Chromatin refers to the complex of DNA and proteins, primarily histones, found within the nucleus of horse cells. It plays a key role in regulating gene expression and maintaining genomic stability. Chromatin structure can influence the accessibility of DNA to transcription factors and other regulatory proteins, thereby impacting cellular function and development in horses. Modifications to chromatin, such as methylation and acetylation, can alter gene expression patterns and are a focus of research in equine genetics and epigenetics. This page compiles peer-reviewed research studies and scholarly articles that explore the structure, function, and modifications of chromatin in horses, as well as its implications for equine health and disease.
Luis-Calero M, Marinaro F, Fernández-Hernández P, Ortiz-Rodríguez JM, G Casado J, Pericuesta E, Gutiérrez-Adán A, González E, Azkargorta M....In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem ...
Peng S, Dahlgren AR, Donnelly CG, Hales EN, Petersen JL, Bellone RR, Kalbfleisch T, Finno CJ.The genomic sequence of the horse has been available since 2009, providing critical resources for discovering important genomic variants regarding both animal health and population structures. However, to fully understand the functional implications of these variants, detailed annotation of the horse genome is required. Due to the limited availability of functional data for the equine genome, as well as the technical limitations of short-read RNA-seq, existing annotation of the equine genome contains limited information about important aspects of gene regulation, such as alternate isoforms and...
Al-Kass Z, Ntallaris T, Morrell JM, Johannisson A.Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or packaging and fertility have not been explored. In the present study, relationships between fertility and DNA fragmentation index, protamine deficiency, total thiols, free thiols and disulfide bonds in stallion spermatozoa were investigated. Ejaculates (n = 36) were collected from 12 stallions and extended to prepare semen doses for insemination. One dose from each ejaculate was sent to the Swedish University of Agricultural Sc...
Ortiz I, Dorado J, Pereira B, Diaz-Jimenez M, Consuegra C, Gosalvez J, Hidalgo M.The objectives of this study were to evaluate the effect of vitrification on the DNA fragmentation rate of equine cumulus cells and to assess its relationship to oocyte in vitro maturation (IVM) after vitrification. Cumulus cells (CC) from 14 mares were recovered from COCs, previously submitted to vitrification (VIT) and IVM. The DNA fragmentation rate of the cumulus cells (CC-DF) was assessed using a chromatin dispersion test. CC-DF rates between vitrified and control COCs were statistically compared by Student's t-test. The rates of CC-DF from control COCs were lower than in vitrified COCs...
Brogna R, Fan J, Sieme H, Wolkers WF, Oldenhof H.Even though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spe...
Al-Kass Z, Brown A, Johannisson A, Ntallaris T, Morrell JM.Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48 hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48...
Li N, Yu J, Yang F, Shao Y, Wu S, Liu B, Li M, Wang T, Li J, Zeng S.The aim of this study was to evaluate the effects of L-proline on the extender quality of frozen and post-thawed jackass semen. Jackass (n = 6) semen samples were collected and cryopreserved in gradient concentrations (0-80 mM) of L-proline in extenders; post-thawed semen samples were cultured in L-proline medium for 10 hours at 37°C. For cryopreservation experiment I, the motile parameters, mitochondrial membrane potential (MMP), and plasma membrane, acrosome, and chromatin structure integrities of post-thawed semen were assessed. For culture experiment II, additional ROS contents were ana...
Paterson YZ, Evans N, Kan S, Cribbs A, Henson FMD, Guest DJ.The transcription factor scleraxis (SCX) is expressed throughout tendon development and plays a key role in directing tendon wound healing. However, little is known regarding its role in fetal or young postnatal tendons, stages in development that are known for their enhanced regenerative capabilities. Here we used RNA-sequencing to compare the transcriptome of adult and fetal tenocytes following SCX knockdown. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes affected in adult tenocytes, indicating that scleraxis-depend...
Loureiro KC, Lima-Verde IB, Johannisson A, Ntallaris T, Jager A, Štěpánek P, da Costa Mendonça M, Severino P, Morrell JM.Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes ...
Johannisson A, Al-Essawe EM, Al-Saffar AK, Karkehabadi S, Lima-Verde I, Wulf M, Aurich C, Morrell JM.The mechanism by which the content of the major groups of seminal plasma proteins in stallion semen changes between the breeding and non-breeding seasons remains unknown. Here, we investigated the proportions of non-heparin-binding, phosphorylcholine-binding, and heparin-binding proteins in seminal plasma with the aim of relating them to sperm quality and testosterone levels in good and bad freezer stallions. Only minor variations in the major protein groups were found between the breeding and non-breeding seasons. In the non-breeding season, a higher content of a subset of non-heparin binding...
Kareskoski AM, Palviainen M, Johannisson A, Katila T.For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expressio...
Mislei B, Bucci D, Malama E, Bollwein H, Mari G.Oxidative stress is regarded as an important cause of sperm damage during cryopreservation. However, seasonal changes in oxidative status in unfrozen and frozen-thawed stallion sperm have not been well established. We tested the hypothesis that sperm ROS concentrations and lipid peroxidation change between breeding and non-breeding seasons and influence quality of unfrozen and frozen-thawed sperm. Eighteen ejaculates from six Warmblood stallions (8-21 y) known to be fertile, were collected in winter and summer and processed for freezing. After 90 min at +4 °C, some straws from each ejacu...
Lodde V, Colleoni S, Tessaro I, Corbani D, Lazzari G, Luciano AM, Galli C, Franciosi F.Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence ...
Pereira BC, Ortiz I, Dorado JM, Diaz-Jimenez MA, Consuegra C, Gosalvez J, Hidalgo M.DNA fragmentation of cumulus cells could be used as an indicator of oocyte vitrification success as an indirect indicator of the quality of the oocyte. This study was designed to compare the DNA fragmentation of post-mortem equine cumulus cells before or after vitrification in the absence of permeable cryoprotectant agents. Cumulus-oocyte complexes (COCs; n = 56) were recovered from slaughterhouse ovaries and subjected to in vitro maturation (42 hr/38.2°C/5%CO ) before (control group) or after a permeable cryoprotectant-free vitrification method using 1 M sucrose (vitrification group). Af...
Al-Essawe EM, Johannisson A, Wulf M, Aurich C, Morrell JM.Freezing and thawing processes induce structural and functional damage to sperm plasma membranes and internal organelles. Adding seminal plasma (SP) has been found to minimize or repair the cryoinjuries in some species. The objective of this study was to investigate whether adding SP from stallions of known freezability after thawing could repair cryoinjuries. Semen was collected from warmblood stallions (n = 8, three ejaculates/stallion) and processed by Single Layer Centrifugation (SLC) to remove SP prior to freezing. Pooled SP (5%) from bad freezer (BF) or good freezer (GF) stallions wa...
Steger K, Balhorn R.Protamines are nuclear proteins which are specifically expressed in haploid male germ cells. Their replacement of histones and binding to DNA is followed by chromatin hypercondensation that protects DNA from negative influences by environmental factors. Mammalian sperm contain two types of protamines: PRM1 and PRM2. While the proportion of the two protamines is highly variable between different species, abnormal ratios within a species are known to be associated with male subfertility. Therefore, it is more than likely that correct protamine expression represents a kind of chromatin checkpoint...
Ferreira HN, Ferreira-Silva JC, Rocha JM, Farras MC, Calixto M, Moura MT, Alvarenga MA, Oliveira AL.Semen cryopreservation causes DNA damage, thus requiring continuous monitoring. Objective: To compare two assays for sperm DNA fragmentation (SDF) from stallions with contrasting semen freezability. Methods: Thirteen stallions were classified as good semen freezers (GSF) or bad semen freezers (BSF). Ejaculates were cryopreserved with three diluents. Semen was subject to SDF evaluation using the sperm chromatin structure assay (SCSA) and Halomax after thawing (0 h) and after a 4 h thermoresistance test. Results: On semen of BSF, analysis by SCSA was similar between evaluations, but Halomax show...
Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Chromosome 11. Here, we investigated the satellite-free centromeres of by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regio...
Read JE, Cabrera-Sharp V, Kitscha P, Cartwright JE, King PJ, Fowkes RC, de Mestre AM.Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCGβ-subunit (), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates promoter activity. Here, studies of the proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equi...
Johannisson A, Figueiredo MI, Al-Kass Z, Morrell JM.An improved fertility prediction for stallions is of importance for equine breeding. Here, we investigate the potential of a combined staining of stallion spermatozoa for superoxide and mitochondrial membrane potential (MMP) for this purpose. Semen samples were analysed immediately after arrival at the laboratory, as well as after 24 h. Superoxide was measured by MitoSOXRed, while MMP was measured with JC-1. Menadione was used to stimulate superoxide production. In addition, other parameters of sperm quality, namely motility, membrane integrity, chromatin integrity, sperm kinematics and Hoec...
Al-Essawe EM, Johannisson A, Wulf M, Aurich C, Morrell JM.Addition of seminal plasma (SP) prior to cryopreservation may influence stallion sperm cryosurvival. The objective of this study was to investigate the addition of pooled SP from "good" or "bad" freezer stallions to spermatozoa selected by single layer centrifugation (SLC) prior to cryopreservation on post-thaw sperm quality. Semen from 12 stallions was collected; 5 mL was frozen as control (C) and the remainder was processed by SLC to remove SP and was divided into three aliquots: i) SLC sample without SP (SLC); ii) SLC plus pooled SP from "good freezer" stallions (SLC-GF); iii) SLC plus po...
Al-Kass Z, Spergser J, Aurich C, Kuhl J, Schmidt K, Johannisson A, Morrell JM.Contamination of semen with bacteria arises during semen collection and handling. This bacterial contamination is typically controlled by adding antibiotics to semen extenders but intensive usage of antibiotics can lead to the development of bacterial resistance and may be detrimental to sperm quality. The objective of this study was to determine the effects of antibiotics in a semen extender on sperm quality and to investigate the effects of removal of bacteria by modified Single Layer Centrifugation (MSLC) through a colloid. Semen was collected from six adult pony stallions (three ejaculates...
Ketchum CC, Larsen CD, McNeil A, Meyer-Ficca ML, Meyer RG.Chromatin remodeling during spermatogenesis culminates in the exchange of nucleosomes for transition proteins and protamines as an important part of spermatid development to give rise to healthy sperm. Comparative immunofluorescence analyses of equine and murine testis histological sections were used to characterize nucleoprotein exchange in the stallion. Histone H4 hyperacetylation is considered a key event of histone removal during the nucleoprotein transition to a protamine-based sperm chromatin structure. In the stallion, but not the mouse, H4 was already highly acetylated in lysine residu...
Oldenhof H, Zhang M, Narten K, Bigalk J, Sydykov B, Wolkers WF, Sieme H.Nonviable freeze-dried sperm have intact chromatin and can be used for fertilization via intracytoplasmic sperm injection. Freeze-dried sperm preferably should be stored at 4°C or lower, because DNA damage accumulates during storage at room temperature. Disaccharides are known to protect biomolecules both during freezing and drying, by forming a glassy state. Their use is challenging because cellular membranes are normally impermeable for disaccharides. In the current study, we demonstrate that membrane impermeable compounds, including lucifer yellow and trehalose, are taken up by stallion sp...
Serafini R, Varner DD, Bissett W, Blanchard TL, Teague SR, Love CC.The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabil...
Oldenhof H, Schütze S, Wolkers WF, Sieme H.Sperm chromatin structure and condensation determine accessibility for damage, and hence success of fertilization and development. The aim of this study was to reveal characteristic spectral features coinciding with abnormal sperm chromatin packing (i.e., DNA-protein interactions) and decreased fertility, using Fourier transform infrared spectroscopy. Chromatin structure in spermatozoa obtained from different stallions was investigated. Furthermore, spermatozoa were exposed to oxidative stress, or treated with thiol-oxidizing and disulfide-reducing agents, to alter chromatin structure and pack...
Leemans B, Gadella BM, Stout TA, Heras S, Smits K, Ferrer-Buitrago M, Claes E, Heindryckx B, De Vos WH, Nelis H, Hoogewijs M, Van Soom A.Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1-5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, t...
Drebert Z, Golke A, Cymerys J, Słońska A, Chmielewska A, Tucholska A, Bańbura MW.Equid herpesvirus type 1 (EHV-1) is a prevalent causative agent of equine diseases worldwide. After primary replication in the respiratory epithelium the virus disseminates systemically through a peripheral blood mononuclear cell (PBMC)-associated viraemia. EHV-1 is the only alphaherpes- virus known so far which is capable of establishing latent infection not only in neurons but also in immune system cells (mainly in lymphocytes and macrophages). Since leukocytes are not the target cells for viral replication but are used to transport EHV-1 to the internal organs, the questionremains how the v...
Choi YH, Velez IC, Macías-García B, Hinrichs K.In nuclear transfer (NT), exposure of donor cell chromatin to the ooplast cytoplasm may aid reprogramming; however, the length of exposure feasible is limited by the developmental life span of the oocyte. We examined the effect of duration of nucleus-cytoplasmic exposure before activation and of in vitro maturation (IVM) in equine NT. In experiment 1, 24 h IVM and a delay of 2, 5, or 8 h between reconstruction and activation yielded 4%, 15%, and 11% blastocysts, respectively. In experiment 2, a 5-h activation delay yielded 17% and 22% blastocysts with two donor cell lines. In experiment 3, usi...
Heutelbeck A, Oldenhof H, Rohn K, Martinsson G, Morrell JM, Sieme H.Equipment for cryopreservation of stallion sperm is not always available. In such cases, diluted semen can be shipped to a facility for later cryopreservation. The aim of this study was to evaluate if selection of sperm via density centrifugation yields higher survival rates when cryopreservation is to be delayed (i.e. carried out after 1 day of storage at 5°C). Two-layer iodixanol as well as single-layer Androcoll density centrifugation were tested and compared with samples prepared with standard centrifugation. Special emphasis was placed on comparing centrifugation on the day of semen coll...
Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Chromosome 11. Here, we investigated the satellite-free centromeres of by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regio...
Sailer BL, Jost LK, Evenson DP.Sperm from four mammalian species were analyzed by the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase assay (TdTA) using flow cytometry. The SCSA quantitates the susceptibility of sperm nuclear DNA to in situ acid denaturation, while the TdTA quantitates the presence of endogenous DNA strand breaks in sperm nuclear chromatin. Correlations were seen between the percentage of sperm cells showing susceptibility to in situ acid denaturation and the percentage of cells showing the presence of DNA strand breaks for humans (r = 0.56, P = 0.004), rams (r = 0.84, P...
Morrell JM, Johannisson A, Dalin AM, Rodriguez-Martinez H.The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically norma...
Hinrichs K, Choi YH, Love LB, Varner DD, Love CC, Walckenaer BE.We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes having compact (Cp) and expanded (Ex) cumuli. In addition, we evaluated the effect of oocyte type, time of recovery, and duration of culture on blastocyst development after intracytoplasmic sperm injection. In oocytes collected within 1 h of slaughter, fibrillar and intermediate chromatin configurations were more prevalent in Cp than in Ex oocytes (68% and 12%, respectively). In Cp oocytes collected after a 5- to 9-h delay, the proportions in the ...
Steger K, Balhorn R.Protamines are nuclear proteins which are specifically expressed in haploid male germ cells. Their replacement of histones and binding to DNA is followed by chromatin hypercondensation that protects DNA from negative influences by environmental factors. Mammalian sperm contain two types of protamines: PRM1 and PRM2. While the proportion of the two protamines is highly variable between different species, abnormal ratios within a species are known to be associated with male subfertility. Therefore, it is more than likely that correct protamine expression represents a kind of chromatin checkpoint...
López-Fernández C, Crespo F, Arroyo F, Fernández JL, Arana P, Johnston SD, Gosálvez J.The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. ...
Johannisson A, Morrell JM, Thorén J, Jönsson M, Dalin AM, Rodriguez-Martinez H.The objective was to investigate the changes in stallion sperm quality (sperm motility, viability, membrane integrity and chromatin integrity) occurring during cool storage, and to study the effect of sperm selection by single layer colloidal centrifugation on these parameters of sperm quality. Spermatozoa from 3 stallions (10 ejaculates, 3-4 per stallion) were selected by centrifugation through a single layer of colloid (SLC). The resulting sperm preparations and the control samples (extended but unselected semen samples) were stored at 5 degrees C for 48h. Assessments of sperm quality, such ...
Livolant F.The fine structure of chromatin in sperm heads was investigated by different microscopic techniques: in vivo examinations in the polarizing microscope, thin sections and freeze-fracture replicas observed by transmission electron microscopy. The freeze-fractured chromatin appears to be formed of superimposed lamellae, each one 330 A thick. These lamellae are parallel to the flattening plane of the sperm head. This situation was already described in other mammal spermatozoa and in particular in the bull and the rabbit. This work presents a new interpretation of this lamellated aspect. The chroma...
Hinrichs K, Schmidt AL.Horse oocytes were collected from an abattoir over a 15-mo period. After classification of follicle size and cumulus morphology, oocytes were either fixed immediately (0 h) or matured in vitro (24 h). There was no effect of season on the number of antral follicles present on the ovaries, or on oocyte maturation rate for any class of oocyte. The proportion of oocytes having condensed chromatin at 0 h increased with increasing follicle size. The oocyte maturation rate also increased with follicle size, and for follicles </= 20-mm diameter, was higher for oocytes initially having expanded cumu...
Franciosi F, Lodde V, Goudet G, Duchamp G, Deleuze S, Douet C, Tessaro I, Luciano AM.Epigenetic modifications are established during gametogenesis and preimplantation embryonic development. Any disturbance of the normal natural environment during these critical phases could cause alterations of the epigenetic signature. Histone acetylation is an important epigenetic modification involved in the regulation of chromatin organization and gene expression. The present study was aimed to determine whether the proper establishment of post-translational histone H4 acetylation at lysine 8 (AcH4K8), 12 (AcH4K12) and 16 (AcH4K16) of equine oocytes is adversely affected during in vitro ma...
Baumann C, Daly CM, McDonnell SM, Viveiros MM, De La Fuente R.Pairing of the sex chromosomes during mammalian meiosis is characterized by the formation of a unique heterochromatin structure at the XY body. The mechanisms underlying the formation of this nuclear domain are reportedly highly conserved from marsupials to mammals. In this study, we demonstrate that in contrast to all eutherian species studied to date, partial synapsis of the heterologous sex chromosomes during pachytene stage in the horse is not associated with the formation of a typical macrochromatin domain at the XY body. While phosphorylated histone H2AX (γH2AX) and macroH2A1.2 are pres...
Oldenhof H, Zhang M, Narten K, Bigalk J, Sydykov B, Wolkers WF, Sieme H.Nonviable freeze-dried sperm have intact chromatin and can be used for fertilization via intracytoplasmic sperm injection. Freeze-dried sperm preferably should be stored at 4°C or lower, because DNA damage accumulates during storage at room temperature. Disaccharides are known to protect biomolecules both during freezing and drying, by forming a glassy state. Their use is challenging because cellular membranes are normally impermeable for disaccharides. In the current study, we demonstrate that membrane impermeable compounds, including lucifer yellow and trehalose, are taken up by stallion sp...
Hinrichs K, Schmidt AL, Friedman PP, Selgrath JP, Martin MG.The chromatin configuration of resting horse oocytes and the time course of in vitro oocyte maturation was characterized using a fluorescent, DNA-specific label. Oocytes were classified as having either compact (CP) or expanded (EX) cumuli at the time of collection. Centrifugation of oocytes was effective in allowing visualization of the germinal vesicle. Two main chromatin configurations were found in oocytes known to have a germinal vesicle: condensed chromatin (CC), in which the chromatin formed a dense mass surrounding the nucleolus; and fluorescing nucleus (FN), in which the entire nucleu...
Minervini F, Lacalandra GM, Filannino A, Garbetta A, Nicassio M, Dell'aquila ME, Visconti A.Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm mo...
Al-Essawe EM, Johannisson A, Wulf M, Aurich C, Morrell JM.Addition of seminal plasma (SP) prior to cryopreservation may influence stallion sperm cryosurvival. The objective of this study was to investigate the addition of pooled SP from "good" or "bad" freezer stallions to spermatozoa selected by single layer centrifugation (SLC) prior to cryopreservation on post-thaw sperm quality. Semen from 12 stallions was collected; 5 mL was frozen as control (C) and the remainder was processed by SLC to remove SP and was divided into three aliquots: i) SLC sample without SP (SLC); ii) SLC plus pooled SP from "good freezer" stallions (SLC-GF); iii) SLC plus po...
Ketchum CC, Larsen CD, McNeil A, Meyer-Ficca ML, Meyer RG.Chromatin remodeling during spermatogenesis culminates in the exchange of nucleosomes for transition proteins and protamines as an important part of spermatid development to give rise to healthy sperm. Comparative immunofluorescence analyses of equine and murine testis histological sections were used to characterize nucleoprotein exchange in the stallion. Histone H4 hyperacetylation is considered a key event of histone removal during the nucleoprotein transition to a protamine-based sperm chromatin structure. In the stallion, but not the mouse, H4 was already highly acetylated in lysine residu...
Li N, Yu J, Yang F, Shao Y, Wu S, Liu B, Li M, Wang T, Li J, Zeng S.The aim of this study was to evaluate the effects of L-proline on the extender quality of frozen and post-thawed jackass semen. Jackass (n = 6) semen samples were collected and cryopreserved in gradient concentrations (0-80 mM) of L-proline in extenders; post-thawed semen samples were cultured in L-proline medium for 10 hours at 37°C. For cryopreservation experiment I, the motile parameters, mitochondrial membrane potential (MMP), and plasma membrane, acrosome, and chromatin structure integrities of post-thawed semen were assessed. For culture experiment II, additional ROS contents were ana...
Leemans B, Gadella BM, Stout TA, Heras S, Smits K, Ferrer-Buitrago M, Claes E, Heindryckx B, De Vos WH, Nelis H, Hoogewijs M, Van Soom A.Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1-5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, t...
Tremoleda JL, Van Haeften T, Stout TA, Colenbrander B, Bevers MM.Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and exami...
Morrell JM, Mari G, Kútvölgyi G, Meurling S, Mislei B, Iacono E, Rodriguez-Martinez H.Some stallions produce ejaculates of low quality and/or low fertility when used for artificial insemination (AI). The purpose of these five case studies was to use Single Layer Centrifugation (SLC) to select the best spermatozoa from 'problem' ejaculates for subsequent use in AI. Sperm quality, in terms of motility, morphology and chromatin integrity, was improved in the SLC-selected samples compared to the corresponding uncentrifuged samples, with the exception of one stallion thought to have ampullary stasis. In this stallion, neither the incidence of spermatozoa with detached heads nor the ...
Morrell JM, Rodriguez-Martinez H, Johannisson A.An improvement in sperm quality after single layer centrifugation (SLC) has been seen in previous studies using small sample sizes (for example, n = 10 stallions). There is a need to investigate whether this improvement is repeatable over several breeding seasons with a larger number of stallions (n ≥ 30 stallions). Objective: To make a retrospective analysis of the results of SLC performed on more than 250 sperm samples (176 ejaculates) from 31 stallions in 3 consecutive breeding seasons. Methods: Sperm quality (motility, proportion of morphologically normal spermatozoa and the proportion o...
Love CC, Kenney RM.A variety of testicular insults can induce changes in the structure of spermatozoal chromatin, resulting in spermatozoal DNA that is more susceptible to acid-induced denaturation. The degree of change in the DNA can be measured using the sperm chromatin structure assay (SCSA). The SCSA measures the relative amounts of single- and double-stranded DNA after staining with the metachromatic dye, acridine orange. Here we used a stallion model (n = 4) to study the effects of scrotal heat stress on spermatozoal DNA. This model was created by insulating stallion testes for 48 h and collecting sperm da...
Paradowska-Dogan A, Fernandez A, Bergmann M, Kretzer K, Mallidis C, Vieweg M, Waliszewski P, Zitzmann M, Weidner W, Steger K, Kliesch S.Highly compacted sperm DNA in protamine toroids and a minor fraction of nucleohistones are prerequisites for the efficient transmission of the paternal genome into the oocyte at fertilization. The objective of this study was to evaluate whether protamines might serve as a prognostic factor for stallion fertility. In situ hybridization detected specific expression of P1 mRNA in the cytoplasm of stage I to VII spermatids, whereas comparable immunohistochemical stainings showed that protein expression was delayed till elongating spermatids in differentiation stages III to VIII. No staining was de...
Hinrichs K, Williams KA.Horse oocytes with expanded (EX) cumuli appear to have greater meiotic competence than do horse oocytes with compact (CP) cumuli but are thought to come from atretic follicles. We evaluated the relationships among cumulus expansion, follicle viability, initial chromatin configuration, and meiotic competence of horse oocytes. Follicle walls were sectioned for histological examination, and the follicles were scraped to obtain the oocytes. Half of the oocytes were evaluated immediately and half were matured for 24 h in vitro. Cumulus expansion was significantly associated with follicle atresia. I...
Dias GM, Retamal CA, Tobella L, Arnholdt AC, López ML.'The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatoz...
Minervini F, Lacalandra GM, Filannino A, Nicassio M, Visconti A, Dell'Aquila ME.The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate t...
Al-Kass Z, Ntallaris T, Morrell JM, Johannisson A.Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or packaging and fertility have not been explored. In the present study, relationships between fertility and DNA fragmentation index, protamine deficiency, total thiols, free thiols and disulfide bonds in stallion spermatozoa were investigated. Ejaculates (n = 36) were collected from 12 stallions and extended to prepare semen doses for insemination. One dose from each ejaculate was sent to the Swedish University of Agricultural Sc...
Neild DN, Gadella BM, Agüero A, Stout TA, Colenbrander B.In general, fertility in breeding stallions is lower and more variable than in the other farm animal species, primarily because selection is based on pedigree, looks and/or athletic performance, with little consideration of fertility or fertility potential. Moreover, because the average stallion breeds only a limited number of mares per year and in-field fertility is influenced significantly by non-stallion factors such as management and mare fertility, meaningful fertility data are hard to come-by. Unfortunately, generating usable figures would involve impractically high costs, time and numbe...
Pedersen HG, Watson ED, Telfer EE.The effects of ovary holding time and temperature on granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology were investigated through a series of experiments. Three experiments were performed to determine the effect of ovary holding time and temperature on granulosa cell apoptosis. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 2h, (2) at 30 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6 or 6-10h, and (3) granulosa cells were held for 0, 1, 2, 3, 5, 12 or 24h in M199 with Hank's salts at room temperature (suboptimal incubation). Granulosa cell DNA was analysed by ...
