bluuebunny/crossref_metadata_2025 huggingface dataset:
27BIRTH OF AFRICA'S FIRST NUCLEAR-TRANSFERRED ANIMAL PRODUCED WITH
HANDMADE CLONING
Authors: Bartels P. , Joubert J. , de la Rey M. , de la Rey R. , Treadwell R. , Callesen H. , Vajta G.
Published: February 2004

Cloning technology has the potential to stimulate the development of the animal biotechnology industry in southern Africa, as well as provide conservationists with an additional tool to possibly assist with conserving critically endangered wildlife species sometime in the future. The aim of this study was to determine whether cloning could produce blastocysts and possibly live progeny in a field-type laboratory without micromanipulators and CO2 incubator. Approx. 1×1-cm ear skin notches were surgically removed from a physically immobilized 9-year-old Holstein cow, a former South African milk production record holder. The tissues were placed into physiological saline and transported to the laboratory at 4°C within 2h, cleaned with chlorohexidine gluconate and sliced finely in
Minimal Essential Medium supplemented with 10% fetal calf serum. The resultant tissue explants were treated as previously described (Bartels et al., 2003 Theriogenology 59, 387) and actively growing fibroblast cultures were made available for the nuclear transfer process. Bovine oocytes from slaughterhouse-derived ovaries were collected and matured for 21h in modified TCM-199 medium supplemented with 15% cattle serum, 10IUmL−1 eCG and 15IUmL−1 hCG. Nuclear transfer was performed using the HMC technique (Vajta et al., 2003 Biol. Reprod. 68, 571–578). At 21h after the start of maturation, cumulus cells and zonae pellucidae were removed and oocytes were randomly bisected by hand. Cytoplasts were selected using Hoechst staining and a fluorescent microscope. After a two-step fusion, reconstructed embryos were activated with calcium ionophore and dimethylaminopurine. Culture was performed in SOFaaci medium supplemented with 5% cattle serum using WOWs (Vajta et al., Mol. Reprod. Dev. 50, 185–191). All incubations including culture of donor cells were performed in the submarine incubator system (SIS; Vajta et al., 1997 Theriogenology 48, 1379–1385). In two consecutive
experiments, 6 blastocysts were produced from 52 reconstructed embryos. On Day 7, 5 blastocysts were selected for transfer into 3 previously synchronized recipients. All three recipients became pregnant, but two of the recipients
aborted at six and seven months, respectively. Post-mortem examination on the first aborted fetus did not reveal any identifiable etiology, but coincided with 6 abortions from natural pregnancies during a heat wave, while the
organism Brucella abortis was isolated from the second aborted fetus. The third pregnancy went to term, and a healthy calf, weighing 27kg, was delivered by Caesarean section. The three-month-old calf is being raised by a surrogate Jersey cow under standard dairy conditions and is expected to join the dairy in eighteen months’ time. The birth of ‘Futhi’, meaning ‘replicate’ in Zulu, is Africa’s first cloned animal and signifies an important milestone in the development of animal biotechnology in Africa.
271INFLUENCE OF ARGININE-GLYCINE-ASPARTIC ACID (RGD) IN
BOVINE SPERM-EGG BINDING, AND FERTILIZATION IN VITRO
Authors: Gonçalves R.F. , Erickson D. , Ealy A.D. , Killian G.J.
Published: February 2004

Osteopontin (OPN), a secretory RGD-containing phosphoprotein, has been identified in cow oviductal ephitelium and fluid, but its role in fertilization is unclear. RGD peptide is capable of blocking fertilization, inducing intracellular Ca2+ transients, and initiating parthenogenetic development when present during bovine fertilization in vitro.This study was conducted to determine whether in vitro sperm binding to the zona pellucida (ZP) and fertilization of bovine oocytes were affected by treating the sperm or oocytes with RGD (arginine–glycine–aspartic acid, a sequence recognized by integrins) or non-RGD-containing peptides. In vitro matured oocytes were incubated (39°C, 5% CO2 in air) for 2 hours in fertilization medium with: (1) no peptides;; (2) 50μgmL−1 RGD (Calbiochem®, San Diego, CA, USA); (3) 1000μgmL−1; (4) 50μgmL−1 non-RGD (Calbiochem®); (5) 1000μgmL−1 non-RGD. The bovine sperm from two differents bulle was collected by artificial vagina, pooled, washed twice with MTM at 500g for 10min and incubated (39°C, 5% CO2 in air) for two h at 5×107 concentration in MTM with: (6) no peptides;; (7) 50μgmL−1 RGD;; (8) 1000μgmL−1; (9) 50μgmL−1 non-RGD; (10) 1000μgmL−1 non-RGD. Following incubation, treated and untreated oocytes were washed and inseminated with 1×105 treated or untreated fresh spermatozoa per 10 oocytes;; after the sperm were recovered from a Percoll gradient (45%/90%). After 18–20h, oocytes were removed from co-culture, and washed in TL-HEPES. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per ZP counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetato-orcein and observed for the presence of pronuclei. For the five replicates, 100–120 oocytes were used for fertilization and 150–170 oocytes were used for sperm-egg binding assays. Data were analized by SAS. Treatment of sperm or oocytes with the RGD peptide significantly decreased (P<0.05) fertilization compared to the non-treated controls or those treated with non-RGD peptides: (1) 80%±3.0; (2) 42%±3.0; (3) 30.2%±3.0; (4) 78.5%±3.0; (5) 79.1%±3.0; (6) 78.9%±3.0; (7) 41.3%±3.0; (8) 29.1%±3.0; (9) 79.2%±3.0; (10) 80.2%±3.0. More sperm bound to the ZP of untreated or non-RGD-treated oocytes or sperm than those incubated with the RGD peptide: (1) 71.2±4.1; (2) 33.2±4.2; (3) 24.2±4.1; (4) 69.5±4.1; (5) 70.2±4.2; (6) 71.9±4.2; (7) 29.8±4.2; (8) 19.8±4.2; (9) 68.9±4.2; (10) 70.6±4.2. These studies demonstrated that incubation of bovine oocytes or spermatozoa with a RGD peptide inhibits sperm-egg binding and fertilization in vitro. These findings support the notion that the role of osteopontin in bovine fertilization may involve inteaction with integrins via its RGD sequence.
273EFFECT OF EPIDERMAL GROWTH FACTOR (EGF) DURING OOCYTE
MATURATION ON IN VITRO PRODUCTION OF BOVINE EMBRYOS
Authors: Hernandez-Fonseca H.J. , Palomares-Naveda R. , Soto-Belloso E. , Gonzalez-Fernandez R. , De Ondiz-Sanchez A.D. , Perea-Ganchou F. , Sirisathien S. , Brackett B.G.
Published: February 2004

Medium components during in vitro maturation (IVM) can significantly influence oocyte maturation and subsequent embryo development in vitro (Rose TA and Bavister BD 1992 Mol. Reprod. Dev. 31, 72–77; Harper K and Brackett B 1993 Biol. Reprod. 48, 409–416). The aim of this experiment was to evaluate the effect of EGF during IVM on further development of bovine embryos in vitro. Bovine ovaries were obtained at a slaughterhouse. Cumulus-oocyte complexes (COC) were aspirated from follicles 2–5mm in diameter. COC were incubated for 24h in either of 3 maturation media: T1 (n=72): modified TCM-199; T2 (n=45): modified TCM-199 supplemented with 10ngmL−1 of EGF;; or T3 (n=46): modified TCM-199 supplemented with 10% fetal bovine serum (FBS). After 24h of IVM, COC were inseminated with 2×106 motile spermatozoa/ml. After 18h of gamete coincubation, presumptive zygotes were denuded and placed in culture in SOF rich in glutamine (g-SOf) for 72h, at which time, cleavage rate (%) wass assesed (embryos with >4 cells). Subsequently, cleaved embryos were incubated for an additional 72h in c-SOF (SOF rich in citrate and glucose). Finally, embryos were cultured in modified TCM-199 for 24–48h, at which time blastocyst formation rate (%) was evaluated. Cleavage rates were similar between T2 and T3 but significantly greater than in T1 (P>0.05; see Table 1). Addition of EGF during IVM (T2;; 11/45, 24.4%) did not yield more blastocysts compared to the other two treatments (6/57, 10.5% and 10/29, 34.5%, T1 and T3, respectively). Nonetheless, T3 (with serum) had a greater yield of blastocysts compared to T1 (P>0.01). Results in this study show that the addition of EGF to chemically defined media results in similar cleavage rates and blastocyst yields to those obtained when using serum during IVM. Key words: in vitro maturation, EGF, cleavage, bovine, embryo.

Table 1
Effect of EGF and serum during IVM on cleavage rate of bovine oocytes
274SIMILARITY OF EMBRYOS PRODUCED BY OVUM PICK-UP AND IN
VITRO FERTILIZATION IN IDENTICAL TWIN CATTLE
Authors: Imai K. , Kobayashi S. , Matoba S. , Tagawa M. , Saito N.
Published: February 2004

The present study was designed to assess the similarity of follicular development, oocyte quality, and their subsequent development on ovum pick-up (OPU)-IVF in identical twin cattle. Four pairs of identical twin Japanese black cows (A, B pairs at 5 years old and C, D pairs at 3 years old) were kept under the same feeding and environmental conditions. OPU was performed for these cows once a week for seven continuous weeks. OPU was done by using a 7.5-MHz linear transducer with needle (17 G, 530-mm length) connected to an ultrasound scanner (SSD-1200, ALOKA, Tokyo, Japan). Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the development, collected COCs were cultured for 20h in TCM-199 supplemented with 5% calf serum (CS) in a microdroplet (volume was adjusted to 5μL/oocyte) at 38.5°C under atmosphere of 5% CO2 in air. After maturation, the COCs were inseminated with frozen-thawed semen collected from the same ejaculation of a single bull. The fertilization was performed with BO solution as described by Imai et al (J. Vet. Med. Sci., 2002, 64(10), 887–891). The zygotes were then cultured in CR1aa supplemented with 5% CS under the same condition of maturation for nine days. Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst production rate on Days 7 to 9 (insemination day=Day 0). Blastocysts were classified according to the IETS creteria. Data were analyzed by ANOVA. A total 56 sessions of OPU were performed in this study. The overall mean number of developing follicles (larger than 2mm in diameter), collected oocytes, and produced blastocysts were 30.3±9.2, 20.1±9.2 and 6.3±3.8 (mean±SD) per session, respectively. The mean number of developing follicles on the day of OPU were significantly different between B and D pairs (38.6±7.5 and 21.9±6.5, P<0.01); however, no significant difference was found within each twin. In oocyte quality, C and D pairs were significantly higher grade than the A pair. The percentages of cleaved oocytes and embryos developed to the blastocyst stage (34±16, 27±10, 41±17 and 39±24) showed no differences among 4 pairs and within each twin. However, the percentage of Grade 1 blastocyst of B pair was significantly lower (P<0.01) than that of other pairs, and C pair was significantly higher (P<0.05) than that of A and D pairs (67±25, 41±22, 93±10 and 71±25; A, B, C and D pairs, respectively). There was no significant difference within twins. These results show little statistical variation between cows of the same genetic background in the production of embryos in vitro.
278EFFECT OF GLYCOSAMINOGLYCAN SUPPLEMENT ON PORCINE PREIMPLANTATION
EMBRYO DEVELOPMENT AND EXPRESSION OF RECEPTORS FOR GLYCOSAMINOGLYCANS IN
PORCINE EMBRYOS
Authors: Kim H.S. , Lee G.S. , Hyun S.H. , Nam D.H. , Jeong Y.W. , Kim S. , Kim J.H. , Kang S.K. , Lee B.C. , Hwang W.S.
Published: February 2004

The present study investigated the effect of glycosaminoglycan (GAG) supplement on developmental competence of porcine in vitro fertilized (IVF) embryos and GAG receptor expression in the porcine embryos. In vitro-matured oocytes were inseminated with frozen-thawed boar semen and cultured in North Carolina State University (NCSU)-23 medium in the absence or presence of various GAGs (hayaluronic acid, heparin or both). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos and blastocysts at Days 2 and 7, respectively. Differential staining was performed in blastocysts at Day 7. All data were analyzed by ANOVA using a Generalized Linear Model (SAS). Inseminated oocytes were cultured in NCSU-23 supplemented with different concentrations (0, 0.1, 0.5 or 1.0mgmL−1) of hyaluronic acid (in Experiment 1) or heparin (in Experiment 2). Supplementing NCSU-23 with 0.5mgmL−1 hyaluronic acid significantly increased (P<0.05) the total number of cells (55.9) and the number of trophectoderm (TE) cells (41.7) compared with the other culture groups (44.7, 45.0 and 31.3, 31.8, respectively). The rate of blastocyst formation was significantly increased (P<0.05) in the 1.0mgmL−1 heparin-supplemented group (21.8%) compared with that in the control culture group (16.4%). In experiment 3, inseminated oocytes were cultured in NCSU-23 supplemented with 0, 0.1mgmL−1 heparin, 0.5mgmL−1 hyaluronic acid, or 0.1mgmL−1 heparin+0.5mgmL−1 hyaluronic acid. The rate of blastocyst formation was significantly increased (P<0.05) in the 1.0mgmL−1 heparin group (21.5%) and the 1.0mgmL−1 heparin+0.5mgmL−1 hyaluronic acid group (22.8%) compared with that of control culture group (16.6%). In experiment 4, total RNA was prepared from oocytes, 2-, 4-, and 8-cell stages, morulae, and blastocysts and reverse-transcribed using the First-strand cDNA Synthesis kit (Amersham Biosciences, Piscataway, NJ, USA). The cDNA was subjected to polymerase chain reaction with primers specific for hyaluronic acid receptor (CD44) and heparin binding protein (HIP). The expression of CD44 gene was detected in oocytes, and in 2- and 4-cell stages;; HIP gene was detected in all preimplanatation-stage embryos. In conclusion, the present study demonstrated the embryotrophic role of GAG and expression of GAGs receptors in porcine IVF embryos. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1).
28PREGNANCIES RESULTED FROM GOAT NT EMBRYOS PRODUCED BY FUSING COUPLETS
IN THE PRESENCE OF LECTIN
Authors: Begin I. , Bhatia B. , Rao K. , Keyston R. , Pierson J.T. , Neveu N. , Cote F. , Leduc M. , Bilodeau A.S. , Huang Y.-J. , Lazaris A. , Baldassarre H. , Wang B. , Karatzas C.N.
Published: February 2004

The procedure of nuclear transfer (NT) using somatic cells remains inefficient partly due to low fusion rates between donor cells and recipient ooplasm. Lectin is a glycoprotein which specifically binds to carbohydrates to induce a tight contact of membrane to membrane (Booth et al., 2001 Cloning and Stem Cells, 3, 139–159). The purpose of this study was to examine the fusion rates and developmental competence of NT embryos following pre-incubation of couplets in medium containing lectin prior to electrical pulsing. Oocytes were collected by laparoscopic ovum pick-up from hormonally primed goats or by aspiration from culled goat ovaries, and cultured for maturation at 38.5°C, 5% CO2. At approximately 24h after the onset of IVM, the cumulus cells were stripped off by brief vortexing in medium containing 0.2% hyaluronidase. Oocytes with first polar bodies were selected for NT. Successful enucleation was confirmed by the absence of MII chromosomes in ooplasm by means of brief exposure of the Hoechst 33342-stained oocytes to UV light. Three cumulus-granulosa cell lines from transgenic goats were used as donor cells. They were cultured to confluency in DMEM+20% FCS for 6 days prior to NT. Individual donor cells were transferred into the perivitelline space of the enucleated oocytes. Couplets were incubated for 15 minutes in TCM199+10% FCS containing 75 or 150μgmL−1 lectin (L-9132 Sigma, St.Louis, MO, USA) prior to being subjected to electrical pulsing (lectin treatment) with one DC pulse at 2.4kV/cm (1st pulsing). The fusion rate was determined 40–60 minutes after the 1st electric pulsing. Non-fused couplets were exposed to a 2nd pulsing. Approximately 30 minutes later, non-fused couplets were exposed to a 3rd pulsing. Couplets without the lectin treatment served as controls. Reconstructed embryos were activated with 5μM ionomycin followed by 5h of incubation in 10μgmL−1 cycloheximide with 7.5μgmL−1 cytochalasin B. A group of 10 to 13 embryos was transferred into a recipient after 12 to 14h of culture in G1.3. Statistical analysis was performed using the χ2 analysis. Results are shown in the following table. This study demonstrated that fusion rate could be improved by pre-incubating couplets in the medium containing 150μgmL−1 lectin prior to electrical pulsing and the embryos derived from the lectin treatment could establish the early pregnancies.
281REDUCTION OF POLYSPERMY IN PORCINE IN VITRO FERTILIZATION BY MODIFIED SWIM-UP METHOD
Authors: Park C.-H. , Koo B.-S. , Yun J.-I. , Kim M.-G. , Lee S.-G. , Son H.-Y. , Lee C.-K.
Published: February 2004

In vitro production (IVP) of porcine embryos facilitates research related to biotechnology and biomedicine. Even though many attempts have been made to optimize the IVP of porcine embryos, the outcome is still unsatisfactory compared to other species, such as mouse and cattle. The high incidence of polyspermic fertilization is one of the major causes lowering the overall efficiency of porcine IVF. The common procedure for fertilization in vitro involves the co-culture of both gametes in the medium drop, which increases sperm concentration and incidence of polyspermy. Therefore, the present study was carried out to increase the efficiency of porcine IVF by reducing polyspermy using a modified swim-up method. This method modifies conventional swim-up washing by placing oocytes directly at the time of washing. Porcine oocytes were aspirated from ovaries and matured. Sperm pellet was prepared in the tube and mature oocytes were placed on a cell strainer with 70-μm pore size (Falcon 2350) at the top of the tube. After fertilization, the oocytes were fixed and stained for examination. Also, the developmental potential of fertilized embryos was measured to evaluate for the feasibility of this method. While penetration rates were similar in both methods (86.67±2.36% to 83.33±1.36%), there was a significant reduction of polyspermy in the modified swim-up method (17.50±1.60%) compared to the control (44.1±3.70%) (P<0.05). Subsequent culture showed higher rate of blastocyst formation in the modified swim-up method (20.44±0.99%) than in the control (15.73±3.26%) (P<0.05), even though the difference was not significant. These results suggest that, by controlling the number of spermatozoa reaching the oocytes, porcine oocytes might be protected from polyspermy in vitro. Also, the developmental potential of the fertilized embryos using this method could be improved by increasing the pool of spermatozoa with better quality. Further optimization of the procedure is required to impliment this method in routine porcine IVF.
282MATURATION MEDIUM EXCHANGE IS EFFECTIVE ON BOVINE EMBRYO DEVELOPMENT IN VITRO
Authors: Park Y.S. , Choi S.H. , Park H.D. , Byun M.D.
Published: February 2004

In vitro embryo development is strongly influenced by IVM conditions. Increased duration of IVM may cause aging of the oocytes, which has a harmful effect on the embryo development. Oocyte maturation depends upon the synthesis of several proteins that may play important roles in the cytoplasmic maturation. These experiments were conducted to determine the effect of IVM duration(18-h or 24-h) and medium exchange (at 18h) on embryo development, and to investigate the protein quantities in IVM medium. Korean Native Cow (KNC) ovaries were obtained from a local slaughterhouse, and cumulus-oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. Groups of 15 COCs were matured in 50-μL drops of TCM-199 supplemented with 10% fetal calf serum (FBS), 1μgmL−1 MFSH, 10μgmLLH and 1μgmL−1 Estradiol-17β for 18h or 24h. In vitro-matured oocytes were fertilized using frozen-thawed percoll separated spermatozoa (Day 0) in fer-TALP medium for 20h and cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (After Day 3). All types of cultures were carried out in an incubator at 39°C, 5% CO2 in air. The total protein quantity in IVM medium at 18h or 24h were compared by 2-dimensional gel electrophoresis using a 10–15% polyacrylamide gradient gels. Data from three replicates were analyzed by chi-square test. The proportions of oocytes reaching the blastocyst stage was significantly higher in 18h IVM group than 24h IVM group (Table 1). However, there was no difference detected in blastocyst rate between 18h IVM group and 18h medium exchange group. Total protein quantity was reduced between 18h and 24h in IVM medium. There were 299 protein spots identified in IVM medium;; there was an increase at 10 spots in the IVM medium analyzed at 18h and a decrease of 20 spots at 24h. This study suggests that duration of IVM affects subsequent embryo development. The total protein quantity was decreased between 18h and 24h in IVM medium. These proteins may be absorbed into the oocytes and reduce development to the blastocyst stage. However, this may be overcome by IVM medium exchange.

Table 1
Effects of duration of IVM and medium exchange on embryo development of KNC oocytes
283ASSESSMENT OF CALVES PRODUCED BY IVP IN A SEMI-DEFINED MEDIUM
Authors: Pugh A. , Hagenson S. , Forsyth J. , Cockrem K. , McMillan V. , Walsdorf W. , Marsh A. , van Wagtendonk A.M. , Tervit H.R.
Published: February 2004

Heavy birth weight, increased calving difficulty, heart function defects, increased perinatal mortality and organ immaturity have been reported for calves produced from IVP embryos compared to those produced from MOET or AI (van Wagtendonk AM et al., 2000 Theriogenology 53, 575–597; Jacobsen H et al., 2002 Anim Reprod Sci 70, 1–11). In this study we examined birth weight (BWT), and blood chemistry at 1 day of age, gestation length and heart function at 7 days, and response to an ACTH challenge at 21 days of calves derived from IVP in a ‘semi-defined’ IVC system (Thompson JG et al., 2000 J. Reprod. Fertil. 118, 47–55) and of contemporary MOET or AI calves. Holstein Friesian (HF) 2- and 3-year-old recipients carrying single HF calves (101×IVP and 21×MOET) were monitored in this study. Within 1 day of birth the calves were weighed and a blood sample taken for analysis. At 7d, ultrasound measurement of the left ventricle diastolic diameter (LVEDd) and % ejection fraction (EF%) was determined. Each calf was then transported to a rearing unit. At 3 weeks of age, 30 IVP and 30 control AI calves of the same age were injected i.v. with Synacthen (synthetic ACTH, Ciba Corporation, 0.1μgkg−1 body weight). Blood samples were collected at −30, 0, 30, 60 and 90min (0min=time of injection) for cortisol measurements. There was no difference in BWT for MOET or IVP calves (40.9±4.7 v. 35.6±4.8kg, respectively). Moreover, gestation lengths (279 days v. 281 days) and calving assistance scores (1.3 v. 1.6) did not differ. Calf mortality at birth was higher for IVP calves (16%) than for MOET calves (5%). All but 7 surviving calves (6×IVP and 1×MOET) had high GGT levels at 1 day. Blood chemistry revealed no differences between the calf types, all measures being within normal ranges. For all calves, heart function analysis revealed no abnormalities with mean LVEDd=4.1±0.6cm and mean EF%=78.5±8.4%. All calves exhibited elevated cortisol following ACTH challenge. There was no difference between control and IVP calves for mean cortisol concentration at any time point (0min, 13.8±5.2; 30min, 46.6±9.8; 60min, 42.8±9.9; 90min, 28.1±8.9ngmL−1). These data suggest that, unlike calves produced in less defined culture systems, calves produced by IVP in a semi-defined culture system have birth weight and gestation lengths similar to those of MOET calves. Moreover, no abnormalities in organ (heart, adrenal) function were detected. However, of concern was the high number of unexplained deaths for IVP calves. This may be due to an overall lack of vigour in IVP calves that, in an unsupervised calving, results in calf death. More vigilence at calving may be needed to ensure calf survival. The authors thank Juliet Jensen, Waikato Hospital, for ultrasound measurements and David Stewart, Morrinsville Veterinary Services, for calf care. This study was funded by Vialactia Biosciences and FRST.
288EFFECTS OF OVIDUCTAL EPITHELIAL AND CUMULUS CELLS ON FERTILIZATION AND
EMBRYO DEVELOPMENT OF PIG OOCYTES IN VITRO
Authors: Yang B.S. , Day B.N.
Published: February 2004

This study was carried out to investigate the effect of adding porcine oviductal epithelial cells (POEC) and also the presence of cumulus cells during in vitro fertilization on fertilization rate and subsequent embryo development of pig oocytes matured in vitro. Cumulus-oocyte complexes (COCs) aspirated from 2- to 6-mm follicles were matured in TCM 199 supplemented with cysteine, EGF, eCG, hCG, and PVA for 20–22h, and cultured in the same medium without hormone for an additional 20–22h. Oviducts attached to ovaries without CL were used to obtain epithelial cells. After removal of the fimbria and utero-tubal junction, oviducts were flushed with MEM supplemented with 10% FBS, and POEC clumps were cultured in the same medium for 48h. After the completion of maturation, COCs were randomly divided into four groups;; cumulus-denuded (D), cumulus-denuded with POEC (DP), cumulus-enclosed (E), and cumulus-enclosed with POEC (EP). Eight to 10 POEC clumps were co-cultured with sperm and oocytes in a 100-μL fertilization drop. Oocytes were fertilized with frozen-thawed spermatozoa for 6h in modified Tris-buffered medium containing caffeine and BSA. Presumptive zygotes were cultured in NCSU23. Oocytes were fixed and stained for the evaluation of penetration at 12h after IVF (n=549 oocytes), and cleavage rate and blastocyst formation were evaluated at 48 and 144h after IVF (n=1531 oocytes), respectively. Results were analyzed by Duncan’s multiple range test using GLM procedure in SAS. Although the sperm penetration rate in group E was lowest among all groups (P<0.05), the monospermic fertilization rate was not significantly different among treatment groups (68.6–81.9%). Although the cleavage rate and percentage blastocyst in group E were significantly lower than other groups (38.1 v. 53.6, 52.0 and 44.6%, and 15.0 v. 21.2, 23.4 and 18.5% in group D, DP, and EP, respectively), blastocyst cell number was not significantly different among treatment groups (24.9–27.3) These results suggested that the presence of cumulus cells alone during fertilization interferes with sperm penetration, cleavage, and blastocyst formation and that POEC may improve both cleavage and blastocyst formation rate.
29HEALTH AND REPRODUCTIVE PROFILES OF NUCLEAR TRANSFER GOATS PRODUCING
THE MSP1-42 MALARIA ANTIGEN
Authors: Behboodi E. , Ayres S.L. , Memili E. , O'Coin M. , Chen L.H. , Meade H.M. , Echelard Y.
Published: February 2004

Somatic cell nuclear transfer (NT) using transfected primary cells is an efficient approach for the generation of transgenic large animals. However, abnormalities associated with the NT process could compromise the usefulness of cloned animals. In order to evaluate whether cloned animals could be used for the production of recombinant therapeutic proteins in milk, we examined the health, reproductive performance and milk production of 4 transgenic does derived from somatic cell NT. Two versions of the MSP1-42 transgene encoding either a glycosylated or a non-glycosylated MSP1-42 protein were constructed. Following somatic cell NT, 4 healthy transgenic does were born (Chen LH et al., 2002 Theriogenology 57, 777 abst). Does 1A and 2A were obtained from the same cell line carrying the glycosylated MSP1-42 transgene, whereas 3A and 4A were from a cell line carrying the non-glycosylated transgene. Health and growth of these NT animals was monitored with the supervision of a veterinarian and compared to age-matched control dairy does. All animals were bred naturally and, except for one control doe, delivered healthy kids. The 4 NT does produced a total of 9 kids of which 8 were live and one was stillborn. The control does delivered 5 kids, one fetus being lost during early pregnancy. Three of the eight kids from the NT animals were transgenic; all kids were normal and healthy. All does were milked after parturition, and recombinant protein expression in the milk of NT does was evaluated. There were no differences in birth and weaning weights or fertility between NT and control animals. The NT does expressing the glycosylated antigen (1A, 2A) produced only small amounts of milk at induction and postpartum. The reduced milk yield for that line is possibly caused by the expression of the transgene. However, normal lactations were achieved with the 2 does expressing the non-glycosylated antigen (3A, 4A), each producing in 100 days enough MSP1-42 protein to generate 5 million doses of the Malaria vaccine. These data demonstrate that transgenic goats produced by somatic cell NT can be used successfully for the recombinant production of therapeutic proteins.
290EFFECTS OF HYALURONAN ON IN VITRO DEVELOPMENT OF PORCINE EMBRYOS
CULTURED IN A CHEMICALLY DEFINED MEDIUM
Authors: Yoshioka K. , Ekwall H. , Rodriguez-Martinez H.
Published: February 2004

Hyaluronan (HA), a glycosaminoglycan present in follicular and oviductal fluids, has been related to sperm capacitation, fertilization and embryo development. We have previously developed an in vitro-production (IVP) system of porcine embryos, where porcine blastocysts can be produced by IVF and IVC in chemically defined media and can develop to full-term by transfer to recipients. The application of a chemically defined medium to IVP in pigs allows the analysis of the physical action of substances on the development of pre-implantation embryos. In the present study, the effects of HA on the development of porcine embryos in a chemically defined medium were investigated. Porcine presumptive zygotes were produced by IVM and IVF of COC from pre-pubertal gilts and frozen-thawed ejaculated boar semen. The zygotes were cultured in Porcine Zygote Medium (PZM)-5 containing different concentrations of HA (0 [control], 1, 2, 5, 10, 20 and 50μgmL−1) until 6 days after IVF, and representative specimens were fixed for cell counting and transmission electron microscopy. Data of percentages and cell numbers were statistically analyzed by one-way ANOVA and Fisher’s PLSD test. The percentage of embryos that developed to the blastocyst stage (15.8% [23/144] to 19.5% [27/139]) did not differ among treatments. However, addition of 5 or 10μgmL−1 HA increased (P<0.05) the total number of cells in blastocysts (56.1 and 58.3 cells [n=22 and 23], respectively) compared to control (no HA, 42.0 cells [n=23]). To evaluate proliferation rates of inner cell mass (ICM) and trophectoderm (TE), embryos were cultured in PZM-5 for various periods of exposure to 10μgmL−1 HA. The numbers of ICM and TE cells in Day-6 blastocysts cultured in the presence of exogenous HA from Day 0 to Day 3 (18.3 and 34.4 cells, respectively [n=38]) or Day 6 (17.9 and 35.9 cells, respectively [n=36]) were significantly (P<0.05) higher than those cultured without HA through the culture period (13.5 and 24.2 cells, respectively [n=26]). In the presence of HA from Day 3 to 6, only the number of TE cells (37.1 cells [n=33]) increased (P<0.05), compared to PZM-5 alone. Differences in ultrastructure were noticed among blastocysts cultured with or without 10mgmL−1 HA. Blastocysts cultured with HA had mainly mature mitochondria while many mitochondria appeared morphologically immature in the blastocysts cultured without HA. Lipid droplets in the blastocysts cultured with HA seemed to be more homogeneous in comparison with those in the blastocysts cultured in PZM-5 alone. Further differences were seen in the numbers of lysosome-like structures, which were greater in blastocysts cultured with HA. This study demonstrates that exogenous HA improves cell proliferation and normality of ICM and TE in porcine embryos cultured in a chemically defined medium, depending on the exposure periods to HA. (Supported by MAFF, Japan and STINT, Sweden.)
295POSSIBLE DOPAMINERGIC MECHANISM REGULATING THE EQUINE CORPUS LUTEUM
Authors: Dille E.A. , King S.S. , Jones K.L. , Roser J.F. , Pearl C.A.
Published: February 2004

Timing of embryo transfer to recipient mares is crucial to the success rate of an embryo transfer (ET) program. Recipient mares need a functional corpus luteum (CL) to maintain the early stages of pregnancy. Interference with luteal function appears to be a significant cause of failure with nonsurgical ET. Little is known about the endocrine control of luteal function in the mare, and the possibility of neuronal control of equine luteal function has not yet been studied. Dopamine (DA) has been shown to affect progesterone secretion in the bovine CL. Prior research in our laboratory suggested the possibility of dopaminergic regulation in the equine CL. The aim of this study was to document the presence of DA D1 receptor (D1r) and DA D2 receptor (D2r) within the equine CL. Immunocytochemistry (ICC) was performed on sections from 9 corpora lutea collected from a local equine abattoir. Tissues were stained using the avidin-biotin complex (ABC) method (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA, USA). Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm sections. Tissue sections were deparaffinized and rehydrated;; endogenous peroxidase activity was quenched in 0.3% hydrogen peroxide in methanol at room temperature. To decrease nonspecific staining, tissue sections were incubated in goat serum (Vectastain ABC Elite kit). Tissue sections were then incubated overnight at 4°C with primary antibodies: Rabbit anti-Dopamine D1 receptor (Calbiochem, San Diego, CA, USA) or Rabbit anti-D2 receptor polyclonal antibody (Chemicon International, Inc, Temecula, CA, USA). Sections were then washed in PBS and incubated in biotinylated goat anti-rabbit IgG secondary antibody. After washing in PBS, sections were incubated in ABC. Sections were washed in PBS and the signal was visualized using 3-amino-9-ethylcarbazole (AEC Red, Vector Laboratories, Burlingame, CA, USA). Tissues were then counterstained with Immunomaster Hematoxylin (American Master Tech Scientific, Inc., Lodi, CA) to visualize the nuclei. As a negative control, tissues were incubated in normal rabbit serum instead of primary antibody, and as a positive control, the ICC procedure was performed on whole rat brain slices. Significant staining of luteal cells was observed using the D1r and D2r antibodies. Positive staining for D1r and D2r was seen throughout luteal cells;; however, no nuclear staining was observed. The presence of these receptors in equine CL tissue suggests a functional significance for DA in luteal function. Further research needs to be performed to determine the mechanistic function of dopamine in mare reproduction.
296LIPIDIC CONTENT IN JERSEY BLASTOCYSTS COMPARED WITH HOLSTEIN AND IVP
EMBRYOS
Authors: Pegoraro L.M. , Barros S. , Sinowatz F. , Palma G.A. , Saalfeld M.H. , Coscioni A.C. , Suita L.A. , Rocha N. , Abrantes V. , Aghnonni L.
Published: February 2004

The objective of this study was to compare the ultrastructure of bovine embryos from different breeds and origin in terms of lipid contents. Jersey and Holstein embryos produced in vivo were obtained from superovulated donors by non-surgical method 7 days after AI. Embryos produced in vitro (Holstein cross breed) were obtained from cumulus-oocytes complexes (COC) aspirated from slaughterhouse ovaries. The COC were matured and fertilized in vitro. The zygotes were cultivated in vitro for 7 days in SOFaa media. Embryos produced in vivo (Holstein n=5; Jersey n=5) and in vitro (n=5) classified as blastocyts grade II were fixed in Karnovsky solution immediately after embryo recovery or embryo culture and prepared for microscopic electronic evaluation. Ultrastructure of inner cell mass and trophoblast cells was analyzed. Morphometry on electron microscopy was performed using a point-count method in random samples of electron micrographs of each embryo category. The data were analyzed by chi square test. The volume density occupied by number of lipid droplets was greater in Jersey and in vitro-produced embryos compared with Holstein embryos (24.3%±11.7; 28.4%±19.6 and 9%±6.68, respectively, P<0,05).
3P66SHC, BUT NOT P53, IS INVOLVED IN EARLY ARREST OF IN VITROPRODUCED BOVINE EMBRYOS
Authors: Favetta L.A. , Robert C. , King W.A. , Betts D.H.
Published: February 2004

High embryo losses occur in the first week of bovine embryo development, before the activation of the embryonic genome, with a high percentage of embryo death and arrest. Arrested embryos appear morphologically normal and do not exhibit any characteristic sign of apoptosis, including DNA fragmentation. We hypothesized that these embryos enter a senescence-like state and that both the cell cycle regulatory protein p53 and the stress-related protein p66shc, which are involved in the onset of senescence in somatic cells, are responsible for this early embryonic arrest of development. The aim of this study was to characterize the expression of p53 and p66shc in 2–4 cell-arrested bovine embryos. Our experimental model consists of in vitro-produced bovine embryos, co-cultured with oviductal cells. In our in vitro production system 86.8±41.4% of embryos cleave, 13.5±0.5% arrest at the 2–4 cell stage and 24.5±0.7% develop to the blastocyst stage. Cleavage occurs between 26 hours post-insemination (hpi) and 48 hpi. Embryos that cleave by 28hpi show only 0.6±0.3% of 2–4 cell arrest and 41.2±2.1% of blastocyst development, whereas 14.2±0.9% of later-cleaving embryos arrest at the 2–4 cell stage and only 26.5±1.7% develop into blastocysts. We compared 2–4 cell embryos collected at 28hpi with those arrested at the 2–4 cell stage and collected at Day 8 post-insemination. Quantification by Real Time PCR showed significantly higher p66shc mRNA levels (P<0.001), but no changes in p53 mRNA levels (P=0.860) in arrested embryos v. 28-hpi embryos (n=3 pools of 100 embryos each). We obtained the same pattern of p53 and p66shc mRNA expression when we compared 28-hpi embryos with later-cleaving embryos (28hpi to 48hpi, n=3 pools of 100 embryos each), and higher p66shc mRNA levels (P<0.050), or similar p53 mRNA levels (P=0.960). We also confirmed higher p66shc protein levels (P<0.001), but no changes in p53 protein levels (P=1.000), in later cleaving embryos compared with 28hpi-cleaving embryos by semi-quantitative immunocytochemistry (n=70). Statistical analysis was carried out using 2-sample t-test or the equivalent nonparametric test (Mann-Whitney test). Taken together, these results demonstrate that the developmental potential of in vitro-produced embryos is related to the time of first cleavage and that p66shc, but not p53, plays a role in early developmental arrest of in vitro-produced bovine embryos. Further experiments are required to investigate the functional role of p66shc in early embryo arrest. Funded by NSERC, CIHR, OGS and OMAFRA.
304INFLUENCE OF ELECTRICAL STIMULUS ON ACTIVATION OF PIG OOCYTES MATURED
IN VITRO
Authors: Park Chang Sik , Jin Dong Il , Chang Young June , Kim Moon Young , Yi Young Joo
Published: February 2004

Electrically induced activation of pig oocytes deserves particular attention for research on parthenogenesis. The aim of this study was to improve electrical activation of in vitro matured pig oocyte. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes involving first polar body were selected for activation. For electrical activation, oocytes were rinsed twice in 0.3M mannitol solution supplemented with 0.1mM CaCl2, 0.2mMMgCl2, 0.5mM HEPES and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1mm apart which were overlaid with the same activation solution. Experiment 1 was conducted to investigate the effect of electrical pulse on oocyte activation. Oocytes were activated with DC pulses of 1.0, 1.5, 2.0 and 2.5kVcm−1 for 30, 60 and 90μs, respectively. Experiment 2 was carried out to investigate the effect of electrical stimulus frequency on oocyte activation. Oocytes were activated one, two and three times, with a DC pulse of 1.0kVcm−1 for 60μs. After activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA and cultured for 20h. Activated oocytes were fixed for 48h in 25% acetic acid (v:v) in ethanol at room temperature, and stained with 1% orcein (w:v) in 45% acetic acid (v:v) to examine pronucleus formation. Data were analyzed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of activation was highest in the DC pulse of 1.0kVcm−1 for 60μs (75.1%) compared with the other durations and strengths (62.5–63.1%). Activation rate by electrical stimulus frequency was highest (76.0%) when oocytes were activated by a one-time pulse. In conclusion, the results suggested that electrical stimulus with a single DC pulse of 1.0kVcm−1 for 60μs might be more efficient than other strengths and durations for activation of pig oocytes.
308MITOCHONDRIA RELOCATION, MICROTUBULE ASSEMBLING AND PARTHENOGENETIC
DEVELOPMENTAL COMPETENCE OF PIG OOCYTES
Authors: Brevini T.A.L. , Vassena R. , Francisci C. , Gandolfi F.
Published: February 2004

Developmental competence of in vitro-produced porcine embryos appears to be limited by specific maternally inherited cytoplasmic factors. We previously reported a relationship between mitochondria distribution during IVM, energy status, and oocyte developmental ability after parthenogenetic activation. The aim of the present study was to investigate the timing of mitochondria relocation during meiosis and the possible relationship with cytoskeleton organization in high and low competence oocytes. To this purpose, homogeneous groups of oocytes were matured in vitro (IVM) with 25% or 0% porcine follicular fluid (pff) to obtain different cytoplasmic competence (high and low, respectively) but similar nuclear maturation (Brevini et al., 2003, Theriogenology, 59, 440). After maturation, oocytes were parthenogenetically activated and cultured as previously described by Grupen et al., 2002 (Mol. Reprod. Dev., 62, 387–396). Active mitochondria were stained with MitoTracker® Orange CMTM Ros (Molecular Probes, Leiden, The Netherlands) at GV, MI and MII meiotic stages. At the same time microtubule organization was determined by immuno-cytochemistry using an antibody raised against α-tubulin (Sigma, St. Louis, MO, USA). Meiotic stages were assessed with DAPI. Specimens were examined with a Leica TCS-NT confocal microscope through an equatorial optical section. Nuclear maturation rate was comparable in the two groups at the end of IVM (46h). Mitochondria relocation from the periphery to the center of the oocyte was evident as early as 20h IVM in the 25% pff group (high competence), while 0% pff oocytes (low competence) did not show any mitochondria relocation at this time point. In agreement with the literature, α-tubulin was not detectable in GV oocytes, while at the end of IVM, α-tubulin was associated with the DNA, forming the meiotic spindle both in high and low competence oocytes. However, oocytes in the 25% pff group displayed a cytoplasmic microtubular organization that co-localized with mitochondria at 20 to 28h IVM. Conversely, α-tubulin was not detected in the cytoplasm of 0% pff oocytes at the same time points and 71% of these oocytes did not undergo any mitochondria relocation at all by the end of IVM. Altogether the present results show that mitochondria relocation takes place at a well-defined time during IVM and is temporally associated with the formation of the microtubule mesh in the oocyte cytoplasm. Low-developmental-competence oocytes display an altered mitochondria distribution and microtubule arrangement or seem to lack the temporal coupling of the two phenomena. We speculate that a tightly linked timing of mitochondria relocation and cytoskeleton microtubule formation in the cytoplasm of the oocyte during IVM may represent a key cytoplasmic factor regulating pig embryo parthenogenetic development.
317EMBRYONIC DEVELOPMENT AFTER HOLDING BOVINE OOCYTES IN UNDILUTED
FOLLICULAR FLUID FOR A 6-HOUR PREMATURATION PERIOD
Authors: Klumpp A.M. , Denniston R.S. , Paccamonti D. , Leibo S.P. , Godke R.A.
Published: February 2004

Follicular fluid has been implicated in follicular growth, initiation of steroidogenesis, regulation of granulosa cell function and oocyte maturation. Although individual components of the follicular fluid have been analyzed, it is still unclear how undiluted follicular fluid may affect subsequent in vitro oocyte maturation (IVM), in vitro fertilization (IVF) and embryo development. The objective of this study was to simulate recovery of bovine oocytes as if under field conditions to determine the effect of holding these oocytes in follicular fluid for a 6-h holding period before performing IVF. Ovaries were obtained from a local abattoir and transported to the laboratory at ∼22°C in a 0.9% saline and antibiotic solution. At the laboratory, ovaries were rinsed with an ethanol solution and randomly allotted to four treatment groups. Follicles ranging in size from 2 to 9mm were aspirated using a 20-gauge needle attached to a sterile plastic syringe. Dominant follicles were not included. Oocytes harvested from the ovaries in Treatment 1 (Control) were placed directly into a standard laboratory maturation medium consisting of TCM-199 supplemented with LH, FSH, fetal bovine serum, estradiol and gentamicin for 22h. Standard laboratory IVF was then performed with frozen-thawed semen from a fertile bull. In Treatment 2, 3mL of pooled follicular fluid was dispensed into a 6-mL conical centrifuge tube and co-incubated with the harvested oocytes at room temperature (22°C) for 6h. Oocytes recovered from the ovaries in Treatment 3 were placed into 3mL of Ringer’s lactate solution for a 6-h holding period at 22°C. Oocytes obtained from the ovaries in Treatment 4 were placed into a mixture of 2mL of Ringer’s lactate plus 1mL of the same pooled follicular fluid and were held for 6h at 22°C. After being held for a 6-h period, oocytes were recovered from each centrifuge tube and were placed into IVM medium for 22h and then subjected to standard IVF. Embryo development in CR1aa culture medium was assessed at 72, 168 and 216h post-insemination in each treatment group. In summary, no significant difference was detected between the standard IVF procedure and oocytes held in follicular fluid 6h prior to IVF (Treatment 1 v. 2). Also, follicular fluid apparently had a positive effect on the oocytes over that of holding oocytes 6h in Ringer’s lactate alone, as indicted by a significantly greater rate of blastocyst development (Treatment 3 v. 4). In conclusion, it should not be overlooked that bovine oocytes, aspirated under field conditions, could be held up to 6h in 22°C undiluted bovine follicular fluid until they can be delivered to a full-service IVF laboratory.