ENHANCING BOAR FERTILITY IN THE FACE OF CLIMATE CHANGE THROUGH THE MITIGATION OF IN UTERO HEAT STRESS

Objective 1) Determine the effects of in utero heat stress on reproductive endocrinology of boars Pigs: Boars (n=40) will be derived from a single sire mating utilizing white crossbred (Large White x Landrace) gilts with divergent genomic breeding values indicating that they are either at Low Risk or High Risk of producing offspring that display negative IUHS phenotypes. Pregnant gilts will be exposed to either thermal neutral (TN) or heat stress conditions. The experimental design is a 2 x 2 factorial with four experimental groups: Low Risk + IUTN, Low Risk + IUHS, High Risk + IUTN, High Risk + IUHS.Briefly, estrous cycles will be synchronized using Matrix® & gilts will be bred via artificial insemination (AI). All pigs will remain at TN conditions (17 to 22°C) for five days post-AI to minimize the risk of embryonic mortality, which is more likely if heat stress occurs in the first week of pregnancy. From days 6-10 post-AI, cyclic HS temperatures of 26°C (night) to 32°C (day) will be applied to pregnant gilts in the HS group. From days 11-59 post-AI, cyclic HS temperatures will be 28°C (night) & 36°C (day) for HS gilts. TN gilts will remain at TN for the entire trial. From day 60 to farrowing, all pregnant gilts will be exposed to TN conditions. After farrowing, sows will nurse their litters ad libitum. At weaning (21 days of age), boars (one per litter) with a median birth weight will be selected for the trial (n=10/experimental group).Examine endocrine function during pubertal development. Throughout pubertal development, we will measure testes and collect blood via jugular venipuncture at key periods in testis development (day 60, 100, 150, 190, 225, 300). Radioimmunoassays (RIAs) for testosterone, cortisol, LH, & follicle-stimulating hormone (FSH) will be performed.Comprehensively assess secretory profiles of reproductive hormones in IUHS boars. After sexual maturity (>300 days), the jugular vein will be surgically cannulated. Hormone secretory patterns will be monitored by collecting blood every 15 minutes for eight hours to accurately capture hormone pulses. Serum will be assayed for ACTH, cortisol, LH, FSH, & testosterone via RIA. A pulse analysis will be performed for each hormone. To comprehensively assess steroidogenic capacity, serum from each timepoint will be pooled within boar & subjected to HPLC/MS-MS at the University of California-Davis to quantify 30 circulating steroid hormone concentrations from all five steroid classes.Evaluate neuroendocrine function in IUHS boars. Assessment of neuroendocrine function will occur by serially collecting blood prior to and after treatment with a GnRH receptor antagonist. Next, the sensitivity of IUHS boars to a GnRH receptor agonist will be tested. Blood will be serially collected before & after treatment with GnRH according to our previous reports to assess the responsiveness of gonadotropes to stimulation. LH, FSH, & testosterone will be measured in serum.Assess testicular steroidogenesis in IUHS boars. To assess steroidogenic capacity of the testis, blood will be serially collected (as indicated above) before & after treatment with human chorionic gonadotropin (hCG); the dose was selected based upon our previous work in boars. Testosterone & LH will be measured in serum.Objective 2) Examine boar semen quality & sperm function after in utero heat stressEvaluate semen quality in boars exposed to IUHS. At 190 days of age, boars will be trained for semen collection once weekly for six weeks. After training, ejaculates will be collected twice weekly for three months. One ejaculate per week (12 ejaculates total per boar) will be subjected to computer-assisted semen analysis (CASA).Examine sperm health & function after IUHS. Previous work showed marked defects in semen quality after IUHS but sperm viability, health, & function have not been assessed. Sperm health will be assessed using flow cytometry-based bioassays to determine DNA compaction, membrane fluidity, viability, acrosome integrity, & mitochondrial function in one semen sample per boar. We will also assess capacitation & the acrosome reaction since both are required for fertilization.Examine sperm fertilization capacity & subsequent embryonic development. To assess fertility, we will determine the effects of IUHS on sperm fertilization capacity & embryonic development. Pig oocytes will be purchased & shipped to UNL. In vitro fertilization will be performed as previously described. Successful in vitro fertilization will be defined by formation of 2 pronuclei & cleavage of the embryo to the 2-cell stage. Percentage of embryos developing to the 4- & 8-cell, compact morula, & blastocyst stage will be determined via stereomicroscope.Objective 3) Elucidate the cellular & molecular mechanisms that regulate porcine testicular function after in utero heat stressPigs: Adult boars (~365 days) from each experimental group will be euthanized. Testis tissue will be preserved (flash frozen, fixed in 4% paraformaldehyde, or stored in RNAlater) for subsequent laboratory analysis. In addition, viable cells will be isolated from a subset of boars (n=4/experimental group) as described below for scRNA-seq.Interrogate the transcriptome of testicular cell types of IUHS boars. scRNA-seq will be utilized to quantify transcriptomic changes that occur in each cell type of the porcine testis after IUHS. scRNA-seq includes the dissection of gene expression at the single-cell resolution unlike traditional RNA sequencing that reflects the average gene expression across thousands of cells. Fresh testis samples will be collected for scRNA-seq as described previously for pig testes.Quantify the number of somatic and germ cells in IUHS boar testes. In addition to gene expression differences, scRNA-seq also reveals the number of each cell type present in the sample thus enabling us to quantify the number of somatic (e.g., Leydig, Sertoli, peritubular myoid) & germ (e.g., spermatogonia, spermatocyte, spermatid) cells in each testis sample.Measure testis composition in mature IUHS boars. To comprehensively assess testis morphology, histological analyses will be performed. Measurements will include total interstitial area, total tubular area, individual seminiferous tubule area, seminiferous tubule numbers per field, height of seminiferous epithelium & Leydig cell area.Efforts: Results will be shared with undergraduate, graduate, and veterinary students in courses that Dr. Desaulniers and Dr. White (Co-PD) teach at the University of Nebraska-Lincoln. We will also share results with pork producers via an article in Pork Talk magazine and seminars at producer-focused venues such as the Boar Stud Managers Conference.Evaluation: 1) Data on pubertal onset and testicular size could be used as a selection tool to non-invasively identify IUHS boars prior to puberty.2) Generation of endocrine data including gonadotropins and testicular steroid hormones on boars during pubertal development and post-puberty in Objective 1. Distinct endocrine profiles could used as biomarkers for IUHS-exposed males.3) Generation of sperm function/health and fertility data on boars post-puberty in Objective 2. Sperm biomarkers (e.g., membrane fluidity) could be used as biomarkers for IUHS-exposed males.4) The generation of single cell RNA sequencing data in Objective 3. This data will be used to determine how IUHS affects each cell type of the testis and could be used to develop new tests for producers. This data will also reveal the cellular/molecular effects of genetic selection for heat tolerance on the testis.5) Publication of manuscripts, preparation of abstracts/presentations, development of Pork Talk article, integration of new knowledge into teaching material.