Fertilization Medium Research Articles

175 EFFECT OF HEAT STRESS ON GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS (BOS TAURUS VS. BOS INDICUS)

Heat stress (HS) reduces the production of bovine embryos, especially taurine embryos, which are not adapted to heat. However, little is known about the competence of embryos produced under HS in breeds adapted or not adapted to heat. The aim of this study was to compare the gene expression of PLAC8, HSF1, COX2 and CDX2, related to competence and implantation, in bovine in vitro-produced embryos (Bos taurus vs Bos indicus), submitted or not submitted to HS. Oocytes from Nelore (zebu) and Jersey (taurine) cows were aspirated by ovum pickup, in vitro-matured in TCM-199 medium with bicarbonate containing 10% FCS, 2 μg mL–1 of pyruvate, 75 μg mL–1 of gentamicin, 20 μg mL–1 of FSH and 10 IU mL–1 of LH for 22 h at 38.5°C in 5% CO2 in air. Matured oocytes were fertilized with semen from Nelore (n = 6) and Jersey (n = 6) bulls, respectively, at 38.5°C in 5% CO2 in air. The fertilization medium was TALP-IVF supplemented with 6 mg mL–1 of fatty acid-free BSA, 2 μL mL–1 of pyruvate, 75 μg mL–1 of gentamicin, 11 μg mL–1 of heparin and 44 μL mL–1 of penicillamine, hypotaurine and epinephrine. The day of fertilization was considered Day 0. Twelve hours post-insemination, presumptive zygotes were denuded and randomly divided into 2 groups, nonstressed or stressed and both were in vitro cultured at 38.5°C in 90% N2, 5% CO2 and 5% O2 in SOFaaci medium supplemented with 5% FCS, 5% BSA and 0,2% sodium pyruvate. In the stressed group, 96-h post-insemination embryos were subjected to HS of 41°C for 6 consecutive hours and then returned to 38.5°C. On Day 7, pools with 5 blastocysts [Nelore (n = 9); Nelore HS (n = 7); Jersey (n = 5); Jersey HS (n = 5)] were subjected to RNA extraction (RNeasy, Qiagen Inc., Valencia, CA, USA). The expression of target genes was analysed by real-time reverse transcription PCR with oligo-dT in reverse transcription and bovine specific-primers in PCR. The expression of cyclophilin A was used as an internal control. The mean mRNA levels of target genes among groups were compared by parametric ANOVA, followed by orthogonal contrast. Heat stress reduced (P < 0.05) mRNA expression of CDX2 and PLAC8 in both breeds; additionally, the expression of these genes was higher in the zebu breed when compared with the taurine breed. Messenger RNA expression of COX2 did not differ between groups, under HS or not, in both the Jersey and Nelore breeds. Moreover, HS reduced the mRNA expression of HSF1 (P < 0.05) in Nelore groups, but not in Jersey groups. The highest levels of PLAC8 and CDX2 in nonstressed Nelore embryos indicate better competence and a higher capacity of implantation of these embryos when compared with Jersey and HS embryos in both breeds. Moreover, low HSF1 levels in stressed Nelore embryos indicate the thermotolerance ability of this breed. In conclusion, the data indicate that HS alters the pattern of gene expression in Nelore and Jersey in vitro-produced bovine embryos. This research was supported by FAPESP.

139 TRANSCRIPTOME ANALYSIS OF SINGLE BOVINE EMBRYOS BY RNA-Seq

Transcriptome sequencing by high-throughput technologies provides global gene expression levels as well as gene structure information. Moreover, analysis of samples from single individuals allows the detection of allele-specific expression. We investigated the possibility of RNA-Seq analysis using single bovine blastocysts. Embryos were in vitro produced using abattoir-derived, in vitro- matured oocytes, TALP-based fertilization and KSOM embryo culture medium. On Day 7 of culture, 5 expanded blastocysts were collected and stored in RNA extraction buffer at –80°C. Total RNA was extracted from each individual embryo using the Arcturus PicoPure RNA isolation kit including DNAse treatment. Approximately 1.5 ng of high-quality total RNA was obtained per embryo. The RNA was amplified using the SPIA-based Ovation RNA-Seq kit (NuGen, San Carlos, CA, USA). After amplification, 6 μg of cDNA was obtained and directly used for library construction with the NuGen Encore NGS Library I kit. Libraries were submitted to the University of California Davis Genome Center for 40-bp single read sequencing on an Illumina GAIIx apparatus. Data analysis was performed using CLC Genomics Workbench. On average, 38 094 173 good-quality reads were produced from each sample. Removing 9 bp from the 5′ end of the sequences greatly improved read alignment. Mapping of trimmed reads to BTAU 4.0 allowing up to 2 mismatches per read resulted in 88.9 ± 0.3% of reads aligning to the genome. Using an SNP discovery algorithm, a total of 31 993 unique SNP were detected with an average of 12 530 ± 496 SNP per sample, 50% of which were heterozygous. Of the total, 45, 21, 14 and 9% were common to at least 2, 3, 4 and 5 samples, respectively. Allelic expression imbalance, defined as 75% of reads corresponding to one allele of a heterozygous SNP with coverage ≥50, was observed in 22% of SNP among those common to at least 2 samples. Mapping the reads to the transcriptome resulted in 71.8 ± 0.4% of reads aligning to genes present in Ensembl. Among those mapping to RefSeq transcripts, 64.4% corresponded to exons, 7.2% to exon-exon boundaries and 0.3% to exon-intron boundaries, with the remainder mapping to introns. An average of 9746 ± 122 genes with RPKM greater than 0.3 were detected in each sample, with 7982 genes expressed commonly among all 5 embryos. The correlation for RPKM between sample pairs was between 0.978 and 0.993. Genes known to be almost exclusively expressed by pre-implantation embryos were present, including OCT4, NANOG and CDX2 among others. Gene ontology analysis of gene groups, divided into quintiles by level of expression, indicated that the most highly expressed genes are enriched in ribosomal proteins and oxidative phosphorylation. Genes with medium-high levels of expression were enriched in structural components including organelles and the cytoskeleton. Genes at the medium level of expression represented nuclear and chromatin proteins, whereas medium-low and low were related to regulation of transcription and DNA metabolism. We conclude that RNA-Seq from a single bovine blastocyst is possible and represents a powerful tool for understanding the biology and pathologies of pre-implantation embryo development.

The novel use of modified pig zygotic medium for the efficient culture of the preimplantation mouse embryos

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94–99%) and blastocyst (90–96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P < 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in M16 or CZB media (P < 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94–98%) and blastocysts (91–93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes.

Ubiquitin‐activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm–egg coat penetration during porcine fertilization

Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.

Quinn’s advantage fertilization medium enhances zona pellucida-induced acrosome reaction compared with human tubal fluid medium

The aim of this study was to compare sperm velocity, hyperactivation, zona pellucida (ZP) binding and ZP-induced acrosome reaction (AR) between Quinn’s advantage fertilization (QAF), human tubal fluid (HTF) and Ham’s F10 media. Semen samples were obtained from normozoospermic men and motile spermatozoa were prepared by gradient centrifugation (PureSperm). Unfertilized oocytes from clinical IVF were used for spermatozoa–oocyte interaction tests. Sperm velocity and hyperactivation were assessed using a Hamilton-Thorn motility analyser. When media were supplemented with human albumin, sperm motility and velocity and sperm binding were not significantly different between QAF and HTF. However, ZP-induced AR was significantly higher with QAF than HTF (42 ± 22 versus 21 ± 18, P < 0.001). Sperm velocity, hyperactivation and sperm binding were also significantly higher in QAF than Ham’s F10 media. Supplementation of media with either human serum or human albumin showed no difference in effect on all sperm test results. In conclusion, QAF medium significantly enhances ZP-induced AR which is essential for sperm penetration. Thus QAF appears to be a better medium than HTF for sperm fertilizing ability in conventional IVF. The aim of this study was to compare sperm velocity, hyperactivated motility, sperm binding and zona pellucida (ZP)-induced acrosome reaction (AR) between Quinn’s advantage fertilization (QAF) and human tubal fluid (HTF) and Ham’s F10 media. Semen samples were obtained from men with normal semen analysis results and all test results were compared in paired control samples between different media. When media were supplemented with human albumin, sperm motility, velocity and sperm binding were not significantly different between QAF and HTF. However, ZP-induced AR was significantly higher with QAF than HTF. Sperm velocity, hyperactivated motility and sperm binding were all significantly higher in QAF than Ham’s F10 media. Supplementation with either human serum or human albumin in media had no different effects on all sperm test results. In conclusion, Quinn’s advantage fertilization medium significantly enhances ZP-induced AR, which is essential for sperm penetration. Thus QAF appears to be a better medium than HTF for sperm fertilizing ability in conventional IVF.

Reduced Glutathione Enhances Fertility of Frozen/Thawed C57BL/6 Mouse Sperm after Exposure to Methyl-Beta-Cyclodextrin1

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. However, freezing the sperm of C57BL/6 mice, the most commonly used genetic background for genetically engineered mice, considerably reduces its fertility. We previously reported that methyl-beta-cyclodextrin dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. Recently, it was reported that exposing sperm to reduced glutathione may alleviate oxidative stress in frozen/thawed mouse sperm, thereby enhancing in vitro fertilization (IVF); however, the mechanism underlying this effect is poorly understood. In the present study, we examined the combined effects of methyl-beta-cyclodextrin and reduced glutathione on the fertilization rate of IVF with frozen/thawed C57BL/6 mouse sperm and the characteristic changes in the zona pellucida induced by reduced glutathione. Adding reduced glutathione to the fertilization medium increased the fertilization rate. Methyl-beta-cyclodextrin and reduced glutathione independently increased fertilization rates, and their combination produced the strongest effect. We found that reduced glutathione increased the amount of free thiols in the zona pellucida and promoted zona pellucida enlargement. Finally, 2-cell embryos produced by IVF with the addition of reduced glutathione developed normally and produced live offspring. In summary, we have established a novel IVF method using methyl-beta-cyclodextrin during sperm preincubation and reduced glutathione during the IVF procedure to enhance fertility of frozen/thawed C57BL/6 mouse sperm.

Combined Application of Methyl-Beta-Cyclodextrin and Reduced Glutathione to the In Vitro Fertilization of Cryopreserved C57BL/6 Mouse Sperm.

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. The benefits of sperm cryopreservation include the saving of space and costs relating to the upkeep of each mouse lineage, the potential avoidance of a genetic drift during the storage term, and the ease of transporting the mice between research facilities. However, embryos need to be obtained using in vitro fertilization (IVF) in order to reproduce live animals from frozen/thawed sperm. In general, the fertility of frozen/thawed sperm taken from C57BL/6 mice, which is mainly used as a base for genetically engineered mice, is severely lower than that of fresh sperm. Previously, we reported that methyl-β-cyclodextrin (MBCD) dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. MBCD effectively induces sperm capacitation by facilitating a cholesterol efflux from the cells. Recently, an interesting report was published on the topic of the supportive effects of reduced glutathione (GSH) on improving the fertility of mouse sperm. However, the action mechanism of GSH during the fertilization process is not fully understood. In this study, we examined the most efficient timing of GSH treatment and the combined effects of MBCD and GSH on the rate of fertilization for IVF using frozen/thawed C57BL/6 mouse sperm. As a result, we found that the presence of GSH in the fertilization medium increased the fertilization rate whereas GSH had no effects in the medium of sperm preincubation, and that the elevation of fertilization rates started at 0.5 mM GSH and reached a peak at 1.0 mM GSH (0 mM: 66%, 0.5 mM: 79% and 1.0 mM: 92%). We also discovered that the MBCD and GSH independently increased the rate of fertilization, while a combination of MBCD and GSH exhibited the highest fertilization rate in each group (control: 14%, MBCD: 65%, GSH: 36% and MBCD + GSH: 87%). In summary, we have demonstrated a novel IVF method using the combined application of MBCD for sperm preincubation and GSH for IVF, in order to achieve a high and stable fertilization rate in frozen/thawed C57BL/6 mouse sperm. (poster)

Soil Resource Database at Village Level for Developmental Planning

A study was carried out in village Mohammadabad, Nalgonda district, Andhra Pradesh to demonstrate the potential of high-resolution remote sensing data in generating soil resource database at village level that would facilitate natural resource developmental planning. For this study, detail soil resource database was generated by interpreting IRS Resourcesat-1 LISS-IV + Cartosat-1 merged data and by undertaking soil survey and soil analysis. This database was integrated with cadastral map and was analysed for preparation of an action plan for the village that included recommendation on suitable crops, soil and water conservation measures and distribution of soil health card to farmers. The study indicates that the LISS-IV + Cartosat-1 merged satellite data helps in mapping soils at phase level and in delineation of more number of pure soil mapping units i.e. consociations at 1:10.000 scale. The plot-wise resource potential and constraints reveals that significant inter and intra-field soil variability exists. Soil fertility assessment indicates that the soils of the village are low to medium in fertility (available NPK Content) and micronutrients like copper, manganese and iron content is above the critical level for optimum growth of crops while zinc content is less than the critical level.

Evidence for the inhibition of fertilization in vitro by anti-ZP3 antisera derived from DNA vaccine

Previously we have found that DNA vaccine, pCMV4-rZPC’ can generate specific antibodies against rabbit ZPC (amino acid 263-415, rZPC’), which binds to ovarian ZP and leads to a significant reduction of fertility in vivo. The purpose of this study was to evaluate the effect of antisera from pCMV4-rZPC’-immunized mice on sperm–oocyte interaction in vitro. The effect of antisera from DNA vaccine-immunized mice on fertilization and early embryonic development was studied using an in vitro fertilization system. The results showed that the antisera supplemented in fertilization medium (10%, v/v) significantly decreased the rate of fertilization compared to that of control groups (P<0.05); whereas the antisera showed no significant effect on the rate of fertilization when ZP-free eggs were used. Moreover, the antisera pre-neutralized with mouse soluble zona pellucida lost the capacity to inhibit fertilization when compared with that of control groups. In addition, the antisera showed no detrimental effect on early developmental potential of mouse embryos in vitro. Taken together, our study provided herein direct evidence showing that antisera generated by DNA vaccine can block sperm–egg recognition during fertilization via targeting the oocyte ZP proteins.

Time Exposure Period of Bovine Oocytes to Sperm in Relation to Embryo Development Rate and Quality

The objective of the study was to determine the effect of different bovine gamete coincubation times on fertilization and embryo development performance. In vitro matured COCs were co-incubated with sperm at a concentration of 1.5 × 106 spermatozoa/ml in TALP medium for 3 hours (T 3, n = 362), 6 hours (T 6, n = 358), or 18 hours (T 18, n = 350). At the end of the coincubation period COCs from times 3 and 6 groups were post-incubated in a new well of fertilization medium without sperm for additional 15 and 12 h, respectively. Cumulus Oocyte Complexes from the T 18 were co-incubated with the sperm suspension for 18 hours. Presumptive zygotes were cultured for 9 days and embryo development was evaluated on days 2, 8, and 9. Thirty blastocysts from each group were stained and total number of nuclei was recorded. The mean (± SEM) percentages of zygotes to develop into ≥2 cell stage were 71.9 ± 5.0; 72.5 ± 5.3 and 81.2 ± 6.1 % for T 3, 6, and 18, respectively, on day 2 and they did not differ (P = .3) among groups. The mean percentage of blastocysts developed on day 8 (25.6 ± 2.8; 24.2 ± 3.3; 28.4 ± 4.2 % for T 3, 6, and 18, resp.) did not differ (P = .4) among groups. The total number of embryonic nuclei was greater (P < .05) for the blastocysts produced from the shortest co-incubation time (T 3).

Optimized Cryopreservation of Mouse Sperm Based on Fertilization Rate

Although procedures for in vitro fertilization with cryopreserved sperm have been published there is a lack of data indicating that the cryoprotectant and cryopreservation procedures used for those procedures were optimal. To redress this, fertilization rate of eggs exposed to sperm in vitro was used as the outcome in the optimization of raffinose concentration in the cryoprotectant (raffinose in water), volume of cryoprotectant, and freezing conditions for C57BL/6J mouse sperm. Sperm were frozen in a cylindrical Dewar with an internal diameter and height of 14.0 cm and 36.0 cm respectively. The optimal concentration of raffinose was 23-24% (510-540 mOsm/kg). The optimal volume of cryoprotectant used to prepare the sperm suspension from a single mouse was 180-400 µl, and sperm proved most fertile when frozen 13-25 mm above liquid nitrogen. Raffinose in the fertilization medium did not inhibit fertilization. Fertilized eggs transferred to oviducts of recipient mice developed into viable offspring.

PENGARUH TEMPERATUR TERHADAP SIFAT MEKANIK PADA BAJA KARBON SEDANG ST 60

Medium carbon fertilizer ST 60 in general is often used in construction of building materials and a variety of fields. Society prefers medium carbon fertilizer because this material is more easily available and relatively affordable prices, and also the strength and tenacity are sufficient. Temperature significantly affects the mechanical properties of carbon steel is ST 60. Formulating of the problem in this study is to determine how the mechanical properties of medium carbon steel ST 60 to temperature 00C, 260C, 500C, 1000C. The purpose of this research to know how much testing on tensile strength, hardness, and price impact. The results of research on testing with 00C yield of 696,68 N/mm2tensile test, hardness test of 201,00 HB, the impact test produces 30,35 J/cm2. In the test with a temperature of 260C produced by 694,47 N/mm2tensile test, hardness test of 200,11 HB, the impact test at 24,52 J/cm2. In the test with a temperature of 500C produced by 685,87 N/mm2tensile test, hardness test of 198,00 HB, the impact test at 41,70 J/cm2. In the test with a temperature of 1000C resulted N/mm2tensile test at 672,89 N/mm2, the hardness of 195,56 HB, the impact test at 37,69 J/cm2. Data analysis in this study using the product moment correlation test and t-test. The conclusion of this research is that there is a negative relationship between temperature with mechanical properties which means the higher the temperature the more down the value of tensile strength, hardness, and the price impact.Keywords: fertilizer, mechanical, temperature.

Effects of Exposure to Zearalenone on Porcine Oocytes and Sperm During Maturation and Fertilization In Vitro

The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZEN-containing (0-1000 µg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 µg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 µg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 µg/l may not affect the fertility of the oocytes.

258 ROLE OF PROGESTERONE AND ITS RECEPTORS ON DEVELOPMENTAL COMPETENCE OF OOCYTES IN CATTLE

Several studies have demonstrated the importance of progesterone (P4) through its receptors (PR) in the regulation of the ovulatory cycle, but its participation in oocyte maturation in mammals has not yet been clarified. Previous results in our group showed changes in the protein expression of genomic (nPR-A/B) and nongenomic (mPRα/β) PR in bovine cumulus–oocyte complexes (COC) during in vitro maturation (IVM), indicating a possible function for these receptors on bovine oocyte maturation. Therefore, we aimed to study the role of P4 and PR in oocyte developmental competence. Good-quality immature COC were placed in maturation medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 of epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of trilostane (0.001, 0.1, and 10 μM), P4 (50 and 100 ng mL–1), promegestone (50 and 100 ng mL–1), RU 486 (0.1, 1, and 10 μM), or antibodies against mPRα or mPRβ. Matured COC were washed and placed in wells containing 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Number of embryos was recorded at day 2, 3, 7, and 8 (Day 0 = day of IVF). Data were analysed using ANOVA analysis using SPSS v. 15.0 software package. Inhibition of P4 production by 10 μM of trilostane during IVM reduced progesterone production in the media, cumulus cells expansion, but had no effect on meiotic resumption in Day 2 and Day 3 embryos; however, there was a significant decrease in the percentage of blastocysts at Day 7 (12 ± 3.5) and Day 8 (14 ± 3.3) compared with the control (30 ± 4.5 and 42 ± 2.5). This reduction on embryo development was overcome by the addition of 50 or 100 ng mL–1 of P4 at Day 8 (33 ± 5.7 and 32 ± 4.1). The same results were obtained with nonmetabolizable P4, where the reduction on blastocysts with trilostane at Day 8 (20 ± 2.0) was completely overcome by 50 or 100 ng mL–1 of promegestone (41 ± 5.1 and 40 ± 6.7). Specific inhibition nPR-A/B with 10 μM of RU 486 produced a significant reduction on blastocysts at Day 7 (24 ± 4.3) and Day 8 (29 ± 5.5) compared with the control (44 ± 2.6 and 47 ± 3.0). Inhibition of mPRα reduced cleavage rate and Day 3 embryos, whereas the inhibition of mPRβ had no effect on meiotic resumption or embryo development. In conclusion, the intracellular signaling of P4 on developmental competence of oocytes in cattle seems to be mainly mediated by nPR-A/B receptors and could be associated with cytoplasmic maturation.

251 SELECTION OF PREPUBERTAL SHEEP OOCYTES USING BRILLIANT CRESYL BLUE TEST

The aim of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth to select competent prepubertal sheep oocytes for in vitro embryo production. The BCB stain allows the determination of glucose–6-phosphate dehydrogenase (G6PD) activity, an enzyme with decreased activity in oocytes that have finished their growth phase. Oocytes were obtained after slicing the surface of lamb ovaries (2–5 months old) obtained from a local abattoir. Oocytes with more than 3 compact cumulus layers and homogeny cytoplasm were selected and stained with different concentrations of BCB diluted in PBS (13-, 26-, 39-, and 52-μM BCB) during 60 min at 38.5°C in a humidified air atmosphere. Oocytes were classified into groups depending on their cytoplasm coloration: oocytes with blue cytoplasm or grown oocytes (BCB+) and oocytes without blue coloration or growing oocytes (BCB–). Oocytes were matured in an enriched TCM-199 medium for 24 h at 38.5°C and 5% CO2 in a humidified atmosphere. Oocyte diameter was also measured. Matured oocytes were partially denuded and transferred to fertilization medium (SOF) supplemented with 10% of oestrous sheep serum. Fresh semen was kept at room temperature (25°C) for 1 h. Highly motile spermatozoa were selected by using Ovipure density gradient (Nidacon EVB S.L.), and oocytes were fertilized with 1 × 106 sp mL–1. After 20 h postinsemination, presumptive zygotes were cultured for 8 days in SOF with 10% of fetal bovine serum at 38.5°C, 5% CO2, and 90% N2. Data was analysed by performing Fisher’s exact test for blastocyst production and ANOVA with Tukey’s post-test for oocyte diameter. Table 1 shows the percentage of BCB-stained oocytes and their embryo development. In this study oocytes exposed during 60 min to 13-μM BCB showed a higher percentage of embryos reaching blastocyst stage than did those in the control group (≤0.01). In other species such as goats (Rodriguez-Gonzalez et al. 2002 Theriogenology 57(5), 1397–1409) and cattle (Alm et al. 2005 Theriogenology 63(8), 2194–2205), the best protocol for the oocyte selection was the use of 26-μM BCB during 90 min. Oocyte diameter showed significant differences between BCB– with BCB+ and control group (110 μm, 134 μm, and 121 μm, respectively, ≤0.001). In conclusion, using 13 μM of BCB during 60 min is a suitable technique for increasing embryo blastocyst rates using lamb oocytes. Table 1.Effect of BCB1 concentration on embryo development of lamb oocytes The grant sponsor was the Spanish Ministry of Science and Innovation, Code: AGL2007-60227-CO2-01.

The Effect of Various Concentrations of Taurine during In vitro Fertilization on the Development of Bovine Embryos Fertilized with Spermatozoa from Three Different Bulls

We investigated the effect of various concentrations of taurine during in vitro fertilization (IVF) on the embryonic development up to the blastocyst stage of bovine oocytes fertilized with three different Japanese Black bulls (Bull A, B and C). In vitro matured oocytes were fertilized with various concentrations of taurine (0, 1, 10, 50 and 100 mM) in the presence of 2.5 or 5.0 mM caffeine plus 25 μg/ml heparin (CH) for 6 hr or 100 μg/ml heparin (H) for 24±2 h. After IVF, the cleavage rates from the 2 to 16 cell stage determined at 3 days and the development rates up to the blastocyst stage determined at 7-8 days from the onset of IVF were assessed. Although the cleavage rates for the taurine concentration groups were not significantly increased in any of the three bulls in the CH groups, the development rates up to the blastocyst stage of the 50 mM taurine group of Bulls A and B, and of the 1 to 50 mM groups of Bull C were increased (p<0.05) compared to those of the control (0 mM taurine) groups. On the other hand, none of the bulls in the H groups showed any significant increase either in the cleavage rates or blastocyst formation rates in any taurine concentrations groups compared with those of the control groups. These results indicate that the addition of 50 mM taurine to a fertilization medium containing caffeine and heparin may stimulate embryonic development up to the blastocyst stage when fertilized with different bull semen.

Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer's disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.

Advances in Swine In Vitro Embryo Production Technologies

Recent advances in new technologies to produce cloned and genetically modified pigs involve manipulating oocytes and/or embryos in vitro. Although a great deal of progress has been made, the current IVM-IVF systems still result in major problems: a high rate of polyspermy; and a low development rate and low quality of blastocysts for in vitro compared with the in vivo-produced embryos. This study summarizes recent advancements in IVM-IVF-IVC porcine systems. Recent methods to select monospermic embryos are also discussed. Finally, achievements in vitrification and in somatic cell nuclear transfer are discussed.

Inhibition of In Vitro Fertilizing Capacity of Cryopreserved Mouse Sperm by Factors Released by Damaged Sperm, and Stimulation by Glutathione

BackgroundIn vitro fertilization (IVF) of eggs by frozen and thawed C57BL/6J mouse sperm is inhibited by dead sperm and enhanced by preincubation of the sperm in calcium-free medium. In other species, the presence of sperm killed by freezing and thawing has been associated with the generation of hydrogen peroxide.Methodology/Principal FindingsThe proportion of eggs fertilized by cryopreserved C57BL/6J mouse sperm was increased significantly by increasing the volume of fertilization medium in which sperm and eggs were coincubated. Enhanced fertilization occurred even though the concentration of potentially fertile sperm was decreased fivefold. This suggested that if a putative soluble factor was inhibiting fertilization, dilution of that factor, but not the sperm, should increase the fertilization rate. This was achieved by coincubation of the gametes in cell culture inserts (Transwells®) that during incubation were transferred progressively to wells containing fresh fertilization medium. Fertilization rates using inserts were high (66.6±2.4% versus 27.3%±2.8% in wells alone). On the assumption that the soluble factor could be H2O2, reduced glutathione was added to the fertilization medium. This enhanced fertilization rate significantly (76.6%±2.0% versus 21.2%±1.9%), while addition of oxidized glutathione did not (82.7%±6.5% with reduced glutathione; 44.5±8.8% with oxidized glutathione; 47.8%±12.1% with no glutathione). Positive effects of reduced glutathione on IVF were also seen with frozen 129S1, FVB, and C3H sperm, and sperm from two lines of genetically modified C57BL/6J mice.Conclusions/SignificanceIVF in cell culture inserts and addition of glutathione to fertilization medium significantly increased the proportion of eggs fertilized by cryopreserved mouse sperm from four inbred strains, suggesting that reactive oxygen species generated during fertilization inhibit fertilization. The modified IVF techniques developed here enhance the feasibility and efficiency of using cryopreserved sperm from genetically modified lines of inbred mice.

279 IMPROVEMENT OF RAT IN VITRO FERTILIZATION PROTOCOL USING CRYOPRESERVED SPERM AND ITS APPLICATION TO OTHER STRAINS: A PRELIMINARY STUDY

In rats, the success of in vitro fertilization (IVF) was reported almost 40 years ago. Although it had been demonstrated in papers that these IVF oocytes using sperm freshly collected from cauda epididymides can be developed to term via embryo transfer, successful IVF with cryopreserved rat sperm has never been reported. Very recently, we reported establishment of a successful IVF system using frozen-thawed spermatozoa treated with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), in Wistar rats (Seita et al. 2009 Biol. Reprod.). The objectives in this study were (1) improvement of the IVF system to a more convenient and simple protocol and (2) a preliminary study for applying our IVF system to inbred rat strains Fischer 344 (F344) and Brown-Norway (BN). In experiment 1, we examined the effect of preincubation time for frozen-thawed sperm on fertilization (2 pronuclei formation). Frozen-thawed sperm were preincubated up to 6 h and then used for IVF according to our previous report. Data showed that sperm preincubated for 5 h contributed to higher fertility than those for other preincubation times. In experiment 2, we examined the effect of co-culture time up to 10 h for IVF on fertility and embryonic development in vitro. The oocytes co-cultured with sperm beyond 6 h showed higher fertilization and blastocyst formation rates than those in 2 and 4 h. In experiment 3, we examined the effect of initial culture period in fertilization medium (310 mOsm modified R1ECM; O h et al. 1998 Biol. Reprod.) on embryonic development in vitro. After IVF, oocytes were cultured in fertilization medium for 6, 12, or 24 h and further cultured in R1ECM up to 120 h. Cleavage rates were not affected by initial culture time in fertilization medium. However, oocytes cultured in fertilization medium for 12 h showed higher blastocyst formation rate than those for 0, 6 and 24 h. In experiment 4, we examined whether the IVF protocol can be applied to F344 and BN rats. Fresh and frozen-thawed sperm collected from cauda epididymides in F344 and BN were used for detection of capacitation-associated tyrosine phosphorylation. In fresh F344 sperm, tyrosine phosphorylation was induced in a time-dependent manner. Although tyrosine phosphorylation was inhibited in frozen-thawed F344 sperm, it was dramatically accelerated by IBMX treatment as well as frozen-thawed Wistar sperm. However, tyrosine phosphorylation in fresh and frozen-thawed BN sperm was suppressed and the phosphorylation in frozen-thawed sperm was not improved by IBMX. Our data demonstrate that (1) a more efficient IVF system using frozen-thawed rat sperm was developed and (2) the IVF system can be applied to at least F344 strain. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.