Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (19 results)

  • Article
  • Peer Reviewed
UC Davis Previously Published Works (2021)
As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of available semen selection techniques. This segment of our two-section review will focus on the selection of spermatozoa based on quality and sex for equine intracytoplasmic sperm injection (ICSI), as well as current and future developments in sperm sorting technologies. Ultimately, novel methods of semen selection will be assessed based on their efficacy in other species and their relevance and future application towards ARTs in the horse.
    Cover page: An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section II.
    • Thesis
    • Peer Reviewed
    UC Davis Electronic Theses and Dissertations (2022)

    In horses, in vitro embryo production is limited to a technique called Intracytoplasmic Sperm Injection (ICSI), in which a sperm is manually selected and injected into the oocyte. Therefore, it is essential to select a sperm that is functionally intact and capable of both fertilizing an embryo and maximizing developmental potential by relying on measurable parameters indicative of sperm quality. However, sperm quality is often oversimplified to prioritize parameters such as motility and morphology, which do not necessarily account for the true ability of the cell to fertilize an oocyte and produce a viable offspring. Thus, understanding more complex properties of sperm is highly beneficial to maximizing the success of ICSI. In non-equid species, researchers have investigated a novel property called Zeta Potential (ZP). ZP is an estimation of surface charge, thought to originate from charged proteins within the sperm glycocalyx, that has been correlated with basic sperm quality parameters, fertilization, and embryo development in other species including humans. This parameter has not been investigated in stallions and is characterized, for the first time, in this thesis. In addition to understanding sperm quality, it is common to select for a population of more fertile sperm based on sperm quality parameters using established sperm selection techniques. Equine breeding facilities utilize sperm selection techniques to enrich the population of sperm portraying superior fertility parameters, including motility and morphology, but also accounting for characteristics such as cell viability, mitochondrial potential, and DNA integrity. Conventional selection techniques include swim up (SU), density gradient centrifugation (DGC), and DGC-SU combination (DG-SU). A novel technique used in humans that has not yet been studied for stallion sperm selection is the microfluidic chip (MF), which selects sperm based upon their rheotactic motion similar to that seen within the female tract. In this work, we will present a thorough comparison of conventional sperm selection methods to MF. Lastly, the sperm transcriptome has become a focus of fertility research, as sperm RNAs are thought to contribute to regulation of the female environment as well as to early embryo development. However, recovery of sperm RNAs is often low, which makes downstream analysis more difficult. As there is success in sperm RNA extraction in non-equine species, we aim to compare sperm RNA extraction methods for both sperm RNA yield and quality. In this thesis, we will be discussing normal sperm morphology and physiology, as well as conventional and novel selection techniques, we will characterize ZP as a novel parameter of equine sperm fertility, and we will compare RNA extraction methods for optimal yield and quality of sperm specific transcripts.

      Cover page: Microfluidic Selection, Zeta Potential Characterization, and Sperm Transcriptomics for the improvement of Equine Embryo Production
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2021)
      As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of semen physiology as it applies to overall fertility. This segment of our two-section review will focus on normal sperm parameters, beginning with development and extending through the basic morphology of mature spermatozoa, as well as common issues with male factor infertility in IVP. Ultimately, the relevance of sperm parameters to overall male factor fertility in equine IVP will be assessed.
        Cover page: An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section I.
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2023)

        Background

        Cerebrospinal fluid (CSF) is highly labile and delayed processing might alter results of analysis.

        Hypothesis/objectives

        To determine the effects of time and addition of autologous serum on cytological evaluation of CSF.

        Animals

        Ten client-owned adult horses requiring euthanasia.

        Methods

        Prospective study. Serum and CSF were collected from each horse before and within 10 minutes after euthanasia. CSF samples were divided into 15 aliquots (2 mL each); 1 aliquot was submitted for routine CSF analysis within 60 minutes of collection. Four drops of autologous serum were added to 7 of the aliquots, and stored at 4°C (serum group); the remaining 7 samples were stored unaltered at 4°C (control group). Total nucleated cell count (TNCC) and cell morphology score were done at T4, T8, T12, T24, T48, T72, and T96 hours after collection. Protein concentration was measured in the control group at T0 and T96 hours.

        Results

        The cell morphology scores were significantly different in the control group at T48 (median 2, range 0-4), T72 (2, 0-4), and T96 (3, 0-4) in comparison to T0 (1). No change was observed in the serum group. TNCC remained stable over time in both groups. No statistically significant difference in CSF protein concentration was found between T0 and T96.

        Conclusions and clinical importance

        The addition of autologous serum to an aliquot of CSF sample before shipping improves the preservation of cell morphology up to 96 hours after collection.
          Cover page: Effect of time and autologous serum addition on the analysis of cerebrospinal fluid in horses.
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2023)

          Background

          Abnormal or undesired mare behaviours are often assumed to be associated with ovarian abnormalities.

          Objectives

          We aimed to determine the incidence of abnormal behaviours and their association with concentrations of one or more ovarian hormones associated with a granulosa cell tumour (GCT).

          Study design

          Retrospective descriptive.

          Methods

          A total of 2914 hormonal profile samples submitted with the words behave, behaviour, or behaving in the submission history were analysed. The association between reported abnormal behaviours and concentrations of testosterone, anti-Müllerian hormone (AMH), inhibins and inhibin-B were assessed. Statistical analysis was performed using a Chi-squared test of association.

          Results

          Of the 2914 cases that were submitted due to behaviour issues, 2506 (86%) did not have any of the measured hormones reach GCT-like concentrations. The remaining 408 cases had either one (63%), two (25.5%), or three (11.5%) hormones with concentrations consistent with those from confirmed GCT cases. Testosterone had the lowest percent of GCT-like values among the cases (7.7%), compared with AMH (9.4%), inhibins (9.6%) and inhibin B (8.7%). Stallion-like behaviour was significantly associated with increased concentrations of all four hormones. In contrast, aggression, oestrous and other abnormal behaviours were significantly less likely to be associated with increased concentrations of the hormones.

          Main limitations

          Retrospective study, using sample submission history.

          Conclusion

          Overall, the abnormal behaviours among mares, except the stallion-like behaviour, were not associated with increased ovarian hormones. These results highlight the common misassumption about the involvement of the ovaries in 'abnormal behaviours' or 'undesirable behaviours' of mares.
            Cover page: Abnormal mare behaviour is rarely associated with changes in hormonal markers of granulosa cell tumours: A retrospective study
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2019)
            The placenta is a dynamic organ which undergoes extensive remodeling throughout pregnancy to support, protect and nourish the developing fetus. Despite the importance of the placenta, very little is known about its gene expression beyond very early pregnancy and post-partum. Therefore, we utilized RNA-sequencing to characterize the transcriptome from the fetal (chorioallantois) and maternal (endometrium) components of the placenta from mares throughout gestation (4, 6, 10, 11 m). Within the endometrium, 47% of genes changed throughout pregnancy, while in the chorioallantois, 29% of genes underwent significant changes in expression. Further bioinformatic analyses of both differentially expressed genes and highly expressed genes help reveal similarities and differences between tissues. Overall, the tissues were more similar than different, with ~ 95% of genes expressed in both tissues, and high similarities between the most highly expressed genes (9/20 conserved), as well as marked similarities between the PANTHER pathways identified. The most highly expressed genes fell under a few broad categories, including endocrine and immune-related transcripts, iron-binding proteins, extracellular matrix proteins, transport proteins and antioxidants. Serine protease inhibitors were particularly abundant, including SERPINA3, 6 and 14, as well as SPINK7 and 9. This paper also demonstrates the ability to effectively separate maternal and fetal components of the placenta, with only a minimal amount of chorioallantoic contamination in the endometrium (~8%). This aspect of equine placentation is a boon for better understanding gestational physiology and allows the horse to be used in areas where a separation of fetal and maternal tissues is essential. Overall, these data represent the first large-scale characterization of placental gene expression in any species and include time points from multiple mid- to late-gestational stages, helping further our understanding of gestational physiology.
              Cover page: Characterization of the placental transcriptome through mid to late gestation in the mare
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2024)
              Embryo-maternal crosstalk is essential to establish pregnancy, with the equine embryo moving throughout the uterus on days 9-15 (ovulation = day 0) as part of this interaction. We hypothesized that the presence of a mobile embryo induces local changes in the gene expression of the endometrium. On Day 12, the endometrial transcripts were compared among three groups: uterine horn with an embryo (P+, n = 7), without an embryo (P-, n = 7) in pregnant mares, and both uterine horns of nonbred mares (NB, n = 6). We identified 1,101 differentially expressed genes (DEGs) between P+ vs. NB and 1,229 DEGs between P- vs. NB. The genes upregulated in both P+ and P- relative to NB were involved in growth factor pathway and fatty acid activation, while downregulated genes were associated with oxytocin signaling pathway and estrogen receptor signaling. Comparing the transcriptome of P+ to that of P-, we found 59 DEGs, of which 30 genes had a higher expression in P+. These genes are associated with regulating vascular growth factors and the immune system, all known to be essential in early pregnancy. Overall, this study suggests that the mobile embryo influences the endometrial gene expression locally.
                Cover page: Embryo-endometrial interaction associated with the location of the embryo during the mobility phase in mares.
                • Article
                • Peer Reviewed
                UC Davis Previously Published Works (2023)
                Bacterial endometritis is among the most common causes of subfertility in mares. It has a major economic impact on the equine breeding industry. The sensitivity of detecting uterine microbes using culture-based methods, irrespective of the sample collection method, double-guarded endometrial swab, endometrial biopsy, or uterine low-volume lavage (LVL), is low. Therefore, equine bacterial endometritis often goes undiagnosed. Sixteen individual mares were enrolled, and an endometrial sample was obtained using each method from all mares. After trimming, quality control and decontamination, 3824 amplicon sequence variants were detected in the dataset. We found using 16S rRNA sequencing that the equine uterus harbors a distinct resident microbiome during estrus. All three sampling methods used yielded similar results in composition as well as relative abundance at phyla (Proteobacteria, Firmicutes, and Bacteroidota) and genus (Klebsiella, Mycoplasma, and Aeromonas) levels. A significant difference was found in alpha diversity (Chao1) between LVL and endometrial biopsy, suggesting that LVL is superior at detecting the low-abundant (rare) taxa. These new data could pave the way for innovative treatment methods for endometrial disease and subfertility in mares. This, in turn, could lead to more judicious antimicrobial use in the equine breeding industry.
                  Cover page: Effect of Sampling Method on Detection of the Equine Uterine Microbiome during Estrus.
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2018)

                  Background

                  The human chromosome 14 microRNA cluster (C14MC) is a conserved microRNA (miRNA) cluster across eutherian mammals, reported to play an important role in placental development. However, the expression kinetics and function of this cluster in the mammalian placenta are poorly understood. Here, we evaluated the expression kinetics of the equine C24MC, ortholog to the human C14MC, in the chorioallantoic membrane during the course of gestation.

                  Results

                  We demonstrated that C24MC-associated miRNAs presented a higher expression level during early stages of pregnancy, followed by a decline later in gestation. Evaluation of one member of C24MC (miR-409-3p) by in situ hybridization demonstrated that its cellular localization predominantly involved the chorion and allantoic epithelium and vascular endothelium. Additionally, expression of predicted target transcripts for C24MC-associated miRNAs was evaluated by RNA sequencing. Expression analysis of a subset of predicted mRNA targets showed a negative correlation with C24MC-associated miRNAs expression levels during gestation, suggesting the reciprocal control of these target transcripts by this miRNA cluster. Predicted functional analysis of these target mRNAs indicated enrichment of biological pathways related to embryonic development, endothelial cell migration and angiogenesis. Expression patterns of selected target mRNAs involved in angiogenesis were confirmed by RT-qPCR.

                  Conclusion

                  This is the first report evaluating C24MC kinetics during pregnancy. The findings presented herein suggest that the C24MC may modulate angiogenic transcriptional profiles during placental development in the horse.
                    Cover page: Kinetics of the chromosome 14 microRNA cluster ortholog and its potential role during placental development in the pregnant mare
                    • Article
                    • Peer Reviewed
                    UC Davis Previously Published Works (2019)
                    Equine chromosome 24 microRNA cluster (C24MC), the ortholog of human C14MC, is a pregnancy-related miRNA cluster. This cluster is believed to be implicated in embryonic, fetal, and placental development. The current study aimed to characterize the expression profile of this cluster in maternal circulation throughout equine gestation. The expression profile of miRNAs belonging to this cluster was analyzed in the serum of non-pregnant (diestrus), pregnant (25 d, 45 d, 4 mo, 6 mo, 10 mo), and postpartum mares. Among the miRNAs examined, 11 miRNAs were differentially expressed across the analyzed time-points. Four of these miRNAs (eca-miR-1247-3p, eca-miR-134-5p, eca-miR-382-5p, and eca-miR-433-3p) were found to be enriched in the serum of pregnant mares at Day 25 relative to non-pregnant mares. To further assess the accuracy of these miRNAs in differentiating pregnant (25 d) from non-pregnant mares, receiver operating characteristic (ROC) analysis was performed for each of these miRNAs, revealing that eca-miR-1247-3p and eca-miR-134-5p had the highest accuracy (AUCROC = 0.92 and 0.91, respectively; p < 0.05). Moreover, eca-miR-1247-3p, eca-miR-134-5p, eca-miR-409-3p, and eca-miR-379-5p were enriched in the serum of Day 45 pregnant mares. Among those miRNAs, eca-miR-1247-3p and eca-miR-409-3p retained the highest accuracy as shown by ROC analysis. GO analysis revealed that these miRNAs are mainly implicated in nervous system development as well as organ development. Using in situ hybridization, we localized eca-miR-409-3p in the developing embryo (25 d) and extra-embryonic membranes (25 and 45 d). In conclusion, the present study is the first to elucidate the circulating maternal profile of C24MC-associated miRNAs throughout pregnancy and to suggest that serum eca-miR-1247-3p, eca-miR-134-5p, and eca-miR-409-3p could be used as pregnancy-specific markers during early gestation (25 and 45 d). Overall, the high abundance of these embryo-derived miRNAs in the maternal circulation suggests an embryo-maternal communication during the equine early pregnancy.
                      Cover page: Expression Profile of the Chromosome 14 MicroRNA Cluster (C14MC) Ortholog in Equine Maternal Circulation throughout Pregnancy and Its Potential Implications
                      Top