bluuebunny/crossref_metadata_2025 huggingface dataset:
174EMBRYO TRANSFER OF VITRIFIED IVF EMBRYOS IN CATTLE: PREGNANCY
COMPARISON AFTER SINGLE AND DOUBLE TRANSFER
Authors: Du F.L. , Dinnyes A. , Sung L.Y. , Xu J. , Jiang S. , Tian C.X. , Yang X.
Published: February 2004

Advancement in vitrification of in vitro-produced bovine embryos will benifit the cattle breeding and production industry. The objective was to evaluate whether bilateral (double) embryo transfers (ET) can improve pregnancy rate compared to ipsilateral (single) transfers. Bovine cumulus-oocyte complexes collected from slaughterhouse ovaries were matured for 20–22h, and subsequently subjected to a standard Brackett and Oliphant in vitro fertilization (IVF). Six hours after IVF, embryos denuded of cumulus were cultured in defined CR1 medium supplemented with essential and non-essential amino acids (CR1aa), plus 6mgmL−1 BSA for 2 days at 39°C under 5% CO2, 5% O2 and 90% N2, and then cultured in CR1aa medium supplemented with 7.5% FBS for a further 5 days on bovine cumulus monolayers. Expanded blastocysts with tighter compaction of the inner cell mass (quality 1) were selected on Day 7 for cryopreservation via modified solid surface vitrification (Dinnyes et al., 2000 Biol. Reprod. 513–8). Vitrification solution contained HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol and dimethylsulphoxide. A droplet of 1–2μL vitrification solution containing 4–5 blastocysts was dropped directly onto a cooled surface within 30s after 3-min incubation in equilibration solution. Prior to ET, embryos were warmed and subsequently washed several times in 0.25M sucrose rehydration solution and M199+7.5% FBS medium. The warmed embryos from initial trials were cultured for 2 to 72h to evaluate their viability after vitrification. During ET trails, vitrified embryos were loaded into transfer straws (one embryo per straw) after warming. The treatments were as following, (1) single transfers, one embryo was transferred into the horn ipslateral to CL; (2) double transfers, one embryo was transferred by non-surgical means into each uterine horn of a synchronous recipient on Day 7. ET trails were conducted in both the USA (double transfers) and China (single v. double transfers). Pregnancy was determined by palpation per rectum around Day 70 after transfer. The data were compared by Student’s t-test. The survival rate of vitrified IVF embryos reached as high as 91.4% (n=256) 2h post-warming, and hatching rate was 70.7% (n=154) 72h after culture in vitro, respectively. The data (Table 1) show that double transfers resulted in a significantly higher pregnancy rate than did single transfers (P<0.05). With double transfers, a higher pregnancy rate was achieved in the USA than in China (76.2% v. 45.6%, P=0.079). This study confirms that double embryo transfers can improve the pregnancy outcome after ET, perhaps because bilateral placement of embryos may increase embryonic signals to the maternal environment. Further evaluation of gestation length, single/twin conception and calving difficulty is under investigation.

Table 1
Pregnancy rate (Day 70) of vitrified bovine IVF embryos following single and double transfer
177HYSTEROSCOPIC BLASTOCYST IMPLANTATION A NOVEL EMBRYO TRANSFER PROCEDURE
Authors: Kamrava M. , Yin M.
Published: February 2004

Various techniques using different types of catheters have been advocated to increase pregnancy rates while reducing side effects from the embryo transfer procedure. However, all of these techniques are ‘blind’ procedures of catheter introduction into the uterus, and the problems of ‘lost embryos’ and the occurrence of ectopic pregnancies persist. A novel hysteroscopic-guided direct embryo transfer procedure with visually directed embryo implantation was developed to improve the current ‘blind’ embryo transfer procedures by increasing chances of success while eliminating tubal pregnancies and decreasing high-order multiple pregnancies from IVF related techniques. At West Coast Infertility Medical Clinic, 57 patients with average age of 28.43±4.54 were analyzed. Stimulation method: controlled ovarian hyperstimulation was initiated with Follitropin β; (Follistim®, Organon Pharmaceuticals, Inc.). Premature endogenous gonadotropin surge (i.e. the prevention of an LH surge) was controlled with ganirelix acitate (AntagonTM, Organon Pharmaceuticals, Inc., West Orange, NJ, USA.). Oocyte retrieval was performed in an office setting under local anesthesia and mild sedation, followed by routine IVF/ICSI, IVC. By Day 5–6, up to 2 best quality blastocyst stage embryos were transferred to patient’s uterus by ‘hysteroscopic embryo implantation’ procedure: a lightweight hybrid (rigid/flexible) mini hysteroscope (Napoli, Inc., Los Angeles, CA, USA) was used for visualization of the endometrial cavity. The scope incorporates a flexible distal end of 3mm in diameter with a straight-through operating channel. In addition, the optic filter is directly connected to a light source, decreasing the weight of the scope and giving a better feel for the scope. The transfer catheter (Napoli, Inc.) is polycarbon based with a tapered tip (to 500μm), beveled to 60°. During embryo transfer procedure, the catheter tip was inserted into a depth of 1mm from the surface of the endometrium under direct hysteroscopic visualization. The loaded embryos with 10μL medium was released underneath the endometrium to produce a ‘bubble’ cushion. Luteal phase support was provided (3000IU of hCG at Day 3 and Day 6 post-retrieval, separately). Pregnancies were determined by serum hCG concentration of 5IUmL−1 or more at Day 16 post-retrieval. Thirty out of 57 (52.6%) women became pregnant. Multiple pregnancy rate was 4 out of 30 (13.3%) and comprised only of twins, and no ectopic pregnancy was found. In conclusion, a newly developed instrument and embryo transfer procedure by mechanical implantation of the embryo was achieved. By implanting the embryos, we have reduced the number of embryos that are transfered, minimized the chances of ‘losing’ embryos, and eliminated ectopic pregnancies.
178EMBRYO QUALITY AND COLOR IN HOLSTEIN FRIESIAN AND BELGIAN BLUE CATTLE
IN RELATION TO DONOR BLOOD CHOLESTEROL AND TRIGLYCERIDES
Authors: Leroy J.L.M.R. , Goossens L. , Geldhof A. , Vanholder T. , Opsomer G. , Van soom A. , de Kruif A.
Published: February 2004

Bovine embryo freezability is closely linked with quality and differs between cow breeds (Visintin JA et al., 2002 Theriogenology 57, 345–359). Dark embryos have a bad freezability, resulting in low pregnancy rates (Hill BR and Kuehner LF, 1998 Theriogenology 49, 168). Excessive accumulation of lipid droplets in dark embryos is suggested to be the cause of this inferior embryo quality and in turn may be influenced by the biochemical composition of the embryonic environment. In a field trial we studied the relationship between donor breed (Holstein Friesian dairy cows v. Belgian Blue beef cows), donor serum total cholesterol and triglyceride concentration, embryo quality and embryo color. The preliminary results are presented. Blood was drawn from Holstein Friesian (HF) (n=74) and Belgian Blue cows (BB) (n=55) prior to embryo flushing on Day 7 after superovulation and subsequent insemination. Serum was analyzed for total cholesterol and triglycerides using commercial photometric assays. Embryos were scored individually by the same operator for quality (excellent to bad: score 1 to 4) and for color: light (L), medium (M) or dark (D) using a binocular stereomicroscope (40X). Student’s t-test, chi-square test and Spearman correlations were used when appropriate. Holstein Friesian cows yielded significantly more embryos (±SEM) per flushing than did BB (6.0±0.59 v. 4.2±0.44) (P<0.05). However, significantly more HF embryos were classified as dark compared to the BB embryos (L, M, D: 39.4%, 43.8% and 16.8% v. 80.5%, 19.5% and 0% of the HF and BB embryos, respectively) (P<0.05). Significantly more BB embryos showed an excellent morphological quality (55.0% of BB embryos compared to 28.4% of HF embryos) (P<0.05). The serum concentrations (±SEM) of total cholesterol (148±5.9mgdL−1 v. 106±4.1mgdL−1) were significantly higher and the serum concentrations of triglycerides (19.6±0.71mgdL−1 v. 28.4±3.34mgdL−1) were significantly lower in HF compared to BB (P<0.05). Within the HF breed, there was a significant positive correlation between donor blood total cholesterol and the color of each embryo (r=0.545) and a negative correlation between serum triglycerides and embryo color (r=−0.484) (P<0.05). In our study, embryo quality and color as well as serum total cholesterol and triglyceride concentrations are clearly influenced by donor breed. Within the HF breed, darker embryos originate from donors with higher serum total cholesterol and lower serum triglyceride levels. These preliminary data suggest that factors influencing total cholesterol and triglyceride concentrations in the donor blood (e.g. breed, milk yield, nutrition) may influence embryo color and thus embryo lipid content, freezability and subsequent pregnancy rate.
182LAPAROSCOPIC EMBRYO TRANSFER IN KOREAN BLACK GOATS
Authors: Shin S.T. , Jang S.K. , Yang H.S. , Lee O.K. , Shim Y.H. , Choi W.I. , Lee D.S. , Cho J.K. , Lee Y.W.
Published: February 2004

In this study, laparoscopic embryo transfer (ET) was conducted to overcome the disadvantages of laparotomic ET including invasiveness, adhesions and duration of surgery in Korean black goats (Capra hircus aegagrus). Transferred transgenic embryos were produced by DNA pronuclear microinjection of in vivo derived zygotes. Recipient goats were synchronized in estrus by using an intravaginal progesterone device CIDR® for 13 days and injection of 400IU PMSG 48hrs before CIDR® removal. Embryos were transferred on day 4 after CIDR® removal. Recipient goats were starved for 48hrs and suspended head down in an operating table at an angle of 45° to the horizontal under general anesthesia. A Veress needle (Vomed, Tuttlingen, Germany) was inserted through the abdominal wall to make a pneumoperitoneum. After obtaining sufficient pneumoperitoneum, a 5-mm laparoscope and grasping forceps (MGB, Berlin, Germany) were inserted through the 5-mm trocar sleeves. After investigation of the ovaries, uterine horns and oviducts, embryos in a polyethylene tube (SP 65, Nastume, Tokyo, Japan) were transferred into the oviduct via the infundibulum in 76 recipients. To compare the pregnancy rates, laparotomic ET was also conducted in 21 recipients. In both groups, two microinjected embryos were transferred per recipient. Pregnancy of the recipient goats was examined by ultrasound on day 30 after embryo transfer. Pregnancy rates of laparoscopic ET were significantly higher than those of laparotomic ET (46.1% v. 28.6%; P<0.05). Our results suggest that laparoscopic ET is a highly efficient method for the transfer of goat embryos.
188FATE OF CHIMERIC EMBRYONIC STEM CELLS RECONSTRUCTED WITH
PARTHENOGENETIC MOUSE EMBRYOS
Authors: Kim J.Y. , Uhm S.J. , Chung K.S. , Lee H.T.
Published: February 2004

Mouse parthenogenetic embryos have been known to be unable to develop to term and are absorbed from Day 11. However, Nagy et al. (1990 Development 110, 815–821) reported that the aggregated tetraploid with embryonic stem (ES) cell developed to term and survived after birth. Thus, our study investigated the developmental capacity of the aggregated ES cells with mouse parthenogenetic embryos. Oocytes obtained from superovulated female mice (BCF1) were treated with 7% ethanol and 5μgmL−1 cytochalasin B for the production of pathenotes and co-cultured with sperm (1×106mL−1) for production of fertilized embryos. The reporter vector (pNeoEGFP) was introduced into ES cells (129S4/SvJae) by electroporation. At the 8-cell stage, pathenotes or fertilized embryos, from which the zona pellucida was removed, were co-cultured with ES cells for 4 h. The aggregated parthenotes or fertilized embryos with 5∼10 ES cells were cultured to the blastocyst stage, and transferred into the uteri of 2.5-day post-coitum pseudopregnant recipients. In experiment I, 144 parthenogenetic blastocysts were transferred into the uterine horns of 9 pseudopregnant recipients, and 5 recipients became pregnant. At Day 9, all fetuses were observed visible in uteri of pregnant fonder mice. At Days 10–11, many fetuses were observed in the progress of absorption in uteri of pregnant fonder mice, but a few fetuses were still alive. However, pathenogenetic fetuses were not detected alive beyond 11 days. In experiment II, the 171 aggregated fertilized embryos with ES cells were transferred (15–20 blastocysts/recipient) into 10 recipients and successfully produced 5 offspring from a recipient. We found that three newborn were chimeric mice derived from ES cells. In experiment III, the 209 aggregated parthenotes with ES cells failed to produce offspring, but inserted pNeoEGFP gene in ES cells was detected in the parthenogenetic 1 of 7 fetuses at 15-days of post-gestation by polymerase chain reactions. Therefore, this result suggests that the parthenotes show restricted development to fetus stage, but the aggregated parthenotes with ES cells might extend their developmental capacity. In the future, we will characterize the mechanism of this unusual phenomenon to understand the role of ES cells during development of chimeric parthenotes with ES cells.
190FEEDERS FROM RHESUS MONKEY SUPPORT PROLONGED DIFFERENTIATED GROWTH OF
RHESUS MONKEY EMBRYONIC STEM CELLS
Authors: Li T. , Xie Y. , Zhang X. , Wang S. , Ji W.
Published: February 2004

Previous study demonstrated that feeders are important for growth and differentiation of rhesus monkey embryonic stem cells (rES)(Thomson JA et al., 1995 PNAS \bf 92, 7844–7848). In this study, we developed 5 rhesus monkey feeder cell lines for rES culture: MESF (ear skin fibroblasts from 2-week-old neonatal monkeys), MAF (adult Fallopian tube cells), MGF (adult follicular granulose fibroblast-like cells), MGK (adult follicular granulose kidney-like cells), and MSC (single-cell cloning from MESF), all of which were fibroblast-like cells. Two experiments were designed to investigate rES cell growth and differentiation potentials. In experiment 1, rES (R366.4) was cultured on MESF, MAF, MGF, MGK, and MSC, with MEF (mouse embryonic fibroblasts) as control. In experiment 2, rES was cultured on the mixed feeders: MSC: MGK at 1:1, 7:3, 8:2 and 9:1, depending on the results of experiment 1. Results of experiment 1 showed that the rES underwent undifferentiated growth on MESF, MAF, MGF and MSC (cultured for 15 passages), but not on MGK, with morphology typical of rES on MEFs, and exhibiting positive alkaline phosphatase staining, positive expression of Oct-4 and GAPDH, and negative expression of PAX6, AFP, BMP4 and hCG of rES as well as normal karyotypes. Embryonic bodies (EBs) were formed on Day 7 to 8 from the rES cultured on MESF, MAF, MGF and MSC. The EBs showed positive expression of hCG, AFP and BMP4. Differentiation of neuron, epithelium and muscle cells was observed after 21 days of continuous culture of the rES. The colonies of rES cultured on MESF, MAF, MGF and MSC were higher than on MEF (1.5 to 3 times). Interestingly, expansion speeds and differentiation rates on MSC were less than on MESF. These results also indicated that fibroblast cells, on the one hand, were alone sufficient for supporting prolonged undifferentiated growth of rES, and on the other hand, other mixed cells except fibroblast cells had the ability to promote the growth and differentiation of R366.4 rES. Results of experiment 2 showed that the mixed feeders (MSC:MGK) at 8:2 or 9:1 supported undifferentiated growth of rES but not feeders at 7:3 or 1:1. Our work demonstrated that the rhesus monkey feeders were better able to support undifferentiated growth and maintain differentiation potentials of rES than were MEFs feeders. The rES could grow if the ratio of mixed other cells serving as the feeders exceeded 20%.
192ISOLATION AND CULTURE OF EMBRYONIC STEM CELL-LIKE CELLS FROM IN VITRO
FERTILIZED PORCINE BLASTOCYSTS
Authors: Son H.-Y. , Park C.-H. , Lee S.-G. , Lee G.-S. , Kim H.-S. , Kim S. , Kang S.-K. , Hwang W.-S. , Lee C.-K.
Published: February 2004

The establishment of porcine embryonic stem (ES) cell lines should be useful for the production of transgenic pigs and studies of developmental gene regulation. Recent development of techniques for production of embryos in vitro could be a useful source for the isolation of ES cells. Therefore, to establish porcine ES cells, this study was conducted to isolate and culture inner cell mass (ICM) from in vitro-fertilized (IVF) porcine blastocysts. Cumulus-oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Oocytes were then fertilized using a modified swim-up method to prevent polyspermy and cultured to the blastocyst stage. Initial culture of ICM was conducted after either culture of whole embryos or isolation of ICM by immunosurgery. Developing IVF embryos were continuously cultured in 50% DMEM and 50% F-10 with 15% fetal bovine serum, 1% non-essential amino acids, 1.7mM L-glutamine, 1% penicillin/streptomycin, 0.1mM α-mercaptoethanol, 1000 unit recombinant human LIF, 40ngmL−1 recombinant human SCF and 20ngmL−1 recombinant human basic FGF on a mytomycin-C-inactivated murine embryonic fibroblast (MEF) feeder layer. Antibodies against porcine cells were produced in rabbit. After removal of zona pellucida, ICMs were isolated by immunosurgery and cultured on feeder cells the same as described above. After IVF, the rates of 2-cell embryos and blastocysts were 70.8% and 20.4%, respectively. Results from the isolation and culture of ICMs of porcine blastocysts are shown in following table. ICM isolated by immunosurgery showed better attachment to feeder cells and ES cell colony formation than cultured whole blastocysts. Morphology of colonies was similar to that of mouse ES cells, showing compact colonies with delineated boundary. Also, these colonies showed alkaline phosphatase activity. Porcine ES-cell like colonies were passed 3 times through physical separation on fresh feeder layers. These results indicated that porcine ES-like cell line can be established from IVF porcine blastocysts. Further characterization of these porcine ES-like cell lines is required.

Table 1
Isolation and culture of ICM from porcine blastocyst produced by IVF
20EFFECTS OF BOVINE SOMATOTROPIN TREATMENT ON AI PREGNANCY RATE IN DAIRY HEIFERS
Authors: Rorie R.W. , Rosenkrans C.F. , Aishman A.J.
Published: February 2004

Treatment of superovulated cows with bovine somatotropin (bST) at the time of insemination has been reported to decrease the number of unfertilized oocytes, while increasing the development rate and morphological quality of embryos (Thatcher WW et al., 2001 Theriogenology 55, 75–89). These findings suggest that bST treatment might be used to improve pregnancy rates in inseminated cattle. The objective of this study was to further investigate the effects of bST (Posilac; Monsanto Co., St. Louis, MO, USA) treatment at or after insemination on subsequent pregnancy rate in diary heifers. Crossbred dairy heifers (n=73) between 14 to 15 months of age and averaging 331±3.6kg, were used for the study. Estrus was induced by a single or repeated (at 14-day intervals) injections of 25mg of PGF2α. Heifers were observed at least twice daily and artificially inseminated about 12h after detection of estrus. At estrus, heifers were randomly assigned across treatments. Treatments were bST (Posilac) injection (500mg, s.c.) at the time of insemination (Day 0), on Day 14, or both at insemination and on Day 14. Untreated animals served as controls. On Day 45 after estrus, ultrasonography was used to determine pregnancy status and measure fetal-crown rump length. Chi-square analysis was used to evaluate the effect of treatment on pregnancy rate. Analysis of variance was used to compare fetal crown-rump length among treatments. Results are presented in the table below. Treatment of heifers with bST at insemination had no effect (P=0.306) on pregnancy rate when compared with the control group. However, bST treatment on Day 14, or on both Days 0 and 14, reduced (P=0.009)pregnancy rate, when compared with the control and Day 0 bST treatment groups. Pregnancy rate was similar (P=0.729) for heifers receiving bST on Day 14v. Days 0 and 14. Fetal growth, as measured by crown-rump length, was unaffected by treatment (P=0.603). Results indicate the timing of bST treatment after the onset of estrus can influence the subsequent pregnancy rate in dairy heifers.
200OVARIAN STIMULATION, LAPAROSCOPIC OOCYTE RETRIEVAL, IVF AND BLASTOCYST
PRODUCTION USING SEQUENTIAL MEDIA IN THE AFRICAN LION (PANTHERA LEO)
Authors: Armstrong D.L. , Crichton E.G. , Dankof S.M. , Schwalbach L.M.J. , Gardner D.K. , Loskutoff N.M.
Published: February 2004

The objective of this study was to test the efficacy of Gardner’s Sequential Medium on in vitro embryo production in lions. Three young (3–4yr) nulliparous lionesses were treated with a total of 50 IU purified porcine FSH (Sioux Biochemical, Sioux Center, IA) in a decreasing regimen over 4 days. At 80h, they were chemically immobilized with a combination of tiletamine and zolazepam (Zoletil; Virbac) and a single deslorelin implant (Ovuplant, Peptech Animal Health Pty, Ltd., North Ryda, New South Wales, Australia) was placed (s.c.) in the lateral thorax over the scapula to stimulate final oocyte maturation. Approximately 24h later, the lionesses were again chemically immobilized (Zoletil); ovaries were viewed, structures recorded and oocytes retrieved laparoscopically (Table 1).
Just prior to oocyte retrieval, four lions were chemically immobilized. Semen was collected by rectal probe electrostimulation and diluted 1:10 in five media (TL HEPES, modified Tyrodes+6mgmL−1 BSA+10% FCS, HEPES-Hams F10+10% FCS, Biladyl and Tyrodes+5% lion serum) and incubated at room temperature and ambient atmosphere for 6h before evaluation and use in IVF. Since there were no differences in sperm motility in any of these media (>90% after 6h), spermatozoa from each male were pooled from all treatments for IVF. A total of 6, 10 and 3 grade A oocytes (73%) from Lionesses #1, 2 and 3, respectively, were inseminated with approximately 1×106 lion sperm/mL in 0.5-mL wells containing G1 Sequential Medium and cultured for 16h in humidified 6% CO2 at 38°C. Then, presumptive zygotes were washed and cultured in 50-μL microdrops of G1 under oil. After 72h, embryos were washed and cultured in 50-μL microdrops of G2 under oil. At 110h post-insemination, 10 had cleaved (53%) and, of these, 5 (50%) developed to the morula stage and 3 (30%) to the blastocyst stage. In conclusion, the present study has shown that lion oocytes can be successfully fertilized in vitro. Furthermore, the in vitro-produced embryos can be cultured to the blastocyst stage in G1/G2 Sequential Media.

Table 1
Ovarian structures observed and oocytes collected from three lionesses
203TIMING OF IN VITRO OOCYTE MATURATION IN SPRINGBOK (ANTIDORCAS MARSUPIALIS), BLACK WILDEBEEST (CONNECHAETES GNU), BLESBOK (DAMALISCUS DORCUS PHILLIPSI), AND REEDBUCK (REDUNCA ARUNDINUM)
Authors: Brad A.M. , Spies T. , Olivier F. , Lane M. , Gardner D.K. , Bartels P. , Krisher R.L.
Published: February 2004

With an increased need for genetic diversity within populations, assisted reproductive technology is becoming an important tool for banking semen and embryos, with the possibility of future AI or embryo transfer between distant populations. Previous research has demonstrated that the broad approach of applying bovine IVM/F/C protocols to African antelope is inefficient for embryo production. The purpose of this study was to determine the timing of oocyte maturation (to telophase or metaphase II) in vitro in springbok (Antidorcas marsupialis; n=84), black wildebeest (Connechaetes gnu; n=18), blesbok (Damaliscus dorcus phillipsi; n=9), and reedbuck (Redunca arundinum; n=1). Ovaries were collected within 4h of death from culled animals on game reserves in South Africa, placed into warm SOF-HEPES, sliced and cumulus-oocyte complexes (COCs) collected. Complexes were placed into GMat medium supplemented with 0.01UmL−1 each FSH and LH, 50ngmL−1 EGF, and 1.0% (v/v) PSA (100U penicillin mL−1, 100μg streptomycin mL−1, 0.25ng amphotercin mL−1). Oocytes were removed from medium after 16, 20, 24, 28 or 32h of maturation in 5% CO2 in air at 39°C. After removal, complexes were denuded with hyaluronidase, mounted on a slide and placed into 3:1 (ethanol:glacial acetic acid) fixative until time of analysis. Oocytes were stained with aceto-orcein, and nuclear maturation was evaluated with a phase contrast microscope. As shown in the Table 1, in springbok (n=311), the percentage of mature oocytes peaked at 28h of maturation and was not different (P<0.05) from oocytes matured for 32h. Fewer springbok oocytes were mature at 16, 20 and 24h. Black Wildebeest (n=88) oocytes were mature by 24h, with no additional increase in maturation at 28 or 32h. Blesbok oocytes (n=42) were not mature at 16 or 20h, and there was no difference (P>0.05) in the percentage of oocytes mature at 24 or 28h. Observational data in reedbuck (n=10) indicated that no oocytes were mature at 20 or 24h, but 25.0% were mature at 28h. The low occurrence of mature oocytes at 28h in blesbok and reedbuck suggests that additional time points should be examined later in maturation. This study demonstrates that oocytes of these species can be successfully matured in vitro, but the rate of maturation and thus the optimal time of insemination for IVF is species specific.

Table 1
Timing of in vitro oocyte nuclear maturation to telophase or metaphase II in springbok, black wildebeest and blesbok
204THE PRODUCTION OF INTRACYTOPLASMIC SPERM INJECTION LION
(PANTHERA LEO) EMBYROS USING SPERMATOZOA COLLECTED BY PERCUTANEOUS EPIDIDYMAL
SPERM ASPIRATION FROM VASECTOMIZED MALES
Authors: Damiani P. , Gomez M. , Cole A. , Pope E. , Aguilar R. , Hammond B. , Nel L. , Cortez C. , Vaccaro J. , Sarrat E. , Markey E. , Dresser B.
Published: February 2004

Contraception and/or sterilization methods have become an essential component of many captive animal management programs. Sterilization techniques are considered to be the last resort if a viable contraceptive cannot be attained and are generally not considered reversible. The African lion (Panthera leo) is one species in which sterilization techniques have been routinely applied. The objective of this study was to develop and evaluate a method for the collection of spermatozoa from male lions that have been previously rendered sterile by vasectomy. Percutaneous epididymal sperm aspiration (PESA) is a technique in which spermatozoa are aspirated from the epididymis and no surgical incision is required. In the present study, two lions (12 and 19 yrs old) were anesthetized and PESA was attempted. A 21-gauge needle attached to a 10-mL syringe (Norm-Ject) filled with 2–3mL of Tyrodes HEPES medium was gently inserted into the head of the epididymis and aspirated gently until spermatozoa were noted. Spermatozoa were visually assessed for motility (grade 1–5; 1=few motile sperm to 5=all motile), sperm concentrations were determined and then the sperm were cryopreserved. The total sperm concentration collected from the older (19yr) male was lower than that obtained from the younger (12yr) lion (0.08×106sperm/mL v. 65.5×106sperm/mL, respectively). Furthermore, more motile spermatozoa (grade 3) were collected from the younger individual compared to the older male (grade 1). Sperm samples from the 12-yr-old lion were frozen by multi-step addition of TEST yolk buffer+glycerol. Lionesses (n=3) were subjected to laparoscopic oocyte retrieval after gonadotropin treatment. A total of 38 oocytes were retrieved and 74% (28/38) were mature as determined by extrusion of the first polar body. Mature oocytes were subjected to ICSI using frozen-thawed spermatozoa obtained by PESA. More than 60% (17/28) of the injected oocytes cleaved and 100% (17/17) reached the morula stage by Day 5 or 6 of IVC. Embryos were cryopreserved and were subsequently transferred (n=15) into one lioness. We have demonstrated that it is possible to collect viable spermatozoa from sterile male lions using the PESA technique. Spermatozoa collected were motile and could be cryopreserved and functional for assisted reproductive techniques. This technique could be applied to other infertile or sterile males whose genetic background would benefit a current conservation program.
212EFFECT OF CRYOPRESERVATION METHODS AND PRE-CRYOPRESERVATION STORAGE ON
BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATES) SPERMATOZOA
Authors: Robeck T.R. , O¿Brien J.K.
Published: February 2004

In conjunction with artificial insemination (AI) and sperm preservation, sperm sexing technology has great potential as a population management strategy for captive bottlenose dolphins. Successful AI using fresh spermatozoa (Robeck TR et al. 2001 CRC Marine Mammal Medicine 193–226) and flow cytometric analysis of bottlenose dolphin spermatozoa (Garner DL and Seidel GE Jr 2002 CSAS Symposium 2–13) support this approach. For sperm sexing, methods for short-term storage of semen in a liquid state are required to enable transport of spermatozoa to the sorting laboratory. In addition, cryopreservation techniques must be optimized for long-term storage of sexed spermatozoa. Our objectives were to assess: (i) 3 cryopreservation methods×2 straw sizes×3 thawing rates (Exp. 1) and (ii) effects of liquid storage for 24h (pre-cryopreservation) and sperm concentration at freezing (Exp. 2) on post-thaw characteristics (PT) of bottlenose dolphin spermatozoa. For Exp. 1 and 2, 4 ejaculates (collected by manual stimulation)×3 males (aged 14–34yr)×4 replicates were used. For Exp. 1, semen was frozen in 0.25-mL (SM) and 0.5-mL straws (LG) by 3 methods (Mt) (Mt1: lactose, egg yolk, −32°Cmin−1; Mt2: lactose, egg yolk, 1.5% Equex STM (Nova Chemical, Calgary, Canada), −19.7°Cmin−1; Mt3: Test yolk buffer (TYB), −116°Cmin−1). All Mt had 3% glycerol. Samples were thawed using a slow (S: 2.8°Cs−1), medium (M: 8.8°Cs−1) or fast (F: 21°Cs−1) rate. In Exp. 2, ejaculates were divided into 4 aliquots for dilution (1:1) and stored at 4°C with EquiPro® (EP4°C, Minitube, Verona, WI, USA) and TYB (TYB4°C) or at 21°C with Androhep EnduraguardTM (AH21°C, Minitube) or no dilution (NEAT21°C). After 24h, samples were frozen and thawed using Mt3×SM×F at 10×106 spermmL−1 (LOW) or 100×106 spermmL−1 (STD). PT evaluations of motility (total motility [TM], % progressive motility [PPM], kinetic rating [KR, 0 to 5]) and acrosomal status (Spermac® , Minitube) were performed at 30min and 6h after dilution (1:1) with AH at 21°C. For statistical analysis (ANOVA), a sperm motility index (SMI=TM×PPM×KR) was calculated and expressed as % of initial SMI. For all ejaculates, initial TM and PPM were greater than 85% and KR was 5. In Exp. 1, at 6h PT, %SMI was highest for Mt3×LG×M (45.5±8.7) and Mt3×SM×F (44.8±11.9). For Exp. 2, %SMI at 0h PT was higher for samples stored at 4°C than at 21°C (TYB4°C 41.0±8.4, EP4°C: 36.7±7.7, NEAT21°C: 23.8±8.6, AH21°C: 14.8±8.6, P<0.001) and, with the exception of AH21°C, was similar between the LOW and STD concentration. At 6h PT, %SMI for all treatments was higher for STD than LOW concentration (P<0.05). Acrosome integrity was similar across treatments. In summary, a semen cryopreservation protocol maintained high levels of the initial characteristics of ejaculated spermatozoa. Transport of semen for sex pre-selection and cryopreservation within 24h may be feasible, but impact of storage time on functional capacity of dolphin spermatozoa is unknown.
22PRODUCTION OF A CLONED CALF USING KIDNEY CELLS OBTAINED FROM A 48-HOUR
COOLED CARCASS
Authors: Adams A.M. , Pratt S.L. , Gibbons J.R. , Arat S. , Respess D.S. , Stice S.L.
Published: February 2004

The ability to produce cloned livestock using postmortem tissue could incorporate an additional application into the field of nuclear transfer. This study examined the feasibility of producing cloned cattle using a primary cell line established from a postmortem beef carcass. A market beef heifer processed at a USDA-certified slaughterhouse was used to develop a primary somatic cell line. Tissue samples were taken from the kidney and forelimb regions either 1) immediately following slaughter (fresh) or 2) 48h postslaughter (cooled) where the carcass was housed at 2 to 4°C. Tissue was removed and placed on ice in PBS+5.0% (v:v) penicillin/streptomycin. A primary culture was established using standard techniques and cultured in supplemented DMEM F-12 medium. Once established, cells were trypsinized and either frozen or continually passaged. Cells used for nuclear transfer (NT) were passaged (48h before use) and cultured with 15μM roscovitine roughly 24h prior to nuclear transfer. Cells were approximately 80% confluent and between passage numbers 1 and 11 at the time of NT. Selected slaughterhouse-derived oocytes were matured in supplemented TCM 199 medium for 18–20h at 39°C in 5.0% CO2 and air. Mature Metaphase II oocytes were vortexed and stained with Hoechst 33342 to help with chromatin removal. Following enucleation, roscovitine-treated carcass cells were placed in the perivitelline space of the oocyte. Reconstructed NT embryos were fused in Zimmermann’s medium and pulsed using needle-like electrodes. This was followed by activation using a combination of calcium ionophore (5μM), cytochalasin D (5μgmL−1), and cycloheximide (10μgmL−1) in TCM+10% FBS. Fused NT embryos were cultured in 50-μL drops of BARC medium (USDA, Beltsville, MD) for 7 days at 39°C in a 5% CO2, 5% O2 and 90% N2 environment. Embryo development for all four groups (Table 1) was assessed with blastocysts (grade 1 or 2) being transferred into recipient cows 7 days post-estrus. Cleavage rates were not significantly different between groups, and the use of either fresh or cooled cells did not impact blastocyst formation. However, there was a significant difference (P=0.05) in % blastocyst based on the source of the donor cell. Overall, one live calf resulted from 34 transferred NTs produced using kidney cells taken from a 48h cooled carcass. These results display the feasibility of producing cloned calves from cells collected post mortem, which ultimately could be used as a tool to select breeding bulls based on their own steer carcass characteristics.

Table 1
Embryo development and pregnancy data for the production of beef carcass clones
223DEVELOPMENTAL PATTERN OF SMALL (1-3mm) ANTRAL
FOLLICLES IN THE BOVINE OVARY
Authors: Jaiswal R.S. , Singh J. , Adams G.P.
Published: February 2004

Knowledge about the developmental pattern of small follicles (<4mm) will be crucial to understanding the endogenous control of folliculogenesis and to developing methods to control it for clinical purposes. This study was designed to characterize the developmental pattern of 1–3mm follicles and to determine, retrospectively, the stage at which the future dominant follicle first attains a size advantage among follicles in the cohort. In Experiment 1, the ovaries of Hereford-cross heifers (n=18) were examined daily by high resolution transrectal ultrasonography for one natural interovulatory interval to evaluate changes in the number of 1–3-mm follicles in relation to the wave pattern of follicles ≥4mm. Interovulatory interval was divided into 2- and 3-wave based on number of waves exhibited by heifers. In Experiment 2, the ovaries of Hereford-cross cows (n=9) were examined every 6h from Day 5 to Day 13 (Day 0=ovulation) to monitor precisely the diameter changes of individual follicles ≥1mm during emergence of the second follicular wave. Data were analyzed by Proc. Mixed procedure for repeated measures (Littell RC et al., 2000 Stat in med 19, 1793–1819) in the Statistical Analysis System software package (SAS version 8.2 for MS Windows;; 2002 SAS Institute Inc. Cary, NC, USA). Results of Experiment 1 revealed a day effect (P<0.05) on the number of small (1–3mm) follicles, with a peak (P<0.05) 1 or 2 days before wave emergence (defined as the day the dominant follicle was first detected at 4mm), followed 3 to 4 days later by a peak (P<0.05) in the number of large follicles (≥4mm). There was an inverse relationship between the number of small and large follicles during Wave 1 (r=−0.66; P=0.05) and Wave 2 (r=−0.62; P=0.04) in 2-wave interovulatory intervals. Similarly, an inverse relation was detected between the number of small and large follicles for Wave 1 (r=−0.79; P=0.01) and Wave 3 (r=−0.90; P<0.01) but not for Wave 2 (r=−0.57; P=0.14) in 3-wave interovulatory intervals. The number of 1–3-mm follicles detected in anovulatory waves did not differ (P=0.53) between 2- versus 3-wave interovulatory intervals;; however, a difference (P<0.05) was observed between anovulatory and ovulatory waves in 3-wave interovulatory intervals but not (P=0.63) in 2-wave interovulatory intervals. Experiment 2 permitted the identification of the future dominant follicle at a diameter of 1mm and its emergence at 6–12h earlier than the largest subordinate follicle (P<0.01). Emergence of the future dominant (r=0.71; P=0.05) and 1st subordinate (r=0.78; P=0.02) follicles was temporally associated with a wave-eliciting rise in circulating concentrations of FSH. Growth rate of the dominant and the 1st subordinate follicle differed (P<0.01) from 2nd subordinate follicle at 84h after their detection at 1mm. It was concluded that small antral follicles (1–3mm) develop in a wave-like manner, and apparent selection of the dominant follicle was manifest much earlier than previously documented.
228DOMINANT FOLLICLE DEVIATION IN GUZERA BREED (BOS
INDICUS) COWS
Authors: Viana J.H.M. , Coutinho G.T.R.M. , Palhao M.P. , Camargo L.S.A. , Sa W.F. , Ferreira A.M. , Nogueira L.A.G.
Published: February 2004

Follicular dynamics of Zebu Cattle are characterized by lower maximum diameter and persistence of dominant follicles, when compared to European breeds. However, the main effects of follicular dominance on subordinated follicle population, and consequently on oocyte recovery and embryo production, have been shown to be similar among breeds. The aim of this study was to evaluate the dominant follicle establishment period, indicated by follicle divergence, in Zebu cows. Pluriparous, non-lactating Guzera breed (Bos indicus) cows (n=5), presenting a mean body weight of 518.0±48.5kg and similar body score condition (ranging from 3.5 to 4, in a 1 to 5 scale), were used. In a first trial, follicular dynamics were evaluated daily during 1 (n=2) or 2 (n=3) estrous cycles, using a portable ultrasound device equipped with a linear rectal 5MHz transducer (Aloka SSD500, Aloka Co., Tokyo, Japan). In a second trial, estrous cycles were synchronized using progesterone intravaginal-releasing devices (CIDR, Pharmacia, Sao Paulo, Brazil), and follicular dynamics were evaluated every 6h after ovulation, during the first follicular wave, until the largest follicle achieved a diameter of 10mm. During each evaluation ovaries were mapped, and evolution of follicles followed individually. Data are normalized for the moment of follicular divergence, and analyzed by ANOVA. Results are presented as mean±SD. Data from the first evaluations confirmed the occurrence of cycles with three (4/8) and four (1/8) follicular waves, despite a mean cycle length of 19.1±1.9 days, with a maximum diameter of 11.6±2.4mm for non-ovulatory follicles, and 14.4±0.5mm for ovulatory follicles. Follicular deviation during the first follicular wave occurred 49.2±5.0h after ovulation, when the largest follicle presented a diameter of 7.3±0.6mm, and the second largest follicle 6.8±0.7mm. Growth rates of the dominant and the largest subordinated follicles were similar before (0.35mm/6h vs. 0.33mm/6h, respectively;; P>0.05), but significantly different after deviation (0.52mm 6h v. −0.04mm 6h; P<0.05). These results shows that the lower maximum diameter and persistence of dominant follicles, in Zebu cattle, is partially compensated by an earlier dominant follicle deviation.
230TRANSCRIPTOMICS ANALYSIS OF CHANGES IN GENE EXPRESSION IN BOVINE
OVIDUCT EPITHELIAL CELLS DURING THE ESTROUS CYCLE
Authors: Bauersachs S. , Rehfeld S. , Koelle S. , Mallok S. , Prelle K. , Wenigerkind H. , Sinowatz F. , Blum H. , Wolf E.
Published: February 2004

The oviduct epithelium undergoes marked morphological and functional changes during the estrous cycle. It has been shown that a dramatic change in the frequencies of ciliated and non-ciliated cells occurs during the estrous cycle. At estrus the epithelium consists of secretory and ciliated cells and at diestrus mainly of ciliated cells. The oviduct provides the microenvironment for sperm capacitation, fertilization, and early cleavage-stage embryonic development. At the molecular level, only a few genes or proteins are known that change during the estrous cycle and which may be important for fertility, so as the bovine oviduct-specific glycoprotein, the major secretory protein in the oviduct. Therefore, we studied systematically the changes in gene expression in bovine ipsilateral oviduct epithelial cells at estrus and diestrus. To identify differentially expressed genes, a combination of subtracted cDNA libraries and cDNA array hybridization was used. Two subtracted libraries were produced to enrich cDNAs of upregulated genes at estrus and at diestrus. A total of 1536 cDNA clones of each library were analyzed with radioactively (33-P) labeled probes generated from the oviduct epithelial cells of six Simmental heifers, three of them slaughtered at Day 0 (estrus) and three at Day 12 after standing heat (diestrus). After normalization of the raw data and statistical analysis, all cDNAs showing significant differences in their expression levels at estrus compared to diestrus were sequenced. Sequencing revealed 84 different cDNAs;; 42 of them matched bovine genes or their human/mouse homologs with known functions, and 42 matched genes without a known function. Half of the genes (n=42) were expressed at a higher level at estrus;; for the other (n=42) expression levels were higher at diestrus. The regulated genes or their products represented a variety of functional classes, such as genes of the secretory pathway, genes involved in transcription regulation, cell-surface proteins, cell–cell interaction proteins, secreted proteins, members of signal transduction pathways, immune-related proteins, and some enzymes. The identification of genes differentially regulated in ipsilateral oviduct epithelial cells at estrus v. diestrus is the first step of a systematic analysis of differential gene expression during the estrous cycle. Further studies will follow, focusing on different compartments of the bovine oviduct and additional times of the estrous cycle.
239IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN PORCINE
PARTHENOTES AT 2-CELL AND BLASTOCYST STAGES USING ANNEALING CONTROL PRIMER
TECHNOLOGY
Authors: Lee H.Y. , Yoon S.J. , Lee K.A. , Kim N.-H.
Published: February 2004

Identification of embryo-specific genes will provide insight into early embryonic development. However, the current methods employed to identify genes expressed at specific stages are labor intensive and produce a high degree of false positives. In the present study we employed an accurate PCR technology controlled by an annealing control primer (ACP, SeeGene, Seoul, Korea) to identify differentially expressed genes in presumptive porcine parthenotes. In vitro-matured porcine oocytes were parthenogenetically activated by square electrical direct current pulses. After 3h of culture in North Carolina State University (NCSU) 23 medium with 0.4% BSA containing 7.5mgmL−1 cytochalasin B, embryos were washed and cultured in NCSU 23 medium with 0.4% BSA. Total RNA was prepared from a pool of 200 2-cell stage and a pool of 100 blastocyst-stage porcine parthenotes using a Dynabeads mRNA DIRECT Kit. First-strand cDNA synthesis was carried out using dT-ACP1, wherein the 3′-end core portion comprised a hybridizing sequence complementary to a poly (A) region of mRNA transcripts. Second-strand cDNAs were synthesized using PCR with an arbitrary ACP whereby the 3′-end core portion of the dT-ACP2 was prevented from annealing to the first-strand. The PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide. The differentially expressed bands were extracted and cloned into a TOPO TA cloning vector (Invitrogen, Seoul, Korea), sequenced and analyzed by BLAST search. To date we have identified 21 differentially expressed genes using 28 arbitrary ACPs. BLAST analysis revealed that most of these genes have strong homology (more than 95%) with known mouse and human genes. Differentially expressed genes in the 2-cell stage include GDP dissociation, putative translation initiation factor (SUI1) and succinate dehydrogenase iron-protein subunit. The blastocyst specific genes include rennin binding proteins, ATPase inhibitor, catenin, ribosomal protein S15a, ribosomal protein large P1, puromycin sensitive aminopeptidase, BMDP gene, arginine N-methyltrasferase p77 isoform, acylamino acid-releasing enzyme, ribosomal protein S14 and KIAA0630 protein. RT-PCR confirmed the expression of these genes in porcine parthenotes during preimplantation development. In conclusion, ACP technology is a specific and sensitive method to locate differentially expressed genes in preimplantation mammalian embryos.
240OVINE PREPUBERTAL OOCYTE SHOWS ALTERATE GENE EXPRESSION
AND LOW DEVELOPMENTAL COMPETENCE
Authors: Leoni G. , Ledda S. , Bogliolo L. , Succu S. , Rosati I. , Bebbere D. , Pintus P.P. , Naitana S.
Published: February 2004

The aim of this work was to evaluate developmental competence and gene expression of prepubertal and adult ovine oocytes. GV prepubertal and adult oocytes were matured, fertilized and cultured in vitro until blastocyst stage;; the time (days) needed to reach this stage was recorded. Blastocysts developed on different days were cultured for hatching to evaluate their quality in relation to cleavage rate. Adult and prepubertal GV oocytes and blastocyst-stage embryos produced, respectively, at 6 and 7 days were compared for quantitative expression of poly(A) polymerase (polyA-P), glucose transporter I (Glut-I), desmocollin II (desmoII), plakofilin (plako) and heat shock protein 70.1 (HSP70) genes. Confirming previous results (Ledda et al., 1996 Zygote 4, 343–348), fertilized prepubertal ovine oocytes developed to blastocyst stage at lower rates than the adult ones (19.9 v. 51.3%, respectively, P<0.001) and this stage was delayed 24h in prepubertal compared to adult embryos (P<0.01), reflecting a lower quality (Fenwick et al., 2002 Hum. Reprod. 17, 407–412) of the former. In fact, 44.7, 25.0, 30.3 and 0% of adult blastocysts were obtained after 6, 7, 8 and 9 days, respectively, of postfertilization culture compared to 0, 48.4, 34.3 and 17.2% of prepubertal ones. Faster-developed blastocysts showed higher hatching rate in both prepubertal (54.8%, 7 days of culture) and adult (89.8%, 6 days). Hatching rate dropped to 18.2% when blastocysts were obtained at 8–9 days in prepubertal and to 54.5% and 32.5% at 7 and 8 days, respectively, in adult embryos. Analysis of gene expression showed that HSP70, plako and desmo genes were not expressed in GV oocytes, and Glut-I mRNA was lower in prepubertal GV oocytes than in the adult ones (P<0.01). All genes were expressed in blastocysts;; we found that Glut-I was at lower levels (P<0.01) in prepubertal-derived blastocysts whereas HSP70 was highly expressed (P<0.05) in prepubertal blastocysts than in those derived from adult oocytes. In conclusion this work shows that prepubertal ovine oocytes have a lower developmental competence compared to the adult ones, correlated to an altered gene expression during the growth phase of the oocyte and early embryonic development. Supported by MIUR (cofin).