Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Animal Sciences
Non Technical Summary
Currently, there is limited knowledge how to avoid use of low or infertile male pigs, called boars. This is an issue because poor fertility boars can be unknowingly used and thus use valuable resources, such as feed and energy, while creating no offspring to be later consumed. Also, when a boar is known to have high fertility, changes can be easily adopted to sire more piglets, thus allowing increased usage of genetically superior boars with higher feed efficiency and other desirable traits to decrease the amount of resources used per pound of pork produced. This project makes use of known and candidate biomarkers to identify Upon implementation of results, such will be beneficial for citizens by the ability to decrease the cost of pork production on a per pound basis and help eliminate unnecessary use of environmental resources. Further, an average increase of an extra piglet/litter could increase US pork farmer annual income by $130 million. Results of such technology can aid in global dissemination of high value genetics to support food security. Outcomes from this study can later provide insight for a similar bull study. Ultimately, insight for future research and validation in human male infertility will be gained which can help reduce the total costs of assisted reproductive technology and help eliminate unnecessary emotional costs for couples struggling with infertility.
Animal Health Component
50%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Goals / Objectives
Goal #1: To develop a boar sperm fertility prediction model.Objective 1: Correlate markers of defective spermatozoa to boarfertilityObjective 2: Correlate markers of acrosomal damage/premature exocytosis to boarfertilityObjective 3: Correlate markers of sperm surface alternations to boar fertilityObjective 4: Correlate markers of capacitation status and/or candidate markers of ability to bind oviductal sperm reservoir binding proteins to boar fertilityObjective 5: Correlate markers of implicated oocyte activation protein, PAWP, to boar fertilityGoal #2: To optimize new sperm nanoparticle purification methods for increasing fertility.Objective 1:Optimize nanoparticle purification success by varyingincubation time with spermObjective 2:Optimize nanoparticle purification successat differenttemperatures with spermObjective 3: Optimize nanoparticle purification success changing incubation setting (rotating vs nonrotating) with spermObjective 4: Observe effect of cocktail combination of nanoparticle labels with sperm and optimize cocktail ratio if successful
Project Methods
Goal #1MethodsSamples of semen from 100 boars with over 50 recorded female/matings/sire will be collected 3 times over the course of 2 months and extended in Minitube Androhep boar semen extender (or other acceptable extender). Samples will be sent from farms overnight to analysis lab in insulated packaging, washed upon receipt and assigned to following treatments: 1) noncapacitated, 2) 3 hour in vitro capacitation, 3) 18 hour in vitro capacitation, and 4) 18 hour capacitation + progesterone (P4) to induce acrosomal exocytosis. Sperm will be fixed in 2% formaldehyde for 40 min, followed by wash and storage in + 4 degrees C. Overview of biomarker labeling: 20-50 million sperm in 100 uL working solution, permeabilization (if necessary; biomarker analysis dependent): in 0.1% Trition X-100 (TrX; included in all further steps if sample is originally premeabilized) PBS; blocked in 5% NGS (normal goat serum) for 25 min; overnight incubation in 1:200 primary antibody (ADAM2, 5, 20-like, NEDL2) (1:20 primary antibody for PAWP) +4 degrees C; secondary antibody (1:2000 DAPI, 1:4000 PNA-Fitc, 1:500 Cy5/Tritc GAR/GAM) (LCA-Fitc in place of PNA-Fitc for additional trials) for 30 minutes at room temperature; wash samples 1 x. FlowSight data acquisition: 488, 405, and 642 nm lasers will be used when appropriate for each biomarker/probe at a standard power setting; 10,000 sperm events will be acquired/sample, including pictures of each cell and biomarker; compensation samples will be included for data compensation prior to analysis. FlowSight data analysis: Samples will be analyzed using a data analysis template with gating based on features previously described and further features as identified as statistically significant by Amnis IDEAS Feature Finder software. Statistical analysis: FlowSight analysis results will be exported to MS Excel with boar field fertility data (conception rates, litter size, etc.). For further detail on multiplex biomarker analysis, refer to our lab. Fertility prediction model formation will be based on previously proposed models . Anticipated results: Creation of a new boar fertility prediction model. EffortsThe above methods will be used to create a change in knowledge. Efforts to deliver science-based knowledge are as described in Milestones and EvaluationData for Goal #1 will be fully acquired at the conclusion of the first year of the fellowship, and analyzed with features, correlations, and a fertility prediction models produced by the end of year two of the fellowship. Indicators of success include correlations made between biomarkers and actual field fertility data, with efforts described above made to disseminate knowledge.Goal #2Methods10 boars will be collected 3 times providing 30 total samples/treatment. Using established methods , 20-50 million live spermatozoa, a dose intended for analysis of nanopurification efficiency, not for AI (requires 1-3 billion) will be incubated with 50 uL nanoparticles (NPs) conjugated with lectin PNA or lectin LCA for 30 minutes, in 2 mL total working volume. Following, NPs will be placed on a magnet, allowed to sediment for 15 min, and free sperm collected. Variables to test: incubation time, sperm:NP ratio, temperature, rotating vs. nonrotating, and cocktail combination of NP labels. Validation of population changes between control and treatment will be acquired and analyzed using Objective 1 methods. Beside Objective 1 biomarkers (PNA, LCA, PAWP, and those found beneficial of ADAMs or NEDL2), vital probes for viability (PI), mito-potential (JC-1), and capacitation status (Fluo-3) will be applied, as reviewed recently . Alternatively, IVF will be used to test fertilizing potential of individual nanopurification protocols. Anticipated results: An optimized procedure for a large boar semen nanopurification AI trial. EffortsThe above methods will be used to create a change in knowledge. Efforts to deliver science-based knowledge are as described in Milestones and EvaluationData for Goal #2 will be fully acquired by the end of the first half of the second year, with results analyzed and conclusions drawn by the end of the fellowship.Citations1. Odhiambo JF, DeJarnette JM, Geary TW, Kennedy CE, Suarez SS, Sutovsky M, Sutovsky P. Increased conception rates in beef cattle inseminated with nanopurified bull semen. Biol Reprod 2014; 91:97. 2. Aarabi M, Balakier H, Bashar S, Moskovtsev SI, Sutovsky P, Librach CL, Oko R. Sperm content of postacrosomal WW binding protein is related to fertilization outcomes in patients undergoing assisted reproductive technology. Fertil Steril 2014; 102:440-447. 3. Odhiambo JF, Sutovsky M, DeJarnette JM, Marshall C, Sutovsky P. Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis. Theriogenology 2011; 76:1168-1176.4. Boe-Hansen GB, Christensen P, Vibjerg D, Nielsen MBF, Hedeboe AM. Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility. Theriogenology 2008; 69:728-736.5. Daigneault BW, McNamara KA, Purdy PH, Krisher RL, Knox RV, Rodriguez-Zas SL, Miller DJ. Enhanced fertility prediction of cryopreserved boar spermatozoa using novel sperm function assessment. Andrology 2015; 3:558-568.6. Broekhuijse ML, Feitsma H, Gadella BM. Artificial insemination in pigs: predicting male fertility. Vet Q 2012; 32:151-157.7. Huo L-J, Ma X-H, Yang Z-M. Assessment of sperm viability, mitochondrial activity, capacitation and acrosome intactness in extended boar semen during long-term storage. Theriogenology 2002; 58:1349-1360. 8. Feugang JML, S. F.; Crenshaw, M. A.; Clemente, H; Willard, S. T.; Ryan, P. L. Lectin-Functionalized Magnetic Iron Oxide Nanoparticles for Reproductive Improvement. Journal of Fertilization: In Vitro - IVF-Worldwide, Reproductive Medicine, Genetics and Stem Cell 2014; 03. 9. Sutovsky P, Kennedy CE. Biomarker-based nanotechnology for the improvement of reproductive performance in beef and dairy cattle. Industrial Biotechnology 2013; 9:24-30.10. Sutovsky P. New Approaches to Boar Semen Evaluation, Processing and Improvement. Reproduction in Domestic Animals 2015; 50:11-19.