bluuebunny/crossref_metadata_2025 huggingface dataset:
238ESTROGEN RECEPTOR ALPHA AND PROGESTERONE RECEPTOR EXPRESSION FROM
REPRODUCTIVE TISSUE AND IN VITRO PRODUCED EMBRYOS OF THE DOMESTIC CAT
Authors: Latino M.W. , Chiang T.C. , Pope C.E. , Gomez M.C. , Giraldo A.M. , Harris R.F. , King A.L. , Dresser B.L. , McLachlan J.A.
Published: February 2004

The in vitro production of cat embryos has been reported by several laboratories, and kittens have been born after transfer of embryos derived by IVF, ICSI, and NT. However, evidence accumulating in other species indicates that in vitro-derived embryos exhibit altered gene expression of developmentally important genes. Because the domestic cat genome is not well defined, the lack of primer sequence information poses a challenge for gene expression profiling. Estrogen, in addition to its essential role in the development and function of the female reproductive tract, is important for maturation of the oocyte. The aim of this preliminary study was to evaluate the expression profile of estrogen receptor alpha (ERα) and progesterone receptor (PR) in domestic cat female reproductive tissue and in vitro-produced (IVP) embryos. Embryos were produced in vitro as described by Gomez et al. (2003 Theriogenology 60, 239–251). mRNA was isolated from pools (n=35–50) of IVP domestic cat embryos at the morula and blastocyst stages (Days 7 and 8) using a modified protocol of the PolyAT Tract System (Promega, Madison, WI, USA). Uterus and ovaries were collected from hystorectomized cats and total RNA was isolated from these estrogen-targeted organs (RNeasy standard protocol, QIAGEN, Valencia, CA, USA). All RNA was reverse-transcribed and subjected to real-time PCR to evaluate expression of ERα and PR. The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as a control for expression in all tissues. Using primers designed to amplify human sequences or multispecies primers, we successfully amplified all three genes from as little as ∼100ng of ovarian or uterine mRNA. We were also able to detect ERα and GAPDH from pools of IVP embryos. Transcripts were cloned and confirmed by sequencing to be homologous to known sequences of the respective genes in a variety of species, including human, mouse, and pig. A 234-bp transcript of ERα (accession #AY349164;; GenBank) corresponding to exons 5 through 7 of the human ERα gene (hormone-binding domain) was identified along with a 110-bp sequence of PR and 98-bp sequence of GAPDH. To our knowledge, this is the first description of ERα or PR cloning in the domestic cat. In summary, we have demonstrated that highly sensitive real-time PCR is effective for the assessment of gene expression in cat ovarian and uterine tissue, as well as in embryos, the latter, of which, are known to have a low transcript copy number. Furthermore, these experiments provide the basis for future studies on the effect of exogenous estrogen on gene expression in cat IVM oocytes and IVP embryos.
241POLYADENYLATION STATUS OF MRNAs CODING FOR PRDX-6, GDF-9, G6PDH AND
CYCLIN B1 BEFORE AND AFTER IN VITRO MATURATION OF BOVINE OOCYTES:
REAL-TIME PCR RESULTS AFTER REVERSE TRANSCRIPTION WITH OLIGO(DT) OR WITH
HEXAMERS
Authors: Lequarre A.S. , Traverso J.M. , Marchandise J. , Donnay I.
Published: February 2004

Changes in length of the polyA tail of an mRNA is determinant for its stability and translation. During oocyte maturation, although some genes are specifically polyadenylated, an important decrease of polyadenylated mRNA is generally observed. Both degradation and de-adenylation can be responsible for this decline but little information about these processes is available yet for in vitro-matured bovine oocytes. Total RNA was extracted from 2 pools of 50 immature and 2 pools of 50in vitro matured bovine oocytes, after addition of 1pg of polyadenylated rabbit globin mRNA to each pool. The RNA of each pool was then divided in 8 samples, half of them were reverse-transcribed using hexamers and the other half using oligo(dT). Six genes were amplified in every sample using real-time PCR and specific molecular beacon probes: (1) rabbit globin, an exogenous gene added as a reporter for the efficiency of extraction and RT;; (2) histone H2A, an endogenous gene reporter for RNA quantity and quality in each pool [This housekeeping gene, that is not adenylated, should be maintained at a constant level through the maturation process (Robert et al., 2002 Biol of Reprod. 67, 1425–1472)]; (3) two antioxidant enzymes, peroxyredoxin 6 (PRDX-6) and G6PDH preventing oxidative stress during maturation [PRDX-6 also has a phospholipase A2 activity that could be implicated in prostaglandins synthesis during maturation]; (4) GDF-9, an oocyte-specific gene involved in cumulus expansion during maturation;; and (5) cyclin B1, one subunit of the MPF factor whose activity is required for meiosis resumption. The PCR result of each gene was normalized with the globin value of the same sample. After this standardization, the variability of the results among the 4 samples of a same pool similarly reverse transcribed did not exceed 15%. Within a pool, the mean PCR signal obtained for a specific mRNA using oligo(dT) was compared with the mean signal obtained with hexamers. Results obtained for immature and mature oocytes are reported in the Table 1. Before maturation, the PCR signals were higher when reverse transcription was performed using oligo(dT) instead of hexamers but that was not the case after maturation. Most messengers coding for these genes probably lost their polyA tails during maturation. This loss was much less pronounced for cyclin B1 messengers. Furthermore, for all genes studied, the PCR results obtained after reverse transcription using hexamers and normalized with the histone H2A value did not show a decrease during the maturation process. In conclusion, deadenylation, more than degradation, would be responsible for the disappearance of maternal polyadenylated mRNA during maturation.

Table 1
Ratio of the PCR signals obtained after RT with oligo(dT) v. hexamers
242GLUTATHIONE CONTENT AND EXPRESSION OF ANTIOXIDANT ENZYME
MRNAS IN SHEEP OOCYTES
Authors: Livingston T. , MacKenzie S. , Edwards L. , Godkin J.
Published: February 2004

Reactive oxygen species (ROS) are normal products of embryo metabolism;; however, oxidative injury may negatively impact development if ROS production exeeds antioxidant defense mechanisms. Protection of the embryo against ROS is dependent, in part, upon the pool of antioxidant enzyme mRNA and products stored in the oocyte. Previous work has demonstrated that retinol administration to superovulated ewes, followed by natural service, resulted in embryos with improved competence to develop in vitro. In other cell systems, retinoids have been shown to participate in the antioxidant network and redox system. The objectives of this study were to analyze glutathione content and characterize antioxidant enzyme mRNA expression in mature ovine oocytes and test the hypothesis that retinol administration affects levels of these products. Ewes were superovulated and administered retinol on the first and last day of FSH injection. Oocytes were collected from oviducts 60h later and either prepared for glutathione (GSH) analysis or subjected to RNA isolation. Glutathione was quantified by the recycling assay of the enzyme 5,5-dithiobis-(2-nitrobenzoic acid)-glutathione reductase using a total of more than 260 oocytes. Transcripts encoding for manganese superoxide dismutase (Mn-SOD), copper zinc superoxide dismutase (Cu-Zn SOD), glutathione synthetase (GS) and glutathione transferase pi (GSTp) were analyzed in a total of 86 single oocytes by semi-quantitative RT-PCR using a histone H2a transcript as the internal control. Chi-square analysis was performed to detect differences due to treatments. Glutathione content did not differ significantly between oocytes collected from retinol-treated (6.78±3.81pmol/oocyte) and control (6.38±1.58pmol/oocyte) ewes. Transcripts encoding for Mn-SOD, Cu-Zn SOD, GS and GSTp were detected in greater than 95% of the oocytes analyzed. Relative abundance of these transcripts did not differ between treatments. For the first time, glutathione content and antioxidant enzyme mRNA expression has been analyzed in in vivo-matured sheep oocytes. Retinol treatment did not appear to affect the expression of these products. Results demonstrate the existence of endogenous antioxidant defense products, stored as mRNA and protein, in sheep oocytes.
247GENE EXPRESSION OF MAMMALIAN RELATIVE OF DNAJ IN BOVINE ENDOMETRIUM
DURING EARLY PREGNANCY
Authors: Oshima K. , Kojima T. , Watanabe H. , Fukushima M. , Dochi O. , Takenouchi N. , Komatsu M. , Yamamoto N.
Published: February 2004

The mammalian relative of DNAJ (MRJ), a member of the molecular chaperones that are known as heat shock proteins, plays an important role in the process of murine chorioallantoic fusion. The objective of this study was to determine the expression pattern of MRJ in the bovine endometrium during early and mid-pregnancy using quantitative RT-PCR. Twenty-eight Japanese Black cows, aged between 1.2 and 15.2 years, with normal estrous cycles, were used in this study. Twenty-one cows were used for study of the level of MRJ during pregnancy. They were artificially inseminated, and their endometrial tissues were collected on Days 16 to 21 (n=7), 30 to 36 (n=6), 48 to 49 (n=4) and 74 to 140 (n=4) of pregnancy. Seven cows were used as controls for the study of cyclic level of MRJ, and their endometrial tissues were collected on Days 13 to 14 (n=4) and 17 to 20 (n=3) of the estrous cycle. The caruncles and the intercaruncles were isolated from the endometrial tissues. All tissues were frozen immediately using liquid nitrogen. Total RNA from these samples was extracted from the tissue using Trizol (Invitrogen Corp., Carlsbad, CA, USA). The first strand of cDNA was synthesized from the total RNA by oligo (dT)12–18 and SuperScript™ II RNase H- Reverse Transcriptase (Invitrogen Corp.) according to the manufacturer’s instructions. Primers and the TaqMan probe for MRJ and glyceraldehyde-phosphate-dehydrogenase (GAPDH) were designed using the primer design software Primer Express™ (Applied Biosystems, Foster City, CA, USA). Bovine GAPDH was used as an internal standard. All PCR reactions were performed using a TaqMan™ PCR Reagent Kit and a MicroAmp Optical 96-Well Reaction Plate and Cap (Applied Biosystems). The assay used an ABI Prism 7700 Sequence Detector (Applied Biosystems). Signals were detected according to the manufacturer’s instructions. The relative level of MRJ expression was calculated on the basis of GAPDH quantity (the method of calculation: relative level=MRJ quantity/GAPDH quantity). Data were analyzed by one-way ANOVA, and means were conpared by Tukey-Kramer’s HSD test. Mammalian relative of DNAJ genes were expressed in all samples examined;; the levels in intercaruncle tended to be greater than those in caruncle. Although MRJ expression level at Days 16 to 21 of pregnancy was greater than at other days during pregnancy, there were no significant differences between the levels at Days 16 to 21 of pregnancy and those of the estrous cycle. These results suggest that MRJ is produced in the endometrium and may play a role in early and mid-pregnancy and the estrous cycle.
248EXPRESSION AND LOCALIZATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND
VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2 IN EQUINE FOLLICLES
Authors: Pedersen H.G. , Greenaway J. , Greve T. , Petrik J.
Published: February 2004

Ovarian follicles undergo pronounced morphological changes, alternating between periods of growth and regression. The equine follicle will grow to an average of 45mm in diameter at ovulation, and during the phase of growth, there is an increase in blood supply to the follicle. Vascular endothelial growth factor (VEGF) is a cytokine that interacts with tyrosine kinase receptors to stimulate angiogenesis, endothelial cell proliferation and vascular permeability. The aim of the study was to evaluate the expression and localization of VEGF and the VEGF-receptor 2 (VEGF-R2) in equine follicles. Ovaries were collected from a slaughterhouse. Granulosa cells from follicles were pooled regardless of the size of the follicles. Western blots were performed using protein extracted from granulosa cells and follicular fluid. Blots were probed with rabbit anti-human VEGF and rabbit anti-mouse VEGF-R2 antibodies and visualized with chemiluminescence. Total RNA was extracted from granulosa cells and integrity of the RNA samples was tested by the amplification of β-actin. Complementary DNA was synthesized by reverse transcription, followed by polymerase chain reaction amplification of cDNA encoding with bovine primer sequences for VEGF and VEGF-R2. The PCR product was resolved on 1% agarose gel and the resulting VEGF and VEGF-R2 bands were sequenced. Immunostaining for VEGF and VEGF-R2 was performed on fixed, paraffin-embedded sections of follicle wall from follicles larger than 30mm. Western blot analysis of granulosa cell lysates revealed 22kDa bands for VEGF, and 210kDa bands for VEGF-R2. VEGF protein was present in follicular fluid, whereas VEGF-R2 was not detectable. RT-PCR experiments revealed the presence of VEGF and VEGF-R2 mRNA in isolated granulosa cells. Sequencing demonstrated 93% and 99% homology to known sequences of equine VEGF and VEGF-R2, respectively. Immunofluorescence experiments performed on dissected equine follicles localized VEGF to the granulosa cell layer and sporadically to the theca cell layer. VEGF-R2 co-localized with VEGF in the granulosa cells, and was relatively absent in the theca layer. The present study detected novel expression patterns for VEGF and VEGF-R2 in equine ovarian follicles. The results of these experiments suggest an extra-vascular role for the VEGF family in follicle development. Future studies will be directed at studying the genomic and proteonomic profiles of follicles during the selection of the dominant follicle in mares.
249BOVINE GRANULOSA CELLS MRNA EXPRESSION OF PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTOR-± AND THE PROTO-ONCOGENE C-FOS
Authors: Rodriguez A. , Royo L.J. , Goyache F. , Diez C. , Moran E. , Salas A. , Gomez E.
Published: February 2004

PPARα and c-Fos are involved in regulation of gene expression and are known to be dependent on retinoic acid (RA), which in turn influences oocyte growth and developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003. Reproduction 125, 409–416), probably acting in part through granulosa cells. Peroxisome proliferator-activated receptor-α (PPARα) heterodimerizes with the retinoid receptor X (RXR), while c-Jun/c-Fos heterodimerizes with liganded retinoic acid receptors (RARs), then preventing formation of transcription factor activator protein 1 (AP-1) complexes capable of DNA binding. Cellular retinoic acid binding protein (CRABP) limits RA excess and regulates the transcriptional potential of RA;; CRABPII has been detected in rat granulosa cells from mature follicles and luteal cells. The aim of this study was to investigate PPARα, c-Fos and CRABPII mRNA expression in bovine granulosa cells. In parallel, other genes whose expression can be influenced by RA were analyzed: luteinizing hormone receptor (LHr), follicle stimulating hormone receptor (FSHr), aromatase and growth hormone (GH). Ovaries were collected at a local abattoir and kept in saline at 30–35°C. Granulosa cells were obtained by aspirating 2- to 7-mm antral follicle contents, pelleted at 700g for 4min and resuspended in RNA-later (Ambion®). Total RNA was isolated with a NucleoSpin® RNAII kit (Macherey-Nagel), and mRNA was reverse transcribed into single-stranded cDNA using a 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche). A PCR standard method was made using 1μL of the cDNA as a template. All PCR primer couples were designed on the basis of the bovine sequence, but c-Fos and CRABPII primers were designed based on the human-murine sequences. Primers within the couple were located in different exons to distinguish DNA from RNA amplification. CRABPII was further investigated in bovine whole ovary, corpus luteum (CL) and liver, in a search for positive controls. Bovine β-actin, 18S and 28S were examined in each sample as positive controls for RNA isolation and cDNA synthesis efficiency. TenμL of product were loaded into an agarose 2% gel in TBE buffer containing ethidium bromide, and were separated by horizontal electrophoresis. Gels were visualized with ultraviolet light and photographed using a digital camera. Gene expression in granulosa was demonstrated for PPARα, c-Fos, LHr, FSHr, aromatase, GH and controls (β-actin, 18S and 28S) but CRABPII gene did not express in granulosa cells, whole ovary, CL or liver under our experimental conditions. While lacking CRABPII expression remains intriguing, the expressed genes support a role of retinoid pathway within granulosa cells under both in vivo and in vitro conditions, because granulosa cells used in the present experiments were derived from follicles providing oocytes for IVM-IVF. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).
25COLD STORAGE OF TISSUES AS SOURCE FOR DONOR CELLS DOES NOT REDUCE THE
IN VITRO DEVELOPMENT OF BOVINE EMBRYOS FOLLOWING NUCLEAR TRANSFER
Authors: Arat S. , Bagis H. , Ergin F. , Sagirkaya H. , Mercan H.O. , Dinnyes A.
Published: February 2004

So far, most calves have been cloned from live adult cows or fresh fetal samples. There are few reports on using cells from a dead mammal for nuclear transfer (NT). This study was conducted to investigate whether different kind of viable cells could be obtained from tissues stored in cold for different duration and whether these cells could be used for NT. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal calf serum (FCS), 50μgmL−1 sodium pyruvate, 1% v:v penicillin-streptomycin (10.000UmL−1 penicillin G, 10.000μgmL−1 streptomycin), 10ngmL−1 EGF, 0.5μgmL−1 FSH, and 5μgmL−1LH. First cell line (CC) was established from articular cartilage of the leg of a slaughtered cow stored at 0°C in a cold storage room for 48h. Second cell line (MC) was established from leg muscle of a cow carcass stored at 0°C for 24h. Tissues from articular cartilage and muscle were cut into small pieces. Tissue explants were cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Bovine granulosa cells (GC) were isolated from ovarian follicles and used for NT as control cells. Prior to NT, all somatic cells were allowed to grow to confluency (G1/G0) in DMEM-F12 supplemented with 10% FBS. Cumulus cells were removed by vortexing with hyaluronidase at 18h after the start of maturation. Matured oocytes labeled with DNA fluorochrome Hoechst 33342 were enucleated under UV to ensure full removal of the chromatin. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused by a DC pulse of 133V/500μm for 25μs. After fusion, NT units were activated using a combination of calcium ionophore (5μM), cytochalasin D (2.5μgmL−1), and cycloheximide (10μgmL−1), and cultured for 7 days. Differences among groups were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. The results suggest that viable cells can be obtained from articular cartilage and muscle of a cow carcass stored at cold temperature for 24 and 48h and these cells have ability to generate NT blastocysts at rates similar to that of the controls. This study was supported by a grant from TUBITAK, Turkey (VHAG-1908-102V048). F Ergin is a volunteer young researcher.

Table 1
In vitro development of NT embroys from different cell lines
256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE
EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS
Authors: Wrenzycki C. , Brambrink T. , Herrmann D. , Carnwath J.W. , Niemann H.
Published: February 2004

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).
260PRELIMINARY RESULTS OF IN VITRO FERTILIZATION FOR THE ENDANGERED
MURCIANO-LEVANTINA BOVINE BREED
Authors: Barajas D.P. , Velasquez J.G. , Poto A. , Coy P.
Published: February 2004

The Murciano-levantina cattle from the Spanish Southeast belong to a bovine endangered breed under the special protection of FAO (WWL-DAD:3)*. Their characteristics of rusticity, longevity, docility and disease resistance make the recovery of the breed interesting. The objective of the present work was to determine the efficiency of in vitro bovine embryo production using oocytes collected from postmortem ovaries fertilized with frozen-thawed sperm obtained from either commercial or Murciano-levantina bulls. Cumulus-oocyte complexes were matured for 24h in TCM199 with 10% FBS, 2mM L-glutamine, 0.2mM sodium pyruvate, 0.3μgmL−1 LH and 5μgmL−1 FSH. Oocytes presumed matured were fertilized with Percoll-prepared sperm (750000/mL) in IVF-TALP, as described by Parrish et al., (1988 Biol. Reprod. 38, 1171–1180). Zygotes (at 18h) or two-cell-stage embryos (at 48h) were stained with Hoechst 33342 (10μgmL−1; 20min) and assessed by means of epifluorescence microscopy. The experiment was replicated on 4 occasions. The results of penetration (PEN-%), average number of spermatozoa inside the oocyte (S/O), monospermy (MON-%), putative embryos (zygotes) with two pronuclei (2 PN-%) and percentages of cleavage at 48 hours were recorded (Table 1). The results show that early embryos can be obtained with no differences in monospermy and cleavage rates regardless whether the sperm source was commercial or Murciano-levantina (P≥0.05). *Lista Mundial de Vigilancia para la Diversidad de los Animales Domésticos (1997). http://www.fao.org/docrep/V8300S/V8300S00.htm.

Table 1
In vitro fertilization results with one commercial and one Murciano-levantina bull
261DIFFERENT TRANSVAGINAL OVUM PICK-UP STRATEGIES TO OPTIMIZE THE OOCYTE
RETRIEVAL AND EMBRYO PRODUCTION OVER A FIXED PERIOD OF TIME
Authors: Chaubal S. , Molina J. , Olrichs C. , Ferré L. , Faber D. , Tian X. , Yang X.
Published: February 2004

The objective of this study was to compare the efficacy of different oocyte retrieval schemes over a period of 10 weeks. Fifteen multiparous Angus cows were randomly assigned (n=3/group) to the following groups: 1) OPU once/week (7-day interval), 2) OPU twice/week (3- and 4-day interval, alternately), 3) Dominant follicle removal (DFR)+OPU once/week. DFR followed by OPU 72h later, 4) DFR+FSH+OPU once/week. DFR followed 36h later by FSH (Folltropin, Bioniche, Belleville, Ontario, Canada) (120mg s.c. and 80mg i.m. administered simultaneously) followed by OPU 48h later, 5) FSH+OPU twice/week. FSH followed by OPU1 30h later and OPU2 96h after OPU1. The interval between OPU2 and next FSH was approximately 42h. The follicles were aspirated using an Aloka ultrasound scanner (Wallingford, CT, USA) and a 5-MHz probe. The COCs were selected based on morphology and matured in TCM-199, supplemented with 10% FCS, 0.01 unitsmL−1 bFSH, 0.01 unitsmL−1 bLH and antibiotics. Fertilization (Day 0) was carried out with TALP-FERT medium containing capacitation factors. Frozen semen from the same bull was used (1×106mL−1) throughout. After 18h the presumptive zygotes were cultured in SOF with 5% FCS (Holm P et al., 1999 Theriogenology 52, 683–700). The embryos were evaluated based on IETS guidelines (Grades 1 and 2 selected). The data were analyzed by chi-square test and ANOVA. In all parameters, the DFR followed by FSH and subsequent OPU once/week protocol gave the best results on a per-cow-per-week as well as total (3 cows over 10 weeks) basis. Though OPU was done only once/week, this group produced more total oocytes (303) than groups where OPU was done twice/week, either with FSH (286) or without FSH (229) and also produced more total embryos on Day 8 (71 blastocysts, 23.4% of oocytes cultured) than the latter two groups (64, 22.4% and 49, 21.4%, respectively). Among the nonstimulated groups, the OPU twice/week group had more total oocytes (236) than groups with OPU once/week, either without DFR (137) or with preceding DFR (160). However, a preceding DFR seemed to have a positive effect on oocyte quality as this group had a better embryo development rate (26.9%), producing more total embryos (42). In comparison, OPU twice/week produced total 49 embryos (21.4%) and OPU once/week produced 26 (19.4%). In conclusion, DFR coupled with single-shot FSH administration can be used effectively over a period of at least 10 consecutive weeks and can increase (P<0.05) the oocyte yield by two-fold and embryo production following IVF by two and half-fold, as compared to routine OPU-IVF done once a week.

Table 1
Per cow per week performance
263BINDING OF STALLION SPERM TO EQUINE AND BOVINE ZONAE
PELLUCIDAE: EFFECT OF MILK, MILK PROTEINS AND GLUCOSE
Authors: Coutinho da Silva M.A. , Seidel G.E. , Squires E.L. , Choi Y.H. , Carnevale E.M.
Published: February 2004

The ability of sperm to bind to zonae pellucidae (ZP) has been correlated with fertilizing capacity of sperm in several species. Limited numbers of equine oocytes are available to perform such assays. Therefore, use of heterologous ZP to perform gamete binding tests with stallion sperm would be useful. We have found that addition of 10% of skim milk-based extender with glucose [EZ-Mixin®, Animal Reproduction Systems, Chino, USA;; (EZ)] to TALP significantly increased the number of stallion sperm bound to bovine ZP. Objectives of the present experiments were to determine: (1) if stallion sperm bind in similar numbers to equine and bovine ZP, and (2) the effects of skim milk, milk proteins and glucose on sperm binding to ZP. Denuded bovine (immature) and equine (mature) oocytes were stored at 5°C in salt solution (1.5M MgCl2, 40mM HEPES, 0.1% PVP). In Experiment I, 4 ejaculates from 2 stallions were centrifuged at 300g for 6min, and sperm pellets were resuspended in 1mL of TALP or EZ. Sperm were stained with Hoechst 33342, centrifuged, and resuspended to 2×106 sperm mL−1. Oocytes were placed into droplets of 45μL of TALP (7 to 10 oocytes/trt/ejac). Extended sperm (5μL) were added to oocytes, resulting in 2times105 sperm mL−1, and the mixutre was incubated for 2h at 38.5°C. Oocytes then were pipetted in TALP to remove loosely attached sperm and observed with fluorescence microscopy;; mean numbers of sperm bound to bovine and equine ZP for TALP were 29±1.9 and 36±2.6 (P>0.1) and for EZ, 149±5 and 152±6.3 (P>0.1), respectively. More sperm bound to ZP with EZ than to ZP with TALP (P<0.001). Experiment II used 4 ejaculates from 4 stallions. After initial centrifugation, sperm were resuspended in 1mL of each of six extenders: TALP, EZ, TALP containing 89.5mM glucose (TG), TALP containing 163.5mM glucose (THG), TALP containing 2.4mgmL−1 of skim-milk powder (TSM), and INRA 96® (IMV Technologies, L’Aigle, France) that contains 27mgmL−1 of native phosphocaseinate. Hoechst 33342-stained sperm and bovine oocytes were processed as described for Experiment I. Treatments containing milk proteins resulted in more sperm binding (P<0.01) than those without milk proteins (Table 1). In conclusion, use of bovine oocytes led to similar results for equine and bovine oocytes;; therefore, bovine oocytes can be used for binding assays with stallion sperm. High concentrations of glucose increased numbers of sperm bound to ZP;; however, presence of milk or milk proteins was more effective in enhancing binding of sperm to ZP. INRA96 contains relatively low glucose (67mM) and one milk protein. Therefore, we hypothesize that native phosphocaseinate may cause increased sperm binding to ZP.

Table 1
Mean sperm bound per ZP±SEM (n=38–40/group)
264IN VITRO FERTILIZATION OF MACACA NEMESTRINA
OOCYTES WITH FRESH AND FROZEN-THAWED EPIDIDYMAL SPERM
Authors: Curnow E.C. , Hayes E.S.
Published: February 2004

In an effort to develop methods to maximize the use of reproductive tissues in our own tissue distribution program (WaNPRC TDP) and to serve as platforms for application of ART to endangered non-human primate (NHP) species, we have examined the effects of collection and cryostorage on epididymal sperm (ES) collected from Macaca nemestrina (Mn). Fresh ES was collected by needle aspiration from the cauda epididymis and prepared by centrifugation in mHTF (Irvine Scientific, Santa Ana, CA, USA)+BSA (3mgmL−1) at 700g for 10min. The resultant pellet was resuspended in fresh mHTF+BSA and held at RT until required. ES subjected to freeze-thaw was collected from the cauda epididymis of testes provided by the WaNPRC TDP by slicing and swim-out into mHTF+BSA. ES was frozen in mTTE medium+5% glycerol (Sankai T et al., 1994 J. Reprod. Fertil. 101, 273). Frozen ES straws were removed from liquid nitrogen and thawed in air at RT for 10min. Thawed ES was centrifuged through an 80% PureSperm gradient (Spectrum Technologies, Healdsburg, CA, USA) at 700g for 15min. The resultant pellet was retrieved and washed in mHTF+BSA at 600g for 5min. For capacitation sperm were incubated in pre-equilibrated HTF+BSA containing 1.0mM caffeine and 0.1mM dbcAMP (Sigma, St. Louis, MO, USA) for 5–10min in a humidified atmosphere of 6% CO2 in air. In vitro-matured Mn oocytes collected from unstimulated ovaries were exposed to fresh or frozen-thawed ES whereas in vivo-matured Mn oocytes collected from superovulated ovaries were exposed to fresh ES in 50-μL Fertilized oocytes were cultured for 24–48h and assessed for cleavage on Day 3 post-insemination. Data were analyzed by single-factor ANOVA or t-test where appropriate and are expressed as mean±SD. Fresh Mn ES (n=4) exhibited very low motility at collection (38±10%) but recovered motility following processing (58±20%). Frozen-thawed ES (n=4) also exhibited low motility post-thaw (53±17%) and recovered after processing (66±28%). The percentage of acrosome-intact sperm was not significantly different (P>0.05) for fresh ES (58.9±8.6%) compared to frozen-thawed ES (70.6±21.9%). Fresh and frozen-thawed ES fertilized in vitro-matured Mn oocytes at similar rates (fresh 68±10.3%, n=170 v. frozen-thawed 71.2±6.7%, n=90; P>0.05). Cleavage rates of fertilized IVM oocytes were not significantly different (fresh 79±7.2% v. frozen-thawed 79.8±5.0%; P>0.05). Fresh ES was also able to fertilize in vivo-matured Mn oocytes collected from superovulated ovaries (95.1±2.5%, n=98) and fertilized oocytes went on to cleave at a high rate (96.1±2.6%). These results suggest that fresh and frozen-thawed ES may be useful for applied ART in endangered species. This work was supported by NIH grant #RR00166 and the WaNPRC TDP.
266IN VITRO-DERIVED EMBRYO PRODUCTION WITH SEXED AND
UNSEXED SEMEN FROM DIFFERENT BULLS
Authors: Ferré L. , Ohlrichs C. , Faber D.
Published: February 2004

The production of pre-sex-selected calves by in vitro fertilization (IVF), using sexed semen, does show some benefits due to the small quantity of sperms needed for the process as compared to other reproductive technologies. The objective of this study was to determine differences among bulls and sperm concentrations in embryo development with sexed and unsexed semen. Follicles ranging from 2 to 6mm in diameter were aspirated from slaughterhouse ovaries. COC were selected and matured in groups of maximum of 30 in 1.8mL of TCM-199, supplemented with 10% fetal calf serum, 0.01UmL−1 bFSH, 0.01UmL−1 bLH and 10μLmL−1 penicillin-streptomycin for 24h at 38.5°C. Fertilization (Day 0) was carried out in micro-drops (50μL) with TALP-FERT medium containing PHE (3μgmL−1 penicillamine, 11μgmL−1 hypotaurine and 0.18μgmL−1 epinephrine), 10μLmL−1 non-essential amino acid and 2μgmL−1 heparin. Frozen/thawed sexed (female) and non-sexed sperms from five bulls were selected in a discontinuous percoll gradient. Sperm concentration was 1×106 for non-sexed semen and 1×106 or 2×106 for sexed semen. After 18–20h, presumptive zygotes were denuded and cultured in groups of 10 in 50-μL micro-drops of SOF citrate with 5% FCS (Holm P et al., 1999 Theriogenology 52, 683–700) under paraffin oil in a 5% O2, 5% CO2, 90% N2 atmosphere with high humidity. On Day 7, blastocysts (BL) were morphologically evaluated and recorded. Results are shown in Table 1. Data was compared by chi-square analysis. Sexed frozen bovine sperm can be used successfully in IVF systems. More research needs to be done to optimize and standardize bovine in vitro fertilization with sexed semen.

Table 1
Results of comparisons between bulls, sperm concentrations, cleavage and embryo development
272OVUM RECOVERY AND BLASTOCYST DEVELOPMENT FOLLOWING
INTRACYTOPLASMIC SPERM INJECTION IN CHIMPANZEES
Authors: Hayasaka I. , Yoshimoto N. , Mori Y. , Suzuki K. , Honda R. , Okamura H. , Ide Y. , Sakamoto W. , Nakashima T. , Nakagata N. , Torii R. , Yoshikawa Y.
Published: February 2004

In the present study, we report on oocyte collection, intracytoplasmic sperm injection and early embryogenesis in chimpanzees. Eight adult female chimpanzees, 11–27 years of age, received a single s.c. injection of 3.75mg GnRH (Leuplin, Takeda Co. Ltd., Osaka, Japan) 1 to 3 days after the beginning of menstruation. Daily i.m. injections of hMG (Humegon, Nippon Organon K.K., Tokyo, Japan) were initiated the following day. The dose of hMG was altered from 75 to 300IU according to serum estradiol levels. When at least one follicle of 17mm or more in diameter was observed, 10000IU of hCG (Pregnyl, Nippon Organon K.K.) were administered by i.m injection. Oocytes were recovered by ultrasound-guided transvaginal follicular aspiration 30.5 to 35.5h after hCG injection. Mature oocytes were denuded of cumulus cells by treatment with 0.1% hyaluronidase, and injected with a frozen-thawed or fresh spermatozoan using a Piezo-driven micromanipulator. Zygotes were cultured in Quinn’s Advantage Fertilization Medium (Cooper Surgical, Inc., Trumbull, CT, USA) with 10 serum protein substitute (SPS) at 37°C in a 5% CO2 atmosphere until the pronucleus stage. The medium was replaced by Quinn’s Advantage Cleavage Medium with 10 SPS from the pronuclear to 8-cell stage, and Quinn’s Advantage Blastcyst Medium with 10 SPS, thereafter. Mild ovarian hyperstimulation syndrome (OHSS) occurred in one female chimpanzee with estradiol levels of 7520pgmL−1. No oocytes were collected from 2 chimpanzees in which large follicles were observed. Thirty-five mature oocytes, one immature oocyte and 6 degenerate/fragmented oocytes were retrieved from 6 chimpanzees, including the one with OHSS. Among 35 mature oocytes injected with spermatozoa, 26 oocytes (74%) produced two pronuclei;; 23 zygotes (66%) cleaved to the 2-cell stage, 22 (63%) to the 4-cell stage, 14 (40%) to the 8-cell stage, and 9 (26%) to the morula stage. Seven zygotes (20%) developed to the blastocyst stage by 120h. There were no differences in fertilization rate or early embryogenesis between frozen and fresh spermatozoa. Results indicate that techniques used for human-assisted reproduction may be applicable to the chimpanzee to help preserve this endangered species.
280LAMBS BORN AFTER IN VITRO EMBRYO PRODUCTION FROM PREPUBERTAL
LAMB OOCYTES AND FROZEN-THAWED UNSORTED AND SEX-SORTED SPERMATOZOA
Authors: Morton K.M. , Catt S.L. , Hollinshead F.K. , Maxwell W.M.C. , Evans G.
Published: February 2004

Developments in sperm sexing technology have resulted in the birth of a number of offspring after IVF of oocytes from adult animals (Johnson LA, 2000 An. Reprod. Sci. 60–61, 93–107). The aim of this study was to combine sperm sexing technology with juvenile breeding. Merino lambs, 2–3 weeks (n=43) were hormone stimulated (Morton KM et al., 2003 Proc. Soc. Reprod. Fert., P18), and COCs were matured in TCM-199 (Sigma) with 10μgmL−1 p-FSH (Folltropin-V; Bioniche Animal Health Australasia), 10μgmL−1 pLH (Bioniche), and 20% sheep serum (v/v) in a humidified 6% CO2, 5% O2, 89% N2 atmosphere for 22h. Semen collected from Merino rams was diluted and frozen as pellets (Unsorted), or stained with H33342, separated into X and Y sperm using a SX MoFlo (Cytomation Inc., Fort Collins, CO, USA), and frozen as pellets (Sorted). Sperm were prepared for IVF by swim-up under 0.5mL of SOF with 2% sheep serum (v/v; SOF+) for 45min (Unsorted), or diluted in 0.5mL of Sydney IVF Sperm Buffer (Cook IVF, Brisbane, Australia) and centrifuged at 650g for 3min (Sorted). After IVM, oocytes were transferred to SOF+, and cultured with 0.5×106mL−1 (Unsorted) or 1.0×106mL−1 (Sorted) motile sperm for 18h. Presumptive zygotes were transferred to Sydney IVF cleavage and blastocyst medium (Cook IVF) for 3 and 5 days, respectively. Oocyte maturation and fertilization were assessed by orcein staining 18h post-insemination (hpi). Two Day-7 blastocysts were transferred to each recipient ewe (n=9; 3 per group) and pregnancies diagnosed by ultrasound on Day 57 of gestation. Data were analyzed by chi-square test. Oocyte maturation was 83.9% (73/87), and monospermic fertilization did not differ for Unsorted (22/32; 68.7%), X- (6/14; 42.8%), and Y-sperm groups (15/27; 55.6%). Polyspermic fertilization was 9.4% (3/32) and 7.4% (2/27) for the Unsorted and Y groups. Cleavage was reduced with X- and Y-sperm compared with Unsorted, but blastocyst formation (from cleaved oocytes) did not differ (Table 1). There were three (100%), zero (0%), and one (33.3%) pregnancies from Unsorted, X- and Y-embryos, respectively, all of which survived to birth, demonstrating that juvenile breeding can be successfully combined with sperm sexing.

Table 1
Cleavage and blastocyst formation after IVF with Unsorted, X- or Y-sperm. Values in parenthesis are percentages
285BIRTH OF PIGLETS AFTER NON-SURGICAL TRANSFER OF PORCINE EMBRYOS
CULTURED IN PZM-4 WITH ALTERED CONCENTRATIONS OF AMINO ACIDS
Authors: Suzuki C. , Yoshioka K. , Iwamura S.
Published: February 2004

We previously developed an in vitro production (IVP) system for porcine embryos and obtained piglets after surgical transfer of blastocysts cultured in Porcine Zygote Medium (PZM)-4. However, the developmental competence of pig IVP embryos to the blastocyst stage is still low and further improvement of IVC medium is needed. In the present study, we evaluated the effects of the addition of glutamine (Gln), hypotaurine (HT), taurine (Tau), BME-essential (EA) and MEM-nonessential (NA) amino acids solutions to PZM-4, and the replacement of polyvinyl alcohol (PVA) with BSA on embryo development to blastocysts. Moreover, the developmental competence of IVP blastocysts after nonsurgical embryo transfer (NS-ET), using a flexible catheter (FC) for deep intrauterine insemination, was investigated. Porcine COC from prepubertal gilts were matured and fertilized in vitro, using frozen-thawed ejaculated boar semen. Presumptive zygotes were cultured in PZM-4, as a basal culture medium, until Day 5 after IVF. Data from six replicates were analyzed by ANOVA. Addition of 0.25 to 4mM Gln to PZM-4 (containing 5mM HT) significantly increased the percentage of embryos that developed to blastocysts (15 to 31%), with addition of 2mM Gln significantly increasing the total cell numbers in blastocysts (43±17 cells) compared with no addition (3% and 20±4 cells, respectively). Addition of 1.25 to 10mM HT to HT-free PZM-4 supplemented with 2mM Gln (named PZM-5) significantly increased the percentage of embryos that developed to blastocysts (22 to 28%) compared with control (no HT;; 4%). In the culture with HT-free PZM-5, addition of 5mM Tau significantly increased blastocyst yield (17%) compared with control (4%). However, Tau addition in the presence of 5mM HT had no effect on development to the blastocyst stage. In combinations of EA and NA added to PZM-5, a single dose of EA significantly increased the percentage of embryos that developed to blastocysts (27%) compared with no dose (19%) or with a double dose of EA (20%), while a double dose of NA significantly increased the total cell numbers in blastocysts (43±16 cells) compared with no NA (37± 6 cells). Replacement of PVA with BSA in PZM-5 had no effect on embryo development to the blastocyst stage. Crossbred sows were used as recipients for NS-ET, and had their estrous cycle synchronized by a described previously method (Yoshioka et al., 2002 Biol. Reprod. 66, 112–119). Five days after hCG injection, a FC was introduced via the cervix into the uterine horn of recipients without sedation. Day-5 blastocysts cultured in PZM-5 were then transferred together with 5ml of TALP-Hepes (45 to 50 blastocysts/recipient). Of 6 recipients, one sow became pregnant and farrowed 7 piglets. Our results indicate that the addition of amino acids to PZM-4 can improve porcine embryo development to the blastocyst stage, and that blastocysts cultured in a chemically defined medium, PZM-5, can develop to full-term following NS-ET.
294EFFECTS OF LONG DAYLIGHT ON CAPRINE SCROTAL CIRCUMFERENCE AND SEMEN QUALITY
Authors: Mahmoud A. , Kouakou B. , Amoah E. , Gelaye S.
Published: February 2004

Seasonal variation of the reproductive activity of small ruminants in the southern USA is controlled by photoperiod. We tested the hypothesis that artificial manipulation of photoperiod has an effect on caprine testicular activity and semen quality. The objective of this study was thus to determine the effects of photoperiod on caprine scrotal circumference and semen parameters during the nonbreeding season. Ten Saanen and Alpine bucks, two to three years old, raised at the Small Ruminant Research and Extension Center of Fort Valley State University (latitude 32°37′ N, longitude 83°39′ W), were used in the study. Throughout the study, the bucks were fed a concentrate diet containing 16% crude protein, and 2.7 Mcal/Kg of digestible energy. Two groups of bucks randomly selected were exposed to long daylight (16L:8D; n=5) or ambient daylight (12L:12D, control;; n=5) for a period of three weeks during the month of March. Scrotal circumference measurements and semen collection were conducted on each buck twice a week for a total of five trials. Semen samples were collected using an artificial vagina and an electroejaculator. Samples were immediately transferred to the laboratory for further evaluation. Data were analyzed as a completely randomized design with repeated measures using SAS program (SAS, 1990). When means were significant by ANOVA (P<0.05), they were separated by least significant difference. Results are presented as the least square means±SEM. Long daylight did not affect semen volume (mL), % motility, sperm concentration (billions/mL), % viability, or % abnormality(P>0.05). However, semen motility, sperm concentration, and abnormality were influenced by treatment in trials 3, 1, and 5, respectively (P<0.05). The least square means±SEM values for these parameters were 35±15, 0.58±0.1, and 20.0±3.7 for long daylight-treated bucks v. 87±15, 0.13±0.1, and 7.8±3.7 for the control, respectively. Further, long daylight affected scrotal circumference (P<0.05). Scrotal circumferences were decreased in trials 1, 2, and 5 (P<0.05). Long daylight decreased scrotal circumference without affecting semen quality in this study.