bluuebunny/crossref_metadata_2025 huggingface dataset:
374 THE EFFECTS OF POLYVINYLPYRROLIDONE ON BOVINE SPERM FUNCTION AND EMBRYO DEVELOPMENT
Authors: Kato Y. , Fukushima M. , Kenmotsu A. , Chikazawa K. , Nagao Y.
Published: December 2006

In assisted reproduction by ICSI, PVP has been successfully used to replicate the viscosity of sperm solution, thus facilitating the handling and immobilization of spermatozoa. Sperm is suspended in medium containing polyvinylpyrrolidone (PVP), then injected into the oocytes together with a small amount of the medium in ICSI. However the effects of PVP on sperm function and embryo development have not been investigated in detail. In the present study, we investigated the effects of PVP solution on sperm function and embryonic development. Frozen–thawed spermatozoa from a Japanese Black bull and immature COCs from slaughterhouse bovine ovaries were used for all experiments. In experiment 1, bovine sperm was cultured in SOF or SOF containing 10% PVP. For detection of sperm acrosomal and chromatin integrity, sperm cultured in each medium were stained by the triple staining method and acridine orange after 0, 15, 30, and 60 min of culture. In experiment 2, zygotes were injected with PVP solution and cultured in vitro; subsequent cleavage and development to blastocysts were examined. In experiment 3, zygote injected with PVP solution was fixed by 4% paraformaldehyde after 1–3 h of PVP injection. The location of PVP solution in zygote was observed. In experiment 4, two-cell embryos were microinjected with a solution of dextran conjugated with fluorescein (FITC-dextran) and cultured in vitro. The location of FITC-dextran in the embryo was examined. In experiment 1, acrosome reactions of the sperm were enhanced after 15 min of incubation in PVP solution (P < 0.05), but chromatin integrity of the sperm was not influenced (P > 0.05). In experiment 2, PVP suppressed the development of the zygote to 2-cell, morula and blastocyst (75.0%, 35.1%, and 26.3% vs. 61.3%, 20.2%, and 12.9% for control and PVP group, respectively, P < 0.05). In experiment 3, the locations of PVP solution in the zygote were observed 1–3 h after injection. In experiment 4, FITC-dextran was observed in ICM at the blastocyst stage. These findings suggest that PVP affects the acrosome but not the chromatin of sperm in ICSI. PVP solution exists locally in embryos injected and affects the developmental capacity of the embryos.
375 UNEXPECTED SEVERE ABNORMALITIES IN MOUSE ROSI OFFSPRING
Authors: Moreira P. N. , Fernández-González R. , Pérez-Crespo M. , Bermejo P. , Hourcade J. D. , Rey R. , Gutiérrez-Adán A.
Published: December 2006

Live offspring resulting from round spermatid injection (ROSI) was first accomplished in the mouse, but similar success has been obtained in rat, hamster, rabbit, mastomys, pig, monkey, and human. ROSI has received clinical attention because some infertile men have no spermatozoa or just a very few in their testes, and these are difficult to harvest and are frequently dead and deformed. Although some clinicians were able to generate healthy children by ROSI, others could not (reviewed in Yanagimachi 2004 Reprod. Biomed. Online 9). The clinical value of ROSI has been widely debated. It remains unclear if post-meiotic and pre-fertilization modifications of sperm cells are necessary to ensure normal development. In order to answer this question, we decided to study and compare mouse offspring generated by ROSI and intracytoplasmic sperm injection (ICSI). ROSI and ICSI with fresh sperm cells were carried out in the B6D2 mouse strain as described (Marh et al. 2003 Biol. Reprod. 69, 169–176; Moreira et al. 2005 Hum. Reprod. 20, 3313–3317). In vitro-produced embryos were transferred at the 2-cell stage into Day 1 pseudopregnant females. As shown in Table 1 oocyte survival after injection was significantly higher (z-test, P < 0.05) with ICSI (91%) than with ROSI (68%). The proportion of live offspring obtained by ICSI was also significantly higher (26% vs. 6%; z-test, P < 0.05). Moreover, fertilization with spermatozoa produced healthy offspring more efficiently than with round spermatids. Out of 30 live offspring generated by ROSI, 6 (20%) presented severe abnormalities during their first 6–8 weeks of age. One ROSI animal presented an abnormally swollen skull (hydroencephaly) with a very thin and soft cranial wall. Another developed a subcutaneous engrossment of the forehead, producing a crest-like appearance. Three others presented deviations in their vertebral columns (hyperkyphosis and scoliosis), and recently a testicular tumor was detected in another animal. These types of malformations were not observed in the control offspring. In our experience, very rarely are they observed after ICSI or in naturally mated animals. To our knowledge, and although the risks of the ROSI procedure have been extensively highlighted in human and other species, the phenotypic abnormalities observed in this study have never been reported. Presently, we keep monitoring these animals as they age, as part of an ambitious plan that is also intended to characterize and understand the origin of the possible phenotypic consequences of the ROSI procedure.

Table 1.
In vitro development and development to term of B6D2
mouse embryos generated by ROSI or ICSI
383 EFFECT OF TIME OF OVULATION INDUCTION BY GONADOTROPIN-RELEASING HORMONE OR PITUITARY LUTEINIZING HORMONE ON OVA/EMBRYO PRODUCTION IN SUPERSTIMULATED BEEF COWS INSEMINATED AT A FIXED TIME
Authors: Chesta P. , Tribulo R. , Tribulo H. , Balla E. , Baruselli P. S. , Bo G. A.
Published: December 2006

It has been shown than it is possible to AI embryo donors at a fixed time (FTAI) by removing the progestin-releasing device at the time of the last FSH treatment and inducing ovulation with gonadotropin-releasing hormone (GnRH) or porcine luteinizing hormone (pLH) at either 12 h (dairy cows: Bos indicus) or 24 h (beef cows: Bos taurus) after device removal (Bo et al. 2006 Theriogenology 65, 89–101). An experiment was designed to determine the best interval from device removal to GnRH or pLH treatment in Angus (beef cow) donors. Red Angus donors (43 cows and 56 heifers) were blocked by parity and randomly assigned to 1 of 4 treatment groups in a 2 × 2 factorial design. On Day 0, all donors received a DIB vaginal device (Syntex, Buenos Aires, Argentina) plus 2.5 mg estradiol-17β and 50 mg progesterone (P4; Laboratorio Rio de Janeiro, Argentina) IM. Superstimulatory treatments were initiated on Day 4, with a total dose of 320 mg (cows) or 200 mg (heifers) NIH-FSH-P1 of Folltropin-V (Bioniche Animal Health Inc., Belleville, Ontario, Canada) in twice daily IM injections over 4 days. All cows received prostaglandin-F treatment (150 µg d-cloprostenol; Bioprost; Biotay, Buenos Aires, Argentina) on Day 6 (AM and PM); DIB devices were removed with the last Folltropin-V treatment on Day 7 PM. Donors received 0.050 mg GnRH (Lecirelina; Gonasyn; Syntex, Buenos Aires, Argentina) or 25 mg Armour pLH (Lutropin-V; Bioniche) 12 h (Day 8 AM) or 24 h (Day 8 PM) later and were FTAI 12 and 24 h after GnRH or pLH. Ova/embryos were collected nonsurgically and classified according to IETS recommendations on Day 15 AM for donors receiving GnRH or pLH on Day 8 AM and on Day 15 PM for those receiving GnRH or pLH on Day 8 PM. Data were analyzed by two-way ANOVA. Although there was no effect of induction agent (GnRH vs. pLH; P > 0.2) and/or interaction between the induction agent and the time of treatment (P > 0.13) on ova/embryo production, treatment with GnRH or pLH on Day 8 AM (i.e. 12 h after DIB removal) resulted in a significantly lower (P < 0.05) number of fertilized ova and Grades 1 and 2 embryos than treatment on Day 8 PM (i.e. 24 h after DIB removal; Table 1). It was concluded that GnRH and pLH were equally efficacious for inducing ovulation in superstimulated donors that were fixed-time AI. Furthermore, a 24-h interval from progestin device removal to GnRH or pLH administration was preferable for Bos taurus beef donors.

Table 1.
Ova/embryo production in Angus donors treated with GnRH or pLH
12 or 24 h after removal of a progestin-releasing device and FTAI 12 and 24 h
later
387 EFFECT OF NUMBER OF INSEMINATIONS ON EMBRYO PRODUCTION IN SUPERSTIMULATED NELORE (BOS INDICUS) COWS SUBJECTED TO FIXED-TIME ARTIFICIAL INSEMINATION
Authors: Martins C. M. , Ayres H. , Torres-Junior J. R. S. , Souza A. H. , Bó G. A. , Baruselli P. S.
Published: December 2006

In embryo transfer programs, 2 inseminations per superstimulated donor are usually recommended. Based on studies of follicular dynamics in Bos indicus donors (Baruselli et al. 2006 Theriogenology 65, 77–88), we tested the hypothesis that a single fixed-time AI (FTAI) at 16 h after pituitary luteinizing hormone (pLH) treatment is as efficacious as 2 FTAI at 12 and 24 h after the pLH treatment. Suckling Nelore cows (n = 10), 60 � 12 days postpartum, were equally distributed in 2 experimental groups: G-1TAI vs. G-2TAI, in a crossover design. All animals received an intravaginal progesterone device (DIB; Syntex S.A., Buenos Aires, Argentina) and 2 mg of estradiol benzoate (IM, Ric Be; Syntex) at random stages of the estrous cycle (Day 0). Superstimulatory treatments started on Day 4 with pFSH (133 mg; Folltropin-V; Bioniche Animal Health Canada, Inc., Belleville, Ontario, Canada) in 8 decreasing doses, given IM 12 h apart. On Day 6, all cows received 2 (AM and PM) treatments of 0.15 mg of d-cloprostenol (Prolise; Syntex). The DIB devices were removed 36 h after the first PGF2� injection. The pLH treatment (25 mg IM; Lutropin-V; Bioniche) was given 48 h after the first PGF2� treatment (12 h after the last pFSH treatment). In the G-2TAI group, all cows received 2 inseminations (12 and 24 h after the pLH). Cows in the G-1TAI group received a single insemination (16 h after the pLH). All inseminations were done with the same batch of semen from a single bull. Ultrasound examinations (PIE Medical Scanner 200; Pie Medical Equipment, Maastricht, The Netherlands) were performed every 12 h from 0 to 72 h after pLH treatment to evaluate the number of ovulatory follicles (<8 mm), the ovulation rate, and the time of ovulation. Variables were analyzed by ANOVA and chi-square test. The homogeneity of the variances (distribution of ovulations) was analyzed by Bartlett's test. Conclusions were based on a 5% level of significance. The results for G-1TAI and G-2TAI were, respectively: number of follicles >8 mm at the time of pLH administration (16.2 � 1.4 vs. 14.8 � 1.2; P > 0.05), ovulation rate (63.8 � 3.8% vs. 64.2 � 4.3%; P > 0.05), interval from the first to the last ovulation (32.4 � 1.8 vs. 33.6 � 1.6 h; P > 0.05), total ova/embryos (8.2 � 0.9 vs. 7.2 � 0.8; P > 0.05), Grade 1 embryos (2.0 � 0.5 vs. 2.3 � 0.4; P > 0.05), transferable embryos (Grades 1, 2, and 3) (4.3 � 0.7 vs. 4.2 � 0.6; P > 0.05), freezable embryos (Grades 1 and 2) (2.9 � 0.6 vs. 2.8 � 0.4; P > 0.05), unfertilized ova (0.6 � 0.2 vs. 0.8 � 0.2; P > 0.05), and degenerated embryos (3.3 � 0.9 vs. 2.2 � 0.3; P > 0.05). These results suggest no significant differences in embryo production between superstimulated Nelore donors inseminated once or twice by fixed time following administration of pLH.
This work was supported by Tecnopec, Brazil.
389 ASSOCIATION BETWEEN WALKING ACTIVITY AND SUPEROVULATORY RESPONSE IN JAPANESE BLACK CATTLE
Authors: Narita M. , Ito M. , Tanimoto Y.
Published: December 2006

Walking activity of cattle changes at estrus, and in this study, we measured the walking activity of Japanese black cattle in a loose-housing barn. Recently, a system (Gyu-Ho; Comtec, Miyazaki, Japan) for determining estrous activity has been developed. This system uses a pedometer equipped with a transmitter, which makes it possible to measure walking activity over a period of time. The objectives of the present study were to compare the walking activity at an induced estrus in superovulating cattle (SOV group, n = 10) to natural estrus in nonstimulated cattle (Natural group, n = 10), and to determine whether there is a correlation between estrus behavior and superovulatory response. A pedometer equipped with a transmitter was placed on the front of the metatarsal bone of each cow. In addition, a sensor (Hatujo-Pitari; Comtec), which determined the duration and frequency of standing estrus by mount pressure, was placed on the tail head of each cow. The walking activity and times of standing estrus were recorded through a receiver every h on a personal computer. In the SOV group, superstimulation was initiated on Days 9 to 13 (Day 0 = day of estrus) with a total dose of 28 mg FSH (Antrin R-10; Kawasaki-Mitaka, Kawasaki, Japan) via twice-daily IM injections in decreasing doses for 4 days. At 72 h after the first FSH injection, 750 µg cloprostenol (PGF; Cropromate-C; Dainippon Sumitomo Pharmaceuticals, Osaka, Japan) was administered. The donors were artificially inseminated at 60 h after PGF. Embryos were collected nonsurgically 6 days after estrus. Walking activity and duration of standing estrus were compared in the SOV and Natural groups. Data were analyzed by t-test (walking activity) and regression analysis. There were no differences between the Natural and SOV groups for the endpoints measured. Mean CL number was 15.7 ± 6.9, mean number of recovered ova/embryos was 10.3 ± 9.9, and mean number of normal (IETS grades 1 and 2) embryos was 6.9 ± 7.1. A significant (P < 0.02) correlation was found between the number of CL and the duration of estrus (r = 0.7). Data suggest that walking activity at estrus did not differ between superstimulated Japanese black cattle and those experiencing a natural estrus. However, superovulatory response appears to be related to the duration of estrous behavior.

Table 1.
Walking activity at estrus of SOV and Natural groups
390 THE EFFECT OF REDUCED FSH DOSE AND NUMBER OF TREATMENTS ON SUPEROVULATORY RESPONSE IN CIDR-TREATED KOREAN NATIVE COWS
Authors: Son D. S. , Choe C. Y. , Cho S. R. , Choi S. H. , Kim H. J. , Kim I. H.
Published: December 2006

Reducing the total dose and numbers of treatments with FSH for superstimulation without decreasing embryo yield may be less stressful and more economical for bovine embryo transfer. The objective of this study was to investigate the effect of dose and the number of days of FSH treatment on superovulatory responses in CIDR-treated Korean native cows. Forty-two cows, at random stages of the estrous cycle, received a CIDR device (CIDRTM; InterAg, Hamilton, New Zealand), 1 mg estradiol benzoate (SY Esrone; Samyang, Seoul, Korea) and 50 mg progesterone (SY Ovaron; Samyang); gonadotropin treatment began 4 days later. Cows were divided into 2 groups based on the dose and numbers of days of treatment with porcine FSH (pFSH): T1 group (n = 20): a total of 28 mg pFSH (recommended dose of Antorin�; Kawasaki Pharmaceutical, Tokyo, Japan) was given in twice daily IM injections in decreasing doses over 4 days (5, 5, 4, 4, 3, 3, 2, and 2 mg); and T2 group (n = 22): a total of 24 mg pFSH given in twice daily decreasing doses over 3 days (5, 5, 4, 4, 3, and 3 mg). Otherwise, all cows received the same treatments. Twenty-five and 15 mg dinoprost (PGF2α; Lutalyse; Pharmacia & Upjohn, Puurs, Belgium) were given with the 5th and 6th injections of pFSH, respectively. CIDR devices were withdrawn with the 6th pFSH injection, and the cows received 100 �g Gonadorelin (GnRH; Fertagyl; Intervet, Boxmeer, The Netherlands) 36 h after CIDR device removal. Cows were artificially inseminated using commercial semen from 4 Korean native bulls twice, at 48 and 60 h after CIDR device removal, and embryos were recovered 6 or 7 days after the 2nd insemination. The number of CL was counted on the day of embryo recovery by transrectal ultrasonography (Sonovet 600 with 5.0 MHz linear-array transducer; Medison Co., Ltd., Seoul, Korea). The recovered embryos were evaluated according to the IETS Manual for stage of development and quality. All data between groups were compared using Student's t-test. The numbers of CL (9.7 � 1.1 vs. 9.4 � 1.3), total ova/embryos (7.2 � 1.1 vs. 6.3 � 1.4), transferable embryos (4.4 � 1.0 vs. 3.6 � 0.9), degenerate embryos (0.9 � 0.3 vs. 1.3 � 0.4), and unfertilized ova (2.0 � 0.6 vs. 1.5 � 0.5) did not differ between groups (T1 vs. T2), respectively (P > 0.05). Data indicate that the reduced dose (24 vs. 28 mg) and numbers of treatments (6 vs. 8) of pFSH for superstimulation of Korean native cows does not affect the embryo yield.
391 EFFECTS OF SEASON AND LACTATION STATUS ON EMBRYO PRODUCTION IN HOLSTEIN COWS
Authors: Vasconcelos J. L. M. , Jardina D. T. G. , Rodrigues C. A. , Guerreiro V. C.
Published: December 2006

The aim of this study was to evaluate effects of season and lactation status on embryo production in Holstein donors (n = 51). Data from 195 embryo collections (94 from lactating cows and 101 from non-lactating cows), at a dairy farm located in Descalvado, Sao Paulo, Brazil, in 2005, were analyzed. The superstimulation protocol included two 3-mg norgestomet ear implants (Crestar�; Intervet, Sao Paulo, Brazil) and an injection of 3 mg of estradiol benzoate IM (EB) on random days of the estrous cycle. Four days later, 500 IU FSH IM (Pluset�; Calier, Buenos Aires, Argentina) were divided into 8 decreasing doses given 12 h apart. With the seventh FSH injection, 0.530 mg IM of sodium cloprostenol (PGFα Ciosin�; Coopers-Brazil, Sao Paulo, Brazil) was given, and implants were removed with the eighth FSH injection. Twelve hours later, 250 �g IM of gonadorelin (Fertagyl�; Intervet) was given, followed by two AI 12 and 24 h later. Ova/embryos were recovered 6.5 days after the first insemination. The total number of ova/embryos, IETS grades 1, 2, and 3 (viable) and grade 4 plus unfertilized (non-viable), were analyzed by General Linear Model (GLM). Effects of donor, lactation status (lactating or non-lactating), sire, season (1: January through March, n = 56; 2: April through June, n = 37; 3: July through September, n = 50; 4: October through December, n = 52), and their interactions were included in the model. There was an effect of donor on all outcome variables (P < 0.01). There was an effect of season on the total number of ova/embryos (1: 10.2 � 1.2; 2: 6.6 � 1.0; 3: 10.0 � 1.1; 4: 11.2 � 1.2; P < 0.02) and of viable embryos (1: 3.7 � 0.5; 2: 2.4 � 0.5; 3: 5.9 � 0.9; 4: 4.4 � 0.7; P < 0.001). Lactation status influenced the total number of ova/embryos (lactating: 10.9 � 0.9 vs. non-lactating: 8.7 � 0.7; P < 0.001) and the number of non-viable embryos (lactating: 6.7 � 0.8 vs. non-lactating: 4.4 � 0.5; P < 0.01). However, there was no effect of lactation status on the number of viable embryos (lactating: 4.14 � 0.5 vs. non-lactating: 4.29 � 0.5; P > 0.10). In summary, the number of viable embryos in Holstein cows was influenced by season and donor, but not by lactation status.
397 LONG-TERM CULTURE OF BOVINE SECONDARY FOLLICLES IN MEDIA CONTAINING BSA, FCS, PLASMA, AND FOLLICULAR FLUID
Authors: Takajo A. , Bao R. M. , Hamawaki A. , Yoshikawa M. , Miyano T.
Published: December 2006

Bovine oocytes within early antral follicles 0.4–0.7 mm in diameter (oocyte: 90–99 �m in diameter) grow to a final size of 120 �m after culture for 2 weeks. However, there has been little success in culturing oocytes in secondary follicles. The aim of this study was to establish a long-term culture system to support the growth of bovine oocytes within secondary follicles. We examined the effect of bovine serum albumin (BSA), heat-treated fetal calf serum (FCS), bovine plasma (Pl), and bovine follicular fluid (FF) on follicular development and oocyte growth. Bovine secondary follicles 150–200 �m in diameter were collected mechanically from bovine ovaries. In the first experiment, secondary follicles were embedded in collagen gels and cultured in �MEM supplemented with 0.1 mg mL-1 sodium pyruvate, 0.08 mg mL-1 kanamycin, 0.05 mM β-mercaptoethanol, 25 or 50 ng mL-1 FSH, and 3 mg mL-1 BSA, 5% FCS, 5% Pl, or 5% FF for 4 weeks. Secondary follicles formed an antrum and the mean diameters of follicles increased significantly in all groups (P < 0.05; Student's t-test) other than the BSA-supplemented one. Integrity of the follicles cultured in BSA- and Pl-supplemented media was maintained better than in other groups, and supplementation with 25 ng mL-1 FSH was more effective than 50 ng mL-1 FSH (P < 0.05; chi-square test). Moreover, the antra were maintained in the Pl-supplemented medium. In the BSA- and Pl-supplemented groups with 25 ng mL-1 FSH, 46% (11/24) and 48% (11/23) of normal-looking oocytes were recovered, and their mean diameters were 75.3 � 3.8 and 91.9 � 2.7 �m, respectively, which were significantly larger than before culture (59.8 � 3.5 �m (P < 0.05); Student's t-test). On the other hand, only 0–7% of the oocytes showed normal morphology in FCS- and FF-supplemented media after 4 weeks. In the second experiment, secondary follicles were cultured in BSA- or Pl-supplemented medium with 25 ng mL-1 FSH for 8 weeks to produce fully grown oocytes. About 30% of the oocytes showed normal morphology in both groups, although they had not reached full size. The diameters of oocytes cultured in BSA- or Pl-supplemented media were 98.8 � 2.1 and 97 � 1.8 �m, respectively. Some of the oocytes from BSA-supplemented medium were denuded, whereas the oocytes were enclosed by cumulus cells in Pl-supplemented medium. These results suggest that bovine secondary follicles can be efficiently cultured in BSA- or Pl-supplemented medium with 25 ng mL-1 FSH in collagen gels over one month.
4 MYOSTATIN GENE KNOCKDOWN THROUGH LENTIVIRAL VECTOR-MEDIATED DELIVERY OF shRNA FOR IN VITRO PRODUCTION OF TRANSGENIC BOVINE EMBRYOS
Authors: Milazzotto M. P. , Feitosa W. B. , Strauss B. E. , Bajgelman M. , Mendes C. M. , Assumpção M. E. O. A. , Visintin J. A.
Published: December 2006

The main goal of husbandry and beef cattle production is to enhance performance rates, for example, weight gain. Myostatin is referred to as a negative regulator of skeletal muscle growth. Genetic engineering of this character in order to produce double muscling animals that can transmit to future progeny will enhance its usefulness. The present research aimed to analyze myostatin inhibition through lentiviral-mediated delivery of shRNA in mouse myoblast culture and the feasibility of the lentiviral-mediated delivery of shRNA into in vitro-produced transgenic bovine embryos. In order to achieve knockdown of myostatin in cell and embryo culture, a lentiviral vector was constructed with ubiquitin C promoter-driven GFP gene (green fluorescent protein) and shRNA to suppress myostatin gene expression driven by the U6 promoter. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR of myostatin and GAPDH genes. Later, bovine oocytes were in vitro-matured and the lentiviral vector was microinjected into the oocyte perivitelline space (2.5 � 106 IU mL-1) after mechanical and chemical cumulus cell removal. Non-microinjected mature oocytes were considered as control. After microinjection, oocytes were fertilized and cultured in vitro. After 4 and 9 days of culture, embryos were evaluated by epifluorescence microscopy. The GFP-positive embryos were green under fluorescence. Cell morphology and embryo development rate data were analyzed by Minitab Release 14 Statistical Software (Minitab, Inc., State College, PA, USA), submitted to ANOVA, and compared by Tukey test (P d 0.05). Real-time PCR data were analyzed by Pair-Wise Fixed Reallocation Randomization Test using REST2005 software. Cell morphology results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells as the transducted group progressed less to myotubes than in the control group. A lower amount of myostatin mRNA after 72 h of differentiation indicated an inhibition tendency by real-time PCR. In relation to the transgenic embryo production, 96.9 � 0.34% (62.65) developed to cleavage, 80.24 � 4.38% (51/65) were GFP-positive, and 50.95 � 3.37% (26/65) achieved blastocyst stage. After hatching, 3.07% (2/65) of GFP-positive embryos maintained fluorescence. In relation to the control group, the cleavage rate was 93.81 � 0.68% (61/65); the blastocyst rate 38.34 � 2.36% (25/65), and none were fluorescent. In conclusion, myostatin gene knockdown was effectively performed by lentiviral vector-mediated delivery of shRNA. Thus, novel studies about the efficiency of this vector on transgenic embryo production can be performed.
This work was supported financially by FAPESP 03/0156-9.
40 TREATMENT OF DONOR CELLS AND ITS EFFECT ON INTERSPECIES NUCLEAR TRANSFER
Authors: Hashem M. A. , Bhandari D. P. , Kang S. K. , Lee B. C.
Published: December 2006

The present study was undertaken to examine the effect of donor cells, under a variety of treatment effects, on the development of goral porcine reconstructed embryos. Three experiments were performed, each with a one-way completely randomized design involving 3 to 4 replicates of all. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused on the effects of cycling, serum-starved (SS), and fully confluent stages of goral cells when reconstructed with porcine enucleated oocytes. In Experiment II, the effects of 2 antioxidants, β-mercaptoethanol (β-ME, 10 �M) and cysteine (2 mM), were examined after cells were fully confluent without serum starvation for 4 h. In Experiment III, the effect of different levels of dimethylsulfoxide (DMSO) at 0%, 0.5%, and 1.0% were tested, after 4 h of treatment, on the development rate after reconstruction with enucleated porcine oocytes. From the results, it appears that there were no significant (P > 0.05) differences from cleavage to morula among cyclic, SS, and fully confluent stages of the cell cycle. None of the treated group reached the blastocyst stage. There were no significant differences at the fused, 2- to 4-cell, and morula stages of embryo development after treatment of the donor cells with β-ME and cysteine before nuclear transfer. However, in the case of 8- to 16-cell stages, there were significant differences between β-ME and cysteine; the donor cells treated with β-ME had a better development rate than those treated with cysteine. No significant differences were observed in fusion, 2- to 4-cell, 8- to 16-cell, blastocyst, and hatching blastocyst stages at the 0.0, 0.5, and 1.0% levels of DMSO. However, there were statistically significant (P < 0.05) differences observed at the morula stage of embryo development. When donor cells were treated for 4 h with 0.5 and 1.0% levels of DMSO, goral-porcine reconstructed embryos reached the morula stage. From the results it can be concluded that goral somatic cells can be de-differentiated in porcine oocytes after treated with antioxidants and DMSO.
401 USE OF FLOW CYTOMETRY TO EVALUATE THE CAPACITY OF BOAR SPERM TO BIND TO EXOGENOUS DNA OF DIFFERENT SIZES
Authors: García-Vázquez F. , Gumbao D. , Gutiérrez-Adan A. , Gadea J.
Published: December 2006

Sperm-mediated gene transfer (SMGT) is based on the ability of sperm cells to bind exogenous DNA and transfer it into the egg at fertilization (Lavitrano et al. 1989 Cell 57, 717–723). SMGT has been reported in several species, but different degrees of efficiency have been reported in different laboratories. In this study, in order to optimize and understand the mechanism of SMGT, the capacity of spermatozoa to bind 2 different sizes of exogenous DNA (previously marked with fluorescein using flow cytometry), the kinetics of DNA binding, and the effect of the male used were evaluated. Semen from 7 fertile boars was recovered and immediately diluted 1 : 10 in SFM (swine fertilize medium) at 37�C and later centrifuged (800g 10 min, 25�C), discarding the seminal plasma to avoid a detrimental effect on DNA binding to cells. Two groups were established depending on the size of DNA (Group A: 5.4 kb; Group B: 20 kb). Linearized plasmid DNA (5.4 kb and 20 kb), marked by random primed DNA labeling with fluorescein-12-dUTP (Roche Diagnostics, Mannheim, Germany) was added (1 � 108 spermatozoa/mL + 5 �g of DNA/mL) in each group and incubated at 16�C. Initial spermatozoa contact with DNA was the starting point, initiating the measurements in the cytometer. Time measurements were taken at 0, 15, 30, 60, 90, and 120 min of incubation. Samples were analyzed on a Coulter Epics XL flow cytometer (Beckman Coulter, Inc., Fullerton, CA, USA). A total of 10 000 sperm cells were counted per sample and 3 measurements in each incubation time, with sample running rates of approximately 600 events/s. Fluorescence was measured using the FL1 sensor, with a 525-nm band-pass filter to detect fluorescein isothiocyanate. The results showed that no significant effect on the parameters studied was associated with the kind of DNA employed, and the main DNA binding to spermatozoa was achieved as soon as 15 min in both groups (Group A: 13.43 � 0.74%; Group B: 13.97 � 0.77%); thereafter, no further significant increase in the binding was detected (Group A: 14.27 � 0.88%, 14.16 � 1.05%, 13.41 � 1.11%, 13.53 � 1.26%; Group B: 13.97 � 0.77%, 15.23 � 0.93%, 15.19 � 0.92%, 15.17 � 1.05%, at 30, 60, 90, and 120 min, respectively). Nevertheless, a significant boar effect was detected. These data suggest that boar sperm is able to bind 20 kb with the same efficiency as 5 kbp and that flow cytometry is a practical tool to evaluate sperm DNA binding capacity in a specific and objective manner and to select the boar that produces better results.
This work was supported by BIOCARM 10BIO2005/01-6463.
404 DNA TAKE-UP BY SPERM AND IN VITRO EMBRYO DEVELOPMENT BY SPERM-MEDIATED GENE TRANSFER IN THE PIG
Authors: Kim T. S. , Cao Y. , Cheong H. T. , Yang B. K. , Park C. K.
Published: December 2006

Sperm mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind and internalize exogenous DNA and transfer it into the oocytes at fertilization. The purpose of this study was to assess introducing exogenous DNA into boar spermatozoa by DNA solution or DNA/liposome complex under different conditions (period of incubation, exogenous DNA, liposome, and concentration of spermatozoa). Genomic DNA of sperm loaded with DNA by treatment was isolated by alkaline lysis. Quantitation of exogenous DNA amplified by PCR was analyzed by agarose electrophoresis densitometry. The quality of treated spermatozoa under the best conditions or no treatment (control) was evaluated during incubation (0, 2, 4, and 6 h) for viability (SYBR-14/PI), motility (Makler counting chamber), morphology (rose bengal staining), and acrosomal status (Coomassie staining). Sperm loaded with DNA also were used for in vitro fertilization. Immature oocytes incubated in TCM-199 medium for 44 h were fertilized in mTBM medium for 6 h and cultured in PZM-3. Cleavage and development of embryos were assessed on Days 2 and 7 of culture, respectively. Transfection rates at the blastocyst stage were assessed by PCR analysis. Data were evaluated by Duncan's multiple-range test using the GLM procedure. In the preliminary experiment, DNA uptake of spermatozoa by DNA solution and liposome/DNA complex was completed within 90-120 min. Transfection efficiency of spermatozoa was significantly (P < 0.05) higher in the 105 spermatozoa group than in the other groups (104, 106, and 107 spermatozoa). The transfection efficiency was gradually increased by increasing the concentration of exogenous DNA. On the other hand, viability of transfected spermatozoa by all treatments (control, DNA solution, and DNA/liposome) at 0 h (72.3 � 0.2, 70.8 � 1.8, and 68.0 � 2.2%, respectively) of storage was significantly (P < 0.05) lower than for fresh spermatozoa (83.3 � 1.7%). Survival and motility of all treatments after 4 h of storage were significantly (P < 0.05) lower than at 0 and 2 h. Both abnormality and acrosome reaction of spermatozoa were gradually increased with prolonged storage periods. On the other hand, the cleavage rate of embryos by DNA/liposome complex (56.3 � 2.3%) was significantly (P < 0.05) lower compared to both DNA solution (64.0 � 1.1%) and control (67.8 � 2.3%). The developmental rates of blastocysts were significantly (P < 0.05) lower in the liposome/DNA complex and DNA solution groups (9.1 � 1.3 and 11.3 � 0.8%) than in the control group (22.2 � 0.6%). The transfection rates of blastocysts were higher in the liposome/DNA group (54.3 � 12.0%) than in the DNA solution group (38.7 � 6.6%). These results show that the SMGT method under the control conditions efficiently transfers exogenous DNA into the porcine oocytes.
This work was supported by the Research on the Production of Bio-organs (No. 2005 03020302) Ministry of Agriculture and Forestry, Republic of Korea
407 AN ATTEMPT TO GENERATE TRANSGENIC CHICKENS BY INJECTION OF EGFP OR YFP GENE-TRANSFECTED BLASTODERMAL CELLS
Authors: Matsubara Y. , Tagami T. , Kagami H. , Nagai T.
Published: December 2006

Transgenic chickens have been generated from blastodermal chimeric chickens created by an injection of the foreign DNA-transfected blastodermal cells into recipient embryos. However, there has been no method that allows the efficient foreign DNA transfection into chicken primary cells without using viral vectors. This method using viral vectors has a limitation on the size of DNA that can be transfected, and homologous recombination is not possible with this method. This study was conducted to establish a DNA transfection method using electroporation, which can transfect up to 5 kb DNAs into cells. Embryos at stage X were obtained from freshly laid eggs (200) of Barred Plymouth Rock chickens. Blastodermal cells (105 cells) were then collected from them and transfected with the linearized EGFP or YFP gene by electroporation using a Nucleofector Device (Amaxa, Inc., Gaithersburg, MD, USA). After transfection, the transfected chicken blastodermal cells were injected into White Leghorn recipient embryos to produce chimeric chickens. By 48 h after gene transfection, EGFP or YFP gene expression in the cells was easily observed under a fluorescence microscope. The gene expression rate, however, could not be determined, because the cultured chicken blastodermal cells were not able to separate and it was impossible to load them on the flow cytometer to determine the rate. The blastodermal cells transfected with the EGFP or YFP gene were cultured over night and injected into recipient embryos. The manipulated embryos were cultured ex ovo until they hatched. Dead embryos before hatch were dissected and observed under the fluorescence microscope to determine whether they had fluorescent organs. EGFP or YFP gene expression was detected at several areas: head, somite, inner tissues, and limb bud of embryos, but not germinal tissues. Six female chicks and 10 male chicks were obtained. Five females and 6 males were raised until they were sexually mature. Five female chickens were artificially inseminated with semen of Barred Plymouth Rock males to obtain progeny from donor transfected cells. However, no progeny was obtained. Thus the contribution of donor cells to the germ cells could not be determined, and it was not clear if the expression of EGFP gene or YFP gene was stable. Furthermore, semen could not be collected from six males because of a technical problem, and males were not mated. Although a large number of cells must be used to transfect a foreign gene, the novel gene transfer method in chicken in this study has great significance in the field of transgenic chicken production. Even if the gene expression is transient, this transfer method will enable us not only to integrate more than 5 kb DNA into the avian genome but also to induce homologous recombination in cultured avian cells.
409 EFFICIENCY OF CO-TRANSFECTION OF TRANSGENE AND Neor GENE INTO GOAT AND PIG FETAL FIBROBLASTS
Authors: Shin Y. M. , Chang S. M. , Kim B. C. , Park C. S. , Jin D. I.
Published: December 2006

Transgenic animals can be generated by nuclear transfer with genetically modified somatic cells in which the essential procedure of transgene transfection is required. Most transgene vectors are constructed to contain transgene and drug-resistant genes to enrich for somatic cells in which transgene integration has occurred. However, construction of transgene vectors along with drug-resistant genes may not be easy, due to inappropriate restriction sites. Therefore, in this study, two separate constructs, human tPA cDNA fused to β-casein promoter sequence as a transgene vector and neomycin-resistant gene (Neor) driven by PGK promoter as a drug-selectable gene, were co-transfected into pig and goat fetal fibroblast cells to estimate the efficiency of transgene transfection following G418 selection. First, goat fetal fibroblasts (GFF) and pig fetal fibroblasts (PFF) were tested for G418 resistance with different concentrations of G418. The pertinent concentrations of G418 were 800 µg mL−1 for GFF and 200 µg mL−1 for PFF. The linearized tPA vector and Neor gene vector were co-transfected into goat fetal fibroblasts and pig fetal fibroblasts with FuGENE6 transfection reagent (Roche Diagnostics, Mannheim, Germany). The cells were selected following exposure of 800 µg mL−1 and 200 µg mL−1 G418 for GFF and PFF, respectively, for 14 days. Cell colonies surviving G418 selection were assayed by PCR amplification with tPA-specific primers. Initially 2 × 106 GFF and PFF were transfected. Resistant colonies were counted and transferred to 24-well plates for expansion and PCR analysis. The results of co-transfection experiments are summarized in Table 1. The transfection of 2 × 106 GFF and PFF yielded an estimated 96 and 93 colonies, respectively, which survived as the G418 selection. However, 54 colonies of GFF and 39 colonies of PFF proliferated during expansion and were subjected to PCR analysis. Twenty-three and 5 of these colonies were identified to contain tPA transgene in GFF and PFF colonies, respectively. Transfection frequencies for tPA gene were 42.6% and 12.8% in GFF and PFF, respectively. These results suggest that co-transfection of transgene vector with Neor gene can be an alternative method for transfection of transgenes into fetal fibroblast cells.

Table 1.
Transfection efficiency of goat fetal fibroblasts (GFF) and pig fetal fibroblasts (PFF) following co-transfection of tPA gene and Neor gene
410 USE OF SPERMATOZOA AS VECTORS OF EXOGENOUS DNA FOR IN VITRO PRODUCTION OF BOVINE TRANSGENIC EMBRYOS
Authors: Simões R. , Binelli M. , Nicacio A. C. , Milazzotto M. P. , Feitosa W. B. , Visintin J. A. , Assumpção M. E. O. A.
Published: December 2006

There are many methods to produce transgenic animals, although when considering bovine species, those methods offer low repeatability, high costs, and low transgene integration efficiency. To overcome these difficulties, sperm mediated gene transfer (SMGT) could be used as an alternative to produce transgenic embryos. This technology allows carrying exogenous DNA into the oocyte during the fertilization period, once spontaneous binding exists between spermatozoa and exogenous DNA. The aim of this study was to compare 4 methods for incorporating DNA into sperm cells—sperm incubation, capacitation, electroporation, and lipofection—to verify the efficiency of embryo production with exogenous DNA, employing spermatozoa as a vector. Cumulus–oocyte complexes (COCs) from abattoir-derived bovine ovaries were randomly divided into 5 groups (4 experimental groups: sperm incubation, sperm capacitation with calcium ionophore, electroporation, and lipofection; and 1 control group). In vitro maturation was performed in TCM-199 medium supplemented with 10% FCS, sodium pyruvate, gentamycin, FSH, hCG, and estradiol in an incubator at 39�C, in an atmosphere of 5% CO2 in air and high humidity for 24 h. After Percoll gradient (45/90%) separation at 600g for 30 min, spermatozoa were washed in Talp Semen medium (200g for 5 min). For in vitro fertilization (IVF), 5 � 106 spermatozoa were used to inseminate microdroplets with 20 matured oocytes from all groups: incubation with exogenous DNA for 1 h, sperm capacitation (250 nM of calcium ionophore) for 5 min followed by sperm incubation for 1 h, electroporation (500V), and lipofection (Effectene�; Qiagen, Mississauga, Ontario, Canada). The EYFP-Nuc plasmid (500 ng mL-1; Clontech, Mountain View, CA, USA) was used as exogenous DNA. Oocytes inseminated with non-treated sperm were considered as the control group. The presumptive zygotes were co-cultured with a granulosa cell monolayer in SOFaa medium in an incubator at 39�C, with an atmosphere of 5% CO2 in air and high humidity. The blastocyst rate was analyzed by ANOVA. Embryos from all experimental and control groups were subjected to PCR to detect internalized EYFP, using primers specific to this exogenous DNA, and considering positive embryos those that showed a fragment of 440 bp after electrophoresis. The fragment of 440 bp from positive embryos was sequenced to check correspondence to the EYFP. Electroporation (17.95%) and sperm capacitation (15.12%) were more efficient for EYFP-positive embryo production when compared to the lipofection protocol (6.25%). Sperm incubation (12.5%) did not show a significant difference from the other groups. Sequenced PCR-positive products for EYFP showed 100% homology with nucleotides of EYFP at GenBank. In conclusion, it is possible to use SMGT to deliver exogenous DNA into an oocyte at the time of IVF.
This work was supported financially FAPESP 03/08542-5 and 03/07456-8.
411 EFFECTIVE PRODUCTION OF ICSI-MEDIATED TRANSGENIC MICE: INTENSITY OF GFP EXPRESSION AS A MARKER GENE AND INCIDENCE OF APOPTOSIS IN BLASTOCYSTS
Authors: Takenaka M. , Kani C. , Horiuchi T.
Published: December 2006

Transgenic animals provide a powerful model to explore the regulation of gene expression, and to study gene functionality. The objective of this study was to improve the production efficiency of transgenic mice in ICSI-mediated transgenesis. We examined the relation between the intensity of green fluorescent protein (GFP) expression as a marker gene in blastocysts and the productivity of green mice after embryo transfer, and the incidence of apoptosis in blastocysts with GFP expression. Spermatozoa were processed fresh (FR) and with 2 methods that cause membrane disruption: freeze–thawing (FT), or freeze–thawing followed by 0.5% Triton X-100 for 0.5 min (TX). The spermatozoa were co-incubated with 4.0 ng �L-1 of pCX-EGFP DNA fragments (3.5 kb) for 1 min on ice and injected into oocytes of B6D2F1 mice. The injected oocytes were cultured in KSOMaa for 96 h. Blastocysts were classified into 3 categories depending on the intensity of GFP expression (strong, moderate, and mosaic/negative) under fluorescence microscopy. The number of apoptotic cells in the blastocysts was determined by the TUNEL method using an in situ cell death detection kit (Roche Diagnostics, Mannheim, Germany). Data were analyzed by the chi-square test in all experiments. Blastocyst rates for FR, FT, and TX were 77% (27/35), 67% (18/27), and 69% (31/45), respectively. The proportion of blastocysts expressing GFP for FR, FT, and TX were 0% (0/27), 22% (4/18), and 52% (16/31), respectively. The percentage of apoptotic cells (the number of apoptotic cells/total cells) in blastocysts with strong GFP expression (7.3 � 0.5%; 4.9 � 0.4/68 � 3) was significantly higher (P < 0.05) than in those with moderate (2.5 � 0.3%; 2.2 � 0.2/88 � 3) or mosaic/negative (1.5 � 0.2%; 1.4 � 0.2/91 � 2) GFP expression. After embryo transfer, the rates of implantation, fetal development, and green fetus production were significantly higher (P < 0.05) in blastocysts with moderate GFP expression (52%, 16/31; 19%, 6/312 and 16%, 5/31, respectively) than in those with strong GFP expression (12%, 4/33; 6%, 2/33; 3%, 1/33). These results suggest that a high amount of GFP expression in blastocyst is detrimental to embryo development and survival.
414 COLOR DOPPLER EVALUATION OF UMBILICAL BLOOD FLOW DURING PREGNANCY IN COWS
Authors: Panarace M. , Jaúregui G. , Garnil C. , Segovia A. , Gutiérrez J. , Lagioia J. J. , Marfil M. , Rodríguez E. , Medina M.
Published: December 2006

There are few reports describing the use of Doppler for reproductive imaging in large animals; only one study (in mares) assessed umbilical blood flow (Bollwein et al. 2004 Theriogenology 61, 499–509). Conditions that constrict the placental vascular bed (i.e. hemorrhage, thrombosis, abnormal development, etc.) increase resistance to incoming blood. Thus, blood flow in umbilical arteries can be used to monitor placental development and function (Giles et al. 1985 Brit. J. Obstet. Gynaecol. 92, 31–38). The objective of the present study was to characterize the Doppler flow velocity waveform in umbilical arteries of cows with apparently normal pregnancies. Twenty-three multiparous, nonlactating Aberdeen Angus cows with pregnancies achieved after transfer of embryos derived by IVF (n = 10) or multiple ovulation and embryo transfer (MOET) (n = 13) were examined weekly from 5 to 38 weeks of gestation. Ultrasonography (Toshiba Nemio 20, Tokyo, Japan) was done using a 5–10 MHz intraoperative finger transducer (transrectal) from 5 to 17 weeks of pregnancy and thereafter using a 3–6 MHz linear-array transducer (transabdominal). The interrogation angle (between the ultrasound beam and the artery) ranged from 45° to 60°. Three resistance indices were calculated: A/B ratio, Resistance Index (RI) = (A − B)/A, and Pulsatility Index (PI) = (A − B)/M. [A = systole, B = diastole, and M = mean maximum Doppler-Shift frequency over the cardiac cycle.] The mean ± SEM duration of pregnancy was 285 ± 1.6 days (range: 269 to 291 days). A repeated measure ANOVA was used to detect differences between groups for every week using the Wald-Wolfowitz test (InfoStat V1.5, FCA, Universidad de Córdoba, Córdoba, Argentina). All 3 resistance indices decreased (by >50%) until 26 weeks, with no substantial change thereafter (Table 1). From 5 to 18 weeks of pregnancy, blood flow was characterized by a systolic pattern (i.e. high resistance with absence of diastolic flow); at 20 weeks, all fetuses had a diastolic flow, consistent with low resistance. There were differences (P < 0.05) between IVF- and MOET-derived pregnancies for RI and PI at 38 and 26 weeks of gestation, respectively, indicating differences in the placental vascular development at these weeks of gestation. In conclusion, umbilical cord blood flow in cattle was characterized by high resistance (5 weeks of gestation), but resistance decreased until 26 weeks; consequently, blood flow was initially systolic but became diastolic. Doppler sonography was useful for assessment of umbilical blood flow from 5 to 38 weeks of pregnancy, and may be useful for assessing placental function in pregnancies under risk, e.g. clone-derived pregnancies (Bertolini et al. 2002 Theriogenology 57, 181–187).

Table 1.
RI and PI at 19, 22, 26, 30, and 38 weeks of pregnancy in IVF- and MOET-derived pregnancies (mean ± sd)