Recipient Organization
UNIVERSAL PIG GENES INC
3354 140TH ST
CLEARFIELD,IA 508408063
Performing Department
(N/A)
Non Technical Summary
Problem: U.S. swine herd reproductive performance is limited more by sire subfertility than dam. Today's boar semen processing and handling techniques have been largely unchanged since the 1980s. The newly identified, but long-time existing technical problem is that current techniques promote non-desired biological events during storage that shorten the in vivo lifespan, deem a considerable portion of spermatozoa subfertile and for pre-mature cell death, and reduces overall pounds/sow/marketed due to inefficient use of superior genetics.Opportunity: Managing the identified problem can 1) increase sow litter sizes, an opportunity cost of $1 billion per 5% increase in piglets per litter marketed and 2) lead to better use of swine genetics, an opportunity cost of $400 million. Most companies in this space have pulled R&D departments from extender development due to fiscally tight times in the pork industry and lack of high impact scientific findings to translate. Consequently, novel and high impact findings from previous USDA NIFA-funded research has gone without implementation. Herein lies the exciting opportunity to translate recent high impact findings to increase U.S. pork production efficiency.Project objectives:1) Optimize a liquid boar semen storage buffer that decreases storage-induced, premature and pathological sperm capacitation.2) Optimize a boar semen buffer that reactivates sperm at time of insemination.Description of the effort: The proposed research seeks to optimize a new semen extender platform with two patented/provisionally patented intellectual properties for which we have secured allocation of rights from the University of Missouri. Proposed Phase I studies will conduct a series of lab experiments to optimize our proof of concept. Our anticipated results are creation of final boar semen extender recipes with technical expected outcomes outlined in the proposal for later phase II animal breeding trials. Commercial applications of the proposed research is high because semen extenders are a mandatory component in the insemination of over 95% of the 115 million pigs produced annually (pig production being a $20 billion industry). Pairing with a world-renowned professor of sperm fertility and having a historical track record as pioneers in the adoption of swine artificial insemination, we enthusiastically look forward to commercialization.
Animal Health Component
0%
Research Effort Categories
Basic
0%
Applied
0%
Developmental
100%
Goals / Objectives
Overarching goals are forfinal lab boar semen extender optimization of previously funded USDA NIFA research for translation to field trials in later Phase II study. These goals and objectives specifically are as follows:Goal #1:Optimize MU Vanguard Sperm Storage bufferObjective 1:Does Zn binding-substrates commonly found in semen extenders affect the Zn signature state and are theirremoval beneficial? If removed, dotheyhave to be replaced with other reagents for optimal buffering or is the current proposed buffer replacementsufficient?Objective 2: What is the optimal concentration of exogenous zinc ions?Objective 3:Is a protein source such as bovine serum albumin (BSA) needed for sperm membrane stabilization?Objective 4:Determine if inorganic pyrophosphate inclusion is better in storage buffer or the activator buffer and optimize final amount.Objective 5:Is dilution effect of seminal fluid motility activating factorssufficient at time of lab processing or does semen need to be collected into the buffer immediately?Objective 6: What is the optimal storage vs. activator buffer pH (ranging from 6.5-7.2)?Goal #2:Develop fixed-time sperm activation buffer, MU Vanguard Sperm ActivatorObjective 1: Determine buffering chemical make up so the final mixture with semen brings semen to physiological pH of ejaculated semen (7.2-7.4).Objective 2: Determine if base activator buffer BTS is sufficient or other identified activators are necessary.Objective 3: Determine if inorganic pyrophosphate inclusion is better in storage buffer or the activator buffer and optimize final amount.
Project Methods
Objective #1All test buffers will be mixed at the University of Missouri and shipped by parcel to IBS with a final buffer osmolality of 290-300 mOsm/L. Semen will be collected following IBS standard operation procedure, maintained at 35°C to prevent temperature shock, and immediately diluted in the lab to a cell concentration of 3.75 x 107 spermatozoa/mL and aliquoted into 80 mL volumes (standard 3.0 billion sperm dose). Only samples with >80% motility upon activation will be used to assure high initial semen quality. Semen will be shipped by overnight parcel to University of Missouri for further testing, carefully mitigating routine stresses of shipping. Analysis will occur on days 1, 3, and 7. Traits to be analyzed include: motility and the ability to reactivate, by computer-assisted sperm analysis (CASA); pH stability; and by multiplex image-based flow cytometry as outlined below with appropriate controls. 10,000 spermatozoa images will be collected and analyzed per boar/biomarker/treatment including brightfield and sidescatter; (for detailed methods, see [16]):Zn signature (probe: FluoZin-3, ThermoFisher F24195), viability (propidium iodide; Acros Organics AC440300010), acrosome health and status (Lectin PNA-AF647; Arachis hypogea/peanut agglutinin, Invitrogen L32460), and live nucleus stain Hoechst 33342 (to sort single sperm for analysis; Calbiochem 382065)Ca2+ probe (Fluo-4 NW, ThermoFisher F36206), intracellular pH (SNARF, ThermoFisher S23921), acrosome health and status (PNA), and Hoechst 33342LCA revealed altered membranes during storage (Lectin LCA-Fitc; Lens culinaris agglutinin, Vector Labs FL-1040), mitochondrial membrane potential (JC-1, IMV 4500-0250)DNA fragmentation (TUNEL assay, Roche 11684795910)A total of 6 boars per treatment will be analyzed in 3 replicates each. SAS 9.4 GLM procedure and Duncan's Multiple Range test will be used. Homogeneity will be checked using Bartlett and Levene tests.Objective #2The base activator buffer will be BTS (with HCO3-) as previously described [4] with increased final concentration of HCO3- so that final concentration once mixed with semen represents 15 mM physiological levels of seminal fluids. The activator will be formulated so final mixture with semen brings semen to physiological pH of ejaculated semen (7.2-7.4). All semen activations will be performed with 20 mL activation buffer and 80 mL of Vanguard Sperm storage extended semen, warmed to 35°C, then analyzed for motility activation underneath the microscope after 5 minutes. Best placement of PPi between the two buffers will also be determined here. The same set of multiplex image-based flow cytometry trials as done in Task 1 will be performed, including controls with semen extended in BTS and IMV NUTRIXcell+. Spermatozoa will undergo 4 hours of IVC as previously described [51] to analyze the ability to acquire the biomarker-defined fertilization competency, comparing between IVC and non-IVC. SAS 9.4 GLM procedure and Duncan's Multiple Range test will be used to analyze three replicates of each. Homogeneity will be checked using Bartlett and Levene tests.Animal Care: The experiments proposed will be done only on boar semen, not on live animals, therefore no ACUC approval is necessary.