bluuebunny/crossref_metadata_2025 huggingface dataset:
296 EFFECT OF FROZEN SEMEN WITH OSTEOPONTIN ON IN VITRO BOVINE FERTILIZATION AND EMBRYO DEVELOPMENT
Authors: Gonçalves R. F. , Bertolla R. P. , Eder I. , Chapman D. A. , Killian G. J.
Published: December 2006

Poor reproductive performance of dairy cattle has a major negative impact on farm profitability. This is in part attributed to the use of semen from bulls of below-average fertility and to early embryonic mortality. Osteopontin (OPN) is an acidic glycoprotein that has been detected in the epithelium of the ampulla, seminal vesicles, and seminal fluid of high-fertility Holstein bulls. The objective of this study was to determine whether freezing semen with purified bovine milk OPN would improve fertilization and embryonic development in vitro. In a first step, frozen semen from 6 bulls was used with different concentrations of OPN (0, 1, 10, and 100 �g mL-1) to evaluate fertilization, and in a second step, semen from 2 of the 6 bulls was frozen with OPN (0, 5, 10, 20, 40, and 80 �g mL-1) to evaluate its effect on in vitro fertilization and embryonic development. In vitro-matured bovine oocytes were inseminated with 1 � 105 frozen–thawed spermatozoa per 10 oocytes. After 18 h (39�C, 5% CO2 in air), oocytes designated for evaluation of fertilization were fixed, stained, and observed to determine the presence of pronuclei. Oocytes destined for embryo culture were placed in 4-well dishes containing 500 �L of synthetic oviduct fluid per well at 5% O2, 5% CO2, and 90% N2 (v/v). Each experiment was repeated 4 times, and data from each experiment were pooled. Analysis of variance using a general linear model was performed using a weighted mean based on the number of oocytes per treatment. Significantly more (P < 0.05) oocytes were fertilized when frozen semen was used with 10 �g mL-1 of OPN (85 � 4.0%, 78 � 4.0%) from 2 of the 6 bulls than when 0 �g mL-1 was used (75 � 4.0%, 69 � 4.0%). Semen from those bulls resultewd in more fertilized oocytes when 20 �g mL-1 (86 � 3.5%, 79 � 3.4%) and 40 �g mL-1 (88 � 3.4%, 81 � 3.9%) of OPN were used during the second phase of the experiment. Oocytes inseminated with 10 �g mL-1 (84 � 2.5%, 77 � 2.3%), 20 �g mL-1 (87 � 2.8%, 79 � 1.9%), or 40 �g mL-1 (89 � 1.9%, 81 � 2.4%) of OPN in the frozen semen had increased cleavage rates at Day 4 compared with those inseminated with 0 �g mL-1 (75 � 3.5%, 69 � 3.4%). At Day 8, blastocyst development was greater for 10 �g mL-1 (40 � 1.8%, 37 � 1.6%), 20 �g mL-1 (42 � 2.1%, 38 � 2.9%), and 40 �g mL-1 (45 � 2.9%, 40 � 2.5%) of OPN than for the semen with 0 �g mL-1 (33 � 2.3%, 29 � 2.8%). We conclude that semen from some bulls that was frozen with purified bovine milk OPN increases IVF, cleavage, and embryonic development, suggesting a facilitative role for OPN in some reproductive technologies.
297 ADDITION OF GLUTATHIONE TO THAWING MEDIUM FOR BULL SPERMATOZOA IMPROVES THE IN VITRO EMBRYO PRODUCTION
Authors: Grullón L. , Gardó J. C. , García-Vázquez F. , Gadea J.
Published: December 2006

The cold shock of spermatozoa is associated with oxidative stress induced by reactive oxygen species (ROS) generation (Chatterjee et al. 2001 Mol. Reprod. Dev. 60, 498–506). Glutathione (l-�-glutamyl-l-cysteinylglycine; GSH) plays an important role as an intracellular defense mechanism against oxidative stress. The process of freezing is associated with a significant reduction in GSH content in novine spermatozoa (Bilodeau et al. 2000 Mol. Reprod. Dev. 55, 282–288). The main objective of this study was to evaluate the effect of GSH supplementation of the thawing extender on bull sperm used for in vitro embryo production. To examine the effect of GSH supplementation during the thawing process, spermatozoa from 6 different bulls were incubated without addition of GSH (control), and with addition of 1 mM or 5 mM GSH to the sperm-TALP medium, and maintained 30 min at 37�C in these media before assay. IVM and IVF were performed as described Coy et al. (2005 Reproduction 129, 19–26, 747–755). Oocytes were fertilized with frozen–thawed semen (106 total sperm/mL). After 18 h co-incubation, they were cultured in KSOM medium for 7 days. Embryos were examined for developmental stages at Day 2 (2–4 cells), Day 3 (8–16 cells), and Day 7 (morula–blastocysts). Finally, the total number of nuclei of blastocysts was determined by staining with Hoechst 33342 (10 mg mL-1; 20 min). The percentage of cleavage in the different embryo stages was higher in the 5 mM GSH group [n = 233, Day 2 (64.63%), Day 3 (56.92%), and Day 7 (31.28%)] than in control [n = 229, Day 2 (47.64%), Day 3 (42.16%), and Day 7 (22.32%)], and the 1 mM group maintained an intermediate position with [n = 246, Day 2 (54.88%), Day 3 (49.56%), and Day 7 (28.98%)]. The bull sperm affected significantly the cleavage parameters studied. The number of nuclei within the blastocysts ranged from 39.93 to 47.71, and no differences were observed between experimental groups. It is known that addition of GSH produces a reduction in ROS generation (Gadea et al. 2005 J. Androl. 26, 749–756). The results showed that in the treatment group there is a reduction in ROS generation in spermatozoa and an improvement in the cleavage rate and blastocyst formation when GSH was used. This study demonstrates that only a short period of incubation of the spermatozoa in an antioxidant could improve the spermatozoa functionality of producing more and better embryos.
This work was supported by AGL2003-03144, BIOCARM 10BIO2005/01-6463 and Seneca 03018/PI/05.
298 EFFECTS OF TRICHOSTATIN A DURING IN VITRO FERTILIZATION OF BOVINE OOCYTES ON SUBSEQUENT DEVELOPMENT, CELL NUMBER, AND ALLOCATION OF RESULTING EMBRYOS
Authors: Ikeda S. , Saeki K. , Tatemizo A. , Iwamoto D. , Kasamatsu A. , Taniguchi S. , Hoshino Y. , Amano T. , Matsumoto K. , Hosoi Y. , Iritani A.
Published: December 2006

Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization or of transferred cell genomes after nuclear transfer to establish a totipotent state for normal development. In the fertilization of bovine oocytes, asynchronous histone acetylation occurs during pronuclear formation in the manner that modification of the paternal genome precedes that of the maternal genome (Wee et al. 2006 J. Biol. Chem. 281, 6048–6057). In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse ovaries were in vitro-matured (IVM) for 21 h in TCM-199 supplemented with 5% v/v FCS, 0.5 mM sodium pyruvate, 0.02 AU mL-1 FSH, and 1 �g mL-1 estradiol-17β at 39�C under 5% CO2 in air. After IVM, the oocytes were subjected to IVF with 3 � 106 mL-1 of Percoll gradient-selected sperm in a defined medium (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were freed from cumulus cells and cultured in mSOF medium until 168 h post-insemination (hpi) at 39�C under 5% CO2, 5% O2, and 90% N2 with high humidity. Cleavage and blastocyst development were assessed at 48 and 168 hpi, respectively. Inner cell mass (ICM) and trophectoderm (TE) of blastocysts were differentially stained by the method of Thouas et al. (2001 Reprod. Biomed. Online 3, 25–29) to assess cell number and ICM/TE ratio. Experiments were replicated 3 times. Data are presented as means � SEM and statistically analyzed by multiple comparison with the Holm method. Rates of cleavage (0 nM: 71.0 � 7%, n = 102; 5 nM: 75.5 � 5%, n = 106; 50 nM: 68.8 � 6%, n = 105; and 500 nM: 71.7 � 4%, n = 98) and blastocyst formation (21.4 � 5%, 22.3 � 6%, 17.8 � 2%, and 18.2 � 2%, respectively) were similar among the groups. However, 500 nM TSA significantly (P < 0.05) increased ICM and total cell numbers (59.8 � 4 and 143.5 � 7, respectively, n = 31) compared with the control (43.1 � 3 and 120.9 � 7, n = 31). In addition, ICM/TE ratios were higher in the 50 nM (0.81 � 0.08, n = 29) and 500 nM (0.92 � 0.2, n = 31) groups than in the control (0.59 � 0.04, P < 0.05). These results suggest that TSA treatment during IVF of bovine oocytes does not affect the blastocyst rate but alters the cell numbers and their allocation to ICM and TE. Overriding epigenetic modification of the genome during fertilization may have a carryover effect on cell proliferation and differentiation in pre-implantation embryos.
This study was supported by a grant from Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST.
299 LEPTIN ACCELERATES PRONUCLEAR FORMATION FOLLOWING INTRACYTOPLASMIC SPERM INJECTION OF PORCINE OOCYTES: POSSIBLE ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE INACTIVATION
Authors: Jin Y. X. , Cui X. S. , Chang W. K. , Kim T. , Kim N. H.
Published: December 2006

Although evidence suggests that leptin modulates oocyte maturation in vitro, few studies have examined the direct effect of leptin on cytoplasmic maturation and pronuclear formation following fertilization of porcine oocytes.The aim of this studywas to determine microtubule and microfilament assembly following oocyte maturation, and to assess blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) of leptin-treated oocytes.A symmetrical shape of the meiotic spindle was considered to be the normal microtubule assembly, and a clear, thick microfilament area containing a second metaphase chromatin was considered to be morphologically normal. Significant differences were determined using Tukey's multiple range test with P < 0.05 being considered significant. Addition of 10 ng mL-1 of leptin to the in vitro maturation (IVM) medium significantly increased the percentage of zygotes that developed to the blastocyst stage (26.1 � 2.1%; P < 0.05) compared with that of the control (0 ng mL-1: 16.3 � 2.8%) or 1 ng mL-1 (18.6 � 2.6%) leptin treatment groups following parthenogenetic activation. After IVM for 44 h, the percentage of oocytes with normal microtubules was higher in the leptin (10 ng mL-1) group than in the control group (89.1 � 4.3% vs. 78.5 � 4.0%, respectively; P < 0.05). However, the percentage of normal microfilament assembly was similar in the leptin-treated and control groups (85.6 � 4.3% and 82.9 � 5.0%, respectively). In oocytes matured in vitro in the presence of 10 ng mL-1 of leptin and subsequently induced by parthenogenetic activation via chemical stimulation, there was a significant increase in the formation of 2 pronuclei (2PN: 52.1 � 5.2%; P < 0.05) at 6 h, compared with the control non-leptin-treated oocytes (40.7 � 4.0%). Similarly, addition of 10 ng mL-1 of leptin to the IVM medium also increased 2PN formation at the same time point following ICSI (leptin treatment: 47.9 � 4.0; control: 36.2 � 3.8%; P < 0.05). The addition of 10 ng mL-1 of leptin to the IVM medium decreased the level of phospho-ERK1/2 at 6 and 9 h and was lower in the leptin-treated group compared with the control non-leptin-treated group (P < 0.01). These results suggest that exogenous leptin enhances cytoplasmic maturation and pronuclear formation following fertilization, possibly via the MAPK pathway.
30 GROWTH AND REPRODUCTIVE SUCCESS OF CLONED MALE CATS DERIVED FROM ADULT SOMATIC CELL NUCLEAR TRANSFER
Authors: Choi E. G. , Yin X. J. , Lee H. S. , Kang S. H. , Lee Y. J. , Kim L. H. , Shin H. D. , Kim N. H. , Kong I. K.
Published: December 2006

There have been very few published reports on the normal growth and fertility of cloned cats derived from adult somatic cell nuclear transfer (SCNT). Here, we report studies investigating the growth and fertility of cloned male cats produced by SCNT. This study was designed to investigate the reproductive success by natural breeding of cloned male cats with domestic female cats and to measure endocrine hormone profiles related to male reproduction such as testosterone, LH, and FSH. Clones A, B, and C produced 3, 2, and 4 kittens, respectively, after natural mating with 4 domestic female cats, and clone D produced 5 kittens, in spite of being further into puberty than the other cloned male cats. All of the females delivered at 64 to 66 days after natural mating. The results obtained are presented as the mean � SEM. Sexual hormones were analyzed by ANOVA. All statistical analysis was conducted with SAS (SAS Institute Inc., Cary, NC, USA), and P < 0.05 was considered significant. Three of the 14 kittens expressed an odd eye color, which was produced from 1 clone A and 2 clone B cats. The eye color of other F1 kittens varied from brown to blue. Body weight at birth ranged from 72.9 to 134 g. Although clone D had a lower libido and entered puberty later than the other cloned males, it produced gonadal hormones within the average range. Four cloned male cats had normal fertility and were free from any reproductive problems. All of the cloned male cats were similar to two control and donor cats with respect to concentration of gonadal hormones. However, the concentration of testosterone in a castrated donor cat was significantly lower than that from clones A, B, C, D, and control cats (0.4 � 0.1 vs. 5.99 � 5.68; 3.46 � 2.81; 6.41 � 2.17; 3.75 � 0.34; 4.0 � 3.63 ng mL-1; P < 0.05). The concentrations of LH and FSH were not significantly different among any of the cats. A total of 14 kittens, 7 male and 7 female, were produced by the natural breeding of cloned male cats with 4 domestic female cats. These results indicate that cloned male cats have normal reproductive success and lie within the normal range of gonadal hormone production. All of the F1 kittens were produced by natural breeding, delivery, demonstrated normal growth, and are still alive and healthy.
This work was supported by KOSEF (grant M10525010001-05N2501-00110).
304 SHORT-PERIOD SPERM - OOCYTE INCUBATION AND FERTILIZATION MEDIA EFFECTS ON THE IN VITRO PRODUCTION OF SWINE EMBRYOS
Authors: Marques M. G. , Nascimento A. B. , Martins L. F. , Barros F. R. O. , Goissis M. D. , D'Avila Assumpção M. E. O. , Visintin J. A.
Published: December 2006

The aim of this study was to evaluate the effect of 2 sperm–oocyte incubation periods (30 min and 6 h) and 3 IVF media (TCM-199, TCM-199 with Ca-lactate, or mTBM). Cumulus–oocyte complexes (COCs) from abattoir-derived swine ovaries were matured in TCM-199 supplemented with 3.05 mM glucose, 50µg mL−1 gentamycin, 0.91 mM sodium pyruvate, 10% swine follicular fluid, 0.57 mM cysteine, 10 ng mL−1 of epidermal growth factor, 10 IU mL−1 eCG, and 10 IU mL−1 of hCG for 22 h, followed by a 22-h incubation without hormones. After in vitro maturation, oocyteswere allocated into 3 IVF media: TCM-199 (supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 50µg mL−1 gentamycin, 1 mg mL−1 BSA, and 3.06 mM mL−1 caffeine-standard medium);TCM-199 Ca (standard medium with 2.92 mM Ca lactate); or mTBM (supplemented with 5 mM mL−1 sodium pyruvate, 0.57 mM cysteine, 50µg mL−1 entamycin, 1 mg mL−1 BSA, and 2 mM mL−1 caffeine). The refrigerated semen at 15°C was centrifuged and capacitated for 2 h at 38.5°C in an atmosphere of 5% CO2 in air and under high humidity conditions in the respective IVF media. Oocytes were denuded, washed with the respective IVF media, and inseminated with the capacitated spermatozoa at a concentration of 6 × 105 sperm mL−1 in 90-µL microdroplets. After 30 min, a group of oocytes from each fertilization medium was gently washed to remove nonbound spermatozoa and returned to a new medium droplet for another 5.5 h (30-min group). The other group of oocytes remained in the same droplet with spermatozoa for 6 h (6-h group). After IVF, oocytes were cultured in porcine zygote medium-3 for 7 days. Data were analyzed by chi-squared test (P < 0.05).With regard to cleavage rates on the third day of embryo development (Day 3), no significant difference was observed among sperm–oocyte incubation periods; however, there was a difference in the TCM-199 group, compared with the mTBM and TCM-199 Ca groups, at the 30-min incubation period. With 6 h of incubation, no differences were observed among groups. When blastocyst (Day 7) rates were evaluated, no significant differences were observed between the 2 TCM-199 media groups; however, the mTBM groups showed higher blastocyst rates. The sperm–oocyte incubation period of 30 min or 6 h of IVF did not interfere with the blastocyst rate. In conclusion, the IVF medium mTBM was more efficient than the TCM-199 media when considering embryo production. Moreover, 30 min of IVF was sufficient for the spermatozoa responsible for oocyte fertilization to bind to the zona pellucida.

Table 1.Cleavage (Day 3) and blastocyst (Day 7) rates of embryos incubated in two sperm–oocyte periods (30 min or 6 h) and cultivated in three IVF media

This work was supported by the FAPESP 05/01420-7.
306 IN VITRO PENETRATION OF SWINE OOCYTES MATURED IN TCM-199 WITH ADDED DIBUTYRYL CYCLIC ADENOSINE MONOPHOSPHATE AND FOLLICULAR FLUID
Authors: Nascimento A. B. , Marques M. G. , Coutinho A. R. de S. , Tavares M. N. , D'Avila Assumpção M. E. O. , Visintin J. A. , Che L. , Bordignon V.
Published: December 2006

During the in vitro maturation (IVM) of pig oocytes, a large variation in the nuclear morphology of the germinal vesicle stage is observed. Thus, some oocytes can start meiosis earlier than others. A reversible alternative to inhibit meiotic resumption is the use of dibutyryl cyclic adenosine monophosphate (dbcAMP) in the early period of IVM, which may synchronize the oocytes to a specific germinal vesicle stage and improve early embryonic development. This study investigated the effects of additional dbcAMP and porcine follicular fluid (PFF) on monospermic and polyspermic penetration rates after IVF. Oocytes from prepuberal females were selected under a stereomicroscope, and those with uniform ooplasm and surrounded by several layers of compact cumulus cells were divided into 2 groups: T1 (control) group: TCM-199 supplemented with polyvinyl alcohol (0.1%), FSH (0.5 �g mL-1), LH (0.5 �g mL-1), epidermal growth factor (10 ng mL-1), pyruvate (0.9 mM), d-glucose (3.05 mM), cysteine (0.1 mg mL-1), and gentamycin (50 �g mL-1); and T2 group: T1 with the addition of 25% PFF and 1 mM dbcAMP. Both the T1 and T2 groups were IVM for 42 to 46 h, with FSH, LH, and dbcAMP used only in the first 22 h. At the end of the maturation period, cumulus cells were chemically removed; the oocytes were washed 3 times in IVF medium (modified Tyrode's buffered medium, mTBM) and placed in petri dishes containing 50 µL of the same medium. The sperm-rich fraction was collected from 2 boars by digital pressure with a gloved hand, extended in Beltsville thawing solution, and incubated for 24 h at 17°C. It was then centrifuged at 1200g for 3 min and standardized for 1 × 105 spermatozoa mL−1. Oocytes were co-incubated with the sperm for 6 h in mTBM at 38.5°C and 5% CO2. After insemination, oocytes were cultured in porcine zygote medium-3 (80 µL), covered with paraffin oil, for 18 h. The presumptive zygotes were fixed and stained with 1% orcein in 45% acetic acid and evaluated under phase-contrast microscopy at a 400× magnification. Differences among groups were determined by one-way ANOVA. In the T1 group, the penetration rate was 39.3 ± 9.6% (162/412), and no difference was observed in comparison with the T2 group, 29.5 ± 4.9% (113/383). The monospermic penetration rate was 31.5 ± 6% (51/162) in the T1 group and differed from that in the T2 group, 71.7 ± 3.3% (81/113). Moreover, the polyspermic penetration was significantly higher in the T1 group, 68.5 ± 6% (111/162), compared with the T2 group, 28.3 ± 3.3% (32/113). These data suggest that the IVM with TCM-199 with added dbcAMP + PFF can improve in vitro production of swine embryos and decrease polyspermic penetration.
308 EFFECT OF GONADOTROPIN TREATMENT ON OOCYTE COLLECTION AND IN VITRO FERTILIZATION IN THE BAN MINIPIG
Authors: Nguyen B. X. , Nagai T. , Kukuchi K. , Uoc N. T. , Ozawa M. , Linh N. V. , Duc N. H. , Thanh D. N. Q. , Hanh N. V. , Huu Q. X.
Published: December 2006

The Ban minipig is a local breed characterized by small ovaries with a scant number of follicles available for in vitro maturation (IVM). The combination of eCG and hCG has been used successfully to control estrus in pig breeding programs. In this paper we present the first results of IVF in this breed in comparison with 2 types of oocyte preparation: (1) from animals not receiving gonatotropin treatment (group 1, n = 9); and (2) from animals receiving an injection of mixed pregnant mare serum gonadotropin and hCG, 300 IU/animal, for 3 days before oocyte collection (group 2, n = 4). All animals were 1 to 3 years old and with body weights that varied from 8 to 12 kg. At the time of collection, the ovaries were observed for follicle development; the cumulus–oocyte complexes (COCs) were aspirated using a 18-gauge needle. COCs of categories A (with more than 4 layers of cumulus cells) and B (with 2 to 4 layers of cumulus cells) were collected and matured in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) at 39�C under 5% CO2 in air. Matured oocytes with expanding cumulus cells were inseminated using male Ban minipig epididymal semen frozen by the methods reported by Kikuchi et al. (1998 Theriogenology 50, 615–623). The frozen–thawed spermatozoa were pre-incubated for 1 h in modified medium-199 adjusted to pH 7.8 in the incubator at 37�C. The capacitated spermatozoa were diluted and added to drops of fertilization medium (Fig-FM; Suzuki et al. 2002 Int. J. Androl. 123, 135–142) containing oocytes; the final concentration of sperm was 106/mL. After 3 h of co-incubation, attached spermatozoa and cumulus cells were removed from oocytes and the oocytes were the cultured in vitro as described previously (Kikuchi et al. 2002). The results obtained from 4 replicates showed that the number of follicles with a diameter larger than 2 mm and the rates of oocytes categorized as A and B were significantly lower (P < 0.05; ANOVA test) in the nontreated animals (0.0 and 67.5%, respectively) than in the treated group (25.5 and 87.1%, respectively). The rates of oocytes with a clearly expanding cumulus obtained after IVM were 78.6 (n = 136) and 88.1% (n = 101) for groups 1 and 2, respectively. The rates of cleaved embryos and embryos developed to the compact morula stage were 47.2 and 9.1% (n = 39), respectively, for group 1; and 89.1 and 18.8% (n = 101), respectively, for group 2. In conclusion, gonadotropin treatment before the collection of oocytes is recommended for application of IVM–IVF to local Ban minipigs.
This work was supported by a grant from the VAST-Japan Society for the Promotion of Science Project.
31 EFFECT OF TREATMENT OF OVINE CYTOPLASTS WITH CAFFEINE ON THE TRANSLOCALIZATION OF HSP27 IN SOMATIC CELL NUCLEAR TRANSFER RECONSTRUCTED EMBRYOS
Authors: Choi I. , Lee J.-H. , Campbell K. H. S.
Published: December 2006

We have previously reported that treatment of enucleated ovine oocytes with 10 mM caffeine for 6 h increases the activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). In addition, when used as cytoplast recipients for somatic cell nuclear transfer (SCNT), the resultant blastocyst stage embryos show an increase in total cell numbers. Furthermore, we have also found that gene expression profiles observed following caffeine treatment more closely resemble those of IVF embryos than non-treated SCNT embryos. In particular, heat shock protein 27 (HSP-27, a molecular chaperon was down-regulated in non-treated SCNT embryos. In contrast, in caffeine-treated SNCT embryos, levels of transcripts were similar to those of IVF embryos (Choi et al. 2006 Reprod. Fertil. Dev. 18, 122). Several studies on heat shock protein 25 (HSP-25)/HSP-27 demonstrated that HSP-27 was underexpressed in SCNT bovine embryos (Pfister-Genskow et al. 2005 Biol. Reprod. 72, 546–555), and phosphorylated HSP-25 becomes sub-localized in nucleus following heat shock condition in mouse embryos (Kim et al. 2002 Mol. Reprod. Dev. 61, 3–13). In cultured cells, HSP-25 phosphorylation was mediated by mitogen-activated protein kinase (MAPK), suggesting that nuclear-translocated HSP-25 might function to protect nuclear structure, thereby preventing apoptosis (Geum et al. 2002 J. Biol. Chem. 277, 19 913–19 921). In this study, we investigated the effects of caffeine treatment on the localization of HSP-27 by fluorescene immunocytochemistry in SCNT and control embryos. HSP-27 was detected primarily in the cytoplasm of non-treated SCNT embryos from the 1-cell stage onwards; however, some nulear staining was observed in some embryos. In contrast, in caffeine-treated SCNT embryos, a more intense immunostaining of HSP-27 was observed in the nucleus than in the cytoplasm. This result indicated that HSP-27 was phosphorylated and translocated into nucleus in SCNT embryos treated with caffeine. In addition, we also examined the levels of transcripts of upstream genes of HSP-27 such as p38MAPKs, and MAPK-activated protein kinase 5 (Mapkapk5) by semi-quantitative RT-PCR. The levels of Mapkapk5 and p38MAPKs transcriptional expression were constant among groups. These observations suggest that the effects mediated by caffeine on HSP-27 occur before the onset of zygotic transcription, are independent of gene expression, but are maintained until the blastocyst stage. Taken together, our results suggest that the down-regulated expression of HSP-27 in SCNT embryos may be linked with the increased incidence of programmed cell death. In contrast, the elevated expression of HSP-27 and increased nuclear translocation observed in caffeine-treated SCNT embryos may contribute to the increase in total cell number we have previously reported at the blastocyst stage.
313 IMPROVEMENT OF FERTILIZATION IN VITRO BY PRE-INCUBATION OF FROZEN - THAWED RAT SPERMATOZOA WITH CAFFEINE-SUPPLEMENTED MEDIUM
Authors: Seita Y. , Sugio S. , Sano D. , Okuda Y. , Kashiwazaki N.
Published: December 2006

We previously reported the successful production of live offspring from cryopreserved rat spermatozoa through IVF (2006 Reprod. Fertil. Dev. 18, 256). The objective of the present study was to improve the IVF system with frozen–thawed spermatozoa for more efficient reproduction of live offspring. We examined the effect of caffeine-supplemented medium on post-thaw sperm fertility in vitro. Epididymal spermatozoa of Wistar rats were cryopreserved in 2.0 mL of freezing medium containing 23% (v/v) egg yolk, 8.0% (w/v) lactose monohydrate, and 0.7% (v/v) Equex STM. Post-thaw spermatozoa were diluted to 0.5 to 1.5 � 106 sperm mL-1 in a droplet of 200 �L of R1ECM as the pre-incubation medium. In Experiment 1, the effect of caffeine-supplemented medium on fertility in vitro was examined. The post-thaw spermatozoa were incubated for 5 h in pre-incubation medium with added caffeine at concentrations of 0, 1, 2, and 4 mM. Ovulated oocytes were introduced into droplets of the pre-incubation medium and co-cultured for 10 h. The oocytes were denuded and fixed to examine for 2 pronuclei formation (2PNF). In Experiment 2, the effect of caffeine-supplemented medium on post-thaw sperm function was examined using the chlortetracycline (CTC) fluorescence assessment. The post-thaw spermatozoa were incubated in pre-incubation medium with added caffeine at concentrations of 0 (Caf+) and 2 mM (Caf-), and CTC patterns were examined hourly until 6 h later. Statistical analyses of the results were carried out by one-way ANOVA. In Experiment 1, the percentages of 2PNF were 14 � 6, 32 � 8, 42 � 4, and 27 � 5% for the 0, 1, 2, and 4-mM groups, respectively, and only the 2-mM group was different from the 0-mM group (P < 0.05). In Experiment 2, the rate of capacitated spermatozoa was not significantly different (P > 0.05) among incubation times in the Caf+ group, but the rates at 5 and 6 h were lower (P < 0.05) than the rate at 1 h in the Caf- group. Although the rates of acrosome-reacted spermatozoa were not significantly different (P > 0.05) in the Caf+ group after 3 h of incubation, the accelerated trend of the acrosome reaction was observed in the Caf- group with increasing incubation time. The results demonstrated that frozen–thawed rat spermatozoa pre-incubated in medium containing 2 mM caffeine could improve in vitro fertility. This positive effect of caffeine would be provided to induce and keep sperm capacitation, and to inhibit the spontaneous sperm acrosome reaction in frozen–thawed rat spermatozoa.
315 SELECTION OF BOVINE OOCYTES BY BRILLIANT CRESYL BLUE BEFORE IN VITRO MATURATION IMPROVES BLASTOCYST DEVELOPMENT
Authors: Sugulle A. , Katakawa S. , Yamamoto S. , Oomori S. , Itou I. , Dochi O. , Koyama H.
Published: December 2006

The morphological identification of immature oocytes has commonly been used to select the bovine oocytes for IVF. However, <30% of the recovered oocytes reach the blastocyst stage after fertilization, and this is probably due to the quality of the oocytes at the beginning of maturation. The brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. The aim of this study was to evaluate the effect of the BCB stain on the selection of bovine oocytes and on the subsequent embryo development for in vitro production (IVP). Cumulus–oocyte complexes (COCs) were collected by the aspiration of 2- to 6-mm follicles. A total of 559 oocytes were divided into 2 groups: (1) a control group, immediately cultured, and (2) a BCB-incubated group. After 90 min of BCB staining (Pujol et al. 2004 Theriogenology 61, 735–744), the oocytes were divided into oocytes with blue cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB−). The COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg mL−1 FSH at 38.5°C under an atmosphere of 5% CO2 in air. The matured COCs were inseminated with 5 × 106 sperm mL−1. After 18 h of gamete co-culture, the presumed zygotes were cultured in CR1aa supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (proportion of ≥5-cell stage, the total cleavage rates) and on Days 7 to 9 (blastocyst rate). The experiment was replicated 5 times, and the data were analyzed by a chi-square test and ANOVA. The results are presented in Table 1. The proportion of embryos with ≥5-cell stage was significantly higher (P < 0.01) in the BCB+ group than in the BCB− group, but not in the control group. The total cleavage rate for the BCB+ embryos was significantly higher than that of either the BCB− or the control group (P < 0.01). There were also significant differences (P < 0.01) in the blastocyst development between the BCB+ and BCB− embryos and between the BCB− and the control embryos (P < 0.05). This result showed that the selection of bovine oocytes by BCB staining before in vitro maturation may be useful for selecting oocytes that are developmentally competent up to Day 9 for IVP.

Table 1.Effect of selection of oocytes by brilliant cresyl blue (BCB) staining on the subsequent embryo development of in vitro-matured/in vitro-fertilized bovine embryos
32 EFFECTS OF FUSION/ACTIVATION METHODS ON DEVELOPMENT OF EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF PORCINE FETAL FIBROBLASTS
Authors: Cong P. Q. , Song E. S. , Kim E. S. , Li Z. H. , Yi Y. J. , Park C. S.
Published: December 2006

Pigs have become increasingly important in the field of biomedical research, and interest has grown in the use of transgenic cloned pigs as potential xenograft donors. The present study were carried out to investigate the effects of intensity of DC pulse, number of DC pulses, and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. Porcine cumulus-oocyte complexes (COCs) were cultured in modified TCM-199 (mTCM-199) medium for 44 h at 38.5�C, 5% CO2 in air. After in vitro maturation (IVM), metaphase II oocytes were selected for enucleation. Porcine fetal fibroblasts were obtained from a porcine fetus on Day 35 of gestation as donor cells. Oocytes were enucleated by removing, with a micropipette, the first polar body along with adjacent cytoplasm containing the metaphase plate; then a donor cell was injected in contact with the cytoplasm of each oocyte. In experiment 1, several different fusion/activation intensities (two DC pulses of 0.4, 0.8, 1.2, 1.6, and 2.0 kV cm-1 for 30 �s) were carried out to investigate the effect on the development of nuclear transfer embryos. In experiment 2, the reconstructed oocytes were fused and activated with 1, 2, or 3 DC pulses of 1.2 kV cm-1 for 30 �s. In experiment 3, reconstructed oocytes were equilibrated in mTCM-199 medium at 38.5�C, 5% CO2 for 0, 1, 2, 3, 4, 5, and 6 h. After equilibration, the reconstructed oocytes were fused and activated with one DC pulse of 1.2 kV cm-1 for 30 �s in fusion medium. The reconstructed embryos were transferred into PZM-3 medium containing 0.3% BSA for further culture. The rates of embryo cleavage and development of blastocyst stage were evaluated at 48 h and 6-7 days, respectively. The cell numbers of blastocysts were counted by using Hoechst 33342 epifluorescence staining. Data were analyzed by ANOVA and Duncan
323 EXPRESSION OF PSP-I AND PSP-II IN THE REPRODUCTIVE TRACT OF THE BOAR BY IMMUNOHISTOCHEMISTRY, WESTERN BLOTTING, AND RT-PCR
Authors: Garcia E. M. , Vazquez J. M. , Parrilla I. , Calvete J. J. , Sanz L. , Martinez E. A. , Roca J. , Rordríguez-Martínez H.
Published: December 2006

In boar seminal plasma, PSP-I and PSP-II form a noncovalent heterodimer, which is known to have a beneficial effect on highly extended boar spermatozoa. These proteins are mainly produced by the seminal vesicles of the male reproductive tract and are mixed with the spermatozoa during ejaculation. This study assessed the epithelial localization and expression of spermadhesin PSP-I and PSP-II subunits using immunohistochemical, western blotting, and RT-PCR methods in porcine testis, epididymis (caput, corpus, and caudal), seminal vesicles, and bulbourethral glands. Tissues were collected from 10 mature boars (Swedish Yorkshire) of proven fertility, frozen or fixed, and embedded in paraffin. Sections of 5 �m were mounted on poly-l-lysine-coated glass slides for immunohistochemistry, and 50 mg from the frozen counterparts were homogenized to isolate proteins (Western blotting study) or RNA (RT-PCR study). Polyclonal antibodies (antiPSP-I and antiPSP-II) against proteins (PSP-I and PSP-II) were used. The immunohistochemistry showed positive labelling for both antibodies in the epithelium of seminal vesicles in all males. Positive immunolabelling, but of variable intensity, was present in the epididymal epithelium (caput, corpus, and caudal), with variation among segments and boars. No labelling was found in the seminiferous epithelium or bulbourethral glands of any boar. After SDS-PAGE and Western blotting, immunoreactive bands were obtained in the extracts of all tissues for both PSP proteins. Among tissues, the highest intensity corresponded to the seminal vesicle lane for both proteins. Epididymis (caput, corpus, and caudal), bulbourethral gland, and testis showed more feeble bands, yet present. The intensity of the bands varied among boars. Amplification products from mRNA were obtained in all tissues explored by RT-PCR, using specific primers for PSP-I and PSP-II. The same trend was obtained regarding intensity bands, corresponding with the intensity obtained by Western blotting. The results indicate that PSP-I and PSP-II are mainly expressed in seminal vesicles and epididymal segments and, in lower amount, in the testis and bulbourethral gland.
This work was supported by Formas, Stockholm, and Fundaci�n S�neca, Murcia, Spain.
326 COMPARING CHANGES IN MEMBRANE LIPID ORDER IN EPIDIDYMAL AND EJACULATED BOAR SPERMATOZOA
Authors: Matas C. , Garcia-Vazquez, F. , Sansegundo M. , Ruiz S. , Gadea J.
Published: December 2006

The diffusion of lipids in the plasma membrane of ejaculated spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium (Wolfe et al. 2001 Mol. Reprod. Dev. 59, 306–313). Merocyanine 540 (M540) is a hydrophobic dye that has been shown to stain cell membranes more intensely if their lipid components are in a higher state of disorder, as is the case of capacitated spermatozoa. It is believed that the membrane fluidity changes detected by M540 precede the calcium influx, making M540 a method for evaluating the early events of capacitation. The aim of this study was to determine if there are differences in the dynamics of lipid disorder in the plasma membrane of ejaculated and epididymal boar spermatozoa under different conditions of capacitation. The sperm capacitation treatments were: washed in Delbucco's PBS supplemented with 0.1 % BSA (PBS-BSA), washed on a Percoll gradient (PG), and unwashed (UW: Control). During measurement, the samples were kept at 38�C and 5 % CO2 to maintain constant incubation conditions. Membrane lipid order and sperm viability were determined by flow cytometry with M540 (2.7 �M) and Yo-Pro-1 (25 nM), respectively. Samples were analyzed on a Coulter Epics XL flow cytometer (Beckman Coulter Co., Inc., Fullerton, CA, USA). A total of 10 000 gated events were collected per sample, with sample running rates of approximately 600 events/s. Data were analyzed by analysis of variance (ANOVA). For the epidydimal vs. ejaculated results, the percentage of low lipid disorder spermatozoa was higher in the epididymal (19.23%) than in the ejaculated (5.84%) groups, and the proportion of high disorder (42.85%) and dead cells (48.59%) was higher in the ejaculated group. In relation to sperm treatment (UW, PBS-BSA, and PG), the percentage of high disorder was similar in all of the treatment groups (UW: 44.62 %; PBS-BSA: 43.08%; PG: 43.41%). Finally, the percentage of low disorder was lower in the PBS-BSA and PERCOLL (10.68% and 12.83%, respectively) groups, and the highest was obtained for the UW group (14.09%). In conclusion, the staining with M540 revealed that the lipid disorder was affected by the source of the sperm and the sperm treatment. A significant increase in membrane lipid low disorder and decrease in high disorder and dead cells were detected when epididymal sperm were compared with ejaculated sperm, so the seminal plasma and the sperm treatment to eliminate disorder have an important effect in the lipid membrane order.
Supported by MEC (AGL2006-03495/GAN) and Fundaci�n S�neca (03018/PI/05).
327 IDENTIFICATION OF OSTEOPONTIN IN WATER BUFFALO SEMEN
Authors: Pero M. E. , Killian G. J. , Lombardi P. , Zicarelli L. , Avallone L. , Gasparrini B.
Published: December 2006

The competitiveness of buffalo breeding will depend on the utilization of reproductive biotechnologies that allows acceleration of genetic progress. A major factor hampering the efficiency of both artificial insemination and in vitro embryo production programs in this species is male hypofertility. Reports for several species suggest that seminal plasma contains factors that influence male fertility. Osteopontin is a glycoprotein found in several biological fluids including seminal plasma, and its presence is associated with spermatozoa concentration. In cattle, expression of osteopontin was highly correlated with bull fertility, and it was proposed to be a marker to predict male fertility (Cancel et al. 1999 Biol. Reprod. 60, 454–460). No data are available about the presence or activity of osteopontin in water buffalo. The aim of this preliminary study was to determine if osteopontin is present in buffalo semen and to evaluate whether freezing procedures cause the loss of osteopontin from spermatozoa. Semen was collected in authorized semen collection centers from 6 buffalo bulls by using an artificial vagina. A collection of bovine semen was used as a positive control. An aliquot from each sample was frozen using standard procedures for semen storage. Each ejaculate was centrifuged at 600g for 10 min at room temperature, and the supernatant was recovered and centrifuged at 10 000g for 1 h at 4�C. The total protein concentration in seminal plasma and spermatozoa was determined by the Bradford method, using ovoalbumin as the standard. Proteins (50 �g) were separated by electrophoresis and analyzed by western blotting (Cancel et al. 1999). Polyclonal antibodies against bovine milk osteopontin were prepared as previously described (Cancel et al. 1997 Biol. Reprod. 57, 1293–1301). The intensities of bands indicated by western blot were quantified by densitometer. Osteopontin was detected in all samples of buffalo semen. Most of the osteopontin detected was in the seminal plasma. Relative amounts of osteopontin detected in spermatozoa were 50% or less of that detected in seminal plasma; furthermore, the protein was not found in sperm from all bulls. These results suggest that most osteopontin is produced by the ampullae and seminal vesicles, similar to what was reported for cattle (Cancel et al. 1999). Semen frozen by standard procedures showed a reduction in amount of osteopontin by up to 50%. These studies suggest that the fertility-associated protein osteopontin may be useful as a sensitive tool to evaluate whether sperm storage procedures are detrimental and result in excess loss of osteopontin from sperm. In conclusion, the results have demonstrated that osteopontin is present in buffalo seminal plasma and sperm. Further studies will examine whether the expression of osteopontin is correlated with the fertility of buffalo bulls, as has been demonstrated in bovine bulls.
332 EFFECT OF DIFFERENT PIG OOCYTE ACTIVATION PROTOCOLS ON EMBRYO DEVELOPMENT IN SOF AND NCSU-23
Authors: Lagutina I. , Lazzari G. , Galli C.
Published: December 2006

Several factors affect nuclear transfer success. These include efficient parthenogenetic activation and embryo culture medium that should efficiently support pre-implantation development of good quality blastocysts. We investigated pig oocyte activation and embryo development in SOFaa in response to ionomycin (Io = 5 µM Io for 4 min; Io° = 15 µM Io for 20 min) and electric impulse (EL; one 30-µs pulse of DC 1.5 kV cm−1 in the presence of 50 µM Ca) in combination with 2 mM 6-DMAP or 10 µg mL−1 cycloheximide (CHX) +5 µg mL−1 cytochalasin B (CB) for 4 h. In addition, we studied the effect of elevated (1 mM) (Cheong et al. 2002 Mol. Reprod. Dev. 61, 488) in comparison with 50 µM Ca during EL activation on embryo development in SOFaa and NCSUaa-23. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon®, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng mL−1 long-EGF, 100 ng mL−1 long-IGF1, 5 ng mL−1 bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. The rates of cleavage, blastocyst formation (BL) and BL cell number on Day 7 (BL-D7) were recorded. All experiments were done with 3 replicates. The data were compared by chi-square test. There was no difference in the ability of Io (all groups) and EL + CB activated oocytes to cleave, whereas the additional treatment of EL-activated oocytes with DMAP and CHX + CB significantly increased cleavage. Io activation resulted in poor blastocyst development in comparison with all EL-activated groups (see Table 1). When calcium levels were elevated during EL activation, significantly more embryos developed in SOFaa (35.6%, n = 191 vs. 26%, n = 192; P < 0.05), but no differences were observed with culture in NCSUaa-23 (about 56%). The BL rate was significantly higher in NCSUaa-23 vs. SOFaa (55.9%, n = 68 vs. 34.8%, n = 69, respectively); however, the BL total cell number was significantly higher in SOFaa (58 ± 18, n = 40 vs. 86 ± 35, n = 56, respectively; P < 0.05). In conclusion, we have found that SOFaa and NCSUaa-23 differ in ability to support pig parthenogenetic embryo development. EL activation combined with elevated Ca significantly increased the embryo developmental capacity in SOFaa but not in NCSUaa-23. NCSUaa-23 was more efficient for embryo culture, whereas SOF produced BLs of higher quality.

Table 1.Effect of activation protocol on the development of pig parthenogenetic embryos in SOFaa

This work was supported by grants ISS-CS11 and Fondazione Cariplo.
334 DNA SYNTHESIS DURING THE FIRST CELL CYCLE OF PORCINE OOCYTES FOLLOWING DIFFERENT ACTIVATION TREATMENTS
Authors: Ock S.-A. , Kang D. , Han J.
Published: December 2006

Inhibitors of protein synthesis and phosphorylation have been widely used for oocyte activation and have been reported to induce abnormalities in nuclear ploidy due to aberrant DNA synthesis (DNAS). The present experiment was designed to compare the DNAS during the first cell cycle of porcine parthenotes following different activation treatments. Cumulus–oocyte complexes were cultured in TCM-199 supplemented with 0.5 �g mL-1 LH and FSH, 10 ng mL-1 EGF, and 0.1% PVA for 22 h, and additionally cultured in media without LH and FSH for 22 h. MII oocytes were then electrically pulsed twice in 0.28 M mannitol containing 0.05 mM CaCl2 and 0.1 mM MgSO4 at 1.8 kV cm-1 for 30 �s (group 1), followed by 7.5 �g mL-1 cytochalasin B (CCB, group 2), 10 �g mL-1 cycloheximide (CHX, group 3), or 1.9 mM 6-dimethylaminopurine (6-DMAP, group 4) for 3 h. Eggs were incubated with 100 �M 5-bromo-222-deoxyuridine (BrdU) for 1 h at 0, 2, 4, 6, 8, 10, and 12 h after activation to evaluate DNAS (Adenot et al. 1997 Development 124, 4615–4625) by determining the BrdU signal under a fluorescence microscope. Experiments were replicated 4 times; results were expressed as mean � SD and analyzed using one-way ANOVA by SPSS 10.0. The percentage of DNAS was calculated by dividing the number of BrdU-positive eggs by the total number of eggs used. DNAS in groups 1, 3, and 4 initiated at 2–3 h post-activation (hpa) but at 4–5 hpa in group 2. In group 1, DNAS was faint until 3 hpa, gradually increasing thereafter until 11 hpa (20.7 � 19.6, 29.4 � 17.0, 41.3 � 16.7, and 64.4 � 6.2, at 4–5, 6–7, 8–9, and 10–11 hpa, respectively). There was a significant (P < 0.05) increase in DNAS at 10–11 h, but a significant (P < 0.05) decrease (34.3 � 6.4) at 12–13 hpa. In groups 2 and 3, after 4–5 hpa, DNAS gradually increased until 7 h (6.2 � 1.4 and 29.8 � 16.6 at 4–5 hpa, and 21.1 � 13.7 and 40.0 � 18.7 at 6–7 hpa, respectively), but a DNAS peak was observed at 8–9 h (44.6 � 9.0) in group 2 and at 10–11 h (40.5 � 22.1) in group 3. Interestingly, group 4 parthenotes showed a different DNAS pattern compared with other groups, as it started at 2–3 hpa (24.8 � 11.7), reached a significantly (P < 0.05) high level at 4–5 hpa (56.3 � 9.0), and gradually decreased at 6–7 hpa (42.7 � 10.3) until 12–13 hpa (29.5 � 14.9). In conclusion, CCB, CHX, and 6-DMAP used for oocyte activation exhibited different patterns of DNA synthesis during the first cell cycle of porcine parthenotes. Therefore, further experiments are required to evaluate the molecular signaling that regulates DNAS, embryonic developmental velocity, and ploidy of 2-cell parthenotes.