bluuebunny/crossref_metadata_2025 huggingface dataset:
131DIFFERENTIAL DNA METHYLATION CHANGES OF THE REPETITIVE SEQUENCES
DURING PREIMPLANTATION DEVELOPMENT IN THE MOUSE
Authors: Kim S.-H. , Kang Y.-K. , Koo D.-B. , Kang M.-J. , Lee K.-K. , Han Y.-M.
Published: February 2004

DNA demethylation as an epigenetic change is a unique event genome-wide, occurring at preimplantation and germ cell stages during mouse development. The paternal genome after fertilization is demethylated first, referred to active demethylation, followed by demethylation of the maternal genome during preimplantation development in the mouse. To examine methylation changes in the early mouse embryo, methylation states of various genomic regions such as intracisternal A-particle (IAP), early retrotransposon (Etn) and centromeric satellite sequences were determined by means of a bisulfite sequencing method. For methylation analysis, genomic DNA was first isolated from each developmental stage of embryo (about 300 cells in total), respectively, and exposed to 1.9M sodium bisulfite overnight. Targeted DNA sequences were amplified from bisulfite-treated genomic DNAs by PCR, cloned into pGEM T-easy vector and sequenced. Results indicated that IAP sequences maintained high levels of methylation until the morula stage and were demethylated in blastocysts. In contrast to the IAP sequences, methylation states of Etn elements were remarkably erased after fertilization, completely demethylated at the 8-cell stage and then remethylated at the morula stage. Centromeric satellite sequences showed low methylation states throughout all preimplantation stages of embryos, indicating that the satellite sequences are substantially demethylated in both paternal and maternal genomes. The results suggest that differential epifenetic changes among the repetitive sequences may be responsible for peculiar chromatin structure of respective genomic loci and/or may regulate gene expression during preimplantation development in the mouse.
132INCIDENCE OF OXIDATIVE STRESS-INDUCED APOPTOSIS IN OVINE EMBRYOS IS
ALLEVIATED IN VITRO BY TROLOX, A WATER-SOLUBLE VITAMIN E ANALOGUE
Authors: Reis A. , Powell K.A. , McCallum G.J. , Rooke J.A. , McEvoy T.G.
Published: February 2004

The ability of ovine embryos to develop in vitro can be curtailed severely by culture conditions that predispose, or medium constituents such as docosahexaenoic acid (DHA; C22: 6n−3) that are vulnerable to oxidative stress (Reis et al., 2003 Reprod. Abs. Ser. 30, 48). This study examined the consequences of culture in such adverse circumstances in terms of resultant blastocyst cell metabolism and survival, and investigated whether presence of a water-soluble vitamin E analogue (Trolox; Sigma, St. Louis, MO, USA) from the zygote stage onward could safeguard ovine blastocyst integrity. Day 6 blastocysts were produced in Synthetic Oviductal Fluid (10 replicates) supplemented with 0.4%w/v fatty acid-free BSA in the absence (SBSA) or presence of DHA (SBSAD) or in the same media with 200μM Trolox (SBSAT, SBSADT). For determination of the functional competence of their mitochondria, individual blastocysts (5 replicates) were incubated for 3h in the presence of [2-14C]pyruvic acid (American Radiolabeled Chemicals, St. Louis, MO, USA; Sp. act. 5.5mCimmol−1) using a ‘hanging drop’ technique, and then fixed for cell counts (Hoechst 33342). In addition, the incidence of apoptosis in corresponding blastocysts (5 replicates) was determined by TUNEL assay (Apoptag Red kit, Serologicals Corporation, Norcross, GA, USA). Cell counts and pyruvate metabolism data (log transformed) were tested using 2-way ANOVA. Blastocyst apoptotic cell indices were analyzed by Generalized Linear Model (binomial distribution; Genstat 6, Version 6.2). Neither DHA nor Trolox significantly affected metabolism of [2-14C]pyruvic acid on a 14CO2 per cell (pmol/3h) basis (Table 1) in Day 6 blastocysts, but there was an interaction (DHA×Trolox, P<0.001). Total cell numbers were lowered (P=0.053) in blastocysts produced in the presence of DHA, and Trolox did not significantly affect this parameter. However, inclusion of Trolox reduced apoptotic indices (mean±SEM; P<0.001) which, for blastocysts from SBSA, SBSAD, SBSAT and SBSADT, were 12±0.8 (n=43), 13±1.1 (20), 9±0.7 (64) and 9±0.8% (36), respectively. The data indicate that Trolox supplementation of DHA-containing media sustained mitochondrial metabolism in Day 6 ovine blastocysts. Moreover, independently of DHA status, Trolox lowered the incidence of apoptosis, presumably by protecting vital but vulnerable cellular constituents. Funded by SEERAD; AR supported by Portuguese Ministry of Science and Technology.

Table 1
Mean (±SEM) pyruvate metabolism indices (pmol/3h) and cell numbers for Day 6 blastocysts
134INCOMPLETE HISTONE ACETYLATION OF SOMATIC CHROMATIN IN BOVINE OOCYTES
Authors: Wee G. , Kim S.-H. , Kim K.P. , Yeo S. , Koo D.-B. , Moon S.J. , Lee K.-K. , Han Y.-M.
Published: February 2004

Histone acetylation as an important regulatory mechanism of chromatin structure preceeding zygotic gene expression in early embryo development. After fertilization, transcriptional activation of the embryo begins during the S/G2 phase of the first cell cycle. However, the precise mechanism underlying activation of zygotic transcription remains to be understood, especially in bovine nuclear transfer (NT) embryos. It is known that acetylation of histone H4 lysine 5 (H4K5) represents hyperacetylation state, which is correlated with gene expression. In this study, the acetylation of H4K5 was observed during pronuclear formation by using immunofluorescence analysis with anti-AcH4K5. Our data were analyzed by the general linear models (GLM) procedure of the SAS. In IVF embryos, acetylation of H4K5 occurred on the paternal chromatin at 8h after fertilization but did not occur on the maternal chromatin until 10h after fertilization. Reconstructed oocytes with deactylated somatic cell nuclei began to show signs of acetylation on chromatin at 3h after fusion. When acetylation intensity was calculated using an image analyzer, IVF embryos presented a higher acetylation signal than NT embryos (P<0.05). To induce hyperacetylation in NT embryos, somatic cells were exposed to trichostatin A (TSA, 1μM for 60h), a specific inhibitor of histone deacetylase (HDAC), prior to NT. Acetylated signals of H4K5 increased significantly in TSA-treated cells as compared with non-treated cells (P<0.05). The reconstructed embryos with TSA-treated cells showed a higher fluorescence intensity than the oocytes with non-treated cells (P<0.05), but weak signals compared to IVF embryos. Thus, the results demonstrated low histone acetylation level of somatic cell nuclei after NT during the zygotic progress. Our findings suggest that developmental failures of NT embryos may be due to incomplete chromatin remodeling of somatic cell nuclei during early embryonic development.
136HIGH PROTEIN DIET INHIBITS INNER CELL MASS FORMATION AND INCREASES
APOPTOSIS IN MOUSE BLASTOCYSTS DEVELOPED IN VIVO BY INCREASING THE LEVELS OF
AMMONIUM IN THE REPRODUCTIVE TRACT
Authors: Gardner D.K. , Stilley K.S. , Lane M.
Published: February 2004

Ammonium is known to adversely affect the development of mouse embryos in culture. Specifically, ammonium has been found to impair inner cell (ICM) mass formation, increase apoptosis, retard fetal development following embryo transfer and induce exencephaly. Significantly, high protein diets in cattle lead to reduced fecundity. This has been linked to elevated urea levels within fluid of the female tract. In this study we have determined the effects of a high protein diet for mice on the levels of ammonium within the female tract and the effects of such a diet on the development and viability of blastocysts developed in vivo. Outbred mice (CF1) were fed a diet of either 25% (high protein) or 14% (control) protein for 4 weeks. Females were superovulated and mated to males of the same strain. In 24 mice, oviduct fluid was collected at 22h post hCG. Ammonium in the oviduct fluid was then quantitated fluorometrically. From other animals, blastocysts were flushed 92h post hCG and analyzed. Blastocyst differentiation and apoptotic indices were determined. Values are mean±SEM. Data were analysed using Student’s t-test. The levels of ammonium in the oviduct were significantly higher (P<0.01) in females fed the high protein diet (356±43μM) compared to the control (68±13μM) (n=12 in each group). Blastocysts (n=139) from females fed the high protein diet had significantly lower total (43.4±1.1; P<0.05) and ICM cell numbers (12.7±0.4; P<0.01), compared to the control group (46.8±0.9 and 15.4±0.4 respectively; n=124). Furthermore, blastocysts from animals fed a high protein diet had a significantly higher apoptotic index (8.7±1.4; P<0.01) compared to the control group (2.0±0.5). These data show that consumption of a high protein diet results in the excess accumulation of ammonium in the fluid of the female reproductive tract of mice. These high levels of ammonium subsequently impair the formation of the fetal progenitor cells and increase cell death at the blastocyst stage. These data from in vivo-developed mouse blastocysts are similar to those for blastocysts developed in culture in the presence of 300μM ammonium. Therefore, it is not advisable to maintain mice on a high protein diet. These data have significant implications for animal breeding, and for patients attempting IVF treatment.
137THE EFFECT OF ANTI-CD44 ON EMBRYO IMPLANTATION IN RABBITS
Authors: Illera M.J. , Bermejo P. , Hernandez J. , Gonzalez A. , Illera J.C.
Published: February 2004

Cellular adhesion molecules are thought to be responsible in embryo attachment in human and animals. Blastocyst attachment to the lining of the mammalian uterus during early implantation involves the initial apposition of the trophoblast to the uterine epithelial surface. A number of cell surface molecules have been implicated in the initial attachment reaction between trophectoderm and maternal surface epithelium, these include heparin-binding epidermal growth factor (HB-EGF), trophinin, CD44, integrins and extra cellular matrix molecules such as osteopontin (OPN) each of which exhibit elevated levels of expression at the time of implantation. CD44 is a membrane glycoprotein that is present in a variety of isoforms and is considered to be the major cell surface receptor for hyaluronan. The aim of these work was to study the effect of CD44 on embryo implantation. The rabbit is potentially an excellent model for study of implantation because it is an obligate ovulator, and therefore pregnancy can be precisely timed. Adult New Zealand rabbit females (n=12) were naturally inseminated with a buck of proven fertility. The day of coitus was counted as ‘Day 0’ of pregnancy. To determine the effect of CD44 at the time of implantation, the animals were divided in three groups: group A (four animals): received 100μg of anti-CD44/mL in an intraperitoneal injection on day 4.5 of pregnancy; group B (four animals), via mid-ventral laparotomies on day 6.5 p.c animals received an injection of 20μg of anti-CD44 on the right horn (0.5mL each, from the ovarian end to the cervix). All these treatments were performed in the right horn. Each animal served as her own control with the left uterine horn receiving 0.5mL of saline. Group C (four animals): This was the control group; two animals received an injection of 2mL of saline and the other two received 0.5mL of saline in each uterine horn. Finally, each animal was sacrificed on Day 10 of the pregnancy, the uterus was removed, and the number of implants were counted in each uterine horn. In the group A: a mean of 4.5±0.47 s.e.m implants were found in the right and on the left side. In group B, a profound reduction in implantation sites was found with the anti-CD44 injected into the uterine horn, a mean of 0 implants in the right uterine horn compared with 3.5 implants on the left side P<0.0001. Group C showed 4.25 implants on the right horn and 5.5 on the left horn; no statistical differences were found. Conclusion: the intraperitoneal injection of the anti-CD44 slightly reduced the number of embryos implanted but this reduction was not statistically significant. Anti-CD44 injected intrauterine affected seriously the embryo survival at the time of implantation.
14OVARIAN FOLLICULAR DYNAMICS IN COWS TREATED WITH A CIDR, ESTRADIOL AND PROGESTERONE LATE IN THE ESTROUS CYCLE
Authors: Kastelic J.P. , Colazo M.G. , Small J.A. , Ward D.R. , Mapletoft R.J.
Published: February 2004

The objective was to characterize ovarian follicular dynamics in beef cows treated with a CIDR (Bioniche Animal Health; Belleville, Ontario, Canada) and an injection of estradiol-17β (E2), with or without progesterone (P4), late in the estrous cycle. Previously synchronized, non-lactating, crossbred beef cows (n=36) received a CIDR (Day 0) 16 to 18 days after ovulation and were randomly allocated to one of three treatment groups: no further treatment (Control, n=12), an injection of 5mg E2 (E2, n=12), or 5mg E2 plus 100mg P4 (E2P4, n=12; both from Sigma Chemical Co., St.Louis, MO, USA) i.m. in 2mL canola oil. On Day 7, CIDR were removed and cows received 500μg i.m. of cloprostenol (Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada). Ovaries were examined once daily by transrectal ultrasonography to detect ovarian follicle growth profiles, and determine the time of ovulation. Blood samples were taken daily for progesterone determination. Data were analyzed by ANOVA (LSD and Bartlett’s tests), Student’s t-test and chi-square procedures. Diameter of the CL and the dominant follicle, and progesterone concentration on Day 0 did not differ among groups (P=0.6; overall mean (±SD), 16.8±2.7mm, 14.1±2.0mm, and 1.5±1.9ngmL, respectively). Thirteen cows ovulated within 3 days of treatment (50% of E2- and E2P4-treated cows and 8.3% of Control cows; P=0.05); cows that ovulated had smaller CL diameters (15.2±1.7 v. 17.7±2.7mm; P<0.004) and lower progesterone concentrations (0.4±0.2 v.2.1±2.2ngmL; P<0.001) at the time of treatment. Follicular wave emergence occurred within 7 days in 4/12 Control cows, 10/12 E2-treated cows, and 10/12 E2P4-treated cows (P<0.01). Although the interval from treatment to wave emergence did not differ among treatments (P=0.8; overall, 3.4±1.5 days), follicular wave emergence was more synchronous (P<0.004) in the E2 group than in the Control or E2P4 groups. At CIDR removal, dominant follicle diameter was larger (P<0.02) in the Control group (15.9±5.5mm) than in the E2 (11.9±1.8mm) or E2P4 (11.5±3.4mm)groups, but dominant follicle diameter was less variable (P<0.003) in the E2 group than in the other two groups. Three cows did not ovulate after CIDR removal; two in the Control group and one in the E2P4 group. Interval to ovulation was shorter (P<0.05) in the Control group (70.8±10.5h)than in the E2 (87.0±9.0h) or E2P4 (86.2±7.2h) groups, and the intervals to ovulation in cows that ovulated following treatment (91.0±8.0h) was longer (P<0.001) than in those that did not (76.6±9.6h). In summary, treatment of cows with an estradiol-progesterone protocol late in the estrous cycle resulted in ovulation (50.0%), atresia (33.3%) or persistence (16.6%) of the dominant follicle present at that time. As length of follicular dominance and timing of ovulation were affected, fertility may be impaired following AI.
143DIETARY CARBOHYDRATES AND LIPIDS AFFECT IN VITRO EMBRYO PRODUCTION FOLLOWING OPU IN HEIFERS
Authors: Adamiak S.J. , Mackie K. , Ewen M. , Powell K.A. , Watt R.G. , Rooke J.A. , Webb R. , Sinclair K.D.
Published: February 2004

The superstimulation protocol of Blondin et al. (2002; Biol. Reprod. 66, 38–43) produces cumulus-oocyte complexes (COCs) of high developmental competence for IVP. Using a similar protocol we assessed the affects of alterations in oocyte donor carbohydrate and lipid metabolism during ovarian stimulation on the production and viability of blastocysts in vitro. A 2×2 factorial experiment offered two diets: Fiber (F) and Starch (S) alone (0) or with 6%w/w (6) protected lipid (calcium soaps of fatty acids). Thirty-two heifers ranked by body condition score (scale: 1=thin, 5=obese) were allocated within score to one of the 4 treatments: F0, F6, S0 and S6. COCs were collected 5 days after estrus by OPU for lipid analysis. Ovarian stimulation (4 doses of FSH (9mg NIADDK oFSH) given 12h apart) commenced 2 days later. COCs were collected 40h after the last FSH injection. GnRH (0.012mg Buserelin) was administered i.v. 6h prior to OPU. A second period of ovarian stimulation and OPU then followed. Following IVM/IVF, zygotes were cultured in SOF with 0.3%w/v fatty acid-free BSA under oil (38.8°C, 5% CO2, 5% O2, 90% N) until Day 8 of development, when blastocysts were subjected to total cell counts and TUNEL analysis. Data were analyzed by ANOVA. Neither follicles aspirated (25.9±1.87) nor oocytes recovered (12.1±0.92) differed between treatments. Total fatty acids in plasma were greater (P<0.001) for the F than for the S diets and increased with the inclusion of protected lipid (0.75, 1.82, 0.50 and 1.39μgmL−1 for F0, F6, S0 and S6, respectively; SED=0.076). The dietary lipid-induced increase in plasma fatty acids was reflected in an increase (P<0.05) in total fatty acids within the oocyte (70.4, 74.7, 69.9 and 78.4 ng/oocyte; SED=3.41). Retrospective analysis by body condition indicated that S diets reduced (P=0.006) blastocyst yields in thin heifers and reduced (P=0.02) cleavage rates in fat heifers (Table 1). Blastocyst yields were lower (P=0.1) for fat heifers on the F0 diet. Total cell numbers were greater for thin heifers on S0 than F0 diet. TUNEL-positive cells averaged 4.2±0.48% and did not differ between treatments. In conclusion, modification of oocyte donor carbohydrate and lipid metabolism prior to OPU can influence IVP outcome in a complex manner dependent on body composition. Supported by Defra and The Perry Foundation.

Table 1
In vitro blastocyst yields and total cell numbers
147ION COMPOSITION OF CULTURE MEDIUM INFLUENCES MITOCHONDRIAL
DISTRIBUTION AND BLASTOCYST DEVELOPMENT OF PREIMPLANTATION PORCINE EMBRYOS
Authors: Conover-Sparman M.L. , Krisher R.L.
Published: February 2004

Elevated intracellular calcium (Ca2+) concentrations impair hamster embryo metabolism and viability (Lane M and Bavister B 1998 Biol. Reprod. 59, 1000–1007). Extracellular magnesium (Mg2+) regulates intracellular Ca2+ by controlling its uptake and release. In the present study, we examined the effects of altering Ca2+ and Mg2+ ion concentrations in Purdue Porcine Medium (PPM1) on porcine embryo mitochondrial distribution, metabolic (glycolytic and Krebs cycle) activity, and in vitro developmental potential. Cumulus-oocyte complexes collected from abattoir ovaries were matured for 40–42h, inseminated with 5×105 sperm mL−1 for 5h, and initially cultured in 1:0.4 or 2:1 ratio of Ca2+ to Mg2+ (concentrations in mM) at 38.7°C, in 6% CO2, 10% O2, balance N2. At 22–26, 46–50, and 70–74h post-insemination, 2-, 4-, and 8-cell embryos, respectively, were removed from culture to evaluate mitochondrial distribution (confocal microscopy after tetramethylrhodamine methyl ester staining) and glycolytic and Krebs cycle activity (5-[3H]-glucose and 2-[C14]-pyruvate, respectively). Remaining embryos were further cultured to determine developmental competence (2:1, n=548; 1:0.4, n=560). Cleavage was assessed on Day 3 (2:1, n=552; 1:0.4, n=560) of culture. All data were analyzed using GLM ANOVA, except mitochondrial distribution data which were analyzed using GLIMMIX. A majority (P<0.05) of 2-cell (65%, 13/20) and 4-cell (67%, 22/33) embryos cultured in 2:1 displayed a homogeneous mitochondrial distribution. More (70%, 21/30; P<0.05) 8-cell embryos cultured in 2:1 had a perinuclear mitochondrial distribution. When cultured in 1:0.4, a majority (61%, 14/23; P<0.05) of 2-cell embryos displayed a cortical mitochondrial distribution, whereas most (P<0.05) 4-cell (66%, 19/29) and 8-cell embryos (69%, 18/26) displayed a homogeneous distribution. Glycolytic and Krebs cycle activities were similar (P>0.05) between treatments and across all cell stages examined. Treatment had no effect (P>0.05) on cleavage or blastocyst total cell number. Unlike hamster embryos, culturing pig embryos in a higher Ca2+ concentration resulted in more embryos developing to the blastocyst stage. Culture medium containing 2mM Ca2+ and 1mMMg2+ best supports in vitro blastocyst development, possibly by supporting a more correct mitochondrial distribution. These results are not mediated via changes in glycolytic or Krebs cycle activity, thus suggesting that another cellular mechanism plays a key role in developmental competence in early pig embryos.

Table 1
Effects of Ca2+:Mg2+ on porcine embryonic development and metabolic activity (mean±SEM).
149GLUCOSE METABOLISM OF IN VITRO AND IN VIVO PRODUCED BOVINE EMBRYOS
Authors: De La Torre Sanchez J.F. , Lane M. , Gibbons J. , Gardner D.K. , Seidel G.E.
Published: February 2004

It has been documented that higher glucose metabolism of bovine blastocysts is correlated with higher pregnancy rates following embryo transfer. The aim of this study was to determine the effect of switching embryos between in vivo and in vitro conditions on glucose metabolism. Four types of embryos were produced: Vivo-vivo: embryos were developed entirely in vivo (7.5 days); embryos were collected from superovulated cows at Day 5 following estrus, transferred to a recipient and collected again 2.5 days later to make an appropriate control. Vivo-vitro: embryos developed 5 days in vivo and then were cultured in vitro for 2.5 days. Vitro-vivo: embryos were produced in vitro using slaughterhouse oocytes and frozen semen, cultured until Day 5, and then transferred to recipients and recovered 2.5 days later. Vitro-vitro: embryos were produced entirely in vitro up to Day 7.5. Embryos were cultured in a chemically defined, sequential system (G1/G2), using recombinant human albumin as a protein source, supplemented with hyaluronan and citrate. At Day 7.5, glucose metabolism was measured by determining the amount of 3H2O released when individual embryos (n=111 for all groups) were placed in a 3-μL hanging drop containing 3H-glucose in a closed chamber for 3h. Embryos were graded 1 (good quality) or 2 (fair or poor). Data were analyzed by ANOVA using a 4×2 factorial design with factors group (vivo-vivo, vivo-vitro, vitro-vivo, vitro-vitro) and quality (1, 2). To validate the vivo-vivo group, 15 embryos produced in vivo (without collection and re-transfer) were analyzed for glucose metabolism, resulting in similar values. Grade 1 embryos metabolized more glucose (P<0.01) than Grade 2 embryos (16.6±1.2 v. 10.7±2.5pmol/embryo/h) The vivo-vivo and vitro-vitro embryos metabolized more glucose (P<0.05) than the vivo-vitro and vitro-vivo embryos (15.4±1.7 and 19.3±2.0 v. 9.0±4.5 and 10.7±1.6pmol/embryo/h, respectively). There was an interaction (P<0.05) due to higher glucose metabolism of Grade 1 over Grade 2 embryos in the vitro-vivo and vitro-vitro groups, but no difference between grades for the vivo-vivo and vivo-vitro groups. We conclude that assessment for visual quality was meaningful, and that changing development conditions (vivo to vitro or vitro to vivo) appears to have a detrimental effect on glucose metabolic capabilities of bovine blasocysts lasting up to 2.5 days, and possibly on their developmental competence. Significantly, bovine blastocysts cultured in sequential media G1/G2 had equivalent glucose metabolism to those embryos developed completely in vivo.
151A SIMPLE AND FAST METHOD FOR CONCURRENT DIFFERENTIAL STAINING AND
TUNEL LABELLING OF BOVINE BLASTOCYSTS
Authors: Fouladi-Nashta A.A. , Alberio R. , Nicholas B. , Campbell K.H.S. , Webb R.
Published: February 2004

Currently techniques of TUNEL labelling for detection of apoptosis and differential staining for counting the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells are used separately for assessment of embryo quality in different species. The majority of these techniques are antibody-based, and time-consuming, frequently giving inconsistent results. Here we report on the development of a simple and fast method for simultaneous differential staining and TUNEL labelling of bovine embryos. Cleaved embryos produced from in vitro-matured and fertilized oocytes were cultured to the blastocyst stage in synthetic oviductal fluid culture medium (SOF) supplemented with 4mgmL−1 BSA and 5% FCS. Embryos were partially permeabilized in 0.5% Triton X-100 solution containing 2μMmL−1 TOTO-3 dye (Molecular Probes, Eugene, OR, USA) for 30s. TOTO-3 is a cell-impermeant nucleic acid dye; thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3mgmL−1 PVA, fixed for 15min at RT in 4% paraformaldehyde containing 10μgmL−1 Hoescht, and TUNEL-labelled using a Cell Death Kit (Roche Applied Science, East Sussex, UI) for 30min at 37°C in a humid chamber. The embryos were then treated with RNase A (50UmL−1) for 30min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3UmL−1)-treated embryos were used as a positive control. After three-dimensional reconstruction using a Leica TCS SP2 confocal microscope, we determined the number of ICM (blue), TE (red) and apoptotic nuclei (green). Only peripheral cells of the blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistently stained blue by the cell permeant dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent differential staining was observed in hatched blastocysts (n=30) than in zona-enclosed blastocysts (n=35); also, more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual staining without Tunel, no differences in cell number (74±22) , and ICM/TE ratio (0.28±0.06) were detected, indicating that the Tunel procedure does not affect the labeling of the DNA. Preliminary observations also indicate that this method can be successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and can be applied for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE differentiation while monitoring the degree of apoptosis in different culture systems and in different species. This work was in part supported by Department of Environment Food and Rural Affairs (Defra) UK.
152EFFECT OF REPLACEMENT OF PYRUVATE/LACTATE IN CULTURE MEDIUM WITH
GLUCOSE ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS IN VITRO
Authors: Karja N.W.K. , Medvedev S. , Fuchimoto D. , Onishi A. , Iwamoto M. , Otoi T. , Nagai T.
Published: February 2004

Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) reported that replacement of pyruvate and lactate with glucose, as energy substrates, at 48h of culture in IVC medium enhanced the quality of IVP porcine blastocysts. However, the exact time during early cleavage stages when the utilization of glucose as an energy source is optimal has not yet been determined. The purpose of this study was to examine the effects of glucose supplementation at different times of culture on the developmental competence of IVP porcine embryos. Porcine cumulus-oocytes complexes were matured in modified NCSU-37 solution and fertilized in vitro according to Kikuchi et al. All cultures were performed at 38.5°C, 5% O2, 5% CO2, and 90% N2. In experiment 1, after being fertilized (Day 0), putative zygotes (1158 in 6 trials) were cultured in NCSU-37 supplemented with 0.4% BSA, 0.17mM sodium pyruvate, and 2.73mM sodium lactate (IVC-pyr/lac). Embryos (30–50 in each group) were then transferred into NCSU-37 supplemented with 0.4% BSA and 5.55mM D-glucose (IVC-glu) at 24, 48, 72, 96, or 118h of culture. As control groups, putative zygotes (391) were cultured in IVC-pyr/lac or IVC-glu for the whole culture period. In experiment 2, after being fertilized, putative zygotes (543 in 4 trials, 30–50 in each group) were cultured in IVC-pyr/lac, and then were transferred into IVC-glu at 48h, 53h, 58h, or 63h of culture, because glycolytic activity of in vitro-derived porcine embryos was reported to increase around the 8-cell stage, and some embryos develop to that stage before 72h of culture in experiment 1. All embryos were cultured for 6 days, and then development to the blastocyst stage and number of cells per blastocyst were assessed. When IVF embryos were cultured in IVC pyr/lac for 24h or 48h and subsequently in IVC-glu until day 6 in experiment 1, the rates of blastocyst formation were significantly higher (P<0.05, ANOVA test) than those of embryos cultured in IVC-pyr/lac for the whole culture period (24.4% and 23.0% v. 14.5%, respectively). However, when IVC pyr/lac was replaced with IVC-glu, there were no significant differences between the energy source replacement groups and the glucose-only group in terms of the proportions of cleavage, development to the blastocyst stage and mean cell number per blastocyst (P>0.05, ANOVA test) (15.2%–24.4%, and 16.8%, respectively). Replacement of pyruvate and lactate with glucose at 58h of culture in experiment 2 significantly enhanced the rate of blastocyst formation (P<0.05, ANOVA test) but not the mean cell number compared with zygotes in which the replacement was done at 48, 53, and 63h of culture (31.3% v. 20.6%, 20.8%, and 21.1%, respectively) (P<0.05, ANOVA test). In conclusion, replacement of pyruvate and lactate with glucose as energy substrates was optimal at 58h of culture for the in vitro development of pig embryos to the blastocyst stage.
153EXPRESSION OF TGF-BETA I AND TYPE I AND TYPE II OF TGF-BETA RECEPTORS IN BOVINE EMBRYOS
Authors: Kim B.K. , Chung H.J. , Yang B.C. , Kim D.H. , Woo J.H. , Choi J.H. , Seong H.H. , Jung J.K. , Chang W.K.
Published: February 2004

Although the effects of TGFβ1, as an important factor in the mice embryo development have been reported, little information relevant to this subject is known in the bovine embryo. The objectives of this study were to investigate the presence and expression patterns of TGFβ1 and TGFβ1 receptors, types I and II, in unfertilized oocytes and fertilized bovine embryos in normal and NT embryo development. We postulated that TGFβ1 may have a beneficial effect on the preimplantation embryo and show different expression patterns at different stages of bovine embryo development. Immature bovine oocytes were aspirated from follicles of ovaries obtained from a local abattoir and they were cultured for up to 24h and fertilized in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were used to investigate the presence of TGFβ1 and type I and type II of TGFβ1 receptors (the essential components of the TGFβ1 signaling pathway) in unfertilized oocytes and preimplantation embryos. Also, mRNA and protein expression patterns of TGFβ1 and their receptors at various stages of embryos were examined. It was found that both receptors, as well as TGFβ1, were present in the unfertilized bovine oocytes, indicating that TGFβ1 is a maternally expressed protein. Although the type I TGFβ1 receptor was present at the morulae and blastocyst stages, the type II TGFβ1 receptor was not present at both stages. It was also confirmed that the expression level of TGFβ1 was high at the 8-cell stage, and mRNA and protein expression patterns of TGFβ1 and their receptors were not coincident. Interestingly, TGFβ1 protein was not detected at blastocyst stage of embryos, whereas the mRNA expression level was high at this stage. The results of this experiment indicate that TGFβ1 protein may be needed by embryos after the blastocyst stage and may be expressed in hatched embryos for implantation. These findings support the hypothesis that there may be an interaction between the TGFβ1 and TGFβ1 receptors in the unfertilized oocytes and preimplantation embryos, and that TGFβ1 signaling may be important for the development of the oocytes and the preimplantation embryos.
157A WATER-SOLUBLE VITAMIN E ANALOGUE (TROLOX) IMPROVES OVINE EMBRYO
DEVELOPMENT DURING SERUM-FREE CULTURE IN THE PRESENCE OF DOCOSAHEXAENOIC ACID
(C22:6n-3)
Authors: McEvoy T.G. , Reis A. , Ewen M. , McCallum G.J. , Rooke J.A.
Published: February 2004

Vitamin E (α-tocopherol) supplementation of culture media can safeguard embryo development in vitro but, as it is fat-soluble, usually serum must be present. This study investigated whether Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; Sigma, St. Louis, MO, USA), a water-soluble vitamin E analogue reported to scavenge peroxyl radicals in a manner similar to that of its parent compound, could provide antioxidant protection for ovine embryos in serum-free culture conditions in the presence or absence of supplementary docosahexaenoic acid (DHA, C22:6n–3), a polyunsaturated fatty acid found in vulnerable membranes. Abattoir-derived oocytes were matured and fertilized in vitro (IVF=Day 0). On Day 1, cleaved eggs were assigned to culture (5% CO2, 5% O2, 90% N2; 38.5°C) to the blastocyst stage in synthetic oviductal fluid plus amino acids (SOF, n=15 replicates, mean±SEM=231±6 per replicate) supplemented with 0.4% w/v fatty acid-free BSA in the absence (SBSA) or presence of (SBSAD) DHA or the same media with 200μM Trolox (SBSAT, SBSADT). Respective DHA concentrations (μg/mL; mean±SEM) in these media were 0.0, 0.5±0.12, 0.0 and 0.7±0.27. Blastocyst lipids were separated into polar and neutral fractions (solid-phase extraction, aminopropyl silica columns), and fatty acid composition of each fraction was determined by gas chromatography (splitless method). Using a 2-way factorial design, blastocyst yields (Generalized Linear Model; binomial distribution) and fatty acid profiles (ANOVA) were analyzed. Days 6+7 blastocyst yields were 37±1.5%, 12±0.7%, 56±2.6% and 34±2.1% (mean±SEM) for SBSA, SBSAD, SBSAT and SBSADT, respectively (+DHA effect, P<0.001; +Trolox effect, P<0.001; Interaction, P<0.001). Corresponding cell counts (86±4, n=64; 72±4, n=19; 89±3, n=103; 79±3, n=62) were affected only by presence of DHA (P<0.01). Fatty acid content in neutral lipids (ng/blastocyst) was 64±3, 73±2, 66±5 and 66±5 and, in polar lipids, 47±3, 56±3, 46±3 and 51±7, for SBSA, SBSAD, SBSAT and SBSADT respectively (NS). These data indicate that addition of Trolox to DHA-supplemented culture medium improved production of blastocysts. Presence of DHA, however, was not associated with alterations in neutral and polar lipid content or fatty acid composition of the blastocysts. The fact that Trolox enhanced blastocyst yields in the absence of supplementary DHA indicates that conventional media may benefit from provision of this antioxidant. Funded by SEERAD; AR supported by Portuguese Ministry of Science and Technology.

Table 1
Fatty acid complement in neutral and polar lipids (ng/embryo) from blastocysts produced in vitro
159EFFECT OF GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF)
AND SERUM, ALONE OR IN COMBINATION, ON INTERFERON-TAU (IFNT) PRODUCTION BY
OVINE EMBRYOS
Authors: Rooke J.A. , Ewen M. , Staines M.E. , McEvoy T.G. , Ashworth C.J.
Published: February 2004

The objectives were to establish whether GM-CSF, which may stimulate IFNT production by blastocysts at implantation, influences IFNT production by ovine embryos in culture and whether GM-CSF can explain stimulation of IFNT production by ovine serum (Rooke et al., 2003 Reprod. Abstr. Series 30:56). Oocytes, aspirated from sheep ovaries obtained from a local abattoir on 4 separate days, were matured and fertilized by standard procedures. On Day 1, cleaved zygotes (approximately 10 per 0.05-mL−1 drop; total, 175 per treatment) were cultured in synthetic oviductal fluid containing either 0.3% bovine serum albumin and amino acids (SOFA) or 10% sheep serum (SOFS) in the presence (+) or absence (−) of 5ngmL−1 ovine GM-CSF. Medium was changed every 2 days. On Days 6 and 7, blastocysts were removed from their group drops and assigned in a balanced manner individually to 0.05-mL−1 drops of one of the 4 media for a further 24h. Embryo grade, diameter and cell number were recorded. IFNT concentrations in conditioned media samples were determined by ELISA. The presence of serum accelerated blastocyst development (cross-tabulation; chi-square analysis; P=0.039; Table 1). Both serum and GM-CSF in group culture media increased (2×2 factorial ANOVA, P<0.001) IFNT concentrations (ngmL−1; SOFA−, 0.5; SOFA+, 1.7; SOFS−, 2.7; SOFS+, 4.2; SED, 0.47) with no interaction between serum and GM-CSF. After 24h individual culture of Day 6 blastocysts, IFNT concentrations (2×2 factorial ANOVA; Table 1) were influenced mainly by the origin of the blastocyst (group treatment covariate, serum, P<0.001; GM-CSF, P<0.001) rather than by individual blastocyst culture media (GM-CSF, NS; serum, P<0.001). The pattern of results was similar for Day 7 blastocysts. Incubation of zygotes in the presence of GM-CSF had no effect on embryo grade or diameter but blastocysts (both day 6 and 7) incubated individually for 24h in the presence of GM-CSF had more cells (128 v. 119; SED, 3.52; P=0.001) than blastocysts incubated concurrently without GM-CSF. However, the pattern of IFNT production after correction for cell number was unchanged. In conclusion, culture of zygotes in medium containing GM-CSF or serum, alone or in combination, from Days 1 to 7 increased IFNT production, and IFNT production by individually cultured blastocysts depended on their origin. SAC receives financial support from the Scottish Executive Environment and Rural Affairs Department.

Table 1
Blastocyst yields (% of zygotes) on Days 6 and 7 and IFNT concentrations (ngML−1) in media after individual culture of Day 6 blastocysts
16FERTILIZING POTENTIAL OF STORED TURKEY SPERM
Authors: Morrell J.M. , Tjellström H. , Persson B. , Holmes P.V.
Published: February 2004

Considerable interest exists in prolonging the viability and duration of the fertilizing capacity of turkey sperm. Commercially available extenders for turkey semen are unable to maintain sperm survival for longer than a few hours and attempts to develop a suitable cryopreservation medium have met with limited success. The objective of the present study was to investigate the use of andrology products designed and optimized for turkey sperm (NidaCon International). In an insemination trial on a commercial turkey farm, semen was collected by abdominal massage and was immediately extended in 4mL of either Turkey Semen Extend (NidaCon International, Gothenburg, Sweden) or Beltsville (Continental Plastic Corp, Delavan, WI). The extended semen (approximately 20μL, representing a dose of approximately 70 million sperm) was used immediately for artificial insemination, with each bird being inseminated once weekly. The two groups of birds were housed in different buildings, with different semen donors for each. Eggs were candled after 5 days. Preliminary results showed that fertilization and hatching rates were similar in the two groups: Group 1 (Turkey Semen Extend) 966 eggs, 84.4% fertilized, 78.7% hatched; Group 2 (Beltsville) 966 eggs, 86.6% fertilized, 79% hatched. A second trial over a longer period showed similar results: Group 3 (Turkey Semen Extend) 18,450 eggs, 93.3% fertilized, 84.6% hatched; Group 4 (Beltsville) 40,873 eggs, 92% fertilized, 84.2% hatched. In a second experiment, turkey semen, extended as described above, was transported to the laboratory in the dark at ca. 34°C. Sperm motility was assessed subjectively at several time points. After 2 hours’ storage, at least 50% of the sperm in Turkey Semen Extend were still motile, compared to only approximately 5% of the sperm in Beltsville medium. Aliquots of semen in Turkey Semen Extend were processed by discontinuous density gradient centrifugation on Turkey Gradient (NidaCon International). At least 40% motility was observed after 24 hours’ storage at room temperature. In conclusion, the new Turkey Semen Extender and Turkey Density Gradient offer exciting possibilities for improving the viability of stored turkey sperm for insemination. Future studies will assess the fertility of both unprocessed stored turkey sperm in Turkey Semen Extend, and gradient-prepared stored turkey sperm.
162INSULIN IMPROVES FREQUENCY OF CLEAVAGE AND IN VITRO DEVELOPMENT OF
BOVINE EMBRYOS IN DEFINED MEDIUM
Authors: Wirtu G. , Pope C.E. , Damiani P. , Dresser B.L. , Godke R.A. , Bavister B.D.
Published: February 2004

Pre-elongation stage bovine embryos contain insulin receptor, ligands and receptors of IGF-I and IGF-II, and possibly EGF ligand. Cumulus cells and blastocysts also express EGF receptor; however, insulin is not produced (Watson AJ et al. 1992 Mol. Reprod. Dev. 31, 87–95; Tetens F. et al. 2000 Anat. Embryol. 201, 349–55; Yaseen MA et al. 2001 Reproduction 122, 601-10). Reported effects of external growth factors during bovine IVC are conflicting. The present study evaluated effects of EGF and insulin on the development of in vitro-produced embryos in a chemically defined IVC medium. IVM medium was TCM199 plus fetal bovine serum, LH and estradiol. IVF was done in Tyrodes solution containing BSA, lactate, pyruvate, heparin, penicillamine-hypotaurine-epinephrine and nonessential amino acids. At 18h post-insemination, ova were vortexed, washed and placed in IVC treatments. Modified KSOM (Yang BK et al. 1995 J. Reprod. Dev. 41, 213–18) with 1X MEM nonessential and 1X BME amino acids was the base IVC medium. Incubations were done at 39°C in a humidified atmosphere of 5% CO2 in air (IVM, IVF) or 5% CO2, 10% O2 and 85% N2 (IVC). Effects of three EGF (0, 50, 100ngmL−1; Experiment 1) or five insulin (0, 5, 10, 15, 20μgmL−1; Experiment 2) doses were tested, in five replicates, by supplementation during the entire IVC period (up to Day 9; Day 0=day of insemination). Embryos were placed in fresh medium on Day 4 post-insemination. Data were analyzed using one-way ANOVA and Tukey’s test. In Experiment 1 (n=646 oocytes), EGF treatment did not affect percentages of Day 2 cleavage (83±3, 82±3, and 81±2%, respectively; mean±SEM.) and Day 9 blastocysts (34±4, 33±4 and 33±5%, respectively) or cell number per blastocyst after Hoechst staining (95±5, 88±4 and 89±5, respectively). In Experiment 2 (n=687 oocytes), insulin improved the frequency of cleavage and percentage of expanding, hatching and total blastocysts. It also increased blastocyst cell number (see Table 1). Lack of effect of EGF on bovine IVC is in contrast to its widely reported beneficial effects in mice. In many bovine IVC studies, the beneficial effects of external insulin were seen at or after the morula stage. The present study demonstrates that insulin can influence embryonic development as early as the initial cleavage stage and before embryonic genome activation. Reseach was funded in part by J. Bennett Johnston Science Foundation.

Table 1
Effects of insulin (μgmL−1) on development to >2-cell (Day 2), expanded, hatched and total blastocysts (Day 9: %±SEM) and on blastocyst cell number (Day 9: mean±SEM)
163IMPROVEMENT OF IN VITRO PRODUCTION OF PIG EMBRYOS BY SYNCHRONISATION
OF OOCYTE MEIOTIC MATURATION WITH CYCLOHEXIMIDE
Authors: Ye J. , Campbell K.H.S. , Luck M.R.
Published: February 2004

It is suggested that the relatively high rates of polyspermic fertilization and poor development of pig embryos produced in vitro are caused by asynchronous oocyte maturation. We have recently shown that pre-treatment of pig oocytes with cycloheximide (CHX) is an efficient way of synchronizing their meiotic maturation in vitro. However, it is not known whether this procedure affects fertilization or further development. The present study examined the effects of CHX-synchronised meiotic maturation on subsequent embryo development and the response to FSH. Pig ovaries were collected from a local abattoir. Cumulus-oocyte complexes (COCs) were aspirated from 3–5mm diameter follicles with a translucent appearance and extensive vascularization. COCs were first pre-incubated in defined maturation medium (DM; M199 with Earle’s salts, 25mM HEPES and sodium bicarbonate, 3mM L-glutamine, 0.1% (w/v) BSA, 0.57mM cysteine, 10ngmL−1 EGF, 0.2μgmL−1 pLH, 100μmL−1 penicillin and 0.1mgmL−1 streptomycin) or in DM supplemented with 50ngmL−1 pFSH (DMF) and 5μgmL−1 CHX for 12h. COCs were then further cultured in the same DM without CHX for 24–30h or in DMF for 36h. For controls, COCs were cultured conventionally in DM for 42h or DMF for 48h. After removal of cumulus cells, all cultured oocytes were inseminated with ejaculated sperm at a final concentration of 300000mL−1 for 6h. The IVF medium was modified Tris-buffered medium containing 0.1% BSA, 20μM adenosine and 0.2mM reduced glutathione. Putative embryos were cultured in NCSU23 without glucose but supplemented with 4.5mM Na lactate and 0.33 mM Na pyruvate for 2 days. Cleaved embryos were further cultured in normal NCSU23 for 4 days. IVM and IVF were performed in 5% CO2 in air and IVC in 5% CO2, 5% O2, 90% N2, all at 39°C and 95% RH. Three replicates with DM, with or without CHX, and one with DMF, with or without CHX, were performed with 30–50 oocytes in each replicate. Statistical comparisons were by t-test. The result with DM showed that the rate for normal cleavage at 2 days after insemination of CHX-treated oocytes (40.6±3.8%) was similar to that of controls (40.4±3.5%). However, the proportion developing to healthy blastocysts at Day 6 was significantly higher in the CHX-treated group (16.9±1.2%) than in controls (9.6±1.3%; P<0.05). A significantly higher number of Day 2-cleaved embryos from CHX-treated oocytes developed to the day 6 blastocyst stage compared with controls (44.7±5.0% and 22.3±2.4%, respectively; P<0.05). Supplementation of the basic maturation medium with pFSH increased the rate of cleavage in both CHX-treated oocytes (73.2%) and controls (76.9%) and increased the proportions developing to healthy blastocysts at Day 6 (CHX-treated: 39.0%; control: 11.5%). We conclude that oocytes pre-treated with CHX retain their developmental competence and that meiotic synchronization with CHX improves the efficiency of in vitro production of pig embryos. (Supported by BBSRC 42/S18810.)
165RE-EXPANSION AND QUALITY OF SPLIT BOVINE EMBRYOS IN VITRO
Authors: Kesseler P. , Mahabir E. , Köster M. , Gilles M. , Wimmers K. , Ponsuksili S. , Schellander K.
Published: February 2004

Transferring split bovine embryos results in a higher number of calves born per embryo. In addition to generating genetically identical progeny, biopsies can be made for molecular biological analyses. We aimed to determine the effect of splitting ratio on the in vitro development of Day 7 (164 to 168h post insemination) IVP bovine embryos. The inner cell mass (ICM) and trophoblast cells were split in three ratios (50:50, 60:40 and 70:30) with a Beaver microblade (Becton Dickinson, N.J., USA.) fixed to a micromanipulator under an inverse microscope at 100 X (Leica, Bensheim, Germany). Split blastocysts were cultured singly in 50μL drops of CR1aa medium at 39°C under 5% CO2 in a moisture-saturated atmosphere. After 1 and 2h culture, the morphology was assessed by judging the shape of the embryos and re-development of the blastocoel. On Day 8 (after 22h culture), the shape of the blastocysts, development of the ICM, blastocoel, proportion of degenerated cells and embryos and re-expanded blastocysts were recorded. Embryos were stained with propidium iodide and Hoechst 33258 for cell counting. The re-expansion status of Group A (50, 60 and 70%) and B (50, 40 and 30%) embryos after 1 and 2h and their quality after 22h culture (1: excellent=<10% degenerated cells, well-defined ICM; 2: fair=<20% degenerated cells) are shown in Table 1. With regards to Group A split blastocysts, a higher (P<0.05) percentage of embryos that re-expanded after 1 and 2h and which yielded Quality 1 and 2 embryos suitable for embryo transfer was observed with the 60% and 70% than with demi-embryos. There were significant differences (P<0.05) between all split blastocysts in Group B after 1h culture. The 30% split embryos showed the lowest re-expansion rate and quality of embryos after 2h and 22h culture, respectively. No differences (P<0.05) were seen in the ratio of the ICM to the total number of cells in both Group A and B. This study showed that the ratio in which blastocysts were split had a significant effect on re-expansion and quality but not on the number of cells.

Table 1