Does Equine Interaction Facilitate Emotional Safety and Learning for College Students within an Agricultural-Based Classroom?

Effective teaching requires an educational environment that promotes learning, and yet, developing such an environment can be challenging within today's agricultural-based classroom for educators due to the trend to a more virtual teaching format and less hands-on learning. Animal interaction, particularly equine activities, has been shown to assist educators in the development of an emotionally safe environment for promoting learning. However, research is lacking as to whether the interaction with the animal needs to be direct or indirect within the collegiate educational environment to observe benefits. Therefore, the objective of this study was to determine the impact of equine interaction, both direct and indirect, within an educational environment on the emotional safety and learning for the college student within the agricultural-based classroom. Three course types were observed within the agricultural-based educational environment that included courses with no equine interaction (Group A) and courses with equine interaction, both direct (Group B) and indirect (Group C) interaction with the horse. Indirect interaction included items such as observation of equine handling via a video or gaining knowledge from reading online materials, but not engaging in direct, hands-on activities with the horse. Development of emotional safety within the students enrolled within these courses was measured using a self-reporting emotional safety evaluation. Due to the structure of the scale, a decrease in emotional safety indicated a positive change. Learning, both development of semantic and procedural memory, was measured using a student-completed knowledge examination and an instructor-completed skill evaluation, respectively. While significant improvement in emotional safety was not observed within any of the course types, a weak negative correlation was found between emotional safety and semantic memory for students enrolled in equine courses, both direct (R = -0.55, R2 = 0.28) and indirect (R = -0.25, R2 = 0.06) interaction, finding as emotional safety scores lowered to the ideal range that knowledge improved. In addition, students within equine courses showed semantic memory development in specific areas of equine sciences (Group B: Grooming/Tacking, p = 0.03; Group C: Equine Behavior, p = 0.04) and direct equine interaction resulted in development of equine-based procedural memory in all four skill areas measured within the study (p = 0.00). As such, learning is promoted through equine interaction, whether direct or indirect interaction, within the agricultural-based classroom, suggesting that both forms of equine interaction can be a valuable educational tool for the instructor within the collegiate setting.

Characterization of Extracellular Vesicle-Coupled miRNA Profiles in Seminal Plasma of Boars with Divergent Semen Quality Status.

Sperm heterogeneity creates challenges for successful artificial insemination. Seminal plasma (SP) surrounding sperm is an excellent source for detecting reliable non-invasive biomarkers of sperm quality. Here, we isolated microRNAs (miRNAs) from SP-derived extracellular vesicles (SP-EV) of boars with divergent sperm quality statuses. Raw semen from sexually mature boars was collected for eight weeks. Sperm motility and normal morphology were analyzed, and the sperm was classified as poor- or good-quality based on standard cutoffs of 70% for the parameters measured. SP-EVs were isolated by ultracentrifugation and confirmed by electron microscopy, dynamic light scattering, and Western immunoblotting. The SP-EVs were subjected to total exosome RNA isolation, miRNA sequencing, and bioinformatics analysis. The isolated SP-EVs were round spherical structures approximately 30-400 nm in diameter expressing specific molecular markers. miRNAs were detected in both poor- (n = 281) and good (n = 271)-quality sperm, with fifteen being differentially expressed. Only three (ssc-miR-205, ssc-miR-493-5p, and ssc-miR-378b-3p) allowed gene targeting associated with cellular localization (nuclear and cytosol) and molecular functions (acetylation, Ubl conjugation, and protein kinase binding), potentially impairing sperm quality. PTEN and YWHAZ emerged as essential proteins for protein kinase binding. We conclude that SP-EV-derived miRNAs reflect boar sperm quality to enable therapeutic strategies to improve fertility.

Intrafollicular injection of nanomolecules for advancing knowledge on folliculogenesis in livestock.

Despite the progress in assisted reproductive techniques, there is still a lack of rapid and minimally invasive in situ approaches for further enhancements of female fertility. Therefore, we synthesized clinically relevant liposome nanoparticles for ovarian intrafollicular injection to allow in vivo cellular imaging for future drug delivery, using the mare as an animal model. Ovarian follicles of living mares were injected in vivo with fluorescently labeled liposomes. Samples of the follicular wall (mural granulosa, theca interna, and theca externa), granulosa cells, and follicular fluid were harvested 24 h post-injection through the follicle wall biopsy (FWB), flushing, and aspiration techniques, respectively, using a transvaginal ultrasound-guided approach. In parallel, post-mortem dissected, and cultured porcine antral follicles were microinjected with doxorubicin-encapsulated liposomes to assess intracellular delivery potential. All injected mare and pig follicles were macroscopically healthy, and fluorescence imaging revealed successful intrafollicular binding to mural granulosa cells and progressive migration of liposomes to other follicle cell layers (theca interna, and theca externa), regardless of the follicle size. Intracellular delivery of doxorubicin was confirmed in all porcine follicle wall cell types. We conclude that the intrafollicular injection of nanomolecules is a promising approach for real-time monitoring of intrafollicular processes and potential utilization of in vivo cellular drug delivery to assist in follicle disease treatments and fertility improvement.

Follicular-fluid proteomics during equine follicle development.

The complex composition of the follicular fluid (FF), the intimate proximity to the oocyte, and the continual changes in their composition have a major effect on folliculogenesis and oogenesis. To date, the profiling of FF proteomes during follicle selection, development, and ovulation has not been comprehensively investigated. Therefore, a shotgun proteomics approach and bioinformatics analyses were used to profile the proteomes of equine FF harvested in vivo from follicles at the following development stages: predeviation (18-20 mm), deviation (22-25 mm), postdeviation (26-29 mm), preovulatory (30-35 mm), and impending ovulation. A total of 294 proteins were detected in FF (FDR <1%), corresponding to 65 common proteins and 124, 142, 167, 132, and 142 proteins in the predeviation, deviation, postdeviation, preovulatory, and impending ovulation groups, respectively. The higher expression of properdin and several other proteins belonging to the complement system during the deviation time and ovulation suggested their contribution in the selection of the future dominant follicle and ovulation. Apolipoprotein A-1 and antithrombin-III appeared to be important throughout folliculogenesis. The "complement and coagulation cascades" was the major KEGG pathway across all stages of follicle development. The significant expression of several proteins belonging to the serine-type endopeptidase indicated their likely contribution to follicle and oocyte development. Our data provide an extensive description and functional analyses of the equine FF proteome during follicle selection, development, and ovulation. This information will help improve understanding of the ovarian function and ovulatory dysfunctions and might serve as a reference for future biomarker discovery for oocyte quality assessment.

Efficacy of hormone stimulation on sperm production in an alpine amphibian (Anaxyrus boreas boreas) and the impact of short-term storage on sperm quality.

The Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas) has disappeared from much of its range in the alpine regions of Central and Western North America, and restoration efforts are compromised by limited knowledge of this species' reproductive biology. This study aimed to establish whether assisted reproductive techniques could be used to improve breeding output in captive boreal toads by determining the most effective concentration of human chorionic gonadotropin (hCG) for induction of spermiation and viability of sperm during cold storage. Male toads (n = 21) were treated with a Low (3 IU g-1), Medium (10 IU g-1), or High (15 IU g-1) concentration of hCG and spermic urine samples were collected over 24 hrs. Treatment effectiveness was evaluated by measuring the response rate, Total Motility (TM), Forward Progressive Motility (FPM), Quality of FPM (QFPM), and concentration. For short-term cold storage, spermic urine samples (n = 13) were stored at 4 °C for 14 days and sperm TM and FPM monitored daily. All treatments induced spermiation; however, a greater number of toads produced sperm in the Medium and High treatments compared to the Low. Overall, TM, FPM, QFPM and sperm concentration were similar across all three treatments, but variation existed in the timing and duration of peak sperm production. Sperm motility was maintained for up to 14 days in cold storage, although the quality slowly decreased over time. An effective reproduction strategy for the boreal toad will provide a means to improve captive breeding efforts and increase our understanding of the reproductive physiology of alpine Bufonids.

Comparative Analysis of Porcine Follicular Fluid Proteomes of Small and Large Ovarian Follicles.

Ovarian follicular fluid is widely used for in vitro oocyte maturation, but its in-depth characterization to extract full beneficial effects remains unclear. Here, we performed both shotgun (nanoscale liquid chromatography coupled to tandem mass spectrometry or nanoLC-MS/MS) and gel-based (two dimension-differential in-gel electrophoresis or 2D-DIGE) proteomics, followed by functional bioinformatics to compare the proteomes of follicular fluids collected from small (<4 mm) and large (>6-12 mm) follicles of pig ovaries. A total of 2321 unique spots were detected with the 2D-DIGE across small and large follicles, while 2876 proteins with 88% successful annotations were detected with the shotgun approach. The shotgun and 2D-DIGE approaches revealed about 426 and 300 proteins that were respectively common across samples. Six proteins detected with both technical approaches were significantly differently expressed between small and large follicles. Pathways such as estrogen and PI3K-Akt signaling were significantly enriched in small follicles while the complement and coagulation cascades pathways were significantly represented in large follicles. Up-regulated proteins in small follicles were in favor of oocyte maturation, while those in large follicles were involved in the ovulatory process preparation. Few proteins with potential roles during sperm-oocyte interactions were especially detected in FF of large follicles and supporting the potential role of the ovarian FF on the intrafallopian sperm migration and interaction with the oocyte.

Proteome changes of porcine follicular fluid during follicle development.

BACKGROUND: Ovarian follicular fluid influences follicle and oocyte growth, but the fluctuation of its protein content during folliculogenesis has not been comprehensively analyzed. Here we used a shotgun approach and bioinformatics analyses to investigate and compare the proteomes of porcine follicular fluid (pFF) obtained from small (< 4 mm), medium (4-6 mm) and large (> 6-12 mm) follicles. RESULTS: Follicular fluid samples containing highest estrogen levels were selected as non-atretic from small (SNA: 26.1 ± 15 ng/mL), medium (MNA: 162 ± 54 ng/mL), and large (LNA: 290 ± 37 ng/mL) follicles for proteomic analyses. We detected 1627, 1699, and 1756 proteins in SNA, MNA, and LNA samples, respectively. Nearly 60-63% of total proteins were specific to each sample, 11-13% were shared in pairwise comparisons, and 247 proteins were shared among all samples. Functional categorization indicated comparable gene ontology (GO) terms distribution per cellular component, molecular function, and biological process categories across samples; however, the ranking of highly significantly enriched GO terms per category revealed differences between samples. The patterns of protein-to-protein interactions varied throughout follicle development, and proteins such as serine protease inhibitor, clade E (SERPINE); plasminogen activator, urokinase (PLAU); and plasminogen activator, urokinase receptor (PLAUR) appeared stage-specific to SNA, MNA, and LNA, respectively. The "complement and coagulation cascades" was the common major pathway. Besides, properdin and fibulin-1 were abundant proteins that appeared absent in LNA samples. CONCLUSION: This study provides extensive and functional analyses of the pFF proteome changes during folliculogenesis and offers the potential for novel biomarker discovery in pFF for oocyte quality assessment.

Silver-coated magnetic nanocomposites induce growth inhibition and protein changes in foodborne bacteria.

Cytotoxicity concerns of nanoparticles on animal or human bodies have led to the design of iron oxide core nanocomposites, coated with elemental silver to allow their magnetic removal from bio-mixtures. Although the antimicrobial effect of silver is well-described, the effects of nanoparticles derived from silver on microorganisms remain unfolded. Here, we characterized a customized magnetic silver nanocomposite (Ag-MNP) and evaluated its effects on bacterial growth and protein changes. The Ag-MNP displayed both longitudinal and round shapes under High-Resolution Transmission Electron Microscopy imaging, while the Energy Dispersive X-ray Spectroscopy and X-ray diffraction analysis confirmed the presence of Ag, Fe3O4 (Magnetite) and FeO2 (Goethite). Optical density, bioluminescence imaging, and Colony Forming Unit assessments revealed that the presence of Ag-MNP induced strong dose-dependent bacteria (Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and S. Anatum) growth inhibition. The TEM imaging showed penetration and infiltration of bacteria by Ag-MNP, leading to membrane degeneration and vacuole formation. The presence of Ag-MNP led to fifteen up-regulated and nine down-regulated proteins (P < 0.05) that are involved in cell membrane synthesis, inhibition of protein synthesis, interference with DNA synthesis, and energy metabolism inhibition. This study provides insights to develop alternative antimicrobials to treat foodborne pathogens with antibiotic resistance avoidance.

Treatment of boar sperm with nanoparticles for improved fertility.

Continuous progress in nanoscience has allowed the synthesis of various nanoscale particles, known as nanoparticles or nanomaterials which, by harnessing unique physico-chemical properties, are crucial for multiple bio-applications. Despite the revealed toxicity (nanotoxicity) of nanoparticles in various in vitro and in vivo studies, their careful design for biocompatibility and effective interactions with single-celled and multi-cellular organisms has permitted their use in several fields of research and biomedicine. The various nanoparticles synthesized and applied in the veterinary sciences, including reproductive biology, have shown potential to influence routine practices in animal production systems. These include post-collection manipulation of semen and the protection of high-quality spermatozoa to extend their preservation, and to improve sperm-related biotechnologies such as sperm-mediated gene transfer, sperm sorting, sex-sorting, and cryopreservation. Therefore, the application of nanotechnology-based tools to semen may enhance assisted reproductive technologies for biomedical applications and improve economic productivity for farmers. Here, we review the efficacy of available techniques and emerging tools of nanotechnology that might be useful for further selection of high quality boar spermatozoa and productivity improvement.

Nanotechnology-based approach for safer enrichment of semen with best spermatozoa.

BACKGROUND: Advances in nanotechnology have permitted molecular-based targeting of cells through safe and biocompatible magnetic nanoparticles (MNP). Their use to detect and remove damaged spermatozoa from semen doses could be of great interest. Here, MNP were synthesized and tested for their ability to target apoptotic (annexin V) and acrosome-reacted (lectin) boar spermatozoa, for high-throughout retrieval in a magnetic field (nanoselection). The potential impacts of nanoselection on sperm functions and performance of offspring sired by sperm subjected to nanoselection were determined. Fresh harvested and extended boar semen was mixed with various amounts (0, 87.5, and 175 µg) of MNP-conjugates (Annexin V-MNP or Lectin-MNP) and incubated (10 to 15 min) for 37 °C in Exp. 1. In Exp. 2, extended semen was mixed with optimal concentrations of MNP-conjugates and incubated (0, 30, 90, or 120 min). In Exp. 3, the synergistic effects of both MNP-conjugates (87.5 µg - 30 min) on spermatozoa was evaluated, followed by sperm fertility assessments through pregnancy of inseminated gilts and performance of neonatal offspring. Sperm motion, viability, and morphology characteristics were evaluated in all experiments. RESULTS: Transmission electron microscopy, atomic force microscopy, and hyperspectral imaging techniques were used to confirm attachment of MNP-conjugates to damaged spermatozoa. The motility of nanoselected spermatozoa was improved (P < 0.05). The viability of boar sperm, as assessed by the abundance of reactive oxygen species and the integrity of the acrosome, plasma membrane, and mitochondrial membrane was not different between nanoselected and control spermatozoa. The fertility of gilts inseminated with control or nanoselected spermatozoa, as well as growth and health of their offspring were not different between (P > 0.05). CONCLUSIONS: The findings revealed the benefit of magnetic nanoselection for high-throughput targeting of damaged sperm, for removal and rapid and effortless enrichment of semen doses with highly motile, viable, and fertile spermatozoa. Therefore, magnetic nanoselection for removal of abnormal spermatozoa from semen is a promising tool for improving fertility of males, particularly during periods, such as heat stress during the summer months.

Reproductive Performance of Mares Fed Dietary Zearalenone.

It is known that zearalenone (ZON) interacts directly with estrogen receptors, and its in vivo effects on reproduction have been well-documented in several species. In contrast, reports of ZON's impact on horse reproduction are conflicting and inconclusive, some studies confounded by the presence of mycotoxins such as deoxynivalenol in the feed. This study assesses the effect of chronic consumption of zearalenone on reproduction in cycling mares fed >95% pure ZON (0, 2, or 8 mg/da; n = 7 mares/treatment) for three estrous cycles, followed by artificial insemination, through 16 days of pregnancy. Animals were on ZON treatment for between 70 and 121 days (average 84) depending on individual cycle patterns. ZON-induced changes in serum concentration of estradiol (E2) and progesterone (P4), and total estrogenicity were measured using RIAs and the E-screen assay, respectively. Effects on reproductive physiology and pregnancy were monitored by ultrasound and clinical parameters. No significant changes were found in reproductive hormone levels of E2, or P4 for mares on ZON treatments compared to controls, although there was a significant (P < 0.01) increase in P4 levels across Cycle number in High ZON (8 mg/da) treated mares. There was also an increasing trend in the interovulatory interval in the High ZON treatment group. The overall estrogenicity was similar across treatments and over time, not differing from controls or between ZON treatment groups. Adverse uterine and ovarian effects were also not observed, but pregnancy rates were mixed with only 4 of 7 mares on Low ZON becoming pregnant, and only 3 maintaining pregnancy and fetal heartbeat by Day 30, compared to 5 of 6 control mares and all 7 mares on High ZON. Because reproductive efficiency and hormone concentrations are highly variable across individuals, this study did not demonstrate that ZON at 2 or 8 mg/da was detrimental to mares' reproduction. Yet, inferring that ZON treatments were completely without effect is also not appropriate, as the absence of measurable significant differences could be attributed to the limited sample size. Most importantly, there were no extreme signs of toxicology, in contrast to previous reports when ZON was fed at these "doses."

Real-Time Bioluminescence Analysis of Escherichia coli O157:H7 Survival on Livestock Meats Stored Fresh, Cold, or Frozen.

Foodborne bacteria such as Escherichia coli O157:H7 can cause severe hemorrhagic colitis in humans following consumption of contaminated meat products. Contamination with pathogenic bacteria is frequently found in the food production environment, and adequate household storage conditions of purchased foods are vital for illness avoidance. Real-time monitoring was used to evaluate bacterial growth in ground horse, beef, and pork meats maintained under various storage conditions. Various levels of E. coli O157:H7 carrying the luxCDABE operon, which allows the cells to emit bioluminescence, were used to inoculate meat samples that were then stored at room temperature for 0.5 day, at 4°C (cold) for 7 or 9 days, or -20°C (frozen) for 9 days. Real-time bioluminescence imaging (BLI) of bacterial growth was used to assess bacterial survival or load. Ground horse meat BLI signals and E. coli levels were dose and time dependent, increasing during room temperature and -20°C storage, but stayed at low levels during 4°C storage. No bacteria survived in the lower level inoculum groups (101 and 103 CFU/g). With an inoculum of 107 CFU/g, pork meats had higher BLI signals than did their beef counterparts, displaying decreased BLI signals during 7 days storage at 4°C. Both meat types had higher BLI signals in the fat area, which was confirmed with isolated fat tissues in the beef meat. Beef lean and fat tissues contrasted with both pork fat and lean tissues, which had significantly higher BLI signals and bacterial levels. BLI appears to be a useful research tool for real-time monitoring of bacterial growth and survival in various stored livestock meats. The dependence of E. coli O157:H7 growth on meat substrate (fat or lean) and storage conditions may be used as part of an effective antibacterial approach for the production of safe ground horse, beef, and pork meats.

Proteomic analysis of antimicrobial effects of pegylated silver coated carbon nanotubes in Salmonella enterica serovar Typhimurium.

BACKGROUND: Synthesis of silver nano-compounds with enhanced antimicrobial effects is of great interest for the development of new antibacterial agents. Previous studies have reported the antibacterial properties of pegylated silver-coated carbon nanotubes (pSWCNT-Ag) showing less toxicity in human cell lines. However, the mechanism underlining the pSWCNT-Ag as a bactericidal agent remained unfolded. Here we assessed the pSWCNT-Ag effects against foodborne pathogenic bacteria growth and proteome profile changes. RESULTS: Measurements of bioluminescent imaging, optical density, and bacteria colony forming units revealed dose-dependent and stronger bactericidal activity of pSWCNT-Ag than their non-pegylated counterparts (SWCNT-Ag). In ovo administration of pSWCNT-Ag or phosphate-buffered saline resulted in comparable chicken embryo development and growth. The proteomic analysis, using two-dimensional electrophoresis combined with matrix assisted laser desorption/ionization time of flight/time of flight mass spectrometry, was performed on control and surviving Salmonella enterica serovar Typhimurium to pSWCNT-Ag. A total of 15 proteins (ten up-regulated and five down-regulated) differentially expressed proteins were identified. Functional analyses showed significant reduction of proteins associated with biofilm formation, nutrient and energy metabolism, quorum sensing and maintenance of cell structure and cell motility in surviving S. Typhimurium. In contrast, proteins associated with oxygen stress, DNA protection, starvation, membrane rebuilding, and alternative nutrient formation were induced as the compensatory reaction. CONCLUSIONS: This study provides further evidence of the antibacterial effects of pSWCNT-Ag nanocomposites and knowledge of their mechanism of action through various protein changes. The findings may lead to the development of more effective and safe antimicrobial agents.

In-depth proteomic analysis of boar spermatozoa through shotgun and gel-based methods.

BACKGROUND: Mature spermatozoa contain numerous epididymal and seminal plasma proteins, which full identification through high-throughput technologies may allow for a better understanding of the sperm biology. Therefore, we conducted a global proteomic analysis of boar spermatozoa through shotgun and gel-based methodologies. RESULTS: The total proteins were extracted from mature spermatozoa and subjecsted to proteome analyses. Functional analyses of gene ontology representations and pathway enrichments were conducted on the shotgun dataset, followed by immunology and gene expression validations. Shotgun and gel-based approaches allowed the detection of 2728 proteins and 2123 spots, respectively. Approximately 38% and 59% of total proteins were respectively fully and partially annotated, and 3% were unknown. Gene ontology analysis indicated high proportions of proteins associated with intracellular and cytoplasm localizations, protein and nucleic acid binding, hydrolase and transferase activities, and cellular, metabolic, and regulation of biological processes. Proteins associated with phosphorylation processes and mitochondrial membranes, nucleic acid binding, and phosphate and phosphorous metabolics represented 77% of the dataset. Pathways associated with oxidative phosphorylation, citrate cycle, and extra-cellular matrix-receptor interaction were significantly enriched. Protein complex, intracellular organelle, cytoskeletal parts, fertilization and reproduction, and gap junction pathway were significantly enriched within the top 116 highly abundant proteins. Nine randomly selected protein candidates were confirmed with gel-based identification, immunofluorescence detection, and mRNA expression. CONCLUSIONS: This study offers an in-depth proteomic mapping of mature boar spermatozoa that will enable comparative and discovery research for the improvement of male fertility.

Progressive erosion of the Relaxin1 gene in bovids.

The relaxin/insulin-like (RLN/INSL) gene family is a group of genes that encode peptide hormones involved in a variety of physiological functions related to reproduction. Previous studies have shown that relaxin plays a key role in widening of the pubic bone during labor and in gamete maturation. Because of these functions, studying the evolution of RLN1, the gene encoding for relaxin, is relevant in livestock species, most of which belong in the group Laurasiatheria, which includes cow, pig, horse, goat, and sheep in addition to bats, cetaceans and carnivores. Experimental evidence suggests that cows do not synthesize relaxin, but respond to it, and sheep apparently have a truncated RLN1 gene. Thus, we made use of genome sequence data to characterize the genomic locus of the RLN1 gene in Laurasiatherian mammals to better understand how cows lost the ability to synthesize this peptide. We found that all ruminants in our study (cow, giraffe, goat, sheep and Tibetan antelope) lack a functional RLN1 gene, and document the progressive loss of RLN1 in the lineage leading to cows. Our analyses indicate that 1 - all ruminants have lost all key regulatory elements upstream of the first exon, 2 - giraffe, goat, sheep and Tibetan antelope have multiple inactivating mutations in the RLN1 pseudogene, and 3 - the cow genome has lost all traces of RLN1. The 5' regulatory sequence plays a key role in activating expression, and the loss of this sequence would impair synthesis of mRNA. Our results suggest that changes in regulatory sequence preceded mutations in coding sequence and highlight the importance of these regions in maintaining proper gene function. In addition, we found that all bovids examined posses copies of the relaxin receptors, which explains why they are able to respond to relaxin despite their inability to produce it.

Bioluminescent magnetic nanoparticles as potential imaging agents for mammalian spermatozoa.

BACKGROUND: Nanoparticles have emerged as key materials for developing applications in nanomedicine, nanobiotechnology, bioimaging and theranostics. Existing bioimaging technologies include bioluminescent resonance energy transfer-conjugated quantum dots (BRET-QDs). Despite the current use of BRET-QDs for bioimaging, there are strong concerns about QD nanocomposites containing cadmium which exhibits potential cellular toxicity. RESULTS: In this study, bioluminescent composites comprised of magnetic nanoparticles and firefly luciferase (Photinus pyralis) are examined as potential light-emitting agents for imaging, detection, and tracking mammalian spermatozoa. Characterization was carried out using infrared spectroscopy, TEM and cryo-TEM imaging, and ζ-potential measurements to demonstrate the successful preparation of these nanocomposites. Binding interactions between the synthesized nanoparticles and spermatozoon were characterized using confocal and atomic/magnetic force microscopy. Bioluminescence imaging and UV-visible-NIR microscopy results showed light emission from sperm samples incubated with the firefly luciferase-modified nanoparticles. Therefore, these newly synthesized luciferase-modified magnetic nanoparticles show promise as substitutes for QD labeling, and can potentially also be used for in vivo manipulation and tracking, as well as MRI techniques. CONCLUSIONS: These preliminary data indicate that luciferase-magnetic nanoparticle composites can potentially be used for spermatozoa detection and imaging. Their magnetic properties add additional functionality to allow for manipulation, sorting, or tracking of cells using magnetic techniques.

Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes.

BACKGROUND: The fertility performance of animals is still a mystery and the full comprehension of mammalian gametes maturation and early embryonic development remains to be elucidated. The recent development in nanotechnology offers a new opportunity for real-time study of reproductive cells in their physiological environments. As a first step toward that goal, we evaluated the effectiveness of a fluorescent and luminescent nanoparticle for in vitro and ex vivo imaging of porcine gametes. METHODS: Freshly harvested boar sperm were labeled with red-shifted (655 nm) quantum dot nanoparticles conjugated (QD+) or not (QD-) with plasminogen antibody and evaluated. Subsets of labeled spermatozoa were loaded into straws and placed within the lumen of gilt reproductive tracts for ex vivo intra-uterine imaging. Porcine cumulus-oocyte complexes (COCs) were matured in the presence of QD- or QD+. Ovarian follicles were microinjected with QD- or QD+ and placed in culture for up to 4 days. After labeling, all samples were supplemented with coelenterazine, the luciferase substrate, and immediately submitted to bioluminescence analysis, followed by fluorescence and hyperspectral imaging. Data were analyzed with ANOVA and P < 0.05 indicated significant differences. RESULTS: All labeled-samples revealed bioluminescence emission that was confirmed by fluorescence and hyperspectral imaging of the QD localization within the cells and tissues. Over 76% of spermatozoa and both immature and mature COCs were successfully labeled with QD- or QD+. The QD- fluorescence appeared homogenously distributed in the oocytes, while found in the entire sperm length with a higher accumulation within the mid-piece. Labeled-follicles exhibited a progressive migration of QD nanoparticles within the follicle wall during culture. In contrast, QD+ fluorescence signals appeared condensed and stronger in the follicle cells, sperm head, and sub-plasma membrane area of mature oocytes. Weaker QD+ signals were detected in the cumulus cells. Fluorescence and hyperspectral microscope imaging showed comparable intracellular QD localization. Ex-vivo intra-uterine bioluminescence imaging of labeled spermatozoa revealed stronger signals captured over the oviducts, with uterine body allowing the lowest signal detection. CONCLUSION: Findings indicate that conjugated and non-conjugated fluorescent nanoparticles can be used for effective labeling of mammalian gametes for in vitro monitoring and potential in vivo targeted-imaging.

Profiling of relaxin and its receptor proteins in boar reproductive tissues and spermatozoa.

BACKGROUND: Relaxin levels in seminal plasma have been associated with positive effects on sperm motility and quality, and thus having potential roles in male fertility. However, the origin of seminal relaxin, within the male reproductive tract, and the moment of its release in the vicinity of spermatozoa remain unclear. Here, we assessed the longitudinal distribution of relaxin and its receptors RXFP1 and RXFP2 in the reproductive tract, sex accessory glands, and spermatozoa of adult boars. METHODS: Spermatozoa were harvested from three fertile boars and reproductive tract (testes and epididymis) and sex accessory gland (prostate and seminal vesicles) tissues were collected post-mortem from each boar. Epididymis ducts were sectioned into caput, corpus, and cauda regions, and spermatozoa were mechanically collected. All samples were subjected to immunofluorescence and/or western immunoblotting for relaxin, RXFP1, and RXFP2 detection. Immunolabeled-spermatozoa were submitted to flow cytometry analyses and data were statistically analyzed with ANOVA. RESULTS: Both receptors were detected in all tissues, with a predominance of mature and immature isoforms of RXFP1 and RXFP2, respectively. Relaxin signals were found in the testes, with Leydig cells displaying the highest intensity compared to other testicular cells. The testicular immunofluorescence intensity of relaxin was greater than that of other tissues. Epithelial basal cells exhibited the highest relaxin immunofluorescence intensity within the epididymis and the vas deferens. The luminal immunoreactivity to relaxin was detected in the seminiferous tubule, epididymis, and vas deferens ducts. Epididymal and ejaculated spermatozoa were immunopositive to relaxin, RXFP1, and RXFP2, and epididymal corpus-derived spermatozoa had the highest immunoreactivities across epididymal sections. Both vas deferens-collected and ejaculated spermatozoa displayed comparable, but lowest immunofluorescence signals among groups. The entire sperm length was immunopositive to both relaxin and receptors, with relaxin signal being robust in the acrosome area and RXFP2, homogeneously distributed than RXFP1 on the head of ejaculated spermatozoa. CONCLUSIONS: Immunolocalization indicates that relaxin-receptor complexes may have important roles in boar reproduction and that spermatozoa are already exposed to relaxin upon their production. The findings suggest autocrine and/or paracrine actions of relaxin on spermatozoa, either before or after ejaculation, which have possible roles on the fertilizing potential of spermatozoa.

Beneficial effects of relaxin on motility characteristics of stored boar spermatozoa.

BACKGROUND: Relaxin is detected in seminal plasma of many species and its association with sperm motility may be beneficial in some aspects of assisted reproduction. Here, we immunolocalized relaxin receptors and investigated the effects of exogenous relaxin on motility characteristics, viability, and cAMP content of boar spermatozoa after storage. METHODS: Commercial doses of boar semen were obtained on the collection day (Day 0) and kept in shipping containers at room temperature for up to 4 days (Day 4). On Day 0, spermatozoa were fixed for immunofluorescence detection of relaxin receptors RXFP1 and RXFP2 (Experiment 1). Semen aliquots were taken from the same dose at Day 0, Day 1, and Day 2 (Experiment 2a), and Day 2 and Day 4 (Experiment 2b) for analyses. Alive spermatozoa were purified and incubated (1 h-37°C) with 0, 50, or 100 ng relaxin/ml (Experiment 2a) and 0, 100, or 500 ng relaxin/ml (Experiment 2b). Afterward, aliquots of each treatment group were subjected to motility (Experiments 2), viability (Experiment 3) analyses, and cAMP quantification (Experiment 4). Data (3-4 independent replicates) were statistically analyzed (ANOVA followed by pairwise comparisons) and p values less or equal to 0.05 was set for significant difference. RESULTS: Both RXFP1 and RXFP2 receptors were immunolocalized on the entire spermatozoon. Relaxin concentration of 100 ng/ml significantly improved the proportions of motile, progressive, and rapid spermatozoa up to Day 2. Only 500 ng relaxin/ml provided beneficial effects on Day 4. The viability of spermatozoa was not affected by relaxin (100 ng/ml) during storage, but the extent of mitochondria membrane damages was significantly decreased. Furthermore, relaxin did not affect the cAMP contents of spermatozoa during storage, in our conditions. CONCLUSIONS: Relaxin could be a valuable motility booster of stored- or aged-spermatozoa for assisted reproduction techniques. However, the related-intracellular signaling cascades of relaxin in boar spermatozoa remain undetermined.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome: a historical perspective.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a congenital defect of the Müllerian ducts characterized by uterovaginal agenesis and underdeveloped female genital organs. This paper is a tribute to the contributors of this condition - August Franz Joseph Karl Mayer, Karl Freiherr von Rokitansky, Hermann Küster and Georges André Hauser. In addition to their contributions, we have discussed findings and reports of similar defects from other important scientists (Hippocrates, Albucasis, etc.) dating as far back as 460B.C. We have also discussed the disease types and different classification systems including VCUAM and AFS/ASRM among others. Even with several surgical and non-surgical treatment options, there are still many questions that remain unanswered and very little is known about the etiology or genetic predisposition of this condition.