bluuebunny/crossref_metadata_2025 huggingface dataset:
304 PRODUCTION OF PORCINE EMBRYOS OF A PREDETERMINED SEX AFTER IN VITRO FERTILIZATION OF IN VITRO-MATURED OOCYTES	WITH SEX-SORTED FROZEN-THAWED BOAR SPERM
Authors: Bathgate R. , Morton K.M. , Eriksson B.M. , Rath D. , Seig B. , Chami O. , Stojanov T. , Maxwell W.M.C. , Evans G.
Published: December 2004

Pre-sexed embryos and offspring have been produced after IVF and embryo transfer (ET) with sex-sorted frozen-thawed sperm in cattle and sheep (Maxwell et al. 2004 Anim. Reprod. Sci. 82–83, 79–95). The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a predetermined sex and that these embryos could be successfully nonsurgically transferred. Ovaries were collected from abattoir slaughtered gilts (n = 138) and selected COCs were matured in vitro (Long et al. 1999 Theriogenology 51, 1375–1390). Sperm were collected from a mature boar and diluted with Androhep (1:3, semen:Androhep; Minitube, Verona, WI, USA), stained with H33342, and separated into X and Y sperm using a SX MoFlo (Cytomation, Inc., Fort Collins, CO, USA). Sex-sorted sperm were cryopreserved in 0.5 mL straws using the Westendorf protocol modified for sorted sperm (Bathgate, unpublished). Thawed sperm (Y sperm only) were prepared for IVF by centrifugation (300g, 10 min) through a Porcipure gradient (Nidacon Int. AB, Gothenburg, Sweden), and washed (centrifugation 300g, 10 min) in mTALP-PVA. For IVF, COCs were denuded and groups of 100 oocytes were transferred to 200-μL drops of mTALP-PVA (Long et al. 1999) and incubated with 5,000 motile sperm for 4–6 (Short) or 18–20 h (Long) . Presumptive zygotes were washed and transferred to 100-μL drops of mNCSU-23 (Long et al. 1999) and cultured until Day 4 (Day 0 = IVF) in humidified 5% CO2, 6% O2, 89% N2. Oocyte cleavage was assessed 48 h post-insemination, and on Day 4 selected morulae were transferred to recipient sows (n = 7 Large White × Landrace; 65 morulae per sow) nonsurgically using a Firflex catheter (Magapor, Zaragoza, Spain). Sex of remaining embryos was confirmed by PCR and restriction analysis (Cong et al. 1993 Hum. Mol. Genet. 2 1187–1191). Data from three replicates were arc sin transformed and analyzed by ANOVA. Oocyte cleavage was similar after Short (724/1547; 46.8%) or Long (598/1528; 39.1%) co-incubation. Resort analysis showed sperm to be >91% purity, and all sexed morulae were of the predicted sex (16/16). Delayed return to estrus (>23 days) was observed in 5 recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine IVP and ET protocols are required to enable full in vivo development.
This work was supported by XY, Inc., Fort Collins, CO, USA.
307 IN VITRO PRODUCTION OF BOVINE EMBRYOS USING FLOW-CYTOMETRICALLY SORTED SPERMATOZOA
Authors: Katska-Ksiazkiewicz L. , Bochenek M. , Rynska B. , Opiela J.
Published: December 2004

There are two practical ways to predetermine the sex of mammalian offspring: sexing pre-implantation embryos and sexing spermatozoa. The only successful and non-invasive method of sexing spermatozoa is quantifying sperm DNA with fluorescing DNA-binding dye, followed by flow cytometry and cell sorting. Our investigations aimed to develop a technology for in vitro embryo production in cattle using fresh and/or frozen-thawed spermatozoa sexed by flow cytometry. Sperm was sorted in a MoFloSX® cytometer using the method of XY, Inc. (Fort Collins, CO, USA; Research Collaboration Agreement). After sorting, the sperm was either used for IVF or frozen and stored in liquid nitrogen. Immature oocytes, recovered from slaughterhouse ovaries, after 22 to 23 h of IVM in TCM-199 containing 20% estrous cow serum and additional granulosa cells (Katska et al. 1998, J. Anim. Feed Sci. 7, 353–362), were fertilized in vitro with fresh or frozen-thawed X and Y fractions of spermatozoa. Simultaneously control, unsorted, fresh and frozen-thawed sperm was used for IVF. The standard protocol of sperm capacitation (Katska and Rynska 1998 Theriogenology 50, 213–222) was applied for both control sperm and fresh fractions of sexed sperm. Briefly, sperm was separated by Percoll gradient centrifugation, washed, and introduced into drops of Tyrode's albumin-lactate-pyruvate (TALP)-IVF (containing 10 μg heparin mL−1 and mixture of penicillamine, hypotaurine, and epinephrine) at a concentration 1 to 2 × 106 spermatozoa mL−1 of medium. Frozen fractions of sorted spermatozoa were centrifuged after thawing (500 g for 10 min) and immediately introduced into the IVF drops at 2 to 3 × 106 spermatozoa mL−1 of medium. Embryos resulting after IVF were co-cultured with Vero cells in B2 medium supplemented with 2.5% fetal calf serum for 8 to 10 days, (i.e., to the hatched blastocyst stage). A total of 2074 IVM oocytes were fertilized with both fresh and frozen-thawed sexed and control sperm of 5 bulls. There were significant differences (P < 0.01) in cleavage rates among fresh control sperm (120/256; 46.9%), the X fraction (66/254; 26.0%), and the Y fraction (58/230; 25.2%). Similar differences in cleavage rates (P < 0.01) were shown for frozen-thawed control sperm (156/335; 46.6%), the X fraction (137/498; 27.5%), and the Y fraction (118/501; 23.6%). No differences were observed in efficiency of embryo development to the blastocyst stage between the fresh control (25.8%) and the Y fraction (25.9%), or among the frozen control (16.7%) and the X fraction (13.1%) or the Y fraction (16.9%). However, significant differences (P < 0.05) were shown between blastocyst rates with the fresh X fraction (10.6%) and the control. Our results suggest that there were differences due to sperm sorting but no differences in efficiency of both fresh and frozen-thawed X and Y fractions of spermatozoa.
Research was supported by the State Committee for Scientific Research as a project 3PO6D 044 23.
312 BLASTOCYST DEVELOPMENT OF MALE AND FEMALE BOVINE EMBRYOS PRODUCED BY IVF WITH FLOW CYTOMETRICALLY-SORTED SPERM
Authors: Zhang M. , Lu K.H. , Seidel Jr G.E.
Published: December 2004

Several studies have demonstrated that male bovine embryos produced in vitro develop faster than female embryos produced in vitro, which results in more male than female blastocysts. The objective of this study was to compare the rate of blastocyst development of male and female bovine embryos derived from sexed sperm and cultured in a chemically defined medium + fatty acid-free (FAF)-BSA. Bovine oocytes (n = 1364) were fertilized with two types of frozen-thawed sperm (X- or Y-chromosome-bearing sperm sorted at 90% accuracy). Oocytes, aspirated from slaughterhouse ovaries, were matured in TCM199 supplemented with 10% fetal calf serum plus hormone additives (15 ng FSH, 1 mg LH, 1 mg 17 β-estradiol mL−1) for 22–24 h at 39°C, 5% CO2 in air with maximum humidity. Semen from one bull was sorted by flow cytometry into X- and Y-chromosome bearing sperm and frozen for later use with IVF. The procedures for IVF and IVC have been previously described (Lu KH, et al. 1999 Theriogenology 52, 1393–1405). Presumptive zygotes were removed from culture and placed in chemically defined medium (CDM-1 [Zhang M et al. 2003 Theriogenology 60, 1657–1663]) 6–7 h after insemination and cultured for 65–66 h. Embryos which had cleaved by 72 h post insemination were further cultured 96 h in CDM-2 (Zhang M et al. 2003 Theriogenology 60, 1657–1663) containing 0.12 IU insulin mL−1. Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7–8 after insemination, respectively. Data were analyzed by ANOVA procedures with replicates and treatments in the model. There was no significant difference in cleavage rate or blastocyst rate between X and Y sperm treatments. These results indicate that embryos produced with Y sperm do not reach the blastocyst stage in significantly higher proportions than embryos produced with X sperm in this chemically defined medium + FAF-BSA. Apparently, this IVC system leads to a more synchronous development of male and female embryos than other methods of producing bovine embryos in vitro.

Table 1.
Cleavage and blastocyst rates with X and Y sperm

This research was supported by XY, Inc., Fort Collins, CO, USA.
319 USE OF PENTOXIFYLLINE AND HYALURONIC ACID FOR STALLION SPERM SEPARATION
Authors: Kutvolgyi G. , Suh T. , Carnevale E. , Seidel Jr G.
Published: December 2004

In this study, we reduced the volume of separating media and the time of centrifugation to increase the yield of viable sperm for ICSI when few sperm are available. Hyaluronic acid (HA) was used successfully in combination with swim-up for separating bull spermatozoa (Shamsuddin and Rodriguez-Martinez 1994 Anim. Reprod. Sci. 36, 61–75). The purpose of this study was to compare the effectiveness of mini-Percoll and swim-up for low numbers of sperm treated or non-treated with HA or pentoxifylline (PX), a molecule that stimulates sperm motility (Gradil and Ball 2000 Theriogenology 54, 1041–1047). Poor-to-medium quality frozen semen (0.5-mL straws, 200 million cells/mL) from 3 stallions was used 3 times each. Two straws were thawed and mixed. Aliquots of 100 μL semen were allocated to 7 treatments. Three aliquots were incubated at 38°C for 20 min before Percoll-centrifugation (P-NT: non-treated; P-PX: 3.5 mM PX; or P-HA: 1 mg/mL HA, each in 0.25 mL chemically defined handling medium (HCDM)); one aliquot was centrifuged through Percoll without incubation (P-CON: control). Our mini-Percoll was a two-layer gradient (0.4 mL 90% and 0.5 mL 45% Percoll) in a 1.5-mL microcentrifuge tube that was centrifuged at 600g for 5 min. Then a 30-μL pellet was washed in HCDM at 300g for 5 min. At the same time, 3 aliquots of 100 μL sperm were placed in 1 mL HCDM for swim-up (S-NT: non-treated; S-PX: 3.5 mM PX; or S-HA: 1 mg/mL HA) for 30 min. A 0.65-mL aliquot of supernatant was centrifuged in HCDM at 300g for 5 min. In every treatment, the final 30-μL pellet aspirated from the bottom was evaluated. Recovery rate was determined using a hemacytometer. Sperm head, tail and acrosome membrane integrity were evaluated with Kovacs-Foote staining (Kovacs and Foote 1992 Biotech. Histochem. 67, 119–124) counting 300 sperm/sample. Cells were classified into 5 categories: intact head, tail and acrosome membrane (Intact); intact head, tail, damaged acrosome (IHITDA); intact head, damaged tail; damaged head, intact tail; damaged head, tail, acrosome. P-CON and P-PX resulted in more intact sperm compared to all swim-ups. IHITDA was nonsignificantly (P > 0.05) higher in P-PX than in P-CON (17 vs. 11%). Better recovery rates were found in P-CON, P-PX, and S-HA than in P-NT, P-HA, S-NT and S-PX. Hyaluronic acid increased recovery rate during swim-up, but not viability in any of the treatments. Pentoxifylline is beneficial if Percoll separation is delayed, but clarification of its effect on acrosome exocytosis is needed.

Table 1.
Intact and recovery rates (LS means ± SE)
32 EFFICIENCY OF FEMALE-DERIVED DONOR CELLS ON HIGH POSTNATAL SURVIVAL IN PIG CLONING
Authors: Cho S.K. , Park M.R. , Kwon D.N. , Lee E.K. , Kang S.J. , Lee S.Y. , Choi Y.J. , Park J.Y. , Son W.J. , Kim J.H.
Published: December 2004

The present study was conducted to investigate the developmental competence of male and female somatic cell derived nuclear transfer (NT) porcine embryos and also the production and survival efficiency of cloned male and female piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1 mM dibutyryl cyclic adenosine monophosphate, and 0.1 IU/mL human menopausal gonadotrophin for 20 h and then culture without dbcAMP and hMG for another 18 to 24 h. Fetal cells were isolated from a male fetus and two female fetuses, and cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/mL BSA under mineral oil at 39°C in 5% CO2 in air for up to 6 days. NT eggs that had been activated with electric pulses and cultured for 1 or 2 days were transported to the experimental station in modified NCSU-23 with antibiotics. NT embryos were surgically transferred into the oviducts of recipients between Day 27 and Day 30; pregnancy was determined by ultrasound. The potential of NT embryos to develop into blastocysts was not different among donor cells of different origins. However, the mean cell number of in vivo female and male blastocysts (83.8 ± 46.2 to 99.2 ± 55.7) was higher than in in vitro culture of NT groups (31.4 ± 8.29 to 33.2 ± 10.15). A total of 11,535 NT embryos (1- to 8-cell stage) were surgically transferred into 66 surrogate gilts. Among fourteen pregnant gilts, four recipients aborted during the period of conception. Five pregnant gilts delivered fifteen female piglets, 1.28 ± 0.33 kg (0.48∼1.83 kg) in female piglets and 0.84±0.25 kg (0.45∼1.25 kg) in male piglets. Nine live cloned female (60.0%) and four male piglets (18.2%) were produced. According to these results, survival rates and birth weights of female cloned piglets were higher than those of cloned male piglets (P < 0.05). This study suggests that use of female, compared with male, fetal fibroblast cells as nuclear donors may increase cloning outcomes.
This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea.
38 HIGH OSMOLARITY AT EARLY CULTURE STAGE IMPROVES IN VITRO DEVELOPMENT OF PRE-IMPLANTATION PORCINE NUCLEAR TRANSFER EMBRYOS
Authors: Im G.-S. , Yang B.-S. , Lai L. , Liu Z. , Hao Y. , Prather R.S.
Published: December 2004

Fragmentation occurs during early developmental stage of electrically activated oocytes and nuclear transfer (NT) embryos, and it might be the cause of the low developmental rate of pre-implantation porcine nuclear transfer embryos. The present study was conducted to investigate whether addition of sugars such as sorbitol and sucrose suppresses fragmentation and supports the development of NT embryos. Oocytes derived from a local abattoir were matured for 42–44 h and enucleated. Fetal fibroblast cells were obtained from a Day 35 porcine fetus. Parthenogenetically activated oocytes or NT embryos were cultured in PZM-3 for 6 days, or in PZM-3 supplemented with sorbitol or sucrose for 2 days and then cultured in PZM-3 for the remaining 4 days. The osmolarity of PZM-3, PZM-3 supplemented with 0.05 M or 0.1 M sorbitol, and PZM-3 with 0.05 M sucrose was 269 ± 6.31, 316 ± 3.13, 362 ± 4.37 and 315 ± 5.03 mOsm, respectively. When the parthogenotes were cultured in PZM-3 supplemented with 0.05 M sorbitol or sucrose for the first 2 days and then cultured in PZM-3 without sugar, a significantly higher (P < 0.05) cleavage rate and blastocyst rate was observed. Interestingly, sugar addition into PZM-3 at early culture stage for 2 days reduced the fragmentation rate compared to the rate in PZM-3 without sugar. In NT embryos, sugar addition into PZM-3 significantly (P < 0.05) increased the cleavage rate (67.6 ± 5.80 vs. 77.3 ± 3.03) and developmental rate to the blastocyst stage (10.2 ± 0.79 vs. 19.4 ± 1.77). There was no significant difference between treatments for the number of nuclei in the blastocysts. In addition the fragmentation rate in sugar-supplemented PZM-3 was reduced compared to that in PZM-3 without sugar (26.1 ± 4.30 vs. 14.5 ± 1.74). In conclusion, the increased osmolarity of PZM-3 with sugar supplementation at an early developmental stage for 48 h could increase the cleavage and developmental rate to the blastocyst stage by reducing the fragmentation rate.
54 COMPOSITION OF ALLANTOIC FLUID IN CATTLE PREGNANT WITH AI-, IVP-, OR NUCLEAR TRANSFER-GENERATED EMBRYOS
Authors: Morrow C. , Berg M. , McDonald R. , Wells D. , Peterson A. , Lee R.
Published: December 2004

Abnormal placentation, pregnancy failure, and hydroallantois are associated with somatic cell nuclear transfer (SCNT) in cattle. Identification of diagnostic markers for abnormal placentation in early gestation would permit therapeutic intervention. Ultrasonography and transvaginal sampling of amniotic and/or allantoic (fetal) fluid enables regular monitoring of fetal health. We report on the composition of serial samples of fetal fluid from individual cows between Days 70–130 of gestation and the potential of steroid and electrolyte composition as an early diagnostic marker for the subsequent occurrence of hydroallantois in SCNT pregnancies in cattle. On Day 70, pregnancy rates were 50% and 60% for cows or heifers implanted with single in vitro-fertilized (IVP, 20/40) or SCNT (25/42) embryos, respectively, and 67% for pregnancies generated by artificial insemination (AI, 12/18). Resulting fetuses were either clones (SCNT) or offspring (IVP/AI) of a donor Holstein bull. Fetal fluids, sampled using ultrasound-guided transvaginal puncture, were collected on Days 70, 100, and 130 of gestation (n = 12 and 139 for amniotic and allantoic samples, respectively). Placental and fetal morphological data were collected following slaughter between Days 135–163 of gestation (n = 14, 20, and 10 for SCNT, IVP, and AI groups, respectively). Fetal fluids were analyzed for progesterone, estrone sulphate, sodium, chloride, potassium, creatinine, urea, calcium, magnesium and phosphate. Pregnancy outcomes for the SCNT group were retrospectively classified as: Fail 100 (pregnancies failing between Days 70–99; n = 6); Fail 130 (failing between Days 100–129; n = 5); Hydrops (greater than 10 L combined amniotic and allantoic fluid at postmortem between Days 135–163; n = 8) and SCNT Pregnant 150 (pregnant between Days 135–163; n = 6). IVP and AI pregnancies were classified as IVP or AI Pregnant 150. Fluid composition was analyzed by ANOVA on log-transformed data. On Day 70, allantoic progesterone and estrone sulphate concentrations were significantly higher (P < 0.05) for the SCNT cows compared to the IVP/AI Pregnant 150 cows. On Day 70, allantoic potassium, chloride, creatinine, and urea concentrations were significantly higher (P < 0.05) for the SCNT Hydrops cows compared to the IVP/AI Pregnant 150 cows. In addition, Day 70 allantoic creatinine and urea concentrations were significantly higher (P < 0.05) for the SCNT Hydrops cows compared to other SCNT groups. By Day 100, allantoic chloride, creatinine, and urea concentrations in SCNT Hydrops cows were significantly lower (P < 0.05) than in IVP/AI Pregnant 150 groups. We conclude that elevated Day 70 allantoic urea and creatinine concentrations are potential early diagnostic markers predicting hydroallantois in recipient cattle carrying SCNT fetuses. Further investigation of these markers in other somatic donor cell lines used for nuclear transfer is warranted to determine their general utility.
57 INVESTIGATION OF CYNOMOLGUS MONKEY (MACACA FASCICULARIS) FETUS FIBROBLAST CELL NUCLEAR TRANSFER
Authors: Narita J. , Tsuchiya H. , Takada T. , Torii R.
Published: December 2004

Use of nuclear transfer (NT) in the cynomolgus monkey to establish tailor-made ES cells with the final goal of cloned embryo production was investigated. Activation stimulus conditions previously confirmed in parthenote production were used. Injection method NT was conducted using cynomolgus monkey fetus fibroblast cells in order to investigated the time it takes, from injection to activation, to reprogram the donor nuclei. Oocytes were collected under laparoscopic observation from mature cynomolgus monkeys 40 h after hCG (400 IU/kg) administration 9 days after follicle stimulation by i.m. injection of FSH (25 IU/kg). Donor cells, 40-day-old fetus fibroblast cells, were cultivated and synchronized at G0/G1 phase. After mature (MII) oocytes were enucleated using a Piezo-drive unit, donor cells were injected. At 2 h (Experiment 1, E1) and 4 h (Experiment 2, E2) after donor cell injection, activation was carried out by 2-min treatment with ionomycin and cultivation by 6-dimethylaminopurine for 4 h. As a control, parthenote production was carried out under the same activation conditions as NT. After activation, in vitro culture was carried out for about 9 days under conditions of 38°C, 5% CO2, and 5% O2. Whole-mount specimens of NT embryos were made immediately post-injection, 2 and 4 h post-injection, and 2 h after activation. Pronuclear formation (PN) and cleavage rates of NT embryos were 82.1% and 95.7% for E1, and 53.8% and 92.8% for E2, respectively. Control PN and cleavage rates were each 100%. Subsequent embryo development arrested at the 6-cell stage (8.7%) in E1 and 5-cell stage (7.1%) in E2 but proceeded to blastocyst stage (27.3%) in the control. For whole mount specimens, donor nuclei caused premature chromosome condensation in enucleated oocyte cytoplasm, and decondensation due to activation was seen, so injected donor nuclei reconstruction had occurred. No difference was seen between E2 and E1 embryo development and whole mount specimens, but E1 PN rate was clearly higher than that of E2. So 2 h of reprogramming time is more appropriate than 4 h. In this study, most NT embryos arrested at the 4-cell stage. These results suggest that development did not proceed beyond MET (maternal-embryonic transition) which is believed to occur between the 4- and 8-cell stage in cynomolgus monkey. Further study will be necessary to find the condition that completely reprograms injected donor nuclei for cloned embryo production.

Table 1.
Development of cynomolgus monkey fibroblast nuclear transfer embryos

This work was supported by grants from the ministry of Education, Science, Sports, and Culture (13358014, 14380382).
59 A PRELIMINARY STUDY OF THE IN VITRO DEVELOPMENT OF ASIAN ELEPHANT, CLONED EMBRYOS, RECONSTRUCTED USING A RABBIT RECIPIENT OOCYTE
Authors: Numchaisrika P. , Rungsiwiwut R. , Thongpakdee A. , Techakumphu M.
Published: December 2004

Interspecies nuclear transfer is an important tool for studying the interaction between the cytoplasm of one cell and the donor nucleus of another (Chen et al. 2002 Biol. Reprod. 67, 637–642). The aim of this experiment was to investigate the possibility of developing in vitro an asian elephant cloned embryo using a rabbit recipient oocyte. The elephant donor cells were obtained from the ear skin of a stillborn Asian elephant (Elephus maximus) and the in vivo-matured recipient oocytes were obtained from FSH-stimulated New Zealand White doe rabbits. Enucleation was accomplished by aspiration of the first polar body and the metaphase II plate together with a small amount of cytoplasm. Successful enucleation was confirmed by UV examination after staining with 5 μg mL−1 Hoechst 33342. The donor cells were introduced into the perivitelline space of the enucleated oocytes immediately after enucleation. The elephant-rabbit reconstructed embryos were fused in 0.3 M manitol with 0.1 mM Ca2+ and Mg2+ using two types of electrical pulses: E1 (n = 61): 3.2 kV/cm, 3 pulses, 20 μs (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369); E2 (n = 69): 2.0 kV/cm, 2 pulses, 20 μs (Chen et al. 2002 Biol. Reprod. 67, 637–642). The fused embryos were activated 1 h after fusion by electrical pulses to those used for fusion and then incubated in 5 μg mL−1 cyclohexamide and 2 mM 6-DMAP for 1 h. Subsequently, the activated embryos were cultured in B2 medium containing 2.5% fetal calf serum. The developmental rate was observed every 24 h for 7 days and the differences in the percentages of embryos developing to a particular stage were determined by chi-square analysis. The results showed that the fusion and cleavage rates of elephant-rabbit cloned embryos fused and activated by E1 were significantly higher than for E2 (P < 0.05; see Table 1). Compared with rabbit-rabbit cloned embryos using adult skin fibroblast as a donor cell and E1 for both fusion and electrical activation, we found that the cleavage and blastocyst rates of elephant-rabbit cloned embryos was higher than for the rabbit-rabbit ones (65% (28/43) versus 58% (28/48) and 7% (3/43) versus 4% (2/48) respectively). Results from this study showed that either of the electrical pulses, 3.2 kV/cm, 3 pulses, 20 μs or 2.0 kV/cm, 2 pulses, 20 μs, can be used to fuse elephant somatic cells to rabbit ooplasm and the rabbit oocytes can be served as recipient oocytes to support the development of elephant cloned embryos up to the blastocyst stage.

Table 1.
Developmental rate of elephant–rabbit cloned embryos after being fused by different electrical pulses

This work was supported by Rajadapisek Sompoj Fund, Chulalongkorn University.
62 RELATION OF INTENSITY OF GENE EXPRESSION IN BOVINE RECONSTRUCTED EMBRYOS TO SUBSEQUENT DEVELOPMENT
Authors: Saeki K. , Tamari T. , Kasamatsu A. , Shirouzu K. , Taniguchi S. , Matsumoto K. , Hosoi Y. , Iritani A.
Published: December 2004

During embryo development, embryonic gene activation (EGA) is the first critical event. We previously showed that EGA is also critical for further development in somatic cell-cloned embryos (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). To show this, we reconstructed bovine embryos with bovine somatic cells transfected with chicken β-actin/firefly luciferase fusion gene (β−act/luc+) and showed that only luminescent embryos at 60 hours post-fusion (hpf) developed to the blastocyst stage. In this study, we examined the relation between the intensity of expression of the same reporter gene in embryos reconstructed with bovine β−act/luc+ fibroblasts and their subsequent development to the blastocyst stage. Bovine fibroblasts were transfected with β−act/luc+ as described earlier (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). The stably transfected and cloned cells were cultured for several passages. The cells were cultured under serum starvation (0.4% FCS) for 7 days and then used as donor cells. In vitro-matured bovine oocytes derived from slaughterhouse ovaries were enucleated at 20 h post maturation. Enucleated oocytes were electrofused with the cells, and activated with a calcium ionophore and cycloheximide. The LUC+ signal (luminescence) in the embryos was detected in medium containing 500 μg mL−1 luciferin with an imaging photon counter (ARGUS 50, Hamamatsu, Japan) for 30 consecutive min at 60 hpf. The intensity of luminescence in embryos (4- to 8-cell stage) was graded as being strong (>10 × 104 pixels/embryo), intermediate (5 to 10 × 104 pixels/embryo), weak (<5 × 104 pixels/embryo), or absent. The embryos were cultured separately until 168 hpf, and examined for blastocyst development. Experiments were repeated four times, and the data were analyzed with Fisher's PLSD test following ANOVA by Stat View software (Ver. 5.0; abacus Concepts, Berkeley, CA, USA). Of 125 embryos that were reconstructed, 74 (59%) developed to the 4- to 8-cell stage at 60 hpf. The luminescence was strong in 29 (39%) of the embryos, intermediate in 12 (16%), weak in 19 (26%), and absent in 14 (19%). Blastocysts were obtained from a group of embryos that exhibited strong luminescence (10/29, 34%), but none of the embryos from the other groups developed to blastocysts. These results suggest that active gene expression in embryos reconstructed with somatic cells is important for their subsequent development.
This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Ministry of Education, Culture, Sports, Science, and Technology, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.
68 REPRODUCTIVE PERFORMANCE OF CLONED BULLS
Authors: Tecirlioglu R.T. , Cooney M.A. , Korfiatis N.A. , Hodgson R. , Williamson M. , Downie S. , Galloway D.B. , Holland M.K. , French A.J.
Published: December 2004

The generation of cloned animals by somatic cell nuclear transfer has been reported in a number of countries worldwide. However, progress has been impeded by the extremely low efficiency of cloning and health of some of the cloned animals. Surprisingly, little is known of the reproductive performance of viable clones when compared to the original cell donor, given that a major motivation of cloning is dissemination of superior genotypes. The aim of this study was to compare semen collected from three cloned bulls (Clones A1, B1, and B2) to that of the original donor bulls (Donors A and B). Parameters examined included ejaculate volume, sperm concentration and the motility characteristics of frozen/thawed semen using computed-assisted semen analysis (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA). The fertilization ability of each semen sample was examined using in vitro matured oocytes derived from abattoir-source ovaries. Frozen/thawed semen samples from donor and cloned bulls were prepared on a Percoll gradient and diluted with fertilization medium to a concentration of 1 million sperm/mL prior to fertilization (IVF). The number of blastocysts and total cell counts were analyzed on Day 7 of culture. Finally, in vitro-fertilized blastocysts (Day 7) were transfer to synchronized recipients (n = 49) to examine in vivo development. Proportional data for the in vitro development of embryos and subsequent pregnancy rates were analyzed by chi-square test, and embryo cell numbers were analyzed using Student's t-test. Progressive motility percentage between donor and cloned bull did not differ: Donor A (62.25 ± 3.89, n = 12); Donor B (66.69 ± 4.47, n = 13); Clone A1 (71.37 ± 8.57, n = 8); Clone B1 (73.75 ± 2.42, n = 12); Clone B2 (72.41 ± 3.26, n = 12). No obvious differences in kinetic motility parameters were evident between cloned and non-cloned donor animals. However, blastocyst rates were significantly higher in cloned bulls (Clone A1: 30.9%, 81/262; Clone B1: 34.4%, 98/285; and Clone B2: 42.9%, 120/280) compared to donor bulls (Donor A: 20.7%, 54/261; Donor B: 20.9%, 76/364). Total embryo cell numbers did not differ significantly between donor bulls (Donor A: 138.3 ± 5.3, n = 39; Donor B: 133.2 ± 5.2, n = 47) and cloned bulls (Clone A1; 126.3 ± 4.4, n = 45; Clone B1: 134.4 ± 7.1, n = 26; and Cloned B2: 140.1 ± 3.9, n = 46). Initial pregnancy rates on Day 30 were also not different between Donor A (42.3%, 11/26) and Clone A1 (47.8%, 11/23). Preliminary observations from the small data set on postpubertal cloned bulls indicate that semen production, semen morphology, and reproductive performance (in vitro and in vivo) were similar in terms of semen characteristics and reproductive performances when compared to their original donor bulls.
78 SURVIVAL OF PORCINE OOCYTES AT GERMINAL VESICLE STAGE AFTER VITRIFICATION WITH OPEN PULLED STRAW METHOD
Authors: Bali Papp A. , Somfai T. , Varga E. , Marosán M.
Published: December 2004

The present study was performed to assess the survival of immature denuded or cumulus-covered porcine oocytes (COCs). Immature porcine oocytes were collected from 2–6 mm follicles of slaughterhouse ovaries and subjected to open pulled straw (OPS) vitrification, according to the method of Vajta et al. (1998 Mol Reprod. Dev. 51, 53–58). After vitrification, oocytes were matured in vitro for 48 h at 39°C, 5% CO2 in air. The maturation medium was TCM199 supplemented with 10% pig follicular fluid, 1.25 mM L-glutamine, 0.9 mM Na pyruvate, 150 μM cysteamine, 0.1 mg/mL streptomycin sulfate, 100 IU/mL PG penicillin g potassium, 10 IU/mL PMSG, and 25 IU/mL hCG. After IVM, to assess nuclear stage, all oocytes were fixed with acetic acid–alcohol (1:3) for at least three days and then stained with 0.1% orcein and examined under a phase-contrast microscope at 100× magnification. All data were analyzed by χ2 test (P < 0.05). Immediately after collection, all oocytes were at the germinal vesicle (GV) stage with an intact GV membrane. After vitrification, significantly fewer oocytes had normal morphology (intact plasma membrane) in the denuded and COC groups (4.7% and 8.5%, respectively) than did the denuded and COC control groups (95% and 92%, respectively). By the end of IVM, significantly fewer oocytes were surrounded by expanded cumulus after vitrification of COCs than were the COC controls (28.1% and 63.5%, respectively). After IVM, more of the COC control oocytes underwent germinal vesicle breakdown than did the denuded controls (95% and 78.2%, respectively); the rate of MII oocytes was higher for the COC controls than for the denuded controls (80% and 54.5%, respectively). After vitrification, the number of oocytes that underwent GVBD was significantly less for both the denuded and the COC groups (2.0% and 7.0%, respectively); the percentage of oocytes that reached MII was also lower (0.64% and 2.78%, respectively). Most of the vitrified oocytes had a damaged GV with disrupted membrane and cluster-like or scattered chromatin in both the denuded and the COC groups (96.4% and 90.7%, respectively). These data suggest that vitrification of cumulus-enclosed immature porcine oocytes is preferable compared to vitrification of denuded ones. Loss of cumulus cells compromises competence of oocytes to resume meiosis, which might result in a lower maturation rate after IVM.
This research was supported by the grants of the Hungarian Scientific Research Fund (T 031758), the Hungarian National Committee of the Technical Development at the Ministry of Education (00796/2003), and the Ministry of Education (OM-KMUFA; BIO-00086/2002).
82 EVALUATION OF CELL DEATH IN CRYOPRESERVED MOUSE EMBRYOS
Authors: Coutinho A.R.S. , Nascimeto A.B. , Mendes C.M. , Simoes R. , Lucio C.F. , Dagli M.L.Z. , Assumpcao M.E.O.A.
Published: December 2004

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnology. Recent evidence indicates that apoptosis may be important in determining the viability of cryopreserved embryos. Our goal was to detect apoptosis and characterize and quantify the embryonic cell death caused by cryopreservation. Mouse morulae were collected, selected, and separated into three groups: fresh, slow-freezing, and vitrification. In the slow-freezing procedure, embryos were exposed to 10% ethylene glycol (EG) for 10 min. After loading, the straws were placed into methanol at −7°C for 5 min, seeded and after 5 min cooled at 0.5°C/minute. After 10 minutes at −31°C, straws were plunged into and stored in liquid nitrogen. Slow-frozen straws were thawed in air for 10 s, and then immersed in a 25°C water bath for 20 s. Embryos were vitrified by exposing them to 10% and 20% EG for 5 min followed by 40% EG + 18% Ficoll + 10% sucrose (EFS) for 30 s and the 0.25-mL straws then plunged into and stored in liquid nitrogen. The vitrified straws were warmed by immersing them in 25°C water for 20 s. Cell membrane integrity was assessed by Hoechst and propidium iodide double staining (H/PI). Fresh and thawed embryos were scored (following IETS recommendations) and then fixed after 30 min in PBS + 10% FCS. Morphology and apoptosis were assessed with Haematoxylin-Eosin staining (HE) and by electron microscopy (MET). The number of Grade I embryos recovered after thawing was higher for slow-frozen embryos (61.5%) than vitrified embryos (29.5%). H/PI detected more membrane permeability in the vitrified embryos (69.7%), than in the slow-frozen (48.4%) or non-frozen (13.8%) groups (P < 0,05, Wilcoxon's test). Nuclear evaluation by HE revealed that vitrification and slow-freezing induced pyknosis and chromatin condensation. Mitotic pattern was observed in the fresh and slow-frozen group, but not in vitrification group suggesting that the embryos were either not randomly allocated to the groups or not-treated and fixed at the same age, or that vitrification changed the nuclear status of the embryos. HE staining revealed weakly staining cytoplasm and degenerated cells in the vitrification group (indicating oncosis), while in the slow-frozen group the presence of cytoplasmic condensation and eosinophilic structures indicated apoptosis. The ultrastructure examination confirmed the HE observations. In conclusion, the results demonstrated that staining with HE allows detection of oncosis and apoptosis in cryopreserved embryos. According to these data, vitrification caused more cellular injuries than slow-freezing, and oncosis was the predominant injury. It is important to point that specific molecular apoptosis tests must be performed to confirm these results.
This work was supported by FAPESP 04/01252-4.
86 CULTURE OF PIG EMBRYOS BEFORE CRYOPRESERVATION
Authors: Gajda B. , Smorag Z.
Published: December 2004

Our previous experiment on cryoconservation of in vitro-cultured porcine embryos (Gajda and Smorag 2000 CryoLetters 21, 231–236) revealed that, unlike for other species, sensitivity to vitrification of such embryos is higher than for those obtained in vivo. Considering that, selection of an optimal in vitro culture medium for pig embryos before cryoconservation becomes more important. Two experiments have been done on 3/4-cell pig embryos. Embryos were obtained from superovulated gilts after flushing the oviduct with PBS medium supplemented with 20% fetal calf serum at 38°C. In experiment 1, the embryos were cultured in three chemically defined media: NCSU-23, NCSU-37, CZB. The culture was performed at 39°C, 5% CO2 in air for 96 to 120 h. The main evaluation criterion was development into blastocysts. Additionally, embryos that developed into blastocysts were stained with Hoechst 33342 and the cells were counted under a fluorescence microscope. Data were analyzed using chi-square and Fisher tests. The highest percentage of embryos developing into blastocysts was observed for those cultured in NCSU-23 (a): 89.2%; lower in NCSU-37 (b): 78.9%; and lowest for those in CZB medium (c): 66.6% (a,c: P < 0.05; a,b and b,c: NS). There were significantly (P < 0.001) more total cells in blastocysts obtained after culture in NCSU-23 (139.5 ± 32.8) than after culture in NCSU-37 (71.9 ± 36.6) or in CZB medium (58.3 ± 8.6). In experiment 2, embryos were cultured in vitro in NCSU-23 medium to the blastocyst stage and then vitrified (Gajda and Smorag 2002 CryoLetters 23, 385–388). Embryos were vitrified in 0.25-mL plastic straws in a mixture of 40% v/v ethylene glycol, 18% w/v Ficoll, and 0.3 M sucrose. Straws with embryos were stored in liquid nitrogen for 3 to 6 months. Dilution after rapid thawing (water bath at 20°C) was done in one step in 0.5 M sucrose solution. Eighty-five thawed blastocysts were surgically transferred into the oviducts of four synchronized recipient gilts. Two recipients became pregnant and farrowed 11 live healthy piglets. These results indicated that NCSU-23 medium provided the best conditions (of those media tested) for in vitro culture of pig embryos before vitrification. Transfer into oviducts of blastocysts that developed from in vitro-cultured and vitrified embryos resulted in full development in vivo.
Research was funded by the State Committee for Scientific Research (Project No. 2 P06D 003 26).
87 VITRIFICATION OF BOVINE OOCYTES TREATED WITH CHOLESTEROL-LOADED METHYL-β-CYCLODEXTRIN
Authors: Horvath G. , Solti L. , Seidel G.
Published: December 2004

Addition of cholesterol-loaded cyclodextrin (CLC) can increase sperm cryosurvival (Purdy et al. 2000 Cryobiology 48, 36–45). The purpose of this study was to determine if cryosurvival of vitrified oocytes could be improved by incubation with CLC prior to vitrification. Slaughterhouse-derived cumulus oocyte complexes were matured in a chemically defined medium with fatty acid-free BSA and hormones for 21 h followed by partial cumulus removal with 100 U/mL hyaluronidase and gentle pipetting. For an additional hour, oocytes were placed into maturation medium supplemented with 0.5% PVA instead of BSA with or without 2.5 mg/mL CLC. At 22 h after the start of maturation, oocytes were transferred to handling media containing 20% FCS or 0.5% PVA in TCM-199 + HEPES (HTCM-199). Oocytes with approximately 3 layers of cumulus were vitrified in two steps. First, they were exposed to VS1 (10% ethylene glycol (EG), 10% DMSO, 6% PVP, or 20% FCS, in HTCM-199) for 30 s, then exposed to VS2 (20% EG, 20% DMSO, 6% PVP, or 20% FCS, 0.48 M galactose in HTCM-199) for 25 s, loaded into cryoloops in groups of five, and plunged into liquid nitrogen. Rapidly warmed oocytes were moved stepwise through 0.5, 0.25, 0.125, and 0 M galactose in HTCM-199 + 20% FCS, 3 min each. All procedures were conducted at 39°C. Warmed oocytes were placed in maturation medium for an additional hour, fertilized with semen from 3 bulls, 3 replicates each, and cultured according to standard procedures (Zhang et al. 2003 Theriogenology 60, 1657–1663). For each replicate, 30 oocytes were assigned to the following treatments: A: chemically-defined media with PVA for the last hour of maturation, handling and vitrification; B: same as A except CLC treatment, for 1 h before vitrification; C: chemically defined media for maturation, but with 20% FCS for HM, VS1 and VS2. Data were analyzed by ANOVA. CLC treatment resulted in higher cleavage rates and 8- to 16-cell embryo production, but not higher blastocyst (bl) production (Table 1). Non-vitrified oocytes developed better than vitrified ones (means: cleavage, 76%; 8- to 16-cell, 64%; bl D8, 21%; bl D9, 24%). Further studies with vitrification of cholesterol-loaded cyclodextrin-treated oocytes and chemically defined media are warranted.

Table 1.
Development of vitrified oocytes (LS means ± SE)
Development of a laboratory protocol for fire performance of landscape plants
Authors: Etlinger Matthew G. , Beall Frank C.
Published: December 2004

Over 2.5 million people and 1 million structures risk destruction from wildland fires in California. One way to mitigate this risk is the manipulation and/or selection of landscape vegetation. In this study, six species were studied for their intrinsic characteristics and tested with a newly-developed laboratory fire protocol at 150 kW to determine heat release rate (HRR). The plants were 2–3 years old and obtained from a local nursery. Whole plants were subjected to desiccation in a dry kiln at 50°C, which was found to be much more effective than simulating fire weather in a greenhouse. This is apparently the first study that has measured plant variables, burned them in a natural vertical position, and related HRR to the plant characteristics. Multiple regression showed the overwhelming importance of foliage and moisture content to peak HRR. Chemical variables were not significant and high moisture contents were found to obscure other plant characteristics.
Mitochondrial substitution rates are extraordinarily elevated and variable in a genus of flowering plants
Authors: Cho Yangrae , Mower Jeffrey P. , Qiu Yin-Long , Palmer Jeffrey D.
Published: December 2004

Plant mitochondrial (mt) genomes have long been known to evolve slowly in sequence. Here we show remarkable departure from this pattern of conservative evolution in a genus of flowering plants. Substitution rates at synonymous sites vary substantially among lineages within
Plantago
. At the extreme, rates in
Plantago
exceed those in exceptionally slow plant lineages by ≈4,000-fold. The fastest
Plantago
lineages set a new benchmark for rapid evolution in a DNA genome, exceeding even the fastest animal mt genome by an order of magnitude. All six mt genes examined show similarly elevated divergence in
Plantago
, implying that substitution rates are highly accelerated throughout the genome. In contrast, substitution rates show little or no elevation in
Plantago
for each of four chloroplast and three nuclear genes examined. These results, combined with relatively modest elevations in rates of nonsynonymous substitutions in
Plantago
mt genes, indicate that major, reversible changes in the mt mutation rate probably underlie the extensive variation in synonymous substitution rates. These rate changes could be caused by major changes in any number of factors that control the mt mutation rate, from the production and detoxification of oxygen free radicals in the mitochondrion to the efficacy of mt DNA replication and/or repair.
Dietary sources of inorganic microparticles and their intake in healthy subjects and patients with Crohn's disease
Authors: Lomer Miranda C. E. , Hutchinson Carol , Volkert Sara , Greenfield Simon M. , Catterall Adrian , Thompson Richard P. H. , Powell Jonathan J.
Published: December 2004

Dietary microparticles are non-biological, bacterial-sized particles. Endogenous sources are derived from intestinal Ca and phosphate secretion. Exogenous sources are mainly titanium dioxide (TiO2) and mixed silicates (Psil); they are resistant to degradation and accumulate in human Peyer's patch macrophages and there is some evidence that they exacerbate inflammation in Crohn's disease (CD). However, whether their intake differs between those with and without CD has not been studied. We aimed to identify dietary microparticle sources and intakes in subjects with and without CD. Patients with inactive CD and matched general practice-based controls (ninety-one per group) completed 7d food diaries. Intake data for dietary fibre and sucrose were compared as positive controls. All foods, pharmaceuticals and toothpastes were examined for microparticle content, and intakes of Ca and exogenous microparticles were compared between the two groups. Dietary intakes were significantly different between cases and controls for dietary fibre (12 (SD 5)v. 14 (sd 5) g/d;P=0.001) and sucrose (52 (sd 27)v. 45 (sd 18) g/d;P=0·04) but not for Ca. Estimated median TiO2and Psil intakes (2·5 and 35mg/individual per d respectively, totalling 1012–1013microparticles/individual per d) were broadly similar toper capitaestimates and while there was wide variation in intakes between individuals there was no significant difference between subjects with CD and controls. Hence, if exposure to microparticles is associated with the inflammation of CD, then the present study rules out excess intake as the problem. Nonetheless, microparticle-containing foods have now been identified which allows a low-microparticle diet to be further assessed in CD.
Intestinal immunity of rats with colon cancer is modulated by oligofructose-enriched inulin combined with Lactobacillus rhamnosus and Bifidobacterium lactis
Authors: Roller Monika , Femia Angelo Pietro , Caderni Giovanna , Rechkemmer Gerhard , Watzl Bernhard
Published: December 2004

Probiotics (PRO) are known to modulate immunity in animals and human subjects and to inhibit colon carcinogenesis in experimental models, but the effects of synbiotics (SYN) are not well understood. Therefore, the effects of PRO (Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12), PRE (inulin-based enriched with oligofructose, 100g/kg) and SYN (combination of PRO and PRE) on the immune system of rats were investigated in the azoxymethane (AOM)-induced colon cancer model. After 33 weeks, rats with and without AOM treatment were killed and immune cells were isolated from spleen, mesenterial lymph nodes (MLN) and Peyer's patches (PP). AOM treatment significantly reduced natural killer (NK) cell-like cytotoxicity in control rats and in PRO- and PRE-supplemented rats. SYN supplementation prevented the AOM-induced suppression of NK cell-like cytotoxicity in PP compared with control rats (P<0·01). SYN and PRE supplementation stimulated IL-10 production in PP in these rats (P<0·01) and in MLN of rats not treated with AOM (P<0·05). Interferon-γ production in PP was decreased by PRO supplementation (PRO and SYN groups combined; P<0·05). Proliferative responsiveness of lymphocytes (PP) from AOM-treated rats was suppressed in SYN-supplemented rats (P<0·01). Overall, SYN supplementation in carcinogen-treated rats primarily modulated immune functions in the PP, coinciding with a reduced number of colon tumours. PRE and PRO provided in combination as SYN may contribute to the suppression of colon carcinogenesis by modulating the gut-associated lymphoid tissue.