bluuebunny/crossref_metadata_2025 huggingface dataset:
158APOPTOSIS IN IN VITRO PRODUCED BOVINE EMBRYOS ACCORDING TO
DEVELOPMENTAL KINETICS
Authors: Meirelles F.V. , Schwarz K.L. , Merighe G.K.F. , Carambula S.F. , Watanabe Y.F.
Published: February 2004

Apoptosis has been previously reported in embryos during late pre-implantation development. Fast-developing embryos are known to present higher developmental competence. The aim of the present work was to evaluate the quality of in vitro-produced bovine embryos with fast (8-cells at 48 hours post-insemination (hpi) and slow (8-cells at 90hpi) cleavage and study the correlation of this phenotype with programmed cell death occurrences. Embryos were produced from immature oocytes obtained from slaughtered cow ovaries, after maturation and fertilization, presumed zygotes were cultured in CR2 medium with 10% FCS, together with granulosa cells under 5% CO2 atmosphere. The number of nuclei in the inner cell mass and trophectoderm (ICM/TE), as well as the number of nuclei with fragmented DNA, were estimated by applying differential staining and TUNEL, respectively; data were analyzed by ANOVA (JMP—SAS Institute). To test the expression of apoptosis regulating genes, a pool of fifty 8-cell embryos from each group (fast and slow) were collected. After RNA extraction and reverse transcriptase reaction, cDNA was amplified with Bax and Bcl2 primers, individually. Results indicated, as expected, higher quality in fast-cleaving embryos, estimated by the number of ICM nuclei (20.8±1.4 and 15.6±2.1—P≤0.05); however, the number of TE didn’t show significant differences (54.9±2.4 and 53.2±3.8); the same was observed for total cell number (75.7±2.8 and 68.8±4.4). The frequency of blastocyst TUNEL-positive nuclei as an estimate of total cell number was significantly larger in the slow group when compared to the rapid development group (19.0±2.5% and 8.5±1.4%, respectively, P≤0.05). The greater proportion of morphologic abnormal nuclei in both groups was located in the ICM, and may explain the lower number of ICM nuclei in slow developing embryos. Hence, embryos of slow development show TUNEL-positive blastomeres at the 8-cell stage, but no fragmented nuclei were observed in embryos at 48hpi. Bax and Bcl2 cDNA amplification showed that both mRNAs were constitutively present at the 8-cell stage in both groups. It can be concluded that in vitro-produced bovine blastocysts, with slow development to the 8-cell stage, present lower quality compared with fast development homologues, estimated by mean number of ICM nuclei, as well as nuclei fragmentation in blastomeres (TUNEL-positive). There is a difference in fragmented nuclei proportion between both groups at the 8-cell stage, but this result may be biased by the numbers of hours in culture. It was possible to demonstrate the presence of mRNA for pro (Bax) and anti-apoptotic (Bcl2) genes in slow- and fast-developing embryos at the 8-cell stage, and the future determination of the ratio between these two transcripts may allow the evaluation of the participation of pre-transcriptional regulation of these genes on the induction of DNA fragmentation. Financial support: Grant 99/12351-3 FAPESP São Paulo, Brazil.
160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF
DOMESTIC CAT IVF EMBRYOS
Authors: Swanson W.F. , Manharth A.L. , Bond J.B. , Bateman H.L. , Krisher R.L. , Herrick J.R.
Published: February 2004

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P>0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P>0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)
176PRODUCTION OF TRANSFERABLE EMBRYOS IN BRAHMAN COWS TREATED WITH BOVINE
SOMATOTROPIN
Authors: Gonzalez-Fernandez R. , Velarde J.C. , Perea-Ganchou F. , Soto-Belloso E. , Palomares-Naveda R. , Hernandez-Fonseca H.J.
Published: February 2004

It is the goal of numerous research laboratories around the world to increase the number of transferable bovine embryos and to improve the viability of these embryos after ET into recipients. Treatment of donor cows with bovine somatotropin (bST) has given satisfactory results that have increased the hopes for an improved commercial production of embryos (Moreira et al. 2002 Theriogenology 57, 1371–1387). In order to demonstrate the effect of bST on the production of transferable embryos, degenerated embryos and non-fertilized oocites, 16 high-value genetic Brahman cows were synchronized and later subjected to a superovulatory (SO) treatment. The SO treatment consisted of decreasing doses of FSH (Folltropin-V®, Vetrepharm Inc, Ontario, Canada) every 12h for 4 days (2.5, 2.0, 1.5, 0.5×2/day). Forty-eight hours after the beginning of the SO treatment, a dose of PGF2α (Lutalyse®, Upjohn, Kalamazoo, MI, USA.) was given to all donors (i.m.). Estrus was detected by careful observation. At the time of first AI (6 hours after estrus detection) all cows were given (i.m. a GnRH (Buserelin, Conceptal®, Intervet, Boxmeer, The Netherlands) analogue and randomly assigned to one of the following groups: bST (n=8), which were treated with 500mg (s.c.) of bST (Boostin-S®, Schering-Plough Caracas, Venezuela) or control group (n=8) not treated. The second AI was done 10 hours after the first AI. Immediately after the uterine recovery, oocytes and embryos were evaluated and classified, according to their morphologic characteristics, into transferable embryos (TE) of good (TE1) or medium (TE2) quality, degenerated embryos (DE) and non-fertilized oocytes (NF). CL were evaluated by rectal palpation to estimate the ovulatory response. The statistical comparison between experimental groups (percentage of each category) was carried out using a chi-square test of SAS. Results are presented in Table 1. Results show that bST donor treatment increases the number and quality of transferable embryos. Key words: bST, ET, donor, embryo, superovulation.

Table 1
Number and percentages of transferrable and degenerated embryos and non-fertilized oocytes in superovulated Brahman cows after treatment with bST
18OVARIAN SUPPRESSION WITH THE PROGESTIN LEVONORGESTREL IMPROVES OVULATION INDUCTION FOR ARTIFICIAL INSEMINATION IN THE DOMESTIC CAT
Authors: Pelican K.M. , Brown J.L. , Wildt D.E. , Ottinger M.A. , Howard J.G.
Published: February 2004

Ovarian response to gonadotropin stimulation for artificial insemination (AI) is variable in the domestic cat. After ovulation induction with gonadotropins, a secondary wave of corpora lutea (CL) is often produced that alters endocrine profiles. This study assessed the impact of ovarian suppression with the progestin, levonorgestrel, before ovarian stimulation with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) on ovarian response in the cat. Queens were assigned randomly to: 1) levonorgestrel (LNG), 6 Norplant® rods implanted for 37d+eCG/hCG (n=6cats); and 2) Control, eCG/hCG alone (n=6). Ovarian response was graded (scale 1–4; 1=excellent, 4=ovulation failure) 36–40h post hCG (Day 5) using laparoscopy. Ovariohysterectomy (OVH) was performed on Day 23 and CL number and CL progesterone (P) content determined. Fecal samples were collected daily and metabolites of estradiol (E) and P quantified from >60d before inhibition through OVH. Estrus and luteal activity were defined as fecal E and P concentrations greater than two or three times baseline, respectively. Time data were analyzed using repeated measures analysis and remaining data were analyzed using ANOVA. LNG abolished ovarian activity in all cats. No E peaks were observed during LNG inhibition compared with pre-inhibition (mean±SEM; 1.8±0.3 peaks/37 days). In contrast, number of E peaks pre- v. during inhibition was similar (P>0.05) in control (pre, 2.2±0.3; during, 2.0±0.0) cats. All LNG cats had baseline E and P concentrations at eCG administration. Conversely, three of six control cats had elevated E and two cats had elevated P concentrations when eCG was given. Ovarian grade was higher (P<0.05) in LNG (1.3±0.2) v. control (2.9±0.4) cats. All LNG cats had ≤Grade 2 responses, whereas two control cats failed to ovulate (Grade 4) or had mature CL (Grade 3) at laparoscopy. For both LNG and control cats, mean peak E (overall mean, 117.4±14.4ngg−1 feces) was higher (P<0.05) and duration of estrus (6.8±0.9d) was longer (P<0.05) after eCG/hCG v. pre-inhibition values (81.4±5.3ngg−1 feces and 3.9±0.3d, respectively). However, P concentrations/luteal phase were higher (P<0.05)after eCG/hCG v. pre-treatment CL in control but not LNG cats. In cats with an ovarian grade of ≤2, control cats had more (P<0.05) CL at Day 23 (14.0±2.9CL/cat) compared to Day 5 (4.5±0.5).LNG cats showed no (P>0.05) accessory CL development on Day 23 (9.2±1.9CL/cat)compared to Day 5 (6.5±1.8). CL P content was not different (P>0.05) across treatments (overall mean, 90.8±18.7ngCL). Results show that inhibition of ovarian activity with levonorgestrel before eCG/hCG improves ovarian response and alleviates accessory CL development in the domestic cat.
183PREGNANCY RATE FOLLOWING TRANSFER OF IN VITRO- AND IN VIVO-PRODUCED
BOVINE EMBRYOS TO LH-TREATED RECIPIENTS
Authors: Small J. , Colazo M. , Ambrose D. , Mapletoft R. , Reeb J. , Kastelic J.
Published: February 2004

The objective was to evaluate the effect of pLH treatment on pregnancy rates in recipients receiving in vivo- or in vitro-produced bovine embryos. Heifers (n=37) and lactating (n=28) and non-lactating (n=150) beef cows were treated at random stages of the cycle with 100μg GnRH i.m. (Cystorelin, Merial Canada Inc., Victoriaville, Quebec, Canada) on Day −9, 500μg cloprostenol i.m. (PGF; Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day —2 and GnRH on Day 0 (66h post-PGF; without estrus detection). Cattle were placed at random, by class, into three groups: no further treatment (Control; n=71), or 12.5mg pLH (Lutropin-V, Bioniche Animal Health, Belleville, Ontario, Canada) on Day 5 (n=72) or on Day 7 (n=72) after the second GnRH. On Day 7, cattle with a CL >10mm in diameter (determined ultrasonically) received in vivo-produced, fresh (Simmental) or frozen (Holstein), or in vitro-produced frozen (Holstein) embryos (embryo type balanced among groups). Embryos were cryopreserved in 10% ethylene glycol; in vivo-produced frozen embryos were thawed 5 to 10s in air, 15s in a water-bath at 30°C and then “direct-transferred” nonsurgically. In vitro-produced frozen embryos (donated by IND Lifetech Inc., Delta, British Columbia, Canada) were thawed in a water-bath at 27°C for 10s and placed in ViGro Holding Plus medium (AB Technology, Pullman, WA, USA) at room temperature, evaluated and then transferred nonsurgically. Pregnancy was determined by ultrasonography on Day 35. Data were analyzed with CATMOD, chi-square and GLM procedures (SAS Institute, Cary, NC, USA.). Twenty cattle (9.3%) did not receive embryos; five heifers had cervical problems, and five heifers and 10 cows did not have a CL >10mm. Overall, 7.1% of the recipients had two CL on the day of embryo transfer. There was no effect (P>0.05) of treatment, embryo type (or interaction) or class of recipient on pregnancy rate (overall, 44.1%, 86/195; Table 1). Similarly, mean (±SD) CL diameter and luteal area did not differ (P>0.05) among groups or between pregnant and open recipients (overall, 22.0±3.4mm and 352.0±108.7mm, respectively). However, recipients with a CL diameter ≥18mm tended (P<0.1) to have a higher pregnancy rate (45.8 vs 25.0%). In a subset of 40 recipients examined ultrasonically on Day 12, 50% of those treated on Day 5 and 70% of those treated with pLH on Day 7 had two CL. In summary, overall pregnancy rate in GnRH-synchronized recipients receiving in vitro- or in vivo-produced embryos by nonsurgical transfer was 44.1%. Embryo survival to Day 35 was not affected by type of embryo or treatment with pLH 5 or 7 days after ovulation.

Table 1
Pregnancy rate in recipients on Day 35 based on pLH treatment and embryo-type
187EFFECTS OF OXYGEN TENSION ON ESTABLISHMENT, LDH ISOZYMES, AND MRNA EXPRESSION OF MURINE EMBRYONIC STEM CELLS
Authors: Gibbons J. , Hewitt E. , Gardner D.K.
Published: February 2004

Orchestrated differentiation of embryonic stem cells into specific tissues or cells will be invaluable for xenotransplantation, biomedicine, and pharmacology. However, the lack of a standardized culture environment for establishment and maintenance of cell lines has hindered the application of this technology. In other cell types, O2 concentration in the culture environment can have a profound effect on proliferation and differentiation. This study, therefore, tested the hypothesis that establishment dynamics, LDH isoforms, and mRNA expression patterns of the resulting cell lines would be affected by the O2 tension in the culture environment. Blastocysts recovered from mice (C57BL/6) uteri on Day 4 (post coitis) were placed in Speciality Medium (4500mgL−1 glucose) DMEM (plus 0.01μgmL−1 LIF) and cultured in a gas environment of 5% CO2 and either ∼20% or 5% O2. More (P<0.05) blastocysts hatched and produced outgrowths (Day 4 of DMEM culture) in the low (76.7±0.1%) compared to the high (58.3±0.1%) O2 group. Although the number of cells per outgrowth was similar between groups (low=58.1±5.2, high=58.8±5.7), a smaller number of colonies in the high O2 group (9/15; 64.3%) stained positive for alkaline phosphatase relative to the low O2 group (14/15; 93.3%). Oxygen treatment had no effect on the patterns of activity of the oxioreductase, lactate dehydrogenase (LDH), in either outgrowths or established stem cell lines. Interestingly, the stem cell lines (both O2 groups) displayed multiple isoforms (Isoforms 3, 4, and 5) of LDH, whereas the outgrowths displayed only Isoform 5. In contrast, two-cell embryos and blastocysts displayed only Isoform 1, and fibroblasts displayed Isoforms 4 and 5. Expression (mRNA) profiles were developed from blastocyst outgrowths, stem cell colonies, and established stem cell lines cultured under either high or low O2 tension, using a RT-PCR for LDH (Isozymes α and β) and a key regulatory enzyme of glycolysis, phosphofructokinase-2 (PFK2; Isozyme 1 and 2). There were no differences between the high and low O2 groups in mRNA expression of LDHα in the outgrowths, or established stem cells. Expression of LDHβ was at a very low level regardless of O2 treatment or cell type. Outgrowths from the low O2 group expressed Isozyme 1 of PFK2 whereas the outgrowths from the high O2 group did not. Enhanced expression of PFK2 is suggestive of increased glycolytic capacity. Reduced O2 environment during the peri-hatching period had significant effects on the resulting embryonic stem cells, supporting the hypothesis that O2 tension can affect stem cell establishment and maintenance.
19POSTPARTUM ANESTROUS TREATMENT WITH INTRA-VAGINAL PROGESTERONE DEVICE OR CALF REMOVAL FOR 120 HOURS IN SUCKLED CROSSBRED DUAL PURPOSE COWS
Authors: Perea-Ganchou F. , Soto-Belloso E. , Palomares-Naveda R. , De Ondiz A. , Hernandez-Fonseca H.J. , Carrillo D. , Castellanos G. , Gonzalez-Fernandez R.
Published: February 2004

Postpartum anestrus is the main reproductive problem that affects crossbred cattle production in Venezuela. (Soto et al., 2002 Theriogenology 57, 1503–1510; Soto et al., 2000 Revista Científica FCV-LUZ 10, 391–398). In order to reduce the postpartum anestrus and lower the calving to conception interval, 167 crossbred (Bos indicus×Bos taurus) anestrous cows between 90 and 130 days postpartum with body condition score 3 (scale 1–5) were randomly allotted to one the following treatments: 1) PH (n=59), intravaginal sponge (IS) impregnated with 250mg of medroxyprogesterone acetate (MAP) during 7 days+50mg of MAP and 5mg of 17β-estradiol (17β-E) i.m. at the day of sponge insertion (Day 0), plus 500 IU of eCG i.m. at Day 5 and1.5mg of 17β-E i.m. at Day 8 (PREGNAHEAT-E, VIATECA, Villa del Rosario, Venezuela); 2) CR (n=57), 120-h temporary calf removal; and 3) CG (n=20), control group. Work was performed in a commercial farm located in a subhumid tropical forrest region. Estrus, first service conception and pregnancy rate were analyzed by chi-square analysis. The intervals to conception were analyzed using analysis of variance (GLM) and the means was compared by least square means method. To evaluate the treatment effect, the interval to conception of each pregnant cow during the first 100 days were considered for the analysis. This interval (days) was significantly (P≤0.05) higher in the PH treatment (155.9±7.3) compared to control (174.4±8.1) but no differences were shown between PH and CR (161.0±7.8). Other results are shown in Table 1. PH treatment improved the estrus and pregnancy rates and shorten the interval to conception indicating that this is a promising anestrous treatment for suckled crossbred dual purpose cows under tropical conditions.

Table 1.

Reproductive performance of postpartum anestrous suckled cows treated with intravaginal progesterone or temporary calf removal
193CHARACTERIZATION OF PORCINE TROPHECTODERM-DERIVED CELLS IN THE
PRESENCE OF HBMP4 IN THE ABSENCE OF A FEEDER LAYER
Authors: Stewart K.B. , Adams A.M. , Pratt S.L. , Stice S.L.
Published: February 2004

A porcine trophoblastic cell line could provide a powerful model for understanding trophoblast cell biology as well as placental gene expression and proteomics in vitro. In this experiment, we derived porcine trophoblastic cells from trophectoderm tissue and assessed their growth on three different extracellular matrix substrates and in three different concentrations of human recombinant bone morphogenetic protein 4 (hBMP4). Human BMP4 has been shown to induce differentiation of human embryonic stem cells into trophoblast lineages. Elongated embryos were flushed using DPBS supplemented with 1% fetal calf serum and penicillin-streptomycin (1X) from the hysterectomized uteri of superovulated and bred prepuberal gilts 15 days post-insemination. The embryonic disc was visualized with a dissecting microscope. The trophectoderm tissue was cut 2–3mm away from the embryonic disc with a scalpel and the trophectoderm tissue was manually dissected into cell aggregates. These aggregates were plated on collagen type IV, Matrigel, and human extracellular matrix (laminin, collagen type IV and heparan sulfate proteoglycan derived from human placenta) in culture medium (DMEM with 15% FCS, 0.1mM 2-mercaptoethanol, 4ngmL−1 basic FGF4 and 1X P/S) in the presence or absence of hBMP4 at 0, 10, or 20ngmL−1. Cell outgrowth was observed within 24 hours of culture. After three days of culture, various cell types (based on size and morphology) were present. Among cultures of predominant large cells were colonies of smaller cells with epithelial-type morphology that had a prominent nucleus and a high nuclear-to-cytoplasmic ratio. The epithelial-type cells grew in tight colonies with definite borders and contained cytoplasmic structures resembling lipid-containing vesicles. These colonies initially appeared on all matrices across all hBMP4 concentrations. After seven days in culture the colonies developed distinct differences across groups. Cell growth on collagen was comprised of tight colonies having definite borders among large cells. Colonies on collagen were larger and more pronounced in both the hBMP4-supplemented groups than when cultured without hBMP4. The Matrigel coated plates contained large sheets of epithelial-type cell growth instead of compact colonies. This type of growth characteristic was present in all hBMP4 treatments on Matrigel. In contrast, few cells survived and propagated on human extracellular matrix. Only small colonies having the desired morphology were among the large cells on human extracellular matrix when cultured in medium containing 10ngmL−1 hBMP4. Cells were passaged and only cells growing on Matrigel could be further cultured. These data suggest that both the cell substrate and hBMP4 affect initial trophoblast outgrowths. Further analysis including immunocytochemistry and RT-PCR is currently being performed to better characterize these cells.
Epidemiology/Diseases
197SUSCEPTIBILITY OF PORCINE MORULAE AND BLASTOCYST STAGE EMBRYOS TO
PSEUDORABIES VIRUS AND PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS
Authors: Mateusen B. , Van Soom A. , Maes D.G.D. , Nauwynck H.J.
Published: February 2004

Porcine preimplantation embryos are refractory to infection with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) during the 2–4 to 16-cell stage as described by Bolin et al. (1981 Am. J. Vet. Res. 42: 1711–1712) and Prieto et al. (1996 Theriogenology 46: 687–693), respectively. Research on the effects of PRV and PRRSV on embryonic cells of morulae, blastocysts and hatched blastocysts is limited. Therefore, the objectives of the present study were (i) to assess the effects of PRV and PRRSV exposure on further embryonic development, and (ii) to determine whether PRV and PRRSV are able to replicate in embryonic cells of porcine morulae and blastocysts. In vivo produced ZP-intact and ZP-free morulae (6 days post-insemination), early blastocysts (7 days post-insemination), and hatched blastocysts (8 days post-insemination) derived from 22 superovulated sows were exposed to 105 TCID50 PRV (strain 89v87, second passage in swine testicle cells) or to 105 TCID50 PRRSV (Lelystad virus strain, 13th passage in swine alveolar macrophages) for 1h at 39°C. Control embryos were incubated under the same circumstances without viruses. Each group of morulae and blastocysts consisted of approximately 20 embryos. Embryonic development was assessed every 12h and differences in rates of development were analyzed using Chi-square analysis or Fisher’s exact test. At 48h post-incubation, embryos were collected and examined for viral antigen by indirect immunofluorescence. Further embryo development of ZP-intact and ZP-free morulae and blastocysts was not affected by exposure to PRV or PRRSV compared to controls (P<0.05). Moreover, using indirect immunofluorescence, no PRV or PRRSV antigen-positive cells were detected. Exposure of hatched blastocysts to PRV inhibited further embryo development as 100% (n=5) of the embryos degenerated 24h after viral exposure. This was significantly different (P<0.05) from the controls and the PRRSV-incubated hatched blastocysts that did not experience any negative influence on embryo development. Based on these results it can be concluded that embryonic cells are not susceptible to a PRRSV infection up to the hatched blastocyst stage. Embryonic cells of morulae and blastocysts are refractory to PRV, but the virus has a detrimental effect on further embryo development of hatched blastocysts. More experiments are necessary to confirm these results and to investigate whether, or at which preimplantation stage, embryos are susceptible to a PRRSV infection.
2POSTHATCHING DEVELOPMENT SYSTEM: A NOVEL IN VITRO CULTURE OF BOVINE EMBRYOS
Authors: Brandão D.O. , Vajta G. , Maddox-Hyttel P. , Stringfellow D. , Lövendahl P. , Rumpf R. , Callesen H.
Published: February 2004

Although high blastocyst rates can be achieved in somatic cell nuclear transfer, abortions and developmental abnormalities still hamper advancement. Reliable and practical methods to evaluate early embryonic development and differentiation are required to understand and overcome the problem. Our aim was to establish an in vitro culture system for monitoring posthatching development (PHD). Slaughterhouse-derived bovine oocytes were matured in vitro, fertilized (Day 0) and cultured (Holm et al., 1999, Theriogenology, 52, 683–700). On Day 8, degenerated embryos were removed from each well and 400L of modified culture medium (SOFaaci plus 0.5% glucose and 10% fetal bovine serum) were added. At Day 11, hatched blastocysts were selected by scoring them as Quality 1 (Q1: >1.0mm, clear trophoblast, compact inner cell mass), Quality 2 (Q2: 0.5mm, dark spots in the trophoblast, less compact inner cell mass), or Quality 3 (Q3: <0.5mm, many dark spots in the trophoblast, spread inner cell mass). The resulting 304 blastocysts in 12 replicates were then loaded into 15mm×1.2 gel tunnels of 2.4% agarose in PBS, supplemented with either 5% (Agar5) or 10% (Agar10) fetal bovine serum, covered with the modified culture medium, and then incubated at 38.5°C in 5% CO2, 5% O2, 90% N2. Embryo morphology and length were evaluated using a stereomicroscope on Days 12, 13, 14 and 15. On Day 14, 75 embryos were removed, biopsed (1mm) for sex determination of each embryo, and processed for light and transmission electron microscopy. Qualitative and quantitative data were analyzed by χ2 test and GLM procedure of SAS, respectively, with P level of 0.05. A total of 170 embryos (56% of total) initiated elongation. This percentage was higher (LSmeansSD, n=12; P<0.05) in Agar10 v. Agar5 in both Q1 (889 v. 637), Q2 (667 v. 485) and Q3 embryos (529 v. 278). Mean embryo length (mm; LSmeansSEM) on Day 13 was higher (P<0.05) in Q1 (2.10.2, n=49) and Q2 (1.71.4, n=98) than Q3 (1.20.3, n=23). On Day 14, Q1 embryos (3.50.2) were longer (P<0.01) than Q2 and Q3 embryos (2.70.1 and 2.00.3). On Day 15, Q1, Q2 and Q3 embryos (4.40.5, n=24, 4.00.3, n=45 and 2.90.6, n=14, respectively) had similar length, probably influenced by the low number of Q3 embryos. The percentage of males was higher (P<0.001) in Q1 (95%; n=40), but similar in Q2 (39%; n=26) and Q3 (71%; n=7). Light microscopy confirmed hypoblast and epiblast formation. Ultrastructural analysis revealed that the latter had penetrated the trophoblast (Rauber’s layer), forming an embryonic disc including many degenerative cells. In conclusion, this culture system represents the first model for rapid growth, elongation, and initial differentiation of bovine posthatching embryos.
205A GAME OF CAT AND MOUSE: XENOGRAFTING OF TESTIS TISSUE FROM DOMESTIC
KITTENS RESULTS IN COMPLETE CAT SPERMATOGENESIS IN A MOUSE HOST
Authors: Honaramooz A. , Snedaker A. , Dobrinski I.
Published: February 2004

Loss of genetic diversity due to infertility or the premature death of valuable individuals is a significant problem in the conservation of rare and endangered felid species, as well as in the maintenance of lines of cats used to study inherited feline and human disease. Attempts to overcome loss of genetic diversity have focused on freezing sperm;; however, sperm cannot be collected from immature males. Previously, we reported completion of spermatogenesis in testis tissue from newborn pigs and goats grafted ectopically into host mice (Honaramooz A et al., 2002 Nature 418, 778–781). Xenografting of testis tissue not only serves as a powerful system for the study of spermatogenesis and testicular maturation, but it also provides a previously unavailable system to obtain sperm from immature animals. The objective of the present study was to extend the technique of testis tissue xenografting to the domestic cat as a model animal for felid species. Testes from 1- to 5-wk-old domestic short-haired kittens (n=8) were cut into small fragments (about 1×3mm each), and up to eight fragments were grafted under the back skin of each castrated immunodeficient host mouse (n=12). Histological examination of the testis xenografts was performed between 14 and 36 weeks post-transplantation. From 8 of the 12 recipient mice, 93% of testis tissue grafts were recovered. No grafts were recovered from the remaining 4 mice. At the time of grafting, the seminiferous cords of the donor testis tissue contained only immature Sertoli cells and gonocytes. At 14 weeks after grafting, tubular expansion was evident, caused by the proliferation of Sertoli cells and tubular lumen formation. By 18 weeks after transplantation, the seminiferous epithelium contained spermatocytes, and by 20 weeks, round spermatids were the most advanced types of germ cells. By 36 weeks after transplantation, xenografts of cat testis tissue had completed spermatogenesis and mature sperm were present. In all recipient animals where xenografts were recovered, the weight of the seminal vesicles in the castrated host mice was restored to pre-castration values, indicating that xenografts were capable of releasing biologically active testosterone. These results demonstrate the potential of xenografting to achieve full spermatogenesis in testis tissue from kittens without the necessity of exogenous hormonal stimulation. It was shown previously that sperm recovered from testis xenografts can support fertilization and development (Shinohara T et al., 2002 Hum. Reprod. 17, 3039–3045; Schlatt S et al., 2003 Biol. Reprod. 68, 2331–2335). Therefore, sperm production in a mouse host can provide an alternative for germ line preservation from immature felids where sperm cryopreservation is not an option. Sperm recovered from xenografts can be used for assisted reproduction, thereby making it possible to produce offspring from immature males.
213EFFECTS OF TIME OF MATURATION AND SHEEP SERUM ON IN VITRO FERTILIZATION IN THE ENDANGERED ELD'S DEER (CERVUS ELDI THAMIN) OOCYTES
Authors: Siriaroonrat B. , Comizzoli P. , Songsasen N. , Spindler R.E. , Monfort S.L. , Berg D.K. , Pukazhenthi B.S.
Published: February 2004

The Eld’s deer, native to Southeast Asia, is threatened with extinction. Although artificial insemination is effective for offspring production, in vitro fertilization (IVF) would be more useful for rapidly disseminating genetic material from valuable founders. The objectives of this study were to: 1) determine if oocytes recovered from exogenous gonadotropin-treated hinds require additional in vitro maturation;; and 2) assess if fertilization is enhanced by supplementing Deer Synthetic Oviduct Fluid (DSOF;; Berg DK et al., 2003 Theriogenology 59, 189–205) with 1-day postestrus sheep serum (SS). Estrous cycles in Eld’s deer hinds (n=10) were synchronized with PGF2α analog (Lutalyse™, 500mg), followed by a 14-day intravaginal CIDR-G insertion;; ovine FSH (Ovagen™; 0.05 unit×8 injections) was administered at 12-h intervals beginning 84h before CIDR-removal. COCs (n=160) were retrieved laparoscopically 40–46h post-CIDR-removal and either fixed or matured in vitro (for 12h v. 24h) in TCM-199 (Earle’s salt) supplemented with 0.33mM pyruvate, 2mM glutamine, 100IUmL−1 penicillin, 100μgmL−1 streptomycin, 10% fetal calf serum, 5μgmL−1 FSH and LH and 1μgmL−1 E2 (5% CO2, 38.5°C). After 12- or 24-h IVM, cumulus cells were partially removed and oocytes (n=110) fertilized in DSOF with pooled frozen-thawed sperm (3 males;; 2×106 motile sperm mL−1), in the absence or presence of SS (20%, v/v). Additional oocytes (n=18) were used for parthenogenetic control. At 20-h postinsemination, presumptive zygotes were fixed and stained (Hoechst 33342) to assess fertilization success (presence of two pronuclei). Data were analyzed by ANOVA. Overall, 16.0±2.6 (mean±SEM) COCs were recovered/female. The majority of COCs were of excellent quality (grade I; 67.7±3.8%). At time of aspiration, 85% of the oocytes (n=11/13) were in metaphase I stage, 7.5% in telophase and 7.5% degenerate. No parthenogenic activation was observed. Likewise, no polyspermy was observed in any treatment. Fertilization was higher (P<0.05) in oocytes matured for 24h and fertilized in the absence (64.4±3.1%) compared to presence (26.9±11.2%) of SS. In the absence of SS, a higher (P<0.05) proportion of oocytes were fertilized after 24h (64.4±3.1%) compared to 12h (27.1±9.0%) IVM. There was no effect (P>0.05) of SS on fertilization among oocytes subjected to 12-h IVM (27.1±9.0% v. 12.5±9.5%). When SS was present during fertilization, no difference (P>0.05) was observed among oocytes matured for 12 or 24h. Results demonstrate that: 1) Eld’s deer oocytes require an additional 24-h IVM to complete maturation;; 2) DSOF supports sperm-oocyte interaction;; and 3) SS is not essential for successful fertilization. (Supported by Morris Animal Foundation.)
216TRANSVAGINAL ULTRASOUND-GUIDED BIOPSY OF THE BOVINE OVARY: A NEW TOOL
FOR THE STUDY OF PREANTRAL FOLLICLES
Authors: Aerts J.M.J. , Bols P.E.J. , Oste Ma. , Van Ginneken C.J.D. , Wechsung E. , Houvenaghel A.
Published: February 2004

Current research on follicular dynamics is focussing more on primordial and preantral follicular populations. Whereas antral follicular development can be studied by ultrasound visualization, preantral follicles can be visualized only on stained ovarian tissue sections obtained through laparotomy or following slaughter, which does not allow dynamic follicular follow-up studies. To our knowledge, only one report has previously been published on a technique for transvaginal corpus luteum biopsy (Kot K et al. 1999 Theriogenology 52, 987–993). Therefore, a new method to repeatedly take ovarian biopsies in living donor cattle through ultrasound-guided transvaginal puncture of the ovarian stroma was developed and tested. Healthy cows with a normal reproductive tract upon rectal examination were prepared as for transvaginal ovum pick-up (OPU) (Bols PEJ et al. 1995 Theriogenology 43, 677–687). The ovary was visualized using a mechanical multiple angle 5-MHz ultrasound transducer (Pie Medical, the Netherlands), fitted next to a needle guidance system in an OPU handle. A specially designed needle carrier with a 14-gauge disposable needle (length 38mm, outer diameter 2.1mm, Kruuse, Japan), acting as a trocar, was inserted through the vaginal wall. A 60-cm-long disposable biopsy needle with an inner stylet having a 20-mm-long specimen notch, a cutting cannula (diameter 1.3mm), and an automated spring-loaded handle with trigger (Quick-Core® Biopsy Needle, Cook, Denmark) was passed through the disposable needle into the ovarian stroma, under ultrasound control. Prior to insertion, the plunger of the biopsy needle was pulled back, with the needle spring-locked in the ready position. The biopsy needlepoint was positioned closely under the surface of the ovary with the stylet retracted and the specimen notch covered by the cutting cannula. With the needle in position, the stylet was advanced to expose the specimen notch within the ovarian stroma. Then, the cutting cannula was fired and a tissue sample was captured. Repeated biopsies were taken in four cows two times a week, over a two-week period, without echographically detectable detrimental effects of the reproductive tract. A tissue specimen was recovered in 25 of 30 attempts, although the dimensions of some of the biopsies were rather small. Biopsies from both the ovarian stroma and the corpus luteum (CL) were taken. They were fixed with 4% paraformaldehyde for 2h at room temperature. Following overnight incubation in a 15% sucrose solution for cryoprotection, 17-μm cryostat sections were made and stained with hematoxylin. Study of the sections revealed small populations of primordial follicles clearly distinguishable from the surrounding ovarian stroma. Distinct groups of luteinized cells could be seen in CL biopsies. Further studies are necessary to standardize the procedure and to evaluate the effects of biopsy-taking on fertility.
221CYSTIC BOVINE FOLLICLES ARE ASSOCIATED WITH AN
ALTERATION IN LOCAL GENE AND PROTEIN EXPRESSION
Authors: Greenaway J. , Linnerth N. , Petrik J.
Published: February 2004

Formation of persistent follicular cysts is a prevalent problem for producers of domestic agricultural animals in North America. The most common group of animals affected is dairy cattle. This condition is problematic as it renders cattle anovulatory and infertile for the duration of the cyst. Various studies have assessed the incidence of follicular cysts in dairy cattle, revealing a range of prevalence of 9–26%. Although there is information regarding the incidence of this disorder, little is known of the etiology. It is known that a complex interaction occurs between the hypothalamus, anterior pituitary, and ovary to regulate normal reproductive function. Dysregulation at any of these sites could contribute to the formation of persistent follicular cysts. The objective of this study was to determine whether local changes in gene and protein expression are present in cystic follicles. Transvaginal aspirations from follicles
that had been greater than 2.5-cm diameter for at least 10 days were collected. Aspirates were centrifuged, and granulosa cells and follicular fluid were separated. Granulosa cells were lysed, and RNA and protein was isolated. For immunohistochemistry, bovine slaughterhouse ovaries with follicles greater than 2.5cm in diameter were dissected, fixed, and processed. Western blot analysis and RT-PCR were performed on
protein and RNA samples, respectively. Cystic and control follicles were analyzed for expression of vascular endothelial growth factor (VEGF) and its receptor VEGF-R2 and members of the insulin-like growth factor (IGF) family, which are known to mediate a host of cellular events during follicular development. Cystic follicles exhibited a significant
increase in VEGF and IGF-I protein concentrations and a reduction in VEGF-R2 and the type 1 IGF receptor. Immunohistochemical analysis demonstrated increased ligand staining and reduced receptor expression in granulosa cells of cystic follicles. These results indicate that there is an altered growth factor profile in cystic follicles and suggest that intra- as well as extra-follicular dysregulation is important in the etiology of this reproductive disorder.
222THE ROLE OF TUMOR NECROSIS FACTOR-RELATED APOPTOSIS INDUCING LIGAND
DURING FOLLICULAR ATRESIA IN PIG OVARIES
Authors: Inoue N. , Manabe N. , Nakayama M. , Matsui T. , Maeda A. , Nakagawa S. , Miyamoto H.
Published: February 2004

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce cell death by binding to its receptors (DR4 and DR5). However, binding to DcR1 or DcR2 cannot induce apoptosis. DcRs compete with DRs. TRAIL has been reported to induce apoptosis in various tumor cells but not in normal cells. However, a recent study revealed that TRAIL induces apoptosis in normal hepatocytes of human but not in those of rat, mouse, or rhesus monkey, indicating that there are species-specific differences in TRAIL and receptor systems. In the present study, we demonstrated Immunohistochemical, Western immunoblotting, and reverse transcription-polymerase chain reaction analyses (RT-PCR) of TRAIL and DR4 in granulosa cells during follicular atresia in pig ovaries. For immunohistochemistry, pig ovaries obtained at a local slaughterhouse were fixed with 20% buffered formalin. For Western blotting and RT-PCR analysis, individual preovulatory antral follicles were dissected from the ovaries. Based on morphological and endocrinological criteria, the antral follicles were divided into three categories as follows: healthy, early stage of atresia, progressed stage of atresia. Significant increases were demonstrated in TRAIL protein and mRNA levels during atresia, but not in DR4 protein. Moreover in an in vitro apoptosis-inducing assay using cultured granulosa cells prepared from healthy follicles, we showed that more than 200ng/mL TRAIL could activate caspase-3 and induce apoptotic cell death in a dose-and time-dependent manner, but less than 100ng/mL of TRAIL could not induce apoptosis. When DcR1 was removed from the cell membrane of granulosa cells, a lower dose of TRAIL could induce apoptosis. The present findings suggested that the TRAIL can induce granulosa cell apoptosis, and that DcR1 blocks TRAIL-induced apoptosis in granulosa cells of healthy follicles in porcine ovaries.
229CHARACTERIZATION OF CONNEXIN 43 IN THE CANINE OVARIAN FOLLICLE
Authors: Willingham-Rocky L.A. , Golding M.C. , Westhusin M.E. , Kraemer D.C. , Burghardt R.C.
Published: February 2004

Connexin 43 (Cx43) is the most abundant gap-junction protein in ovarian follicle cell interactions and has been well characterized for several mammalian species. However, the involvement of Cx43 in canine follicular development has not been extensively investigated. The objective of this study was to 1) characterize the spatial patterns of Cx43 localization within the canine ovarian follicle at various physiological states and during the estrous cycle (prepubertal, anestrus, proestrus, estrus and diestrus), and 2) to characterize the canine Cx43 mRNA transcript. The spatial expression pattern of Cx43 protein was evaluated by immunofluorescence microscopy in canine ovaries. Cx43 was not detected in primordial follicles, but was detected in primary and secondary follicles at each physiological state at all granulosa cell borders. In secondary follicles, definitive, punctate staining patterns were localized along mural granulosa cells and also in the surrounding granulosa-cumulus cell borders. Notably, more intense staining was observed in the corona radiata cells immediately surrounding the oocyte, as well as in trans-zonal projections and at the perivitelline membrane. Patterns of localization were most similar between proestrus and diestrus, and between prepubertal and anestrus in secondary follicles. Estrus-stage follicles showed a decrease in localization at the corona-oocyte cell borders as compared to proestrus and diestrus. In large, healthy antral follicles from proestrus and estrus stages, Cx43 was present in the stroma, theca, and granulosa layers. However, antral follicles from estrus-stage ovaries showed more intense staining in the mural granulosa and theca layers, and less intense in the stroma as compared to those of proestrus stage. The most intense pattern of punctate staining was observed in the corpora lutea of diestrus-stage ovaries. Additionally, gene-specific primers were designed from highly conserved regions of Cx43 mRNA among bovine, human and mouse. RNA was isolated from canine uterus and used as a template for RT-PCR. The PCR products were then sequenced and verified in GenBank to assess homology. The sequenced coding region for canine Cx43 mRNA shares 93% sequence homology with bovine vascular smooth muscle. Canine specific primers for this sequence have been designed, and expression analysis studies in canine ovarian follicles are currently underway. These results indicate that the pattern of localization of Cx43 is similar to that reported for the cow and the pig, except that in canine ovarian preantral follicles, Cx43 is also localized to the peri vitelline membrane. Additionally, these results suggest that the localization of Cx43 is dependent on the physiological state of the ovary, and is likely necessary for folliculogenesis and subsequent oocyte development in canines.
235RETINOID-DEPENDENT MRNA EXPRESSION IN BOVINE OOCYTES
PREMATURED AND/OR MATURED IN VITRO
Authors: Gomez E. , Diez C. , Moran E. , Rodriguez A. , Royo L.J. , Goyache F. , Facal N. , Gutierrez-Adan A.
Published: February 2004

As a transcription factor, retinoic acid (RA) can activate or silence a wide number of genes, thus inducing differentiation in cell systems and playing a role in cell cycle regulation. However, little is known of RA-dependent gene expression in the oocyte. Bovine oocytes and cumulus cells express most RA receptors, and the presence of 9-cis-RA during in vitro maturation (IVM) is beneficial to oocyte development (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). The present work analyzes the relative abundance of various developmentally important gene transcripts in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were manipulated in defined medium with polyvinyl-alcohol (DM-PVA). Those COCs undergoing prematuration were cultured for 24h in DM-PVA with 25μM roscovitine. For IVM, some prematured COCs were cultured for 24h in DM-PVA containing pFSH, LH and E2. Incubations were made at 39°C in an atmosphere of 5% CO2 in air and high humidity. Within experiments, COCs were cultured with nM 9-cis-RA 5, in 1% ethanol (both as vehicle and inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Using Real Time PCR (10 oocytes per group) (Rizos et al., 2003 Biol. Reprod. 68, 236) we examined the relative mRNA expression of genes involved in protection against free oxygen radicals (Mn-superoxide dismutase, MnSOD), glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH) and cell cycle events (Cyclin B1 and H1). Data (of 4 replicates) were analyzed by ANOVA and Duncan test (P<0.05). Regarding immature oocytes, prematuration in 1% ethanol increased cyclin B1 expression and decreased cyclin H1, while 9-cis-RA increased G6PDH. Maturation without additives increased cyclin B1 and G6PDH, but decreased cyclin H1 and MnSOD expression;; opposite trends were observed under increasing ethanol dosages (3% and 5%). Maturation with 1% ethanol or 9-cis-RA enhanced cyclin B1 and G6PDH, while reducing cyclin H1 and MnSOD expressions. The presence of 9-cis-RA during both prematuration and maturation processes tended to show more prominent effects than the ones observed when it was present only during prematuration or maturation alone. In our study, in presence of 9-cis-RA during both prematuration and maturation processes, the expression of cyclin B1 and G6PDH tended to increase, while cyclin H1 and MnSOD tended to decrease. However, the differences with the control group without additives were not significant. Our study during both prematuration and maturation processes show that beneficial effects of RA on oocyte developmental competence may not be related to the alteration of mRNA expression of the four genes analyzed. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175; 2003-05783).