Vivo-derived Bovine Embryos Research Articles

229 Machine learning identifies differences in morphokinetics of in vivo-derived bovine embryos between hot and cool seasons

229 Machine learning identifies differences in morphokinetics of in vivo-derived bovine embryos between hot and cool seasons

86 Difficulty of Transfer of In Vivo-Derived Bovine Embryos and Route of Administration of Flunixin Meglumine at the Time of Transfer may Affect Pregnancy Rate

Recipient handling during embryo transfer (ET) induces prostaglandin F2α (PGF2α) production in 2 periods: an early transient and rapid increase around the time of ET, followed by another 2 to 4 h later. This PGF2α is associated with embryonic loss during early gestation by affecting both the embryo and the corpus luteum. To control this, antiprostaglandins such as flunixin meglumine (FM) have been applied IM at the time of ET with varying results. In such studies, the interaction of IM administration of FM and difficulty of transfer has not always been evaluated, possibly confusing the interpretation of the results. Furthermore, IV FM injection at ET and its relationship with pregnancy rates (PR) has not been determined. The objectives were (1) to determine the relationship between difficulty of ET and PR; and (2) to evaluate the efficacy of IM v. IV FM on pregnancy outcomes. One hundred and ten crossbred (Bos taurus × Bos indicus) heifers (18-24 months old) from 3 farms were used as recipients. Two evaluation systems of ET difficulty were used: (1) duration of transfer (objective determination of the elapsed time measured in seconds between the introduction of the catheter and embryo release), and (2) level of difficulty experienced by the practitioner (subjective determination; 1 = minimum and 2 = medium to extreme manipulation). Quality 1 and 2 fresh embryos from superovulated cows were transferred by the same practitioner. At ET, recipients were randomly divided into 3 groups: (1) Control (no treatment, n = 31); (2) FM-IM (n = 39): injected IM with 2.2 mg kg−1 FM at ET; and (3) FM-IV (N = 40): injected with 2.2 mg kg−1 FM IV at ET. Pregnancy was diagnosed at 30 to 40 and 60 to 90 days after ET. Spearman’s test was performed to determine the correlation between duration and difficulty at ET and Chi-square test was used to compare PR. The mean duration of transfer for all heifers was 62.3 ± 57.5 s (11 to 357 s; median: 44.5 s). There was a high correlation (0.8; P < 0.001) between the ET difficulty evaluation systems. Overall, ET difficulty 1 had higher PR than ET difficulty 2 (64.2 v. 40.7; P = 0.013). The PR was significantly improved (P < 0.01) in the FM-IV group (75 and 70% at 30 and 60 days after ET) compared with control (45.2 and 32.3%) and FM-IM (33.3 and 30.7%). In conclusion, results indicate that the difficulty of transfer affects PR achieved following the transfer of in vivo-derived bovine embryos. Treatment with FM-IV following transfer resulted in significantly higher PR compared with control and FM-IM recipients. The IV injection of FM may antagonize the very early and transient increase of PGF2α caused by genital tract manipulation (even gently performed) at embryo transfer. Further research is necessary to confirm the results of the present study.

141 EXPRESSION STATUS OF AQUAPORINS 3, 7, AND 9 IN BOVINE PRE-IMPLANTATION EMBRYOS

The aquaporins (AQP) are a family of small integral membrane proteins that work as channels for rapid water transport. In mouse embryos, AQP3, AQP7, and AQP9 were found to play important roles in the pre-implantation development or adaptive cellular response to osmotic stress. In addition, because AQP3, AQP7, and AQP9 permeate not only water but also cryoprotectant, such as glycerol, these AQP thought to be involved in the process of cryopreservation. However, little information is available for AQP in bovine embryos. The understanding of the mechanisms mediated by AQP that embryos utilise to survive during culture and the process of cryopreservation will contribute to development of in vitro culture systems and cryopreservation procedures for bovine embryos. The aims of present study were to clarify the expression status of AQP3, AQP7, and AQP9 in bovine pre-implantation embryos, and to evaluate the expression level of AQP3 in in vivo-derived (IVD) and IVF bovine embryos. For production of IVF embryos, cumulus-oocyte complexes (COC) were aspirated from ovaries collected at a local slaughterhouse. The COC were in vitro matured, fertilized, and then cultured for 7 days. The IVD embryos at early blastocyst (EB) and blastocyst (BC) stage were obtained from donor cows treated with superovulation and AI. In experiment 1, in order to clarify the expression of AQP3, AQP7, and AQP9 mRNA in bovine pre-implantation embryos, total RNA was extracted from pools of 30 IVM oocytes, pools of 15 IVF embryos at 2- to 4-cell, 8- to 16-cell, compaction morula (CM), EB, BC, and expanded blastocyst (ExBC) stage, and RT-PCR was performed followed by agarose gel electrophoresis. In experiment 2, in order to clarify the expression status of AQP3, AQP7, and AQP9 during bovine pre-implantation development after zygotic gene activation, total RNA was extracted from pools of 15 IVF embryos at 8- to 16-cell, CM, EB, BC and ExBC stage (n = 5), and relative quantifications of AQP mRNA were performed using real-time RT-PCR. Data were analysed by Scheffé’s method. In experiment 3, AQP3 transcript levels in single IVD and IVF embryos at EB and BC stages (n = 10) was evaluated by real-time RT-PCR. Data were analysed by Mann-Whitney’s U test. In experiment 1, AQP3 and AQP7 transcripts were detected in IVM oocytes and all stages of embryos. AQP9 mRNA was detected in IVM oocytes and in 2- to 4-cell, 8- to 16 cell, CM, and EB stage embryos, but was not detected in BC and ExBC stage embryos. In experiment 2, AQP3 and AQP7 transcript levels were significantly increased from 8- to 16-cell to CM and EB stage, and significantly decreased from EB to BC and ExBC stages (P < 0.05). AQP9 transcript level was significantly decreased from 8- to 16-cell to CM and EB stage (P < 0.05). In experiment 3, AQP3 transcript level in IVF embryos was significantly lower than that in in vivo embryos at the BC stage (P < 0.05). Our results indicate that AQP3 and AQP7 may have specific roles at morula and EB stage in bovine embryos. In addition, AQP3 expression is influenced by developmental condition of bovine embryos.

The Two-Cell Embryo—Two Sides of the Same Coin?

Every month, numerous papers appear in the popular scientific journals, including Biology of Reproduction, describing the expression of candidate genes, or the global gene expression pattern, in mammalian embryos. Many of these studies are very interesting and cleverly designed, and all aim to give us better insight into the regulatory mechanisms underlying embryo development. Most studies, however, are by their nature invasive, and the findings therefore can be difficult to extrapolate beyond the developmental stages examined. Furthermore, partly because of the challenges of extracting sufficient mRNA for transcript analysis, such studies are normally based on pools of embryos; this can potentially mask differences between treatment groups. In this month’s Biology of Reproduction, Held and colleagues [1] describe a study that examined the relationship between the transcriptome of a single blastomere from a twocell bovine embryo and the developmental competence of the sister blastomere with the aim of identifying potentially important candidate genes as markers of developmental capacity. A few years ago, the same group published a novel model in which a biopsy sample was taken from an in vitroproduced blastocyst, following which the blastocyst was transferred to a recipient [2]. Based on pregnancy outcome, the biopsy samples were pooled for gene expression analysis, thus identifying genes associated with development to term and early pregnancy loss. This model was later replicated by the same group using in vivo-derived bovine embryos [3] and by other investigators using human embryos [4]. These studies [1] clearly indicate that developmental competence of bovine blastocysts is associated with distinct gene expression signatures. Interestingly, other recent studies have shown that following blastocyst transfer at Day 7, different types of embryos (in vivo-derived, in vitro fertilization (IVF)-derived, cloned) elicit different responses from the endometrium around the time of initiation of implantation, and these responses are associated with different developmental outcomes [5, 6]. In other words, to some extent, the embryo is master of its own destiny. The studies described in the current paper [1] address a similar question but go further back along the developmental trajectory. The authors search for an answer by modifying the biopsy technique referred to above; separating sister blastomeres of bovine two-cell embryos allows for the correlation of the transcriptome in one blastomere with the developmental capacity of the other. Developmental fates of sister blastomeres were highly correlated (if one blastomere developed to the blastocyst stage, the same applied to the other in 94% of cases), suggesting that this is a reasonable strategy. A large number of differentially expressed genes were observed between blastomeres whose sister blastomeres reached the blastocyst stage and those which did not cleave after separation. Likewise, the transcription differed between those that formed blastocysts and those that stopped developing before embryonic genome activation (8to 16-cell stage). Downstream analysis revealed clustering of genes associated with cell response to oxidative stress. The expression of a selected group of candidate genes was then confirmed by comparing expression in early cleaving and late cleaving two-cell embryos and in embryos cultured under high and low oxygen conditions. Why are such studies important? In cattle, most embryonic loss occurs very early during embryogenesis, typically around Day 16 post conception, before maternal recognition of pregnancy [7]. Fertilization rates in vivo are normally high (;90%). Recent studies in high-producing dairy cows suggest that a significant proportion of loss may occur by as early as Day 7 (the blastocyst stage); approximately 50% of embryos flushed from single-ovulating cows on Days 6–7 were viable [8]. In vitro, despite .80% of oocytes typically cleaving to the two-cell stage after IVF, only approximately 30% develop to the blastocyst stage. This has been convincingly demonstrated to be primarily the result of compromised oocyte quality rather than suboptimal culture conditions, as oocytes matured in vivo develop to the blastocyst stage in vitro at high rates after IVF [9]. Thus, understanding what regulates developmental competence may contribute to our understanding of embryonic mortality and survival. This has potential implications for improving conception rates in dairy cows, for genomic selection of embryos in breeding programs, and having ramifications for human assisted reproduction, where embryos are typically transferred at early cleavage stages.

112 BLUETONGUE VIRUS INFECTION IN CATTLE AFTER TRANSFER OF BOVINE IN VIVO-DERIVED EMBRYOS

Bluetongue virus (BTV) has been categorized by the OIE as a category 1 disease agent, for which proper handling between collection and transfer is thought to be sufficient to prevent transmission through embryo transfer. For bovine viral diarrhoea virus, it was shown that effectiveness of washing procedures depends on virus strains (Waldrop et al. 2004 Theriogenology 62, 45–55). Also BTV-8 has unique characteristics in comparison with other strains (De Clercq et al. 2008 Transbound. Emerg. Dis. 55, 352–359). The aim here was to investigate whether embryo transfer of in vivo-derived bovine embryos after in vitro exposure to BTV-8 can be performed without risk for infection of the recipients if IETS washing and trypsin treatment procedures are followed. Donor cows (n = 2) were synchronized and superovulated using Stimufol® (Ulg, Liége, Belgium) and subsequently inseminated. At 6.5 days post-insemination (dpi), flushed embryos (n = 14 and n = 3) were placed in 800 μL of minimal essential medium (MEM), containing 104.9 50% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/2 P5, VAR, Brussels, Belgium) and incubated for 1 h at 39°C in 5% CO2 in air (Vandaele et al. 2011 Vet. Res. 42, 14–21). Next, embryos were washed in pairs in 5 consecutive Petri dishes containing PBS with antibiotics and 0.4% BSA, w/o Ca and Mg. Then, embryos were exposed to 2 consecutive trypsin (Invitrogen, Carlsbad, CA, 25050-014) washes of 45 s each at 39°C in 5% CO2 in air and finally, another 5 consecutive washes in PBS with 2% FCS. Each Petri dish contained at least 2 mL of medium and was gently agitated between washes. Embryos were transferred in a maximum of 7 μL of medium and a new tip was used after every wash step. Washes 1 to 5 and washes 6 to 10 were pooled and analysed for BTV-8 (RT-qPCR). After these washes, 3 pairs of embryos (n = 6) were loaded in straws and transferred to 3 BTV-8 negative recipients. Two sentinel cows served as control. Cows were bled twice weekly and blood and serum samples were analysed for BTV-8 (RT-qPCR) and BTV-8 antibodies. Viral BTV-RNA was detected in all 3 recipient cows at 7 days after transfer and viraemia was confirmed by the establishment of high antibody titers at 14 days after transfer. Viral BTV-RNA was detected in washes 1 to 5 for each pair of embryos (Cp-value around 29), whereas washes 6 to 10 had Cp-values around the cut-off value (40), indicating that probably the last wash was BTV-8 negative. None of the recipients was pregnant at 28 days post-transfer. In conclusion, washing and trypsin treatment did not succeed in removing BTV-8 from in vitro-spiked in vivo-derived bovine embryos. These unexpected results stress the need for further in vivo research, e.g. what is the virus load in vivo embryos may be exposed to in utero during viraemia? Does BTV-8 react differently with the zona compared with other strains? Are alternative washing procedures needed to remove BTV-8 from the zona?

177 GENE EXPRESSION PROFILES OF IN VITRO- AND IN VIVO-DERIVED BOVINE EMBRYOS

Microarray technology is one of the most powerful tools for gene expression profiling in animal sciences. The objectives of this study were to determine the effect of vitrification on gene expression in in vitro- and in vivo-derived bovine embryos, and to identify differential mRNA expression patterns between embryos produced by in vivo v. in vitro conditions. Three pools of in vivo- and in vitro-derived blastocyst-stage embryos were used for microarray analysis. Total RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus Bioscience, Mountain View, CA). Bovine ovarian tissue total RNA was used as the reference. Total RNA samples were amplified using an Ovation® Pico WTA System (NuGEN Technologies, San Carlos, CA). The bovine 16 846-member microarrays spotted with 70-mer oligonucleotides were purchased from the Bovine Genomics Laboratory, University of Missouri. Amplified cDNA samples were labeled with Alexa Fluor 647 and 546 dyes (Molecular Probes, Eugene, OR), respectively. Combined, labeled samples were dried and resuspended in hybridization buffer containing 50% formamide (vol/vol), 5× SSC, and 0.1% sodium dodecyl sulfate (wt/vol). After denaturation and cooling, cDNA was applied onto a microarray slide. Microarrays were hybridized overnight at 42°C. Following hybridization, the slides were washed with different stringency buffers and water. After drying by centrifugation, the arrays were scanned on a GenePix 4000B scanner (Axon Instruments, Union City, CA). GenePix Pro4.1 software was used for griding and analysis of spot intensities. Good-quality spots were analyzed using the GeneSpring 7.3 software (Agilent Technologies, Inc., CA, Santa Clara, CA). The data were normalized per spot and per array by Lowess normalization. When comparing two treatments, the Welch t-test with Benjamini and Hochberg multiple testing correction was performed to determine the differentially expressed genes between embryo groups. Microarray experiments were performed in 3 biological and 2 technical replicates for all embryo samples. Differentially expressed genes between all embryo groups were identified. The DAVID Functional Annotation Tool was used to analyze the genes that were differentially expressed. The DAVID Functional Annotation Tool determined the co-occurrence probability and provided gene-GO term enrichment analysis to highlight the most relevant GO terms associated with a given gene list. Differentially expressed Kyoto Encyclopedia of Genes and Genomes pathways are as follows: Ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis were significantly upregulated in the fresh embryos, whereas sphingolipid and purine metabolism was the upregulated in the vitrified in vitro-derived embryos. Gene expression was very similar between fresh and vitrified in vivo-derived, as opposed to in vitro-derived, embryos. This study was funded by the TUBITAK (Project no. KAMAG107G027) and startup funds to Yuksel Agca at the University of Missouri.

Assessing Epidemiologic Risk of the Quantity of Bovine Viral Diarrhea Virus found to be Associated with in vivo-derived Bovine Embryos

Bovine viral diarrhea virus (BVDV) has been shown to be associated with single transferable in vivo-derived and in vitro-produced bovine embryos despite washing. However, a thorough risk assessment of the potential for transmission of BVDV via transfer of in vivo-derived embryos has not been undertaken. Hence, the primary objective of this study was to evaluate the potential of BVDV to be transmitted during the transfer of in vivo-derived embryos.

147 PREGNANCY RATES OF RECIPIENT ANIMALS FOLLOWING APPLICATION OF A SELECTIVE PROSTAGLANDIN F2α RECEPTOR ANTAGONIST DURING EMBRYO RECOVERY

Companion research presented at this meeting has indicated that addition of a prostaglandin2α (PGF2α) receptor (FPr) antagonist to culture medium prevented the detrimental action of PGF2α on embryo development. The aim of this study was to evaluate addition of an FPr antagonist to the collection medium on pregnancy rates after transfer of bovine embryos to recipient animals. An initial experiment was performed to determine in vitro development of in vivo-derived morula-stage frozen-thawed embryos cultured in KSOM-PVA medium with 1000 nm AL-8810 (Cayman Chemical Inc., Ann Arbor, MI, USA) (AL, n = 94), 1000 nm AL-8810 and 10 ng mL–1 PGF2α (Cayman Chemical Inc.) (AL+PGF, n = 94), 10 ng mL–1 PGF2α (PGF, n = 94), or serving as controls (CON, n = 91). Embryos remained in their treatment for a 30-h period until blastocyst development was recorded. In a subsequent experiment, embryos were recovered (n = 783) from superovulated donors on Day 7 after artificial insemination with medium containing 1000 nm AL-8810 (AL), 100 nM AL-8810 (AL100), or with vehicle (VEH: 1 mL DMSO; Sigma-Aldrich, St. Louis, MO, USA) in a double blind study. Following collection, embryos were classified by stage and quality, and then transferred fresh to recipients or frozen (ethylene glycol, direct transfer). Frozen embryos, following thawing, were transferred during the subsequent breeding period. Pregnancy rates were determined by ultrasonography (28–35 days post-transfer) and confirmed by calving date. Data were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Results from the initial experiment indicated that culture of in vivo-derived bovine embryos in medium containing AL-8810 improved blastocyst development compared to PGF (58.5% v. 45.7%; P = 0.05). In addition, a strong tendency to increase embryo development was observed in AL+PGF compared to PGF treatment group (57% v. 45.7%; P = 0.07). Overall pregnancy rates of fresh and frozen embryos were increased in the AL and AL100 groups (55% and 58%, respectively) compared to VEH (43%; P = 0.009). Since AL treatments did not differ in pregnancy rates, subsequent analysis combined AL and AL100 data. Transfer of frozen embryos collected with medium containing AL-8810 (n = 238) increased pregnancy rates (AL, 45%) compared to embryos recovered without (n = 221) AL-8810 (VEH, 34%; P = 0.01). Transfer of fresh embryos collected with medium containing AL-8810 (n = 241) tended to have increased pregnancy rates (AL, 76%) compared to control (n = 83; VEH, 66%; P = 0.09). Although data collection continues, no abnormalities in calf health, birth weight, or weaning weight have been observed between any treatments. In conclusion, recovery of embryos with flushing medium containing an FPr antagonist improved pregnancy rates after transfer. Funding was provided by Ultimate Genetics and the Tennessee Agricultural Experiment Station for completion of these studies.

167 EXPRESSION PATTERN OF GAP JUNCTIONAL CONNEXINS DURING IN VITRO AND IN VIVO PREIMPLANTATION EMBRYO DEVELOPMENT IN BOVINE

During preimplantation development, several connexin proteins are expressed and assembled into gap junctions in the plasma membrane at compaction but the functional significance of connexin diversity remains controversial. The present investigations were (i) to compare the expression pattern of a panel of gap junctional connexin (Cx) gene transcripts from in vitro-produced (IVP) under low (5%) and high (20%) oxygen (O2) concentrations and in vivo-derived (IVD) bovine embryos during various preimplantation stages, and (ii) to evaluate the expression of the same set of gene transcripts in blastocysts derived from IVP (low O2 concentration) and in vivo embryos following a conventional cryopreservation method using 1.5 m ethylene glycol (EG) (Hasler et al. 1997 Theriogenology 48, 563–579). Cumulus-oocyte complexes were matured in TCM199 supplemented with 10% FBS and hormones for 22 h at 39�C, 5% CO2 in air, and then inseminated and cultured in SOF medium for 7 days. IVD embryos were collected from 18 superovulated and artificially inseminated cows. In Experiment I, five pooled embryos from each developmental stage (2-, 4-, 8-, 16-cell, morula, and blastocyst) and embryo source (IVP under low and high O2 concentrations and IVD) and, in Experiment II, Day 7 IVP (low O2 concentration) and IVD blastocysts following cryopreservation and storage for at least 1 week were used for analyzing the expression pattern of gap junctional connexin (Cx30, Cx31, Cx32, Cx36, Cx43, and Cx45) gene transcripts using real-time RT-PCR (four replicates). Normalization of mRNAs at each developmental stage of bovine preimplantation embryos was performed by employing a similar amount of RNA at the reverse transcription (RT) step. This was followed by analyzing by qRT-PCR the target genes using GAPDH as a reference gene. Significant differences in gene expression were analyzed byANOVA and Student's t-test. Relative abundances (RA) of Cx30, Cx31, and Cx32 of IVD embryos were significantly (P < 0.05) higher at all stages compared to that of IVP embryos, except at the blastocyst stage for Cx32. Differences in Cx36 and Cx45 were observed at all stages, with the levels being higher (P < 0.05) in IVD than in IVP embryos. However, the differences at the 4-cell stage between the two embryo sources were not significant. The RA of Cx43 transcript in IVD embryos at 4- and 16-cell stages was higher (P < 0.05) than in IVP embryos, but did not differ at the 2-cell stage. Furthermore, there were differences at the 8-cell and blastocyst stages among IVD, and low and high O2 IVP embryos. Following cryopreservation, RA of all analyzed connexin transcripts of IVD blastocysts were significantly (P < 0.05) higher than that of the low O2 concentration. However, the expression levels in both embryo sources were lower compared to blastocysts before cryopreservation, except for Cx36. To the best of our knowledge, this is the first report on the differences in the expression pattern of a panel of gap junctional connexin gene transcripts during the key developmental stages of IVP embryos with direct comparisons of IVD counterparts.

Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos

In vivo-derived bovine embryos are of higher quality than those derived in vitro. Many of the differences in quality can be related to culture environment-induced changes in mRNA abundance. The aim of this study was to identify a range of mRNA transcripts that are differentially expressed between bovine blastocysts derived from in vitro versus in vivo culture. Microarray (BOTL5) comparison between in vivo- and in vitro-cultured bovine blastocysts identified 384 genes and expressed sequence tags (ESTs) that were differentially expressed; 85% of these were down-regulated in in vitro cultured blastocysts, showing a much reduced overall level of mRNA expression in in vitro- compared with in vivo-cultured blastocysts. Relative expression of 16 out of 23 (70%) differentially expressed genes (according to P value) were verified in new pools of in vivo- and in vitro-cultured blastocysts, using quantitative real-time PCR. Most (10 out of 16) are involved in transcription and translation events, suggesting that the reason why in vitro-derived embryos are of inferior quality compared with in vivo-derived embryos is due to a deficiency of the machinery associated with transcription and translation.

234 QUANTITATION OF BOVINE VIRAL DIARRHEA VIRUS ASSOCIATED WITH SINGLE ZONA PELLUCIDA-INTACT IN VIVO-DERIVED BOVINE EMBRYOS

Quantitation of bovine viral diarrhea virus (BVDV) associated with individual transferable embryos is prerequisite to a thorough assessment of the risk for transmission of BVDV via embryo transfer. One objective of this study was to determine the proportion of in vivo-derived bovine embryos that remained virus-positive after artificial exposure to a high-affinity strain of BVDV and thorough washing. A second objective was to determine the quantity of virus associated with these individual embryos. A total of 87 zona pellucida-intact, Day 7, in vivo-derived bovine embryos were exposed to a type 1 noncytopathic strain of BVDV (SD-1) and washed according to International Embryo Transfer Society recommendations. Subsequently, individual embryos were sonicated, and the RNA was extracted from the sonicate fluids and stored at -80�C until assayed using a real-time quantitative polymerase chain reaction (QPCR). Twenty-six percent (23/87) of the embryos contained virus. The average quantity of virus associated with individual embryos after viral exposure and washing was 1.12 viral copies per 5 �L (SD = 1.57 copies 5 per �L-1; SEM = 0.33 copies 5 per �L-1). Assessment of data using tolerance intervals (P = 0.05) indicates that 90% of contaminated embryos will be associated with ≤4.64 viral copies per 5 �L, whereas 99% of contaminated embryos will be associated with ≤6.62 copies per 5 �L. Obviously, only extremely small quantities of virus were associated with less than one-third of the embryos tested. Based on previous research, it is presumed that this virus is associated with the outer layers of the zona pellucida. A logical next step in the risk assessment would be to determine if these quantities of zona-associated virus are sufficient to infect na�ve recipients and/or embryonic cells after embryos are transferred. Further, similar efforts should be made to estimate the quantity of virus associated with in vitro-derived, zona pellucida-intact, bovine embryos after exposure to the same high-affinity strain of virus and washing.

140 LOCALIZATION OF PRIMORDIAL GERM CELLS IN DAY 21 BOVINE EMBRYOS

In most animal species, germ cell precursors, i.e., primordial germ cells (PGCs), arise from the epiblast and then migrate to the future gonadal ridge during development. At least in the mouse, PGCs may be cultured as embryonic germ cells that remain pluripotent. PGCs are the only cells in which OCT4 expression is maintained after gastrulation. The present study aimed at identifying the localization of PGCs in Day 21 in vivo-derived bovine embryos by immunohistochemical staining against OCT4. Six embryos were obtained after slaughter of superovulated heifers 21 days after insemination. The uterine tracts were flushed and embryos fixed, paraffin-embedded, and processed for immunohistochemistry. Embryos were sagitally sectioned, and selected serial sections were immunohistochemically stained for OCT4 to identify potential PGCs. Two embryos were at the neural groove stage. At this stage of development, the primitive gut had not yet been abstricted from the yolk sac and the allantois was not visible. A weak homogeneous OCT4 staining was localized to nuclei in a well-defined region of the epiblast, which was in the process of a gradual anterior to posterior differentiation into neural and surface ectoderm. Moreover, a strong OCT4 staining was localized to a few scattered cells found in the visceral mesoderm associated with the yolk sac in the region of the endoderm-hypoblast transition at some distance from the embryo proper. Four embryos were at the neural tube/somite stage. At this stage of development, the primitive gut had been defined and only the midgut was connected to the yolk sac. Furthermore, the allantois was visible as an anchor-shaped structure at the posterior end of the embryo. A strong OCT4 staining was found in nuclei of solitary cells in the endoderm and its associated visceral mesoderm of the ventral aspect of the mid and hindgut. The described OCT4 staining corresponds well with previous findings in the pig, in which presumptive PGCs are found in the endoderm epithelium during the neural groove stage. Later, during the early somite stages, they are localized in the endoderm and visceral mesoderm of the yolk sac and allantois, and in later somite stages, they are found in the developing genital ridge. This is, however, the first study to demonstrate the localization of these cells, at least by OCT4 staining, in bovine embryos at the neural groove and neural tube/somite stages.

134 CONCEPTION RATES OF IN VITRO-PRODUCED BOVINE EMBRYOS CRYOPRESERVED IN 6% GLYCEROL AND TRANSFERRED BY THE DIRECT METHOD

Ethylene glycol has been used as the standard cryoprotectant for direct transfer of bovine embryos due to its high permeability. But Merton et al. reported that cryoprotectivity of glycerol for bovine embryos was superior to that of ethylene glycol (2001 Theriogenology 55, 312 abst). We previously reported that nonsurgical transfer of in vivo-derived bovine embryos cryopreserved in a lower concentration (5%) of glycerol and thawed by stepwise method resulted in a 55.4% conception rate, whereas direct transfer without removal of cryoprotectant showed only a 45.1% conception rate (Takada et al. 2005 Jpn. J. Embryo Transfer 27, 59–64). In this experiment, survival and conception rates of in vitro-produced (IVP) bovine embryos cryopreserved in 6% glycerol solution (GLY) were compared to those of embryos cryopreserved in 10% ethylene glycol plus 0.1 M sucrose solution (EG). Cumulus–oocyte complexes were matured and fertilized according to Numabe et al. (2000 Theriogenology 54, 1409–1420). Presumed zygotes were cultured in mSOF supplemented with 5% calf serum (CS) and 0.25% linoleic acid albumin at 38.5�C under 5% CO2, 5% O2, 90% N2 for 7 days. At the expanded blastocyst stage, embryos were placed in GLY or EG in PBS supplemented with 20% CS for 15 min at room temperature and loaded into 0.25-mL straws. Straws were placed directly into an alcohol freezer. When the cryoprotectant was GLY, straws were seeded at -4.0�C, held for 10 min, cooled at 0.5�C min to -30.5�C, and then plunged into liquid nitrogen. When the cryoprotectant was EG, the seeding point was -7.5�C, and the plunging point was -34.0�C, but the rest of the protocol was the same as for GLY. In Exp. 1, thawing in both groups was done in a 30�C water bath, and the contents were directly rehydrated in PBS with 20% CS. Thawed embryos were cultured in mSOF with 5% CS for 24 h to assess embryo survival rate, based on the re-expansion of the blastcoele and on their hatching ability. In Exp. 2, embryos in both groups were thawed and transferred to synchronous recipients without removing the cryoprotectant. Data were analyzed using chi-square analysis. In Exp. 1, the developmental rates of post-thaw embryos were similar in GLY (46/52, 88.5%) and EG (45/52, 86.5%); however, the hatching rate was significantly higher (P < 0.05) in embryos cryopreserved in EG (26/52, 50.0%) than in GLY (15/52, 28.8%). In Exp. 2, the conception rates of embryos were similar in both groups, GLY (7/15, 46.7%) and EG (6/15, 40.0%). In conclusion, after direct rehydration of embryos, the developmental ability of IVP bovine embryos cryopreserved in EG was superior to that of embryos cryopreserved in GLY in vitro. However, conception rates in vivo were similar in both groups. These results suggest that a lower concentration of glycerol might be still useful as a cryoprotectant for direct transfer of IVP bovine embryos.

212 TRYPLE SELECT (10X) EFFECTIVELY REMOVES BOVINE HERPESVIRUS-1 ASSOCIATED WITH IN VIVO-DERIVED EMBRYOS

Porcine-origin trypsin will effectively remove bovine herpesvirus-1 (BHV-1) from in vivo-derived embryos. It is not known if TrypLE (Invitrogen, Carlsbad, CA, USA) could be used to remove BHV-1, but this recombinant porcine sequence trypsin-like protease would be an attractive alternative because it is highly stable at room temperature and does not pose the same threat for contamination as animal-origin trypsin. Thus, the objective of this study was to determine if TrypLE Express (1X) for 1.5 min of exposure or TrypLE Select (10X) for 10 min of exposure would be effective at removing BHV-1 from Day 7 zona pellucida-intact, in vivo-derived embryos after they had been exposed to the virus. Day 7 bovine in vivo-derived morulae and blastocysts and non-fertile degenerate (NFD) embryos were collected and shipped overnight to our facility. Upon arrival, the zona pellucida intact embryos were washed according to the International Embryo Transfer Society protocol. Developed embryos were washed separately from NFD embryos. One group of 5 or 10 NFD or developed embryos was not exposed to virus and served as the negative control. The remaining embryos and 10 NFD were exposed to 106 PFU/mL BHV-1 (Colorado strain) for 1 h. Following exposure, one group of 5 or 10 NFD or developed embryos was washed and served as the positive control. One group of 10 developed embryos was washed and treated with porcine origin trypsin. The remaining developed embryos were divided into groups of 5 or 10 and washed and treated with TrypLE Express for 1.5 min or TrypLE Select (10X) for 10 min. Following treatment, the embryos were sonicated in groups of 5 or 10 and assayed by virus isolation. The negative control embryos, porcine origin trypsin treated embryos, and TrypLE Select treated embryos were negative for virus. The positive control embryos and the TrypLE Express treated embryos were positive on virus isolation (Table 1). When it was determined that TrypLE Express was not effective at 1.5 min, TrypLE Select (10X) was used for 10 min. These preliminary results indicate that use of TrypLE Select (10X) for 10 min is effective for removal of BHV-1 associated with Day 7, zona pellucida-intact, in vivo-derived embryos. In addition, TrypLE Select has the advantage of being an animal-origin-free product. However, use of TrypLE Express (1X) for 1.5 min was not effective. Because it is not practical to expose embryos to trypsin for 10 min, further research is needed to determine the ideal treatment concentration and time that will effectively remove BHV-1 without harming in vivo-derived bovine embryos. Table 1. Virus isolation results

Seroconversion of calves following intravenous injection with embryos exposed to bovine viral diarrhea virus (BVDV) in vitro

Two recent studies demonstrated that a high-affinity isolate of BVDV (SD-1), remained associated with a small percentage of in vivo-derived bovine embryos following artificial exposure to the virus and either washing or trypsin treatment. Further, the embryo-associated virus was infective in an in vitro environment. Therefore, the objective of this study was to determine if the quantity of a high-affinity isolate of BVDV associated with single-washed or trypsin-treated embryos could cause infection in vivo. Twenty zona-pellucida-intact morulae and blastocysts (MB) were collected on day 7 from superovulated cows. After collection, all MB were washed according to International Embryo Transfer Society (IETS) standards, and all but 4 MB (negative controls) were exposed for 2 h to 10 5–10 6 cell culture infective doses (50% endpoint) per milliliter (CCID 50/mL) of viral strain SD-1. Following exposure, according to IETS standards, one half of the MB were washed and one half were trypsin treated. All MB were then individually sonicated, and sonicate fluids were injected intravenously into calves on day 0. Blood was drawn to monitor for viremia and(or) seroconversion. Seroconversion of calves injected with sonicate fluids from washed and trypsin-treated embryos occurred 38% and 13% of the time, respectively. Therefore, the quantity of a high-affinity isolate of BVDV associated with single-washed or trypsin-treated embryos was infective in vivo.

134 EVALUATION OF APOPTOSIS IN BOVINE EMBRYOS BY FLUORESCENT LABELING OF CASPASES-3 AND -7

Reports indicate that apoptosis is more prevalent in in vitro-produced (IVP) embryos than in in vivo-derived (IVD) embryos. These studies most often utilize the TUNEL procedure to label fragmented DNA. DNA fragmentation, however, is not unique to apoptosis as cells undergoing necrosis also exhibit substantial DNA fragmentation. Also, improper tissue handling has been reported to induce enough DNA fragmentation for TUNEL-positive staining of nuclei. Thus, the objective of this study was to evaluate the presence of caspases-3 and -7 as indicators of apoptosis in bovine fresh IVD embryos (F-IVD; n = 32), frozen-thawed IVD embryos (FT-IVD; n = 32) and IVP embryos cultured in CR1aa (n = 64). Embryos (quality grades, QG = 1 to 4) were evaluated for apoptotic cells by using a fluorescence-based assay for the detection of active caspaces (Caspatag®, Caspase 3,7 In Situ Assay Kit, Chemicon International, Inc., Temecula, CA, USA). To validate this assay, positive controls (PC) were prepared by incubating IVP-embryos in cyclohexamide for 3 to 4 h prior to staining. After staining, embryos were placed on slides and the number of apoptotic cells/embryo were counted. Embryos in which individual fluorescent points were not apparent were categorized as having profuse staining patterns (PSP) and were assigned an arbitrary apoptotic cell value of 20. Data were analyzed using PROC GLM of SAS with a post hoc LSD test. More than 65% of all embryos displayed at least 1 fluorescent cell, regardless of treatment. PC embryos consistently exhibited PSP. Overall, culture conditions did not markedly affect the incidence of apoptosis. FT-IVD embryos had significantly more (P < 0.05) apoptotic cells (8.2 ± 2.9) than F-IVD (0.9 ± 0.3). However, when embryos exhibiting PSP were excluded, the statistical difference was no longer present (1.5 ± 0.9 vs. 0.9 ± 0.3, respectively). It is unclear why some embryos exhibit specific staining patterns while others exhibit PSP, but we hypothesize that PSP embryos are likely degenerate. Interestingly, different degrees of fluorescence were often present within an embryo. Often, small pinpoint areas of fluorescence were detected while other times the fluorescence appeared to consume the entire cytoplasm of the cell. The capase protein has been localized in the cytoplasm of the cell, so the different degrees of fluorescence may represent different degrees of apoptosis. There was no major effect of culture environment on apoptosis when embryos were categorized by QG. Furthermore, within a treatment, there was no significant difference in apoptosis across embryo QG. To our knowledge, this is the first report of apoptosis evaluated in relation to embryo QG. Based on these findings, apoptosis does not appear to be heavily involved in the reduced developmental potential of IVP bovine embryos. The authors would like to sincerely thank Dr. Neil Schrick for his generous donation of frozen in vivo-derived bovine embryos.

153 EFFECTS ON SEX RATIO AND PREGNANCY RATES OF IN VIVO-DERIVED BOVINE EMBRYOS USING LOOP-MEDIATED ISOTHERMAL AMPLIFICATION SEXING METHOD

In this study, we examined the effects of developmental stages and quality grades on sex ratio of in vivo-derived bovine embryos. Furthermore, pregnancy rates of fresh frozen-thawed sexed embryos or intact (non-sexed) fresh and frozen-thawed embryos were compared in order to efficiently carry out the sexing of embryos in the field. Embryos were collected from donors at 7 days after estrus following a routine superovulation protocol, and classified into four stages (late morula, early blastocyst, blastocyst, and expanded blastocyst) and two quality grades (Grade 1 and Grade 2–3) by the IETS manual. Embryos were frozen by direct transfer method from 1 to 3 h post-collection in 0.25-mL straws as described previously (Aoyagi et al. 1996 Theriogenology 45, 165 abst). Frozen embryos were thawed in 30°C water for 20 s following 7 s in air. They were then squeezed out into PBS + 5% FCS (PBS), washed twice, and incubated in CR1aa + 5% CS (CR1aa) or PBS. Recently, a commercial embryo sexing program was performed at our laboratory using loop-mediated isothermal amplification (LAMP). The procedure takes 5 min to perform each embryo biopsy and only 40 min for the LAMP process. A few cells of fresh (F; n = 105) and frozen-thawed (Z; n = 143) embryos of Grade 1 (H), and fresh (F; n = 77) embryos of Grade 2–3 (L) were biopsied with a microsurgical blade, and sex was determined by the LAMP method. Embryos were transferred non-surgically into heifers on Day 7 of the estrus cycle. Pregnancies were determined by ultrasonography on Day 30. Data were analyzed by the chi-square test. The sexing of all 325 embryos yielded 148 female (46%), and only 2 embryos were indeterminant (1%). There was no evidence of any effect of developmental stage on sex ratio (female embryos: late morula 69/157 (44%), early blastocyst 42/94 (45%), blastocyst 29/53 (55%), and expanded blastocyst 10/21 (48%)). However, when the sex ratio was examined for embryos of different quality grades, significantly more females were found in the embryos appearing more degenerated (female embryos: FH + ZH vs. FL; 42% vs. 57%, P < 0.05). Pregnancy rates on Day 30 with FH embryos (38/45, 84%) were similar to rates obtained with non-sexed fresh (60/81, 78%) and frozen-thawed embryos (44/54, 82%). The pregnancy rates on Day 30 with ZH embryos incubated in CR1aa (18/40, 45%) were lower than those of FH, non-sexed fresh, and frozen-thawed embryos. However, pregnancy rates of ZH embryos incubated in PBS (13/16, 81.3%) were significantly higher than for those frozen embryos that were thawed and incubated in CR1aa (P < 0.05). After the transfer of embryos sexed by the LAMP method to recipient animals, all 55 calves born were of the predicted sex. In conclusion, the present results showed that with the LAMP method for sexing of the embryos, there were only a few samples for which sex could not be determined. Examination of in vivo-derived Day 7 embryos indicated that female embryos graded lower than male embryos. Furthermore, the removal of a few cells from a fresh or frozen-thawed embryo did not impact its subsequent viability.

247 MESSENGER RNA EXPRESSION PATTERNS OF DNA AND HISTONE METHYLTRANSFERASES IN PREIMPLANTATION DEVELOPMENT OF IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS

The effects of in vitro production (IVP) and/or somatic nuclear transfer on mRNA expression patterns have mostly been determined in morulae and blastocysts, i.e. after embryonic genome activation. Comparative data regarding mRNA expression patterns throughout the oviductal phase of pre-implantation development are scarce. Here we studied mRNA expression for genes related to DNA methylation and modification of histones which account for the major epigenetic reprogramming during development. Pertubated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases associated with assisted reproduction technologies. The objective of the present study was to compare mRNA expression of DNA methyltransferases Dnmt1, -3a, and -3b and histone methyltransferases SUV39-h1 and G9a between in vivo-derived bovine embryos and their IVP counterparts using a semiquantitative RT-PCR assay (Wrenzycki et al. 2002 Biol. Reprod. 66, 127-134) employing two embryos for each assay. In vivo-derived embryos were collected from 28 superovulated heifers by endoscopic flushing of oviducts (zygotes to 8-cell stages) (Besenfelder et al. 2001 Theriogenology 55, 837-845) or by uterine flushing (16-cell stages to blastocysts). Endoscopic flushing at different time points after AI (Days 1, 1.5, 2, 3, 4, and 4.5) yielded 31 zygotes; 15 two-cell, 5 three-cell, 13 four-cell, 1 five-cell, 2 six-cell, and 11 eight-cell embryos; 4 degenerated embryos; and 18 unfertilized ova. The recovery rate (corpora lutea counted per recovered embryos) was 58% and 62% for the endoscopic and uterine flushing, respectively. Differences in the relative abundance of each gene transcript between groups were tested using ANOVA with the main effects being origin (in vivo/in vitro) and developmental stage (zygote to blastocyst) and their interactions followed by multiple pairwise comparisons using a Tukey test (P < 0.05). Origin of embryos affected the relative abundance of transcripts for Dnmt1, Dnmt3a, and SUV39-h1, and developmental stage affected the relative abundance of transcripts for Dnmt1, -3a, -3b, SUV39-h1, and G9a. No interactive effects were observed for origin and developmental stage in the relative abundance of all transcripts. The relative abundance of Dnmt1 transcripts differed significantly between in vivo- and in vitro-produced morulae and blastocysts. For Dnmt3a, mRNA differences were determined between in vivo- and in vitro-produced 10-16-cell stages and morulae. Suv39-h1 transcripts differed significantly between in vivo- and in vitro-derived zygotes, 2-cell embryos, 8-cell embryos, 10-16-cell embryos, and blastocysts. The results suggest that IVP alters mRNA expression of genes related to epigenetic modifications very early in development, even before the embryonic genome has been activated.

Different strains of noncytopathic bovine viral diarrhea virus (BVDV) vary in their affinity for in vivo-derived bovine embryos

Washing procedures (without trypsin treatment) recommended by the International Embryo Transfer Society (IETS) for use on in vivo-derived embryos effectively removed a cytopathic strain (NADL) of bovine viral diarrhea virus (BVDV) after artificial exposure. However, these washing procedures have not been evaluated using other isolates of BVDV, including representative non-cytopathic strains. Thus, the objective of this study was to evaluate the efficacy of the IETS procedures following artificial exposure of in vivo-derived bovine embryos to two different strains and biotypes of BVDV. One hundred and twenty-nine zona pellucida-intact (ZP-I) morulae and blastocysts (MB) and 56 non-fertile and degenerated (NFD) ova were collected 7 days following exposure to bulls from 32, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to IETS standards. Subsequently, half of the MB and NFD ova were exposed for 1 h to approximately 106-cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1, and the other half were exposed to the same concentration of CD-87. After exposure, groups of ≥3 and ≤10 MB or NFD ova were washed using methods that met or exceeded IETS standards. Then, the washed groups were sonicated, and sonicate fluids were assayed for presence of virus using virus isolation and a reverse transcription nested polymerase chain reaction. No virus was detected in any group of MB or NFD ova that had been exposed to the CD-87 isolate. However, virus was detected in association with 50% of the groups of MB and 33% of the groups of NFD ova that had been exposed to the SD-1 isolate. Therefore, standard embryo-washing procedures recommended by the IETS are more effective for removal of some isolates of BVDV than for others. It remains to be determined if the quantity of a high-affinity isolate of BVDV associated with individual washed embryos would infect recipients via the intrauterine route. Further, it should be determined if an alternative embryo processing procedure, washing and trypsin treatment, would be more effective for removal of high-affinity isolates.

Infectivity of bovine viral diarrhea virus associated with in vivo-derived bovine embryos

Early research indicated that bovine viral diarrhea virus (BVDV) would not adhere to zona pellucida-intact (ZP-I), in vivo-derived bovine embryos. However, in a recent study, viral association of BVDV and in vivo-derived embryos was demonstrated. These findings raised questions regarding the infectivity of the embryo-associated virus. The objectives of this study were to evaluate the infectivity of BVDV associated with in vivo-derived bovine embryos through utilization of primary cultures of uterine tubal cells (UTC) as an in vitro model of the uterine environment and to determine if washing procedures, including trypsin treatment, were adequate to remove virus from in vivo-derived embryos. One hundred and nine ZP-I morulae and blastocysts (MB) and 77 non-fertile and degenerated (NFD) ova were collected on day 7 from 34, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to International Embryo Transfer Society (IETS) standards and exposed for 2h to approximately 106 cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1. Following exposure, some groups of <10 MB or NFD ova were washed in accordance with IETS standards. In addition, an equivalent number of MB and NFD ova were subjected to IETS standards for trypsin treatment. Subsequently, NFD ova were immediately sonicated and sonicate fluids were assayed for presence of virus, while individual and groups of MB were placed in microdrops containing primary cultures of UTCs and incubated. After 3 days, embryos, media, and UTCs were harvested from each microdrop and assayed for BVDV. Virus was detected in the sonicate fluids of 56 and 43% of the groups of NFD ova that were washed and trypsin-treated, respectively. After 3 days of microdrop culture, virus was not detected in media or sonicate fluids from any individual or groups of MB, regardless of treatment. However, virus was detected in a proportion of UTC that were co-cultured with washed groups of MB (30%), washed individual MB (9%) and trypsin treated individual MB (9%), but no virus was detected in the UTC associated with groups of trypsin-treated embryos. In conclusion, virus associated with developing embryos was infective for permissive cells. Further, the quantity of virus associated with a proportion of individual embryos (both washed and trypsin treated) was sufficient to infect the UTC. In light of these results, an attempt should be made to determine if the quantity of a high-affinity isolate of BVDV associated with an individual embryo would infect recipients via the intrauterine route.