bluuebunny/crossref_metadata_2025 huggingface dataset:
210INCREASE IN OVULATION RATE AFTER IMMUNIZATION OF MALPURA EWES AGAINST
A SYNTHETIC PEPTIDE SEQUENCE OF THE ±-SUBUNIT OF BOVINE INHIBIN
Authors: Naqvi S.M.K. , Palta P. , Joshi A. , Gulyani R. , Paul V. , Maurya V.P. , Manik R.S.
Published: February 2004

Unlike many other breeds of sheep (e.g. Boroola, Romney or Merino) which have high fecundity, the Malpura ewe, an Indian breed of sheep, is marked by an ovulation rate of one and a low incidence of twinning. Active immunization against a number of inhibin-based synthetic peptides has been reported to increase ovulation rates in these high fecundity breeds of sheep. The objective of the present study was to explore the possibility of increasing ovulation rates in Malpura ewes by active immunization against a synthetic peptide replica of the N-terminal sequence of the bovine inhibin. Adult Malpura ewes (n=5) were actively immunized against a synthetic peptide that corresponded to the N-terminus of the α-subunit of bovine inhibin [bIα(1–29)Tyr30]. The peptide was conjugated to ovalbumin, with a peptide-to-ovalbumin ratio of around 20 moles mole−1, to increase its antigenicity. Control ewes (n=5) were immunized against ovalbumin. On the day of primary immunization, 400μg of peptide-ovalbumin conjugate or ovalbumin were dissolved in 1mL of isotonic saline, emulsified with an equal volume of Freund’s complete adjuvant and injected at four sites in each ewe. Following this, boosters 1, 2 and 3 were given on Days 28, 56 and 84, respectively, of the experiment (Day 0=day of primary immunization); boosters were 200μg of peptide-ovalbumin conjugate or ovalbumin dissolved in 1mL of isotonic saline and emulsified with an equal volume of Freund’s incomplete adjuvant. Estrus was synchronized by a double injection schedule of PGF2α (7.5mg Lutalyse, once each on Days 35 and 45). The animals were subsequently allowed to undergo normal cyclicity until the end of the experiment. Ovulation rate was determined by counting the number of corpora lutea observed during laparoscopic examinations approximately 5 days after estrus during three estrous cyles following treatment. The ovulation rate between control and immunized groups was compared by repeated measures ANOVA. Immunization of the Malpura ewes against the synthetic peptide sequence of the α-subunit of bovine inhibin [bIα(1–29)Tyr30] increased ovulation rate over 5-fold compared to that of controls (Table 1). In conclusion, we have shown that inhibin-based fecundity vaccines have the potential of increasing ovulation rates in the Malpura breed of sheep.

Table 1
211QUALITY CHANGES IN BLUE WILDEBEEST (CONNOCHAETES TAURINUS) EPIDIDYMAL SPERMATOZOA MAINTAINED AT 4°C
Authors: Olivier F. , Spies T. , Martinez-Pastor F. , Barry D.M. , Bartels P.
Published: February 2004

Wildlife management in southern Africa often involves the harvesting of animals on ranches and reserves, providing unique opportunities to collect and assess the quality of epididymal spermatozoa for possible future conservation actions. The black wildebeest (Connochaetes gnu) is facing renewed threats to its survival, including the production of fertile hybrids from crossing with the more common blue wildebeest (Connochaetes taurinus). The close relationship between the two wildebeest species allows for the blue wildebeest to be used as a model to assess epididymal sperm quality over time while maintained at 4°C. Field conditions often preclude the immediate availability of liquid nitrogen, necessitating the development of alternative short-term storage methods. All chemicals were provided by Sigma (South Africa) unless otherwise stated. Testes were harvested from 6 blue wildebeest bulls at a local game farm, Savannah, and kept at 5°C during transportation to the lab. Epididymides were dissected out and spermatozoa were flushed out of the cauda epididymis using 1mL of Tris-citrate egg yolk extender (Fraction A, Biladyl, Minitub, Germany), followed by storage at 4°C and assessment at 12h intervals. At each interval, an aliquot was removed, washed with a modified buffered HEPES solution (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH;; 400mOsm/kg, pH 7; Sigma) and visually assessed with a phase contrast microscope (×200, at 37°C) to determine the percentage of motile (MS) and progressive motile (PS) spermatozoa. In addition, plasma membrane integrity (PMI) was assessed with eosin-nigrosin staining and active mitochondrial status (MIT) assessed with an epifluorescent microscope (×400) using the fluorescent probe JC-1 (Molecular Probes, The Netherlands;; 7.5μM; 30min at 37°C). Resilience to hypo-osmotic shock was also evaluated by incubating the sample in a modified hypo-osmotic medium (100mOsmkg−1; 15min RT), and staining with PI to assess plasma membrane integrity (HOSPMI). A summary of results is presented in the table 1. The MS, MIT and HOSPMI did not decrease significantly during the 48h storage period. The only parameters that showed a significant decrease were PS and PMI (P<0.01, Kruskall-Wallis test). However, PMI showed a slow but steady decrease (13%), whereas PS underwent a significant drop (52%). In conclusion, epididymal spermatozoa from the blue wildebeest, kept at 4°C for 48h, may still be useful for some assisted-reproduction techniques. The use of spermatozoa from a common but closely related wildebeest species allows for the development of assisted-reproduction techniques that may one day aid the conservation of threatened wildebeest species. Additional research is needed to confirm these findings and to test the effect of longer storage times on spermatozoa of this species as well as closely related endangered species.

Table 1
Parameters measured during the 12-h time periods (mean±SD)
215SUCCESSFUL OUT-OF-BREEDING SEASON ESTRUS SYNCHRONIZATION FOLLOWED BY
FIXED TIME INSEMINATION IN WATERBUFFALO (BUBALUS BUBALIS)
Authors: Züge R.M. , Rodacki U. , Grandi A.T. , Aerts J.M.J. , Bols P.E.J.
Published: February 2004

The most important barrier to the increase of buffalo productivity is an overall poor reproductive efficiency, characterized by late sexual maturity, seasonal anestrus and long periods of postpartum ovarian inactivity resulting in extended calving intervals and poor expression of estrus behavior (Singh J et al., 2000, Anim. Reprod. Sci. 60–61, 593–604). Buffaloes are seasonal breeders with the highest reproductive activities during winter (short day lengths) and a high frequency of anestrus during the summer months (Singh G et al. 1985, Ind. J. Anim. Res. 19, 57–60). Recent research demonstrated that a combination of progesterone, estradiol benzoate and equine chorionic gonadotropin (eCG) was effective for estrus induction and synchronization in buffalo heifers under Mediterranean conditions (Barile et al. 2001, Livestock Prod. Sci. 68, 283–287). The aim of the present study was to investigate the impact of an estrus synchronization protocol on reproductive efficiency of water buffalo during out of the normal breeding season. A total of six heifers (21 to 23 months of age) and three cows (5, 6 and 18 years of age) were enrolled in an estrus synchronization protocol lasting for 12 days. All animals were kept under tropical conditions in the coastal part of Paraná (Antonina), about 450km south of São Paulo. The experiment was performed in December, 2002, during the Brazilian summer season, when reproductive efficiency of buffaloes is greatly reduced. On the first day of the protocol (Day 0), animals were implanted with an intravaginal device containing 1g of progesterone (DIB, Syntex SA, Buenos Aires, Argentina) and injected with 10mg estradiol benzoate (Estrogin, Famavet, São Paulo, Brazil). On Day 9, the DIB implant was removed and the animals received 150μg (i.m.) of cloprostenol (Prolise, Syntex SA, Buenos Aires, Argentina) and 2500 IU of eCG (Novormon, Syntex SA, Buenos Aires, Argentina). On Day 11, all animals received 1500 IU of hCG (Vetecor, Lab. Calier, Spain). Artificial insemination (AI) was performed on Day 12 using frozen-thawed semen from a bull of proven fertility. Only one AI was performed per heifer/cow. Pregnancies were determined by ultrasound examination at 53 days following AI and confirmed by rectal palpation at 90 days post AI. The use of this estrus synchronization protocol, followed by fixed-time insemination, resulted in four pregnant heifers (66%) and three pregnant cows (100%). Our results demonstrate that buffalo reproduction can be successful during out-of-breeding season when adequate hormonal treatment is used. Additional experiments should be done to validate the protocol.
218ULTRASOUND-GUIDED TRANSVAGINAL INJECTION OF A LOW DOSE OF FSH-LH INTO
THE BOVINE OVARY AS AN ALTERNATIVE WAY TO STIMULATE FOLLICULAR GROWTH:
PRELIMINARY RESULTS
Authors: Bols P.E.J. , Vanholder T. , Leroy J.L.M.R. , Aerts J.M.J. , Van Soom A.
Published: February 2004

While some researchers claim a positive influence of FSH on the number of punctured follicles and retrieved oocytes in stimulated cows (Looney CR et al. 1994 Theriogenology 41, 67–72), others found comparable results between protocols with one stimulated v. two unstimulated ovum pick-up (OPU) sessions a week (Stubbings RB and Walton JS 1995 Theriogenology 43, 713–721). The use of FSH/LH causes explosive follicular growth and a substantial increase in ovarian blood supply when given at the superovulation dose. These drawbacks limit the repeated use of FSH stimulation prior to OPU in a twice per week puncture protocol. The aim of this experiment was first, to investigate the feasibility of injecting FSH directly into the ovary at a substantially lower dose and, second, to study the local effects of intra-ovarian FSH injection on follicular dynamics. Six donor cows with normal reproductive tracts were prepared as for transvaginal OPU (Bols et al. 1995 Theriogenology 43, 677–687). The ovary was visualized using a Multiple Angle 5MHz ultrasound transducer (Pie Medical, Maastricht, The Netherlands), fitted next to a needle guidance system in an OPU handle. A vial of freeze-dried FSH/LH (Stimufol, 500μg porcine FSH and 100μg porcine LH, Ulg FMV, Liège, Belgium) was restored and diluted with saline to a solution containing 10μg pFSH and 2μg pLH per mL. A total dose of 160μg pFSH and 32μg pLH, divided over 8 injections of 1mL diluted FSH/LH in each ovary, was given to 4 animals by transvaginal ultrasound-guided injection with a 19-gauge needle. The ovaries of one negative control cow were injected with 1mL saline using the same protocol. The sixth cow received a classical FSH stimulation as prescribed by the manufacturer by means of i.m. injections at a total dose of 500μg FSH and 100μg LH as a positive control. During the first session, all follicles with a diameter of >5mm were aspirated, while prior to each injection (morning and evening), ovarian activity was checked by ultrasound examination and taped on video for all cows. Intra-ovarian FSH injection was successful since, in most cases, a small echographically dense area was seen during and immediately following injection. This area moved around following displacement of the ovary, indicating intra-ovarian disposition of the FSH. Following the four-day stimulation treatment, the average (±SD) number of follicles with a diameter >5mm was 5.5±4.2 on the ovaries of intra-ovarian injected cows, 21 in the case of the positive control cow and only 1 follicle in the negative control. These results suggest that transvaginal, ultrasound-guided injection of a low FSH/LH dose directly into the ovary might be an alternative way for ovarian stimulation prior to OPU. Additional dose-titration experiments are ongoing.
224FOLLICULAR GROWTH IN SHEEP SUPEROVULATED WITH FSH AFTER
PRE-TREATMENT WITH GNRH ANTAGONISTS
Authors: Lopez-Alonso C. , Encinas T. , Veiga-Lopez A. , Garcia-Garcia R.M. , Ros J.M. , Gonzalez-Añover P. , Cocero M.J. , Gonzalez-Bulnes A.
Published: February 2004

In sheep, the injection of a single dose of 1.5mg of the GnRH antagonist Teverelix (Antarelix™, Zentaris, Frankfort, Germany) eliminates large dominant follicles and increases the number of smaller follicles (2–3mm in size) in a short period of time (Lopez-Alonso et al., 2003. Reprod. Abstr. Ser., 30:71). This treatment would be beneficial for increasing the efficiency of ovarian stimulatory protocols, since embryo output is enhanced in the presence of a high number of small follicles, coincidentally with the absence of large follicles, at starting the FSH treatment. However, possible effects of this single high dose of GnRH antagonist on the capacity of follicles to develop in response to FSH treatments has not been determined. In this study, we have characterized patterns of follicular development during a superovulatory treatment with purified ovine FSH (Ovagen™, ICPbio, Auckland, NZ) in sheep treated i.m. with 1.5mg of Antarelix (n=6) or saline (n=4) on Day 9 after the insertion of a progestagen sponge (Chronogest®, Intervet Int, Boxmeer, The Netherlands). Ewes were superovulated with eight decreasing doses (1.5×3, 1.25×2 and 1×3mL) of Ovagen™ injected twice daily from Day 12 of sponge insertion. Number and size of all >2-mm follicles was determined by transrectal ultrasonography just prior to every FSH injection from the first dose to the withdrawal of progestagen sponges. At the start of the gonadotrophin treatment on Day 12, ewes treated with GnRH antagonist showed a higher number of 2–3mm follicles than control ewes (16.2±3.8 v. 5.3±0.3, P<0.05), and a lower number of >4mm follicles (2.2±0.5 v. 5.0±0.6, P<0.01). Thereafter, administration of Ovagen™ induced a significant rise in the number of >4mm follicles at sponge removal in both groups (P<0.0005 for treated ewes and P<0.01 for controls). This number was higher in females treated with GnRH antagonist than in control sheep, although differences did not reach statistical significance (19.3±3.8 v. 12.7±5.4). Current data confirm that administration of a single dose of GnRH antagonist decreases presence of large follicles and increases the number of smaller follicles at the first day of FSH injection. This pre-treatment does not affect competence of follicles to grow in response to superovulatory FSH treatments. Moreover, the number of preovulatory follicles at sponge removal was higher than in untreated ewes. We conclude that the pre-treatment with a single dose of 1.5mg of Antarelix™ on Day 9 of sponge insertion, three days before starting the FSH treatment in a superovulatory protocol, might increase the ovarian response and, thereafter, the yields of follicles.
226HEAT SHOCK OF GERMINAL VESICLE-STAGE BOVINE OOCYTES
REDUCES EMBRYO DEVELOPMENT
Authors: Payton R.R. , Saxton A.M. , Lawrence J.L. , Edwards J.L.
Published: February 2004

Culture of germinal vesicle (GV)-stage oocytes at an elevated temperature occurring in heat-stressed dairy cattle reduced ability of oocytes to progress to metaphase II after resumption of meiosis (Payton RR et al., 2003 Biol. Reprod. 68, 343 abst). The objective of this study was to evaluate embryo development of oocytes heat-shocked at GV stage. To prevent cumulus-oocyte complexes from resuming meiosis after removal from follicles, oocytes were cultured in roscovitine (cell cycle inhibitor of p34cdc2/cyclin B kinase;; 50μM) for 24h (McCann LM et al., 2000 Biol. Reprod. 64, 141 abst and Payton RR et al., 2003 Biol. Reprod. 68, 343 abst) showed that roscovitine is effective for maintaining >90% of oocytes at GV-stage in a reversible manner. Germinal vesicle-stage oocytes were cultured at 38.5°C for 24h (experimental control) or 41.0°C as follows: HS 0–6 (41°C for 6h, 38.5°C for 18h), HS 0–12 (41°C for 12h, 38.5°C for 12h), HS 12–24 (38.5°C for 12h, 41°C for 12h), HS 18–24 (38.5°C for 18h, 41°C for 6h), or HS 0–24 (41°C for 24h) in 5.5% CO2 and humidified air. In addition, a group of COC were not cultured in roscovitine but placed in maturation medium (lab control). After a total of 24h, COC were washed extensively of roscovitine and cultured for an additional 24h in maturation medium. Oocytes presumed mature were fertilized with Percoll-prepared frozen-thawed semen. Presumptive zygotes were cultured in KSOM containing 1X nonessential amino acids in 5.5% CO2, 7% O2, and 87.5% N2 at 38.5°C in humidified air. Cleavage and development to blastocyst were recorded on Days 3 and 8 post-insemination, respectively. Data were collected in 7 replicates and analyzed as an incomplete block using mixed models of SAS (2000) after testing for normality. Use of roscovitine for maintaining oocytes at GV-stage for 24h did not alter cleavage (80.5 and 73.4%; SEM=5.8; lab and experimental controls), development to 8–16 cell (50.4 and 52.6%; SEM=4.6; lab and experimental controls), or blastocyst (29.7 and 24.8%; SEM=3.2; lab and experimental controls) stages. Culture of GV-stage oocytes at 41°C for up to 24h did not increase lysis (8.0–11.1%; SEM=2.7). Heat shock of GV-stage oocytes for as few as 6h reduced the proportion developing to 8–16 cell stage after release from inhibitor (Table 1). When experimental control and HS 0–6 were pooled for comparison to HS 0–12, effects of heat shock for reducing development to blastocyst were noted (P<0.005). Moreover, negative effects of heat shock for reducing developmental competence of GV-stage oocytes increased as duration of heat shock increased (linear contrast;; experimental control, HS 0–12, and HS 0–24; P<0.04). Results indicate that a physiologically relevant elevated temperature for as few as 6h compromises continued development of GV-stage oocytes. Seasonal depressions in fertility of heat-stressed cattle may be due in part to direct effects of elevated temperature on GV-stage oocytes.

Table 1
227OVARIAN FOLLICULAR DYNAMICS IN POSTPARTUM MEDITERRANEAN
ITALIAN BUFFALOES
Authors: Presicce G.A. , De Santis G. , Senatore E.M.
Published: February 2004

In cattle and buffaloes resumption of ovarian activity, as expressed by first post partum ovulation and follicle turnover leading to periodic estrus cycles and ovulations, can be affected by many factors among which uterine infections and dietary supplementation play a leading role. In addition, buffaloes are considered seasonal animals characterized by a tendency to reproduce in correspondence to a decrease of day length. This aspect may further affect their reproductive efficiency by delaying or interrupting ovarian activity if calving occurs in transitional (i.e., from end winter into beginning of spring) or unfavourable (spring-summer) periods of the year, when an increase of daylight hours occurs. The aim of this study, carried out in the months of February to May, was to understand the effect of increasing daylight hours over resumption of postpartum ovarian follicular dynamics and estrus cycles. Mixed parity Mediterranean Italian buffaloes (n=20) were monitored daily by ultrasonography from calving until first postpartum ovulation and following cycle or until 60 days if no ovulations had been recorded. Ultrasound monitoring was performed by using a 7.5MHz linear-array probe and an Aloka SSD-500 monitor. No pharmacological intervention was allowed during the course of the study and animals were kept under similar feeding and farm management conditions. All animals exhibited a mild to moderate uterine inflammation (n=13; 65%) or aspecific infection (n=7; 35%) after calving. Infections were manifested as purulent discharge and as ultrasound detection of highly echogenic debries in the uterine lumen. Only one animal retained pathological accumulation in both horns for the entire duration of the study. Among the 20 buffaloes under study, 12 (60%) exhibited postpartum ovulations, ranging from 9 to 45 days. In eight buffaloes (40%), such ovulations occurred in the ovary contralateral to the ovary bearing the gravidic CL. Following postpartum ovulations, seven buffaloes (35%) underwent a complete cycle leading to ovulation. Three of such cycles (15%) culminated with a second ovulation in the same ovary where post partum ovulations had occurred. Among the full cycles following postpartum ovulations, four of them were one-wave, with a range of 8 to 19 days, whereas the remaining three cycles were two-wave with a range of 17 to 22 days. Eight buffaloes (40%) were reported in anovulatory condition following parturition, although in four of them both ovaries displayed a typical follicle turnover. In conclusion, resumption of ovarian activity and reproductive efficiency in water buffaloes can be affected by increasing day length as evidenced by a high rate of postpartum anovulatory conditions and reduced follicle turnover.
23REPROGRAMMING MAMMALIAN CELLS IN XENOPUS EGG EXTRACTS: ROLE OF EMBRYONIC LAMIN B3
Authors: Alberio R. , Campbell K.H.S.
Published: February 2004

The generation of animals by nuclear transplantation has demonstrated that a fully differentiated cell can be reversed into totipotency when transferred into an oocyte. Identification of oocyte specific molecules responsible for the reprogramming of somatic cells may contribute to the understanding of cell differentiation and embryo development. We have developed a heterologous system to investigate the effect of lamin B3, a major component of Xenopus laevis egg cytoplasm, on DNA replication of mammalian somatic cells. Bovine fetal fibroblasts were arrested at G1/S by incubation in aphidicolin for 18h. After permeabilization with digitonin, the cells were incubated in either (1) lamin B3 depleted, or (2) whole Xenopus egg extracts (1000 cells μL−1 extract) supplemented with an energy regenerating system for a period of 3h at 21°C. Xenopus lamin B3-depleted egg extracts were prepared by three rounds of incubation with Dynabeads coated with a mouse monoclonal lamin B3 antibody (mAbLB3). Immunodepletion was confirmed by western blotting. Purified lamin B3 was obtained by dialysis of the beads after immunodepletion, and the purified lamin B3 was used for rescue experiments. DNA replication of cells incubated in the extracts was assessed by adding 25μM Biotin-11-dUTP for 3h. After treatment cells were fixed in 70% methanol at −20°C and incubated in mAbLB3 for 30min at 37°C. This was followed by incubation in FITC-conjugated sheep anti-mouse antibody and in 5mgmL−1 Texas Red-conjugated Streptavidin for 40min at 37°C. After three hours’ incubation in egg extracts, DNA replication was detected in 60% of cells and more than 95% of cells were lamin B3 positive. In contrast, DNA replication in immunodepleted extracts was significantly lower (P≤0.01, by one-way ANOVA) than in cells incubated in whole extracts and was coincident with the few lamin B3-positive cells observed. More than 95% of cells were lamin B3-negative and did not replicate DNA. When purified lamin B3 was re-added to depleted extracts, DNA replication was detected in 60% of cells. DNA synthesis resumed in 93% of control cells 3h after release from aphidicolin into culture medium at 39°C. These experiments show that somatic nuclei, which possess a nuclear envelope with somatic variants of lamins, are able to synthesize DNA in egg extracts only when Xenopus lamin B3 is incorporated into the nuclear envelope. This heterologous system provides new information on the role of an embryonic molecule, namely Xenopus lamin B3, in the reprogramming of DNA replication of somatic cells incubated in egg environment. These results open new questions as to whether embryonic lamins also exist in mammals, and whether failure in development of cloned animals is in part due to abnormal or incomplete replacement of somatic variants of proteins with their embryonic counterparts.
231EXPRESSION PATTERN OF CERTAIN DEVELOPMENTALLY IMPORTANT
GENES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING CELL LINES OF
DIFFERENT EFFICIENCY
Authors: Beyhan Z. , First N.L.
Published: February 2004

Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this respect is the analysis of gene expression patterns in nuclear transfer embryos to dissect the processes that failed and to develop means to overcome the limitations imposed by these factors. In this study, we investigated the expression patterns of histone deacetylase-1,-2,-3 (HDAC-1,-2,-3), DNA methyltransferase-3A (DNMT3A) and octamer binding protein-4 gene (POU5F1) in donor cells with different cloning efficiencies (low: no-pregnancy, medium: pregnancy but no live birth and high: live birth) and nuclear transfer embryos derived from these cell lines using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay with SYBR green chemistry. Employing standard protocols, we produced nuclear transfer embryos from three different cell lines categorized as having varying efficiencies in supporting development to term. Embryos were collected at morula, blastocyst and hatched blastocyst stages and total RNA was extracted from pools of 4–5 embryos using Absolutely RNA nanoprep kit (Stratagene, La Jolla, CA, USA). Relative level of expression at these stages was analyzed using ΔΔCT method with HH2A as the reference gene and in vitro-fertilized embryos as the control samples. Statistical analysis was performed on ranked expression data employing SAS statistical analysis software procedure ANOVA. Same set of genes were also analyzed on donor cells using standard curve method. All genes investigated were affected by nuclear transfer and followed somewhat altered expression patterns. In general, expression of HDAC genes was elevated especially at the compact morula stage but became comparable to control embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than in IVF embryos at all stages. POU5F1 transcript levels were also reduced in nuclear transfer embryos at the compact morula and blastocyst stages. The difference, however, disappeared at the hatched blastocyst stage. There was a cell line effect on the expression patterns of all genes investigated. Cell lines efficient in producing offspring tended to resemble control embryos in gene expression patterns compared to inefficient cell lines. These results agree with several studies reporting altered gene expression patterns for certain genes in cloned embryos. Our data also suggest that cell line differences in developmental competency observed in cloning experiments might be related to physiological differences in transcriptional regulation and nuclear remodeling, DNA methylation, and lineage differentiation in embryos cloned from these cell lines.
232SEARCH FOR THE BOVINE HOMOLOG OF THE MURINE PED GENE
Authors: Fair T. , Gutierrez-Adan A. , Murphy M. , Rizos D. , Martin F. , Boland M.P. , Lonergan P.
Published: February 2004

The aim of the current study was to identify the bovine homolog of the murine Ped (preimplantation embryo development) gene, which regulates mouse preimplantation embryonic growth, including cleavage rate and embryo survivability, and to characterize the expression pattern of this gene during bovine preimplantation embryo development. Experiment (I): The National Center for Biotechnology Information (NCBI) GenBank/EBI EST databases were searched for bovine-expressed sequence tags (EST) that were homologous with the murine Ped gene (Accession number: NM_010394). The resulting ESTs were aligned and assembled in to one complete sequence (841bp), which was shown to be homologous with the Murine Ped gene and the Bovine Major Histocompatibility Complex class I 4221.1 gene (Accession No.: AJ010865, length 1090bp). The expression of the protein product of the Ped gene by bovine tissue was confirmed using Western Blot analysis. Experiment (II): The expression pattern of the Ped gene homolog during in vivo and in vitro bovine preimplantation embryo development was characterized using real time PCR. Embryos at the same stage for age were compared (Day 1: 2-cell; Day 2: 4-cell; Day 3: 8-cell; Day 4: 16-cell; Day 5: early morula;; Day 6, compact/late morula;; Day 7: blastocyst). The relative transcript abundance was consistently lower in the in vitro-cultured embryos at all stages of preimplantation development the differences were significant on Days 2, 4, 5, 6, and 7. The relative transcript abundance was significantly lower on Days 1, 2, and 3 of in vivo culture than on Days 4, 5, 6, and 7 and was significantly higher in Day 7 blastocysts than in Day 5 early morula. In in vitro-cultured embryos the relative transcript abundance was significantly higher in Day 7 blastocysts compared to all other stages of the preimplantation period. Experiment (III): A quantitative analysis of the Ped gene homolog was carried out on replicates of pools of ten 2-cell embryos collected at 25, 28, 32, and >36hpi from three different fertilizations. Transcript relative abundance was highest in those embryos that had cleaved by 25hpi. By 28hpi abundance had decreased slightly;; as time to cleavage increased further to 32 and >36hpi, the relative abundance decreased significantly. In conclusion, we have successfully identified a potential bovine homolog of the murine Ped gene. Furthermore, we have characterized the expression pattern of this gene during preimplantation embryo development in cattle and we have shown that a greater relative abundance of the gene transcript is associated with embryos of higher quality and greater developmental potential.
234IMPRINTING STATUS OF DEVELOPMENTALLY IMPORTANT GENES IN
BOVINE PREIMPLANTATION EMBRYOS
Authors: Gebert C. , Wrenzycki C. , Herrmann D. , Lucas-Hahn A. , Carnwath J.W. , Niemann H.
Published: February 2004

Several imprinted genes have been identified in different species such as mouse and man but in cattle imprinting has only been confirmed for the Insulin-like growth factor 2 receptor gene (Igf2r). DNA methylation is one of the most common mechanisms of imprinting. Imprinting is correlated with the methylation of normally unmethylated CpG islands, and aberrations in methylation are thought to be involved in the Large Offspring Syndrome (LOS) which is frequently observed in offspring derived from in vitro-produced and/or cloned embryos. The imprinting of the bovine Insulin-like growth factor 2 gene (Igf2), the Igf2r gene and the Mammalian achaete scute homologue 2 gene (Mash2) was analyzed in bovine preimplantation blastocysts cultured in SOF+BSA. mRNA levels of the Igf2r gene and the Mash2 gene in in vitro-produced (IVP) and parthenogenetic expanded single blastocysts were analyzed by semi-quantitative RT-PCR (Wrenzycki C et al., 2001 Biol. Reprod. 65, 309–317). Genes expressed exclusively from the maternal allele (i.e. paternally methylated) should be represented by a higher relative abundance of transcriptional products in parthenogenetic embryos whereas paternally
expressed genes (i.e. maternally methylated) should be correlated with a higher gene expression in IVP embryos carrying one paternal and one maternal allele. For determination of methylation patterns by bisulfite sequencing, sequences of several DNA fragments from the bovine Igf2 gene were identified in samples from bovine uterus and kidney. The primer pairs were generated from the ovine and bovine Igf2 sequences available in the Genebank database (accession number U00664/X53553). DNA fragments identified were from the 5′ untranslated region and the 3′ translated region of the bovine Igf2 gene. All fragments consist of a high number of CG dinucleotides, and computer analysis using the CpGwin program (Anbazhagan R et al., 2001 BioTechniques 30, 110–114) revealed CpG islands within these fragments. Relative abundances of transcriptional products were statistically analyzed using the SigmaStat 2.0 (Jandel Scientific, San Rafael, CA, USA) software package. After testing for normality, an ANOVA followed by multiple pairwise comparisons using the Tukey test was employed. Results from the semi-quantitative RT-PCR analyses revealed no difference (P>0.05) in gene expression pattern of the Igf2r gene, suggesting that the Igf2r gene is
biallelically expressed during bovine preimplantation development. In contrast, there was a significant difference (P<0.05) in the relative abundance of mRNA of the Mash2 gene, with a lower relative abundance of transcriptional products in parthenogenetic expanded blastocysts. Thus, in cattle as reported in mice (Guillemot F et al., 1995 Nat. Genet. 9, 235–242), the paternal allele of the Mash2 gene seems to participate in the expression of the gene prior to implantation.
236LOCALIZATION OF INTERFERON-TAU IN BOVINE EMBRYOS AND CUMULUS CELLS BY
CONFOCAL MICROSCOPY
Authors: Johnson K.M. , Alvarez X. , Kubisch H.M.
Published: February 2004

Interferon-tau (IFN-t) is a protein secreted by the conceptus of ruminant species and thought to be the primary signal in maternal recognition of pregnancy. Experiments were conducted to detect IFN-t in bovine oocytes, cumulus cells and embryos by use of immunocytochemistry and laser-scanning confocal microscopy. Embryos were produced by in vitro fertilization of in vitro-matured oocytes. Oocytes and embryos were fixed in formaldehyde at various stages of development, and stored in PBS until staining and microscopy. Cumulus cells were stripped from immature oocytes and cultured in M-199 (10% fetal calf serum) on coverslips treated with poly-D-lysine. They were divided into four treatment groups: (1) without hormones (control), (2) with the addition of FSH, (3) estradiol, or (4) FSH and estradiol. Bovine MBDK cells (ATCC CCL 22) and primary fibroblasts were cultured as controls on coverslips but without addition of hormones. A polyclonal antibody raised against bovine IFN-t was used, followed by a secondary conjugated antibody (AlexaFluor 488, Molecular Probes, Eugene OR). Actin was stained with phalloidin (AlexaFluor 568, Molecular Probes, Eugene OR). Cumulus, MDBK cells and fibroblasts were further stained with propidium iodine to visualize nuclei. Imaging was performed on a Leica laser-scanning confocal microscope. IFN-t was detected in hatched and unhatched Day 7 and Day 9 blastocysts, where its expression was restricted to the trophectoderm. IFN-t was also found in Day 5 and 6 morulae, but not at earlier stages. Furthermore, IFN-t was detected in the cumulus cell masses of oocytes before and after IVM, but not in the oocyte itself. Controls, in which the primary antibody was omitted, were negative regardless of developmental stage. IFN-t was also found in cultured cumulus cells regardless of whether hormones had been added to the medium;; however, the protein was localized in the nuclei of cells only if they had been cultured with FSH, whereas in cells cultured with estrogen alone or without hormones IFN-t was restricted to the cytoplasm. In contrast, no IFN-t was detected in MDBK cells or fibroblasts. These results extend previous findings by showing that IFN-t is expressed as early as the morula stage. Moreover, these results demonstrate that IFN-t is also produced by cumulus cells where FSH appears to initiate a translocation of IFN-t into the nucleus, suggesting a role in regulation of gene expression.
246IMPROVEMENT OF THE DEVELOPMENTAL CAPACITY OF OOCYTES FROM PREPUBERTAL
CATTLE BY INTRAOVARIAN IGF-I APPLICATION
Authors: Oropeza A. , Hadeler K.G. , Herrmann D. , Wrenzycki C. , Niemann H.
Published: February 2004

The developmental potential of oocytes from prepubertal cattle is lower compared to their adult counterparts. Differences between oocytes from calves and cows have been found with regard to size, ultrastructural characteristics and metabolism. GH and IGF-I receptor mRNA have been identified in the cumulus cells and in oocytes (Izadyar F et al. 1997 Mol. Reprod. Dev. 47, 175–180; Armstrong D. et al., 2002 Reproduction 123 (6), 789–797). It is known that IGF-I and GH affect mRNA expression of glucose transporter 1 (Glut1) as well as glucose uptake by embryos. The goal of this study was to improve the developmental capacity of oocytes from prepubertal cattle and to determine whether an induced increase of GH and IGF-I levels affects the mRNA expression pattern of Glut1, UBF and eIF1A. Holstein calves (n=30), 6–7 months old, were randomly divided into three groups. One group received a single s.c. injection of 500mg of somatotrophin (rbST Posilac, Monsanto Company, St. Louis, MO, USA) and during the subsequent 2 weeks their follicles were aspirated 4 times via transvaginal ultrasound-guided ovum pick-up (OPU). The second group received an intraovarian injection of 6μg rhIGF-I (R&D Systems, Wiesbaden-Nordenstadt, Germany). The third group served as control and received an intraovarian injection of 0.6mL of 10mM acetic acid. All animals were i.m. injected with 60mg FSH (Folltropin®; Vetrepharm Inc., Ontario, Canada) 48h prior to aspiration. The treatments were repeated with the same animal groups at the ages of 9–10, 11–12 and 14–15 months. Five adult cows were i.m. injected each with 100mg FSH. For mRNA expression analysis (RT-PCR), embryos were collected with 2–4 cells, 8–16 cells and as blastocysts. The relative abundance of mRNA for Glut 1 and eIF1A was higher (P<0.05) in 8–16 cell embryos from IGF-I-treated calves and cows than in control and rbST treated calves. The in vitro embryo production (IVP) results are shown in the Table 1. Our data show that IGF-I increased mRNA expression of Glut1 and eIF1A which may improve the developmental capacity of embryos produced from calves.

Table 1
OPU/IVP-Results for calves (6–15 months old) in comparison with cows
259PRODUCTION OF TROPICAL DAIRY CALVES BY EMBRYO TRANSFER USING
LOCAL LAISIND (BOS INDICUS) RECIPIENTS AND INTERCONTINENTAL FRESH
IVF SHIPPED EMBRYOS
Authors: Nguyen B.X. , Uoc N.T. , Ty L.V. , Monson R.L. , Leibfried-Rutledge M.L. , Duc N.H. , Bui L.C. , Huu Q.X. , Hanh N.V. , Thanh N.T. , Linh N.V. , Rutledge J.J.
Published: February 2004

Increasing the diary population and milk production is a goal of many tropical developing countries. We report in this paper an attempt to develop a system of intercontinental shipping for transfer of fresh crossbred Bos taurus×Bos indicus IVF embryos into local Laisind (Bos indicus) recipients as a way to produce tropical dairy calves with highly improved milk productivity. The production of embryos was done at BOMED, Inc (Madison, WI, USA). Oocytes collected from ovaries of Holstein (Bos taurus) at slaughter and semen from milking Gir (Bos indicus) were used for IVP. Cleaved embryos were selected for air shipping in portable incubators at Day 4 (Group 1), Day 3 (Group 2) or Day 2 (Group 3) after IVF. The duration of shipping varied from 60 to 65h. Embryo transfer was done in Vietnam. Laisind cows (Yellow cattle×Red Sindhi) with body weight more then 280kg and normal reproductive activity were selected for treatment of estrous synchronization with double 11-day interval injection of PG2α (Intervet, Boxmeer, The Netherlands) and single injection of eCG (SABC Vietnam) two days before the second injection of PG2α. Timing of injections was calculated according to the IVF schedule. Embryos collected from portable incubators were transferred to a CO2 incubator for further culture at 39°C. Two experiments were carried out: (1) transfer of embryos without sexing;; (2) transfer of embryos after biopsy and sex determination by PCR. In experiment 2, compact morulae or morula-blastocysts were selected for sex determination. Four to five blastomeres were aspirated from each embryo using a cutting pipette and an aspiration pipette of 30-μm diameter. PCR was done as previously described (Uoc et al., 1999 J. Biology). After biopsy, embryos were kept in culture for one day to observe the development in vitro. Embryos developed to morula-blastocyst or hatching blastocyst at Day 7 or Day 8 after IVF were transferred nonsurgically to recipients with estrus detected in the period from 0 to 12h before or after the starting IVF. Pregnancy was confirmed by rectal palpation 3 months after embryo transfer. The average rate of embryos developed into morula-blastocyst was more then 50% (Table 1) and there were no significant differences among different shipping groups. For experiment 2, more then 87 % of embryos biopsied developed in vitro to expanding and hatching embryos. The average rate of female embryos was 56.3%. The pregnancy rate at 3 months was more then 44% (n=188). The first group of calves was born without unusual birthing problems. In conclusion, the system of embryo transfer using intercontinental shipping of fresh IVF embryos and local Bos indicus recipients can be applied for production of dairy calves. Supported by grant from the AIRE-Development agency.

Table 1
Development in vitro of IVP embryos
26ABERRANT REPROGRAMMING OF IMPRINTED GENE EXPRESSION IN ENLARGED
PLACENTAS OF MICE CLONED FROM ES CELLS TREATED WITH TSA OR 5AZAC
Authors: Baqir S.G. , Zhou Q. , Jouneau A. , Renard J.-P. , Betts D.H. , Smith L.C.
Published: February 2004

The success rate of producing cloned animals is very low, and in many cases is associated with the formation of enlarged placentas. Increasing evidence has pointed towards epigenetic deregulation of imprinted genes due to incomplete or abnormal resetting of DNA methylation and/or histone acetylation patterns during development. It has previously been shown that drugs that alter DNA methylation (5AzaC) and histone acetylation (TSA) over-express imprinted genes in mouse ES cells (Baqir and Smith, 2001, Theriogenology 55, 410). Our objective in this study was to determine whether nuclear transfer is able to reprogram imprinted gene expression patterns in the placenta of mice cloned from ES donor nuclei exposed to 5AzaC and TSA. ES donor cells were treated with either TSA or 5AzaC prior to injection into enucleated oocytes. Total RNA was extracted from placentas of day 14–15 fetus clones, and reversed transcribed; the expression pattern of imprinted genes (Ipl, Mash2, Igf2, H19, Igf2r, p57, Peg1), non-imprinted placental-specific genes (Esx1, Dlx3, Tpbp) and a housekeeping gene (Gapdh) was examined by Real Time PCR. Samples were standardized with an exogenous control (Globin) and expressed as fold changes in relation to placentas of cloned fetus derived from non-treated donor cells. Data were analyzed by ANOVA and mean gene expression values were compared using the Tukey-Kramer test. Our results show that several imprinted genes (Mash2, H19, Ipl) and placenta-specific genes (Esx1 and Dlx1) were properly reprogrammed in non-enlarged (71mg) placentas of fetus clones derived from the TSA and 5AzaC treated ES donor cells. Although Gapdh expression did not differ among normal and enlarged 210mg) placenta groups, the expression level of Igf2 and Mash2 was higher in enlarged placentas from fetus clones produced from TSA-treated ES donor cells (4.6 and 3.5 fold) compared to non-enlarged placentas from non-treated ES cells (1 fold). Conversely, oversized placentas from cloned fetuses derived from TSA-treated donor ES cells under-expressed Peg1, H19 and Ipl (0.5, 0.2 and 0.2 fold, respectively) compared to control placentas (1 fold). In addition, enlarged placentas from the TSA- and 5AzaC-treated group displayed down-regulation of placenta specific genes Esx1 and Dlx3 and up-regulation of Tpbp, suggesting the presence of abnormal distribution of placental layers. These results indicate that while several imprinted and non-imprinted placenta specific genes were correctly expressed in normal size placentas of fetus clones derived from TSA and 5AzaC treated donor ES cells, enlarged placentas displayed aberrant gene expression patterns, suggesting that improper resetting of the epigenetic program after nuclear transfer is directly related to altered DNA methylation and histone acetylation patterns. Funded by NSERC & CIHR.
269IN VITRO FERTILITY OF BOAR SPERMATOZOA PRESERVED AT
10^°C FOR 22 DAYS
Authors: Funahashi H.
Published: February 2004

Fertility of boar spermatozoa as determined following artificial insemination seems to be maintained during liquid preservation at 10–15°C for several days, although prolonged liquid preservations reduce the pregnancy rate rapidly. However, it is not clear if spermatozoa can penetrate into oocytes in an IVF system even after a prolonged liquid preservation. Oxidative stress could also be one of the possible detrimental factors in liquid preservation of spermatozoa. In the present study, fertility of liquid-preserved spermatozoa was examined using an IVM-IVF system. Whether cysteine can improve the fertility was also determined. Spermatozoa (from four Berkshires) was resuspended at 1×108 cells mL−1 in Modena solution containing 15% (v/v) boar seminal plasma and 0 or 5mM cysteine after washing 3 times. Sperm suspensions (1mL) were then preserved at 10°C for 22 days following a program for cooling down (to 15°C for 4h, keeping at 15°C for 12h and then to 10°C for 6h). At Days 1, 8, 15 and 22 after the start of preservation, spermatozoa (5×105 cells mL−1) were co-cultured with IVM oocytes in an IVM/IVF system (Funahashi et al., 1997 Biol Reprod 57, 49–53). Viability and functional status of spermatozoa were also examined at Days 8 and 15 of preservation by using LIVE/DEAD sperm viability kit and CTC fluorescence assay. Data (mean±SEM) from 4–6 replicates were analyzed by ANOVA and Fisher’s protected LSD test. When spermatozoa that had been preserved without cysteine (Cys−) were used, penetration rates were not different (P>0.05) from those with cysteine (Cys+) at Day 8 of preservation (91.4±3.4% in Cys− and 99.3±0.7% in Cys+), but lower (P<0.02) at Days 15 and 22 (72.6±13.6% and 33.8±8.4% in Cys−; 94.8±2.1% and 71.1±10.8% in Cys+, respectively). Both viability and proportion of uncapacitated live cells were higher (P<0.05) in Cys+ than Cys− at Days 8 and 15. These results demonstrate that boar spermatozoa can penetrate into oocytes in vitro even after a liquid preservation at 10°C for 22 days and that cysteine can improve the viability and penetrability in vitro of spermatozoa during liquid preservation. Supported by the Ito Foundation.