bluuebunny/crossref_metadata_2025 huggingface dataset:
335 EFFECT OF VITAMIN E ON THE DEVELOPMENT OF BOVINE OOCYTES AFTER CHEMICAL ACTIVATION
Authors: Park J. I. , Jang Y. , Lee E. S.
Published: December 2006

Oxidative stress is known to induce apoptotic cell death by reactive oxygen species (ROS) generated from in vitro culture systems. This study was conducted to evaluate the effect of Vitamin E (VitE), as antioxidant, on development of bovine embryos activated in vitro. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL-1 l-cysteine, 20 mg/mL-1 sodium pyruvate, gonadotropins (250 IU each of eCG and hCG/mL), 10 mg/mL-1 epidermal growth factor, and 100 �M VitE. Oocytes were cultured at 38.9�C in 5% CO2 in humidified air. After 22 hours of culture, oocytes with polar bodies were selected and subjected to activation treatments. Oocytes were exposed to calcium ionomycin (5 �M for 5 min), followed by incubation with 6-DMAP (2 mM) for 3.5 hours in medium supplemented with or without VitE (100 �M). After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9�C in 5% CO2, 5% O2 in humidified air for 7–8 days. Cell numbers were counted by the number of nuclei of blastocysts stained with Hoechst 33342, and apoptosis was detected by TUNEL assay using a MK500 kit (Takara Bio, Inc., Otsu, Shiga, Japan). Total cell and apoptotic cell number were determined under a fluorescence microscope. Data were analyzed using Student's t-test and chi-square test. The cleavage and blastocyst rates were significantly higher (P < 0.05) after activation with VitE (78.1% and 16.3%, n = 80) than without VitE (66.7% and 11.0%, n = 60). Total cell numbers were also significantly higher (P < 0.05) in blastocysts after activation with VitE (143.0 ± 34.02, n = 21) than in those without VitE (127.63 ± 40.25, n = 20). However, the percentage of TUNEL-positive (apoptotic) cells was similar between blastocysts activated with VitE (5.38 ± 2.22) and those without VitE (6.76 ± 1.98). The results of the present study demonstrate that vitamin E added to activation medium promoted further development of activated embryos, although its role in the alleviation of apoptosis remains unclear.
338 INFLUENCE OF OOCYTE DIAMETER AND MORPHOLOGY ON APOPTOSIS AND BLASTOCYST DEVELOPMENT IN PREPUBERTAL GOAT
Authors: Anguita B. , Paramio M. T. , Jimenez-Macedo A. R. , Romaguera R. , Morato R. , Mogas T. , Izquierdo D.
Published: December 2006

In vitro embryo production from prepubertal females is lower than from adult females. There are different hypotheses to explain this fact. The aim of this study was to analyze the apoptosis of prepubertal goat oocytes and its relationship to embryo development according to oocyte diameters. Oocytes from slaughtered prepubertal goats were recovered by slicing, and classified as: healthy (H: compact cumulus cells and homogeneous cytoplasm) and early atresic (EA: granulated cytoplasm and/or initial cumulus expansion), and by oocyte diameter: 110–125 µm, 125–135 µm, and >135 µm. They were matured in TCM-199 for 27 h at 38.5°C and 5% CO2 in air. After maturation, a sample of oocytes was denuded, and oocytes and cumulus cells, separately, were analyzed by TUNEL assay (In Situ Cell Death Detection Kit; Roche Diagnostics SL, Barcelona, Spain) to study the apoptosis. The rest of oocytes were fertilized in vitro and the presumptive zygotes were cultured for 8 days in SOF at 38.5°C, 5% CO2 and 90% N2. Results are shown in Table 1. Fisher's exact test showed a significantly higher percentage of blastocyst formation in the largest oocytes than in those with smaller diameters; moreover, the largest healthy oocytes produced a higher rate of blastocyst formation than the early atretic oocytes of the same diameter group. TUNEL assay showed that the percentage of apoptotic oocytes was lower in the largest healthy oocytes, whereas in early atresic oocytes, apoptosis was not related to oocyte size. After maturation, the percentage of apoptotic cumulus cells was low (10% of cells) in all oocyte categories. However, in the early atretic group, the percentage of apoptotic cumulus cells increased in oocytes < 125 µm. In conclusion, in prepubertal goat oocytes, the percentage of blastocysts formed depends on oocyte diameter and the percentage of apoptotic cumulus cells.

Table 1.Effect of oocyte diameter and morphology on apoptosis and blastocyst development
This work was supported by a grant from Generalitat de Catalunya (2006FIC 00187) and a grant from the Universitat Autonoma de Barcelona (EME-2004-25).
34 PIGLETS BORN FROM HANDMADE CLONING
Authors: Du Y. , Zhang Y. , Li J. , Kragh P. M. , Schmidt M. , Bøgh I. B. , Zhang X. , Purup S. , Jørgensen A. L. , Pedersen A. M. , Villemoes K. , Yang H. , Bolund L. , Vajta G.
Published: December 2006

Somatic cell nuclear transfer (SCNT) is probably the most efficient way to produce pigs with targeted genetic modification. Handmade cloning (HMC) is a new technology for SCNT developed recently (Vajta et al. 2005 Reprod. Fertil. Dev. 17, 97–112). However, HMC that resulted in births in cattle was regarded technically difficult in pigs due predominantly to the fragility of MII phase porcine oocytes. The purpose of our present work was to use optimized porcine HMC for production of cloned piglets. Data were analyzed by t-test using SPSS (11.0; SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. Oriented handmade enucleation was performed as described elsewhere (Li et al. concomitant abstract). Briefly, oocytes that were partially digested with 3.3 mg mL-1 of pronase for 20 s were removed of polar bodies (PB) and adjacent small volume of cytoplasm by manual bisection in HEPES-buffered TCM-199 medium supplemented with 2% calf serum and 2.5 �g mL-1 of cytochalasin B. Halves without PB were collected as putative cytoplasts. In vitro-cultured porcine fetal fibroblasts were used as donor cells. After cytoplast-fibroblast pairing, fusion and activation of fused cytoplast-fibroblast pairs (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Clon Stem Cells 7, 199–205), reconstructed embryos were cultured in a modified well of the well (WOW) culture system (Feltrin et al. 2006 Reprod. Fertil. Dev. 18, 126 abst) with porcine zygote medium-3 (PZM3) supplemented with 4 mg mL-1 of BSA. The cumulative effect of the optimization steps has resulted in considerably improved in vitro efficiency, shown as 64 � 2.3 (mean � SEM) reconstructed embryos from 151.3 � 4.8 oocytes could be obtained per day after 3–4 h manual work, including a 1-h pause between fusion and activation. One-half (50.1 � 2.8%) of the reconstructed embryos developed to the blastocyst stage after 7 days, a rate that was significantly higher than that obtained with traditional cloning (TC; 27.7 � 2.2%; P < 0.01). To compare the transfer efficiency between HMC and TC, blastocysts from both HMC and TC produced by using nuclear donor cells of different origin, respectively, to identify the offspring were transferred surgically to synchronized recipients. Of 6 pregnancies produced, 2 are ongoing, 2 were lost, and 2 term litters of 3 and 10 piglets were born. Live birth/transferred embryo efficiencies for HMC and TC are 17% (10/58) and 15% (3/20). According to our knowledge, a litter size of 10 cloned healthy piglets, as achieved in this study, is the highest one that ever has been reported. Our data suggest that porcine HMC is a very promising method for SCNT and may promote its widespread application for various purposes.
340 EFFECT OF GLUCOSE ON THE MORPHOLOGY OF GERMINAL VESICLE AND MEIOTIC PROGRESSION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM
Authors: Funahashi H. , Koike T.
Published: December 2006

Glucose metabolism through the pentose phosphate pathway (PPP) seems to play a critical role in meiotic resumption in mouse oocytes (Downs et al. 1998 Biol. Reprod. 58, 1084–1094). However, the role is not clear in porcine oocytes. In the present study, we examined whether glucose affects morphological change of germinal vesicles and the resumption of meiosis in porcine oocytes in a chemically defined medium. In the first experiment, porcine cumulus–oocyte complexes (COCs) were collected from 3–6-mm follicles of slaughterhouse ovaries and cultured in a chemically defined medium, mNCSU37-PVA with/without 5.55 mM glucose in the presence of eCG, hCG, and dibutyryl cAMP for 20–22 h and then in the absence of eCG, hCG, and dibutyryl cAMP for 24 h. In the second experiment, 5.55 mM glucose in the maturation medium was replaced with the same concentration of Na pyruvate. In the third experiment, the PPP inhibitor 6-aminonicotinamide (6-AN) was added to the maturation medium at various concentrations (0, 10, 50, and 100 �M). To determine the activity of glucose-6-phosphate dehydrogenase (G6PD), OCCs were fixed, blocked, and treated with anti-G6PD polyclonal antibody and the secondary antibody labeling a fluorescent material. Results from 3–5 replicates were analyzed by ANOVA and Duncan's multiple range test. When OCCs were cultured in glucose-free chemically defined maturation media, regardless of the presence of hormones and dibutyryl cAMP, germinal vesicle breakdown (GVBD) of oocytes was inhibited (10.0–21.3%), as compared with OCCs cultured in the presence of glucose and hormones (91.4–92.0%). In a majority of oocytes in which GVBD was inhibited, the arrest occurred at the GV-I stage. When OCCs were cultured in maturation media in which glucose was replaced with Na pyruvate, GVBD was not inhibited any more than in control samples that were cultured in the presence of glucose (97.4% vs. 97.1%). However, the incidence of oocytes developing to the metaphase II stage was significantly lower in this condition than in controls (4.8% vs. 49.9%, respectively). A majority of the oocytes were at the metaphase I stage (86.0% vs. 45.5% in controls). The presence of 6-AN in maturation media significantly inhibited GVBD of oocytes (77.3, 29.0, 7.4, and 8.4% at 0, 10, 50, and 100 µM, respectively) and arrested the oocytes at the GV-I stage. Immunocytochemistry with anti-G6PD demonstrated the activity of G6PD in cumulus cells of OCCs. In conclusion, these results demonstrate that glucose plays a critical role in the release of porcine oocytes arrested at the GV-I stage, probably through PPP of cumulus cells. The current results also suggest the possibility of gluconeogenesis in porcine OCCs when glucose in maturation media was replaced with Na pyruvate.
341 IN VITRO MATURATION OF GOAT FOLLICULAR OOCYTES SUBSEQUENT TO THEIR CRYOPRESERVATION BY VITRIFICATION
Authors: Garg N. , Purohit G. N.
Published: December 2006

The in vitro maturation, fertilization, and morphological changes of goat cumulus–oocyte complexes (COCs) cryopreserved by vitrification using DPBS + 0.5% sucrose + 0.4% BSA and 4 M, 6 M, 8 M, or 10 M concentrations of glycerol (G) or ethylene glycol (EG) were studied. Oocytes were cryopreserved by vitrification by holding them over LN vapor for 2-3 min and then plunging them into LN. After 7–10 days of storage, the COCs were rewarmed and used for in vitro maturation or fertilization in 2 experiments to record morphological damage due to vitrification, nuclear maturation 28 h after culture (9 replicates), and fertilization 24 h after insemination (10 replicates). Oocytes were matured in vitro in 50–100 �L microdrops of TCM-199 supplemented with 5 �g mL-1 FSH, 5 �g mL-1 LH, and 1 ng mL-1 estradiol, 25 mM HEPES, 0.25 mM pyruvate, and antibiotics for 28 h at 38 � 1�C and 5% CO2 in a CO2 incubator. Matured oocytes were fertilized using frozen–thawed buck semen prepared as per previous methods (Palomo et al. 1999 Theriogenology 51, 927). Freshly collected COCs were matured and fertilized simultaneously and kept as controls. The arcsin transformed data of the proportions of oocytes matured or fertilized was compared by Duncan's new multiple range test. The proportions of COCs that were recovered in morphologically normal form were highest for oocytes vitrified in 6 M concentrations of both G (90.8%) and EG (95.0%). The proportions of oocytes in morphologically normal form increased with increasing concentrations of both G and EG up to 8 M but at 10 M, the proportion of normal oocytes decreased significantly (70.7% for G and 72.0% for EG; P < 0.05). At the end of experiments 1 (n = 856) and 2 (n = 1204), nuclear maturation and fertilization of oocytes was significantly higher (P < 0.05) for fresh oocytes (M-II: 65.62%; fert: 40.8%) than for vitrified oocytes. Nuclear maturation and fertilization of vitrified oocytes increased with increasing concentration of both G [M-II: 4 M (22.7%), 6 M (34.2%), 8 M (41.9%); fert: 4 M (15.2%), 6 M (25.7%), 8 M (31.5%)] and EG [M-II: 4 M (8.0%), 6 M (21.7%), 8 M (25.0%); fert: 4 M (5.5%), 6 M (14.7%), 8 M (17.2%)] up to 8 M concentration, but at 10 M concentration, the proportion of oocytes matured (6.4% for G, 8.4% for EG) or fertilized (4.2% for G, 5.6% for EG) decreased significantly (P < 0.05). EG was found to be a better cryoprotectant for vitrification of goat oocytes as evident by significantly higher proportions (P < 0.05) of oocytes maturing and fertilizing in EG compared to G at all concentrations tested. It was concluded that vitrification brings about morphological damage to oocytes. The optimum vitrification cryoprotectant is up to 8 M concentration of G and EG, with EG being better.
343 DISTRIBUTION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND IN VITRO MATURATION OF PORCINE OOCYTE - CUMULUS COMPLEXES FROM SMALL AND MEDIUM FOLLICLES
Authors: Ishida Y. , Funahashi H.
Published: December 2006

Glucose metabolism through the pentose phosphate pathway (PPP) plays a critical role in meiotic maturation and fertilization. However, the relationship between the distribution of a PPP key enzyme, glucose-6-phosphate dehydrogenase (G6PD), in cumulus–oocyte complexes (COCs) and the in vitro maturation (IVM) of the oocytes is not clear. In the present study, we examined the distribution of G6PD, the morphological characteristics in OCCs derived from small (d2 mm in diameter) and medium (3 to 6 mm in diameter) follicles, and the rate of oocyte maturation. Porcine COCs were collected from small or medium follicles of slaughterhouse ovaries. The COCs were cultured in a maturation medium, BSA-free NCSU37 supplemented with 10% porcine follicular fluid, eCG, and hCG, for 20 h and then in the absence of hormones for 24 h. To determine the distribution of G6PD, the COCs were treated with 13 �M brilliant cresyl blue (BCB) in TL-HEPES-PVA for 90 min. Results from 3–6 replicates were analyzed by ANOVA and Duncan's multiple range test. The mean diameters for COCs collected from small follicles (136.7 µm for the outer zona and 103.1 µm for ooplasm) were significantly less than for those derived from medium follicles (164.1 µm and 122.0 µm, respectively). G6PD activity was detected in the cumulus cells for most of the COCs derived from medium follicles, but it was not significantly different from that of COCs derived from small follicles. In the second group of COCs, very little G6PD activity was found in both the cumulus cells and the oocytes (34.7 ± 11.5% and 18.0 ± 6.7% in COCS derived from small and medium follicles, respectively). After stimulation by eCG and hCG, the percentages of COCS in which G6PD activity was detected in the cumulus cells, but not in the oocytes, were 56.2 ± 23.8% and 72.9 ± 6.1% for small and medium follicles, respectively. The percentage of oocytes at the metaphase II stage (53% and 63.9% in oocytes from small and medium follicles, respectively) was higher for the COCs that showed higher G6PD activity in their cumulus cells. In conclusion, although no statistical differences were detected in the distribution of G6PD between COCs from small and medium follicles, due to a large variation, a higher percentage of mature oocytes seems to be the result of COCs where the G6PD activity is detected in the cumulus cells, but not in the oocyte, during IVM.
344 FOLLICULAR FLUIDS COLLECTED FROM LARGE FOLLICLES IMPROVED THE PROGRESSION OF IN VITRO NUCLEAR MATURATION OF PIG OOCYTES
Authors: Ito M. , Kitagawa M. , Iwata H. , Kuwayama T. , Monji Y.
Published: December 2006

The present study examined the effects of supplementation of in vitro maturation medium with porcine follicular fluids (pFFs) on the progression of nuclear maturation, subsequent fertilization, and development of pig oocytes. The pFFs were collected from follicles of different diameters: small follicles (SFs, 3–4 mm in diameter) and large follicles (LFs, 5-6 mm). In experiment 1, oocytes were collected from follicles 3–6 mm in diameter. The oocytes were cultured in maturation medium (NCSU-37) supplemented (10% v/v) with pFF from SFs (SpFF) or LFs (LpFF); the kinetics of nuclear maturation, fertilization rate, and developmental competence of oocytes were evaluated. In experiment 2, oocytes were collected from SFs or LFs, and the effects of supplementation of SpFF and LpFF on the nuclear maturation of these oocytes were examined. In experiment 3, each pFF was denaturated by heat treatment (60�C for 30 min) or trypsin treatment (200 �g mL-1), and the effects of these pFFs on the nuclear maturation of pig oocytes were examined. In experiment 4, pFFs were ultrafiltered, and separated into fractions greater and less than 30 kDa; the effects of the separated pFFs on nuclear maturation were examined. We used Student's t-test to examine the differences among the results; a P value less than 0.05 was considered to be significant. In experiment 1, the progression of nuclear maturation was significantly faster when the maturation medium was supplemented with LpFF than with SpFF (MI rates at 36 h after culture: LpFF, 20.0%, and SpFF, 35.0%; MII rates at 36 h after culture: LpFF, 33.3%, and SpFF, 20.0%). The rates of polyspermic fertilization were significantly lower in the maturation medium with LpFF than in that with SpFF (LpFF, 31.7%; SpFF, 66.7%). Moreover, the rates of monospermic fertilization (LpFF, 45.0%; SpFF, 21.7%) and blastocyst formation (LpFF, 15.2%; SpFF, 3.0%) were significantly greater in the medium supplemented with LpFF than with SpFF. In experiment 2, we observed that LpFF accelerated the progression of nuclear maturation of pig oocytes collected from LFs (MII rates at 36 h after culture: LpFF, 61.7%; SpFF, 35.5%), but not those collected from SFs (MII rates at 36 h after culture: LFs, 15.0%; SFs, 11.7%). In experiment 3, the accelerating effect of pFF on nuclear maturation was eliminated by heat (MII rates at 36 h after culture: LpFF, 30.0%; SpFF, 25.0%) or trypsin treatment (MII rates at 36 h after culture: LpFF, 20.0%; SpFF, 21.7%). In experiment 4, only LpFF with a molecular weight greater than 30 kDa had positive effects on the progression of nuclear maturation (MII rates at 36 h after culture with LpFF: >30 kDa, 30.0%; <30 kDa, 11.7%). The present study suggests that LpFF contains some proteins that improve the progression of nuclear maturation and the developmental competence of oocytes.
345 EFFECT OF FOLLICLE STIMULATING HORMONE AND HUMAN CHORIONIC GONADOTROPHIN ON THE IN VITRO MATURATION OF CANINE OOCYTES
Authors: Kim M.-K. , Oh H.-J. , Fibrianto Y. H. , Jang G. , Kim H.-J. , Hong S.-G. , Park J.-E. , Kang S.-K. , Lee B.-C. , Kim D.-Y.
Published: December 2006

Growth, maturation, and ovulation of the Graafian follicle depend on appropriate patterns of secretion, sufficient concentrations, and adequate ratios of various reproductive hormones, especially follicle stimulating hormone (FSH) and luteinizing hormone (LH) (Van Tol et al. 1996 Mol. Reprod. Dev. 45, 218–224). The present study investigated the effects of FSH and human chorionic gonadotrophin (hCG) on the nuclear maturation of canine oocytes. In addition, in order to investigate the effect of stage of the estrous cycle on the meiotic competence of canine oocytes matured in vitro, oocytes were collected from various reproductive states and matured in vitro in the presence of the gonadotrophins. Estrous cycle stage was evaluated for each bitch by ovarian morphology, and bitches were categorized according to the stage of the estrous cycle (anestrus, follicular, or diestrus) prior to oocyte collection. Recovered oocytes were cultured in serum-free tissue culture medium (TCM)-199 supplemented with various concentrations of FSH (Exp. 1: 0, 0.1, 1.0, or 10 IU) or hCG (Exp. 2: 0, 0.5, 1.0, or 10 IU) or both (Exp. 3: 1 IU FSH + 1 IU hCG) for 72 h to determine the effective concentration of these hormones, and to examine their combined effect. After maturation culture, oocytes were denuded in PBS containing 0.1% (w/v) hyaluronidase by gentle pipetting. The denuded oocytes were stained with Hoechst 33342 in glycerol and the nuclear state of oocytes was evaluated under UV light. The rates of maturation to the MII stage were significantly higher (P < 0.05) when follicular-stage oocytes were supplemented with 1 IU FSH (6.2%) compared with the other FSH-supplemented groups (0.0 to 3.3%) or to the control (1.8%), or 0.1 or 10.0 IU FSH (0 to 1.2%). Significantly higher (P < 0.05) maturation rate to MII stage was observed in follicular-stage oocytes supplemented with 1.0 IU hCG (7.2%) compared with the control or other hCG-supplemented groups (0 to 1.5%). However, FSH and hCG together did not improve the nuclear maturation rate of canine oocytes (2.4%) compared with FSH (6.2%) and hCG alone (7.2%). In conclusion, FSH or hCG alone significantly increased the maturation of canine oocytes to the MII stage.
This work was supported by grant No. M1062503005-06N250300510 from KOSEF, Republic of Korea.
35 RELATIONSHIP BETWEEN THE ONSET OF ENUCLEATION DURING MEIOTIC MATURATION OF RECIPIENT OOCYTES AND DEVELOPMENTAL ABILITY OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PIGS
Authors: Fahrudin M. , Kikuchi K. , Karja N. W. K. , Ozawa M. , Somfai T. , Nakai M. , Ohnuma K. , Noguchi J. , Kaneko H. , Nagai T.
Published: December 2006

In somatic cell nuclear transfer (SCNT), maturation promoting factor (MPF) is believed to be one of the factors involved with nuclear envelope breakdown and chromatin condensation of the transferred nucleus. Although MPF activity is high both in metaphase-I or -II oocytes (M-I and M-II, respectively), only M-II oocytes have been used exclusively as recipient cytoplasts in SCNT. In this study, we examined the effect of different onset of (1) enucleation of recipient oocytes at the M-I and M-II stages, and (2) fusion and activation of the couplets on their developmental ability to the blastocyst stage in pigs. The primary cultured cumulus cells were used as donor karyoplasts, and recipient cytoplasts were prepared by enucleation of in vitro-matured oocytes using gradient centrifugation in percoll solution. A karyoplast and a cytoplast were fused by 2 DC pulses of 1.5 kV cm-1 for 20 �s, and then the couplets were activated by 2 DC pulses of 0.8 kV cm-1 for 30 �s. The reconstructed embryos were cultured according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) except for the addition of 5% FCS to NCSU-37 during Days 2–7 (Day 0 is the day of SCNT) of embryo culture using the WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 258–264). Some of the embryos were fixed at 1, 10, and 24 h after activation and examined for morphology of nuclei. After 30 h of IVM, oocytes (mainly at the M-I stage) were enucleated. Then the couplets were fused immediately (Group A) or at 48 h after the onset of IVM (Group B); activation was conducted at 48 h of IVM (Group A) or at 1 h after fusion (Group B). As a control group, oocytes were enucleated after 48 h of IVM and then the couplets were fused and activated. None of the embryos in Group B developed to the blastocyst stage. However, a few of the embryos [2/117 (1.7%)] in Group A developed to the blastocyst stage; however, the rate was significantly lower than that of the control group [10/112 (8.9%); chi-square; P = 0.03]. The rates of embryos undergoing premature chromosome condensation (PCC) in Group B at 1 h and 10 h after activation were significantly lower than those in Group A [1 h: 51/69 (73.9%) vs. 76/76 (100%); 10 h: 24/76 (31.6%) vs. 45/91 (49.5%), respectively); some of them had pseudo-pronuclei. By 24 h after activation there were no detectable differences in the rates of cleavage [2/70 (2.9%) vs. 2/61 (3.3%)]; however, the rates were significantly lower than that of the control group [23/90 (25.6%); chi-square; P < 0.05]. These results suggest that MPF activity might be changed in oocytes without nucleus during the maturation culture. Thus, a specific nucleus-associated factor(s) that may present in the cytoplasm seems to be essential for the successful remodeling of the transferred nucleus and the development of SCNT embryos to the blastocyst stage.
357 CALCIUM RELEASE INDUCED BY THIMEROSAL AND INOSITOL 1,4,5-TRIPHOSPHATE IN HEAT-SHOCKED PORCINE OOCYTES
Authors: Tseng J.-K. , Tang P.-C. , Ju J.-C.
Published: December 2006

Elevated ambient temperature has been known to be deleterious to the developmental competence of mammalian oocytes and embryos, although the mechanism is still unclear. The objective of this study was to determine the effect of heat shock (HS) on the alteration of intracellular calcium concentrations ([Ca2+]i) of matured pig oocytes by two different calcium releasing agents. Porcine cumulus–oocyte complexes were aspirated from the follicles (3–6 mm) and subjected to standard in vitro maturation procedure for 42 h. Matured oocytes were then randomly allocated to different heat treatments at 41.5°C for 0 (Control, C0h), 1 (HS1h), 2 (HS2h), or 4 h (HS4h). An additional control group was cultured for 4 h without heat shock (C4h). Oocytes were incubated with 2 µM fura-2 acetoxymethyl ester (AM) and 0.02% pluronic F-127 in Ca2+-free PBS (40 min) following heat shock, and then washed with Ca2+-free PBS (30 min) for detection of [Ca2+]i. Fluorescent images were captured with alternative excitation wavelengths at 340/380 nm by a rotating chopper disk equipped with an Axon imaging system. Data from both experiments were analyzed by ANOVA using the General Linear Model (GLM) of the SAS (SAS Institute, Inc., Cary, NC, USA). In Experiment 1, matured oocytes were activated by 200 mM thimerosal (10 min) following heat treatment. The maximal [Ca2+]i in the HS2h group was the highest among all treatment groups. The lowest maximal peak of [Ca2+]i was observed in the HS4h group, but it was still higher than that in the C4h group (P < 0.05). The total amount of Ca2+ release represented by the total area of the peaks in C4h was lower than in any other groups except HS4h (P < 0.05). In Experiment 2, each matured oocyte was injected with approximately 10 pL of inositol 1,4,5-triphosphate (IP3, 0.5 mM); the Ca2+ transient was recorded as described in the previous experiment. The maximal value of [Ca2+]i in the C4h group was still the lowest among the heat-shocked and C0h groups (P < 0.05). The total Ca2+ release in the HS2h group was the highest among all treatment groups, but only significantly higher than the HS1h and C4h groups (P < 0.05). A similar pattern of Ca2+ release in HS-oocytes was induced by thimerosal and IP3 stimulations. These results indicate that Ca2+ releasing capacity of matured pig oocytes is enhanced by a shorter duration of heat shock, but declines after prolonged exposure of heat shock and/or in vitro culture. The differential Ca2+ releasing capacity of heat-shocked oocytes prior to fertilization revealed physiological changes of pig oocytes after heat shock. This finding provides further insight for the low fertilization and developmental competence that occurs in farm species during hot seasons.
358 MORPHOLOGICAL CHANGES IN VITRIFIED WATER BUFFALO CUMULUS - OOCYTE COMPLEXES AND THEIR IN VITRO NUCLEAR MATURATION
Authors: Yadav R. C. S. , Sharma A. , Purohit G. N.
Published: December 2006

Morphological changes and in vitro nuclear maturation of buffalo cumulus–oocyte complexes (COCs) was evaluated subsequent to their cryopreservation by vitrification in solutions containing 4 M, 6 M, 8 M, and 10 M concentrations of glycerol (G) or ethylene glycol (EG) or their combination. COCs collected from buffalo ovaries by aspiration (n = 1342) were equilibrated in 50% of the vitrification solution and then placed in the vitrification solution (Dulbecco's phosphate-buffered saline+0.5M sucrose + 0.5% BSA + cryoprotectant). COCs were transferred to empty semen straws, kept
over LN vapor for 2–3 min, and then plunged into LN. After 7–10 days of storage, COCs were warmed and evaluated for morphological damage. Morphologically normal COCs were cultured in vitro (9 replicates each with 5–10 oocytes in 50–100-µL culture drops) in TCM-199 medium supplemented with 5µgmL-1 FSH, 5µgmL-1 LH, and 1 ngmL-1 estradiol with 25mM HEPES, 0.25mM pyruvate, and antibiotics. The COCs were incubated for 24 h at 38±1°C and 5% CO2 in humidified air in a CO2 incubator and evaluated for nuclear maturation at the end of 24 h of culture. Freshly collected COCs were also matured in vitro and kept as controls (n=142). The proportions of COCs retrieved in morphologically normal form were compared by chi-square test; the arcsin transformed data of the proportions of oocytes matured was compared by Duncan's new multiple range test. The proportions of oocytes recovered in a morphologically normal form were highest in the 6M EG group (95.23%), followed by 8M EG (94.0%) and 6M G (90.6%) groups. At 10M concentration, a significantly (P <0.05) lower percentage of oocytes was morphologically normal. The morphological abnormalities recorded were change in shape, rupture of zona pellucida, and leakage of oocyte contents. A significantly higher (65.62%; P <0.05) proportion of fresh oocytes reached metaphase-II compared to oocytes vitrified in all concentrations of G and EG. The proportion of oocytes reaching metaphase-II increased with increasing concentrations of both G and EG, but at 10M concentration the proportion of oocytes reaching metaphase-II decreased. The proportions of COCs reaching metaphase-II in 4M, 6 M, 8M, and 10M glycerol were 6.9%, 21.2%, 25.7%, and 5.5%, respectively. The respective proportions of COCs reaching metaphase-II in 4M, 6 M, 8M, and 10M ethylene glycol were 21.9%, 34.3%, 40.8%, and 7.5%. No significant benefit of in vitro maturation of oocytes was seen for oocytes vitrified in a combination of both G and EG. It was concluded that although vitrification brings about some damage to the oocytes, yet it appears to be a good tool for oocyte cryopreservation, and 8M concentration of either G or EG appears to be optimum for vitrification of buffalo oocytes.
362 SEX RATIO OF IN VITRO-PRODUCED BLASTOCYSTS OBTAINED USING FROZEN SEMEN FROM A CLONED BULL OR ITS ORIGINAL CELL DONOR
Authors: Heyman Y. , LeGuienne B. , Audouard C. , Charreaux F. , Humblot P. , Vignon X.
Published: December 2006

Bulls obtained by somatic cell nuclear transfer (SCNT) have proved to develop normally, produce sperm, and be fertile (Shiga et al. 2005 Theriogenology 64, 334–343; Tecirlioglu et al. 2006 Theriogenology 65, 1783–1799). However, due to epigenetic variability encountered with clones, it cannot be excluded that major alterations may affect the X chromosome and especially the dosage of X inactivation in female embryos. This could result in a deviation of the sex ratio in offspring. To test this hypothesis, the sperm from a cloned bull was used in IVF to assess its ability to induce a different proportion of male and female embryos when compared to results obtained with the sperm of the original donor before cloning. In the present experiment, semen was collected twice weekly during 3 months from a 3-year-old cloned bull of the Charolais breed (H-2) and frozen-stored. Samples from 3 different ejaculates were used in comparison with control frozen straws from the original bull (H-0), prepared at the same age, to produce in vitro embryos. A total of 6 replicate IVF experiments were performed on the same batches of in vitro-matured oocytes (n = 654) using the standard swim-up and IVF technique in the laboratory. Twenty hours after insemination, presumptive zygotes were vortexed and cultured for 7 days in microdrops of B2 medium with Vero cells. Fertilization, cleavage, and blastocyst formation was assessed, and by Day 7, all of the blastocysts were individually frozen after grading. Two groups of 40 representative blastocysts derived from each bull (clone or donor) were used to determine the sex ratio using the sexing kit developed by UNCEIA R&D (Maisons-Alfort, France). Percentages between groups were compared by chi-square test. Semen from the cloned bull resulted in significantly lower fertilization and cleavage rates than that from the original donor (82.7% and 70% vs. 94.6% and 91%, respectively; P < 0.01), but further in vitro development was not different, as the proportions of blastocysts/cleaved were, respectively, 31.5% (74/235) vs. 38.7% (112/290). Sex determination was achieved in 79/80 of the in vitro-produced embryos and indicated that 55% of the blastocysts derived from the clone were male (22/40) and 45% female (18/40). This was not different from the proportion of male (61.5%, 24/39) and female (38.5%, 15/39) embryos in the group derived from semen of the original donor bull. In conclusion, these preliminary results indicate that the semen from this cloned Charolais bull has the same potential for in vitro embryo production as its original cell donor, and there is no evidence that SCNT could induce any deviation of the sex ratio. This observation needs to be confirmed with other sets of cloned bulls. (Shiga et al. 2005 Theriogenology 64, 334–343. Tecirlioglu et al. 2006 Theriogenology 65, 1783–1799.)
363 FACTORS INFLUENCING PREGNANCY RATES FOLLOWING TRANSFER OF BOVINE IN VIVO EMBRYOS BIOPSIED FOR SEX DETERMINATION
Authors: Li S. , Yu W. , Fu J. , Bai Y. , Jin F. , Shangguan B.
Published: December 2006

Data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 was analyzed to examine the effects of various factors (biopsy, freezing, sample size, embryo development and quality, in vitro culture, and recipient quality) on pregnancy rates of in vivo-biopsied embryos. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sexing determination using protocols and sexing kits supplied by AB Technology Ltd. Fresh embryos were implanted on the same day or frozen with AG freeze medium (AB Technology Ltd., Pullman, WA, USA) for later transfer. Recipients were synchronized with CIDA + PG protocols. Embryos were cultured in 6-well dishes containing 1.3 mL of holding medium (AB Technology Ltd.) in each well at room temperature (20–25�C) for examination of embryo survival in vitro. The chi-square test was used in statistic analysis. The implantation of fresh embryos after biopsy did not affect pregnancy rates (49.6%, 257/518) compared to that of non-biopsied fresh and frozen–thawed embryo groups (52.9%, 47/140 and 46.6%, 177/380, respectively). However, for biopsied embryos subjected to frozen and thawed procedures before implantation, particularly for those subjected to the removal of a larger biopsy, a reduced pregnancy rate was observed (41.8%, 297/710; P < 0.01). Pregnancy rates among biopsied embryos at 3 different development stages (morula-early blastocyst, blastocyst, and expanded blastocyst) were not different. Similar results were found between embryo groups of grade 1 and 2. A significant decrease in pregnancy rate (0/10) was observed with embryos held in vitro for a longer period of time (>5 h), suggesting detrimental effects of in vitro conditions on embryo survival. The highest pregnancy rate (68.0%) was observed in recipients synchronized for the first time before being implanted with biopsied embryos. Significant decreases in such rates were found in recipients synchronized for the second or third times or those with an abortion history at the first or second synchronization-implantation treatment (P < 0.01). Better pregnancy rates (45.6%, 41/90; 46.1%, 76/165; and 45.5%, 5/11) were obtained for recipients implanted with biopsied embryos at Days 7.5, 8.0, and 8.5 post-heat detection, respectively, compared to 16% at Day 7 (3/18, P < 0.05). It is concluded that mechanical treatment (cutting) does not reduce the survival of biopsied embryos; however, cryopreservation reduces their ability to survive in vivo. The analyses also suggest that holding embryos in vitro should not be longer than 5 h unless more favorable in vitro conditions can be provided. To achieve better results of implantation of biopsied embryos, embryo transfer should be performed during 7.5–8.5 days post-estrus, and the healthy recipients synchronized for the first time should be used.
369 AMPLIFICATION OF THE SRY GENE FOR SEXING OF BUFFALO (BUBALUS BUBALIS) EMBRYOS USING NESTED MULTIPLEX PCR
Authors: Zhang M. , Fu Q. , Qin W. S. , Zheng H. Y. , Lu Y. Q. , Lu S. S. , Lu K. H.
Published: December 2006

In mammals, the Y chromosome-linked SRY gene is responsible for male sex determination. Therefore, a logical approach for embryo sex identification is to amplify the male-specific single-copy SRY gene. The objective of this study was to design specific primers for amplification of buffalo SRY gene and develop a reliable PCR method for sex identification of buffalo embryos. Genomic DNA was extracted from swamp buffalo (Bubalus bubalis) peripheral blood. A pair of primers based on the sequence of Holstein bovine SRY gene (forward, 52-GTTTGCCTTATGGATTTATT-32; reverse, 52-TCTACTTTAGCCTATTTG-32) was used to amplify whole buffalo SRY gene. This amplified fragment was isolated and constructed into plasmids for sequencing. Two pairs of primers, S1/S2 (forward, 52-CCATGAACGCCTTCATTTTGTG-32; reverse, 52-ACGAGGTCGATATTTATAGC CC-32) and S3/S4 (forward, 52-AAGCAGCTGGGCTATGAGTGGAA-32; reverse, 52-ACGAGGTCGATATTTATAGCCC-32), were designed based on the SRY sequence above. Simultaneously, the G3PDH gene was amplified to serve as an internal control for both male and female embryos. A multiplex-nested-PCR system was optimized by varying the following parameters individually: concentration of Mg2+, dNTPs, primers, and different cycles number. Twenty-seven IVF morulae were identified with the optimal PCR procedure after biopsy. Accuracy of PCR amplification was verified by dot blotting. The sex of 24 embryos fertilized with Y-sperm separated by flow cytometry was also examined. Results indicated that the optimal procedure of Nested-Multiplex-PCR consisted of 1.5 mM Mg2+, 100 �M dNTPs, 0.5 �M SA3/SA4 primers, and 0.25 �M GA3/GA4 primers, and 35 cycles. Accuracy of identification was 100% for 27 IVF morulae; 14 were judged as males and 13 were females. The result of blotting confirmed that the accuracy of amplification was 100%. The proportion of males was 83.3% (20/24) in embryos fertilized with Y-sperm. This confirms that the PCR system targeting the SRY gene can be used for accurate sex identification of buffalo embryos.
This study was supported by grants from the Foundation of Guangxi Key Laboratory for Subtropical Bio-Resource Conservation and Utilization (SB0403) and the Guangxi Department of Science and Technology (0626001-3-1).
370 BILATERAL DIFFERENTIAL TESTICULAR BIOPSIES IMPROVE TESE AND ICSI OUTCOMES IN NON-OBSTRUCTIVE AND OBSTRUCTIVE AZOOSPERMIC INFERTILE PATIENTS
Authors: Al-Ansari S. M. , Ridha-Albarzanchi M. T. , Kanan Z. A. , Al-Badri A. A. , Al-Atraqchi S. K.
Published: December 2006

The goal of the present study was to evaluate the clinical significance of bilateral differential testicular biopsies (BDTB) to improve testicular sperm extraction (TESE) and ICSI outcomes in non-obstructive azoospermic (NOA) men. The male patients were divided into an obstructive azoospermic group (OAG, n = 40) and an NOA group (n = 50). The women in both groups had normal ovulatory cycles and reproductive hormone concentrations. BDTB were taken from upper, middle, and lower zones of the testes. At least 50 transverse histologic sections of seminiferous tubules were examined in each biopsy. The FSH, LH, and prolactin concentrations were significantly higher in the NOA group compared to the OAG (18.3 vs. 6.2 mIU mL-1, 8.5 vs. 4.1 mIU mL-1, and 9.6 vs. 6.4 ng mL-1, respectively; P < 0.01), whereas the testosterone level and testicular size were significantly lower in the NOA group vs. the OAG (3.7 vs. 5.3 ng mL-1 and 3.3 vs. 4.4 cm, respectively; P < 0.01). A new method (BDTB) for scoring testicular biopsies was used. This method is different from Johnson's method as SCO or TF or complete MA. Following positive BDTB results, TESE and ICSI were performed. The oocyte recovery rate/patient was 3.8% (154/40) and 3.6% (178/50) in the OAG and the NOA group, respectively (P > 0.05). The TESE-ICSI rates were 75.9% (117/154) in the OAG and 59.5% (106/178) in the NOA group (P < 0.005). The embryo cleavage rate was significantly higher in the OAG compared to the NOA group (98/117 = 83.8% vs. 66/106 = 62.3%, respectively; P < 0.005). The number of embryos transferred/patient in the OAG was significantly higher compared to that in the NOA group (2.5 vs. 1.3, respectively; P < 0.01). The embryo implantation rate per embryo transfer in the OAG was 52% (51/98) vs. 45.5% (30/66); P > 0.05. The clinical pregnancy rate per embryo transfer was 52.5% in the OAG and 40% in the NOA group (P > 0.05). It was concluded from the results of this study that both the BDTB and TESE were useful diagnostic and prognostic predictors of successful sperm retrieval for ICSI treatment in the non-obstructive and obstructive azoospermic male patients. The clinical embryo implantation rates were not significantly different between both groups (NOA group and OAG) which may indicate that the embryos of both groups have similar implantation potentials. Bilateral differential testicular biopsies were found to be positively correlated with the TESE and ICSI outcomes.
371 INTRACYTOPLASMIC SPERM INJECTION OF EQUINE OOCYTES USING AIR-DRIED SPERM OR SPERM STORED IN A HIGH OSMOLARITY MEDIUM
Authors: Alonso A. , Miragaya M. , Losinno L. , Herrera C.
Published: December 2006

The generally accepted method of long-term sperm preservation is freezing in liquid nitrogen. However, it is not always available. Other techniques have shown to preserve sperm for a short period of time that can be used for intracytoplasmic sperm injection (ICSI). Mouse offspring have been produced after ICSI with sperm stored in a high osmolarity medium. Also, human embryos were obtained by ICSI with air-dried sperm. Recently, equine blastocysts have been obtained by ICSI with lyophilized sperm. In our study, cleavage rate was evaluated after ICSI of equine oocytes using air-dried sperm or sperm stored in a high osmolarity medium. Oocytes were obtained from slaughterhouse ovaries by scraping individual follicles and transported in a portable incubator at 38�C for 15 h in TCM-199 buffered with HEPES and supplemented with glutamine, sodium pyruvate, LH (Bioniche Animal Health, Inc., Beltville, Ontario, Canada), FSH (Bioniche Animal Health, Inc.), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and 10% FBS (GIBCO, Grand Island, NY, USA). On arrival, they were cultured for 6 more hours in 5% CO2 in air at 38�C in microdroplets of the same medium, without HEPES. Sperm was collected from 2 stallions of proven fertility. The ejaculate was diluted in Kenney extender, centrifuged, and the pellet was resuspended in HEPES-TALP (H-TALP). Three sperm treatments were used: (C) Control: ejaculated motile sperm processed as described above; (1) Air-dried sperm: obtained from spreading sperm on a sterile slide and drying it for 10 min in a laminar flow chamber; (2) Sperm in a high osmolarity medium: ejaculated motile sperm resuspended in H-TALP with high osmolarity (800 mOsmol). Samples from groups 1 and 2 were stored at 5�C for 2 to 3 days before being used for sperm injection. Only oocytes with an intact cytoplasm and a visible polar body were selected for injection and randomly assigned to each experimental group. Each MII oocyte was injected with 1 sperm cell and activated in Ionomicin for 10 min and DMAP for 3 h. Injected oocytes were cultured in DMEM : F10 1 : 1 with 10% FBS at 38�C in 7% O2 and 5% CO2 for 48 h. The number of cleaved embryos was recorded. Data was analyzed by chi-square test. A total of 135 MII oocytes were injected. The cleavage rate in group 1 was significantly lower than in the control group (31/71, 43.66% vs. 28/38, 73.68%) (P < 0.05). No differences were observed between group (2) and control (19/29, 73.07% vs. 28/38, 73.68%) (P > 0.05). This is the first report of equine oocytes fertilized by ICSI with air-dried sperm or with sperm kept in high osmolarity medium. These simple sperm preservation techniques might be an alternative option when liquid nitrogen is not available. Further studies will determine if it is possible to obtain pregnancies or even healthy offspring.
372 SPERM ASTER FORMATION AND BLASTOCYST DEVELOPMENT OF IN VIVO-MATURED BOVINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION
Authors: Horiuchi T. , Takenaka M. , Kani C. , Emuta C. , Ogata Y. , Matushige T.
Published: December 2006

In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and blastocyst rates (Horiuchi et al. 2002 Theriogenology 57, 1013–1024). The reason why the exogenous activation treatment in bovine ICSI is needed to promote cleavage and blastocyst development is not clear. The objective of this study was to examine the effect of activation treatment on sperm aster formation, cleavage, and blastocyst development of in vivo- and in vitro-matured bovine oocytes following ICSI. In vivo-matured oocytes were collected using transvaginal devices under ultrasound guide at about 29 h after GnRH injection from Japanese Black cows superstimulated with a total 19 mg FSH (Antrin�; Denka Pharmaceutical Co., Kanagawa, Japan) divided into twice daily over 3 days, and treated with 750 �g cloprostenol (Estramate�; Sumitomo Chemical Co., Tokyo, Japan). In a total of 8 aspiration sessions, 131 oocytes were collected; of 116 oocytes with expanded cumulus cells, 84 (72%) had a first polar body and were used for ICSI. On the other hand, in vitro-matured bovine oocytes were prepared by culturing immature follicular oocytes derived from abattoir ovaries. Bull spermatozoa, immobilized by scoring their tails, were injected into in vivo- or in vitro-matured oocytes. At 4 h after ICSI, the oocytes were treated with or without 7% ethanol for 5 min for activation. The injected oocytes were fixed at 8 h after ICSI, and sperm aster formation was examined by using specific antibodies and immunofluorescence microscopy. Data were analyzed by the chi-square test in all experiments. The rate of sperm aster formation in in vivo-matured oocytes was similar regardless of activation treatment (71% vs. 65%), but the rate in in vitro-matured oocytes was significantly (P < 0.05) higher in the group receiving activation treatment than in the non-activation group (57% vs. 19%). Cleavage (88% vs. 88%) and blastocyst rates (59% vs. 47%) of in vivo-matured oocytes after ICSI were also similar, regardless of activation treatment, but cleavage (72% and 20%) and blastocyst rates (19% and 7%) of in vitro-matured oocytes were significantly (P < 0.05) higher in the group receiving activation treatment than in the non-activation group. Moreover, the blastocyst rate of in vivo-matured oocytes was significantly (P < 0.05) higher than the rate in in vitro-matured oocytes. These results show that activation treatment after ICSI of in vivo-matured bovine oocytes is not necessary for cleavage and blastocyst development, and suggest that the necessity of activation treatment in bovine ICSI has relevance to in vitro maturation of bovine oocytes.