bluuebunny/crossref_metadata_2025 huggingface dataset:
135 IN VITRO DEVELOPMENT OF IN VIVO PRODUCED EMBRYOS FROZEN AT MORULA OR BLASTOCYST STAGES
Authors: Sartori R. , Machado G. M. , Guardieiro M. M. , Bastos M. R. , Leme L. , Siqueira Filho E. , Rumpf R. , Dode M. A. N.
Published: January 2008

This study was designed to compare cryotolerance between morulae and blastocysts collected from superovulated heifers. Twenty pubertal beef heifers (10 Nelore and 10 crossbred Nelore � Simmental) were superovulated with 100 mg of FSHp (Folltropin-V, Bioniche, Ontario, Canada), and embryos were collected and evaluated 7 days after estrus. Grades 1 and 2 embryos (IETS) were divided into four groups: morulae cryopreserved (MC) in liquid nitrogen (n = 24); blastocysts cryopreserved (BC; n = 19); morulae fresh (MF; n = 23); and blastocysts fresh (BF; n = 18). For freezing, embryos were immersed in ethylene glycol (Ethylene Glycol Freeze Plus with 0.1 m sucrose, Bioniche, Pullman, WA, USA), and a standard protocol (cooling rate of –0.5�C/min) was used. Prior to in vitro culture, embryos were removed from nitrogen, kept at room temperature for 5 s, and put in a water bath at 30�C for 20 s. Within 5 h after recovery, thawed and fresh embryos were washed five times in holding solution (Holding Plus, Bioniche), transferred to synthetic oviduct fluid medium (SOF, Nutricell, Campinas, SP, Brazil), and cultured for 72 h. Embryos were evaluated at 48 and 72 h of culture. After the last evaluation, degenerate and non-hatched embryos were removed from culture, and the remaining embryos were measured by a graduated ocular coupled to the Motic Images Plus 2.0 program. Hatched blastocysts were kept in culture for an additional 48 h for post-hatching development assessment. For post-hatching culture PHD medium (Brand�o DO et al. 2005 Biol. Reprod. 71, 2048–2055) was added into each well, to have a final composition of 50% SOF and 50% SOF PHD. At 120 h of culture (48 h of PHD culture) only morphologically normal blastocysts were measured. Comparison among groups was performed by ANOVA or chi-square test. Data are presented as mean � SEM. After 48 h of culture, hatching rate (%) was significantly lower in cryopreserved (MC = 8.3 and BC = 21.5) than in fresh (MF = 56.5 and BF = 77.8) embryos (P < 0.05). However at 72 h, hatching rate was similar among BC (75.9), MF (78.3), and BF (88.9), being MC (41.7) still lower (P < 0.05). The diameter (µm) of hatched embryos after 72 h of culture was 272.8 � 27.1a (n = 8), 320.6 � 18.6ab (n = 14), 385.3 � 14.2c (n = 17), and 378.0 � 22.0bc (n = 16) for MC, BC, MF, and BF, respectively (a–cP < 0.05). After 120 h of culture, the diameter of MC (379.0 � 39.9; n = 8), although similar to BC (495.4 � 59.6; n = 10), was smaller than MF (509.1 � 36.5; n = 11) and BF (511.8 � 41.2; n = 14). The results of this study with zebu cattle suggest that morulae are less resistant to cryopreservation in liquid nitrogen than blastocysts. Moreover, frozen/thawed embryos, when put in culture, present a slower development compared with fresh embryos.
Financial support from CNPq and FAPESP from Brazil.
140 UTILIZATION OF LASER-ASSISTED HATCHING TO IMPROVE THE PREGNANCY RATE OF IN VITRO-FERTILIZED BOVINE EMBRYOS
Authors: Menges S. , Wei H. , Faber D. , Kraemer D. , Long C.
Published: January 2008

In vitro-produced (IVP) bovine embryos are known to produce a lower pregnancy rate when compared to conventional in vivo-produced embryos. The inability of the IVP embryo to hatch from the zona pellucida (ZP) after embryo transfer is thought to be one contributing factor. This study was designed to evaluate the utilization of a microscope objective-mounted laser to cut the ZP to assist hatching prior to transfer into the recipient. Preliminary data were acquired to evaluate the effect of laser treatment on in vitro development and blastomere survival following treatment. In six replicates, bovine oocytes were in vitro-matured, fertilized, and cultured as per standard laboratory procedures (TransOva Genetics, Sioux Center, IA, USA). On Days 5, 6, and 7 of in vitro culture, embryos were randomly divided into 3 treatment groups: no treatment (Control; n = 63), sham ZP cut (Sham; n = 68), or ZP cut (Cut; n = 70). Control embryos were immediately returned to the incubator following selection. Sham embryos were exposed to all conditions as Cut except laser-assisted hatching. The XYClone� system is a 300-mW, class 1 laser that emits a 3.5-µm beam at a wavelength of 1480 nm (Hamilton Thorne Biosciences, Beverly, MA, USA). This laser was used to produce the Cut group, using a pulse strength of 90% and pulse length of 600 µs. Embryos were returned to culture until Day 8 when rates of embryonic development and the percentage of live cells were determined. Chi-square was used to analyze all data. No significant effect of treatment or day of exposure was noted in either the total number of developing embryos or the ratio of live cells in each embryo. Mean live cells ranged from 89 to 96% across all treatments regardless of day of treatment. To investigate IVP embryo viability after laser-assisted hatching, commercially produced embryos (TransOva Genetics, Sioux Center, IA, USA) were randomly divided into two groups on the day of transfer, Control or Cut. The ZP of treated embryos were cut with slightly reduced laser exposure of 80% pulse strength and pulse length of 500 µs on Day 7, immediately prior to transfer into estrus-synchronized recipients. Pregnancy rates were determined via ultrasonagraphy at Day 30 (n = 337) and, due to the commercial nature of this project, only a subset of the Day 30 pregnant cows was checked at Day 60 (n = 289). The 30-day pregnancy rates were 49.2% and 54.1% for Control (n = 189) and Cut (n = 148) embryos, respectively, and were not statistically different (P > 0.05). However, at Day 60, the pregnancy rates for the Control (45.7%; n = 166) and Cut groups (57.7%; n = 123) were statistically different (P < 0.05). These results demonstrate that laser-assisted hatching using the XYClone system can improve 60-day pregnancy rates for in vitro-produced embryos.
143 IMPROVEMENT OF A SUPERSTIMULATORY PROTOCOL IN NELORE COWS: REPLACING THE LAST TWO DOSES OF pFSH BY eCG
Authors: Barros C. M. , Barcelos A. C. Z. , Gouvêa L. M. , Meneghel M. , Barcelos D. S. , Barcelos L. N. , Trinca L. A.
Published: January 2008

In the present study, the superstimulatory protocol coined P-36 (Nogueira et al. 2007 Theriogenology 67, 655–660) was modified in order to test if replacement of pFSH by eCG in the last day of superstimulatory treatment would improve follicular growth, ovulation rate, and embryo yield. Nelore cows (n = 20) were randomly allocated to two groups: P-36/LH48 and P-36/LH48/eCG. Each female received both treatments in a cross-over design; the first experimental period was in October (Spring 2006, both groups) and the second in February (Summer 2007, both groups). At a random stage of the estrous cycle (D0), animals received a progesterone intravaginal device (DIB�, 1.0 g; Syntex S.A., Buenos Aires, Argentina) plus estradiol benzoate (EB, Estrogin�, 2.5 mg IM; Farmavet, Sao Paulo, Brazil). The animals were superstimulated with pFSH (Folltropin-V�, Bioniche Animal Health, Ontario, Canada), administered twice daily in decreasing doses of 53.2, 39.9, 26.6, and 13.3 mg (total dose = 133 mg), from Day 5 to Day 8, except the P-36/LH48/eCG group, where the last two doses of pFSH were replaced by two doses (200 IU) of eCG (Novormon�, Syntex, Buenos Aires, Argentina). All cows were treated with D-cloprostenol (150 µg IM;Veteglan�, Calier, Barcelona, Spain) on Day 7 at 7:00 h, and DIBs� were removed 36 h after PGF2alpha administration. On Day 9, ovulation was induced by pLH administration (Lutropin�, 12.5 mg IM; Bioniche Animal Health) at 7:00 h. The animals were inseminated at 12 and 24 h after pLH administration, without estrus detection. Embryos were recovered on Day 16 or 17. Data were analyzed by ANOVA (Proc Mixed, SAS; SAS Institute, Inc., Cary, NC, USA), and the difference was considered significant when P < 0.05 or not significant (NS). Results for animals from groups P-36/LH48 and P-36/LH48/eCG were compared and are reported in this order. The number of follicles with diameter larger than 6 mm at the time of pLH administration (15.25 � 2.06 and 21.05 � 2.76; P < 0.01), the ovulation rate observed up to 48 h after pLH administration (77.7 � 5.6 and 83.9 � 2.6; NS), the total number of oocytes/embryos recovered (6.65 � 1.18 and 10.0 � 1.48; P < 0.03), the number of embryos recovered (6.05 � 1.24 and 8.35 � 1.30; NS), and the number of viable embryos (5.10 � 1.10 and 7.30 � 1.20; NS) are reported. The embryo quality (excellent, good, fair, and poor) was NS among groups P-36/LH48 and P-36/LH48/eCG. It is concluded that replacement of pFSH by eCG, on the last day of the superstimulatory protocol P-36, was beneficial since there was a significant increase in the number of follicles (>6 mm at the time of pLH administration) and the total number of structures recovered. Experiments are in progress to confirm these beneficial effects of eCG on the P-36 protocol.
This work was supported by FAPESP (Sao Paulo, Brazil). A. C. Z. Barcelos received a fellowship from CAPES (Brazil).
149 LAPAROSCOPIC EMBRYO TRANSFER IN PIGS
Authors: Terlouw S. L. , Ewerling S. , Didion B. A. , Dobrinsky J. R.
Published: January 2008

In an effort to optimize the number of live offspring from cloned and transgenic pig embryos, embryos are surgically transferred into the isthmus region of the oviduct soon after micromanipulation. Surgical embryo transfer in pigs is successful but still an invasive process. Laparoscopic embryo transfer (Besenfelder et al. 1997 Theriogenology 47, 1051–1060) is much less invasive than surgery and is more adaptable to a variety of commercial embryo transfer conditions. Our goal was to develop the use the laparoscope as an alternative method of embryo transfer for micromanipulated embryos. Naturally cycling maternal white line donor and recipient females were used for laparoscopic embryo transfer. Donors were selected to be in estrus 0 to 24 h before recipients. Two- to four-cell embryos were surgically recovered via mid-ventral laparotomy and immediately prepared for laparoscopic transfer into the oviduct through the infundibulum or through puncture of the oviduct into the ampulla. Sixty-nine embryos (range, 13–20) were transferred into the oviduct via the infundibulum of four recipients. Two recipients farrowed (50%), one recipient spontaneously aborted on Day 27, and one returned to estrus on Day 25 of the estrous cycle. Twenty-one pigs were born (10.5 pigs/farrowed sow) resulting in 30% (21/69) of transferred embryos becoming live offspring. Ninety-seven embryos (range, 10-21) were transferred into the ampulla of the oviduct of six recipients. Four recipients farrowed (67%) and 2 returned to estrus on Day 20 and Day 28. Thirty-nine pigs were born (9.5/sow farrowed) resulting in 39% (38/97) of transferred embryos becoming live offspring. Observations indicate that it is much easier to find the oviduct and deliver embryos via puncture into the ampulla than find and insert the transfer catheter into the infundibulum. Using the efficiencies generated from these data, 1.1 more pigs can be expected per transfer by transferring embryos to the oviduct via puncture than into the oviduct via the infundibulum. These data demonstrate the effective use of the laparoscope for oviductal embryo transfer. Further work is needed to determine if the laparoscope improves the production of live offspring compared to surgical embryo transfer.
157 VIRAL RISK ASSESSMENT OF BOVINE OOCYTES HARVESTED FROM ABATTOIR ORIGIN
Authors: Pommer J. , Nichols M. , Kasinathan P. , Sullivan E. , Robl J. , Griffin J. , Boerma P. , Vos C.
Published: January 2008

Bovine oocytes derived from abattoir origin are mainly used for artificial reproductive techniques in both agricultural and biomedical applications. Regulatory agencies have expressed concern for potential transmission of adventitious viruses by sourcing bovine oocytes from abattoir origin. To evaluate this concern, a viral risk assessment was conducted on batch samples collected from follicular fluid, cumulus cells, oocytes, and Day 8 embryos. These batch samples were collected from ovaries on seven randomly selected days in a 2-week period and they were frozen and stored at –80°C until tested. All samples were tested by 9 Code of Federal Regulations (9CFR) part 113.53c (animal viral testing) at a GLP compliant laboratory (American BioResearch Laboratories, Sevier, TN, USA). The 9CFR viral testing includes bovine viral diarrhea virus (BVDV), bovine parvovirus, bovine adenovirus type 3 and 5, bovine rabies virus, bovine bluetongue virus, bovine respiratory syncytial virus, bovine reovirus, viral cytopathic effect, and hemadsorption on permissive cell cultures. Batch samples were also tested for BVDV and bovine leukemia virus (BLV) by polymerase chain reaction (PCR) and follicular fluids for BVDV antibody neutralization activity at an accredited diagnostic laboratory (Animal Disease Research and Diagnostic Laboratory, SDSU, Brookings, SD, USA). The 9CFR viral testing results on all the batch samples were negative. The BVDV PCR test had a low positive (37.89 cycles) with one follicular fluid batch sample (1/7) and a low positive (37.9 cycles) on all cleaved embryos (7/7). However, BVDV virus isolation was negative for both batch samples by 9CFR testing. The BLV PCR had a positive follicular batch sample (1/7), with all other samples testing being negative. BVD serum neutralization antibody assay demonstrated that all follicular fluid had significant titers of 1:128–1:1024. Although some of the viral particles may have been detected in follicular fluid and cleaved embryos by PCR, none of the batch samples collected were positive for viral growth in this study. The BVDV PCR indicated low levels of BVDV RNA. It is speculated that the positive BVDV PCR results on cleaved embryos could possibly be attributed to the use of irradiated fetal calf serum (contaminated BVDV virus) used in culture media. Follicular fluids also have high titers to BVDV which may have neutralized the virus. These results indicate that virus-free bovine oocytes can be derived from the abattoir. Thus, with appropriately applied quality assurance testing, abattoir-origin oocytes might be used effectively in agricultural and biomedical applications.

Table 1. Summary of test results on batch samples from abattoir-derived oocytes
158 PRODUCTION OF RECOMBINANT LH FOR USE IN TIGER (TIGRIS ALTAICA) ASSISTED REPRODUCTION:A PRELIMINARY REPORT
Authors: Aaltonen J. T. , Bedows E. , Estes K. A. , Butnev V. Y. , Bousfield G. , Loskutoff N. M.
Published: January 2008

Genetically, porcine LH is the closest commercially available gonadotropin analog to tiger LH (93% homologous); however, its use may lead to possible autoimmune reactions, lessening ovarian responses in stimulated tigresses over time (Crichton et al. 2005 Biol. Reprod. 68, 105–113). To overcome this problem for use in assisted reproduction, we produced recombinant tiger LH (tLH), and tested the bioactivity of several tLH constructs using heterologous (rat) and homologous (cat) Leydig cell assays. To clone tLH, mRNA was isolated from an Amur tiger pituitary by TRIzol extraction (Invitrogen, Carlsbad, CA). DNA was synthesized from the mRNA using reverse transcriptase (Stratagene, La Jolla, CA) and PCR was performed using tiger-specific primers for glycoprotein hormone α subunit or LH β subunit. The α subunit was cloned into the double-expression vector pIRES (Invitrogen). The tLH β subunit was cloned into the second site of pIRES and also into the plasmid pGS. Chinese hamster ovary (CHO) K1 and human granulosa cell tumor (COV 434) cells were transfected with plasmid DNA by calcium-phosphate precipitation: (1) pIRES containing α and pGS containing LH β, or (2) pIRES containing α and LH β. Cells were grown in selection media (250 µg mL–1 geneticine for pIRES, or 25 µm methionine sulfoximine for pGS). Media was collected and clarified at 1500g for 30 min. An immature rat Leydig cell assay protocol (Bousfield et al. 2001 Biol. Reprod. 64, 136–147) detected biological activity (testosterone production) by RIA. Of 14 tLH constructs created, 1 wild-type construct (LH WTCHO8) had LH activity 3 times greater than any other. A domestic kitten Leydig cell assay was performed in order to assess comparative sensitivities and specificities. Domestic kitten testicles, obtained from a local spay clinic, were disassociated with collagenase (225 U mg–1, Worthington Biochemical, Lakewood, NJ); however, the cells were more difficult to disperse than rat testicles, leading to low Leydig cell yields as determined histologically. Modification of the Leydig cell collagenase protocol for the cat was achieved by increasing the temperature and surface area, and agitating the minced tissue in medium on a stir plate. Samples of the 14 tLH constructs were run in parallel using rat and cat Leydig cell assays. Although rat Leydig cell testosterone concentrations (3.4 ng mL–1) were nearly 10-fold greater, the same trend for the different constructs was found in the cat Leydig cells with the same wild-type construct (LH WTCHO8, 0.37 ng mL–1) having greater LH activity than any other. The lower testosterone concentrations in the cat bioassay may be explained by insufficient Leydig cell numbers, age (sensitivity), damaging effect(s) of collagenase, or felid specificity. Still, these results lend validity to the use of the heterologous rat Leydig cell bioassay for recombinant tiger LH.
16 COMPUTER-ASSISTED SPERM ANALYSIS SPERM PARAMETERS OF BOS TAURUS CRIOLLOS BULL SEMEN v. THOSE OF OTHER BOS TAURUS BREEDS IN ARGENTINA
Authors: Lüssenhoff M. G. , Larraburu G. , Cavia R. , Garcia Guerra A. , Brogliatti G. M.
Published: January 2008

Criollos is the oldest cattle breed in America and the world at large. The origin dates back to the first cattle brought by C. Columbus. They are well known for their toughness and longevity. Their genetic variability is another advantage to be taken into account in crossbreeding because it ensures high hybrid vigor. The objectives of the present study were to determine computer-assisted sperm analysis (CASA) motility parameters and sperm morphology of Criollos bull semen (brought from Parque Nacional Los Glaciares, Patagonia, Argentina) v. other Bos taurus semen. A total of 29 ejaculates of different adult bulls (Angus and Hereford) and 25 ejaculates of Criollos bulls were evaluated in an artificial insemination center between December 2004 and December 2005. Semen collection was done by electroejaculation (EE) in all breeds. Fresh semen was diluted in a semi-defined semen extender (Andromed; Minitüb, Tiefenbach, Germany) and frozen in an automatic freezer (Digicool, IMV, Paillette Crista, France). Stain morphology was done using eosin/nigrosin, and evaluated in an optic microscope with phase contrast. Parameters of volume, total concentration (CONC), motility %, motile progressive sperm %, velocity average path (VAP, μm s–1), velocity straight line (VSL, μm s–1), curvilinear velocity (VCL, μm s–1), and linearity (LIN, %) were determined by CASA (HTM-ceros 12.1, Berkley, CA, USA). The results obtained were analyzed statistically with a one-way ANOVA test and are summarized in Table 1. Results from the sperm motility analysis indicate that there were no significant differences (P > 0.05) in CONC, motility %, motile progressive %, or VSL between Criollos and other B. taurus bull semen. VAP and VCL rates were significantly higher in Criollos bull semen than in other B. taurus semen, and LIN was lower. Also, there was a significant difference in volume collected between both breeds. Regarding sperm morphology, Criollos bull semen is at the maximum limit acceptable for head defects (detached heads: 15%). These results suggest that Criollos bull semen is not different from other Bos taurus semen; however, it does show different velocity and linearity rates.

Table 1. CASA sperm motility parameters for semen from Criollos v. other Bos taurus bulls

This research was supported by Centro Genetico Bovino Eolia S.A.
163 SEARCH FOR GENES OF WHICH THE AMOUNTS OF TRANSCRIPTS OSCILLATE EVERY 24 h IN THE MOUSE OVARY
Authors: Amano T. , Hatanaka Y. , Saeki K. , Hosoi Y. , Iritani A. , Matsumoto K.
Published: January 2008

Perturbation of circadian rhythm is believed to be detrimental to the physiology of organs, including the mammalian ovary. However, the molecular mechanisms that are regulated by circadian rhythm in the ovary have not been identified. To identify the molecular mechanisms that are regulated by circadian rhythm and to speculate on the physiologies that are likely to be damaged by perturbation of circadian rhythm in the ovary, we searched for genes in which the amount of transcripts oscillates every 24 h in the mouse ovary. To achieve this, expression profiles of circadian genes (per1, per2, and bmal1) that code transcription-regulation factors for which transcription activities are known to oscillate every 24 h in almost all organs, and wee1, the transcription activity of which circadian genes regulate and which is known to elongate the G2 phase in the cell cycle, were analyzed in this study. Six-week-old female ICR mice were kept individually under a lighting schedule with lights on for 14 h followed by lights off for 10 h. A vaginal smear of each mouse was collected every day to determine its estrous cycle. Ovaries of 3 mice were collected continuously every 4 h over a 4-day period from the start of the light period on the day of proestrus. Total RNA was extracted from each ovary, and 500 ng each was used for cDNA synthesis. Transcripts of each gene and of tbp were quantified by real-time PCR, and the amount of the transcripts of each gene in each sample was divided by the amount of tbp transcripts. The obtained relative values in each sample were used as the representative data of the amount of transcripts of each gene. The amounts of per1, per2, and bmal1 clearly oscillated every 24 h. The maximum and minimum values of per1 and per2 were observed at 16 and 4 h, respectively, after onset of the light period each day. The maximum and minimum values of bmal1 were observed at the time of onset of the light period and at 12 h after onset of the light period each day. Averages of the maximum values of per1, per2, and bmal1 each day were significantly greater than averages of the minimum values (per1, 3.60 � 0.10 and 1.38 � 0.09; per2, 0.82 � 0.08 and 0.27 � 0.06; bmal1, 0.61 � 0.05 and 0.17 � 0.01; P < 0.05). The cyclicity in the oscillation of the amount of wee1 transcripts was weaker than that observed in circadian genes, but the average of values that were obtained from 12 to 20 h after onset of the light period each day was significantly greater than that obtained from 0 to 8 h (0.29 � 0.02 and 0.22 � 0.01; P < 0.05). Our results suggested that the cell cycle of ovarian cells is regulated in a circadian manner through wee1 transcription, which is regulated by circadian genes of which the amounts of transcripts oscillate every 24 h. Because an abnormal cell cycle seems to trigger the development of tumors or follicular cysts, perturbation of circadian rhythm may cause those ovarian diseases.
168 miRNA LEVELS DURING BOVINE PREIMPLANTATION EMBRYONIC DEVELOPMENT
Authors: Berg D. K. , Beaumont S. E. , Pfeffer P. L.
Published: January 2008

MicroRNAs (miRNAs) are a class of naturally occurring non-coding RNAs that play a role in gene regulation. They are highly conserved, single-stranded RNAs, 22 nucleotides in length, that are cleaved from larger inactive hairpin precursor transcripts, and use the RNA interference-related pathways to repress their mRNA targets. They play diverse regulatory roles in cellular proliferation, morphogenesis, apoptosis, and differentiation. Maternal miRNAs are crucial for early mammalian development (Murchison et al. 2007 Genes Dev. 21, 682–693; Tang et al. 2007 Genes Dev. 21, 655–648), while sperm-borne miRNAs do not contribute significantly to miRNAs in the zygote (Amanai et al. 2006 Biol. Reprod. 75, 877–884). Our objective was to identify miRNAs that are expressed during bovine in vitro oocyte maturation (MII) and blastocyst stages as well as during parthenogenic development. MII oocytes (n = 1680) were generated from abattoir-derived oocytes and matured in vitro for 24 h. Cumulus cells were removed and the first polar body was visually assessed before the oocytes were frozen in liquid N2. Parthenogenic blastocysts (n = 575) were produced using ionomycin/6DMAP activation, and IVF blastocysts (n = 1150) were produced using standard in vitro fertilization followed by in vitro culture in synthetic oviduct fluid (Thompson et al. 2000 J. Reprod. Fertil. 118, 47–55). Blastocysts (grades 1 and 2) were selected on Day 7 post-activation/insemination and frozen in liquid N2. RNA was isolated using the mirVana miRNA isolation kit (Ambion, Scoresby, Victoria, Australia). miRNAs were quantified using the TaqMan� MicroRNA Human Panel-Early Access Kit (Applied Biosystems, Scoresby, Victoria, Australia) following the manufacturer's protocol. Absolute copy numbers per embryo were estimated. Of the 157 miRNAs in the panel, 102, 136, and 118 were detected above background in oocytes, IVF, and parthenogenic blastocysts, respectively. Only 28 miRNAs were present at over 100 copies in MII oocytes, with maximum levels reaching 1300 copies. Levels were generally much higher at blastocyst stages, with 21 miRNAs present at more than 10 000 copies. miR-16 was one of the most abundant miRNAs in all samples tested. Copy numbers per blastomere cell were 5-fold higher in IVF blastocysts compared to parthegenotic blastocysts for miR-19a, 21, and 30b. The low copy numbers of mature miRNAs before embryonic genome activation may have implications for somatic cell nuclear transfer experiments in that exogenously added miRNAs from the donor cell could impact on the embryonic gene expression profiles.
173 SIMULTANEOUS QUANTIFICATION OF MULTIPLE PROTEINS IN PORCINE PARTHENOTES BY MULTIPLE SELECTED REACTION MONITORING
Authors: Gupta M. K. , Jang J. M. , Jung J. W. , Uhm S. J. , Kim K. P. , Lee H. T.
Published: January 2008

Simultaneous quantification of multiple proteins could be a very useful tool in cell signaling studies. Parthenogentic embryos do not develop to term, although their in vitro developmental ability to the blastocyst stage and to form pluripotent stem cells upon culture is comparable to those of sperm- or somatic nucleus-introduced embryos. This study was designed to quantify the expression of eight selected proteins by multiple selected reaction monitoring (mSRM) of trypsinized preparations from porcine zygotes (n = 1500) produced by parthenogenesis. The trypsinized samples were prepared, spiked with isotopically labeled synthetic MAP3K (IPTGTV*HNQAK) peptides as internal standards for quantification (asterisk denotes isotopic amino acid residue), and analyzed by reverse phase LC-MS/MS. Upon the appearance of the target peptide, SRM data were acquired within fragmention mass �1.5 m/z and each SRM transition, with its respective retention time, was validated as indicative of each specific peptide. Data were processed by integrating the appropriate peaks for the native and internal standard peptide, followed by calculation of the ratio of peak areas to estimate the relative abundance of the peptide. Analysis of data showed that the relative abundance of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), STAT2, calpastatin, complement cytolysis inhibitor, plakoglobin, serotransferrin, and epsilon-globin were 203.72, 138.52, 60.50, 18.63, 31.23, 0.85, 10.33, and 0.12, respectively. These data were consistent with those observed by reverse phase LC-MS/MS combined with 1-dimensional (1-D) sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of cellular lysates from porcine embryos (n = 6000). The data presented here, therefore, provide mSRM of embryos for the first time in any species and suggest that mSRM in combination with reverse-phase LC-MS/MS combined with 1-D SDS-PAGE could be a powerful tool for proteome analysis of porcine embryos to obtain vital new information for understanding the basic biology of embryonic development.
This study was supported by grants from Biogreen 21, RDA, and the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea.
174 EPIDERMAL GROWTH FACTORS AND CALBINDIN-D9k GENE EXPRESSIONS DURING PREGANCY IN THE PORCINE UTERUS
Authors: Kim Y.-J. , Jung E.-M. , Lee G.-S. , Hyun S.-H. , Jeung E.-B.
Published: January 2008

To stably maintain pregnancy, several genes are expressed in the uterus. In particular, the endometrial expression of genes encoding growth factors appears to play a key role in maternal–fetal communication. Previous studies have characterized the endometrial expression kinetics of the genes encoding epidermal growth factor (EGF), its receptor (EGFR), transforming growth factor-alpha (TGF-α), amphiregulin (Areg), heparin-binding (Hb) EGF, and calbindin-D9k (CaBP-9k) in the pig during implantation. Here, we further characterized the expression patterns of these molecules during the entire porcine pregnancy. Porcine (n = 3 per PD) were collected at pregnancy days (PD) 12, 15, 30, 60, 90, and 110 and subjected to semi-quantitative RT-PCR. The data were analyzed with a nonparametric one-way analysis of variance using the Kruskal-Wallis test, followed by Dunnett's test for multiple comparisons to the negative control. EGF and EGFR showed similar expression patterns, being highly expressed around implantation time and then disappearing. TGF-α and Areg expression levels rose steadily until they peaked at PD30, after which they gradually decreased to PD12 levels. The Areg mRNA expression pattern was confirmed by real-time PCR, and similar Areg protein expression patterns were observed. Immunohistochemical analysis of PD60 uteri revealed Areg in the glandular and luminal epithelial cells. Hb-EGF was steadily expressed throughout the entire pregnancy while CaBP-9k was expressed strongly on PD12, and then declined sharply in PD15 before recovering slightly for the remainder of the pregnancy. Thus, the EGF family may play a key role during implantation in pigs. In addition, CaBP-9k may help maintain uterine quiescence during pregnancy by sequestering cytoplasmic Ca2+.
176 DOXYCYCLINE-INDUCIBLE EXPRESSION OF THE hEPO GENE IN A RETROVIRUS VECTOR
Authors: Kwon M. S. , Koo B. C. , Choi B. R. , Lee H. , Kim T.
Published: January 2008

The use of 'bioreactors' to address the growing demand for large quantities and increasing numbers of biopharmaceuticals is of prime strategic relevance to medical advancement. However, it is well known that constitutive overexpression of some cytokine genes may cause serious physiological disturbances in vivo and in vitro. The main objective of this study was to test the feasibility of controllable gene expression of hEPO, a glycoprotein hormone that stimulates red blood cell production. We constructed and tested retrovirus vectors designed to express the hEPO (human erythropoietin) gene under the control of tetracycline-inducible promoters. Advantages of this system over other counterparts, whose gene expressions are controlled by steroid hormones or heavy metals, are little or no cytotoxicity of the inducer and efficient turn-on/off transition. However, the most critical problem of the tetracycline-controllable gene expression system is that overall gene expression level is low. As a solution for this problem, we introduced a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence into the retrovirus vector at a downstream region of either the hEPO gene or the sequence encoding rtTA (reverse tetracycline-controlled transactivator) of Tet-On system. In cooperation with tetracycline, the rtTA activates the tetracycline response promoter which controls the expression of the hEPO gene. Transformed cells derived from pig and chicken were cultured in medium supplemented with or without 1 µg mL–1 of doxycycline (a tetracycline derivative) for 48 h, and induction efficiency was measured by comparing the hEPO gene expression level using RT-PCR, Western blotting, and ELISA. Western blotting analysis showed that, among several vectors tested, the vector carrying the WPRE sequence at the 32 side of the hEPO gene was the best in both chicken and porcine cells. Quantitative analysis using ELISA resulted in a more than 10-fold increase in overall hEPO gene expression without sacrificing the efficiency of turn-on/off transition. These results should be very helpful in establishing transgenic livestock as bioreactors, making it possible to produce commercially valuable and biologically active proteins in large quantities.
179 MODULATION OF RHOPHILIN-2 MAY REGULATE THE PROGRESSION OF CELL DIVISION IN FERTILIZED MOUSE EGGS
Authors: Matsuoka T. , Tokoro M. , Shin S. , Amano T. , Hosoi Y. , Saeki K. , Iritani A. , Matsumoto K.
Published: January 2008

It has been reported that activity of Rho, one of the GTPases, is essential for division of nuclei and cytoplasm of fertilized mouse eggs. Since it has been reported that alteration of activities of GTPases modifies their ability to attach to each of their effector proteins in somatic cells, effector proteins seem to be able to control not only progression but also repression of cell division by changing their cellular localizations through activities of GTPases. For this reason, Rhophilin-2, one of the effector proteins of Rho, seems to be involved in the decision of progression of division of fertilized mouse eggs. To examine whether this involvement works in fertilized mouse eggs, cellular localization of Rho and Rhophilin-2 in fertilized mouse eggs that were treated with Rho inhibitor were analyzed. Moreover, cellular localization of GABA A receptor association protein (GABARAP), which was identified in our previous study (Matsuoka et al. 2006 Reprod. Fertil. Dev. 18, 176–177) as a protein that interacts with Rhophilin-2, was also analyzed. Fertilized mouse eggs were obtained from in vitro fertilization technique. One group of fertilized eggs was obtained at 24 h after insemination as experimental control. To obtain the mouse eggs in which Rho activities were inhibited, Clostridium botulinum C3 exoenzyme (C3-CB), an inhibitor of Rho activity, was injected into the other group of fertilized mouse eggs at 12 h after insemination, and were collected after 12 h of subsequent culture. Cellular localization of Rho (n = 100), Rhophilin-2 (n = 10). and GABARAP (n = 10) in the collected oocytes was analyzed by using immunofluorescence. Our results showed that Rho and Rhophilin-2 were co-localized at the midbody microtubule, which is an important device for cytoplasmic division in control eggs. However, the inhibition of Rho activity did not modify the co-localization of Rho and Rhophilin-2. On the other hand, localization of GABARAP was modified by the inhibition of Rho activity, and GABARAP was detected around the nuclei of fertilized eggs in which Rho activity was inhibited. In the next experiment, we examined whether interaction of Rhophilin-2 and GABARAP was modified by the inhibition of Rho activity by using a co-immunoprecipitation assay (co-IP) (n = 100). The interaction of Rhophilin-2 and GABARAP was found to disappear after inhibition of Rho activity. These results suggest that activity of Rho seems to regulate cytoplasmic division through Rhophilin-2 modification. Moreover, Rho seem to modulate the nuclear division of fertilized mouse eggs by regulating the interaction between Rhophilin-2 and GABARAP.
This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.
18 PREGNANCY RATES IN DUAL-PURPOSE CROSSBRED (BOS TAURUS × BOS INDICUS) HEIFERS TREATED WITH DIFFERENT OVSYNCH-BASED PROTOCOLS
Authors: Perea-Ganchou F. , De Ondiz A. , Ramirez C. , Palomares-Naveda R. , Hernandez-Fonseca H. J. , Portillo-Martinez G.
Published: January 2008

In the American tropics, the majority of cattle populations are based on crossbred animals resulting from the mating of zebu (Bos indicus) with dairy breeds (Bos taurus). In Venezuela, as in other tropical regions, crossbred herds have been developed rapidly due to superior characteristics over other genetic groups, such as adequate heat and insect tolerance, milk production based mainly on pastures, and good quality meat to satisfy the animal protein demand of the human population (Soto et al. 2002 Theriogenology 57, 1503–1510). In order to compare the effectiveness of three ovulation synchronization protocols, a study on follicular characterization was carried out at a commercial farm in Zulia State, Venezuela. Seventy-seven crossbred heifers were randomly selected and assigned to the following treatments: OVSYNCH (T1, n = 27), Day 0: 20 mg of gonadotropinreleasing hormone (GnRH) IM; Day 7: 25 mg of prostaglandin F2α (PGF2α) IM; Day 9: 20 mg of GnRH IM; and Day 10: timed AI (TAI). Modified OVSYNCH (T2, n = 25), similar to T1, but without GnRH on Day 9 and adding 1 mg of estradiol-17β on Day 8. Progestagen + E + PGF2α (T3, n = 25), Day 0: 50 mg of progesterone and 5 mg of estradiol-17β IM; Day 7: 25 mg of PGF2α; Day 8: 1 mg of estradiol-17β IM; Day 10: TAI. The results were analyzed by the Proc Freq and chi-square tests of the Statistical Analysis System (SAS; SAS Institute, Inc., Cary, NC, USA). Cows treated with T1 and T2 showed higher pregnancy rates than those treated with T3 (P < 0.05); 40%, 32%, and 12% for T1, T2, and T3, respectively. There was no difference between T1 and T2. Results of the parameters considered during follicular characterization examinations through ultrasound were: TALINIFID (initial size of the dominant follicle: 5.5 mm), DIAULFD (last day size of the dominant follicle: 10.2 mm), and TALFINFO (final size of the ovulatory follicle: 10.9 mm). No differences between treatments were found for follicular sizes during the evaluated times. In conclusion, Modified Ovsynch showed an effectiveness similar to that of the Ovsynch protocol. However, the combination of progestagen, estradiol, and PGF2α produced a decreased pregnancy rate in dual-purpose crossbred heifers.
184 INHIBITION OF HUMAN COMPLEMENT-MEDIATED CYTOTOXICITY IN MINIPIG CELLS BY EXPRESSION OF hCD59
Authors: Park K. W. , Kim E. J. , Choi K. M. , Hong S. P. , Han G. S. , Yoo J. Y. , Kim S. H. , Park Y. C. , Seol J. G.
Published: January 2008

Xenotransplantation of a pig organ to a human is a possible solution for the shortage of donor organs for transplantation. However, hyperacute rejection (HAR) due to natural antibodies (Nab) present major obstacle in pig-to-human xenotransplantation. To overcome this, much effort has been dedicated to preparing transgenic pigs that express human complement regulatory proteins (CRPs). One of CRPs, the human CD59 gene, can prevent the terminal polymerization of the membrane attack complex by complement. In this study, we investigated the inhibitory effect of hCD59 on complement-mediated cytotoxicity in hCD59-transfected minipig cells. To generate cell lines expressing hCD59, we transfected the hCD59 gene into minipig fetal fibroblasts and established seven transgenic clonal cell lines. The integration of hCD59 gene was confirmed by PCR and expression levels were measured by RT-PCR, fluorescence-activated cell sorting (FACS), Western blot, and immunohistochemistry. FACS analysis of hCD59 clonal cell lines demonstrated a substantial increment of hCD59 expression. The level (82% to 95%) of hCD59 protein expression was increased relative to that of the control. Human complement-mediated cytotoxicity was measured using a CytoTox96� (Promega Corp., Sydney, Australia) non-radioactive cytotoxicity (LDH) assay using normal minipig cells as a negative control. In the LDH release assay, human complement-mediated cytotoxicity was also significantly reduced to 39.6 � 17.8% in comparison to that of the control group (73.6 � 19.1%; P < 0.05; n = 6). These results indicate that the expression of hCD59 gene in minipig cells can efficiently control complement-mediated cytotoxicity.
19 THE EFFECT OF PROGESTERONE ON FOLLICULAR DEVELOPMENT AND PREGNANCY RATE IN BEEF CATTLE
Authors: Pfeifer L. F. , Mapletoft R. , Adams G. , Kastelic J. , Small J. , Dias F. , Singh J.
Published: January 2008

The objective was to determine the effect of the level of progesterone (P4) during the ovulatory wave on fertility. Low v. high progesterone concentrations which were expected to result in high v. low LH pulse-frequency were compared. Lactating, postpartum beef cows (n = 69) at random stages of the estrous cycle were given cloprostenol (PGF; Schering-Plough Animal Health, Pointe-Claire, QC, Canada) twice at an 11-day interval to synchronize ovulation (Day 0 = day of 2nd PGF). Cows were given 1.5 mg estradiol benzoate on Day 10 (7 days after expected ovulation). On Day 10, the Low-P4 group was given PGF and an intravaginal CueMate (Bioniche Animal Health, Belleville, ON, Canada) containing one P4 pod (expected to achieve a P4 plasma level of 1 ng mL–1). The High-P4 group also received a CueMate with one pod on Day 10, but no PGF was administered (i.e., cows were allowed to retain their CL for 4 days after expected emergence of the estradiol-induced wave). On Day 18, CueMates were removed and PGF was administered to both groups. Fifty-four to 56 h later, all animals received 12.5 mg of Lutropin-V (Bioniche) IM and were timed AI (TAI). Dominant follicle diameter was measured on Day 18 and at the time of AI. Pregnancy diagnosis was done by ultrasonography 27 days after AI to compare fertility between groups. Follicle diameter data were analyzed by Student's t-test and pregnancy rate data were compared by the chi-square test. The dominant follicle in the Low-P4 group was larger (P < 0.05) than in the high-P4 group on the day of the CueMate removal (Day 18) and on the day of the TAI (11.8 � 0.4 and 13.7 � 0.5 v. 13.3 � 0.6 and 15.3 � 0.6 in the High-P4 and Low-P4 groups, respectively). Pregnancy rate was similar (P = 0.16) between the groups (High-P4: 55.6% (20/36) and Low-P4: 44.4% (16/33)); however, a larger study is in progress to confirm these results. To conclude, Low-P4 resulted in a larger dominant follicle without any apparent effect on pregnancy rate.
The project was funded by Saskatchewan ADF and NSERC. L. Pfeifer is funded through a CAPES scholarship. We acknowledge Schering-Plough and Bioniche for donation of pharmaceuticals.
197 DOES DURATION OF BOVINE OOCYTE MATURATION IN VITRO AFFECT THE SPEED OF EMBRYO DEVELOPMENT IN VITRO AND SEX RATIO AT THE TWO-CELL OR BLASTOCYST STAGE?
Authors: Bermejo-Álvarez P. , Gutiérrez-Adán A. , Lonergan P. , Rizos D.
Published: January 2008

The faster-developing blastocysts in IVC systems are generally considered more viable and better able to survive following cryopreservation or embryo transfer than those that develop more slowly. However, evidence from several species indicates that embryos that reach the blastocyst stage earliest are more likely to be males than females. The aim of this study was to determine whether the duration of maturation could affect early embryo development and, furthermore, the sex ratio of early- or late-cleaved embryos and blastocysts. Cumulus–oocyte complexes were matured in vitro for 16 h (n = 2198) or 24 h (n = 2204). Following IVF, presumptive zygotes from each group were examined every 4 h between 24 and 48 h postinsemination (hpi) for cleavage, and all embryos were cultured to Day 8 in synthetic oviduct fluid to assess blastocyst development. Two-cell embryos at each time point and blastocysts on Days 6, 7, and 8 from both groups were snap-frozen individually for sexing. Sexing was performed with a single PCR using a specific primer BRY. There was a significantly lower number of cleaved embryos from the 16-h compared with the 24-h maturation group at 28 (10.0 � 1.51 v. 28.8 � 3.57%), 32 (35.3 � 1.48 v. 57.6 � 3.33%), 36 (54.8 � 1.76 v. 67.4 � 2.81%), 40 (63.3 � 1.82 v. 72.0 � 2.54%), and 48 (70.6 � 1.78 v. 77.1 � 2.18%) hpi, respectively (mean � SEM; P d 0.05). However, the blastocyst yields on Day 6 (17.1 � 3.11 v. 16.4 � 2.11%), 7 (30.6 � 4.10 v. 34.6 � 3.51%), or 8 (34.1 � 3.90 v. 39.4 � 4.26%) were similar for both groups (mean � SEM; 16 v. 24 h, respectively). Significantly more 2-cell early cleaved embryos (up to 32 hpi) were male compared with the expected 1:1 ratio from both groups (16 h: 1.24:0.76 v. 24 h: 1.17:0.83, P ≤ 0.05); however, the overall sex ratio among 2-cell embryos was significantly different from the expected 1:1 in favor of males only for the 16-h group (1.18:0.82, P ≤ 0.05). The sex ratio of blastocysts on Day 6, 7, or 8 from both groups was not different from the expected 1:1. However, the total number of male blastocysts obtained after 8 days of culture from the 24-h group was significantly different from the expected 1:1 (1.19:0.81, P ≤ 0.05) and approached significance in the 16-h group. These results show that the maturational stage of the oocyte at the time of fertilization has an effect on the kinetics of early cleavage divisions but not on blastocyst yield. Furthermore, irrespective of the duration of maturation, the sex ratio of early-cleaving 2-cell embryos was weighted in favor of males, and this observation was maintained at the blastocyst stage.
199 EFFECT OF HEPARIN CONCENTRATION ON BOVINE PREIMPLANTATIONAL DEVELOPMENT IN VITRO USING SEX-SORTED SPERM
Authors: Chaubal S. A. , Nedambale T. L. , Xu J. , Shaffer C. , Kilmer T. , Shefler A. , Yang X. , Tian X. C. , Du F.
Published: January 2008

The objective of this study was to examine the effect of heparin on bovine IVF and to improve the efficiency of IVF production by using sex-sorted sperm. The fertility performance of sex-sorted and unsorted semen from 4 bulls was compared to determine the optimal heparin concentration during preimplantational embryo development. A total of 7615 matured bovine oocytes were randomly allocated among different heparin concentrations (0, 2.5, 5, 10, 20, 40, 60, 80, and 100 μg mL–1) in Brackett-Oliphant medium and coincubated with either sex-sorted or unsorted sperm for 6 h. Presumptive zygotes were cultured in CR1aa+ 6 mg mL–1 of BSA in 5% O2 , 5% CO2 and 90% N2 at 39°C until Day 8 (Day 0, culture post-IVF). Cleavage rates at Day 2 and embryo development to blastocyst (BL) at Day 8 were recorded. Data (4 replicates) were analyzed by a general linear model (SPSS 11.0, SPSS Inc., Chicago, IL). The optimal heparin concentration for each treatment was determined as the lowest value from those groups that resulted in the highest BL rates. The results (Table 1) demonstrated that a differential requirement of heparin concentration was important for the highest preimplantational BL development between sexed sperm and unsorted control within each bull. By optimizing heparin concentration, in 3 out of 4 (75%) bulls, the in vitro BL development with sex-sorted sperm could be increased to a level that was comparable to the highest BL rate from unsorted sperm (bulls A, B, and C, P > 0.05). A higher heparin concentration was required for optimal BL development in bulls A and C; however, a lower concentration was desirable for bulls B and D, indicating that a partial capacitation to the sperm may have taken place in bulls B and D during the sorting process, as reported by Lu and Seidel (2004 Theriogenology 62, 819–830). The fertility of sorted sperm from bull D (1 out of 4, 25%) was adversely affected, even after heparin optimization for BL development (P < 0.05). This result suggests that sperm sorting could affect the IVF fertility of sorted sperm in a bull-specific manner, but it was not significant for all bulls.

Table 1. Blastocyst (BL) development in bovine IVF after heparin optimization using sorted and unsorted sperm

This project was supported by the SBIR program under a USDA Cooperative State Research, Education, and Extension Service (CSREES) grant to F. Du (USDA #2006-03069).