bluuebunny/crossref_metadata_2025 huggingface dataset:
148 PRODUCTION OF PUPS BY OVARIAN TRANSFER IN THE SYRIAN HAMSTER
Authors: Suzuki O. , Koura M. , Takano K. , Noguchi Y. , Uchio-Yamada K. , Matsuda J.
Published: January 2008

Instead of gametes, ovaries could be used as alternative resources for cryopreservation. In this study, we attempted to produce pups by transplantation of fresh ovaries to foster mothers as a first step to establish a preservation method of hamster strains on the basis of the cryopreservation of ovaries in combination with ovarian transfers. We also checked the possibility of inter-strain transfers of ovaries, since immunological tolerance among hamster strains is suggested by the fact that most Syrian hamsters were derived from only a small number of animals. Fresh ovaries collected from 3-weekold females of HAW (white coat) and APA (albino) strains were transferred into ovarian bursae of 3-week-old recipient females with agouti-coat (Slc:Syrian) under anesthesia with xylazine and ketamine. Halves of ovaries of recipients were removed immediately before the transfers, i.e., both donor and recipient ovaries were present in each ovarian bursa. Successful transplantation of ovaries was checked by the coat color (non-agouti color) of offspring produced by test matings of recipients with Slc:Syrian males after the recipients became mature. Five and six recipients received HAW and APA ovaries, respectively. All 11 recipients tested became pregnant and delivered pups. According to the coat color of the pups, three of five recipients which received HAW ovaries and one of six recipients which received APA ovaries delivered pups derived from transplanted ovaries (two out of eight pups, one out of seven pups, and three out of 11 pups for HAW; one out of five pups for APA). Our results indicate that transplantation of fresh ovaries to foster mothers could be used for production of pups from grafted ovaries in the Syrian hamster. In particular, immunological tolerance for ovarian transfers among at least three strains suggests that recipient strains might not need to be the same as donor strains for practical ovarian transfers in the Syrian hamster. Our results would facilitate the development of a strain preservation system for the Syrian hamster based on ovarian cryopreservation.
This work was supported by a grant from the Ministry of Health, Labor, and Welfare of Japan.
160 PREGNANCY RATES AFTER USING THE CIDR-G DEVICE AND FIXED-TIMED ARTIFICIAL INSEMINATION IN WHITE-TAIL DEER
Authors: Gentry Jr G. T. , Saenz J. , Forbes W. , Whitehead K. , Sanders D. , Olcott B. , Chenevert J. , Bondioli K. R. , Godke R. A.
Published: January 2008

Two synchronization protocols for fixed-timed AI (FTAI) in White-tail deer were evaluated over a 2-year period. InYear 1, White-tail does (n = 38), with a mean body weight of 55 kg and mean age of 2.4 years, were stratified by weight, age, and last fawning date randomly across 2 estrous cycle synchronization treatment groups. Does received either a CIDR-G device for 14 days (CIDR 14) and underwent AI 60 h post-CIDR removal, or received a CIDR-G device for 7 days (CIDR 7) with 1 mg of estradiol benzoate (i.m.) at CIDR insertion (Day 0), 25 mg of prostaglandin F2α (PGF2α; Dinoprost) on Day 6 and 1 mg of estradiol benzoate (i.m.) on Day 7 with AI occurring 52 h post-CIDR removal. In Year 2, White-tail does (n = 36), with a mean body weight of 60 kg and mean age of 3.9 years, were stratified as described for Year 1 and allotted to 2 treatments. Does received a CIDR-G device for 14 days and were inseminated 60 h post-CIDR removal (Treatment A) or were synchronized in the same way as does inTreatment A but also received 200 IU (i.m.) of eCG (Sigma, St. Louis, MO) at CIDR removal (Treatment B). All does received 1 mg of Domosedan (i.v.) before initiating the AI procedure. In both years, electroejaculated semen was collected from a single buck and frozen for AI. In both years, clean-up bucks were introduced into the pen with does for natural cover at no less than 14 days following AI. A gestation length of 195 � 7 days was used to determine whether the fawn(s) resulted from AI or from natural mating. In Year 1, FTAI pregnancy rates were not different between the CIDR 14 (56%) and CIDR 7 (24%) treatment groups. Likewise, pregnancy rates were not different between CIDR 14 FTAI (56%) and clean-up bucks (75%). However, the FTAI pregnancy rate was lower (P < 0.001) for the CIDR 7 treatment group (24%) compared with the clean-up bucks (100%). Treatments did not affect fecundity rates; however, those females pregnant from FTAI had lower (P < 0.001) fecundity rates compared with those females pregnant from natural cover (1.2 v. 1.9 fawns/doe). In Year 2, treatment did not affect FTAI pregnancy rates across treatments (33% for Treatment A and 55% Treatment B); however, fewer (P = 0.024) does in Treatment A were pregnant following FTAI (33%) compared with those pregnant from clean-up bucks (75%). There was no difference in FTAI fecundity rates across treatment groups (1.3 v. 1.7 for Treatments A and B, respectively) or between FTAI treatments and natural cover (1.3, 1.7, and 1.5 for Treatments A, B, and clean-up bucks, respectively). These results indicate that the use of a 14-day CIDR synchronization protocol with FTAI occurring 60 h after CIDR removal yields acceptable pregnancy rates of White-tail does following transcervical AI.
165 EXPRESSION OF Oct-4 IN BUFFALO (BUBALUS BUBALIS) EMBRYOS GENERATED THROUGH IN VITRO FERTILIZATION OR PARTHENOGENETIC ACTIVATION
Authors: Kumar D. , Anand T. , Singh K. P. , Chauhan M. S. , Palta P. , Manik R. S.
Published: January 2008

Octamer-4 (Oct-4) is a member of Class V of the POU transcription factors family, which is involved in transcriptional regulation during early embryonic development and cell differentiation. It is expressed in the inner cell mass of blastocysts and in embryonic stem cells (ESCs), and its expression is widely used as a marker of pluripotency in ESCs in many species. This study was, therefore, carried out to examine the expression of Oct-4 in embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages generated through IVF or parthenogenetic activation. A total of 100 embryos were used in the study, 10 for each embryonic stage from both methods of embryo production. Immature oocytes obtained from slaughterhouse buffalo (Bubalus bubalis) ovaries were subjected to IVM in TCM-199 + 10% FBS + 5 µg mL–1 pFSH + 0.81 mm sodium pyruvate for 24 h in a CO2 incubator (5% O2, 5% CO2, 90–95% relative humidity) at 38.5�C. IVF was carried out immediately after IVM; the cleaved embryos were cultured for 8 days in CR2 medium containing 0.6% BSA and 10% FBS for production of embryos at different stages. For production of embryos through parthenogenesis, after 24 h of IVM, oocytes were denuded of cumulus cells by incubation in 0.2% hyaluronidase in Dulbecco's phosphate-buffered saline for 2 min. The denuded oocytes with a prominent polar body were parthenogenetically activated by exposure to 7% ethanol for 7 min, followed by incubation with 2 mm 6-dimethyl aminopurine in CR2 medium for 3.5 h in a CO2 incubator (5% O2, 5% CO2, 90–95% relative humidity) at 38.5�C, and then subjected to IVC as described above. A two-step RT-PCR was carried out using Cells-to-cDNA Kit-II (Ambion, Austin, TX, USA), using bovine primers 52-GTT CTC TTT GGA AAG GTG TTC-3' and 5'-ACA CTC GGA CCA CGT CTT TC-3' for the amplification of Oct-4. For this, the embryos were washed with PBS, transferred to 30 µL of cold cell lysis buffer and incubated in a thermal cycler at 75�C for 10 min. The cell lysate was treated with DNase-I at 37�C for 30 min to degrade genomic DNA and then heated at 75�C for 5 min to inactivate the DNase-I. The cell lysate (10 µL) was used for making cDNA using random primer. The PCR cycle included heating to 94�C for 2 min, followed by 33 cycles of 94�C for 30 s, 57�C for 30 s, and 72�C for 45 s. A final extension at 72�C for 10 min was carried out to complete the amplification of the Oct-4 gene. Transcripts of Oct-4 were detected at all of the embryonic stages, from the 2-cell through the hatched blastocyst stage in both IVF and parthenogenetically generated embryos. These results indicate that Oct-4, which is believed to be a reliable marker for pluripotency of ESCs in a number of species, is expressed in early cleavage-stage buffalo embryos and continues to be expressed in preimplantation-stage blastocysts.
175 TRANSCRIPTOME ANALYSIS OF BOVINE DAY 150 FETAL LIVER AND COTYLEDON REVEALS GENES INVOLVED IN FETAL GROWTH
Authors: Krebs S. , Bauersachs S. , Reichenbach H. D. , Weppert M. , Hiendleder S. , Blum H. , Wolf E.
Published: January 2008

Major problems in breeding of modern dairy cows include increasing rates of stillbirth and dystocia, associated with elevated costs for veterinary intervention (caesarean section) and loss of production animals (cows and calves). Rates of stillbirth as high as 12% have been reported, primarily caused by fetal overgrowth and increased birth weight. In order to discover molecular markers for selection against stillbirth, we developed a model for the inheritance of fetal growth traits. A Fleckvieh bull segregating for paternal stillbirth was used for insemination of 36 cows that were slaughtered at Day 150 of pregnancy in order to recover and phenotype the fetuses. The mode of inheritance indicated involvement of imprinting. Mapping results suggested an imprinted region on chromosome 9 as candidate for the stillbirth QTL. Due to the complexity of the trait, we opted for a holistic approach that is not restricted to the QTL candidate region but allows identification of genes and networks that influence fetal growth. Transcriptome profiles of liver and cotyledon samples from the fetuses with the highest (n = 10) and the lowest total weight n = 10) were analyzed using Affymetrix Bovine GeneChips (Affymetrix, Inc., Santa Clara, CA, USA). Analysis with the program SAM showed 41 up- and 4 down-regulated genes in liver samples of the heavy-weight group. Most of these genes are involved in immune response. Interestingly, many of these genes are reported to be regulated by vitamin D. Furthermore, vitamin D is closely connected to the IGF1 system and thus the most important fetal growth regulation circuit. Seasonal effects on vitamin D levels could mostly be excluded by the experimental design and did not correlate with growth traits. Most likely, the mRNA levels of our candidate genes were influenced by alterations in the IGF1/vitamin D circuit and did not cause the observed weight differences. The imprinted candidate genes showed no correlation with fetal weight. Gene set enrichment analysis indicated enhanced metabolic activity in the liver of heavy-weight fetuses. Genes from the QTL region showed a clear enrichment in correlation with fetal weight, confirming their involvement in fetal growth. The gene with the best correlation, GHITM (growth hormone inducible transmembrane protein), could give an explanation for the enhanced metabolic activity, as it is reported to function as a metabolic regulator. Simultaneous analysis of the data sets for liver and placenta in a linear model (R-package LIMMA; Smyth 2004 Stat. Applic. Genet. Mol. Biol. 3, art. 3) yielded essentially the same differentially expressed genes for liver and a higher number of differentially expressed genes for placenta (89 up- and 114 down-regulated), with little overlap between the two tissues.
This work was supported by Grant BMBF FUGATO-Fertilink.
180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS
Authors: McHughes C. E. , Springer G. K. , Spate L. D. , Li R. , Woods R. J. , Green M. P. , Korte S. W. , Murphy C. N. , Green J. A. , Prather R. S.
Published: January 2008

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development.

Table 1. Differentially represented transcripts between developmental stages
191 ESTROGEN REGULATES THE LOCALIZATION AND EXPRESSION OF CALBINDIN-D9k IN THE PITUITARY GLAND OF IMMATURE MALE RATS VIA THE ESTROGEN RECEPTOR ALPHA PATHWAY
Authors: Tinnanooru P. , Jeung E.-B.
Published: January 2008

Estrogen (E2; estradiol) plays a key role in the regulation of many pituitary hormones. It exerts its effects by binding to the intracellular estrogen receptor (ER), which then functions as a transcription factor. Although E2 has been shown to regulate calbindin-D9k (CaBP-9k) in the female reproductive system of rodents, the effects of E2 on the regulation of CaBP-9k in male rats remains to be elucidated. For investigation of E2-induced regulation of the pituitary CaBP-9k gene, immature male rats were injected with E2 daily for three consecutive days with a dose of 40 µg kg–1 body weight (BW). The expression levels of CaBP-9k mRNA and protein were analyzed by RT-PCR and Western blot analysis, respectively, in the absence and presence of ICI 182 780 (ICI), an E2 antagonist. In addition, the tissue localization of CaBP-9k was determined by immunohistochemistry. CaBP-9k was localized in the cytoplasm of a specific cell type (acidophils) in the anterior lobe of the pituitary gland and highly expressed in the intermediate lobe. Exposure to E2 increased the number of cells that stained positive for CaBP-9k. For determination of which ER subtype is involved in CaBP-9k regulation in the pituitary, the immature rats were treated with 1 mg kg–1 BW propyl pyrazole triol (PPT, an ER-α-selective ligand) or diarylpropionitrile (DPN, an ER-β-selective ligand) for three days. The data were analyzed by the non-parametric procedure of the Kruskal-Wallis test, followed by Dunnett's test for two-pair comparisons. Pituitary CaBP-9k expression was mainly mediated by PPT in immature male rats, whereas no significant alteration of pituitary CaBP-9k gene expression was observed after DPN treatment. In addition, the estrogenicity of PPT in the induction of CaBP-9k expression was completely blocked by an estrogen antagonist, ICI 182 780, indicating that pituitary CaBP-9k expression is solely induced by ER-α. Taken together, these results suggest that pituitary CaBP-9k is induced by estrogen in male rats and its expression is predominantly regulated by ER-α, but not ER-β.
194 EXPRESSION OF microRNA-15a, 16, AND 21 AND THEIR POSSIBLE ROLES IN MOUSE EMBRYOS DEVELOPING IN VITRO
Authors: Zhang D. X. , Shen X. H. , Cui X. S. , Kim N.-H.
Published: January 2008

MicroRNAs (miRNAs) are small (~22 nucleotides) non-coding RNA molecules that can regulate gene expression by base-pairing with fully or partially sequence-complementary target mRNAs. Hundreds of miRNAs have been identified in various multicellular organisms and many miRNAs are evolutionarily conserved. While miRNAs play an important role in animal development, little is known about their biological function during early mammalian development. In order to obtain insight into the role of miRNAs in early embryogenesis, we first determined the expression levels of three apoptosis-related miRNAs, miR-15a, -16, and -21 in mouse preimplantation embryos using TaqMan� MicroRNA Assays. Five embryos of each developmental stage were snap-frozen and amplified by stem-loop RT primer and TaqMan Universal PCR Master Mix (Applied Biosystems Inc., Foster City, CA, USA). The miRNA concentrations (10–X) in embryo samples were calculated by standard curve from synthetic lin-4 miRNA and the absolute copy number per embryo was obtained based on the formula of 6.02 � 10(8–X). All three miRNAs had low expression levels from the zygote to the 8-cell stage and were up-regulated thereafter. In general, among the three miRNAs, miR-15a exhibited the lowest expression in preimplantation embryos, while miR-16 exhibited the highest. Because of the low levels of miRNA-15a, we determined developmental ability and apoptosis of embryos following microinjection of miRNA-15a. The microinjection of miR-15a into zygotes did not affect embryo development up to the blastocyst stage (miR-15a, 90 � 4.5% v. buffer 94.6 � 5.8%); however, it did induce a significant degree of apoptosis (P < 0.05; Tukey's multiple range test). Furthermore, the expression levels of miR-15a and -16 were increased in microinjected blastocysts compared to the control group (copy number per blastocyst, miR-15a, 6991 � 1223 v. 3098 � 592; miR-16, 196216 � 958 v. 133514 � 6059). Real-time RT-PCR data showed that the gene expression levels of the housekeeping gene GAPDH, the anti-apoptotic gene Bcl-xL, and the miRNA pathway-related genes GW182 and Dicer remained unchanged in miR-15a-injected blastocysts compared to the control group. In contrast, the expression of the stem cell-specific transcriptional factor Oct-4 (fold change, 1.451 � 0.12), the pro-apoptotic gene Bax (1.418 � 0.12), and Caspase 3 (1.314 � 0.19) were significantly increased in microinjected blastocysts. In addition, treatment of 2-cell embryos with 600 µm H2O2 induced apoptosis and increased the expression level of miR-16 at the blastocyst stage (P < 0.05). Taken together, the changes in the expression levels of miR-15a, -16, and -21 in various embryonic developmental stages indicate a possible role for them in early embryogenesis. Furthermore, the high expression levels of miR-15a and miR-16 seem to be linked to apoptosis in blastocyst-stage embryos; this may be due to an increase in the expression of pro-apoptotic genes.
196 COMPARING OOCYTE LIPID CONTENT WITH CIRCULATING CHOLESTEROL AND TRIGLYCERIDE LEVELS OF BOS TAURUS AND BOS INDICUS DONOR COWS
Authors: Ballard C. B. , Looney C. R. , Lindsey B. R. , Pryor J. H. , Lynn J. W. , Bondioli K. R. , Godke R. A.
Published: January 2008

Intracellular lipid content has been found to be higher in Bos indicus oocytes compared with Bos taurus oocytes (Ballard et al. 2007 Reprod. Fertil. Dev. 19, 170). The objective of this study was to further determine whether there was a correlation between oocyte lipid content and circulating cholesterol (CHL) and triglyceride (TRG) levels of mature purebred Brahman (BR; n = 11) and English breed (EN; n = 13) beef cows. All donor cows were maintained on the same ration 30 days prior to and during the oocyte collection interval. Oocytes were collected from donors at 30-day intervals (3 replicates/female) by transvaginal ultrasound-guided aspirations. Blood samples were collected to evaluate total circulating CHL and TRG levels just prior to CIDR insertion and again prior to oocyte collection. Follicle-stimulating hormone (Folltropin-V; Bioniche, Belleville, Ontario, Canada) was given twice daily for 3 days in decreasing doses beginning on Day 4 after CIDR insertion, with oocyte recovery on Day 8. The mean number of follicles aspirated/donor and oocytes recovered/donor were 12 and 11 for the BR and 18 and 7 for the EN donors, respectively. Oocytes were matured in TCM-199 with 10% fetal bovine serum + bovine LH and bovine FSH (0.01 U mL–1). After 20 h, mature oocytes with a visible polar body (BR = 242/391 or 61%, and EN = 170/258 or 65%) were denuded by vortexing in Tyrodes HEPES medium + BSA (0.03 mg mL–1). Visual color scores (1 = light, 2 = medium, 3 = dark) were then assigned to the oocytes. Buoyancy of mature oocytes was then measured by using a sucrose step-density gradient column prepared with sucrose and Dulbecco's PBS. Results from the sucrose gradients ranged from 23% (high lipids) to 35% (lower lipids). The oocytes were stained with Nile Red and evaluated by fluorescent microscopy. Oocyte images were evaluated by using Scion Image software to calculate the Nile Red units (NRU; higher NRU = higher lipid content). Serum was analyzed (Texas Veterinary Medical Diagnostic Laboratory) for CHL and TRG concentrations. Data were analyzed by one-way ANOVA. There was no difference between visual color scores for oocytes from BR and EN donors. However, BR oocytes had significantly (P < 0.05) lower buoyant density and significantly higher mean NRU scores (32% and 173) compared with oocytes harvested from EN donors (33% and 152), respectively. Serum CHL and TRG levels were both significantly higher (P < 0.05) in the BR donors (150 and 33.2) compared with the EN donors (110 and 25.5), respectively. These results suggest that serum cholesterol and triglyceride levels may be predictive of donor oocyte lipid content.
20 ADMINISTRATION OF FOLLICLE-STIMULATING HORMONE AT CIDR REMOVAL DOES NOT AFFECT THE PREGNANCY RATE IN A CIDR-BASED, COSYNCH PROTOCOL IN LACTATING BEEF COWS
Authors: Small J. , Dias F. , Pfeifer L. , Lightfoot K. , Colazo M. , Kastelic J. , Mapletoft R.
Published: January 2008

In previous studies, giving eCG at CIDR removal significantly increased the pregnancy rate after timed-AI in beef cows. However, eCG is not universally available. Therefore, we tested the hypothesis that giving pFSH at CIDR removal might improve the pregnancy rate in a CIDR-based, Cosynch protocol in postpartum, suckled Bos taurus beef cows; a secondary objective was to compare pregnancy rates when GnRH v. pLH was used to synchronize wave emergence and ovulation. This work was conducted as two experiments (separate locations). All cows were given a CIDR (containing 1.9 g progesterone; Pfizer Animal Health, Montreal, QC, Canada) on Day 0 (without regard to stage of estrous cycle or cyclicity). On Day 7, CIDRs were removed, all cows were concurrently given 25 mg PGF (Lutalyse; Pfizer Animal Health, Groton, CT, USA), half were given 20 mg pFSH (Folltropin-V; Bioniche Animal Health, Belleville, ON, Canada), and all cows were timed-AI 54 h later (Day 9). In Experiment 1, 240 cows [94 � 10.8 days postpartum; body condition score (BCS: 1 = emaciated, 9 = obese; mean � SD): 5.8 � 0.4] were used; at CIDR insertion and AI, cows were allocated to receive either 100 µg GnRH (n = 160; Cystorelin, Merial Canada Inc., Victoriaville, QC, Canada) or 12.5 mg pLH (n = 80; Lutropin-V, Bioniche Animal Health). In Experiment 2, 109 cows (59.2 � 19.5 days postpartum; BCS: 5.6 � 1.1) were used; all received 100 µg GnRH (Cystorelin) at CIDR insertion and AI. In Experiment 1, three cows that lost their CIDR were excluded; pregnancy rates after timed-AI (logistic regression, backward selection: parity, pFSH, synchronizing treatment, and their interactions) were not different between cows given pFSH v. control cows (64.7 v. 65.2%; P > 0.80), nor between cows given GnRH v. pLH (62.7 v. 69.6%; P = 0.91). However, there was an interaction (P < 0.04) between parity and the synchronizing treatment; in primiparous cows, pregnancy rates were significantly lower in those given GnRH v. pLH (59.3 v. 83.3%). In Experiment 2, pregnancy rates after timed-AI (logistic regression, backward selection: parity, pFSH, and their interaction) were not different between cows given pFSH v. control cows (38.2 v. 42.6%; P > 0.6). In conclusion, our hypothesis was not supported; giving pFSH at CIDR removal did not significantly improve the pregnancy rate in a CIDR-based, Cosynch protocol in postpartum, suckled Bos taurus beef cows.
200 EFFECT OF EXOGENOUS PROGESTERONE DURING IN VITRO CULTURE ON EARLY EMBRYO DEVELOPMENT IN CATTLE
Authors: Clemente M. , Lonergan P. , Borque C. , de La Fuente J. , Rizos D.
Published: January 2008

Preimplatation embryos grown in vitro are sensitive to their environment, and the conditions of culture can affect developmental potential. Progesterone (P4) is the key hormone responsible for maintenance of pregnancy in mammals, and circulating levels in the early postconception period have been associated with pregnancy success. It is not clear whether P4 acts directly or indirectly on the embryo to alter gene expression and development. The aim of this study was to assess the effect of varying levels of exogenous P4 on the development of bovine zygotes to the blastocyst stage in vitro. A preliminary study was conducted to analyze the media used for culture (stock of P4, SOF, SOF + 1 × 10–7 M P4) on Days 1 (day of culture), 4, and 7 for P4 concentration in 25-μL droplets overlain with mineral oil or 500 μL in wells with or without mineral oil. P4 was measured using an ELISA kit, prepared for human serum or plasma (DE1561 Dimeditec Diagnostics GmbH, Kiel, Germany). Inter- and intra-assay coefficients of variation were 6.63 and 6.42%, respectively, and recovery was 95%. P4 concentration on Day 1 in all media was the expected (40 ng mL–1). However, on Days 4 and 7 in media under mineral oil, the level of P4 was nearly zero (0.1 to 1.6 ng mL–1) compared with the media without mineral oil, which remained unchanged (39 to 40 ng mL–1) through the 7 days of culture. Zygotes (n = 1467) were produced in 8 replicates by in vitro oocyte maturation and fertilization, and were cultured in groups of 40 to 50 in wells of 500 μL without mineral oil in (1) SOF supplemented with 5% fetal calf serum (control–), (2) SOF with ethanol (control+), (3) SOF with P4 0.1 × 10–7 M, (4) SOF with P4 1 × 10–7 M, and (5) SOF with P4 10 × 10–7 M at 39°C, 5% CO2 and 5% O2, with maximum humidity. No significant difference was found between groups in cleavage rate or blastocyst yield on Days 6, 7, and 8 (Table 1). These results indicate that the addition of P4 to the in vitro culture medium (SOF) did not enhance the development of bovine embryos to the blastocyst stage. However, further studies on the quality of these embryos in terms of gene expression are in preparation.

Table 1. Effect of P4 on bovine in vitro early embryo development
204 AMINO-REACTIVE CROSSLINKER BIS(SULFOSUCCINIMIDYL)SUBERATE INDUCES ZONA PELLUCIDA HARDENING AND REDUCES PENETRATION IN PIG IN VITRO FERTILIZATION
Authors: Grullón L. , Cánovas S. , Matás C. , Romar R. , Coy P.
Published: January 2008

Zona pellucida (ZP) hardening is considered to be the final step in the prevention of polyspermy during fertilization in mammals. However, unfertilized pig oviductal oocytes show a resistance of hours or days to pronase digestion (Broermann et al. 1989 J. Anim. Sci. 67, 1324–1329). We previously demonstrated that the amino-reactive crosslinker DSP is effective in inducing ZP hardening and improves the monospermy levels at pig IVF (Coy et al. 2007, Reprod. Dom. Anim., in press). In this study, a different chemical crosslinker, BS3 [bis(sulfosuccinimidyl) suberate], which also forms stable amide bonds among proteins, was used to evaluate its effect on ZP digestion time, penetration, male pronuclear formation and monospermy percentages, and the mean number of sperm per oocyte. In experiment 1, porcine in vitro-matured oocytes (n = 300) were incubated for 30 min at 0, 0.06, 0.30, or 0.60 mg mL–1 of BS3 in TALP medium and assessed for ZP digestion time in 0.5% pronase solution. The results (analyzed by ANOVA in all the experiments) showed a significant (P ≤ 0.01) dose-dependent increase in ZP hardening, from 69.0 s in the control to 426.3, 2028.3, and 2979.2 s, respectively, for the different BS3 concentrations. In experiment 2, oocytes (n = 473) were fertilized in vitro after no treatment or treatment with BS3 at 0.06, 0.30, and 0.60 mg mL–1. Fresh ejaculated spermatozoa were selected by Percoll� gradient 45:90. Oocytes were inseminated with 105 sperm mL–1, which resulted in high penetration and polyspermy percentages in the control group (83.1 and 89.9%, respectively). However, for the BS3-treated oocytes, significant differences compared with the control group (P ≤ 0.001) were observed in all 3 groups, showing penetration percentages of 22.2, 18.1, and 21.5%, respectively, and monospermy percentages of 100, 88.2, and 95.0%, respectively. The mean numbers of sperm per oocyte were 1.0, 1.1, and 1.05 for the BS3 groups, which were significantly different from 5.0 for the control group. In conclusion, BS3 can be used to induce ZP hardening in the pig and regulate polyspermy in IVF systems, although additional experiments are necessary to find the optimal concentration to improve the penetration percentages with high levels of monospermy.
Granted by MEC and FEDER (AGL2006-03495).
205 CAPACITATION OF STALLION SPERMATOZOA EVALUATED BY FERTILIZATION OF BOVINE OOCYTES
Authors: Hudson M. R. , Seidel Jr G. E. , Squires E. L. , Spizzirri B. E. , Walker D. J. , Graham J. K.
Published: January 2008

In vitro fertilization in the horse does not work reliably. Several methods of capacitating sperm in other species fail in the horse. The goal of this experiment was to develop a method to capacitate equine spermatozoa using calcium ionophore A23187 or phosphatidylcholine 12 (PC12). We also studied effects of maturing bovine oocytes for 24 or 28 h on fertilizability by capacitated equine sperm, hypothesizing that longer maturation would yield oocytes more easily fertilized by equine spermatozoa. Two sets of bovine oocytes were aspirated from 3 to 8 mm follicles of abattoir ovaries 4 h apart, but fertilized at the same time. On the day of fertilization, semen from 1 of 3 stallions was collected, evaluated, and centrifuged through 33% Percoll to remove seminal plasma. The resultant pellet was extended to 5 × 107 cells mL–1 in M199 containing 0.6% BSA, 2 mm caffeine, and 5 mm CaCl2. Sperm were treated with A23187 (1 or 3 μm) or PC12 (40 or 70 μm) or both A23187 and PC12 (1 μm/40 μm) in 500- μL aliquots. Sperm were incubated at 39°C for 10 min (for A23187 and combination treatments) or 15 min (for PC12 treatments), and then diluted 1:20 for fertilization. Oocytes from each maturation time were fertilized using the same semen preparation for each treatment. Oocytes and sperm were incubated together for 18 h in FCDM in 5% CO2 at 39°C (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596). Presumptive zygotes were cultured for 30 h in CDM-1, vortexed to remove cumulus cells, and evaluated for cleavage. Oocytes were also co-incubated with killed sperm to determine the level of parthenogenesis. Cleaved embryos were stained with orcein to ensure that each cell had a nucleus. Number of cell divisions were recorded as 0 for a 1-cell, 1 for a 2-cell, 1.5 for a 3-cell, etc. More oocytes cleaved after 28 h (18%) than 24 h (14%) maturation (P < 0.01). Sperm of Stallion 1 resulted in higher overall cleavage (24%) than Stallions 2 or 3 (11 and 12%; P < 0.01). Highest cleavage was seen with 28 h maturation and 70 μm PC12 and 3 μm A23187 (27 and 24%, respectively). The most cell divisions were seen with 28 h maturation and 70 μm PC12 (0.48); 28 of the 49 cleaved in this treatment reached ≥4-cell stage. In conclusion, both A23187 and PC12 were able to capacitate equine sperm in a dose-dependent manner as determined from cleavage of bovine oocytes matured for 28 h; maturation for the conventional 24 h was an inferior model for this purpose.

Table 1. Mean responses of bovine oocytes fertilized by equine sperm
215 APPLICATION OFA MICROFLUIDIC SPERM SORTER TO THE IN VITRO FERTILIZATION OF PORCINE OOCYTES
Authors: Sano H. , Matsuura K. , Naruse K. , Funahashi H.
Published: January 2008

In porcine IVF, polyspermy is a persistent obstacle to the efficient production of normal embryos in vitro. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) has been developed to isolate motile human spermatozoa (from diluted semen by 2 laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. In addition, we have succeeded in reducing the incidence of polyspermic penetration by a transient coincubation of oocytes with spermatozoa in the presence of caffeine, followed by culture of the oocytes in the absence of caffeine (2004 Reproduction 128, 789–800). If porcine oocytes could be cocultured with motile spermatozoa in the chamber of the MFSS, the gradual accumulation of motile spermatozoa might further improve the efficiency of production of monospermic fertilized porcine embryos. The current study was undertaken to apply the MFSS technology to porcine IVF. The sperm-rich fraction from 3 Berkshire boars was diluted at 1 � 108 cells mL–1 with modified Modena containing 20% seminal fluid, cooled to 15�C for 4 h, and kept at the same temperature until use within 2 days. Stored, diluted semen with greater than 80% viability was flowed with Tyrode lactate-HEPES-polyvinyl alcohol medium for 5 or 10 min at room temperature. The concentration and viability of spermatozoa mixed in the flowed medium were determined. In the next experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine for 5 min at room temperature. Before flowing, porcine IVM oocytes were put into the chamber of the MFSS, where motile spermatozoa will accumulate with modified TCM-199 containing 5 mm caffeine. After flowing for 5 min, those oocytes were transferred into a 500-µL droplet of caffeine-free modified TCM-199, and culture was continued for 8 h. Statistical analyses were carried out by ANOVA and Fisher's PLSD post hoc test. After flowing for 5 min, the concentration of spermatozoa recollected from the chamber was 5.7 � 105 cells mL–1. The viability of recollected spermatozoa was significantly higher than the original flowed semen. However, when spermatozoa were flowed for 10 min, the viability of recollected spermatozoa was not different from the original flowed semen, whereas the concentration of recollected spermatozoa was 9.7 � 105 cells mL–1. When IVM oocytes were cocultured with spermatozoa gradually accumulated in the chamber of MFSS for 5 min, 35% (22 to 48%) of oocytes were penetrated, and 95% (90 to 100%) of the penetrated oocytes were monospermic. These observations demonstrate that the MFSS can separate penetrable boar spermatozoa with a high viability and sufficient concentration for IVF. Furthermore, the current data suggest the possibility of improving the efficiency of monospermic fertilization of porcine IVM oocytes by a transient coculture with MFSS.
218 DEVELOPMENT OF EVENLY AND UNEVENLY CLEAVED TWO-CELL PORCINE EMBRYOS
Authors: Thanh D. N. Q. , Kikuchi K. , Somfai T. , Ozawa M. , Nakai M. , Maedomari N. , Nguyen B. X. , Nagai T.
Published: January 2008

Mammalian eggs are so microlecithal that the embryos would be expected to divide in unison and that each division would lead to 2 equal blastomeres, which are believed to have a greater competence for further development than zygotes with unequal cleavage. However, some studies have shown that uneven blastomere size commonly occurs from the very first division in mammals, and it seems to be concerned with the generation of the first cell lineages of the blastocyst cells: trophectoderm and the inner cell mass (Gueth-Hallonet and Maro 1992 Trends Genet. 8, 274–279). In our study, we produced porcine embryos in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1031–1041), and newly formed 2-cell embryos were collected. Based on the timing of the first cleavage (30 or 36 h after insemination), the cleavage pattern (E: equal; U: unequal) and the presence or absence of a second cleavage (+ or –) within the first 2 days of IVC was classified into groups: 30E(–), 30E(+), 30U(–), 30U(+), 36E(–), 36E(+), 36U(–), or 36U(+). There was no difference between the 30E and 30U groups in proportions of the 2-cell stage, which had a nucleus in both blastomeres (99.0 � 0.8% and 91.4 � 3.6%, respectively) or between the 36E and 36U groups (98.2 � 1.1% and 88.0 � 7.2%, respectively). Comparison of further development between the 30E and 30U groups showed that there was no difference in blastocyst rates (70.7 � 5.7% and 61.7 � 7.8%, respectively) and total cell numbers (39.1 � 2.1 and 31.7 � 2.3, respectively). Although the blastocyst rate in the 36E group (37.3 � 6.7%) was significantly higher (P < 0.05) than that of the 36U group (12.0 � 5.1%), the total cell number was not different (26.3 � 5.5 and 25.3 � 5.2, respectively). The timing of the first division, however, had a great influence on further development of the embryos; the 30-h cleaved embryos had a greater rate of blastocyst development (68.2 � 6.3%) than did the 36-h embryos (28.2 � 4.8%, P < 0.01 by ANOVA). The cell numbers of blastocysts derived from 30-h cleaved embryos (37.2 � 2.6) were significantly higher than those of the 36-h embryos (26.2 � 2.3, P < 0.01) as well. Two-cell embryos that were newly formed at 30 h and underwent the next cleavage within the first 2 days of IVC (30 + group) had a higher blastocyst rate (74.8 � 7.0%) and greater cell numbers (40.6 � 2.6) than those not showing a second division during this period (30– group; 46.8 � 5.0% and 19.9 � 2.2, respectively). In contrast, for embryos showing the first cleavage at 36 h of insemination, the presence of the next cleavage within 2 days after the first cleavage did not have any effect on embryonic development. These results suggest that the developmental ability of porcine embryos was influenced by the timing and shape of the first cleavage and by the subsequent occurrence of the second cleavage.
223 ENZYMATIC ACTIVITY LEVEL OF DIFFERENT GLYCOSIDASES IN INTACT AND ACROSOME-REACTED PORCINE SPERM
Authors: De Ondiz A. , Avilés M. , Garca-Vázquez F. A. , Carrasco C. , Grullón L. , Ruiz S.
Published: January 2008

The sperm–egg interactions are species-specific forms of cell recognition and the binding event which are a necessary prerequisite for fertilization (Park et al. 2002 Anim. Reprod. Sci. 72, 83–94). Glycosidase enzymes that remove carbohydrates could play an important role in the reproductive tract, modulating decisive physiological events mediated by carbohydrates, which play a key role in sperm–oocyte recognition. The aim of this study was to analyze the presence of the glycosidases α-D-mannosidase, α-L-fucosidase, β-D-glucosaminidase, and β-D-galactosaminidase in intact and acrosome-reacted sperm from fertile matured boars. Sperm were washed three times in PBS by centrifugation at 800g for 10 min. The pelleted sperm were resuspended in the same buffer to obtain a final concentration of 250 � 106 spermatozoa mL–1. The acrosome reaction was induced by incubation of the sperm with 10 µm of calcium ionophore A23187 at 37�C for 30 min. Different enzymes were detected by incubating 8 µL (for α-Dmannosidase) or 80 αL (for the rest of the enzymes) of sperm sample with the corresponding substrate conjugated to 4-methylumbelliferil for 2 h at 37�C in PBS at pH 7.3. Fluorescences were read on a Fluostar Galaxy fluorimeter (BMG LabTech GmbH, Offenburg, Germany), using wavelengths of 340 and 450 nm for excitation and emission, respectively, and were corrected by subtracting tissue and substrate blanks. The results were analyzed using a one way ANOVA. An average of fluorescence units of 9685.86 � 1081.75, 7394.63 � 874.29, 3154.17 � 514.10, and 1666.40 � 117.86 was detected in the intact sperm sample for the α-D-mannosidase, α-L-fucosidase, β-D-glucosaminidase, and β-D-galactosaminidase, respectively. For the acrosome-reacted sperm sample (60–65% acrosome-reacted sperm in the samples measured by fluorescence microscope), an average of 9756.14 � 1011.45, 7026.93 � 771.48, 1185.70 � 277.51, and 1111.60 � 176.70 for α-D-mannosidase, α-L-fucosidase, β-D-glucosaminidase, and β-D-galactosaminidase, respectively. Statistically significant differences (P < 0.05) between intact and acrosome-reacted sperm were detected only for the β-D-glucosaminidase and β-D-galactosaminidase. These results suggest that the four different enzymes detected are mainly present in the sperm plasma membrane. Under the conditions used in this study, α-D-mannosidase is the main enzyme activity present in the sperm. Importantly, β-D-glucosaminidase and β-D-galactosaminidase activity detected in the intact sperm is decreased after the induction of the acrosome reaction.
239 OOCYTE MATURATION IN VITRO: PROTEOME ALTERATIONS ANALYZED BY ULTRA-SENSITIVE FLUOROCHROME LABELING AND NANO-ESI-MS-MS
Authors: Berendt F. , Froehlich T. , Boelhauve M. , Guengoer T. , Wolf E. , Arnold G. J.
Published: January 2008

In mammals, oocytes are arrested in the diplotene stage of the first prophase until the beginning of the estrous cycle. Directly before ovulation, the oocyte completes the first meiotic division and progresses to metaphase II. This process is mimicked by oocyte maturation in vitro, which is critical in assisted reproduction techniques in humans and animals. A sensitive 2-dimensional (2D) difference gel electrophoresis (DIGE) saturation labeling approach including an internal pooled standard was used for quantitative proteome profiling of immature v. in vitro-matured bovine oocytes. A mixture of 0.25 µg of oocyte sample and 0.25 µg of the internal pooled standard was separated on each analytical 2D gel, corresponding to the protein content of approximately five oocytes. In total, the study comprised 48 2D gel images representing 24 DIGE experiments [6 independent samples � 2 gels per stage (immature/mature) � 2 pH gradients]. DeCyder differential in-gel analysis (DIA; Amersham Biosciences, Piscataway, NJ, USA) detected 2244 spots (SD = 100) in pH 4–7 images, and 1291 spots (SD = 65) in pH 6–9 images. In total, 38 spots with different intensity were detected (abundance ratio ≥2; Student's t-test, P ≤ 0.01). Differently abundant spots were identified by nano-LC-MS/MS analysis of matched spots in a preparative gel of saturation-labeled protein extract from 2200 immature oocytes. Ten spots could be unambiguously identified and comprised, e.g., interesting proteins such as clusterin, 14-3-3 ε, redox enzymes, and new polymorphic forms of glutathione-S-transferases. Clusterin is suggested to interact with components of the complement membrane attack complex, and 14-3-3 ε is a mediator of the activation of the maturation promoting factor, a key enzyme of the meiosis. Our study demonstrates the feasibility of differential oocyte proteomics using saturation labeling. Several of the proteins identified in this study are already known to play an important role in oocyte maturation, which reflects the relevance and reliability of our approach. Furthermore, we found proteins which are so far unknown in the context of oocyte development. A more detailed knowledge of the role of these candidates during oocyte maturation may help to optimize the in vitro-maturation process in order to increase the rate of successful in vitro fertilization and other assisted reproduction techniques in cattle and other mammals.
This work was supported by grant Deutsche Forschungsgemeinschaft (FOR 478, GRK 1029).
24 DEVELOPMENT OF BOVINE CLONED EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF EMBRYONIC CULTURED CELLS ISOLATED FROM SOMATIC CELL NUCLEAR TRANSFER BLASTOCYSTS
Authors: Bai X. J. , Yu J. L. , Murakami M. , Zhang Y. , Dong Y. J.
Published: January 2008

Embryonic stem (ES) cells derived from somatic cell nuclear transfer (NT) bovine embryos would increase the utility of the cow as a large animal model for human cell therapy. They would also be useful for studies of cell differentiation. Such cells exhibit full pluripotency, and cloned offspring were obtained from them following a second NT in mice, indicating that the reprogramming that produced pluripotent ES cells could be reversed (Wakayama et al. 2001 Science 292, 740–743). The objective of this study was to examine if there would be any beneficial effects of using somatic cell NT-derived embryonic cultured cells as donors for cloning in cattle. Cloned embryos were produced from a single cell line of bovine fetal fibroblasts (FF) and adult ear-tip cells (AEC) (passages 1 to 5) by NT, as described previously (Dong et al. 2004 Asian–Aust. J. Anim. Sci. 17, 168–173). NT embryos that reached the blastocyst stage were cultured separately to isolate embryonic cultured cells derived from FF (NT-FF) and AEC (NT-AEC) according to previous methods (Dong et al. 2003 Acta Genet. Sin. 30, 114–118). More than 80% of the generated embryonic cultured cells stained positive for alkaline phosphatase. Embryonic cells cultured for 7 to 35 days were used as the donor cells for NT in the NT-FF and NT-AEC groups. Cloned embryos were produced using individual cell lines of FF, AEC, NT-FF, and NT-AEC (passages 1 to 5, putative cell cycle stage of G0 or G1) as donor cells, and their development in vitro is summarized in Table 1. The FF and AEC groups include data from the initial round of NT. The rates of fusion and embryo development were compared by chi-square analysis. Duncan's multiple range test was used to compare the mean cell numbers of blastocysts. The percentage of embryos that developed into blastocysts was significantly higher (P < 0.05) in the FF group than in the AEC group. Interestingly, we observed that the developmental potential in vitro and the mean cell number of blastocysts tended to be higher in the NT-FF and NT-AEC groups than in the FF and AEC groups. A total of 15 and 6 good quality Day 7 embryos in the NT-FF and NT-AEC groups were nonsurgically transferred to 5 and 3 synchronized recipients (2 to 3 embryos/female), respectively. On Day 30 of gestation, 3 (60%) and 1 (33%) females in the NT-FF and NT-AEC groups, respectively, were diagnosed as pregnant via ultrasonography. One (20%) recipient cow in the NT-FF group remained pregnant at Day 60 of gestation, but lost the pregnancy by Day 90. These results suggest that cloning of bovine embryonic cultured cells generated from fetal and adult somatic cells by NT can produce transferable embryos and initiate pregnancies, although none of the pregnancies has developed beyond the first trimester at this time.

Table 1. Development in vitro of bovine NT embryos produced from different donor cell types