Brief Summary of Minutes of Annual Meeting

Minutes of the W-2171 Multi-State Project Meeting January 6, 2012 Renaissance Glendale Hotel and Spa Phoenix, Arizona Attendees (Station): Milan Shipka (AK) Rick Rorie (AR) Patricia Berger (CA) Pablo Ross (CA) George Seidel (CO) Curt Youngs (IA) Ken Bondioli (LA) Carol Keefer (MD) Erdogan Memili (MS) Brett White (NE) Clay Isom (UT) Mark Mirando USDA by conference call The meeting was called to order by the project Chair Erdogan Memili at 1:00 PM. Following correction of several name spellings the minutes of the previous meeting were approved. Attending stations presented brief summaries of the reports included in the distributed Technical Report. Dr. Mark Mirando joined the meeting through teleconference and reviewed the handouts concerning the National Institute of Food and Agriculture (NIFA) that had been distributed to the members. The first Director of NIFA resigned in May 2011 and Dr. Chavonda Jacobs-Young is serving as Acting Director. Interviews for a replacement have been conducted and results sent to the White House. Current priorities for funding will remain the same or 2012. It was summarized that NIFIA faired fairly good in the 2012 appropriated budget. Funding for the AFRI competitive programs remained essentially the same compared to last year. 2012 RFAs for Global Food Security, Food Safety, Sustainable Bioenergy and the Fellowships Grant Programs have been released. The RFA for the Foundation Program is expected to be released in March 2012. Letters of intent would be due approximately 6 weeks after the release and applications would be due approximately 3 months after the release. The informal group of Rosenkrans, Memili, Ross and Bondioli reported on their efforts to create a collaboration from within the group to apply for an anticipated large integrated grant for reproduction. This group met after the previous meeting and on several occasions throughout the year by telephone or email. An outline of potential topics for such an application was generated and distributed to the project members. During these discussions it was anticipated that the 2012 Food Security RFA would include an opportunity to apply for a planning grant to assemble a team and develop a proposal. When the 2012 RFA was released there was not a provision for a planning grant but called for full proposals addressing Translational Genomics for Improved Fertility in Animals. The group decided that there was not sufficient time to assemble a competitive team and proposal for this opportunity. Additional opportunities for collaborations within the project membership were discussed and encouraged. One opportunity that was discussed includes collaboration between groups with experience and availability of deep sequencing technology and groups with physiological models where this technology would be appropriate. Pablo Ross (CA) and Erdogan Memili (MS) reported that they had established such collaboration. Trish Berger (CA) suggested the possibility of a collaboration to establish a proposal addressing the Climate Change Priority. Such a proposal could address heat stress and mitigating this effect of climate change through genetics and Biotechnology. The meeting next year cannot be associated with the annual IETS meeting because this meeting is outside of the US. It was agreed that the meeting would be held at Utah State University with a target date of late February. It was tentatively planned that the meeting would be 1.5- 2 days over Friday-Saturday and include a keynote speaker and short presentations from the attending stations. Pablo Ross was unanimously selected as the incoming project secretary. The meeting was adjourned at 5:00 PM. Respectfully submitted. Ken Bondioli 2011-2012 Secretary

ACCOMPLISHMENTS. Objective 1: Understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis. Osteopontin is a fertility-associated protein found in higher concentrations in the seminal plasma of bulls that produce higher conception rates. Bulls were evaluated for polymorphisms within the osteopontin gene promoter as potential markers for fertility. A modified long-term progestin-Select Synch estrous synchronization protocol was developed and evaluated for use in for beef cattle. Semen collected during hot weather has poor viability after cryopreservation. A study evaluated the effects of organic and inorganic trace mineral supplementation on semen collected and cryopreserved during hot weather. Data suggest porcine oocyte fertilizability is very sensitive to short-term preovulatory heat stress. Together with previous observations on detrimental effects of slightly elevated temperature on in vitro maturation, these data suggest elevated ovarian temperature contributes to the reduced oocyte quality. RNASeq analysis in single blastocyst was successfully achieved, allowing for global gene expression profiling, SNP discovery, allelic specific expression analysis, and of characterization of unannotated bovine genes. MicroRNAs clearly are present in bovine oocytes and preimplantation embryos, and furthermore likely have important regulatory functions in these cells. We also demonstrated that the antioxidant cysteamine could counteract the negative effects of culturing embryos in 5% CO2 in air relative to using lower oxygen concentrations. We showed that vitrification of in vivo-produced bovine embryos could be accomplished satisfactorily in 0.25-ml plastic straws suitable for direct transfer, although results were not satisfactory for in vitro-produced embryos. We collected liver and placental tissue samples from in vitro produced and in vivo control bovine fetuses at days 90 and 180 of gestation. We used a bovine 13K oligonucleotide microarray to investigate the transcriptional profiles in both tissues of IVP fetuses, and compared them with those of their age-matched in vivo counterparts. We cultured mouse oocytes under microgravity condition simulated by NASAs rotary cell culture system, examined the maturation rate and observed the spindle morphology (organization of cytoskeleton) during the mouse oocytes meiotic maturation. The presence of osteopontin (SPP1) mRNA in oocytes and cumulus cells and the larger mRNA abundance before maturation suggests a role of this protein prior maturation of oocytes. Microfluidic devices provide the opportunity to culture oocytes and embryos individually, with development equal to that of standard culture drops. These results demonstrate that this microfluidic system provides an efficient way to successfully mature oocytes individually. The post-thaw motility of stallion spermatozoa was higher in 5% egg yolk than 20% egg yolk, but jack spermatozoa exhibited higher post-thaw motility when cryopreserved in high than low egg yolk. For both species, post-thaw motility was higher for spermatozoa cryopreserved in 60 mM ²-CD than 0 mM. Spermatozoa cryopreserved in low egg yolk exhibited better CASA parameters than those cryopreserved in high egg yolk for both species, and the use of 60 mM ²-CD likewise resulted in poorer post-thaw CASA parameters in both species. All post-thaw treatments designed to induce acrosome reaction caused a decrease in viability compared with the control. Post-thaw viability of stallion spermatozoa remained relatively constant over time whereas viability of jack spermatozoa decreased by nearly 30% from 0 to 90 min. Post-thaw treatment of stallion sperm cells with b-CD increased sperm acrosome reaction from 5% (90 min) to 22% (90 min). The percentage of acrosome-reacted control jack sperm cells went from 11% (0 min) to 31% (90 min), whereas those treated with b-CD went from 12% (0 min) to (85% (90 min). It was demonstrated that bovine epididymal sperm had lower levels of cryopreservation induced acrosome reaction but similar levels of cryopreservation induced capacitation compared to ejaculated sperm and that epididymal sperm were less dependent upon the capacitation agent, heparin to support in vitro fertilization. It was demonstrated that cat epididymal sperm could be frozen in a completely defined extender and that addition of cryoprotectant after gradual cooling was beneficial to post-thaw survival. Increasing fertility in dairy cattle is an important goal. Male infertility represents a part of the overall infertility in dairy cattle, and can be partitioned into compensatory and non-compensatory components where compensatory refers to infertility which can be overcome by increasing sperm number and non-compensatory infertility represents the remainder, presumably due to molecular and genomic defects. Through estimation of single nucleotide polymorphism (SNP) association with non-compensatory bull fertility, identifying regions of the genome influential to this trait is possible. Use of this information in selection can allow for an increase in cattle fertility resulting in economic benefits. In this study, high density SNP genotypes and non-compensatory fertility data from 795 Holstein sires were used to examine SNP association with fertility. A Bayes B analysis was performed to develop information for genomic selection and to identify genomic regions associated with non-compensatory fertility. A cross-validation approach was used to assess effectiveness of the models within the original set of 795 bulls. Correlations of predicted and observed fertility values were approximately 0.145 in cross-validation. Fertility is one of the most economically important traits controlling animal reproduction. Despite significant economic impact, there are no reliable markers to predict semen quality. The objective of this study was to identify spermatozoal proteins associated with bull fertility, ability of the sperm to fertilize the oocyte and support embryonic development. To accomplish our objectives, we isolated total spermatozoal proteins from four Angus bulls with different fertility records. Next, differentially expressed proteins were determined using two dimensional in gel electrophoresis (2D-DIGE) followed by sequencing of the most differentially expressed proteins. Immunoblotting experiments were conducted to confirm expression of key proteins detected by 2D-DIGE. Our results from 2D-DIGE experiments showed around 2000 detectable spermatozoal proteins. Of these comprehensive lists of proteins, we identified 80 of the proteins with highest differentially expression in spermatozoa from high and low fertility bulls. Diverse sets of differentially proteins known to play roles in sperm motility, metabolism and cell morphology were identified. These proteins included Outer dense fiber of sperm tails 2 (ODF2) and Manganous superoxide dismutase (MnSOD), Heat Shock Protein, Tubulins, Alpha enolase, and Acrosomal vesicle protein-1. Expression profiles of ODF2 and MnSOD were confirmed using immunoblotting. The findings are significant because they help us understand fundamental biology of the male gamete and early development. In addition, the identified proteins can be used as molecular markers to predict bull fertility, an economically important trait. Transgenic pigs were produced expressing a spermatogonial marker gene. The full-length bAIRN ncRNA has been characterized as a transcript whose length is approximately 65kb that is transcribed from a promoter located approximately 440bp upstream of DMR2 on the IGF2R antisense strand. Analysis of serum progesterone levels at the time of embryo transfer in relation to conceptus development at Day 17 of gestation was performed and was found to vary based on embryo production source (in vivo vs. in vitro). The results of immunohistochemistry indicate a co-localization of FAK protein clusters that involves specific oocyte membrane integrin proteins during fertilization. These findings illustrate the formation of focal adhesions via colocalization of signalling molecules with integrin ² subunits in bovine oocytes and further support the hypothesis that induction of signalling pathways is initiated with sperm-oocyte binding in the bovine. These data illustrate the formation of focal adhesions indicated by colocalization of ±V and FAK at the site of spermatozoa binding to the vitelline membrane. These results provide evidence that spermatozoa interact with integrins, known outside-in signaling complexes, at the level of the vitelline membrane in bovine oocytes. These events could serve as the initiation of signaling cascades associated with bovine oocyte activation and require further investigation. A defined culture medium, USU6 was compared to three other successful undefined embryo culture media/conditions: mSOFaaci, CR1aa, and CR1aa with bovine cumulus cell co-culture. There was no significant difference among cleavage rates for USU6 (88.5%), CR1aa with co-culture (85.1%), CR1aa (82.2%), and mSOFaaci (76.4%). The day 7 blastocyst rates for CR1aa with co-culture (35.7%) was not different than CR1aa alone (31.1%) but was significantly higher than USU6 (27.3%) and mSOFaaci (7.5%). The day 9 hatched blastocyst rates were significantly higher for USU6 (20.0%) and CR1aa with co-culture (17.4%) compared to CR1aa alone (2.6%) and mSOFaaci (0.0%). These data indicate a defined media, USU6 is equivalent or better for bovine IVF embryo development in vitro to the hatched blastocyst stage compared to other common undefined embryo culture media/conditions. The results of the study are summarized as following: 1. Histone H3K9me3 and H3K9/18ace levels were very prominent in GVs. 2. H3K9/18ace levels in fibroblast nuclei in GV oocyte cytoplasts were enhanced 2 h post fusion, and then declined by 4-6 h post fusion. 3. H3K9me3 level of fibroblast nuclei in GV oocyte cytoplasts was enhanced 4 h post fusion, and then declined or restored to original level 6 h post fusion. 4. H3K9me3 level of fibroblast nuclei in non-enucleated GV oocyte cytoplasm was similar to that of the changes in enucleated GV oocytes. 5. GV oocyte extract-treatment reduced the H3K9me3 level of fibroblast nuclei, especially when treated cells were cultured overnight. We have performed co-immunoprecipitation experiments to evaluate the interaction of importin ±8 with oocyte-specific nuclear factors (such as Oct-4 and Nobox). Hela cells were co-transfected with constructs expressing Nobox-flag and importin ±8-myc. Whole cell lysates were immunoprecipitated with anti-flag antibody and analyzed by western blotting using anti-myc antibody. Our data indicate that importin ±8 interacts with Nobox, an oocyte-specific transcription factor important for folliculogenesis and early embryonic development. Objective 2: Refine methods for production of genetically modified animals to improve livestock production efficiency. cAMP levels during sheep oocyte in vitro maturation were characterized and reported. The levels of cAMP in sheep oocytes were successfully elevated by treatment with forskolin and IBMX, resulting in delayed oocyte maturation without compromising the rate of oocytes reaching the MII stage; however, no difference in parthenogenetic blastocyst production was observed between treated and control oocytes. We investigated the possibility of replacing reprogramming viral factors with small molecules for the purpose of induce pluripotency or trans-differentiation. To this end, we evaluated the effects of carcinogens at nongenotoxic levels on somatic cells by identifying 16 candidate chemicals through biology-oriented in silico high-throughput screening, and established a reprogramming protocol of 16-day treatment. We evaluated the reprogramming potency of purified mouse Klf4 protein linked with the cell-penetrating peptide of HIV transactivator of transcription (TAT) or Drosophila Penetratin protein at the N- or C-terminus in conjunction with the other 3 pluripotent factors (Oct3, Sox2 and C-myc) carried by retrovirus. We have secured a $1.5 Million grant from the Bill and Melinda Gates Foundation to develop naturally resistance chickens. Overall data uncovered a high similarity at the transcriptional level between ADSC and BMSC both prior differentiation and during differentiation. Those data support ADSC being particularly similar to BMSC. Results indicate that CD34+ cells were similar to CD34- in the in vitro osteogenic differentiation but have lower in vivo healing capacity. The uADSC have a greater healing capacity than sorted cells. Overall our data indicate that the sorting of ADSC cells is not of clinical relevance. Circular defects in the posterior region of the pig mandible with diameters equal or greater than 25 mm will result in limited healing without additional medical intervention and can be termed critical-sized defects. This porcine model will allow for the rapid development and testing of new approaches for the repair of damaged bone, which is especially prevalent in the craniofacial area. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. It was demonstrated that treatment of bovine adipose stem cells with the epigenetic modifying agents zebularine and valproic acid for 5 days resulted in increased expression of the pluripotency associated transcription factors Oct 4 and Nanog but the transcription factor Sox 2 was not affected. It was demonstrated that exposure of bovine fetal fibroblasts to cellular extracts from bovine adipose stem cells resulted in increased expression of the pluripotency associated transcription factor Sox 2. Exposure of fetal fibroblasts to extracts for human embryonic stem cells had no affect on any of the pluripotency associated transcription factors.