Location: Reproduction Research2011 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
Results are beginning to become available from genomic analysis for reproductive biology traits. The collection of reproductive phenotypes has been a significant activity of this project, and reliable genomic association results are now available for puberty failure, litter size, stillbirth rate, and average birth weights. Analyses for several other reproductive traits are ongoing including birth intervals, age at puberty, ovulation rate, and postweaning return to estrus. A substantial effort was needed to develop the appropriate analysis for this data, resulting in significant delays; however, now that these issues are resolved the remainder of the analyses should be forthcoming. All efforts to examine boar fertility as described in the project ended with the retirement of the scientists involved in that work. In the past year, a new scientist was hired to perform research in gilt development, which is a stated reproductive priority of the National Pork Board. The scientist was notified that he received funding for a NIFA grant within weeks of joining USMARC. Several experiments in the area of gilt development have been initiated. A Research Associate was hired to conduct an experiment to define the transcriptome of two different placental trophoblast cell types. Since hiring, the needed placental trophoblast cells have been isolated using laser capture and we are about to perform high throughput sequencing on RNA isolated from these cells. This experiment addresses one of the experiments described in our project. We have initiated collaborations with two commercial pig producers for which the immunocrit assay is being used to explore colostrum acquisition of newborn piglets. In one experiment, the immunocrit is being used to measure the effect on colostrum of removing a portion of the litter from the sow for a short period of time on the colostrum status of the remaining piglets. Preliminary results indicate that preweaning mortality is decreased and the number of piglets receiving inadequate colostrum is reduced by this treatment. In the other experiment, the relationship between the timing of the birth process and colostrum acquisition by piglets is being measured. Preliminary results suggest that delays in the birth process reduce IgG intake from colostrum, but even when delays are significant, piglets still receive relatively sufficient IgG. We have also initiated collaboration with a commercial producer to examine seasonal infertility in pigs. We are exploring the relationships between progesterone, estrogen and seasonal abortions. Results so far indicate that progesterone does not appear to be associated with the losses.
1. Replacing antibiotics in young swine feed with natural alternatives. Antibiotics have been fed at subtherapeutic levels as growth promoters for more than 50 years and the majority of swine produced in U.S. receive antibiotics in their feed at some point during the production process. The addition of antibiotics to swine diets benefits producers by improving feed efficiency and decreasing susceptibility to bacterial infections. However, because of concerns over antibiotic resistance, alternatives to antibiotics are a high priority for U.S. swine producers. ARS researchers in Clay Center, Nebraska, determined that feeding a natural antimicrobial agent called lysozyme to young pigs consuming a liquid diet was as effective as antibiotics in increasing growth performance, improving gastrointestinal health, and decreasing pathogen shedding in feces. Thus, this natural antimicrobial agent is a suitable alternative to antibiotics for young pigs consuming manufactured liquid diets. The identification of suitable alternatives to antibiotics will enable the swine industry to effectively transition away from dietary antibiotic use.
2. New culture system to improve reproduction in pigs. The pre-implantation period of pregnancy is highly influential on sow productivity. During this period, the pig embryo undergoes a significant morphological change known as elongation, which is critical for maternal recognition of pregnancy, embryo spacing, placental development, fetal growth and ultimately piglet survival. An effective culture system is key to developing knowledge about the pre-implantation period of pig embryos, in particular elongation. ARS researchers in Clay Center, Nebraska, in collaboration with the University of Nebraska-Lincoln, developed an effective culture system that supports embryo elongation in a culture dish. This culture system provides a three-dimensional matrix that supports early stage embryos and facilitates morphological changes, increases in gene expression, and hormone production that mimic elongated embryos in sows. The system will be used to identify specific factors critical for embryonic development, which could improve pregnancy outcomes, piglet production, net returns for US pork producers, and meet the demand of consumers and exporters for US pork.
3. Identification of chromosomal regions associated with weaning-to-estrus interval in swine. Weaning-to-estrus interval (WEI) contributes to the number of non-productive days of females within a breeding herd. ARS researchers in Clay Center, Nebraska, determined the chromosomal regions that are associated with this trait in Landrace-Duroc-Yorkshire females using WEI data from 0 to 14 days post-weaning and the Illumina Porcine 60K SNP Beadchip to detect DNA sequence variation in individual animals. Nine chromosomal regions approaching or surpassing threshold levels for association with WEI were identified on seven chromosomes. Genetic markers within these regions will be useful in marker-assisted selection of sows that rebreed efficiently after weaning, improving retention of sows in the breeding herd.
4. Metabolically relevant candidate gene associations with reproduction, production, and body condition traits in female pigs. Excessive weight and/or body condition loss during lactation is an indicator of tissue breakdown in exchange for maintaining milk output and can have adverse effects on subsequent reproductive performance. ARS researchers in Clay Center, Nebraska, selected candidate genes involved in metabolic pathways and processes to identify and genotype single nucleotide polymorphisms (SNP, variations in DNA sequence) and generate associations with reproductive and performance traits in sows. Milk production, as measured by litter weight at weaning and body condition traits in dams at weaning (backfat thickness and loin eye area), was nominally associated with several SNPs in genes involved in fat synthesis. Weaning-to-estrus interval was nominally associated with several SNP in these genes as well. These discoveries will be made publicly available and can be used in marker-assisted selection and marker-assisted management practices enabling producers to provide a higher quality, low input product for consumers by improving identification of young animals better suited to handle the demands of milk production.
5. Creatine supplementation had no effect on birth intervals, stillbirth rates or preweaning survival in pigs. Prolonged delivery of piglets and reduced piglet vigor at birth are associated with stillbirth and poor preweaning survival, respectively. Creatine supplementation of athletes has been reported to improve physical performance by providing increased energy substrates for muscle activity. To test whether creatine could accelerate the delivery of piglets during birth and reduce piglet stillbirth rate and preweaning mortality, the diet of pregnant pigs was supplemented with creatine or no treatment from day 110 of gestation until farrowing. The birth of each piglet was video recorded and the time required for each birth and whether each piglet was born alive or dead were recorded. To measure creatine effects on piglets, the development of specific brain regions was measured in the largest and smallest piglets in each litter, and the survival to weaning of the remaining piglets in each litter was recorded. Creatine supplementation improved development of the brain stem in the smallest piglets but did not accelerate the birth process or reduce stillbirth rates or preweaning mortality. Thus, creatine treatment is not useful for reducing piglet losses.
6. A single treatment with synthetic colostrum of compromised piglets identified with the immunocrit did not significantly improve their survival. Previous research indicated that piglets that do not receive colostrum from the sow within the first 24 hours after birth are predisposed to high mortality. A simple rapid procedure for measuring serum immunoglobulin, the immunocrit, was used to identify these piglets on day 1 of age while they were still alive, and the effect of a single treatment with 25 ml of a synthetic colostrum formulated by APC Inc. was compared to no treatment to determine whether the single treatment reduced subsequent mortality. Results indicated that a single dosing of piglets was relatively quick and simple but did not improve the mortality of compromised piglets. These results indicate that either a more extensive intervention is needed to reduce mortality of these piglets or the deficits present in piglets failing to acquire sufficient colostrum cannot be reversed.
7. Placental gene expression pathways revealed by breed and line differences. Novel biological pathways related to placental development in pigs were defined in greater detail by pathway analysis of gene expression profiles of placental tissue collected from highly prolific Meishan pigs, a line of pigs selected for increased uterine capacity (defined as the number of fetuses that can be supported by the uterus until the end of pregnancy), and control western breed pigs. The placenta serves as the critical transport organ between the developing fetus and mother to regulate nutrient exchange and this information has developed insight into new biological processes and exposed genetic variation with the potential to manipulate prenatal growth and improve survival. Key differences in placental physiology were identified by pathway analysis showing increased sterol (cholesterol) biosynthesis and transport in Meishans compared to control pigs. Cholesterol is the precursor of many steroid hormones that control reproductive processes. Additionally, upregulation within the integrin signaling pathway was significantly associated with improved placental growth and fetal survival in selection lines for uterine capacity compared to control pigs. Taken together, this work identified key differences in placental physiology between breeds or lines with marked differences in prolificacy, fetal and placental birth weights, and embryonic survival. Improving fetal survival represents an opportunity to improve efficiency and profitability of the swine industry by targeting genetic variation to manipulate these key pathways.Barb, C.R., Hausman, G.J., Lents, C.A., Lkhagvadorj, S., Qu, L., Cai, W., Couture, L., Wang, L., Rekaya, R., Nettleton, D. 2010. Gene expression in hypothalamus, liver and adipose tissues and food intake reponse to melanocortin-4 receptor (MC4R) agonist in pigs expressing MC4R mutations. Physiological Genomics. 41:254-268.