-cell stage onwards. Interestingly, JMJD1A, which is positively regulated by POU5F1, was not detected in GV and MII oocytes but was present in blastocyst stage embryos of both groups. Taken together, these results suggest that incomplete epigenetic modifications of genomic DNA and histones lead to a delayed onset of ZGA which may affect further development and establishment of totipotency. Subsequently, aberrant expression patterns reported previously in SCNT embryos may be attributed to improper expression of histone H3K9 and H3K4 demethylase genes during early embryo development.
255 DIURNAL RHYTHMS OF GLUCOSE AND NON-ESTERIFIED FATTY ACID CONCENTRATIONS IN BLOOD IN HEAT-STRESSED DAIRY CATTLE IN EGYPT
Authors: Shehab-El-Deen M. A. M. M. , Leroy J. L. M. R. , Fadel M. , Saleh S. , Maes D. , Van Soom A.
Published: December 2008

The hyperthermia associated with summer heat stress (HS) is a major cause of reduced fertility in high yielding dairy cows in the tropics and subtropics. The hyperthermia directly affects reproductive function. Furthermore, HS induces indirect effects (e.g. through a dropped dry matter intake and thus an altered energy balance), which may also have an impact on cow fertility. The extent of exposure to HS and HS-associated metabolic changes, such as hypoglycemia and high levels of non-esterified fatty acids (NEFA), may affect cattle fertility by impairing the developmental capacity of the oocyte. It is known that changes in glucose and NEFA concentrations in the blood may be reflected in the follicular fluid of the dominant follicle (Leroy et al. 2004). Therefore, the aim of the current study was to estimate to what extent oocytes may be exposed to HS-associated metabolic changes in dairy cattle during summer by assessing the diurnal changes in plasma. Respiration rate (RR) and rectal temperature (RT) along with plasma concentrations of glucose and NEFA were examined in 10 lactating Holstein cows at 4-h intervals from 7:00 AM to 3:00 AM for three successive days in August 2007. Temperature humidity indices (THI) were calculated as well. Linear mixed effect models with cow as random factor including the effect of daytime were used to investigate possible differences among studied parameters at different daytimes. At 3:00 PM, THI and RT were the highest (83.7 ± 0.6 and 40.1 ± 0.1, respectively) and the lowest value for THI recorded at 3:00 AM (74.2 ± 1.4) and for RT recorded at 7:00 AM (39.04 ± 0.08). RR increased gradually from 66.1 ± 3.1 resp min–1 at 3:00 AM to 93.0 ± 4 resp min–1 at 11:00 AM. Glucose concentrations decreased significantly (P < 0.001) at 11:00 AM and 3:00 PM. Decreased glucose concentrations coincided with increased NEFA concentrations at 11:00 AM and 3:00 PM (P < 0.001). The NEFA concentrations measured at 3:00 PM were significantly higher than at all other time points (Table 1). In conclusion, high producing dairy cows may be exposed to diurnal fluctuation of HS-associated metabolic changes such as significantly decreased glucose and increased NEFA concentrations in plasma for about 12 h per day, which may be reflected in the follicular fluid of the dominant follicle and as such directly affect oocyte and granulosa cell quality.

Table 1.Mean ± SEM of glucose and non-esterified fatty acids (NEFA) concentrations (mmol L–1) in blood plasma of 10 lactating Holstein cows at 6 different daytimes during August 2007 in Egypt
257 DEVELOPMENT OF HEAT-STRESSED OVA MATURED IN THE PRESENCE OF A PROSTAGLANDIN F2ALPHA RECEPTOR ANTAGONIST
Authors: Ward A. M. , Schrick F. N. , Payton R. R. , Peixoto E. , Edwards J. L.
Published: December 2008

Studies in the literature have shown that cumulus–oocyte complexes produce PGF2α, that ova and cumulus cells have PGF2α receptors, and that PGF2α addition to maturing ova, above what would normally be produced, decreases blastocyst development. Because previous studies have shown elevated systemic and tissue levels of PGF2α as a consequence of heat stress, it was hypothesized that detrimental effects of exposing maturing ova to elevated temperatures may be mediated in part through heat-induced increases in PGF2α. To test this hypothesis, cumulus–oocyte complexes were matured at 38.5 or 41.0°C in the presence of a PGF2α receptor antagonist (AL-8810). Preattachment embryo development of AL-8810-treated ova was compared with development of ova matured in media with or without diluent (DMSO added at the same concentration as AL-8810; diluent and developmental controls, respectively), resulting in 6 total treatment combinations. Data were analyzed as a randomized block design (blocking on oocyte collection date) with fixed effects of maturation temperature, AL-8810, and the respective interaction included in the statistical model. In experimental replicates in which the effects of heat stress decreased blastocyst development greater than 10% (n = 14), a significant maturation temperature × AL-8810 interaction was noted when evaluating blastocyst development (P = 0.05). Specifically, when ova were heat stressed during the first 12 h of in vitro maturation, blastocyst development was reduced in developmental and diluent controls (26.2 v. 18.8 and 24.4 v. 19.9, respectively; SEM = 1.6). In contrast, when ova were matured under the same conditions but in the presence of a PGF2α receptor antagonist, the effects of heat stress to reduce blastocyst development after in vitro fertilization were no longer observed (22.5 v. 22.5; SEM = 1.6). When using abattoir-derived ovaries, it is not uncommon to collect, on occasion, ova that are developmentally challenged (i.e. blastocyst development is less than the 20 to 50% expected). In this experiment, this occurred on 5 occasions. Data from these experimental replicates were analyzed and reported separately because previous efforts had shown that the responsiveness of ova to changes in culture environment differs depending on the level of developmental competence. Relevant to this study, addition of AL-8810 to developmentally challenged ova matured under thermoneutral conditions increased cleavage (60.4 v. 55.4%, respectively; P = 0.06) and blastocyst development (17.7 v. 13.7%, respectively; P = 0.07). In summary, data illustrate that developmentally challenged ova, heat-stressed or otherwise, are susceptible to detrimental effects of PGF2α. The ability to increase blastocyst development approaching or exceeding the values expected for competent ova suggests the usefulness of a PGF2α receptor antagonist during in vitro maturation to improve the efficiency of in vitro production procedures.
259 A RAPID, NON-PCR-BASED BOVINE EMBRYO BIOPSY SEXING ASSAY
Authors: Bleher R. , Erwin W. , Paprocki A. M. , Syverson C. M. , Koppang R. , Didion B. A.
Published: December 2008

Sex determination of bovine embryos in conjunction with embryo transfer is an established method to maximize efficiency by producing offspring with the preferred gender. Present methodology for sexing embryos is based on PCR, which requires molecular laboratory equipment, trained personnel, and several hours before a result becomes known. We developed a simple, rapid, non-PCR procedure for identification of Y-chromosomes in bovine blastomeres recovered via biopsy. A biopsy (n = 5 to 8 blastomeres) was taken from a blastocyst-stage embryo and transferred onto a plastic slide. After drying and fixation, the cells were denatured and incubated with a peptide nucleic acid probe designed to target unique Y-chromosome specific sequence. The probe was conjugated to a fluorescent dye (CY-3) which enables Y-chromosome detection as a bright spot within blastomere nuclei when using a fluorescent microscope. The absence of signal indicates female embryonic DNA. From placement of the biopsy onto a plastic slide, the methodology required approximately 75 minutes to determine embryo gender. The accuracy of the biopsy sexing procedure was demonstrated by parallel gender determination of the same embryo using an established PCR method designed for the bovine amelogenin locus. Based on 18 in vitro-produced bovine embryos generating a result for both assays, there was a 94.4% match (17/18) of gender assignment. The present technology represents a simple alternative to PCR-based embryo sexing technology. Research is ongoing for future development of live embryo sexing determination.
262 ICSI OF EQUINE OOCYTES WITH SEX-SORTED FROZEN-THAWED SEMEN RESULTS IN LOW CLEAVAGE RATE BUT NORMAL EMBRYO DEVELOPMENT AND PREGNANCIES
Authors: Colleoni S. , Spinaci M. , Duchi R. , Merlo B. , Tamanini C. , Lazzari G. , Mari G. , Galli C.
Published: December 2008

Sorting of sperm by flow cytometer has allowed selection of offspring of predetermined sex in several species by artificial insemination, although the success rate is often lower than with non-sexed semen. In horses, the problem was partially overcome with hysteroscopic insemination using sex-sorted fresh sperm. However, when sex-sorted frozen–thawed sperm were used the pregnancy rate was heavily reduced in comparison with non-sexed frozen–thawed semen. Because it has been demonstrated that in vitro assisted reproductive techniques, namely intracytoplasmic sperm injection (ICSI), has permitted live foals to be obtained using sperm with low fertility in the field, in this study we investigated the possibility of using ICSI with sexed-sorted frozen–thawed sperm for equine embryo production in vitro. Briefly, semen was collected from two Standardbred stallions of proven fertility (Stallions A, B), sorted using a MoFlo SX flow cytometer and frozen (Johnson LA and Welch GR 1999 Theriogenology 52, 1323–1341). Sex-sorted and control non-sexed frozen semen (two stallions of in vitro proven fertility: C, D) was thawed, centrifuged on a Percoll gradient, washed and diluted 1:1 in PVP before ICSI. Oocytes were collected from ovaries of slaughtered mares and matured in vitro. Metaphase II oocytes were injected with sperm, subsequently cultured up to the blastocyst stage and frozen conventionally in 10% of glycerol (Galli et al. 2002 Theriogenology 58, 713–715). Six embryos from sexed-sorted sperm were thawed and non-surgically transferred in naturally cycling synchronous recipient mares. Results are summarized in Table 1. Overall, 70 and 58 (stallion A, B) and 30 and 15 (stallion C, D) oocytes were injected with sex-sorted or control frozen–thawed sperm, respectively. Mean cleavage rates were 20.3% for sorted sexed sperm and 71.1% for control, showing a significantly lower cleavage rate for sexed sperm. This difference was reflected in the number of blastocysts obtained (4.7% v. 20.0%). From the 6 frozen–thawed embryos derived from sexed sperm, that were transferred, 4 pregnancies resulted. One pregnancy was lost around 21 days, a second was pharmacologically aborted, and two were maintained (one from male and one from female sorted semen are currently in the 11th month of gestation). In conclusion, this study demonstrates that ICSI with sex-sorted sperm can be used for producing equine blastocysts able to establish pregnancies at a high rate following embryo transfer. However, the overall efficiency of the system is limited due to the very low cleavage rate obtained with sexed-sorted frozen–thawed sperm.

Table 1.Development of embryos produced by ICSI with sorted and non-sorted frozen–thawed semen

This work was supported by an RFO (ex 60%) and Camera di Commercio Cremona grant. The Authors wish to thank Società Italiana Produttori Sementi.
265 SEXING OF BOVINE EMBRYOS BASED ON PCR TECHNIQUE
Authors: Koban E. , Tas A. , Aslan Ö. , Akkoc T. , Arat S. , Bagis H.
Published: December 2008

Turkish cattle breeds are well adapted to harsh environmental and poor feeding conditions. However, their productivity is low. Increasing fertility rate and obtaining a high number of progeny from high-quality animals are important parameters in animal husbandry. The objective of the present project is to produce sexed embryos and cryopreserve them for subsequent transfer. The birth of the calves produced from the transferred embryos with sex determined prior to transfer by PCR are additional objectives of the study. To develop and optimize the PCR method, DNA was first isolated by using standard phenol-chloroform extraction from blood samples of cows and bulls to use as positive control. Then two multiplex PCR methods were developed using one autosomal (bovine 1.715 satellite locus which produces 216 bp long PCR product), and two Y-chromosome specific loci BRY4.a (300 bp) and BRY1 (300 bp). Both multiplexes include bovine 1.715 satellite locus, and they either include BRY4.a or BRY.1 as the second locus. Female individuals produce one PCR band, whereas male individuals produce two PCR bands. Bovine parthenogenetic blastocysts were used to test these two multiplex PCR methods. Immature bovine oocytes were aspirated from slaughterhouse material and in vitro matured in tissue culture medium-199 (TCM-199) supplemented with 10% FCS, sodium pyruvate, EGF, bLH, bFSH and penicillin/streptomycin for 18 h at 39°C and 5% CO2 in humidified air. After removing the cumulus cells of matured oocytes (MII), meiotic spindles and first polar bodies were removed. Oocyte-cell complexes were fused by one 30 μs pulse of 133V/500 μm. All fusion units were subjected to chemical activation. Afterwards, parthenogenetic oocytes were cultured in Sage cleavage® medium supplemented with 8 mg mL–1 BSA for 72 h and then developing embryos were cultured in Sage blastocyst® media supplemented with 4 mg mL–1 BSA + 5% FCS for 4 additional days. Then they were stored at –20°C until DNA extraction. For DNA extraction two methods (Park et al. 2001 Theriogenology 55, 1843–1853; Tshimangadzo et al. 2004 Biol. Reprod. 71, 1671–1676) were employed to test their efficiency in our laboratory conditions, and we got better results with the former method. Repeated PCR tests of these parthenogenetic blastocysts were carried out and tests revealed only one PCR product of 216 bp corresponding to the 1.715 satellite locus as expected. The multiplex PCR methods will also be employed for Nuclear Transfer and IVF embryos.

This project is supported by Turkish Scientific and Technical Research Council-TOVAG (project no: KAMAG 107G027).
266 WHOLE GENOME AMPLIFICATION ON BLASTOMERS OF POST-BIOPSY BOVINE EMBRYO
Authors: Polisseni J. , Machado M. A. , Souza A. L. , Domingues R. , Guerra M. O. , Serapiao R. V. , Pereira M. M. , Sa W. F. , Carvalho B. C. , Camargo L. S. A. , Viana J. H. M. , Peters V. M.
Published: December 2008

Biopsy of embryos is very useful for choosing the desired sex and for production of cloned and transgenic livestock. However, only a small amount of genomic DNA is available to perform genetic studies. Alternatively, methodologies using whole genome amplification (WGA) have been developed. The aims of this study were to evaluate the effect of WGA on blastomeres removed from 8- to 16-cell bovine embryos and to determine the sex of blastomeres. Oocytes obtained from slaughterhouse ovaries were in vitro matured and fertilized. On the fourth day after fertilization, 8- to 16-cell bovine embryos were biopsied, and one-fourth of an embryo was removed. The blastomeres (n = 56) were submitted to WGA followed by PCR. Prior to the whole genome amplification, male and female bovine DNA samples were serially diluted (30 ng μL–1, 3.0 ng μL–1, 0.3 ng μL–1, 0.03 ng μL–1, 0.003 ng μL–1, 0.0003 ng μL–1) and embryos of various development stages (2, n = 6; 4–7, n = 5; ≥8-cell, n = 5; blastocyst n = 27) were used to standardize PCR protocols and set the amplification limits. To digest the cellular cytoplasm and release the genomic DNA, embryos and blastomeres were submitted a 3 mg mL–1 proteinase K before PCR. Next, blastomeres were submitted to the GenomiPhi DNA Amplification Kit (GE Healthcare) according to manufacturer’s instructions. The product (1 μL) was electrophoresed on a 1% agarose gel stained with 3.0 μg mL–1 ethidium bromide. The reaction mixture was added to the material to be amplified (2 mm MgCl2, 5X PCR buffer, 0.2 mm each dNTPs, 0.05 U μL–1 GoTaq DNA polymerase, 0.25 μm of primer). The products were submitted to electrophoresis on 8% polyacrilamide gel and stained with silver nitrate procedure. The chi-square test was used for statistic evaluation of the results to test the WGA efficiency and to determine the sex rates of bovine embryos and biopsied samples submitted to PCR. It was possible to achieve 98% efficiency in amplifying blastomeres using the WGA kit. Amplified samples showed approximately 400 ng of DNA generated from an estimated initial amount of 12 pg of DNA resulting from two cells per embryo. In whole embryos from different stages, no diffference was detected in the proportion of sexes (P > 0.05). However, a greater number of female samples was noted in biopsied material (76%, 25/33) (P < 0.05). PCR efficiency in blastocysts (93%, 2/27) was statistically greater (P < 0.05) than embryos in early stages of development (83%, 5/6), and biopsied material to 2, 4–7 and ≥8-cell (40%, 2/5; 60%, 3/5; and 59%, 33/56; respectively). These differences could be related to a sex-chromosomal mosaicism or absence of a nucleus in biopsied samples. The WGA creates a DNA stock sample that could be used for various gene profiling and sex determination analyses.

Financial support: Fapemig, CNPq.
267 DIFFERENCES IN BULL SPERM NUCLEAR SHAPE BETWEEN FLOW-SORTED AND NON-SORTED SPERM USING FOURIER HARMONIC ANALYSIS
Authors: Schindler J. R. , Monson R. L. , Willenburg K. L. , Parrish J. J.
Published: December 2008

The objective of the study was to evaluate sperm nuclear shape in sex-sorted and non-sorted semen using Fourier Harmonic Amplitudes (FHA). Frozen semen was obtained from a single commercially available source. Mature bulls (n = 15) from the same breed with an average age of 4.42 ± 2.08 years were collected and the semen was either frozen or X-chromosome sorted using a flow cytometer and then frozen. Frozen straws were transported to the lab and analyzed for FHA. Briefly, straws were thawed and cells were incubated with 1.6 μm Hoechst 33342. Cells were then washed, fixed, dried to a slide and analyzed for nuclear head shape. Harmonic amplitudes 0 to 5 (HA0–HA5), derived from FHA, were previously shown to be an accurate, objective, and repeatable measure of sperm nuclear shape. HA0 describes the overall nuclear size of the sperm, whereas HA1 describes the anterior head, HA2 the length of the sperm along the longitudinal axis, and HA3 to 5 the distal, post-nuclear curvature of the sperm head. Each unit of semen was evaluated for motility and FHA. There was a significant decrease in motility in the sorted group (77 ± 1% v. 54 ± 3%; P < 0.0001). Multivariate ANOVA showed that there were differences between the sorted and non-sorted groups in HA1 to 4 (P < 0.02). Harmonic amplitude means ± SD (microns) for sorted and non-sorted treatments are as follows: HA1 (0.117 ± 0.003 v. 0.109 ± 0.003), HA2 (1.087 ± 0.005 v. 1.063 ± 0.005), HA3 (0.139 ± 0.003 v. 0.130 ± 0.003), and HA4 (0.201 ± 0.004 v. 0.191 ± 0.004), respectively. The nuclear shape of X-sorted sperm is longer and more pinched in both the anterior and posterior head. Interestingly there was no difference in HA0 (P = 0.119) indicating that the overall size of the sperm head is not affected by the sorting process. The differences in harmonic amplitudes may be due to the size and a restricted location of the X v. Y chromosome in the sperm nucleus.
269 DEMETHYLATION DYNAMICS OF PATERNAL GENOMES IN PRONUCLEAR BOVINE ZYGOTES PRODUCED BY IN VITRO FERTILIZATION AND INTRACYTOPLASMIC SPERM INJECTION WITH NONDRIED OR FREEZE-DRIED SPERMATOZOA
Authors: Abdalla H. , Umayahara I. , Ohnami N. , Hirabayashi M. , Hochi S.
Published: December 2008

Epigenetic reprogramming in early preimplantation embryos, which refers to erasing and remodeling epigenetic marks, including DNA methylation, is essential for differentiation and development. In many species, the paternal genome is subjected to genome-wide active demethylation before DNA replication commences, whereas the maternal genome maintains its methylation status until being demethylated passively during the subsequent cleavage divisions. This study was designed to investigate the dynamics of paternal genome demethylation in pronuclear-stage bovine zygotes produced by conventional IVF or intracytoplasmic sperm injection (ICSI) using conventionally frozen, nondried and freeze-dried (FD) bull sperm stored at either +4 or –196°C for 1 year. Zygotes were fixed and immunostained using anti-5 methyl cytosin (Calbiochem, San Diego, CA, USA) at 8, 10, 14 and 18 h post IVF (hpi) and at 6 and 12 h post ICSI (hpic). After digital optical sections of the zygotes had been collected by a Bio-Rad confocal laser scanning microscope equipped with a krypton/argon ion laser, the fluorescence emitted by each pronucleus was quantitatively analyzed by Image-J software. The relative methylation of the male pronucleus to the female pronucleus in each zygote (RM) was calculated. Differences among groups regarding proportions of zygotes showing different levels of RM were analyzed using Fisher’s exact probability test, whereas differences among groups regarding the overall average of RM were analyzed using Student’s t-test. In conventional IVF, the overall average RM significantly decreased (P < 0.05) from 0.92 at 8 hpi to 0.69 at 10 hpi with no additional significant decrease at 14 and 18 hpi (0.67 and 0.64, respectively). This decrease was accompanied by significantly higher proportions of zygotes showing RM < 0.60 (45.5, 37.5, and 38.2% at 10, 14, and 18 hpi, respectively) compared with 3.7% at 8 hpi. Because spermatozoa take about 2 h to penetrate into oocytes in a bovine IVF system, zygotes at 6 and 12 hpic may be developmentally comparable with those at 8 and 14 hpi. The overall averages of RM in ICSI-derived zygotes (0.79 and 0.66 at 6 and 12 hpic, respectively) were similar to those in corresponding IVF-derived zygotes (8 and 14 hpi), but a significantly higher proportion of 6 hpic zygotes (37.8%) than 8 hpi zygotes (3.7%) showed RM < 0.60. Surprisingly, the proportions of 12 hpic zygotes produced with FD sperm showing RM < 0.60 (60.6 and 62.4% for +4 and –196°C storage, respectively) were significantly higher than the proportions of those produced with nondried sperm (39.4%). These findings indicate that demethylation of the bovine paternal genome occurred early after IVF and ICSI, and that the freeze-drying of the paternal genome resulted in a higher demethylation level.
272 LEUKEMIA INHIBITORY FACTOR SIGNALING PATHWAY IN PIG PARTHENOGENETIC PLURIPOTENT CELLS
Authors: Brevini T. A. L. , Pennarossa G. , Antonini S. , Gandolfi F.
Published: December 2008

Leukemia inhibitory factor (LIF), its receptor heterodimer (LRβ-gp130), and the related signal transducer and activator of transcription-3 (STAT3) constitute a system controlling self-renewal and pluripotency of embryonic stem cells (ESC) in the mouse, where LIF withdrawal or direct inhibition of STAT3 causes ESC differentiation. By contrast, several studies have demonstrated that LIF is not required to maintain human ESC pluripotency. Scattered information is available in other species, and data on the role of LIF in pig ESC are scanty. The aims of the present study were (a) to characterize the expression profile of gp130, LRβ, and STAT3 in pig parthenogenetic cell lines (ppC), previously derived in our laboratory and shown to be positive for the main pluripotency related markers; (b) to evaluate the role of LIF pathway in maintaining the pluripotency of these cells. To this purpose, ppC were cultured as previously described (Brevini et al. 2007 Theriogenology 68, 206–214) and screened by RT-PCR for the two LIF receptor subunits (LRβ and gp130) and STAT3. Pig granulosa cells were used as positive controls. To better investigate the possible role of LIF in maintenance of pluripotency in ppC, the formation of embryoid bodies (EB) was induced in the presence or in the absence of the cytokine. Undifferentiated cells were cultured in hanging drops either with or without LIF for 12 days. The EB formation and the expression of molecular markers specific for the three germ layers was evaluated at the end of the differentiation period. Molecular analysis allowed us to detect transcription of STAT3, whereas no signal for LRβ and gp130 was detected in ppC. These results seem to indicate that LIF does not play a role in the maintenance of pluripotency in the pig. However, after removal of LIF, ppC routinely formed EB that expressed molecular markers specific for the three germ layers. On the other hand, when LIF was added to the differentiation medium, pig cells were unable to form EB. They kept proliferating in an undifferentiated state, and no expression of molecular markers specific for the three germ layers was detected. Moreover, when re-plated on inactivated feeder-layers, they formed distinct colonies that maintained expression of pluripotency markers. Our results show that a role of LIF in pluripotency maintenance through a classical LRβ-gp130 and STAT3 activation pathway is unlikely. However, interaction with an alternative nonclassical activation signaling pathway cannot be ruled out. Indeed, the presence of the cytokine in the medium used for differentiation experiments actively inhibited EB formation, indicating a possible role in preventing differentiation in the porcine species. Further studies are needed to elucidate these aspects.

Supported by: PRIN2005; PRIN2006; First 2006; First2007.
276 EXPRESSION OF CD9 AND ALPHA6-INTEGRIN IN PORCINE BLASTOCYSTS
Authors: Goissis M. D. , de Barros F. R. O. , Marques M. G. , Mendes C. M. , Milazzotto M. P. , Viana C. H. C. , Assumpção M. E. O. A. , Visintin J. A.
Published: December 2008

Establishment of embryonic stem cell (ESC) culture in pigs has not been achieved. Verification of pluripotency markers is necessary for validation of a pluripotent cell line. Not all markers observed in ESC from other species are characterized in swine embryos. The objective of this study was to characterize CD9 and α6-integrin expression in porcine blastocysts and to derive porcine ESC using Matrigel. In vitro or in vivo porcine blastocysts were submitted to total RNA extraction for RT-PCR, fixation for immunocytochemistry or immunosurgery for culture of inner cell mass. Expression of Oct-4, CD9, and α6-integrin was detected by PCR. CD9 and α6-integrin PCR products had their nucleotide sequence assessed and compared with public nucleotide database. CD9 product was identical to CD9 porcine sequences and α6-integrin product was similar to human and equine α6-integrin. Immunocytochemistry revealed Oct-4 expression in cytoplasm of the inner cell mass (ICM) and trophoblast cells. CD9 and α6-integrin were observed preferentially on trophoblast cells. No ESC colonies were obtained using co-culture on mouse embryonic fibroblasts (MEF) or on Matrigel. This study describes for the first time expression of CD9 and α6-integrin in porcine blastocysts.

Financial support: Fapesp 05/57314-0.
279 COMPARISONS OF BONE MARROW AND PUTATIVE SKIN-DERIVED MESENCHYMAL STEM CELLS AS DONOR FOR NUCLEAR TRANSFER IN MINIATURE PIG
Authors: Kang E. J. , Park B. U. , Song H. J. , Yang Y. I. , Kim M. J. , Maeng K. H. , Jeon B. G. , Lee S. L. , Rho G. J.
Published: December 2008

Since the birth of the first cloned sheep was reported, fibroblasts are commonly used as donor cells for nuclear transfer. However, in some species there is a higher incidence of abnormal fetuses, still births, and neonatal deaths as a consequence of reprogramming disorders including DNA methylation and histone acetylation. Cloned embryos with mesenchymal stem cells (MSC) have shown higher developmental ability compared to fibroblast, suggesting that undifferentiated genome can required ease reprogramming. Because MSC are relatively difficult to collect from bone marrow, skin is an alternative source of the donor cells. However, molecular and functional analyses remain uncertain between MSC derived from bone marrow and skin stem cells isolated from ear. The present study compared the expression of early transcription factors (Oct-4, Nanog and Sox-2), and differentiation capability to osteocytes, adipocytes and chondrocytes of MSC isolated from bone marrow and skin-derived putative stem cells from miniature pig. Bone marrow was isolated by Ficoll density gradient method, and skin separated from epidermis and dermis was diced into 2-mm diameter explants, and attached to tissue culture dishes. Cells were then cultured in DMEM/F12 supplemented with 10% FBS, 10 ng mL–1 bFGF, 10 ng mL–1 EGF at 38.5°C, in a humidified atmosphere of 5% CO2 in air. Expression of Oct-4, Nanog, Sox2 was analysed by RT-PCR. Osteogenic and adipogenic differentiation were induced following the protocols described previously (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90; Mohana Kumar et al. 2007 Mol. Cells 24, 343–350) and compared by histological staining and RT-PCR. Osteocytes were defined by the formation of the mineral nodules of deposition of calcium by Von Kossa staining and differentiations into adipocytes and chondrocytes were identified by oil red O staining of lipid vacuoles and alcian blue of proteoglycan, respectively. Skin-derived MSC were revealed to similar mRNA expression of Oct-4, Nanog, Sox2 compared to bone marrow derived MSC. However, bone marrow derived MSC were higher mRNA expression about of osteocytic genes (osteoclacin and osteonectin), chondrocytic gene (collagen type) and adipocytic genes (aP2) than those of skin-derived MSC. In addition, bone marrow derived MSC were revealed greater deposition of calcium, proteoglycan, and lipid vacuoles than those of skin derived MSC by histological staining. The results of present study suggest that cells isolated from skin have lower potential than MSC isolated from bone marrow. However, skin-derived stem cells have properties of multi-lineage differentiation and can be obtained easily. These stem cells, therefore, can serve as easily accessible and expandable source possessing donor cells for cloning, potential animal model, and clinical applications.

This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.