Brief Summary of Minutes of Annual Meeting

Meeting convened by Dr. Ken Bondioli (LSU) at 1 PM on February 14, 2013. Dr. Mark Mirando was unable to join by telephone due to illness. Initial discussion included issues about loss of intellectual capital, e.g. limited appointment of new faculty with retirements and funding. This was followed by station reports (Arkansas-Rosenkrans, California-Berger, Colorado- Winger, Connecticut-Tian, Georgia-Stice, Louisiana-Bondioli, Missouri-Rivera, Montana-Schmidt, and Nebraska-White). Utah (Bunch, Isom, Polejaeva, Wang, and Aston) deferred most of their discussion to the following day. Lively discussion of research followed presentations with collaborations potentially developing from these exchanges and continued into dinner and lunch. The meeting was reconvened at 8:07 on Friday, February 15. Following the presentations from Utah, and comments from Dr. Milan Shipka, liaison to USDA, on impact statements, the group as a whole discussed the wording of our impact statements. Following extensive discussion of these drafts, a copy was to be submitted to all members by email after the meeting. The group then proceeded to the topic of the rewrite, due January 15, 2014. Since this deadline for submission occurs during the IETS meeting in 2014, the decision was made to have the W2171 meeting on Friday, January 10, before the IETS meeting. The rewrite will need to be complete before our next meeting. Pairing of a more experienced and a relatively new member to the group was made for each objective. For Objective 1 related to efficieny of gamete and embryo development, Charles Rosenkrans and Clay Isom agreed to be the writing team. For objective 2 related to efficiency of transgenic animal production, Matt Wheeler (not present) and Rocio Rivera were appointed as the writing team. Clay Isom was nominated, seconded, and voted in as Secretary of the group. The group requested that Ken Bondioli and Milan Shipka on behalf of the group write a letter thanking Clay Isom for the much appreciated organization for this meeting. The meeting was adjourned and members shared lunch at the union and several participated in afternoon tours of Utah State facilities generously provided by the Utah members.

PROGRESS OF WORK AND PRINCIPAL ACCOMPLISHMENTS: OBJECTIVE 1 Understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis OBJECTIVE 2 Refine methods for production of genetically modified animals to improve livestock production efficiency ACCOMPLISHMENTS: 1) The success of an artificial insemination program utilizing sorted semen might be improved by delaying insemination beyond the typical 10 to 14 hours after onset of estrus that is currently used for conventional, unsorted semen. 2)Managing beef cows in such a manner that they are not consuming toxic forage during the breeding season and have adequate body condition during pregnancy will result in heifers that have greater fecundity. 3) Genotyping bulls based on mutations associated with the heat shock protein 70 gene may lead to dairy cows with improved reproductive rates. 4) Preliminary sow breeding trials build upon previous basic research and suggest very short term cooling in the weaning to breeding interval may alleviate summer subfertility in swine. 5) RNA-Seq analysis of single bovine oocytes enabled quantification of polyadenylated transcript abundance and subsequent UTR analysis providing insight into the mechanisms facilitating posttranscriptional regulation during oocyte maturation and pre-implantation embryogenesis. RNA-seq analysis of single equine ICM and TE provides a comprehensive characterization of genes expressed in these compartments of the blastocyst. 6) Characterization information on goat MSC, and show that significant differences can exist between MSC isolated from different tissues and from within the same tissue. 7) Determined the expression patterns of Lin28 in the sheep placenta. 8) Timing of expression suggest unique roles of Lin28A and Lin28B. Identified cell-secreted vesicles in ovarian follicular fluid and demonstrated their uptake by granulosa cells. 9) Identified ACVR1 protein in exosomes isolated from follicular fluid of equine ovaries. 10) Identified gross morphological, histological and molecular abnormalities in placentomes from prenatal androgenization in ewes. 11) Identified gene expression alterations in placentomes, of prenatal androgenization of ewes, in genes that regulate epigenetic DNA modifications like histone demethylase KDM4D that could contribute to environmental programming of the placenta. 12) Detection of Lin28A and Lin28B in sheep trophectoderm cells suggests a pluripotent stem cell population exists in sheep placenta. 13) Identified four reprogramming factors will not reprogram somatic cells without LIF. LIF in fact actively reprogram the epigenetic modifications of the somatic cells in addition to maintaining the pluripotency of already reprogrammed cells. 14) With the understanding on the role of LIF in nuclear reprogramming, we now have more understanding of the reprogramming mechanism. The knowledge obtained from our study will bring us closer to producing safer and more completely reprogrammed cells using chemical approaches. 15) Although avian ESC and primordial germ cell lines have been established they have not been used in gene targeting studies, mostly likely because they have not been clonally isolated nor are they highly proliferative in extended cultures. 16) Overall data indicated that the transcriptomes of the two MSC are similar under both conditions. In addition, despite the limited differentially expressed genes between the two MSC, the enrichment of several functions/pathways might indicate differences in therapeutic application. 17) It was demonstrated that cryopreserved ejaculated and epididymal bovine sperm could be used within the same in vitro fertilization protocol. The inclusion of the capacitating agent heparin increased blastocyst production for both epididymal and ejaculated sperm. 18) Studies demonstrated that unlike for red deer, increased bicarbonate and Ca2+ are not necessary to induce capacitation of white-tailed deer epididymal spermatozoa. Although sheep serum did not affect the number of live acrosomeintact spermatozoa, its presence did alter the lipid architecture of the sperm plasma membrane. 19)Initial parameters for the utilization of synthetic mRNA for induction of bovine pluripotent stem cells were determined and it was demonstrated that transfection of bovine fetal fibroblasts with synthetic mRNA encoding human Oct 4 resulted in the stimulation of endogenous bovine Oct 4. 20) A pluripotency reporter gene consisting of the bovine NANOG promoter fused with a nuclear localized green fluorescent protein (GFP) gene was demonstrated to faithfully reflect regulatory mechanisms controlling bovine NANOG expression in embryos and following iPSC reprogramming. 21) The abundance of miRNAs in the spermatozoa and the differential expression in sperm from high vs. low fertility bulls suggests that the miRNAs possibly play important functions in the regulating mechanisms of bovine spermatozoa Identification of specific microRNAs expressed in spermatozoa of bulls with different fertility phenotypes will help better understand mammalian gametogenesis and early development. 22) It was demonstrated that misregulation of the KvDMR1 imprinted cluster occurs in LOS. 23) It was demonstrated that culturing bovine embryos on a collagen matrix improves development to the blastocyst stage. 24) Carried out experiments to determine the effect of adipocytokines on the expression maternal effect genes associated with cumulus cell maturation in cumulus-oocyte complexes isolated from obese (Lethal Yellow, high fat fed C57BL/6) and normal-weight (C57BL/6) female mice after ovulation. 25) Provided important evidence that fluctuations in endocrine hormones and proinflammatory cytokines associated with an obese phenotype alters either the transcriptional activity of target genes or post-transcriptional stability of target mRNAs in the female reproductive tract which likely contributes to poor oocyte quality and reduced fertility. 26) Studies performed in a bovine model of fertility provide evidence that the ability of the follicle to convert androgens to estrogen uniquely regulates gene expression in granulosa cells and the oocyte. Likewise, the consequence of androgen excess is a direct or indirect negative effect on oocyte mRNA abundance, which likely impacts the developmental competence of the oocyte. 27) Constructed lentiviral vectors containing a constitutively active promoter fused to selected siRNA designed to reduce GnRHR II mRNA levels in porcine cells and tissues. 28) Established a genetically altered line of swine with reduced levels of GnRH II receptors using a non-replicative lentiviral delivery system. 29) Performed immunohistochemistry and immunofluorescence procedures using an antibody specific for the GnRH-II receptor on fixed testicular tissue. 30) Examined serum LH and testosterone concentrations in boars treated with either a GnRH-I or GnRH-II agonist. 31) Transgenic pigs were produced expressing a spermatogonial marker gene. 32) Differences in mRNA expression were identified between in vivo produced conceptuses recovered on Day 15 of gestation that were classified based on length. 33) 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. 34) In vitro exposure of bovine embryos to TNFa resulted in an inhibition of development to the blastocyst stage. 35) 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). 36) GFP marker in aggregated embryos could track the development of cloned blastomeres. 37) The PLC isotypes ´3, ´4, and ³2 decreased Ca2+ ioscillations and cleavage rates, indicating they are involved in both IP3R and RyR activation. PLC ´4 and ·2 did not impact Ca2+ i but did significantly decrease cleavage rates.

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