bluuebunny/crossref_metadata_2025 huggingface dataset:
166MOUSE EMBRYONIC DEVELOPMENT FROM ONE-CELL STAGE TO BLASTOCYST, UTILIZING
KSOM SUPPLEMENTED WITH AMINO ACIDS IN A MICROCHANNEL DEVICE
Authors: Lopes P.H.C. , Malusky S.A. , Lima A.S. , Beebe D.J. , Wheeler M.B.
Published: February 2004

Great efforts have been made to improve in vitro culture for enhancement of embryonic development. However, in vitro development of mammalian embryos still remains a challenge for the scientific community. Recently, the use of microfluidic culture devices, as an alternative technique compared to the standard drop, has allowed mammalian embryos to develop to the hatched blastocyst stage. With the use of a different medium, mouse strain, and microchannel device than previously reported (Raty S et al., 2001 Theriogenology 55, 241 abst), this study was undertaken to determine if a microchannel device fabricated from borosilicate and poly-dimethylsiloxane would support development of mouse embryos from one-cell to blastocyst, as an alternative to standard microdrop culture. Mice (F1 inbred C57BL/6CRL X SJL) from 3 to 8 weeks old were superovulated with 5IU of PMSG and 5IU of hCG. The female SJL strain of the mice has demonstrated low reproductive performance. One-cell embryos were collected in M2 medium (Sigma, St. Louis, MO, USA.). For each treatment, 240 embryos in 24 replicates were cultured. Groups of 10 embryos were cultured in the microchannel device using 500μL of KSOM with amino acids (MR-106-D, Speciality Media, Phillipsburg, NJ, USA.); no additional supplements were added. Groups of 10 embryos were cultured in standard microdrops (control) using 30μL of the same medium covered with mineral oil. Embryos were cultured in a 100% humidified, 5% CO2 in air atmosphere at 37°C for 96h. Embryos were allocated to the control treatment or the microchannel device treatment using a randomized block design. The percentage of embryos at each stage of development was evaluated at 24-h intervals. The stage of embryo development at each observation was analyzed by ANOVA using the general linear model in SAS (PROC GLM, type I sum of squares). Blastocyst development in the microchannel device was not different when compared to results obtained in the standard drop. The percentage of blastocysts developing, when analyzed from one-cell stage, was 29±5% for the control and 26±6% for the microchannel. The percentage of blastocysts, when analyzed from cleavage, was 35±5% for the standard drop and 31±7% for the microchannel device. The results obtained are encouraging, when considering the non-optimized medium and mouse strain utilized in this experiment. In conclusion, the results show the microchannel device may be considered an alternative technique for use in embryo culture as it supports development of mouse embryos from one-cell stage to blastocyst.
168FACTORS AFFECTING ON EMBRYO TRANSFER PREGNANCY RATES OF IN
VITRO-PRODUCED BOVINE EMBRYOS
Authors: Aoki S. , Murano S. , Miyamura M. , Hamano S. , Terawaki Y. , Dochi O. , Koyama H.
Published: February 2004

The objective of this study was to analyze factors affecting the pregnancy rates after transfer of IVF-derived Japanese Black embryos. Holstein cows and heifers (n=7250) were selected as recipients, and embryo transfers were performed for 3yr (between 1998 and 2000). The IVM-IVF procedure was performed according to a method previously described (Hamano S and Kuwayama M 1993 Theriogenology 39, 703–712). IVF-derived embryos that developed into expanded blastocysts (grade 1, manual of IETS) after 7 to 8 days (insemination=Day 0) were used for this study. Some of these embryos were frozen in TCM-199 supplemented with 1.4M glycerol, 20% calf serum, and 0.25M sucrose. The embryos were seeded at −6°C, held at −6°C for 10min, and then cooled to −25°C at a rate of 0.33°Cmin−1. Frozen embryos were thawed in a 30 to 35°C water bath after 10s of air thawing. Fresh (n=3952) or frozen-thawed (n=3298) embryos were nonsurgically transferred to recipients on Days 6 to 9 of the estrous cycle. Data collected at the time of embryo transfer included recipient parity (cow or heifer), whether recipient estrus was natural or synchronized with PGF2α, cloprostenol or CIDR, methods of estrous confirmation (showing standing heat, rectal palpation of ovary without standing heat, or showing only mucous vulvular discharge), number of examinations of the CL by palpation per rectum (twice on the day before embryo transfer and the day of embryo transfer, or once on the day of embryo transfer), type of embryos (fresh or frozen), and day of the estrous cycle at the time of embryo transfer. CATMOD procedures of SAS were used to determine the factors affecting the pregnancy rate. Overall pregnancy rates were 37.3% (n=2704). Whether recipient estrus was natural or synchronized and the type of embryos did not influence the pregnancy rates. Heifers had significantly higher pregnancy rates than cows (44.0% v. 33.0%, respectively, P<0.05). Pregnancy rates among the subset of heifers and cows showing standing heat were significantly higher than those showing only mucous vulvular discharge (39.5% v. 33.5%, respectively, P<0.05). Examining the CL twive had a significantly higher pregnancy rate than did a single examination of the CL (41.1% v. 35.6%, respectively, P<0.05). Pregnancy rate on Day 8 (38.4%, 1358/3533) of the estrous cycle at the time of embryo transfer was significantly higher than on Days 6 (27.7%, 23/83) and 7 (36.2%, 1235/3408) (P<0.05), and the pregnancy rate on Day 6 of the estrous cycle at the time of embryo transfer tended to be lower than on Day 9 (38.9%, 88/226) (P<0.08). These results demonstrate that confirming standing heat, performing CL examination twice before embryo transfer, freezing high quality embryos, and performing embryo transfers on Day 8 resulted in an improved pregnancy rate for the transfer of IVF-derived embryos.
17PREVENTION OF ANESTRUS USING AN INTRAVAGINAL PROGESTAGEN DEVICE COMBINED WITH 17²-ESTRADIOL, GNRH, AND PGF2± AT 60 DAYS POSTPARTUM IN DUAL PURPOSE CROSSBRED ZEBU COWS
Authors: Palomares-Naveda R. , Perea-Ganchou F. , Hernandez-Fonseca H.J. , Sandoval-Martinez J. , De Ondiz-Sanchez A. , Goicochea-Llaque J. , Gonzalez-Fernandez R. , Soto-Belloso E.
Published: February 2004

In order to study the effect of an intravaginal device (sponge) impregnated with Medroxyprogesterone acetate (MAP) on estrus induction and fertility, an assay was carried out on a commercial farm located in Zulia state, Venezuela. Eighty-four noncyclic (milk progesterone level <0.5ng/mL) suckled crossbred cows (B. taurus×B. indicus) at 60 days postpartum and 3–3.5 body condition score (scale 1–5) were randomly allotted to one of the following treatments: T1 (n=22), intravaginal device impregnated with 250mg of MAP during 7 days+50mg of MAP and 5mg of 17β-estradiol (17β-E) i.m. on the day of device insertion (Day 0) and 1.5mg of 17β-E i.m. on Day 8 (Pregnaheat-E®; VIATECA, Venezuela); T2 (n=28), 17β-E given at Day 0 was replaced by 20μg of busereline (Conceptal® Hoescht, A. Germany)+injection of 25mg of dinoprost (Lutalyse® Pharmacia Upjohn, Kalamazoo, MI, USA) on Day 7 and 1.5mg of 17β-E 12h after device removal (Day 8); T3 (control group; n=34), without treatment. Animals detected in heat were inseminated in the uterine body 9 to 12 hours later in routine AM-PM. Pregnancy diagnose was carried out at 60days after AI by transrectal examination. Studied variables were: estrus rate (ER), pregnancy rate (PR), anoestrus rate (AR), and partum-conception interval (PCI). Variables were analyzed by frequency procedures and chi-square test of Statistical Analysis System (SAS). PCI was analyzed by variance model (PROC GLM) and compared by Ls means. Results are shown in Table 1. T1 and T2 showed higher ER and PR (P<0.05) than T3. AR was lower (P<0.05) in T1 and T2 than T3. PCI was significantly reduced (P<0.05) in T1, but not in T2 versus T3. The cows treated with MAP showed greater estrus and pregnancy rate, shorter partum-conception interval and lower anoestrus rate than the control group. Treatment with the intravaginal device was effective to prevent postpartum anoestrus and to improve reproductive performance in dual purpose zebu crossbred suckled cows.

Table 1.
Effect of an intravaginal progestagen device on reproductive performance in crossbred Zebu cows
172ELECTIVE SINGLE EMBRYO TRANSFER (ESET) IN HUMANS DOES NOT AFFECT THE CHANCES OF A SUCCESSFUL PREGNANCY
Authors: Catt J. , Wood T. , Henman M. , Jansen R.
Published: February 2004

Improvements in human IVF have led to increased pregnancy rates but at the expense of increasing twinning rates. Twins are a bad outcome for the offspring, parents and the healthcare system. An obvious solution to this is to transfer only one embryo and freeze the rest for potential further treatment. This study looked at the effect of doing this on the cumulative live birth rate (when the cryopreserved embryos were thawed and transferred). Patients less than 38 years of age presenting for IVF treatment and with more than two embryos suitable for transfer were offered the chance of transferring only one embryo (elective single embryo transfer, eSET) and freezing the rest. Those patients declining a single embryo transfer had two transferred and served as the controls. Patients not achieving a pregnancy returned for a frozen embryo transfer but were not restricted on the number transferred (to a maximum of two). Cumulative live birth rates were recorded over the ensuing two years. Statistical comparisons were made using paired chi-square tests. The live birth rates from the initial fresh transfer was 41% for eSET (41/111) and significantly higher (53%, P<0.05) for the two-embryo transfer group. These differences were eliminated when the frozen embryos were factored in, both groups rising to 61% of patients treated (68 and 172 live births, respectively). The twinning rate was significantly reduced (P<0.01) from 33% in the two-embryo transfer group to 6% (arising from 4 sets of twins in the frozen embryo transfers) in the eSET group. eSET in the fresh embryo transfer cycle does not affect the chances of a live birth and reduces the twinning rate at least fivefold. Currently, 70% of patients under the age of 38 are electing to have eSET.
173TRANSFER OF VITRIFIED BLASTOCYSTS FROM ONE OR TWO SUPEROVULATED LARGE
WHITE HYPERPROLIFIC DONORS TO MEISHAN RECIPIENTS: REPRODUCTIVE PARAMETERS AT
DAY 30 OF PREGNANCY
Authors: Cuello C. , Berthelot F. , Martinat-Botté F. , Guillouet P. , Furstoss V. , Boisseau C. , Manceau P. , Locatelli A. , Martínez E.A.
Published: February 2004

The present study was designed to determine the effect of pooling embryos from two donors on the reproductive success of transfer of vitrified/warmed porcine blastocysts. Superovulated Large White hyperprolific gilts (n=24) were used as embryo donors. Gilts were artificially inseminated 12 and 24h after initial detection of estrus using fresh semen, and slaughtered on Days 5.5 to 6 of the estrous cycle (Day 0=Onset of estrus). Embryos were recovered by flushing the uterine horns, and unhatched blastocysts were selected. Vitrification and warming were performed as reported previously (Berthelot et al., 2000 Cryobiology 41, 116–124). Embryo transfers were conducted in asynchronous (−24h) Meishan gilts (n=20). Twenty vitrified/warmed blastocysts were surgically transferred into one uterine horn. Ten recipients received embryos from one donor (group 1) and the other ten transfers were performed with mixed embryos from two donors (group 2). Pregnancy was assessed ultrasonographically at Day 25 after estrus and recipients were slaughtered five days later. The pregnancy rate from the different groups was compared using Fisher exact test. The GLM procedure of SAS was used to determine the effect of the origin of embryos (one or two donors) on the number of developed fetuses and viable fetuses at Day 30 of pregnancy. The ovulation rate was 32.5±11.8 (mean±SD). The total number of embryos collected was 634, of which 57 (9.0%), 36 (5.7%), 513 (80.9%) and 28 (4.4%), were unfertilized oocytes and degenerated embryos, morulae, unhatched blastocysts and hatched blastocysts, respectively. The ratio of collected embryos to the number of corpora lutea was 81.3%. The pregnancy rate for group 1 (70%) was not different (P>0.05) than that for group 2 (90%). No significant differences were detected between group 1 and group 2 for in vivo embryo development (number fetuses/transferred embryos in pregnant recipients; 33.3% v. 40%) or in vivo embryo survival (number viable fetuses/transferred embryos in pregnant recipients; 27.9% v. 33.9%). However, the in vivo efficiency (number viable fetuses/total transferred embryos) was higher (P<0.05) when transfers were performed with embryos from two donors (19.5% v. 30.5%). These results indicate that pooling embryos from two donors increases the in vivo efficiency after transfer of vitrified/warmed porcine blastocysts. This study was supported by grant from SENECA (FPI/99, Spain).
179IMPORTANCE OF EMBRYO TRANSFERS IN TRANSGENIC MOUSE FACILITIES
Authors: Mahabir E. , Mayer A. , Marschall S. , Hrabe de Angelis M. , Schmidt J.
Published: February 2004

With the increasing demand for and production of transgenic and mutant mice for biomedical research, embryo transfer plays a paramount role. The purpose of performing embryo transfer in this species is to generate transgenic mice via blastocyst injection of embryonic stem cells or pronuclear injection of DNA constructs, to revitalize cryopreserved sperm and embryos, and to generate mouse lines that meet specific pathogen-free health standards for breeding in barrier areas (rederivation). We present results from two years of carrying out embryo transfers for rederivation purposes in the large mouse breeding facility of the GSF—National Research Center for Environment and Health, Neuherberg, Germany. Pathogens to be eradicated from inbred transgenic (C57BL/6 background) and mutant (C3H background) mouse lines included mouse hepatitis virus, mouse minute virus, and mouse parvovirus. In vitro- and in vivo-produced two-cell embryos were washed 3 times in M2 medium. A total of 20 embryos each were transferred to the oviduct of 8- to 12-week-old specific pathogen-free pseudopregnant (Day 0.5) Swiss recipients under aseptic conditions. Mice were then kept singly in individually ventilated cages and manipulated in a Class II laminar flow hood. From each transfer to one to five recipients with embryos originating from the same mouse line, one recipient was tested for the presence of microorganisms 6 to 12 weeks after embryo transfer, i.e. at 0 to 6 weeks after weaning, according to the FELASA (Federation of European Laboratory Animal Science Associations) Guidelines. A total of 290 embryo transfers were performed for revitalization of cryopreserved sperm from 52 mouse lines, cryopreserved two-cell embryos from 18 mouse lines and rederivation of 12 mouse lines using freshly collected two-cell embryos. From these 290 embryo transfers, 59 mouse lines were re-established (40 from cryopreserved sperm, 7 from cryopreserved embryos and 12 from in vivo-produced embryos). Health monitoring of 54 recipients showed that all mouse lines generated were free of all pathogens stated in the FELASA list. The results presented here show that all 12 (100%) mouse lines were re-established after transfer of freshly collected two-cell embryos whereas 77% and 39% success rates were observed for revitalization of cryopreserved sperm and embryos, respectively. The success of embryo transfer in eradicating pathogens depends on the inability of these pathogens to transverse the zona pellucida and enter and/or infect embryonic cells. In our mouse facility, embryo transfer provided an efficient method to successfully revitalize cells of the mouse germ line as well as to eradicate prevalent murine pathogens. Furthermore, the results demonstrate the efficiency of transferring embryos of different origins and thereby obtaining and maintaining specific pathogen-free health standards in our mouse colonies.
181COMPARISON OF THE PREGNANCY RATES AFTER SYNCHRONIZATION OF
OVULATION USING GnRH AND PGF2± IN RECIPIENT DAIRY CATTLE
Authors: Nishisouzu T. , Sugawara M. , Aoki S. , Kishida K. , Moriyoshi M. , Moriyoshi M. , Dochi O. , Koyama H.
Published: February 2004

Treatments with GnRH and PGF2α for synchronization of ovulation has resulted in acceptable pregnancy rates after fixed-time artificial insemination in dairy cows without estrus detection. The objective of the present study was to evaluate the practicability of ovulation synchronization (Ovsynch, Pursley JR et al. 1995 Theriogenology 44, 915–923) in dairy cattle using GnRH and PGF2α for the embryo transfer recipients. Dairy cattle (cows; n=100, heifers; n=88) were randomly allocated to one of two groups. The control group (cows; n=45, heifers; n=37) was composed of cows in natural estrus. The ovulation synchronization group (cows; n=55, heifers; n=51) was treated with an intramuscular injection of 100μg of GnRH at a random stage of the estrous cycle. Seven days later, the cattle received PGF2α (Cows; 25–30mg) or PGF2α analog (Heifers; 0.5mg) in order to regress the corpora lutea (CL). Forty-eight hours later, cows and heifers received a second injection of 100μg GnRH. Embryo transfer was carried out 7 days after the second injection of GnRH in the ovsynch group and 7 days after estrus in the control group. The cattle judged to have CL 17mm were classified as acceptable recipients. The size of the follicles and the CL were determined to be of estrus stage and embryo transfer by means of ultrasonography. The mean numbers of follicles and CL were analyzed by ANOVA, while pregnancy rates were analyzed by chi-square test. The results are presented in the Table. The proportion of cows and heifers determined to be acceptable embryo transfers was not different between the control group and the ovsynch group. There were no differences in the proportion of acceptable embryo transfers between the control group and the ovsynch group. Follicle diameter at the time of estrus in the control group (cows; 20.7±0.7mm, heifers; 16.8±0.5mm) were significantly larger than that of the ovsynch group (cows; 18.0±1.0mm, heifers; 14.7±0.2mm) (P<0.05). Although CL diameter at the time of embryo transfer in heifers showed no differences between the control group and the ovsynch group (25.0±1.0mm v. 22.8±1.5mm), The CL diameter of the control cow group was larger than that of the ovsynch group (29.8±0.7mm v. 26.1±1.0mm, P<0.05). However, no differences in pregnancy rate were seen between the control group and the ovsynch group. These results suggest that ovsynch can be effectively applied in an embryo transfer program for cattle.

Table 1
Proportion of acceptable embryo transfer recipients and pregnancy rate in dairy cattle in the control ovsynch groups
184PREGNANCY RATES AFTER ESTRUS SYNCHRONIZATION TREATMENT WITH NEW AND REUSED CIDR-B DEVICES
Authors: Solorzano C.W. , Mendoza J.H. , Oden J. , Romo S.
Published: February 2004

It is not well known whether used CIDR devices containing progesterone (P4) combined with estradiol benzoate (EB) and prostaglandin F2α (PGF) can provide acceptable estrus synchronization rates (ESR) and pregnancy rates (PR) in ET or AI programs. Three experiments were designed to study the effect of new and used CIDR-B, with different P4, EB and PGF treatments on ESR and PR in a reproductive program in beef cattle in a tropical climate. Experiment 1 was a control to evaluate ESR and PR in lactating recipient females. All cows (n=284) were treated with a new 1.9-g CIDR (CIDR-B, InterAg, New Zealand), combined with 2mg EB and 50mg P4 on Day 0. CIDR devices were removed on Day 8 and all cows received 0.25mg cloprostenol at that time. Estrus was expected to occur 24h later. Seven days after estrus, all cows showing heat were examined by rectal palpation and those with a CL 15mm in diameter or larger were selected to receive a frozen/thawed embryo (1.5M ethylene glycol=EG) by nonsurgical direct transfer (DT). PR were determined by rectal palpation 60d after estrus. Ninety percent of the cows displayed signs of estrus (256/284) and 40% of those that received a frozen embryo were pregnant (96/239). Experiment 2 was designed to evaluate ESR and PR in dry recipient cows treated with a used CIDR-B (first reuse). All cows (n=274) were treated with a reused 1.9-g CIDR combined with 2mg EB and 50mg P4 on Day 0. CIDR devices were removed on Day 8 and all cows received 0.25mg cloprostenol at that time. Estrus was expected to occur 24h later. Seven days after estrus, all cows that showed estrus were rectally evaluated and those with a CL 15mm in diameter or larger received a frozen/thawed embryo (1.5M EG) by DT. A total of 93% of the treated cows showed signs of estrus (254/274) and 51% of those that received an embryo were pregnant (110/217). Experiment 3 was designed to evaluate ESR and PR in virgin heifers, treated with a used CIDR (second reuse). All heifers (n=414) were treated with a reused 1.9-g CIDR combined with 1mg EB on Day 0. CIDR devices were removed on Day 8 and all heifers were expected to show estrus 24h later. Approximately 12h after estrus, all heifers that showed signs of estrus were inseminated, using frozen/thawed semen from a single bull. Of the treated females, 78% showed signs of estrus (323/414) and 69% of the inseminated were pregnant (223/323). These results suggest that in a CIDR that was used in two previous occasions, there is still a remaining amount of P4 that allows estrus synchronization in heifers. Furthermore, the reutilization of CIDR-B devices can contribute to reduce the costs related to ET or AI programs in cattle. However, the diverse existing conditions among the 3 experimental groups in this study make a statistical comparison impossible. Therefore, further studies are needed, under controlled experimental conditions, to confirm the results obtained.
186SEROCONVERSION OF CALVES TO BOVINE VIRAL DIARRHEA VIRUS (BVDV) FOLLOWING
INTRAVENOUS INOCULATION WITH ARTIFICIALLY EXPOSED IN VIVO-DERIVED EMBRYOS
Authors: Waldrop J.G. , Stringfellow D.A. , Givens M.D. , Galik P.K. , Riddell K.P. , Riddell M.G. , Carson R.L.
Published: February 2004

An early study demonstrated that washing embryos was effective for removal of virus after artificial exposure of in vivo-derived embryos to a cytopathic isolate of BVDV (Singh et al., 1982 Theriogenology 17,437–444). However, a recent study using representative noncytopathic isolates of BVDV demonstrated that washing was less beneficial for removing some isolates of BVDV than for others (Waldrop et al., 2000 Theriogenology 57, 575). Thus, the objective of this study was to determine if the quantity of a high affinity isolate of BVDV that remains associated with single-washed or trypsin-treated embryos is sufficient to cause infection in vivo. Twenty zona pellucida-intact Day 7 morulae and blastocysts (MB) were collected from superovulated cows. After collection, all MB were washed according to International Embryo Transfer Society (IETS) standards, and all but 4 (negative controls) were exposed for 2h to approximately 106 cell culture infective doses (50% endpoint) per mL (CCID50/mL) of viral strain SD-1. Following exposure, one-half of the MB were washed and one-half were trypsin-treated according to IETS standards. All MB were then individually sonicated, and sonicate fluids were injected intravenously into seronegative calves. Blood was collected from each calf on Days 0, 3, 6, 9, 12, 15, 20, 25, and 30, and sera were assayed for BVDV and anti-BVDV antibodies. All cattle used in the study were determined to be virus- and antibody-negative 30d prior to and the day of intravenous inoculation of sonicate fluids into calves. Viremia was not detected in any calf following injection, possibly due to intermittent sampling and/or small amount of embryo-associated virus present. However, seroconversion of 38 and 13% of the calves occurred following injection with sonicate fluids from washed and trypsin-treated embryos, respectively. Findings demonstrated that the quantity of a high affinity isolate of BVDV associated with single-washed or trypsin-treated embryos is sufficient to be infective in vivo as evidenced by seroconversion. These results emphasize the need for studies to determine if the virus associated with exposed individual embryos constitutes an infective dose when placed into the uterus.
189ESTABLISHMENT OF BUFFALO EMBRYONIC STEM-LIKE (ES-LIKE) CELL LINES FROM
DIFFERENT SOURCES OF DERIVED BLASTOCYSTS
Authors: Kitiyanant Y. , Saikhun J. , Kitiyanant N. , Sritanaudomchai H. , Faisaikarm T. , Pavasuthipaisit K.
Published: February 2004

In the pleuripotent embryonic stem (ES) cells in mammalian species are derived from the inner cell mass (ICM ) of preimplantation embryos. In the current study we report the successful isolation of pleuripotent undifferentiated buffalo ES-like cells from the ICMs of in vitro fertilization (IVF), somatic cell nuclear transfer (NT)-reconstructed and parthenogenetic (PA) embryos. The ICMs were isolated from hatched blastocysts that had spread out after 3–5 days of culture on mouse embryonic fibroblast (MEF) feeder cell layer in the presence of leukemia inhibiting factor (LIF). The production of MEF and ES-like cells were the same as previously described in bovine ES cells (Kitiyanant Y et al., 2000 Science Asia 26, 81–86). The primary culture and propagation of the cell lines were performed every 2–3 days. The cell lines appeared to be normal diploid karyotype and expressed the cell specific markers of alkaline phosphatase, stage-specific embryonic antigen 3 and 4 (SSEA-3 and SSEA-4), TRA-1-60 and TRA-1-81 similar to those characterized in monkey ES cells (Thomson JA et al., 1995 PNAS 92, 7844–7848 and Kuo HC et al., 2003 Biol. Reprod. 68, 1727–1735). The buffalo ES-like cells from these three different sources were able to be cultured in vitro for more than 20 passages on the feeder cell layer without differentiation. They were aggregated to form the embryoid bodies (EBs) in suspension culture. When EBs were plated on 0.1% gelatin-coated culture dishes, the cells differentiated to be neural-, epithelial- and fibroblast-like morphologies. These results suggest success in establishment of buffalo ES-like cells from either IVF, NT or PA preimplantation embryos and also their differentiation in vitro. Buffalo ES-like cells should be a useful source of cells for gene targeting and for the studies of the mechanism of gene expression in the preimplantation embryos obtained from different methods. These results are encouraging for the feasibility of cell transplantation studies in the future. This research was funded by Thai Government and The National Center of Biotechnology and Genetic Engineering.
191IN VIVO AND IN VITRO DIFFERENTIATION OF EMBRYONIC STEM CELLS DERIVED
FROM PARTHENOGENETIC EMBRYOS IN MICE
Authors: Mitani T. , Teramura T. , Tada T. , Hosoi Y. , Iritani A.
Published: February 2004

Availability of embryonic stem (ES) cells opens the prospect for regenerative medicine. However, ES cells genetically mismatched to diseased individuals cause immunological rejection. In this study, we established ES cells from parthenogenetic embryos in mice and examined their pluripotency. Oocytes were collected from (C57BL/6xDBA)F1 mice (BDF1) by superovulation. Parthenogenetic diploid embryos were produced by activation treatment in 5mM SrCl2 in Ca2+-free KSOM medium for 2h, followed by cultivation in 5μgmL−1 cytochalasin B for 6h. The zonae pellucidae of embryos developed to the blastocyst stage in vitro were removed by a 5-min incubation in 0.5% pronase. Inner cell masses (ICMs) isolated immunosurgically were seeded on the feeder layers (mitomycin C-treated mouse embryonic fibroblasts) in DMEM supplemented with 15% Knock-Out Serum Replacement (Invitrogen), 2mM L-glutamine, non-essential amino acids, β-mercaptoethanol and 103UmL−1 of Leukemia inhibitory factor (LIF) at 37°C in a humidified atmosphere with 5% CO2 in air. The attached ICM cells were mechanically disaggregated and seeded on the fresh feeder layers. After several passages, parthenogenetic ES (PnES) cell lines were established. The efficacy of establishing PnES cell lines was 66% (37/56). To examine the characteristics of PnES cell lines, seven lines were subjected to histochemical and immunohistochemical analysis. All showed alkaline phosphatase activity and immunoreactivity to anti-SSEA-1 and anti-Oct4 antibodies. They maintained euploid sets of choromosomes at 29; 59%. PnES cells from two of the seven lines were injected into 59 host blastocysts obtained from ICR mice, resulting in 16 chimeric offspring (27%). In another experiment, injection of ICM cells and ES cells obtained from fertilized BDF1 blastocysts and ICM cells obtained from BDF1 parthenogenetic blastocysts also produced chimeric offspring (35%, 7/20; 46%, 6/13; and 53%, 10/19, respectively). However, no chimeric mouse with germline transmission was obtained from PnES cells. Injection of 1×107 of PnES cells into SCID mice formed teratocarcinomas. Immunohistochemical analysis showed cells positive for nestin (specific to neuroepitherial stem cells), Tu-J (class III β-tublin), NF-M (neurofilament), desmin (muscle), and albumin (hepatocytes), which indicated their differentiation potency to the cells derived from all three germ layers. Simple embryoid bodies produced from these cell lines were plated on tissue culture dishes under conditions for induction of differentiation. Immunohistochemistry and RT-PCR analysis showed their differentiation into neurons (NF-M, nestin), cardiomyocytes and hepato-like cells (albumin, α-fetoprotein). Our results indicate that PnES cells are pluripotent similar to the ES cells from fertilized embryos except for germline transmission and should be tested in cell replacement animal models.
194THE EFFECT OF OVIDUCTAL INCUBATION OF OOCYTES AND EMBRYOS ON ADHERENCE
OF BOVINE VIRAL DIARRHEA VIRUS
Authors: Bielanski A. , Lutze-Wallace C.L. , Nadin-Davis S.
Published: February 2004

The zona pellucida (ZP) plays a major role as a protective shell against infection of embryonic cells and as a carrier of infectious agents in the spread of livestock diseases through embryo transfer practices. It has been demonstrated that pathogenic agents are more likely to adhere to the surface of ZP of IVF embryos than to that of in vivo fertilized embryos. It has been suggested that divergent conditions for the production of these two types of embryos may lead to changes in their morphology and to differences in the interaction of the ZP with pathogens. The objective of this study was to investigate whether or not experimentally induced changes in hardening ZP (HZP) affect adherence of BVDV to the ZP of oocytes and embryos produced in vitro. To induce HZP, the oocytes or IVF embryos in groups of 30–50 were incubated in the ligated, ampullar part of the oviduct at 38°C in 5%CO2, 5%O2 and 90% nitrogen for 5h. Following incubation, a proportion of oocytes/embryos was exposed to 1% pronase to determine HZP (the time of ZP lysis), while the remaining oocytes and embryos were incubated with 106 TCID50/mL of either noncytopathic (NY-1) or cytopathic (NADL) strain of BVDV at 38°C for 3h. Subsequently, oocytes and embryos were washed according to the method recommended by IETS and then tested for the presence of BVDV (virus isolation and PCR tests). At the end of experiments the oviductal tissues were tested by PCR and proven free of BVDV. For immature and matured oocytes and embryos not exposed to the oviduct, the ZP dissolution times were 3.6±0.24 (mean±SEM, n=20), 3.8±0.24, and 4.0±1.24min, respectively (Chi-square test; P>0.05). Corresponding times for those incubated in the oviduct were 393±47, 431.0±50, and 467±61min, respectively (P>0.05). There was no difference between the number of virus-positive oocytes and embryos (n=965 in 193 samples) following experimental exposure to BVDV regardless of whether or not they were previously incubated in the oviduct (P>0.05). Lower, but not significant, differences in percentages of samples associated with the infectious cytopathic strain of BVDV as compared to a noncytopathic strain were detected (P>0.05). It was concluded that the modification in proteolytic resistance properties of ZP during in vitro oviductal incubation did not influence the adherence of BVDV to ZP of oocytes or IVF embryos. Further studies are warranted to determine why IVF embryos are more prone to the adherence of pathogenic agents than are in vivo fertilized embryos.
196PREVENTION AND TREATMENT OF BOVINE VIRAL DIARRHEA VIRUS INFECTIONS IN
FETAL FIBROBLAST CELLS
Authors: Givens M.D. , Stringfellow D.A. , Riddell K.P. , Galik P.K. , Sullivan E. , Robl J. , Kasinathan P. , Dykstra C.C. , Boykin D.W.
Published: February 2004

Unnoticed infections with bovine viral diarrhea virus (BVDV) can occur in cultured cells used for somatic cell nuclear transfer. Aromatic cationic molecules have exhibited inhibitory activity against in vitro replication of BVDV. The purpose of this research was to evaluate the ability of aromatic cationic compounds to prevent or treat noncytopathic BVDV infections of fetal fibroblast cells. Aromatic compounds tested were 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606); 2-(2-benzimidazolyl)-5-[4-(2-imidazolino) phenyl]furan dihydrochloride (DB772); and 2-(1-methyl-2-benzimidazolyl)-5-[4’-(2-imidazolino)-2’-methylphenyl]furan dihydrochloride (DB824). To evaluate prevention of BVDV infections, 10 cell lines in the absence or presence of 7 dilutions of each of the 3 compounds were inoculated with BVDV. The concentrations of BVDV in medium and cell lysates were determined by serial dilution and virus isolation. Samples were obtained 72 hours post-inoculation. Bovine viral diarrhea virus in cell culture medium and cell lysate samples was evaluated by comparison to equivalent samples from control cultures in which no compound was added (percent of control=cell culture infective doses (50%; CCID50) of BVDV in compound sample/CCID50 of BVDV in control sample lacking compound). The viral inhibitory concentrations (99%) of compounds were calculated with JMP software by least-squares regression techniques. Cumulatively, the 99% endpoints for inhibition of viral replication in fetal fibroblast cell lines for the 3 compounds were 0.1μM, 0.007μM and 0.028μM, respectively. To evaluate therapeutic treatment of established BVDV infections, the concentration of BVDV in medium and cell lysates of 2 fetal fibroblast cell lines were evaluated. The cell lines were previously determined to be infected with a genotype 1a strain of BVDV. Samples were obtained during 4 sequential passages in the absence or presence of 0.04μM and 4μM concentrations of DB772 or DB824. Presence of BVDV was determined by reverse transcription nested polymerase chain reaction and virus isolation. While BVDV persisted in cultures supplemented with no aromatic compound or 0.04μM, both DB772 and DB824 effectively cured BVDV infections after 1 passage in 4μM, and cells remained viable. Results indicate that BVDV infections can be effectively prevented or treated in fetal fibroblast cultures. Further research is needed to determine if exposed cells are competent for production of normal embryos via nuclear transfer.
198BOVINE VIRAL DIARRHEA VIRUS (BVDV) IN CELL LINES USED FOR SOMATIC CELL CLONING
Authors: Stringfellow D. , Riddell K. , Givens M.D. , Galik P. , Sullivan E. , Robl J. , Kasinathan P.
Published: February 2004

Most isolates of BVDV cause unapparent infections in cultured cells. Fetuses, postnatal animals or fetal bovine serum are possible sources of the virus for cultivated cells used as karyoplasts in cloning. Routine screening by veterinary diagnostic laboratories of 39 fetal fibroblast cell lines used in cloning research had revealed that 15 (39%) were positive for BVDV by various assays including RT-nPCR. As some were valuable transgenic cell lines, a rigorous protocol for evaluation of each line was undertaken to confirm infection with BVDV. A cryopreserved vial of each line was thawed, medium discarded and cells incubated (38.5°C in 5% CO2 and air) through 2 passages (6–10 days) in α-MEM supplemented with 10% equine serum. At the end of the second passage, cells were separated from medium, washed and assayed for presence of BVDV using virus isolation in 2 sequential passages in Madin Darby Bovine Kidney Cells and RT-nPCR. Available lots of fetal bovine serum and medium that had been used to culture the cells also were tested for BVDV. When the virus was detected, the RT-nPCR products were sequenced and compared. Also, an attempt was made to evaluate the earliest available cryopreserved passage of any positive cell lines. Results indicated that just 5 of 39 of the original cells tested (13%) were positive. Since cryopreserved earlier passages of 4 of the cell lines were available, they were assayed with the result that 2 of the 4 were not infected at the earliest passage. Further, BVDV was isolated from one lot of fetal bovine serum that was used to culture one of the cell lines. Sequence analysis verified that only 2 of these 4 cell lines were infected with the same isolate of BVDV, and one isolate was identical to the virus found in the fetal bovine serum used in medium to culture it. The discrepancy between our viral detection and that of the diagnostic laboratories is explained in part by the presumed test protocols. All BVDV-positive cells, as reported by the diagnostic laboratories, were positive by RT-nPCR. We presume that they did not separate medium from cells before assays. Thus, any noninfectious viral RNA that was in the medium (e.g. as would be expected in many lots of irradiated serum) would have been reported positive. The only possible sources for BVDV in these cell lines were the fetuses from which they originated or fetal bovine serum used in medium. Sequence analysis confirmed that serum was the source of viral infection in one line. The likely source of virus for 2 other lines was serum, since they were not infected at earlier passages. The 2 remaining cell lines were positive at the earliest available passages, so the fetuses from which cells were harvested could not be discounted as the source of BVDV. This report highlights the risks of introducing BVDV in embryo technologies and the difficulties that can be encountered in attempting accurate diagnosis of the presence of infectious virus.
201RED DEER (CERVUS ELAPHUS) CALVES BORN FROM
IN VITRO-PRODUCED BLASTOCYSTS FERTILIZED AND CULTURED IN DEER SYNTHETIC OVIDUCT FLUID
Authors: Berg D.K. , Beaumont S.E. , Berg M.C. , Asher G.W.
Published: February 2004

Deer Synthetic Oviduct Fluid medium (DSOF;; Berg and Asher 2003 Theriogenology 59, 189–205), based upon the composition of red deer oviduct fluid, has been shown to support routine in vitro fertilization and blastocyst development (15%) of in vitro-matured red deer oocytes without the use of somatic cell co-culture or serum supplementation. However, pregnancy establishment and fetal survival remained to be determined. The objectives of this study were to transfer in vitro red deer blastocysts produced by the DSOF culture system, follow fetal survival through calving and investigate trans-cervical embryo transfer for red deer. Red deer hinds of mixed age were synchronized using a 12-day CIDR (Pharmacia & Upjohn, Auckland, NZ) synchrony program (Berg DK et al. 2002 Ani. Reprod. Sci. 70, 85–98). Onset of estrus was synchronized to the day of IVF and embryos were transferred 7 days later. in vitro red deer blastocysts were produced after aspirating oocytes from abattoir-sourced ovaries. Selected COCs were matured and fertilized and presumptive zygotes cultured in vitro (Berg and Asher 2003 Theriogenology 59, 189–205) with modified Ca2+ concentrations: 3.0mM and 1.5mM for early and late DSOF, respectively. in vitro blastocyst development was 14.7% (21/143) on Day 7 and 22.4% (32/143) on Day 8. Ten blastocysts (grade 1 and 2) were selected for transfer on Day 7 (post-IVF) and placed into Emcare embryo holding medium (ICPbio, Auckland, NZ). Blastocysts were loaded into 0.25-cc straws (n=5) or tom cat catheters (n=5) and transported to the Ruakura Deer Unit at 25°C. Hinds were restrained and sedated as described for OPU (Berg and Asher 2003 Theriogenology 59, 189–205) and an attempt was made to pass a cattle transfer pistolette through the cervix. If unsuccessful, the hind underwent laparoscopic uterine transfer. Serial serum progesterone values diagnosed pregnancies at Day 21 and fetal survival was determined using rectal ultrasonography on Day 35, 45, 60 and 90. Seven single-embryo transfers were completed;; 2 of 5 trans-cervical attempts and 5 using the laparoscopic method. Serum progesterone levels confirmed 57% (4/7) of the hinds were pregnant on Day 21;; 2/2 (100%) from trans-cervical and 2/5 (40%) from laparoscopic transfers. No pregnancy losses occurred after Day 21. Four calves, 1 male and 3 female, were born unassisted after 230 to 233 days of gestation. Birth weights ranged from 7.3 to 10kg. Our results indicate that in vitro red deer blastocysts produced using the DSOF culture system can establish pregnancies after transfer and result in normal healthy calves.
206POSTNATAL MANAGEMENT OF CHRYPTORCHID BANTENG CALVES CLONED BY NUCLEAR
TRANSFER UTILIZING FROZEN FIBROBLAST CULTURES AND ENUCLEATED COW OVA
Authors: Janssen D.L. , Edwards M.L. , Koster J.A. , Lanza R.P. , Ryder O.A.
Published: February 2004

A multi-institutional collaboration was developed to evaluate the potential for conservation management of genetic variation in captive banteng calves via nuclear transfer cloning of a deceased genetically valuable individual. Fibroblast cultures frozen in 1978 were fused with enucleated cow ova and resulting embryos were transferred to domestic beef cattle for gestation. Two banteng (Bos javanicus) calves produced by heterologous nuclear transfer were delivered by standing Cesarean section on April 1 and 3, 2003. Birth weights for the two calves were 23.13 and 38.64kg, respectively. The larger calf failed to gain strength and died on April 8. The surviving calf was transferred from Sioux Center, IA, to the San Diego Wild Animal Park in Escondido, CA on April 18, 2003. The calf was offered a goat’s milk-based formula containing 21.9kJ metabolizable energy (ME) and 22% total solids. The total solids fraction consisted of 26.3% crude protein, 29.8% crude fat, 33.5% total carbohydrate. Intake and growth rate for this calf was compared to other hand-reared bovid calves produced by normal mating. Average daily gain from birth to 16 weeks of age was 767g day−1. A routine neonatal physical examination revealed that the surviving calf was bilaterally cryptorchid. The large calf that did not survive was noted to have one abdominally retained testicle, whereas the other testicle was scrotal. Ultrasound examination of the healthy banteng calf confirmed abdominal localization of the retained testicles. Both testes were located in the abdominal cavity about 4–6cm from the caudal pole of the kidneys. Therapeutic intervention was considered because of the genetic value of the calf and because the cell donor exhibited normal scrotal testicles. In July, 2003, orchipexy was conducted on this calf. The left testis was exteriorized through a small abdominal flank incision. After freeing it from tissue holding it in place, it was relocated through a subcutaneous tunnel to a pouch created just in front of the scrotum. The attachments to the testis were not long enough to allow the testis to be placed fully in the scrotum. At a follow-up examination conducted four weeks after the surgery, the left testicle was palpable in the subcutaneous pouch just cranial to the scrotum. Under ultrasound examination, the left testicle was easily identified less than 1 cm deep with echogenic texture of a testis. Color flow Doppler ultrasound analysis demonstrated pulsatile flow in the center of the testis synchronous with the heart beat. These findings indicate testicular viability and blood supply.
207OVARIAN STIMULATION, TRANSVAGINAL, ULTRASOUND-GUIDED OOCYTE RETRIEVAL,
ICSI AND BLASTOCYST PRODUCTION IN SEQUENTIAL MEDIA IN THE WESTERN
LOWLAND GORILLA (GORILLA GORILLA GORILLA)
Authors: Loskutoff N.M. , Bowsher T.R. , Chatfield J.A. , Stones G.A. , Ramey J.W. , Zhang L. , Putman M. , Boland C. , Wharton D. , Gardner D.K.
Published: February 2004

Two young (ages: 15 and 16 yr;; studbook # 947 and # 939, respectively) parous female gorillas were initially primed with daily oral monophasic estrogen/progesterone treatment (Ovcon 35: Bristol-Myers Squibb Co., Princeton, NJ, USA) for 3 mo to synchronize their menstrual cycles. After withdrawing treatment, urine was tested daily for occult blood (Hemastix;; Baxter Healthcare Corp., Deerfield, IL, USA). On days 3–10 following the onset of menses (Day 1), ovarian activity was stimulated with 225 IU human FSH im (Repronex;; Ferring Pharmaceuticals, New South Wales, Australia). Then on Days 6–8 this treatment was combined with 25mg GnRH antagonist im (Antagon;; Organon, West Orange, NJ, USA) to prevent premature endogenous LH release. Final oocyte maturation was stimulated 36h after the last FSH/GnRH treatment with 10,000IU hCG im (Profasi;; Serono Lab., Hingham/Rockland, MA, USA) and oocyte retrieval was performed 36h post-hCG administration in sternal recumbency (knee-chest) using a 3–6mHz probe, 17-ga needles and 87mmHg (VMAR-5000 Regulated Vacuum Pump;; Cook Veterinary Products). Both gorillas displayed a thickened endometrium, and approximately 6 (# 947) to 10 (# 939) maturing follicles (10–15mm) were detected in each animal. In female # 947, one oocyte was collected but peritoneal fluid and pathology (hydrosalpinx) were also diagnosed and the right ovary showed no follicular development. A total of 3 oocytes were recovered in highly viscous follicular fluid containing massive amounts of cumulus cells. They were transported in a HEPES-buffered transport medium in a portable incubator (CryoLogic, Mulgrave, Victoria, Australia) at 37°C by airline immediately from Brownsville to Dallas, TX, USA, and within 6-h post-retrieval were fertilized by ICSI using cryopreserved sperm collected by rectal probe electrostimulation (age: est. 40yr ; studbook # 268). The fertilized oocytes were cultured in Gardner’s Sequential Medium at 37°C in 6% CO2 all three cleaved and developed to blastocysts by 115h post-ICSI. One high-quality expanding blastocyst was transported back to Brownsville, TX, and transferred transcervically into the oocyte donor (studbook # 939), and two fair-quality blastocysts were transported overnight to Omaha, NE, and transferred into a synchronized gorilla recipient (age: 29yr; studbook # 543/91). Weekly urine samples from the two embryo transfer recipients are being tested for pregnancy diagnosis using OvuQuick test strips (Quidel, San Diego, CA, USA). Acknowledgments: This research was supported in part by the Morris Animal Foundation (Ruth Morris Keesling Animal Health Study).