Project : USDA ARS

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Research Project: Improving Lifetime Productivity in Swine using Systems Biology and Precision Management Approaches

Location: Livestock Bio-Systems

2025 Annual Report

Objectives
Objective 1. Improve postnatal survival of preweaning piglets by identifying factors that contribute to within-litter variations on piglet growth and development. Sub-objective 1.A: Determine the influence of ovarian responses (OR and serum P4 levels) during early gestation on within-litter variation in embryo elongation and uterine environmental responses in young females (Exp. 1) and multi-parous sows (Exp. 2). Sub-objective 1.B: Evaluate the influence of regulatory factors (i.e., miRNAs) and nutrient transfer (i.e., metabolites) from maternal to fetal plasma across the placenta on divergent-sized fetal growth and within-litter variation during late gestation using a global approach (i.e., RNA-seq and non-targeted metabolomics, respectively). Objective 2. Discover nutritional and environmental influences on gilt development and productivity to minimize reproductive failure of replacement gilts. Sub-objective 2.A: Improve gilt development by understanding how growth relates to prebreeding anestrus. Sub-objective 2.B: Minimize pubertal failure in gilts by identifying mechanisms in the anterior pituitary gland that are mediating the effects of nutrient balance on secretion of gonadotropin hormones necessary for initiation and maintenance of reproductive cycles. Sub-objective 2.C: Improve neonatal management of replacement gilts by identifying how colostrum intake impacts early ovarian development. Objective 3. Identify and evaluate biological predictors of sow performance and longevity within the breeding herd. Sub-objective 3.A: Identify plasma biomarkers and blood transcript profiles from young pre-breeding females and associate those profiles with their subsequent breeding herd longevity. Sub-objective 3.B: Evaluate USMARC swine population for effects of seasonal climate upon production parameters over the past decade. Sub-objective 3.C: Generate hypomethylated, hypermethylated, or control pregnancies during summer or winter months to determine epigenetic impact upon placental, fetal, and piglet production. Objective 4. Utilize and develop precision management technologies to improve preweaning piglet survival, gilt development, and sow longevity and increase efficiency of pork production. Sub-objective 4.A.: Utilize ESF data to develop prediction models for gilt fertility (e.g., behavioral estrus) and sow welfare during gestation (e.g., abortion/miscarriage, lameness, appropriate body weight).

Approach
Pork is the most consumed meat animal product globally. Improving lifetime efficiency of swine is critical to support an increasing global population. Improved lifetime efficiency will provide a high-quality source of protein while reducing the impact of swine production on the environment and ensuring the social welfare of animals. Lifetime efficiency is a complex trait that is influenced by genetic, environment, and management components. The comprehensive goal for this project is to further our understanding of these traits using physiology-, biology-, and technology-based approaches to provide improvements for fetal and neonatal health, gilt development, and sow longevity. We will accomplish this goal utilizing independently, and in combination, transcriptomics, metabolomics, proteomics, environmental data, and automated precision measurements (Figure 1). Within objective 1, we will investigate molecular pathways and signals that improve production of consistently sized piglets initiated shortly after conception. Objective 2 will delve into the influence nutrition has on the young female and the impact upon neuroendocrine gene expression as the gilt transitions into the active state of reproduction. Identifying biological markers that can assist in selecting females with longevity in the breeding system and investigating climate and therapeutics to assist with stayability are the central themes of Objective 3. In the fourth objective, feeding behavior and activity measurements of young gilts and gestating females will be collated and activity patterns will be generated to predict reproductive success or failure. The projects designed herein will clarify and contribute to the existing complex knowledge gap of swine productive life. Application of these studies will result in breeding females that are consistently well adapted, produce large litters of uniform piglets and remain fertile and healthy in the swine herd increasing production efficiency, improve economic competitiveness of U.S. pork producers, and contribute to basic understanding of biological and environmental influences upon swine production.

Progress Report
Within the project, Improving Lifetime Productivity in Swine using Systems Biology and Precision Management, significant progress has been made towards the four objectives over the past year. For Objective 1, we have focused on evaluating potential regulatory factors that play a role in the initiation of embryo elongation. Specifically, we have begun to characterize extracellular vesicles (EVs), such as exosomes and microvesicles, that play a role in cell-cell communication within the uterine environment during embryo elongation. This has involved developing a standardized workflow for evaluating EVs from uterine luminal flushing (ULF), including isolation (e.g., ultrafiltration and ultracentrifugation) and characterization (e.g., Nano-flow cytometry and cytogenic electron microscopy) techniques. In addition, we have performed a temporal RNA-Seq evaluation of miRNAs isolated from within these EVs from ULF at different time points during embryos elongation. We have also developed a luminal epithelial explant culture system in conjunction with our already established 3D-hydrogel culture systems from embryos that will be used to characterize specific maternal and embryonic EVs that are secreted from these two culture systems, respectively. For evaluation of the improvement of uterine capacity and corresponding reduction in within-litter fetal weight variation, we have continued to evaluate feed additives in gestation diets that are specifically formulated to target placental vascular and structural development. These feed additives have been expanded to evaluate additives in gestational diets that can modify the methylation status of placentas and potentially improve pregnancy outcomes during seasonal dips in fertility. In support of Objective 2, approximately 500-800 pubertal phenotypes and electronic sow feeder (ESF) body weight data have been added to the database. Initial analysis revealed that gilts that were heavier at birth were at greater risk of failing to reach puberty. Samples collected from gilts fed different levels of dietary energy in normal and delayed puberty gilts have been processed for proteomic assays. We continue to evaluate early life plasma markers and the predictability of breeding herd longevity in support of Objective 3. Transcriptomic data captured from young gilts that subsequently transitioned through the breeding system are being analyzed. Similar to previous work using liquid chromatography-mass spectrometry data, we plan to identify differentially expressed gene targets from young gilts in relation to lifetime production and longevity traits in the same animals. Our hypothesis is that we will be able to identify young females prior to entering the breeding system that will either be successful or have an increased risk of premature culling. We are in the final stages of completing tissue collections for the hypo- and hyper-methylated placenta during early pregnancy in either winter or summer. Upon completion of tissue collections, we will begin processing samples in the lab. A second subset of animals have completed farrowing and those offspring are being followed through the swine system to determine if a hypomethylated or hypermethylated environment during early pregnancy improved growth, development, and sexual maturation traits in the offspring. In support of Objective 4, electronic sow feeder data in gilt development and gestation pens continues to be processed and uploaded onto the relational database. A collaborative project with academic partners using various methods (e.g., caliper, visual, ultrasound, and digital, depth, and IR images) to assess body condition of females throughout gestation has been initiated. These data will allow researchers to develop an automated imaging system to assess body condition. In support of all objectives, the most recent scientific hire has been participating in trainings and courses to support -omics research. A bioinformatics pipeline for miRNA-seq analysis has been developed and is in the final stages of approval for use by IT. All scientists within the project plan are engaged in projects supported by external funding that provide beneficial information in support of swine lifetime productivity. In support of Objective 1, along with academic partners, a continuation of a funded proposal investigating extracellular vesicles within the uterine environment of the early pregnant pig and how those compounds support embryo establishment and development. Objective 2 compliments an externally funded proposal investigating male fertility in a knockout pig model. And finally, an externally funded proposal investigating the microbiome of lactating sows contributes to factors impacting sow longevity within Objective 3.

Accomplishments
1. An alternative to castration in swine. Male piglets are castrated to prevent aggressive behaviors that increase the risk of injury and to improve pork quality by eliminating skatole, which produces an unfavorable taste and odor in pork. Gene editing provides an opportunity to develop alternatives to castration. ARS scientists at Clay Center, Nebraska, and collaborators used precision gene editing to recode a small number of bases in the genome to render the KISS1 gene nonfunctional. Scientists measured hormones and testicular development in pigs that had no functional copies of the KISS1 gene or only one functional copy and compared them to control pigs that had two functional copies. Results indicate that when only one functional copy of the KISS1 gene was present, pigs had normal reproductive development. Pigs with no functional copies of the KISS1 gene had low levels of reproductive hormones and did not develop testicles large enough to require castration. Scientists also found that boars without both functional copies of the KISS1 gene produced very little skatole. This research demonstrates the potential to use gene editing to develop alternatives to castration in swine. In turn, this will improve the efficiency of pork production for producer stakeholders and improve animal well-being.

2. Improving pregnancy rate in pigs. Failure of young female pigs (gilts) to become pregnant and produce a litter is the main cause of culling in the swine industry, costing producers over $1500 for each female gilt removed. The GnRH2 hormone receptor (GnRHR2) is important for reproduction in male pigs, however, its role in reproduction of gilts is completely unknown. ARS scientists in Clay Center, Nebraska, and collaborators used a unique line of gilts that were genetically modified to produce less GnRHR2. They examined the ovaries and measured reproductive hormones in the GnRHR2-modified gilts and compared them to unmodified gilts. They found that the GnRHR2-modified gilts had fewer ovulations and their ovaries produced less progesterone, a critical hormone that supports pregnancy. This research provides important new insights into hormonal factors contributing to pregnancy in gilts. The refined understanding in hormonal contributions to pregnancy will lead to new methods to increase fertility of gilts, thus improving the efficiency of pork production for producer stakeholders.

3. Improving boar fertility. Pork producers in the United States require 30 million doses of semen every year to breed sows. However, certain boars have poor semen quality and fertility, increasing costs and reducing production efficiencies for pork producers. ARS scientists at Clay Center, Nebraska, and collaborators discovered a GnRH2 hormone receptor (GnRHR2) on boar sperm. The location of GnRHR2 is associated with a special region in sperm that controls its motility, the ability of sperm to efficiently propel itself towards the egg, which is a marker of semen quality. Treating boar semen with a compound that blocked the GnRHR2 inhibited sperm mortility and morphology but this was reversed by treatment with GnRH2 hormone to activiate GnRHR2. These data provide a crucial new discovery that GnRH2 hormone and its receptor regulate sperm function and will lead to new methods to improve semen quality and fertility.

4. Identifying young female pigs (gilts) predisposed to future lameness. Decreasing culling of gilts in breeding for health reasons would reduce economic losses, improve production efficiencies, and improve animal well-being. The use of precision management tools provides an opportunity to identify gilts at risk of premature removal before the producer incurs the costs to develop these females. ARS scientists at Clay Center, Nebraska, and collaborators used pressure sensitive mats and video monitoring to measure walking traits of gilts before and after they entered the breeding herd. Mobility, posture, and weight distribution on each leg and hoof were measured and gilts were followed throughout their lifetime until removal from the herd. Scientists identified several walking traits measured early in life that were predictors for premature removal of gilts from the herd later in life due to lameness. This research is identifying new methods for producers to effectively select breeding gilts that will remain in the herd with reduced risk of lameness and improve animal well-being.

U.S. DEPARTMENT OF AGRICULTURE

Livestock Bio-Systems: Clay Center, NE