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United States Department of Agriculture

Agricultural Research Service


Location: Reproduction Research

2009 Annual Report

1a.Objectives (from AD-416)
1] Develop techniques to predict boar fertility and potential for sperm production at an early age and discover seminal plasma protein markers associated with successful cryopreservation of boar sperm.

2] Develop strategies to improve uterine capacity, the farrowing process, and neonatal piglet survival to increase the number of piglets weaned per sow.

3] Improve sow longevity by determining the role of prepubertal growth and development of gilts and mammary involution after weaning on the efficient return to estrus of the postpartum sow.

1b.Approach (from AD-416)
Changes in testicular composition and volume will be monitored during pubertal development to establish their relationship with testicular size and sperm production at maturity. Histological approaches and ultrasonography in combination will be used to develop a non-invasive protocol. Genomic scans on these same boars will identify QTL associated with sperm production. In a sub-population of these boars, individual differences in viability of sperm cells will be assessed during cold storage and following cryopreservation. A series of experimental approaches will investigate uterine capacity. RNA from placental tissues collected between days 25 to 45 of gestation from lines of pigs selected for ovulation rate or uterine capacity will be hybridized to porcine arrays to yield expression differences in the placental transcriptome related to line and stage of gestation. Identification of polymorphisms in differentially expressed genes will establish haplotype associations for uterine capacity and fetal survival. A catalog of imprinted genes will be established for porcine placenta and evaluated for coding region polymorphisms identified in Meishan x White Composite embryos. The contribution of placental fold development to placental efficiency will include gene sequence variation in hyaluronidase and heparanase and the association of this variation with differences in litter size and piglet birth weights. Laser capture micro-dissection combined with suppressive-subtraction hybridization will define expression differences between placental trophoblast cell types during late gestation. Impact of farrowing intervals on stillbirths will be monitored with 24-hr video recording of parturition and correlated with piglet survival and growth during lactation. A second experiment will investigate the effect of dietary creatine on farrowing intervals, stillbirth, and preweaning survival. Further investigation will include treatment of late pregnant sows with monoclonal antibodies against the alpha subunit of the porcine insulin receptor to interfere with insulin binding to its receptor with the goal of increasing fetal, blood glucose concentrations. Additionally, the variation among sows in their ability to recover from a glucose challenge will be associated with subsequent piglet fat levels and liver glycogen levels at birth and preweaning survival. Reciprocal transfer of embryos from Meishan and crossbred gilts will explore the contributions of embryonic and maternal genotypes on development of neonatal pigs and growth of piglets prior to weaning. This will define molecular markers (genes and proteins) within uterine, fetal, and placental tissues during late gestation to examine in subsequent studies for their association with perinatal piglet survival. The association of weaning to estrous intervals with growth characteristics and age at puberty will be assessed in conjunction with determination of plasma urea nitrogen and creatinine as markers of muscle turnover. Plasma leptin and glucose will be monitored as markers for metabolic status. DNA will be genotyped and analyzed for QTLs associated with age at puberty and days to estrus after weaning.

3.Progress Report
Several experiments are ongoing within this research project. Scientists assigned to the project continue to measure various reproduction and production related phenotypic traits and have contributed significantly to this effort. This effort is awaiting genotyping using the Illumina 60K swine chip, which began arriving in January of 2009; this genotyping will be complete by December 2009. We have collected average birth interval data on approximately 600 female pigs and plan to collect an additional 200 in the coming year. A dataset comprised of gilts that fail to cycle by the time breeding season begins (~240 days of age) along with their normally cycling littermates has been collected to allow a case-control genomic study designed to discover markers associated with failure to attain puberty. Temperament scores and their association with production traits, including post weaning return to estrus, have been collected. The association between plasma creatine and post weaning return to estrus in first parity sows is being determined. A study to examine the effect of creatine treatment on farrowing intervals and preweaning survival was undertaken, however the results indicate that a further replicate of this experiment during the 2010 04 farrowing season is necessary to fully examine the possible effects of this treatment. The Pork Board agreed to support an experiment to determine the effect of fatty acid and/or zinc supplementation on piglet brain myelination; the first replicate of this experiment was successfully accomplished, and we are in the middle of the second replicate. Preliminary results examining differences in uterine capacity between Meishan and White Crossbred gilts during late gestation suggest that the Meishan may have greater uterine capacity but that both breeds respond similarly to crowding with regard to placental microscopic fold development. However, more data is needed to provide conclusive results, and further experimentation is planned.

1. Factors encoded on the X chromosome influence pubertal age of boars. Pubertal age in males is difficult to align with an exact physiological event. However, diameter of the seminiferous (sperm producing) tubules within the testes during pubertal development in boars increases in association with differentiation of Sertoli cells, which are cells that support developing sperm cells. The increase in tubule diameter is indicative of sexual maturity. Tubular diameter was determined in littermate boars that differed in two allelic variants of thyroid-binding globulin (TBG) to determine the effect of this genetic marker on sexual maturity. The gene for TBG is located near the centromere of the X chromosome, a location previously determined to affect the size of the testis in boars. Boars with the C allele of TBG had seminiferous tubules of larger diameter at defined chronological ages during pubertal development than boars with the A allele. These findings document that TBG or genes that reside near TBG on the X chromosome are involved in regulation of pubertal development of boars.

2. Sequence differences in genes expressed by placental tissue that are associated with selection for uterine capacity. Uterine capacity in pigs, defined as the number of fetuses capable of being supported by the uterus until farrowing, was improved by direct selection over 11 generations, but the underlying mechanisms of that change are not known. Scientists at USMARC generated experimental data using Affymetrix porcine microarrays to document the patterns of expressed genes by placental tissue from day 25 to day 40 of gestation and between the line selected for uterine capacity and a contemporary unselected control line. A method was developed to identify RNA sequence differences between the two lines. This analysis method yielded a large number (5617) of putative RNA sequence differences between the lines. Merging these data with the gene expression results indicated that 179 RNAs with sequence changes were also differentially expressed between the selected and control line. This overlapping list of targets offers evidence of expression differences in the placenta as well as sequence variation in those genes that could be exploited to develop genetic markers for associations with uterine capacity. This information is of value to the industry to improve litter size, because uterine capacity is known to contribute to this trait. Improving litter size represents an opportunity to improve efficiency and profitability of the swine industry.

3. Effect of zearalenone-contaminated feed on prepubertal gilt development. Mycotoxins are produced from molds in feedstuffs and can reduce animal performance. Of particular note in swine is the mycotoxin zearalenone, which has estrogen-like activity and is known to alter the reproductive performance of sows. In addition, zearalenone down-regulates proteins associated with protein synthesis and cellular proliferation in some model systems. The goal of this experiment was to determine the effect of zearalenone-contaminated feed on prepubertal gilt reproductive development, growth performance, and potential mechanisms affecting these outcomes. Feeding zearalenone-contaminated feed to gilts results in a 50% increase in size of the reproductive tract, which does not appear to be associated with increases in estrogen receptor. No differences in growth performance were observed. However, preliminary results indicate that skeletal muscle from these gilts has modified cellular signaling for decreased protein synthesis. These findings improve our understanding of the effect of zearalenone on pig performance and will aid in the development of approaches to alleviate the effect of mycotoxin-contaminated feedstuffs in swine.

4. Molecular cloning and characterization of heparanase mRNA in the porcine placenta throughout gestation. The placenta plays a direct role in regulating fetal growth and survival in the pig. The regulatory function of the pig placenta has implications for uterine capacity, litter size, and postnatal piglet health. As a result, developing an understanding of the mechanisms that regulate placental development and function can provide approaches to improve sow productivity (i.e., number of pigs weaned per sow). Factors affecting placental development are complex and have not been comprehensively characterized. The objective of the current study was to characterize the expression of heparanase (HPSE) in the pig placenta throughout gestation to assess the potential involvement of HPSE in the development of the pig placenta. Heparanase is known to participate in the development of other tissues, including both tissue structure changes and blood vessel formation, which are both relevant to pig placental development. This study identified a full-length cDNA sequence for HPSE that putatively codes for a functional HPSE protein. In addition, the chromosomal location of the HPSE gene was found to be similar to the location of previously identified regions associated with differences in litter size and prenatal survival. The expression pattern and localization of HPSE mRNA in the pig placenta throughout gestation is consistent with a role for HPSE in the changes in placental structure that occur during pregnancy. Taken together, these results demonstrate that HPSE likely plays a role in the development and modification of the pig placenta. This knowledge will contribute to efforts to improve placental function, and therefore improve litter size and postnatal survival of piglets.

5. Brain myelination is impaired in small fetuses during late pregnancy in the pig. Preweaning mortality of piglets ranges from 10 to 20% of piglets born alive, representing a significant loss to the swine industry. Crushing by the sow is a primary reason for preweaning loss of the piglet, suggesting that interventions that improve the ability of the piglet to avoid the sow when she lays down could reduce preweaning loss. Piglet reflexes and coordination are likely to be influenced by the degree of myelination of specific regions of the brain because myelin in the brain improves the speed of nerve impulses. Myelination of the cerebellum (a brain region that controls coordination), brain stem and spinal cord (regions controlling reflexes) were examined in the largest and smallest piglet fetuses in litters collected on days 93, 100, and 110 of pregnancy. Myelination increased in all three brain regions during this period of pregnancy and was less in the brain stem of the smallest fetuses compared to their largest littermates. These results suggest that nutritional strategies during late gestation focused on improving piglet brain stem myelination could be useful in decreasing preweaning mortality of piglets, thereby improving sow productivity and the profitability of swine production.

Review Publications
Miles, J.R., Freking, B.A., Blomberg, L., Vallet, J.L., Zuelke, K.A. 2008. Conceptus development during blastocyst elongation in lines of pigs selected for increased uterine capacity or ovulation rate. Journal of Animal Science. 86(9):2126-2134.

McDaneld, T.G., Smith, T.P., Doumit, M.E., Miles, J.R., Coutinho, L.L., Sonstegard, T.S., Matukumalli, L.K., Nonneman, D.J., Wiedmann, R.T. 2009. MicroRNA Transcriptome Profiles During Swine Skeletal Muscle Development. Biomed Central (BMC) Genomics. 10:77.

Miles, J.R., Vallet, J.L., Freking, B.A., Nonneman, D.J. 2009. Molecular Cloning and Characterisation of Heparanase mRNA in Porcine Placenta Throughout Gestation. Reproduction, Fertility and Development. 21(6):757-772.

Ford, J.J., Wise, T.H. 2009. Sertoli Cell Differentiation in Pubertal Boars. Journal of Animal Science. 87(8):2536-2543.

Trott, J.F., Horigan, K.C., Gloviczki, J.M., Costa, K.M., Freking, B.A., Farmer, C., Hayashi, K., Spencer, T., Morabito, J.E., Hovey, R.C. 2009. Tissue-specific Regulation of Porcine Prolactin Receptor Expression by Estrogen, Progesterone and Prolactin. Journal of Endocrinology. 202:153-166.

Corbin, C.J., Berger, T., Ford, J.J., Roselli, C.E., Sienkiewicz, W., Trainor, B.C., Roser, J.F., Vidal, J.D., Harada, N., Conley, A.J. 2009. Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars. Biology of Reproduction. 81(2):388-395.

Last Modified: 4/18/2014
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