bluuebunny/crossref_metadata_2025 huggingface dataset:
340 IN VITRO MATURATION OF PREPUBERTAL BOVINE OOCYTES ON A GRANULOSA CELL MONOLAYER FROM ADULT ANIMALS IMPROVES THEIR DEVELOPMENTAL COMPETENCE
Authors: Ponebsek S. , Wrenzycki C. , Hadeler K.-G. , Herrmann D. , Korsawe K. , Niemann H.
Published: December 2006

Oocytes from prepubertal calves have a decreased developmental competence compared with oocytes from adult animals. The goal of this study was to improve the developmental competence of juvenile oocytes by maturation on granulosa cell (GC) monolayers from adult animals. Oocytes were recovered by ovum pickup (OPU) from 48 Holstein Friesian calves at 7-8 months of age and 18 adult cows. Animals received intramuscular
injections of 60 mg FSH 48 h prior to each OPU session. Follicles were punctured twice per week in six consecutive OPU sessions. Cumulus oocyte complexes (COCs) recovered from calves were divided into three quality groups (classes I-III) and were then randomly distributed into three maturation groups: COCs were matured for 24 h on either GC or fibroblasts or without co-culture. Cow oocytes were matured without co-culture. TCM-199 supplemented with BSA (0.1%), hCG (5 IU/mL), and eCG (10 IU/mL) served as the medium in all groups. After maturation, all COCs were fertilized in vitro; after 18 h, presumptive zygotes were cultured in SOF+BSA for 8 days (37�C, 5% CO2). On Day 3, cleavage rates and, on Day 8, blastocyst rates were determined. The relative mRNA abundance of the following transcripts, critically involved in early embryonic development was determined: growth differentiation factor-9 (GDF-9), heat shock protein 70 (Hsp-70), and glucose transporter-3 (Glut-3). Single immature and matured oocytes (for GDF-9 and Hsp-70) and 8-16-cell embryos and expanded blastocysts (for Hsp-70 and Glut-3) from calves and cows were examined by semiquantitative RT-PCR. Cleavage and blastocyst rates were similar in oocytes derived from cows and calves matured on GC (74.3% vs. 70.0% and 22.3% vs. 22.3%, respectively), but were significantly higher (P < 0.05; one way ANOVA, Student-Newman-Keuls
Method) than in the group without co-culture on fibroblasts (55.2% vs. 53.6% and 11.7% vs. 5.5%, respectively). GDF-9 expression was similar in immature calf and cow oocytes. After maturation, a significant decrease in GDF-9 expression was observed in calf oocytes. Matured cow oocytes
showed a significantly higher mRNA abundance of GDF-9 than matured calf oocytes. The relative abundance of Hsp-70 was decreased in matured oocytes of all groups. Expanded blastocysts derived from adult oocytes expressed Hsp-70 significantly higher than blastocysts derived from oocytes of the control calves. The relative abundance of Glut-3 mRNA was similar in 8-16 cell embryos and expanded blastocysts in all groups. Overall, mRNA expression pattern for Hsp-70 and Glut-3 in blastocysts from GC matured oocytes were similar to that of cow blastocysts. Results indicate that maturation of juvenile calf oocytes on granulosa cells from adult animals improves their developmental competence. These findings provide clues toward identification of factors critically involved in acquiring full developmental capacity at puberty.
341 OXYGEN TENSION AND EGF AFFECT METABOLISM OF IN VITRO-MATURED CUMULUS-ENCLOSED MOUSE OOCYTES
Authors: Preis K. A. , Seidel Jr G. E. , Gardner D. K.
Published: December 2006

In vitro maturation of immature oocytes results in limited success in both clinical and research laboratories. Although reduced oxygen concentration is beneficial to embryo development, the optimal concentration for oocyte maturation has yet to be determined. The objective of this study was to determine whether oxygen tension (20% or 5% O2) affects oocyte physiology. Additionally, the effect of epidermal growth factor (EGF) in maturation medium on oocyte metabolic activity and subsequent embryo development was determined. Cumulus–oocyte complexes (COCs; n = 231) were collected from 28-day-old unprimed F1 (C57BL/6 × CBA/ca) mice. COCs were individually matured in defined medium at 37°C in 6% CO2 in one of four groups (Table 1). For the metabolism study, COCs were further divided into two groups: individual maturation in a 2-µL drop of medium for 16 h (n = 131); or individual maturation in 5-μL for 12 h and then placed in a 0.5-μL drop of medium for 4 h (n = 100), the time of greatest metabolic activity of the COC. At 17 h of maturation, COCs were individually fertilized, and zygotes were individually cultured until 96 h, at which time blastocyst development was assessed. Metabolic profiles were analyzed by ANOVA, and blastocyst rates were analyzed by Fisher's exact test. Maturation rates and blastocyst development were not different between groups. However, at 12–16 h of maturation, metabolism of COCs was affected by both oxygen tension and EGF (Table 1). Concerning metabolism over the entire course of maturation, glucose uptake and lactate production were higher in COCs in 5% O2 + 100 ng EGF (P < 0.05) than in the remaining three groups. There was no difference between 5% O2 and 20% O2 + 100 ng EGF, but 20% O2 caused less glucose uptake and lactate production than did the other three treatment groups (P < 0.05). Results of this study are the first to show that oxygen tension alters COC metabolism: COCs matured under 5% O2 were more active metabolically than COCs matured under 20% O2. The effect of oxygen tension is to some extent moderated by the presence of EGF, as metabolic activity of COCs matured under 20% O2 + 100 ng EGF was closer to that of COCs matured under 5% O2 conditions. Although blastocyst rates were similar across the four groups, embryos derived from oocytes matured in different oxygen tensions may exhibit different developmental potential. In conclusion, results of this study have implications for the improvement of maturation conditions in both clinical and research laboratories.

Table 1.
Carbohydrate metabolism of individual COCs at 12–16 h of maturation
343 EFFECTS OF EPIDERMAL GROWTH FACTOR SUPPLEMENTATION ON IN VITRO MATURATION AND GENE EXPRESSION OF CANINE OOCYTES
Authors: Song H.-J. , Lee S.-H. , Maeng G.-H. , Kim J.-G. , Balasubramanian S. , Choe S.-Y. , Rho G.-J.
Published: December 2006

Despite many efforts to improve canine in vitro maturation (IVM), the efficiency is still low compared to that of other mammalian species (Marie et al. 2004). Epidermal growth factor (EGF) has stimulatory effects on the resumption of oocyte maturation and cumulus expansion in vitro and on prei-mplantation embryonic development in mammals by either an autocrine or a paracrine pathway, or a combination of both systems (Paria et al. 2001 PNAS 98, 1047-1052). The present study investigated the effects of EGF supplementation on in vitro maturation and gene expression of canine oocytes. Oocytes were recovered by slicing ovaries recovered from 40 bitches after ovariohysterectomy at random stages of the estrous cycle. Cumulus-oocyte complexes (COCs) were matured in TCM-199 containing 10% FBS, 1 �g/mL FSH and LH, and EGF (0, 10, or 30 ng/mL) for 48 or 72 h at 39�C in a humidified atmosphere of 5% CO2 in air. In Experiment I (n = 2520 oocytes), the nuclear maturation status was assessed by fluorescence microscopy after bisbenzimide (Hoechst 33342) staining (10 �g/mL) at 0, 48, and 72 h of incubation. In Experiment II (n = 90 oocytes), expression of transcripts such as EGF receptor (EGFR), luteinizing hormone receptor (LHR), and gap junction protein (GJA5) were determined in 10 intact COCs each at 0, 48, and 72 h, respectively, by reverse transcription-polymerase chain reaction (RT-PCR). At 0 h 10-20% of the oocytes had undergone resumption of meiosis (GVBD<MII). After 48 h of IVM, rate of meiotic resumption for 0, 10, and 30 ng/mL EGF were 28, 35, and 30%, respectively. At 72 h of IVM, oocytes in the 10 ng/mL EGF group had resumed meiosis at a higher frequency (55%; P < 0.05) than in the 30 ng/mL EGF or the control group (39 and 42%, respectively). At 72 h of IVM, the frequency of maturation to the MII stage was significantly higher in the 10 ng/mL EGF group (9.6%) than in the 30 ng/mL EGF or the control group (4.2 and 3.3%, respectively). The expression of EGFR was significantly higher (P < 0.05) in 0 h oocytes than in the 48- or 72-h oocytes. Further EGFR expression levels were decreased in the presence of EGF in a dose dependent manner. Transcripts for LHR were detected at all maturation intervals and its expression patterns were not altered by supplementation with 10 ng/mL EGF. Expression of GJA5 was observed only after 48 h of IVM, and levels of expression were similar in oocytes supplemented with both 10 and 30 ng/mL EGF. In summary, our results indicate that supplementation of canine IVM medium with 10 ng/mL EGF had a positive influence on the progression of maturation to MII at 72 h. The effect may not be related to the alteration of mRNA expression of genes analyzed in the present study, due to the complex patterns regulating meiotic arrest in canine oocytes.
This work was supported by Grant no. 204119-03-1-LG000 from ARPC, Republic of Korea.
348 DEVELOPMENT OF A REAL-TIME POLYMERASE CHAIN REACTION ASSAY FOR QUANTIFICATION OF Y-CHROMOSOME-BEARING SPERM IN CATTLE
Authors: Maschari A. S. , Bettegowda A. , Smith G. W. , Pursley J. R.
Published: December 2006

Recent data from our laboratory indicate that increasing the time from AI until ovulation in cattle affects the sex ratio of offspring in favor of females (Pursley et al. 1998 J. Dairy Sci. 81, 2139-2144, and unpublished data). One possible explanation for this phenomenon is that the population of X- and Y-chromosome-bearing sperm (X-sperm, Y-sperm) deviate over time to the detriment of Y-sperm. Thus, at the time of ovulation, fewer Y-sperm would constitute the population available for fertilization. Co-culture of sperm and oviductal cells is one way to test whether a greater percentage of Y-sperm detaches from oviductal cells following prolonged incubation. In order to test this, a reliable high-throughput method to determine numbers of Y-sperm is required. The objective of this study was to develop and validate a quantitative PCR (q-PCR)-based assay to accurately determine numbers of Y-sperm in samples from cattle. Primers were designed to amplify a 57-base pair (bp) portion of the SRY gene (for quantification of Y-sperm) and a 21-bp section of autosomal DNA from the 1.715 satellite (for estimation of total sperm numbers). Standard curves were developed from plasmids containing cloned fragments of the SRY gene and the 1.715 satellite region from which primers were designed. Average R2 of 0.9977 and 0.9886 were obtained for the 1.715 satellite and SRY standard curves. Assay of fixed amounts of male genomic DNA revealed between and within assay coefficients of variation of 2.17% and 1.54% for the 1.715 satellite and 8.79% and 9.08% for the SRY assay, respectively. Specificity of the assay for the SRY gene was verified using female and male genomic DNA. Similar copy numbers per unit DNA were obtained for the 1.715 satellite sequence in both male and female DNA samples, but the SRY sequence was undetectable in female DNA samples. Samples of separated semen with known amounts of X- and Y-sperm (donated by XY, Inc., Fort Collins, CO, USA) were used to determine accuracy of the q-PCR assay in quantification of numbers of Y-sperm. DNA was extracted from sorted semen samples containing 16.8%, 32%, 66.5%, and 79% Y-sperm and copies of SRY and 1.715 satellite DNA determined in duplicate samples. Percent recovery of genomic DNA from sorted semen samples was calculated using 1.715 satellite primers and was used to adjust expected number of SRY copies for each sample of sorted semen analyzed. Differences in observed copies of SRY gene versus expected copies of SRY gene were determined by Student's t-test. No differences in observed versus expected copies of the SRY gene were detected for each sample (P > 0.05). In conclusion, an assay that accurately quantifies numbers of Y-sperm was developed and validated. This assay will be utilized in future studies to determine if the proportions of Y-sperm released from cultured oviductal cells deviate over time.
351 INSEMINATION OF HOLSTEIN HEIFERS WITH SEXED SPERM PROCESSED WITH OR WITHOUT EGG YOLK IN THE GLYCEROL-CONTAINING FREEZING MEDIUM
Authors: Schenk J. , Crichton E.
Published: December 2006

Preparation of media for use in processing sorted sperm is laborious and expensive. The objective was to determine whether pregnancy rates in heifers following AI of frozen/thawed X chromosome-bearing sperm were equivalent for sperm processed for cryopreservation using glycerol-containing medium with or without egg yolk. X chromosome-bearing sperm from each of two Holstein bulls were isolated on the basis of DNA content using a flow cytometer. Sorted sperm were collected in 50-mL plastic tubes containing 2-mL of 20% egg yolk-TRIS extender without glycerol until each tube contained 12.4 mL and approximately 12 million sperm. Sorted sperm were cooled (5�C) over 90 min before the addition of 12% glycerol-containing TIS medium with or without 20% egg yolk, as two equal volumes (6.5 mL) at 15-min intervals. Tubes containing 25 mL were centrifuged at 850g for 20 min (5�C). After the supernatant was removed, sperm pellets within treatments and bulls were pooled. Sperm concentration was adjusted to 10 � 106/mL with medium containing 6% glycerol and 20% egg yolk-TRIS. Sorted sperm were packaged and frozen in 0.25 mL-coded straws on freezing racks in LN2 vapor. Straws from each bull-treatment combination were equally packaged in goblets to assure random use. Post-thaw sperm progressive motility, determined with time-lapse photography ('track motility'), was 44% for non-egg yolk and 43% for 20% egg yolk-glycerol-containing media. Nonsynchronized Holstein heifers (n = 119) were inseminated 4, 12, or 24 h after observed standing estrus. Sperm were deposited into the uterine body anterior to the internal cervical os. Approximately 1 month after insemination, pregnancy was determined using ultrasound. Data were subjected to ANOVA. The pregnancy rate and 95% confidence interval for sorted sperm processed with non-egg yolk-glycerol-containing medium was 56% (49-73; n = 59), and 55% (38-62; n = 60) for the 20% egg yolk-glycerol-containing medium (P > 0.1). Bull or inseminator effects on pregnancy rates were not significant (P > 0.01). Pregnancy rates as a function of time of insemination at 4, 12, or 24 h relative to observed estrus were 62% n = 65; 48% n = 48; and 50% n = 6, respectively (P > 0.1). Even though the scope of the field trial was limited, omission of egg yolk from the glycerol-containing medium did not adversely affect pregnancy rates. Preparation of non-egg yolk-glycerol-containing medium greatly reduces associated time, labor, and expense. This study also resulted in quite acceptable pregnancy rates with insemination doses of 2 � 106 total sorted frozen/thawed sperm.
356 BLASTOCYST DEVELOPMENT FROM DOMESTIC CAT OOCYTES INJECTED WITH DEHYDRATED SPERMATOZOA
Authors: Moisan A. , Leibo S. , Lynn J. , Gómez M. , Pope C. , Bondioli K. , Dresser B. , Godke R.
Published: December 2006

Live offspring have been produced by intracytoplasmic sperm injection (ICSI) of dehydrated spermatozoa into mouse and rabbit oocytes (Wakayama and Yanagimachi 1998 Nature Biotechnol. 16, 639; Liu et al. 2004 Biol. Reprod. 70, 1776). The objective of the present study was to determine the capability of dehydrated domestic cat spermatozoa to fertilize oocytes after ICSI. Spermatozoa were collected from three toms by ejaculation into an artificial vagina. Freshly collected samples were layered under 1 mL of Tyrode's salt solution supplemented with HEPES, BSA, glutamine, pyruvate, lactate, and 50 mM ethyleneglyeotetraacetic acid (EGTA) pH = 8.2-8.4, and allowed to swim-up during a 12-min incubation at 38�C. From the upper 600 �L of the sperm suspension, four 100-�L aliquots were collected and placed into 2-mL glass ampoules, plunged into liquid nitrogen (LN2), freeze dried at -43�C to -45�C and 44 to 76 � 10-3 mbar pressure, and stored at 4�C. Four to twelve 10-�L aliquots of the same suspension were placed onto glass slides, air-dried for 30 min, and stored in a desiccator at room temperature. Domestic cat oocytes were collected from minced ovaries and allowed to mature in vitro for 22 to 24 h. After maturation, oocytes were either fertilized in vitro (IVF; n = 36), or sperm injected (ICSI) with fresh or refrigerated (n = 74), freeze-dried (n = 45), or air-dried (n = 45) spermatozoa. After ICSI or IVF, oocytes were cultured in a three-step-sequential medium (G�mez et al. 2004 Cloning Stem Cells 6, 247) for up to 8 days. Cleavage and development to the blastocyst stage was assessed on Days 2 and 8 of culture, respectively. Cleavage rates after IVF (56%), ICSI with freeze-dried (60%), or ICSI with fresh spermatozoa (59%) were higher than those obtained after ICSI with air-dried spermatozoa (35%; P < 0.05). Blastocyst development after ICSI treatments was obtained only with fresh spermatozoa (9%), and was lower than that obtained after IVF (25%; P < 0.05). Recently, one hatching blastocyst was produced when oocytes (n = 18) were exposed to calcium ionophore 2 h after ICSI with freeze-dried sperm. This is the first report that domestic cat embryos can be produced in vitro by injecting oocytes with dried spermatozoa.
357 FERTILIZATION AND EMBRYONIC DEVELOPMENT OF PORCINE OOCYTES FOLLOWING INTRACYTOPLASMIC SPERM HEAD INJECTION ENHANCED BY ELECTRIC STIMULATION TO OOCYTES BUT NOT BY PRE-TREATMENT OF SPERMATOZOA WITH DITHIOTHREITOL
Authors: Nakai M. , Kashiwazaki N. , Takizawa A. , Maedomari N. , Ozawa M. , Noguchi J. , Kaneko H. , Shino M. , Kikuchi K.
Published: December 2006

Failure of sperm nuclear decondensation has been reported after injection into oocytes in pigs (Kren et al. 2003 J. Reprod. Dev. 49, 271-273). We examined the effects of pretreatment of spermatozoa with Triton X-100 (TX-100) and dithiothreitol (DTT) and electric stimulation of oocytes after injection on sperm decondensation, male pronuclear formation, and in vitro development to the blastocyst stage. We performed three replicates in each experimental group, with a total of about 70 oocytes per group. In Experiment 1, spermatozoa were pretreated with 1% TX-100 and 5 mM DTT (T+D), and injected into IVM oocytes that were collected from crossbred gilts. Electric stimulation (1.5 kV/cm, 20 �s; Nakai et al. 2003 Biol. Reprod. 68, 1003-1008) was applied 1 h to the oocytes after the injection (the stimulated group) or was not applied (the nonstimulated group). Some of the oocytes in each group were evaluated for morphological changes of sperm nuclei at hourly intervals until 10 h post-injection. Of nonstimulated oocytes, those injected with untreated spermatozoa showed a delayed peak in nuclear decondensation (39.4 to 44.1%, 3-6 h after the injection) compared to that of oocytes injected with T+D treated spermatozoa (57.0 to 52.6%, 1-1 h). The rate of male pronuclear formation increased after 4 h post-stimulation (by 40 to 60%) when the injected oocytes were stimulated, whether or not spermatozoa were pretreated. In nonstimulated oocytes, the rate of male pronuclear formation stayed at the basal level (less than 20%) throughout the culture period regardless of sperm treatments. Thus, the T+D treatment of spermatozoa did not affect decondensation and pronuclear formation. In Experiment 2, the effects of electric stimulation and sperm treatments with T+D on the rate of blastocyst formation and the mean numbers of cells per blastocyst were evaluated. Oocytes that were stimulated after injection of either T+D-treated or untreated spermatozoa showed significantly higher percentages of blastocyst formation (24.8% and 27.1%, respectively) than did nonstimulated oocytes (1.1% and 4.1% for T+D-treated and untreated, respectively; P < 0.01). The rate of blastocyst formation was not different between the T+D-treated and the untreated groups. The mean number of cells per blastocyst was not different among all groups (14.0-29.4). In conclusion, the pretreatment of sperm with TX-100 and DTT shifted the timing of sperm nuclear decondensation forward. However, pronuclear formation and development to the blastocyst stage in vitro were not improved by the sperm treatment. Electric stimulation to the injected oocytes enhances in vitro development to the blastocyst stage in pigs.
359 ICSI AND ACTIVATION OF OOCYTES RECOVERED FROM TRANSITIONAL AND CYCLING MARES
Authors: Suh T. , Purcell S. , Seidel Jr G.
Published: December 2006

Ovarian follicular development in mares during the transitional period before the breeding season leads to an accumulation of antral follicles of various sizes. The quality of oocytes at this stage may be compromized until the first seasonal ovulation. In this study, we evaluated the developmental competence of oocytes recovered from transitional and cyclic mares, and the effect of zygote activation after intracytoplasmic sperm injection (ICSI). A 2 × 2 × 2 factorial experiment consisting of oocytes from transitional and cyclic mares, two follicle sizes (10 to 20 and 20+ mm), and two treatments (control and activated) was conducted. Follicular oocytes of 14 mares were aspirated in March and April (transitional) and May to July (cyclic) five times per each period at 10-day intervals, without use of hCG. Oocytes aspirated from mares were matured in vitro in a defined medium similar to SOF plus FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF), estradiol (E2), prostaglandin (P4) and 10% FCS, for 30 ± 1 h under 5% CO2 in air at 38.5°C; oocytes with a first polar body were used for ICSI. Motile sperm from frozen-thawed semen were used for sperm injection with a piezo-driven pipet. For activation after ICSI, presumptive zygotes were cultured in G1.3 containing 0.02 µM phorbol 12-myristate 13-acetate (PMA) for 2 h, and then in 2 mM 6-dimethylaminopurine (6-DMAP) for 3 h under 6% CO2 in air at 38.5°C. Zygotes were cultured in 50 µL drops of DMEM/F12 containing 10% FCS for 9 days at 38.5°C in 5% CO2/5% O2/90% N2. Medium was replaced every 3 days. Cleavage and blastocyst rates were calculated based on non-degenerating injected oocytes. Data were analyzed by Fisher's exact test. A total of 115 and 78 oocytes were recovered from cyclic and transitional mares. Average maturation rates to MII in the respective groups were 76.5 and 65.4%, respectively (P < 0.07), and those of 10 to 20 and 20+ mm follicle groups were 70.6 and 80.0%, respectively (P > 0.05). The average cleavage rate in cyclic mares was higher than in transitional mares, and that of the activated group averaged over follicle sizes was higher than that of controls (P < 0.05; Table 1); those of 10 to 20 and 20+ mm follicle groups were not different (P < 0.05; Table 1). Blastocyst rates per oocyte within main effects were not different (P < 0.05; Table 1). Oocytes from transitional mares had lower cleavage rates than those of cyclic mares, but blastocyst development was similar. Activation of zygotes clearly improved cleavage rates of in vivo-derived immature equine oocytes after ICSI.

Table 1.
Main effect means of responses after ICSI
365 THE EFFECT OF ADMINISTERING PROGESTERONE AND ESTRADIOL PRIOR TO eFSH ON THE SUPEROVULATORY RESPONSE OF MARES
Authors: Logan N. , McCue P. , Alonso M. , Squires E.
Published: December 2006

eFSH has been used to induce multiple ovulation in cycling mares. However, the response to eFSH is variable. Generally, eFSH is initiated 5 to 7 days after ovulation at a time when the follicular population on the ovaries may be variable. The objective of this study was to determine whether administration of progesterone and estradiol for 10 days prior to eFSH (Bioniche Animal Health, Athens, GA) would enhance the response to eFSH administration. Thirty normal cycling mares were assigned to 1 of 2 groups. Group 1: Control eFSH treatment - mares were examined daily with ultrasonography beginning 5 days after ovulation. Once the follicles in these mares reached 20 to 25 mm in size, eFSH treatment (12.5 mg, i.m.) was administered twice a day. Cloprostenol (Schering-Plough, Union, NJ, USA) treatment (250 �g) was administered on the second day of eFSH treatment. eFSH treatment continued until the majority of the cohort of follicles reached e35 mm. Treatment was stopped and after approximately 36 h hCG (2500 IU, i.v.; Chorulon; Intervet, Millsboro, MD, USA) was administered. Group 2: Injectable progesterone + estradiol (150 mg of progesterone and 10 mg of estradiol; P+E) treatment was initiated in diestrus (5 to 7 days post-ovulation) for 8 mares and in early estrus for 7 mares in this group. Injectable progesterone was continued for 10 days and Cloprostenol (250 �g) was administered on Day 10. Mares were then examined daily with ultrasonography and, once they had acquired 20- to 25-mm follicles, eFSH treatment was initiated. Twice-daily injections of eFSH (12.5 mg, i.m.) were continued until a majority of the cohort of follicles was e35 mm. hCG was administered approximately 36 h later. All mares were inseminated with 1 billion progressively motile spermatozoa from one of two stallions on the day of hCG administration and on the following day with cooled semen (1 billion progressively motile spermatozoa) from the same stallion. Data were analyzed by t-test and Fisher's Exact Test. The number of days of eFSH treatment was similar for the P+E (2) vs. the control (1) group (4.2 � 2.0 vs. 4.9 � 1.3 days, respectively). However, the number of ovulations induced in response to eFSH was greater for mares in the eFSH control (1) group (5.6 � 2.0) than for those in the P+E (2) group (3.0 � 1.9). Embryo recovery per flush was also greater for eFSH control (1) mares (2.7) vs. P+E (2) mares (1.1). Embryo quality was excellent or good for all embryos in both groups. Seventy-three percent of the mares (11 of 15) in both groups gave at least one embryo at each recovery attempt. However, more mares in the eFSH control (1) group gave two or more embryos (60%) compared to those in the P+E (2) group (20%). In summary, treatment of mares with P+E prior to eFSH treatment resulted in fewer ovulations, fewer embryos recovered, and fewer mares providing e2 embryos. Thus, there was no advantage in pretreating mares with P+E prior to eFSH treatment.
366 THE BOVINE MODEL FOR REPRODUCTIVE AGING: SUPEROVULATORY RESPONSE
Authors: Malhi P. S. , Adams G. P. , Mapletoft R. J. , Singh J.
Published: December 2006

Two experiments were done to test the hypotheses that aging in cattle is associated with a reduction in the number of follicles recruited into an ovarian follicular wave, and a reduction in the ovarian response following gonadotropin treatment for superovulation. Old cows (13 to 16 years old; n = 6 to 9) and their young daughters (3 to 6 years old; n = 7 to 9) were treated to induce an ovarian superstimulatory response twice in each of two consecutive years (i.e. 31 and 33 superstimulatory cycles, respectively). In Experiment 1, follicular wave emergence was synchronized using a single treatment of estradiol (5 mg) combined with progesterone (100 mg) at the time of CIDR-B (Bioniche Animal Health, Belleville, Ontario, Canada) insertion. Superstimulatory treatment consisted of a total dose of 50 mg NIH-FSH-P1 (Folltropin-V; Bioniche Animal Health) per 100 kg body weight divided into twice daily intramuscular injections over 4 days. CIDR devices were removed at the time of the last FSH treatment and 500 �g cloprostenol (Estrumate; Schering-Plough Animal Health, Montreal, Quebec, Canada) was administered twice along with last two FSH treatments to induce luteolysis. Ovulation was induced using 25 mg porcine LH (Lutropin-V; Bioniche Animal Health) administered 24 h after the last FSH treatment. Ovulations were detected by ultrasonography at 24, 48, and 72 h after LH treatment. In Experiment 2, follicular wave emergence was synchronized with ultrasound-guided follicle ablation in the presence of a functional corpus luteum. Superstimulatory treatment was given as in Experiment 1. Ultrasound-guided follicle aspirations were performed 48 to 72 h after the last FSH for the purpose of another experiment. In both experiments, the ovarian follicular response was monitored daily by ultrasonography. There were no differences (P > 0.3) in mean body weight and dose of FSH treatment between old cows and their daughters. Fewer 2-5-mm follicles (P < 0.01) were detected at the expected time of wave emergence in old cows than in their daughters. In Experiment 1, the difference in mean number of ovulations in old and young cows (32 � 4 vs. 40 � 3) did not reach significance (P = 0.11), but fewer follicles e6 mm (P < 0.01) were detected on the day before ovulation in old cows than in their daughters. The number of follicles e6 mm detected just before ovulation (Experiment 1) or follicular aspiration (Experiment 2) in successive superstimulatory cycles was correlated within animals (r > 0.8; P < 0.0001). Similarly, the number of ovulations in successive superstimulatory cycles (Experiment 1) was correlated within animals (r = 0.6; P = 0.04). In conclusion, aging was associated with fewer 2-1 mm follicles at the time of wave emergence, a lower follicular response, and a tendency for a lower ovulatory response subsequent to gonadotropin treatment. The follicular and ovulatory response to superstimulatory treatment was repeatable within individuals.
369 ASSESSMENT OF SEMEN QUALITY FOR PREDICTING RECOVERY OF VIABLE EMBRYOS IN SUPEROVULATED CATTLE
Authors: Schrick F. N. , Saxton A. M. , Stroud B. K.
Published: December 2006

Several studies have illustrated a relationship between male fertility and subsequent pregnancy rates following natural service and artificial insemination. However, few studies have evaluated the relationship of semen quality to recovery of viable embryos within a commercial embryo transfer setting. The objective of the current study was to retrospectively evaluate the relationship of semen quality, determined immediately before insemination, and subsequent embryo recovery from superovulated cattle. All donor animals were superovulated and then inseminated (same technician throughout the study) with frozen-thawed semen at 12 and 20 h after onset of estrus with one straw of semen per insemination. Since data were collected from a commercial setting, several different bulls (n = 277) and breeds (n = 33) were included within the analyses; however, the same semen (bull and collection date of semen) was used for each insemination for that respective flush. Immediately following thawing of semen and before insemination, 5 �L of semen was obtained from each straw and evaluated by the same individual for concentration, motility, rate of forward movement, direction of movement, and morphology using a phase contrast microscope. Approximately 7 days later, ova/embryos were recovered and defined as unfertilized (not cleaved), degenerate, or viable. Semen was assessed and utilized in 742 recoveries resulting in the evaluation of 9732 ova/embryos. Chi-square analyses were used to explore relationships among semen and fertilization/embryo variables. A complex model addressing combinations of semen variables was developed with logistic regression. Total ova/embryos recovered had a significant impact on embryo data and was included in the model during statistical analyses with the significance level set at P < 0.05. Analyses were conducted only on uterine recoveries in which ova/embryos were collected. Effects of bull and breed were significant on embryo data evaluated but at a very low level of relevance (F < 2.0). Total ova/embryos collected per recovery highly influenced the percentage of cleaved embryos (P < 0.0001). Results indicate that all semen parameters evaluated affected various aspects of embryo development (percent fertilized, viable, or degenerate). In particular, semen morphology was more associated with quality of embryos recovered than the other semen factors measured. Semen classified as excellent-to-good morphology resulted in 59% Quality 1 embryos (percent of fertilized embryos) whereas semen rated fair was intermediate (53.5%) and semen rated poor produced only 29.5% Quality 1 embryos (P < 0.05). In conclusion, semen evaluation within a commercial embryo transfer setting provides some expectation of resulting embryo quality.
375 PRODUCTION OF GERMLINE TRANSGENIC CHICKENS USING MURINE LEUKEMIA VIRUS-BASED RETROVIRUS VECTOR SYSTEM
Authors: Koo B. C. , Kwon M. S. , Choi B. R. , Chang W. , Jeon I. , Kim T.
Published: December 2006

We report here successful generation of germline transgenic chickens expressing the enhanced green fluorescence protein (EGFP) gene throughout whole bodies. The founder chickens were produced by injecting replication-defective recombinant retroviruses encapsulated with the vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped retrovirus vector system beneath the blastoderm of non-incubated chicken embryos (stage X). Of 129 injected eggs, 13 chickens hatched. These 13 chickens were analyzed at 6-16 weeks post-hatching and all were found to emit green fluorescence in at least one part of the body. One cock of the 13 Go chimeric founder cockerels was mated with several non-transgenic hens, and one of 102 G1 siblings was found to emit a green fluorescent signal in its whole body. Successful germline transmission of the EGFP transgene was further confirmed in the G2 generation chickens: Crossing of the G1 cock and several non-transgenic hens resulted in about 50% (18/37) of the G2 cockerels being transgenic. The results of this study would be very helpful in establishing a useful transgenic chicken model system for generation of transgenic chickens as bioreactors producing therapeutic proteins, as well as for studies on embryo development.
377 DEVELOPMENT OF A VIBRATORY MICROINJECTION SYSTEM TO INCREASE THE PRODUCTION RATE OF TRANSGENIC ANIMALS
Authors: Miyawaki F. , Hasegawa J. , Kikuchi T. , Tsuchiya M. , Saito Y. , Suzuki H. , Ippongi T. , Kobayashi K. , Itohara S.
Published: December 2006

Pronuclear injection is the standard method to deliver genes to zygotes, especially of medium- to large-size animals, but the production rate of transgenic animals is still low. To increase it, we developed a vibratory microinjection system (VMS) that vibrates a micropipette (Femtotip�; Eppendorf Co., Ltd., Tokyo, Japan) longitudinally to facilitate its penetration of the zona pellucida and nuclear membrane. The VMS can provide a wide range of frequencies and amplitudes of vibration, and is different from the piezo impact micromanipulator that uses an internal force resulting from a rapid deformation of piezoelectric elements. P Vibratory microinjection (VM) at a frequency of 5 kHz and an amplitude of 0.5 �m, which was not at the tip of micropipettet but at the vibrator's level, was compared with ordinary microinjection (OM). No fragmentation of the Venus gene encoding a variant of yellow fluorescent protein was confirmed after it had been exposed to this vibration for 1 h. A total of 910 eggs obtained from 36 BDF-1 mice were used. This experiment consisted of 15 sets of injections. In each set, the same number (d40) of eggs was allotted to the VM and OM groups. The eggs in each group were injected with the Venus gene using a single micropipette. The two groups were treated simultaneously with two sets of injection apparatuses. Embryonic development was assessed after 4-day culture. The following results were achieved: (1) VM made the micropipette penetrate zona pellucida with much less cellular deformation; the deformation rates were 28.7 � 7.2% for VM and 35.4 � 7.3% for OM (P < 0.0001). (2) Ninety-six of 455 embryos (21.1%) in the VM group developed to the blastocyst stage, whereas only 41 of 455 eggs (9.0%) in the OM group did (P < 0.0001). The numbers of Venus-expressed blastocysts were 29 for the VM group and 19 for the OM group (P = 0.14). However, the death rates in VM and OM groups were 39.1% and 32.7%, respectively (P = 0.045). This higher death rate in the VM group might have been due to transverse vibration at the tip of micropipette because it was bent approximately 20�. (3) Two of 15 micropipettes in the VM group pulled out a part of the nuclear DNA and/or cytoplasm before finishing the given number of injections while 9 of 15 micropipettes in the OM group did (P = 0.0063). We conclude that the VMS achieved zygote injection with significantly less cellular deformation and also resulted in significantly better embryonic development, suggesting anticipated improvement in the production of transgenic animals. The VMS can also cut the cost of micropipettes and save the trouble of frequent replacement of micropipettes.
378 EFFECT OF TRANSGENE CONCENTRATION AND FLANKING MATRIX ATTACHMENT REGIONS ON THE EFFICIENCY OF ICSI-MEDIATED TRANSGENIC MOUSE PRODUCTION
Authors: Moreira P. N. , Pozueta J. , Valdivieso F. , Montoliu L. , Gutiérrez-Adán A.
Published: December 2006

The efficiency of ICSI-mediated transgenesis can be influenced by many different factors. In this study, we have assessed the impact of transgene concentration and of the presence of transgene-flanking matrix attachment regions (MARs) on the efficiency of this procedure. Presented data include assays with three DNA constructs; a plasmid EGFP of 5.4 kb (pEGFP, Clontech Laboratories, Inc., Palo Alto, CA, USA); this plasmid EGFP construct flanked with two MAR elements (M-EGFP-M; 2.3 kb of the human �-interferon domain boundaries) (Guti�rrez-Ad�n et al. 2000 Transgenic Res. 9, 81-89); and a yeast artificial chromosome (YAC) of 520 kb. ICSI-mediated transgenesis was done in the B6D2 hybrid mouse strain as previously described (Moreira et al. 2004 Biol. Reprod. 71, 1943-1947), using two concentrations (1 and 15 ng/�L) of the M-EGFP-M transgene, three concentrations (1, 6, and 15 ng/�L) of the pEGFP transgene, and three concentrations (2, 3.6, and 5 ng/�L) of the YAC construct. Embryos were cultured in vitro until the 2-cell stage and transferred into Day 1 pseudopregnant females. Live offspring or Day 14 fetuses were analyzed by PCR for the detection of the genomic presence of the integrated transgene. The proportion of transgenics (PT) obtained with 2, 3.6, and 5 ng/�L of the YAC construct was 10, 21, and 13% respectively. A Z-test analysis of the data generated with this construct indicated that the number of transgenic offspring, when expressed as a proportion of injected ova (efficiency rate of the procedure, ER), increased with DNA concentration up to a threshold of 3.6 ng/�L, and suggested that higher concentrations of this transgene (and maybe of co-purifying contaminants) decreased the ER of the procedure. Interestingly, although much higher transgene concentrations were used for the pEGFP construct, this threshold was not reached. With an increase in transgene concentration (from 1 to 15 ng/�L), the PT increased (from 39 to 70%), as well as the ER of the procedure (from 3 to 7%). In addition, independently of the transgene concentration tested, when M-EGFP-M was used, the ER of the procedure and the PT observed were similar to the ones obtained with pEGFP. These results indicate that, regardless of the importance of other factors such as transgene size, purity, and copy number, the concentration to be used on ICSI-mediated transgenesis should be optimized for each construct, in order to maximize the PT among the liveborn offspring and the overall efficiency of the procedure. Our results also indicate that transgene-flanking MARs do not have a significant impact on the efficiency of this transgenesis method.
379 LACTATION INDUCTION IN PREPUBERTAL BULLS AND HEIFERS AS A TOOL FOR PREDICTING MAMMARY SPECIFIC TRANSGENE EXPRESSION IN CATTLE
Authors: Powell A. , Kerr D. , Wall R.
Published: December 2006

The bovine's long generation interval results in a lapse, from the time of birth, of two to three years before mammary-specific transgenes can be assessed in genetically engineered animals. This experiment was conducted in an attempt to reduce that waiting period by up to two years. Lactation was induced in prepubertal bull and heifer calves, 3 to 8 mo of age, as a means of predicting transgene behavior during subsequent normal lactations in the heifers and daughters of bulls. Transgenic animals tested were either founder animals, produced by somatic cell nuclear transfer, or G1 offspring of founder bulls. The transgene consists of a lactation specific sequence encoding lysostaphin, an antimicrobial protein targeted against Staphylococcus aureus, a mastitis-causing pathogen. Estrogen, progesterone, and dexamethasone were administered as previously described (Ball et al. 2000 J. Dairy Sci. 83, 2459) to nine heifers (transgenics = 5) ranging in weight from 90 to 165 kg. Eight bull calves (transgenics = 7) weighing from 81 to 178 kg received additional estrogen and progesterone injections as well as reserpine prior to dexamethasone treatment. Animals were hand-milked twice daily for 4 to 7 days. All nine heifers responded to the milk induction scheme, yielding between 19 mL and 4.5 L. Milk volume from the three responding males (100 �L to 2.5 mL) was significantly less than that harvested from females (P = 0.025). Only bull calves over 150 kg had a positive response. Transgenic females produce less milk then non-transgenics (313 � 494 vs. 2276 � 552 mL, respectively; P = 0.033). Most importantly, there was no detectable difference between the concentration of lysostaphin in milk from induction (8.1 � 2.7 �g/mL) and natural lactations (3.5 � 2.6 �g/mL) in the four transgenic heifers tested (P = 0.229). The result was the same when lysostaphin was analyzed as a percentage of total protein (P = 0.427). Induction of a G1 heifer and a bull calf from the same founder bull produced similar lysostaphin concentrations in their milk (5.6 � 0.9 and 5.2 � 0.5 �g/mL; respectively). �-lactoglobulin concentration was also similar during induced and natural lactation (P = 0.165) for all animals studied. However, total protein was greater in induced milk samples compared to natural lactation samples (28.4 � 1.7 vs. 21.2 � 1.7 mg/mL; P = 0.007) as was lactoferrin (707 � 51 vs. 213 � 51 �g/mL; P < 0.001). Conversely, compared to that in induced milk samples, lactose was more concentrated in the natural lactation samples (34.6 � 2.5 vs. 46.0 � 2.1 g/L). In this study transgene expression was detected in milk from induced lactations and its concentration in those samples was generally predictive of product concentration in the natural lactation milk. The induction protocol was effective in male (>150 kg) and female calves.
381 PRODUCTION OF A TRANSGENIC PIGLET BY A NEW SPERM INJECTION TECHNIQUE
Authors: Yong H. Y. , Murphy C. , Rieke A. , Lai L. , Hao Y. , Li R. , Wax D. , Samuel M. , Korte S. , Whitworth K. , Prather R.
Published: December 2006

The technique for intracytoplasmic sperm injection (ICSI) has, until now, focused on scoring the tail of the sperm prior to catching and aspiration into the injection pipette. This is in spite of the fact that damage to the head would more closely simulate what occurs during normal fertilization. In addition, to aid in visualizing the injection process so that a reduced volume can be injected, the oocyte is generally centrifuged to clear a portion of the cytoplasm. Thus, with conventional ICSI, the sperm are immobilized with polyvinylpyrrolidone, repeatedly frozen and thawed, treated with DTT or Triton X-100, and severed between the head and tail; the oocyte is centrifuged or activated. All of the above treatments are designed to compensate for the intrinsic defects in conventional ICSI. Our objective was to use a modified ICSI procedure whereby aggressively motile sperm were captured onto the broken tip of an injection pipette and then injected into noncentrifuged oocytes. Damage to the head of the sperm occurred on the pipette or while pushed through the zona pellucida. These procedures are based on the work of Yong et al. 2003 Hum. Reprod. 18, 2390, where they achieved an improvement in development in vitro as compared to conventional methods. Ovaries were collected from prepubertal gilts, and oocytes were aspirated and matured in vitro. Sperm were collected from a transgenic boar carrying the green fluorescent protein (GFP) and frozen. After thawing, aggressively motile sperm were captured and injected through the zona pellucida and into the cytoplasm of the in vitro-matured oocytes. A total of 452 injected oocytes (43-171 oocytes per recipient) were surgically transferred into the oviduct of six surrogate gilts. Two gilts (33%) became pregnant. One gave birth to a healthy male piglet. GFP expression was observed in the nose and hooves by direct epifluorescent examination of the newborn piglet. This pattern of GFP expression is identical to that in non-ICSI-derived GFP pigs in this line. This result showed for the first time that this new sperm injection technique could be used for production of a viable transgenic piglet using in vitro-matured oocytes and frozen-thawed sperm.
39 DEVELOPMENTAL COMPETENCE OF RECONSTRUCTED EMBRYOS PRODUCED BY NUCLEAR TRANSFER BETWEEN CYNOMOLGUS MONKEY FIBROBLAST CELLS AND RABBIT OOCYTES
Authors: Hosoi Y. , Yamochi T. , Kawata N. , Takenoshita M. , Ohta S. , Matsumoto K. , Saeki K. , Iritani A.
Published: December 2006

Interspecies nuclear transfer has been used as an invaluable tool for studying nucleus-cytoplasm interactions and it may also be used for rescuing endangered species whose oocytes are difficult to obtain. In this study, we investigated interaction of the cynomolgus monkey cell as a nuclear donor with the rabbit oocyte as a host cytoplasm. Whole cynomolgus fibroblast cells were injected into the rabbit enucleated oocytes (cynomolgus-rabbit cloned embryos) and cultured in TCM-199 and RPMI 1640 culture media. Rabbit-rabbit cloned embryos we used as control in this study. Karyotype analyses confirmed that genetic material of blastocysts was derived from the cynomolgus donor cells at blastocyst stage. Mitochondrial constitution analysis of the cynomolgus-rabbit cloned embryos indicated that mitochondria from both donor cells and enucleated oocytes coexisted. After culture for 168 h post-nuclear transfer, all cynomolgus-rabbit cloned embryos in TCM-199 were arrested at the 8-cell stage, but some of them developed to the blastocyst stage in RPMI 1640 (11/59, 18.6%). In this experiment, the nutrition requirement in vitro and the cleavage rate at each 24 h were examined. When TCM-199 was supplemented with lactate, some of these embryos developed to the blastocyst stage (15.3%, 2/13). This means that cynomolgus-rabbit cloned embryos might be controlled by the donor nucleus even in these early developmental stages. However, the timing of cleavage of cynomolgus-rabbit cloned embryos is very similar to that of the rabbit-rabbit cloned embryos. Time of cleavage may depend on the protein accumulated in the cytoplasm. In the prolonged culture of reconstructed embryos on feeder cells, adhesion cells were observed. These cells are also very similar to the cells derived from cynomolgus embryos by the same method. Our results suggest that: (1) a cynomolgus nucleus can co-ordinate with rabbit oocyte cytoplasm in early embryo development, (2) the 8- to 16-cell stage block in the cynomolgus-rabbit cloned embryos may due to the same reason as that in the cynomolgus embryos, and (3) ooplasmic factors that control time of cleavage are highly conservative between the species.