bluuebunny/crossref_metadata_2025 huggingface dataset:
360 EVALUATION OF QUALITY OF IN VITRO-MATURED BOVINE OOCYTES BY SEDIMENTATION WITH PERCOLL
Authors: Yotsushima K. , Shimizu M. , Kon H. , Izaike Y.
Published: December 2006

A simple method to evaluate the quality of in vitro-matured bovine oocytes is available for development of an in vitro embryo production system. Oocyte quality relates closely to oocyte fatty acid composition and mitochondrial distribution. The purpose of this study was to examine the influence of the quality of cumulus–oocyte complexes (COCs) and serum supplementation in IVM medium on the distribution of bovine oocyte specific gravities by sedimentation with Percoll before and after IVM. COCs were aspirated from abattoir-derived ovaries and were classified as classes A to D by the morphology of their cumulus cell layers as follows: class A, compact and more than 3 layers thick; class B, compact but <3 layers; class C, partially naked and <3 layers; and class D, naked or expanded. The classified COCs were cultured in TCM-199 supplemented with 0.1% BSA, 5 µg mL−1 insulin, 10 µg mL−1 transferrin, and 10 ng mL−1 transforming growth factor-α (M199-BITT) for 22–24 h. To evaluate the influence of serum supplementation, oocytes from classes A and B were also incubated in M199-BITT as serum-free culture or TCM-199 supplemented with 10% fetal calf serum as serum-supplemented culture. Percoll solutions were prepared by diluting Percoll with PBS supplemented with 0.3% BSA, 1 mg mL−1 glucose, and 0.2 mM sodium pyrvate to 20, 17.5, 15, 12.5, 10, 7.5, and 5% solutions. After removal of cumulus cells, denuded oocytes were put on the surface of Percoll solution for 3 min, and the precipitated oocytes were transferred to stepwise high density solution. The percent of Percoll solution just before buoyancy was considered as the oocyte specific gravity value. Statistical analysis was performed by one-way ANOVA. Oocytes from class A had the highest specific gravities before and after IVM in all classes (Table 1). After IVM, oocyte specific gravities from classes A and C were higher than those of oocytes before IVM (class A: P < 0.05, class C: P < 0.001). The specific gravities of in vitro-matured oocytes cultured in serum-free medium were higher than those cultured in serum-supplemented medium (15.3 ± 0.3%, n = 71, and 14.0 ± 0.3%, n = 58; P < 0.01). These results show that the specific gravity was affected by the morphological quality of COC, and the culture conditions for IVM may profile the metabolic activity of oocytes during IVM.

Table 1.Specific gravities of the bovine oocytes classified by morphology of COC before and after IVM
364 AMPLIFICATION AND APPLICATION OF THE HMG BOX OF BOVINE SRY GENE FOR SEX DETERMINATION
Authors: Lu W. , Rawlings N. , Zhao J.
Published: December 2006

A fast and reliable method for bovine sexing has been developed through amplification of the bovine high motility group (HMG) box of the sex-determining region of the Y chromosome gene (SRY). We describe a similar PCR-based method, which involves amplification of the HMG-box sequence of the bovine SRY gene with optimized PCR parameters and apply it to bovine sex-determination. Oligonucleotide primers were designed according to the conserved bovine SRY HMG box sequence motif (Gen-Bank Accession NM-011564); the forward primer was 52-CACGCATCTTACTTCCTCCCCTTTTAAAAG-32 and the reverse primer was 52-AGACGCCTTTGTTGAGCGAGAGTAAGGAAG-32. In agarose gel electrophoresis, a normal bull showed 1 SRY band, and a normal cow showed no SRY band. After optimization, the PCR procedure for sex determination was applied to 14 embryo biopsies. The biopsied embryos were transferred into 14 recipient cows on the same day (Day 7 of the estrus cycle) that the embryos were collected and sex of the calf was confirmed after parturition. Nine calves were born and anatomical sex corresponded to the sex determined by PCR in all cases (100% accuracy). Thus, this study showed for the first time that the present method can be applied in bovine breeding programs to facilitate manipulation of the sex ratio of offspring and also allows a quick diagnosis for the XY-bovine offspring by amplification of the HMG box of the bovine SRY gene. This method also differs from that of Thomsen and Poulsen working with pigs and Mara in which the sex of in vitro-produced ovine embroys was determined by duplex PCR.
385 RELATIONSHIP AMONG ESTROUS BEHAVIOR, SUPEROVULATORY RESPONSE, AND EMBRYO SEX RATIO IN SUPERSTIMULATED HOLSTEIN HEIFERS
Authors: Jung Y. G. , Ideta A. , Lim K. T. , Lee W. Y. , Song H. B. , Hayama K. , Urakawa M. , Aoyagi Y.
Published: December 2006

It is desirable to control the sex of embryo transfer offspring in the dairy industry. The relationship between estrous behavior, superovulatory response, and embryo sex ratio after superovulation has not been investigated. The objective of the present study was to compare estrous behavior, superovulatory response, and sex ratio of embryos resulting from superstimulated donors in herds in which the female ratio was naturally high (>50%, FR-H, n = 9) or low (d50%, FR-L, n = 7). Holstein heifers (14 to 18 months of age, n = 16) were given decreasing doses of FSH IM (total of 28 Armour units, Antrin R-10; Kawasaki-Mitaka, Kanagawa, Japan) twice daily for 4 days between Days 8 and 10 of the estrous cycle. Five (25 mg) and 3 (15 mg) mL of dinoprost (PGF; Veterinary Pronalgon; Pfizer Animal Health, Tokyo, Japan) were administered IM with the seventh and eighth FSH treatments, respectively. The length of estrus and numbers of standing mounts were determined by an electric heatmount sensor (HeatWatch; Comtec Co. Ltd., Miyazaki, Japan), which was attached to the tailhead of the animals. Heifers received 50 �g of GnRH IM (fertirelin acetate; Conceral; Schering-Plough Animal Health KK, Tokyo, Japan) 48 h after the first PGF treatment, and were inseminated with frozen–thawed semen into the uterine body 12 and 24 h later. Seven-day embryos were recovered nonsurgically and Grade 1 embryos (IETS classification) were selected for this study. Male and female embryos were identified using the loop-mediated isothermal amplification procedure (Hirayama et al. 2004 Theriogenology 62, 887–896). The data (mean � SEM) were compared with Fisher's protected least significant difference test following ANOVA. Mean values for female sex ratio in FR-H and FR-L embryos were 72.2 � 4.6% (32/46) and 30.6 � 7.1% (23/68), respectively (P < 0.01). The duration of estrus in the FR-H group (13 h 10 min � 3 h 51 min) was shorter than that of the FR-L group (24 h 51 min � 6 h 32 min; P < 0.05). Moreover, the numbers of standing mounts in the FR-H group (27.8 � 5.9) were less than in the FR-L group (75.0 � 18.5; P < 0.05). Although the mean number of ova/embryos collected from FR-H heifers (8.4 � 1.0) was significantly less than from FR-L heifers (19.7 � 3.1; P < 0.05), the percentage of Grade 1 embryos did not differ (63.7 � 8.2%, 46/76 vs. 54.5 � 8.3%, 68/138, respectively; P > 0.05). In summary, superstimulated heifers from the FR-H herd had a shorter duration of estrus, a lower superovulatory response, and a higher proportion of female embryos than heifers from the FR-L herd. However, data do not demonstrate a cause and effect relationship.
39 BOVINE SOMATIC CELL NUCLEAR TRANSFER USING CUMULUS - OOCYTE COMPLEXES COLLECTED FROM THE IDENTICAL INDIVIDUAL BY OVUM PICKUP
Authors: Hasegawa K. , Takahashi S. , Akagi S. , Takeda K. , Imai K. , Shimizu M. , Okazaki T. , Abe S. , Izaike Y.
Published: December 2006

We previously produced a cloned calf by nuclear transfer (NT) using cumulus cells removed from cumulus–oocyte complexes (COCs) after IVM. If both cumulus cells and oocytes are obtained identically and individually, and can be used simultaneously for NT, the production of cloned cows will be more expedient. And the cloned offspring produced from them will not exhibit the heteroplasmic mixed mtDNAs of donor cells and recipient oocytes. In this study, we examined the developmental potential of NT embryos using cumulus–oocyte complexes (COCs) collected from cows individually by ovum pickup (OPU). The cumulus cells were removed from COCs after IVM. The cumulus cells and cumulus-free MII oocytes derived from the same cow were used as donor nuclei and recipient oocytes, respectively. NT was performed as previously described (Akagi et al. 2003 Clon Stem Cells 5, 101–108). In Experiment 1, we examined the in vitro development of NT embryos using COCs collected by OPU. The aspiration of the follicles was performed once a week consecutively for 6 weeks in 6 cows (Cows A, B, C, D, E, and F) without hormone stimulation. In Experiment 2, we examined fetal development after the transfer of NT embryos. A Japanese black cow (Cow G) was used for OPU. On Day 7, 13 NT blastocysts were transferred to 7 recipient cows. The mtDNA genotypes of the donor cow and the cloned calf were analyzed by PCR-mediated single-strand conformation polymorphism analysis as previously described (Takeda et al. 2003 Mol. Reprod. Dev. 64, 429–437). The results of Experiment 1 are summarized in Table 1. Fusion rates did not differ among individual cows. However, the developmental rates of NT embryos at the blastocyst stage varied widely among individual cows, with a range of 19 to 64%. In Experiment 2, 2 of 7 recipient cows became pregnant on Day 30. One pregnant cow aborted on Day 60, and another cow calved a healthy calf. The mtDNA genotype of the cloned calf was confirmed to be identical with that of the donor cow. These results indicate that COCs from an identical individual can be used as donor nuclei and recipient oocytes for NT in order to produce female clones with the same mtDNA as that of the donor cow.

Table 1.In vitro development of NT embryos using COCs collected by OPU
398 EFFECT OF INCUBATION PERIOD ON TRANSFECTION RATES IN BOVINE SPERMATOZOA
Authors: Assumpção M. E. O. A. , Feitosa W. B. , Rovegno M. , Marques M. G. , Nicacio A. C. , Simões R. , Visintin J. A.
Published: December 2006

Current data about the kinetics of exogenous DNA interaction with spermatozoa are controversial. The amount of DNA associated with the spermatozoa is proportional to the incubation period. However, a great amount of exogenous DNA, associated or internalized, can activate endonucleasis, which cleaves the exogenous and/or endogenous DNA similarly to apoptosis. The aim of this work was to evaluate the effect of the incubation period of bovine spermatozoa with exogenous DNA on transfection rate and induction of apoptosis, oncosis, or initial oncosis and/or late apoptosis of sperm cells. For that, 5 × 106 spermatozoa/mL were incubated with 500 ng of plasmid pEYFP-Nuc (Clontech, Mountain View, CA, USA) for 60, 90, and 120 min. Sperm cells were then subjected to electrophoresis to remove non-associated exogenous DNA. DNA (endogenous and exogenous) was extracted and real-time PCR was performed to quantify exogenous DNA/spermatozoa association. Each reaction was performed with 12.5 µL of Platinum SYBR Green; 0.5 µL of ROX; 5.6 µL of H2O; 5 µL of sample (1 µg); 0.7 µL of primer sense (5′-ATGGCCGACAAGCAGAAGAAC-3′) and 0.7 µL of primer anti-sense (5′-TGCCGTCCTCGATGTTGTG-3′); 500 nM each. Real-time PCR was conducted for 40 cycles of 95°C for 15 s and 64°C for 1 min. Apoptosis and oncosis analyses were evaluated by flow cytometry with 106 sperm/mL stained with 2 µL of Yo-pro (100 µM) for 20 min and with 10 µL of propidium iodide (6 µM) for 10 min at room temperature. Data were analyzed by MINITAB Realease 14 Statistical Software (Minitab, Inc., State College, PA, USA), subjected to ANOVA, and compared by Fisher's or Tukey's test (P ≤ 0.05) for transfection rate and apoptosis/oncosis, respectively. Linear regression obtained by the amplification of pEYFP-Nuc at different concentrations showed efficiency of R2 = 0.9987. The results summarized in Table 1 show that increase in length of the incubation period increases the amount of exogenous DNA associated with spermatozoa and does not affect the average of live spermatozoa in apoptosis, oncosis, and initial oncosis and/or late apoptosis groups.

Table 1.Quantification of exogenous DNA associated with bovine sperm cells and percentages of live, apoptotic, and oncotic spermatozoa

This work was supported financially by FAPESP 03/07456-8 and 03/10234-7.
399 SPERMATOZOA-MEDIATED GENE TRANSFER IS INFLUENCED BY SIZE, STRUCTURE, AND CONCENTRATION OF EXOGENOUS DNA
Authors: Feitosa W. B. , Milazzotto M. P. , Martins E. A. L. , Rocha A. M. , Avanzo J. L. , Visintin J. A. , Assumpção M. E. O. A.
Published: December 2006

Mammalian sperm cells have a spontaneous ability to take up exogenous DNA in a process regulated by specific protein. However, little is known about the effect of exogenous DNA on sperm cell association. The aim of this work was to evaluate the effect of size, concentration, and structure of exogenous DNA sequence in apoptosis and oncosis induction and the association with spermatozoa. To evaluate the effect of size and concentration of exogenous DNA, sequences of 2.2, 5.5, and 8.5 kb were used at the concentrations of 500 ng (Experiment 1) or 5 × 1011 molecules (Experiment 2). To assess the effect of structure (Experiment 3), 3 sequences of approximately 500 bp were used, containing 65.7% of AT (AT sequence), 46.4% of AT (IN sequence), and 38.6% of AT (GC sequence). To evaluate apoptosis and oncosis in all treatments, sperm cells were incubated with exogenous DNA for 1 h, stained with 2 µL of Yo-Pro (100 µM) for 20 min, and 10 µL of propidium iodide (6 µM) for 10 min, and then analyzed by flow cytometry. To evaluate the transfection rates, sperm cells, after incubation, were separated from non-associated exogenous DNA by 2.5% gel electrophoresis. DNA (genomic and exogenous) was extracted and association rates obtained by real-time PCR. Data were submitted to ANOVA and compared by Tukey's test (P ≤ 0.05). The number of viable, apoptotic, oncosis, or in initial apoptosis/late oncosis spermatozoa incubated with different concentrations or sequences of exogenous DNA did not differ among groups. In Experiment 1, a higher DNA association with sperm cells was observed in the 5.5 kb sequence in comparison with the 2.2 and 8.5 kb. In Experiment 2, the 8.5 kb sequence had the lower association with the spermatozoa in comparison with the 2.2 kb and 5.5 kb. In Experiment 3, there was a higher interaction of the AT sequence when compared to IN and GC sequences. Reference data suggest that big sequences of exogenous DNA have advantages in the interaction with spermatozoa. The present work is the first one to show that the advantage of the big sequences on interaction became a disadvantage in relation to the ability of these cells to internalize this DNA.

Table 1.
Quantification of different exogenous DNA associated to sperm cells

Financial support was provided by FAPESP 03/10234-7 and 03/07456-8.
406 EFFECTS OF SUCKLING INTENSITY ON MILK YIELD AND PIGLET GROWTH IN LACTATION-ENHANCED ALPHA-LACTALBUMIN TRANSGENIC GILTS
Authors: Marshall K. M. , Hurley W. L. , Shanks R. D. , Wheeler M. B.
Published: December 2006

The objective of the present study was to determine the effects of suckling intensity on milk yield and piglet growth. Lactation capacity of the gilts was enhanced through over-expression of a mammary-specific transgene, bovine alpha-lactalbumin. Lactational response to increased suckling stimulation was determined by fostering litters of either the same age (day 1) or 7 days older (day 7) than the day of lactation to gilts either non-transgenic (C) or transgenic (T) for bovine alpha-lactalbumin. Twenty first-parity gilts were allocated between 4 treatments depending on gilt genotype and age of litter fostered (CDay1, CDay7, TDay1, TDay7). Litters were standardized to 10 piglets within 24 h postpartum, and non-birth piglets were fostered to subject gilts with an equal litter weight within age groups at 36 h postpartum. Milk yield was determined by the weigh-suckle-weigh method on Days 6, 9, 12, 15, and 18 of lactation. Mean daily milk yield was greater (P = 0.031) for T gilts compared with C gilts, and tended to be greater (P = 0.056) for all gilts with Day 7 piglets compared to those with Day 1 piglets. Daily milk yield of TDay7 gilts increased rapidly to peak at Day 9 and was greater than the milk yield of all C gilts at Days 9 (P < 0.01), 12 (P < 0.02), and 15 (P < 0.02). Mean daily milk yield of TDay7 gilts was 2.1 kg greater (P = 0.002) than for CDay7 gilts and 2.0 kg greater (P = 0.004) than for TDay1 gilts. Daily milk yield of TDay1 gilts was not different (P = 0.491) from that of CDay1 gilts. Daily milk yield of CDay7 gilts did not differ (P = 0.630) from that of CDay1 gilts. Piglet weight gain between Days 3 and 6 was greater (P < 0.01) in the TDay7 group than in all other groups, and was greater (P < 0.05) than in the C groups from Days 6 through 9. No difference was found in comparing accumulated weight gain of the piglets between the day of age at foster (Day 1 vs. Day 7; P = 0.606) or between the CDay1 and CDay7 groups (P = 0.759). Accumulated weight gain of piglets suckling TDay7 gilts from Days 3 through 6 and from Days 3 through 9 was greater (P < 0.02) than in the other groups, and continued to be greater (P < 0.05) than in either of the C groups through Day 15. However, by Day 15, accumulated weight gain of the piglets suckling TDay1 gilts was not different (P = 0.395) from that of the TDay7 group and was greater (P = 0.049) than that of the CDay1 group by Day 15. The enhanced lactation potential of the transgenic gilts synergizes with suckling intensity to stimulate increased milk production during early lactation, and also stimulates piglet growth. This synergy may help regulate milk yield during lactation in bovine alpha-lactalbumin transgenic swine.
412 VIABILITY OF ENZYMATICALLY ISOLATED BOVINE PRIMORDIAL AND PRIMARY OVARIAN FOLLICLES COLLECTED BY THE BIOPSY PICK-UP TECHNIQUE
Authors: Aerts J. M. J. , Martinez-Madrid B. , Flothmann K. , De Clercq J. B. P. , Bols P. E. J.
Published: December 2006

Our institution recently developed a new tool for transvaginal, ultrasound-guided collection of ovarian biopsies from living donor cows (Aerts et al. 2005 Theriogenology 64, 947–957). The biopsy pickup (BPU) device consists of a modified needle guidance system, which is equipped with a trocar needle and carries a 60-cm-long true-cut biopsy needle. In a previous experiment, the presence of primordial and preantral follicles in BPU-derived biopsies was demonstrated. As follicular integrity is a prerequisite for further culture or research, the aim of the present study was to assess the viability of enzymatically isolated primordial and primary ovarian follicles collected by the BPU instrument. Four cows were subjected to a one-time BPU procedure. Four cortical biopsies were collected per ovary from each animal. Follicle viability was determined by the dual fluorescent probe technique described by Cortvrindt and Smitz (2001 Fertil. Steril. 75, 588–593). In live cells, calcein-AM is converted into calcein by intracellular esterase enzymes, producing an intense green fluorescence. In dead cells, ethidium homodimer-I is able to penetrate the damaged plasma membrane and, upon binding to nucleic acids, undergo a 40-fold enrichment of fluorescence, thereby producing an intense red signal. Upon retrieval, the biopsies were immersed in HEPES-buffered minimum essential medium (GIBCO, Grand Island, NY, USA) at 4�C. In the laboratory, the tissue samples were transferred to 50-mL conical tubes containing 20 mL PBS supplemented with 1 mg mL-1 collagenase type IA (Sigma-Aldrich NV/SA, Bornem, Belgium) and were incubated in a water bath at 37�C for 60 min. The digestion was terminated by adding an equal volume of cold PBS. The resulting suspension was centrifuged at 300g for 10 min at 8�C. The pellet was resuspended in PBS and transferred to a Petri dish. Each Petri dish was examined under a phase-contrast inverted microscope (Olympus CHX41) equipped with an eyepiece micrometer, and the primordial and primary follicles were collected with a glass micropipette. Isolated follicles were mounted on a slide in 50-�L droplets of PBS containing 2 �mol L-1 calceine-AM and 5 �mol L-1 ethidium homodimer-I (Molecular Probes, Leiden, The Netherlands), and incubated in the dark for 45 min at 37�C. After incubation, the follicles were examined under a fluorescence microscope (Olympus BX61). A total of 95 enzymatically isolated follicles were analyzed for viability with fluorescent probes; 89 (93.7%) of these were viable. Viable follicles were retrieved from all animals. This experiment confirms that viable primordial and primary follicles can be retrieved from living donor cows by the BPU procedure, which makes the technique suited for further culture or research.
415 USE OF COMPUTER-ASSISTED ULTRASOUND IMAGE ANALYSIS IN EMBRYO RECIPIENT SELECTION
Authors: Siqueira L. G. B. , Viana J . H. M. , Souza E. D. , Camargo L. S. A. , Fonseca J. F. , Fernandes C. A. C. , Torres C. A. A.
Published: December 2006

Ultrasonography has been extensively used in the study of reproductive function in areas such as antral follicular dynamics, ovulation, luteal function, and early pregnancy. However, most studies with ultrasound evaluation are limited to measurement of diameter, length, or area of internal structures, and few groups use differences in image density (echotexture) as an evaluation parameter. For corpora lutea, echo-texture differences reflect variations in vascularization and luteal cell proportions. The aim of the present study was to evaluate image analysis as a tool for embryo recipient selection. Grades I or II embryos produced by conventional superovulation (n = 55) or IVF (n = 87) were nonsurgically transferred to primiparous cows or heifer recipients (n = 142). Estrous synchronization was performed using the heat-synch protocol (Day 0: CIDR�; Day 5: 400 IU of eCG; Day 7: 500 �g of cloprostenol plus CIDR removal; Day 9: 2 mg of estradiol benzoate). Sonographic corpora lutea images were obtained on the day of embryo transfer (Day 7), using a portable ultrasound device (Aloka SSD 500; Aloka Co., Sao Paulo, Brazil) equipped with a linear rectal 5 MHz probe. Images were digitalized in .TIFF format at a resolution of 1500 � 1125, using a video capture board (Pinnacle DC10; Pinnacle Systems, Mountain View, CA, USA). A representative elementary area (REA) of 2.704 pixels (25 mm2) was defined in the luteal tissue, using the criteria proposed by Van den Bygaart et al. (1999 Can. J. Soil Sci. 79, 149–160). Image analysis was performed using a custom software package (QuantPro�). Each image dot (pixel) received a numeric value ranging from 0 (black) to 255 (white). Data from cows and heifers later diagnosed as pregnant or not in the groups receiving SOV or IVF embryos were analyzed by ANOVA, and results are presented as means � SEM. As expected, the pregnancy rate was higher for conventional than for IVF embryos (58.2% vs. 31.0%; P < 0.05). There was a low correlation (R = 0.33) between luteal tissue area and pixel value for Day 7 corpora lutea. There was no difference (P > 0.05) in luteal tissue area or echotexture between pregnant and non-pregnant animals in the groups receiving superovulation embryos (3.12 � 0.26 cm2 vs. 2.63 � 0.25 cm2, and 78.03 � 2.25 vs. 79.73 � 3.73, respectively) and IVF embryos (3.25 � 0.24 cm2 vs. 3.03 � 0.14 cm2, and 74.81 � 1.93 vs. 70.82 � 1.62, respectively). However, in the IVF embryo group, no pregnancy was established in cows bearing a CL with mean pixel value lower than 60.25 or greater than 89.27 (total values ranged from 44.72 to 99.79). Corpora lutea image analysis, using the REA proposed in this approach, had limited value to predict pregnancy rate of embryo recipients. Further studies will investigate the accuracy of the REA established and other image characteristics, including the pixel distribution pattern.
416 EFFECTS OF THE CORPUS LUTEUM WITHIN THE OVARY ON THE FOLLICULAR DYNAMICS AFTER FOLLICULAR ASPIRATION AND ON THE DEVELOPMENTAL COMPETENCE OF ASPIRATED OOCYTES
Authors: Takuma T. , Otsubo T. , Kurokawa Y. , Otoi T.
Published: December 2006

The use of ultrasound-guided transvaginal follicular aspiration combined with in vitro embryo production is increasing in the commercial cattle breeding industry. However, frequent puncture of follicles can induce the dysfunction of corpora lutea (CL) (Petyim et al. 2001 J. Vet. Med. A 48, 449–463), resulting in irregular estrous intervals or absence of estrus. This study was conducted to compare follicular and oocyte characteristics in ovaries ipsilateral vs. contralateral to the CL after follicular aspiration. The experiment was performed using a crossover design with each Japanese Black cow (n = 7) assigned to each of the 3 groups. The treatment period was 30 days for each group, and successive treatments were separated by a 2-month rest period. Hence the study spanned a period of 7 months (3 treatment periods and 2 rest periods). To verify ovulation prior to each treatment, the stage of the estrous cycle in each cow was synchronized with an ovulation synchronization (Ovsynch) program. Follicular aspirations were performed 6 times (every 3 or 4 days) between Day 3 and Day 20 after GnRH administration (Day = 0) of the Ovsynch program. All cows in each group ovulated the follicle before the initiation of follicular aspiration treatment. Follicles (e3 mm in diameter) from the ovary ipsilateral (Group 1) or contralateral (Group 2) to the CL were aspirated a using B-mode ultrasound scanner equipped with a 7.5 MHz probe. As a control (Group 3), the follicles from both ovaries were aspirated, irrespective of the presence of CL. In all groups, follicular development in each cow was monitored daily from Days 0 to 31. The cumulus–oocyte complexes (COCs) collected from each cow in Group 1 (n = 158), Group 2 (n = 140), and Group 3 (n = 277) were subjected to IVM/IVF/IVC. The numbers of embryos cleaved and developed to the blastocyst stage were recorded 3 days and 8 days after IVF, respectively. The mean profiles of total visible follicles (e3 mm in diameter) in ovaries with follicular aspiration in Groups 1 and 2 were similar to those in Group 3, irrespective of the presence of CL. The average numbers of visible follicles in the ovaries with follicular aspiration 7 days after the end of follicular aspiration treatment were not different from those in the ovaries without follicular aspiration (P > 0.05; one-way ANOVA). Moreover, there were no significant differences among the groups with respect to the percentages of cleavage (57.5–65.7%) and development to the blastocyst stage (23.6–32.1%) of collected COCs (P > 0.05; chi-square analysis). These results indicate that the presence of CL within the ovaries does not affect the ovarian follicular dynamics after follicular aspiration and the developmental competence of collected COCs.
43 INFLUENCE OF DONOR CELL DIFFERENTIATION ON CLONING EFFICIENCY IN PIGS
Authors: Hornen N. , Kues W. A. , Lucas-Hahn A. , Petersen B. , Hassel P. , Niemann H.
Published: December 2006

We recently reported the discovery of a novel type of stem cells which could be derived from primary cultures of fibroblasts by high density culture (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). The goal of the present study was to analyze the suitability of this specific stem cell population (fetal somatic stem cells, FSSCs) in NT and to test their ability to produce normal offspring upon transfer of cloned embryos. In the first of 4 experiments, FSSCs from isolated attached colonies were compared with fetal fibroblasts in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups [FSSCs: 75.3 � 10.5% (mean � SD) vs. 83.7 � 9.2%; fetal fibroblasts: 64.8 � 17.3% vs. 82.5 � 5.6%, respectively]. Blastocyst rate differed significantly between the two groups (6.4 � 3.5% vs. 24.9 � 8.6%). In the second experiment, FSSCs of 3 different sizes (<14 �m, 15–20 �m, >21 �m), obtained from dissociation of spheroids, were compared in their ability to form blastocysts upon use in NT. No differences were found among the 3 groups (fusion rates: 93.0 � 3.1 vs. 91.3 � 10.1 vs. 92.3 � 5.1; cleavage rates: 83.5 � 7.9 vs. 83.1 � 1.6 vs. 83.2 � 5.8; blastocyst rates: 15.3 � 7.9 vs. 17.6 � 6.8 vs. 10.4 � 2.7, respectively). In the third experiment, FSSCs 15–20 �m in size, derived from spheroids, were compared with fetal fibroblasts. No differences were detected between groups (fusion rates: 83.3� 7.3% vs. 86.8 � 5.3%; cleavage rates: 86.1 � 6.7% vs. 80.7 � 5.9%; blastocyst rates: 21.4 � 5.6% vs. 18.4 � 5.6%, respectively). In the final experiment, 70–100 nuclear transfer complexes cloned from FSSCs were transferred immediately after activation to prepubertal gilts to evaluate their in vivo developmental potential. Pregnancies were established in 3 of 7 recipients, which delivered 7 piglets, of which 3 piglets were vital and showed normal development. Four piglets were lost due to dystocia. These results show that FSSCs are able to generate cloned embryos, and pregnancies can be established and vital piglets can be produced.
45 EFFECT OF OSMOLARITY IN CULTURE MEDIUM ON THE PRE-IMPLANTATION DEVELOPMENT OF PORCINE NT AND IVF EMBRYOS
Authors: Hwang I. S. , Moon H. J. , Shim J. H. , Park M. R. , Kim D. H. , Yang B. C. , Ko Y. G. , Yang B. S. , Cheong H. T. , Im G. S.
Published: December 2006

In vitro production of the pig embryo is very important as an initial step to improve its application in biotechnology. The in vitro production system for pig embryos, however, has been plagued by the high incidence of polyspermy and poor embryo quality. The present study was conducted to examine the relationship between apoptosis and osmolarity of culture medium in pre-implantation development of porcine NT and IVF embryos. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 40–44 h. Fresh semen was diluted and equilibrated at 16�C. The final concentration of motile spermatozoa was adjusted to 5 � 105 cells/mL in fertilization medium. Fetal fibroblasts were prepared from a 35-day-old porcine fetus for use as donor cells. The NT and IVF embryos were cultured in PZM-3 supplemented with 0.05 M sucrose or a final concentration of 138 mM NaCl (280–320 mOsmol) for the first 2 days, and then cultured in PZM-3 (250–270 mOsmol) for the remaining days. For the control, NT and IVF embryos were cultured in PZM-3 for whole culture period. After 6 days of culture, the developmental ability of embryos, total cell numbers, ratio of ICM/TE, and apoptosis of cells in blastocysts were examined. The developmental rate to the blastocyst stage of NT embryos was significantly higher (P < 0.05) in the sucrose and NaCl groups than in the control [14.7% (21/153) and 21.7% (34/154) vs. 11.5% (18/152), respectively]. Also, the developmental rate to the blastocyst stage after IVF was slightly higher in embryos cultured in the medium supplemented with NaCl than in the control group [21.8% (49/235) and 26.4% (61/237) vs. 18.9% (44/247)]. For apoptosis, both NT and IVF blastocysts produced in the sucrose and NaCl groups showed slightly lower frequency of apoptosis compared to that of the control (2.2% and 2.8% vs. 3.1% for NT; 0.9% and 0.7% vs. 1.1% for IVF). These studies suggest that the high osmolarity in the early embryo culture stage could enhance the in vitro development of both porcine NT and IVF embryos to the blastocyst stage and could reduce the apoptosis of cells.
46 EFFECT OF MANIPULATION MEDIUM ON THE DEVELOPMENT OF RECONSTRUCTED DOMESTIC CAT EMBRYOS
Authors: Imsoonthornruksa S. , Lorthongpanich C. , Srirattana K. , Sripunya N. , Laowtammathron C. , Ketudat-Cairns M. , Parnpai R.
Published: December 2006

Domestic cat can serve as a valuable model for assisted reproductive techniques studies of endangered felid species. Therefore, this study was conducted to examine the effect of different manipulation medium on in vitro development of reconstructed domestic cat oocytes. The oocytes were recovered by slicing the ovaries of the cats that had been superstimulated with 200 IU eCG (Intervet, Boxmeer, The Netherlands). The procedures for SCNT were described previously (Lorthongpanich et al. 2004 Reprod. Fertil. Dev. 16, 149 abst). The manipulation medium for SCNT procedures was evaluated between HEPES-buffered TCM-199 (Sigma-Aldrich Corp., St Louis, MO, USA) + 10% FBS (199H) and Emcare embryo holding solution (ICPbio, Ltd., Auckland, New Zealand) (Emcare) during the denuding, enucleation, injection, activation, and holding steps. Parthenogenetic activation (PA) embryos were used as a control for both media. There was no significant difference in fusion rate when either 199H (63%) or Emcare (76%) was used. The cleavage, 8-cell, and morulae development rates of SCNT and PA were not significantly different when using either 199H or Emcare (Table 1). However, the blastocyst formation rates of SCNT and PA in Emcare (44% and 29%, respectively) were significantly greater than those of 199H (14% and 18%, respectively; P < 0.05). These results indicated that the manipulation medium is important for SCNT blastocyst development.

Table 1.In vitro development of cloned domestic cat and parthenogenetic activation of embryos from different manipulation media
48 EFFECTS OF TRICHOSTATIN A ON DEVELOPMENT OF BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
Authors: Iwamoto D. , Saeki K. , Kishigami S. , Kasamatsu A. , Tatemizo A. , Abe Y. , Ikeda S. , Taniguchi S. , Mitani T. , Kato H. , Matsumoto K. , Hosoi Y. , Wakayama T. , Iritani A.
Published: December 2006

Although cloning by somatic cell nuclear transfer (SCNT) has been achieved in various mammalian species, its efficiency has been very low (Han et al. 2003 Theriogenology 59, 33–44). Successful cloning requires conversion from differentiated donor nuclei to embryonic nuclei after transfer of the somatic nuclei into enucleated oocytes. Reprogramming of the transferred somatic nuclei must be completed by the time when normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, both full-term development and pre-implantation development of mouse SCNT embryos were significantly enhanced by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). The objective of this study was to investigate the effects of TSA on the development of bovine SCNT embryos. Bovine fibroblasts were cultured under serum starvation (0.4% FCS) for 7 days and then used as donor cells. The cells were electro-fused with bovine enucleated matured oocytes, and activated with a calcium ionophore and cycloheximide. They were subsequently cultured in mSOF medium until 168 h post-activation (hpa). The NT embryos were exposed to 0 (control), 5, 50, and 500 nM TSA from the start of activation to 48 hpa. Experiments were repeated 3 times, and the data were analyzed with Fisher's PLSD test following ANOVA. The cleavage rates were the same among the groups (60 to 80%; P >0.05). However, the blastocyst rate of NT embryos treated with 50 nM TSA was higher than that of control embryos (40% vs. 19%, respectively; P < 0.05). On the other hand, the blastocyst rate was lower with 500 nM TSA than with 5 or 50 nM TSA (7% vs. 33% or 40%; P < 0.05). These data suggest that proper TSA treatment after somatic cloning improves the rate of development of bovine cloned embryos to the blastocyst stage. Further research is needed to examine whether NT embryos derived from different cell lines or types have similar susceptibility to TSA.
6 TIMING OF DEADENYLATION OF GDF-9 AND CYCLIN 3′ UTR CONSTRUCTS IN BOVINE OOCYTES
Authors: Walker D. J. , Wilusz C. J. , Seidel Jr G. E.
Published: December 2006

The maternal pool of mRNA undergoes major changes during oocyte maturation and early embryonic development. Specific genes are activated or degraded in response to changes in poly-(A) tail length. However, little is known about how the oocyte targets specific transcripts for degradation or translation in a timely manner. The objective of this study was to determine how poly-(A) tail length of different transcripts is affected in bovine oocytes by time of in vitro maturation. Cyclin B1 and GDF-9 32 untranslated regions (UTRs) were cloned into modified p-GEM plasmids containing a poly-(A) tract of 60 or 0 adenosines (A60 or A0, respectively). Each 32 UTR was transcribed in vitro with (A60) or without (A0) a poly-(A) tail to generate UTP32-labeled RNA. Transcriptions producing at least 200 000 counts per min (cpm) per �L were used for subsequent injections into denuded bovine oocytes. Cumulus-oocyte complexes (COCs) recovered from slaughterhouse-derived ovaries (n = 216) were vortexed to remove cumulus cells immediately after aspiration, after 3 h of in vitro maturation, or after 19 h of maturation in a chemically defined medium supplemented with FSH, LH, EGF, and cysteamine. After vortexing, denuded oocytes were injected and snap frozen, or matured in vitro for 1 or 3 h. Eight oocytes were injected with ~0.5 nL (~100 cpm/oocyte) labeled RNA at each time point in 3 replicates. Total RNA was isolated from injected oocyte pools and loaded onto a 5% denaturing acrylamide gel for size separation. Radiolabeled A0 was used as a control point of reference for deadenylation. Gels were dried, and RNA was visualized on a phosphoimager after 24 h exposure to a phosphor screen. Changes in polyadenylation status (transcript size) were evaluated by comparing shifts in bands from gene-specific A60
74 STUDY OF TRANSGENIC GOATS EXPRESSING HUMAN GRANULOCYTE - MACROPHAGE COLONY-STIMULATING FACTOR IN THE MAMMARY GLAND
Authors: Park H. S. , Jung S. Y. , Park S. H. , Park J. K. , Lee J. S. , Kim T. S. , Hong S. P. , Choi K. M. , Seol J. G. , Park K. W.
Published: December 2006

Somatic cell nuclear transfer (SCNT) is one of the most useful methods for production of transgenic animals. While both adult and fetal fibroblasts have been used in SCNT, adult cells provide the opportunity to use donor cells from an animal with a proven high yield of milk production. In this study, in vitro development and pregnancy rates were compared following the use of transfected fibroblasts that were obtained from adult and fetal tissues. Ear and fetal fibroblasts were collected from Saanen goats and cultured in serum-starvation condition (TCM-199 + 0.5% FBS) until cell confluence. Linearized DNA containing the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene that is targeted for expression in the mammary gland by the β-casein promoter (pBC1/hGM-CSF) was transfected into the cells using lipofectamine 2000 (Invitrogen, Inc., Carlsbad, CA, USA). Transfected colonies were selected with G418 for 14 days. Well-separated colonies were isolated and screened for the presence of transgene by PCR and southern blotting. Recipient oocytes were surgically collected by flushing the oviducts of FSH-stimulated goats at 35 h after hCG injection. The zonae pellucidae of the oocytes were partially drilled using a laser system and each somatic cell was individually transferred into the enucleated oocyte. The couplets were electrically fused and activated by ionomycin + 6-DMAP. The reconstructed embryos were cultured in mSOF medium containing 0.8% BSA at 39�C in an atmosphere of 5% CO2, 5% O2, 90% N2 for 12 to 15 h. Nuclear transfer embryos (2- to 4-cell stages) were surgically transferred into the oviducts of recipients that were in either natural or induced estrus. Pregnancy was diagnosed by progesterone assay and ultrasound on Days 21, 42, and 60 of pregnancy. The type of donor cells, ear or fetal fibroblasts, affected neither the fusion rate (54/65, 83.1% vs. 89/116, 76.7%, respectively) nor the cleavage rate (19/54, 35.2% vs. 37/89, 41.6%, respectively). A total of 55 embryos derived from ear fibroblasts and 84 embryos derived from fetal fibroblasts were transferred into 9 and 14 recipients, respectively. No pregnancy was observed in recipients that received NT embryos derived from ear fibroblasts (0/9); however, 5 (2 in natural estrus and 3 in induced estrus; 35.7%), 3 (1 in natural and 2 in induced estrus; 21.4%), and 2 (2 in induced estrus; 14.3%) of 14 recipients that received NT embryos derived from fetal fibroblasts were confirmed pregnant on Days 21, 42, and 60, respectively. These results imply that the type of donor cells for nuclear transfer may be directly correlated with the success rate. More studies will be needed to examine factors affecting production of transgenic goats and to improve results obtained using adult donor cells.
76 PREOVULATORY EMBRYO TRANSFER INCREASES SUCCESS OF PORCINE SOMATIC CLONING
Authors: Petersen B. , Lucas-Hahn A. , Lemme E. , Hornen N. , Hassel P. , Niemann H.
Published: December 2006

Somatic cloning has emerged as a valuable tool for the production of transgenic animals. However, the technology still suffers from low efficiencies (<1%). In the present study, we used 2 different recipient synchronization protocols and assessed the effects on the cloning efficiency in pigs using triple transgenic donor cells within a bigger study on xenotransplantation. Double transgenic adult fibroblasts (hCD59/DAF) were transfected with a construct coding for human thrombomodulin (hTM). After selection with 800 µg mL−1 G418 for 14 days, cells were analyzed for integration of the construct by PCR and visually selected for expression of the hTM-GFP fusion protein. One positive cell clone was used for all nuclear transfers. Nuclear transfer (NT) was performed using in vitro-matured oocytes, as previously described (Hoelker et al. 2005 Cloning Stem Cells 7, 35–44). Peri-puberal German Landrace gilts were used as recipients in 2 different synchronization protocols. Group 1 received 1200 IU pregnant mare serum gonadotropin (PMSG) IM 117 h prior to embryo transfer, followed by 500 IU human chorionic gonadotropin (hCG) 72 h later. Nuclear transfer complexes were transferred surgically 46 h later, approximately 6 h post-ovulation. Group 2 was synchronized by administration of 5 mL Altrenogest for 12 days (20 mg gilt/day). At the end of treatment, the animals received 1000 IU PMSG IM, followed by an injection of 500 IU hCG 80 h later. Cloned embryos were transferred surgically approximately 20 h prior to ovulation. Maintenance of pregnancy was supported by injections of 1000 IU PMSG on Day 11 and 500 IU hCG on Day 14 of gestation. Animals were checked for pregnancy by ultrasound 25 days after transfer (Table 1). In group 1, only one pregnancy could be maintained, leading to the birth of one piglet after caesarean section. In contrast, 12 pre-ovulatory transfers resulted in 9 pregnancies with a total of 47 cloned piglets born. All offspring had normal birth weights (1.0–1.5 kg) and showed no malformations. Average litter size in group 2 was 5.2, and the overall efficiency was 3.1% and 4.4% when related to pregnant recipients. The results show that the transfer of NT-complexes into oviducts of pre-ovulatory recipients significantly improved the success rates of porcine cloning. This is an important feature for the production of transgenic pigs for biomedical and agricultural application.

Table 1.
Results of porcine nuclear transfer

This project is funded by the Deutsche Forschungsgemeinschaft (FOR 535).
77 LOSS OF SAND CAT CLONED EMBRYOS DURING EARLY PREGNANCY MAY BE ASSOCIATED WITH ABERRANT Oct-4 EXPRESSION
Authors: Pope C. E. , Gómez M. C. , Truhe M. , López M. , Dumas C. , Cole A. , Lyons L. , Dresser B. L.
Published: December 2006

Genetic incompatibilities between the donor nucleus and the recipient cytoplast of inter-species cloned embryos may be associated with a low frequency of development to the blastocyst stage and/or in vivo developmental competence. We have established pregnancies after the transfer of inter-species Black-footed cat (BFC; Felis nigripes) cloned embryos and live offspring after the transfer of inter-species African Wildcat (AWC; Felis silvestris lybica) cloned embryos into domestic cat recipients (G�mez et al. 2006 Theriogenology 66, 72; 2004 Cloning Stem Cells 6, 217). However, many of the pregnancies were re-absorbing before Day 30 of gestation. According to Arat et al. (2003 Mol. Reprod. Dev. 66, 334), inter-generic mouse–bovine cloned embryos do not express the POU transcription factor Octamer-4 (Oct-4), and this abnormal gene expression may compromise pluripotency and contribute to the failure of cloned embryos to undergo post-implantation development (Boiani et al. 2002 Genes Dev. 16, 1209). The purpose of the present study was to evaluate (1) the in vivo developmental competence of nuclear transfer embryos derived by fusion of Sand cat (SC; Felis margarita) fibroblasts with domestic cat (DSH; Felis catus) cytoplasts after transfer into domestic cat recipients, and (2) protein distribution of Oct-4 in derived cloned blastocysts. Fibroblast cell lines were established from skin biopsies of 2 adult SC (both males). Cloned embryos were produced as described by G�mez et al. (2004 Cloning Stem Cells 6, 217). Reconstructed SC (n = 1113) Day 1 NT embryos were transferred by laparoscopy into the oviducts of 29 gonadotropin-treated DSH recipients on Day 1 after induced ovulation. Pregnancy was assessed by ultrasonography on Day 22. Seven (24%) cats receiving somatic cell nuclear transfer (SCNT) embryos became pregnant. Two SC cloned embryos developed to term; one kitten died in utero on Day 61 of gestation and the second kitten died 2 h after being delivered by caesarean section on Day 62 of gestation. The clonal status of the SC kittens was assessed by multiplex PCR amplification of 20 microsatellite markers. Immunocytochemical localization of Oct-4 was determined on Day 8 SCNT (n = 16) and DSH/IVF (n = 10) blastocysts. Embryos were incubated in 1 : 100 anti-human Oct-4 polyclonal antibody for 1 h, followed by incubation in 1 : 128 FITC-labeled anti-goat IgG for 1 h. Embryos were then counter-stained with propidium iodide, and examined with epifluorescence microscopy. Most cloned blastocysts had a detectable signal of Oct-4 (69%), but some exhibited aberrant expression in the trophectoderm (TPD; 44%). In contrast, the signal in most DSH/IVF blastocysts was restricted to the inner cell mass (ICM; 80%). Oct-4 expression was not detected in 2-cell DSH/IVF embryos. These observations suggest that aberrant Oct-4 expression may contribute to the embryonic loss observed in the present study.