Advances in genome editing technologies, such as the CRISPR/Cas9 system, have facilitated gene manipulation and the generation of pigs resistant to disease, models for studying human diseases and for xenotransplantation. However, double-strand breaks generated by the CRISPR/Cas9 system are preferentially repaired by the non-homologous end joining (NHEJ) pathway compared with high-fidelity homology-directed repair (HDR). All reports of pigs created by zygote injection of the CRISPR/Cas9 system result from NHEJ rather than HDR. The molecule known as RS-1 was found to stimulate RAD51 and thus enhance HDR. Thus, our goals are to understand this pathway by evaluating the response, as well as dosage and temporal effects, of RS-1 on porcine embryo development, and to determine a safe concentration to achieve high HDR rates without affecting embryo development. As RS-1 was added to culture medium from a 7.5mM stock solution in DMSO, we also evaluated whether RS-1 in solution could have affinity and migrate to the mineral oil overlay placed in the media during embryo culture. After IVF, embryos were cultured without RS-1 (control groups) or in the presence of RS-1 at 7.5 and 15 µM. At 7.5 µM, RS-1 improved HDR in rabbits created by zygote injection of the CRISPR/Cas9. Based on that, we designed five experimental groups to evaluate short- (A, 20h) and long-term (B, 144 h) effects of RS-1: two groups in the presence of RS-1 at 7.5 µM (7.5 µM A and 7.5 µM B), two control groups (control A and control B), and a group in the presence of RS-1 at 15 µM A for short-term exposure only. First, embryos in the groups containing (7.5 µM A, 7.5 µM B, and 15 µM A) and without RS-1 (control A and control B) were cultured for 20h without the mineral oil overlay. Then, embryos from control A, 7.5 µM A, and 15 µM A groups were washed and cultured without RS-1 until Day 6 in the presence of the mineral oil overlay. To evaluate long-term effects of RS-1, embryos from control B and 7.5 µM B groups were washed and transferred to medium without or with RS-1 at 7.5 µM, respectively, and cultured until Day 6 with no mineral oil overlay. We report the mean values for each set of data±s.e.m., and the degree of statistical significance in all analyses was defined at P<0.05. We observed that the presence of RS-1 at 15 µM decreased cleavage rates (control A 79.1±2.7%; 7.5 µM A 83.6±1.6%; 15 µM 70.9±4.4%; control B 80.8±1.4%; 7.5 µM B 80.0±2.8%), and long-term exposure to RS-1 decreased development to the blastocyst stage (control A 46.5±3.0%; 7.5 µM A 42.7±3.4%; 15 µM 41.8±4.7%; control B 44.9±3.3%; 7.5 µM B 28.8±4.9%). In addition, RS-1 exposure decreased the total cell number compared with the controls (control A 61.4±2.8; 7.5 µM A 49.2±3.0; 15 µM 48.5±2.3; control B 59.9±3.4; 7.5 µM B 41.5±2.5), however no differences in apoptosis rates were observed between the treatments. Our work will serve as a basis to understand the effects of RS-1 and RAD51 during embryonic development and to improve the applicability of customizable nucleases for the production of genetically modified pigs.

One barrier to successfully making embryo transfer viable in the swine industry is an inability to consistently cryopreserve oocytes and embryos. This process is made difficult by the high lipid content of porcine oocytes and embryos. The objective of this study was to test the in vivo fertilized embryo’s sensitivity to vitrification. Gilts were inseminated on the first day of standing oestrus (Day 0) and then again 12 h later. On Day 2 the oviducts and tip of the uterine horns were flushed with PVA-treated TL-HEPES and 2-cell stage embryos were collected and placed into PVA-treated TL-HEPES and centrifuged at 17 000 × g. The treatment groups were 1) 300 mOsmo centrifuged for 6 min, 2) 500 mOsmo centrifuged for 6 min, 3) 500 mOsmo centrifuged for 12 min, and 4) 500 mOsmo centrifuged for 18 min. After centrifugation the embryos were transferred to Porcine Zygote Medium 3 (PZM3) and cultured to Day 6 or 7 at which point blastocysts were vitrified using 10% DMSO, 10% ethylene glycol in M199 supplemented with 20% FBS (holding medium) for 2 min. Embryos were transferred to holding media with 20% DMSO and 20% ethylene glycol and drawn into an open pulled straw via capillary reaction; it was then submerged into LN2. Embryos were thawed using a step down concentration of 0.33 mM and then 0.2 mM sucrose in holding media each for 6–7 min and then were moved to holding medium alone for 6 to 7 min. The embryos were washed in PZM3, then transferred to 500 μL of PZM3 and cultured for 18 h. Re-expanded embryos were observed, and the nuclei of all embryos were stained with Biz-benzimide and visualised with UV light to determine total cell number. After the embryos were centrifuged and cultured, there was no difference in development to blastocyst (SAS Institute, Cary, NC, USA; Proc GLM) with a mean percentage blastocyst of 85.1% and an N of 54, 51, 53, and 51, respectively, for each treatment. After thawing, percentage of embryos re-expanded was 23.5a, 26.4a,b, 43.2a,b, and 45.6b, respectively. Data was analysed using a PROC GLM in SAS (P < 0.05), with 37, 43, 30, and 36 embryos in each group, respectively. No difference in total cell number across treatments was detected after analysis using PROC GLM in SAS (P < 0.05) with a mean cell number of 29.0. These data suggest that in vivo matured and fertilized blastocysts can survive high osmolarity treatment, centrifugation, and vitrification. The data also show that a high osmolarity treatment centrifuged for 18 min leads to a greater number of re-expanded embryos post-thaw, which may be attributed to better separation of the lipid. Funded by the NIH NCRR R21RR025879 and Food for the 21st Century.

Most mammalian embryo culture media contain some form of unidentifiable biological contaminant, usually associated with fetal bovine serum (FBS) or bovine serum albumin (BSA). Such factor(s) confound experiments attempting to evaluate culture media composition and decrease the repeatability of experiments when different lots or batches of FBS or BSA are used. The goal of this study was to formulate a completely chemically defined culture media for development of early porcine embryos based on adding ligands for which there is the presence of mRNA for the corresponding receptors in the blastocyst. Cumulus–cell oocyte complexes were matured for 42 h in M199 supplemented with EGF, FSH, and LH. Metaphase II oocytes were selected and fertilized in modified Tris-buffered media for 4 h. Presumptive zygotes were then placed into porcine zygote media with 0.3% BSA (PZM3) or 0.1% polyvinyl alcohol (PZM4). At 28 h post-fertilization, 2- to 4-cell stage embryos were selected and placed into treatment groups for 5 days. Fifteen embryos were put into 25 μL of media and cultured in 5% CO2, 5% O2, and 90% N at 38.5°C. The treatment groups were as follows: 1. PZM3, 2. PZM4, 3. PZM4 + 0.5 mM N-methyl D-aspartic acid (NMDA), 4. PZM4 + 0.5 mM NMDA + 10 μM homocysteine (HC), and 5. PZM4 + 10 μM HC. There were 120, 135, 120, 135, and 120 embryos for each treatment, respectively. The percentages of embryos that developed to the blastocyst stage were 63.3%a, 29.7%b, 46.1%c, 55.6%ac, and 49.2%ac, respectively [SAS; SAS Institute, Cary, NC, USA) Proc GLM (a,b,cP < 0.05)]. Total cell number was determined using bisbenzimide to stain the nuclei, and the data were analysed by SAS Proc GLM. There was no difference in cell number among treatments with a mean cell number of 31.4. To further investigate the equality of the chemically defined media, the surface area of the blastocysts was measured by using Nis Elements BR3.0 software under 20× magnification. There was less surface area in treatment 5 compared with 4 [296 180ab, 295 114ab, 303 451ab, 271 913b, and 316 773a arbitrary units (a,bP < 0.05), with n = 33, 26, 37, 31, and 32 embryos in each treatment, respectively]. Because HC has been shown to affect global DNA methylation of bovine embryos, we stained our embryos for 5-methylcytidine (Eurogentec anti 5-MECY-0100) and embryos were visualised by UV light with a Texas red filter, and intensity was measured by Nis Elements BR 3.0 software under 20X magnification. The mean intensities were lower for the NMDA treatment [26.7a, 19.4a, 13.6b, 17.0a, and 19.4a arbitrary units (a,bP < 0.05)] compared with the other treatments. When embryos were cultured without BSA development decreases, but adding NMDA and HC returns development to control levels as measured by percentage of blastocysts, surface area, and global DNA methylation. We conclude that PZM4 supplemented with 0.5 mM NMDA and 10 μM HC may replace PZM3 as a chemically defined culture media for early porcine embryos. Embryo transfer experiments will be necessary to confirm that these embryos have equal developmental competence. Funded by the NRI (2006-35203-17282) and Food for the 21st Century.

In vitro culture systems are suboptimal as compared to in vivo. Previous next-generation sequencing analysis of in vivo fertilized and in vitro cultured (IVC) or in vivo cultured (IVV) porcine blastocyst stage embryos identified an arginine transporter (SLC7A1) expressed 63 fold higher in IVC compared to IVV blastocysts (Bauer et al. 2010 Biol. Reprod. Epub ahead of print). Arginine catabolism may play important roles in placental and conceptus growth and development as it is a substrate for synthesis of nitric oxide synthase and polyamines. The objective of this study was to determine the effects arginine had on both embryo development and mRNA expression in in vitro fertilized embryos. Cumulus–oocyte complexes were matured for 44 h in M199 supplemented with EGF, FSH, and LH. Oocytes with a visible polar body (metaphase II) were selected and fertilized in modified Tris Buffered Medium for 5 h and then placed into one of 5 treatment groups (Porcine Zygote Medium 3 (PZM3) with 0 mM, 0.12 mM (current concentration of arginine in PZM3), 0.36 mM, 0.72 mM, or 1.69 mM arginine). Twenty-eight hours post-fertilization, cleaved embryos were selected and moved into 25 μL drops of respective culture media and cultured to day 6 in 5% CO2, 5% O2, 90% N2 at 38.5°C. To determine the effect arginine had on development the percent of embryos that made it to the blastocyst stage for each treatment group were analysed using PROC GLM in SAS (SAS Institute, Cary, NC). A least significant difference post test comparison was completed to determine if significant differences existed between treatment groups (a,b,cP < 0.05). The percentage of cleaved embryos on Day 6 that developed to blastocyst was 57.2%b,c, 50.2%c, 67.3%a,b, 67.3%a,b, 70.4%a (N = 147, 163, 150, 120, and 134) in 0 mM, 0.12 mM, 0.36 mM, 0.72 mM, and 1.69 mM arginine, respectively. Real-time PCR was then completed to assess the affect arginine supplementation had on SLC7A1 mRNA expression. Three biological replicates, each containing 10 blastocyst pools to ensure enough starting material, were collected for each treatment group. RNA was isolated from each sample and 5 μL was linearly amplified (NuGEN Ovation Pico WTA System) so multiple genes could be compared and then purified using Bio-Rad MicroSpin Columns. Expression levels were calculated relative to the reference sample and the housekeeping gene, YWHAG. The ΔΔCT values were log-transformed and analysed using PROC GLM in SAS. The expression of SLC7A1 mRNA was decreased (P = 0.0006) compared to PZM3 in the 1.69 mM arginine group. These results illustrate the positive effects that additional arginine may be having on porcine embryo development during culture from the 2-cell to the blastocyst stage. Supplementing arginine to a final concentration of 1.69 mM during culture increases development of porcine embryos to blastocyst compared to PZM3 and also decreases the expression of SLC7A1. Evaluation of the transcriptional profile appears to be a good method of letting the embryo tell us what it needs for development, and in this case arginine. Funded by F21C.

cDNA derived from trophectoderm (TE) and embryonic disc (ED) of a single day 12 porcine embryo was subjected to next-generation sequencing using the Illumina platform. The short sequencing reads from triplicate sequencing runs were aligned to a custom database designed to represent the known porcine transcriptome. As expected, genes involved in epithelial cell function and steroid biosynthesis were more abundant in cells from the TE; genes involved in maintenance of pluripotency and chromatin remodeling were more highly expressed in cells from the ED. Quantitative real-time PCR was used to confirm the validity of the approach. We conclude that gene expression profiles of even extremely small samples (<= 1,000 cells) can be adequately described without RNA/cDNA preamplification. We also demonstrate the utility of pre-genome genomics resources-such as EST repositories-in the analysis and application of next-generation sequencing data in the absence of an appropriately annotated reference genome.

In vitro embryo culture systems promote development at rates lower than in vivo systems. The goal of this project was to discover transcripts that may be responsible for a decrease of embryo competency in blastocyst-stage embryos cultured in vitro. Gilts were artificially inseminated on the first day of estrus, and on Day 2, one oviduct and the tip of a uterine horn were flushed and the recovered embryos were cultured in porcine zygote medium 3 for 4 days. On Day 6, the gilts were euthanized and the contralateral horn was flushed to obtain in vivo derived embryos. Total RNA was extracted from three pools of 10 blastocysts from each treatment. First and second strand cDNA was synthesized and sequenced using Illumina sequencing. The reads generated were aligned to a custom-built database designed to represent the known porcine transcriptome. A total of 1170 database members were different between the two groups (P < 0.05), and 588 of those had at least a 2-fold difference. Eleven transcripts were subjected to real-time PCR that validated the sequencing. There was an overall decrease in inner cell mass (ICM) and trophectodermal (TE) cell numbers in embryos cultured in vitro; however, no difference in the ICM:TE ratio was found. Interestingly, the transcript SLC7A1 was higher in the in vitro cultured group. This difference disappeared after addition of arginine to the 4-day culture. Illumina sequencing and alignment to a custom transcriptome identified a large number of genes that yield clues on ways to manipulate the culture media to mimic the in vivo environment.

Skin-derived progenitors (SKP) are neural crest derived and can generate neural and mesodermal progeny in vitro, corresponding to the multipotency of neural crest stem cells. Likewise, neural stem/progenitor cells (displaying as neurospheres) have the capacity of self-renewing, and can produce most phenotypes in the nervous system. Both form spheres when cultured with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Although the "stemness" of neural stem/progenitor cells has been extensively investigated, the molecular comparison of SKP spheres and neurospheres has not been elucidated. Here, SKP spheres and neurospheres from the same individual porcine fetuses were isolated with the same culture medium, and the multipotency was tested by in vitro differentiation assays. Microarray analysis was used to illustrate the "stemness" of SKP spheres and neurospheres. The upregulated genes that were in common in the SKP spheres and neurospheres are involved in ribosome, tight junction, gap junction, cell communication, calcium signaling, ErbB signaling, JAK-STAT signaling, MAPK signaling, etc. The differentially expressed genes between SKP spheres and neurospheres are mainly involved in ECM-receptor interaction and the transforming growth factor-beta (TGF-b) signaling pathway. Finally, treatment with leukemia inhibitory factor (LIF) or MEK inhibitor results in a distinctive impact on the "stemness" and differentiation genes of SKP spheres and neurospheres. Thus, the cell-intrinsic genetic program may contribute to the innate "stemness" of SKP spheres and neurospheres in a similar local microenvironment.

One major obstacle in mammalian embryo culture has been unidentifiable biological contaminants in the media due to the inclusion of Bovine Serum Albumin (BSA) or Fetal Bovine Serum. The goal of this study was to remove BSA from culture media and develop chemically defined media based off the embryo’s biological and physiologic makeup. We evaluated the presence of message in various stages of porcine embryos and found that the message for the ionic glutamate receptor, N-Methyl-D-aspartic acid (NMDA) increased about 3-fold from oocyte to blastocyst. Thus, this study was conducted to determine if the addition of NMDA (0.5 mm) would improve development of embryos in an already chemically defined medium. Slaughterhouse derived ovaries were aspirated, cumulus–oocyte complexes were identified and then matured for 42 h in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were selected and fertilized in modified Tris buffered medium with 0.25 × 106 mL–1 frozen–thawed porcine semen for 5 h. Presumptive zygotes were then transferred to Porcine Zygote Medium with 0.3% BSA (PZM3) or 0.1% PVA (PZM4). After 28 h, cleaved embryos were selected and embryos were placed into treatment groups: (1) PZM3, (2) PZM4, or (3) PZM4 + 0.5 mm NMDA. Embryos were cultured in 5% CO2, 5%O2, 90% N2 until Day 7. For this experiment the number of cleaved embryos cultured in each treatment group were 260 for group 1, 220 for group 2 and 300 for group 3. Percentage of development to blastocyst was determined and analyzed with SAS Proc GENMOD Procedure (a,b P < 0.05). The percentage developed to blastocyst was (1) 47.5% a, (2) 29.6% b, and (3) 36.1% a,b, respectively. Total cell number of the blastocysts was determined by using Hoechst nuclear stain and statistically analyzed by SAS Proc GENMOD Procedure. The average cell number for the treatment groups was (1) 25.8 a, (2) 19.6 b, and (3) 22.9 a,b, respectively. Culture without BSA significantly reduced development to blastocyst and total cell number; however, with the addition of 0.5 mm NMDA there was no significant difference from media containing BSA. This indicates that NMDA can be used to partially replace BSA to form a chemically defined media. Funded by a grant from the USDA NRI 2006-35203-17282.