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.
Martino NA, Dell'Aquila ME, Filioli Uranio M, Rutigliano L, Nicassio M, Lacalandra GM, Hinrichs K.Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle's/Hank's' M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic su...
Morrell JM, Johannisson A.The effect on sperm motility and chromatin integrity of adding homologous or heterologous equine seminal plasma (SP) to fresh stallion spermatozoa selected by single layer centrifugation (SLC) was studied. No statistical difference in mean progressive motility was seen after adding SP at time 0 h, although there were differences for individual stallions. The proportion of spermatozoa with high velocity was increased compared to untreated SLC-selected spermatozoa (P < 0.05), with significant differences between individuals (P < 0.01). When the SLC samples were stored for 24 h before a...
Johannisson A, Lundgren A, Humblot P, Morrell JM.The decrease in foaling rates after artificial insemination with cooled semen warrants the search for new predictors of fertility. The objectives were to investigate levels of naturally occurring reactive oxygen species (ROS) in cooled, stored stallion semen doses for artificial insemination (AI), and their relationship with parameters of semen quality and with pregnancy rate. Semen was collected from warmblood stallions (n=15) and used to prepare commercial semen doses for AI. Sperm quality was evaluated after cooled transport to the laboratory overnight. The results were correlated with obse...
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...
Morrell JM, Stuhtmann G, Meurling S, Lundgren A, Winblad C, Macias Garcia B, Johannisson A.Many attempts have been made to identify laboratory tests that are predictive of sperm fertility, both to improve the quality of stallion semen doses for artificial insemination (AI) and to identify potential breeding sires if no fertility data are available. Sperm quality at the stud is mostly evaluated by assessing subjective motility, although this parameter can be poorly indicative of fertility. Sperm morphology and chromatin integrity in Swedish stallions are correlated to pregnancy rate after AI. Because single layer centrifugation (SLC) selects for spermatozoa with normal morphology and...
Cortés-Gutiérrez EI, Dávila-Rodríguez MI, López-Fernández C, Fernández JL, Crespo F, Gosálvez J.The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the AL...
Gutiérrez-Cepeda L, Fernández A, Crespo F, Ramírez MÁ, Gosálvez J, Serres C.Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of e...
Vieira LA, Gadea J, García-Vázquez FA, Avilés-López K, Matás C.After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4°C up to 96h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing-tha...
Morrell JM, Pihl J, Dalin AM, Johannisson A.There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin i...
Oldenhof H, Friedel K, Akhoondi M, Gojowsky M, Wolkers WF, Sieme H.Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures...
Potocki L, Bugno-Poniewierska M, Tischner M, Wnuk M.The development of molecular techniques with fluorochromes has had an invaluable impact on discovering the nature of chromatin structure. Here, we show the application of a locus specific identifier probe (LSI) for precise and selective visualization of the horse IGF2 gene in the metaphase, interphase nuclei and sperm cells. Our study may be helpful for interpretation of results of interphase fluorescence in situ hybridization (I-FISH). We analyze and discuss the variation in the number and localization of FISH signals in somatic and sperm cells of horse.
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...
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...
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...
Morrell JM, Rodriguez-Martinez H, Johannisson A.This study compared the effect on semen quality of different handling methods used in the preparation of stallion semen doses for artificial insemination. The three methods were (i) extending the ejaculate to 30-50 x 10(6)/ml, (ii) single layer centrifugation (SLC) and (iii) sperm washing (centrifugation without a colloid). An additional treatment was to add seminal plasma (SP) in various proportions to some SLC preparations. The resulting samples were evaluated for sperm motility by computer assisted sperm analysis, membrane integrity using the Nucleocounter SP-100 and chromatin integrity by ...
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...
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 ...
Cortés-Gutiérrez EI, Crespo F, Serres-Dalmau C, Gutiérrez de las Rozas AL, Dávila-Rodríguez MI, López-Fernández C, Gósalvez J.Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present....
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...
Cavalcanti MC, Rizgalla M, Geyer J, Failing K, Litzke LF, Bergmann M.In eukaryotic cells, the major protein constituents of the chromatin are histones, which can be divided into five classes, identified as H1, H2A, H2B, H3 and H4. During normal spermatogenesis, a testis-specific H1t is expressed in primary spermatocytes and believed to facilitate histone to protamine exchanges during spermiogenesis. In equine testes we detected the H1 protein at 22kDa by western blot analysis while H1t was detected at 29kDa. H1 protein was found to be expressed in all germ cells up to elongating spermatids (Sc) at stage IV. In peripubertal animals, there was a prolonged express...
Morrell JM, Johannisson A, Dalin AM, Hammar L, Sandebert T, Rodriguez-Martinez H.Artificial insemination is not as widely used in horses as in other domestic species, such as dairy cattle and pigs, partly because of the wide variation in sperm quality between stallion ejaculates and partly due to decreased fertility following the use of cooled transported spermatozoa. Furthermore, predictive tests for sperm fertilising ability are lacking. The objective of the present study was to assess sperm morphology and chromatin integrity in ejaculates obtained from 11 warmblood breeding stallions in Sweden, and to evaluate the relationship of these parameters to pregnancy rates to i...
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. ...
Bertelsmann H, Sieme H, Behne D, Kyriakopoulos A.In the sperm nuclei, of mammalian species selenium has been found only in the form of sperm nuclei glutathione peroxidase (snGPx) where it is most likely bound to the chromatin of spermatozoa. Over 80% of selenium in sperm is bound to the selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the midpiece of rat sperm. Zinc in sperm is mainly contained in the outer dense fiber (ODF) proteins of the flagella of mammalian spermatozoa. In the sperm nuclei, zinc is predominately located in the chromatin to the protamine proteins. In order to investigate if the insertion of zinc...
Choi YH, Love LB, Varner DD, Hinrichs K.Holding immature oocytes before the onset of maturation simplifies oocyte transport and aids in scheduling later manipulations. We report here a method for holding equine oocytes in the absence of meiotic inhibitors. In Experiment 1, immature oocytes with expanded cumuli were cultured at 38.2 degrees C in medium containing cycloheximide, or were held at room-temperature in M199 with Hanks' salts, for 16-18 h before maturation. Control oocytes were matured immediately after recovery. Oocytes were fertilized by intracytoplasmic sperm injection and cultured for 4d. Embryo development was not diff...
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...
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...
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 ...
Morrell JM, Pihl J, Dalin AM, Johannisson A.There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin i...
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...
Choi YH, Love LB, Westhusin ME, Hinrichs K.Early development of embryos produced by transfer of equine nuclei to bovine cytoplasts is superior to that of intraspecies equine nuclear transfer embryos. This may be related to differences in chromatin remodeling or efficiency of activation between the two oocyte types. The pattern of donor nucleus remodeling was examined in equine-equine and equine-bovine reconstructed oocytes. Chromosome condensation occurred in equine cytoplasts by 2 h but was not seen in bovine cytoplasts until 4 h. We investigated the effect of activation of equine-equine reconstructed oocytes at <30 min or at 2 h a...
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...
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...
Hinrichs K.When recovered from the follicle, horse oocytes may be categorised as having either a compact or an expanded cumulus. Cumulus expansion is strongly associated with follicle atresia. Oocytes with expanded and compact cumuli have similar proportions in the germinal vesicle stage when recovered from the follicle. However, during in vitro culture, a higher proportion of oocytes with expanded cumuli mature, and they do so more quickly, than do oocytes with compact cumuli. Using Hoechst 33258 to label chromatin, in the germinal-vesicle stage horse oocytes can be divided into those in which the nucle...
Cavalcanti MC, Rizgalla M, Geyer J, Failing K, Litzke LF, Bergmann M.In eukaryotic cells, the major protein constituents of the chromatin are histones, which can be divided into five classes, identified as H1, H2A, H2B, H3 and H4. During normal spermatogenesis, a testis-specific H1t is expressed in primary spermatocytes and believed to facilitate histone to protamine exchanges during spermiogenesis. In equine testes we detected the H1 protein at 22kDa by western blot analysis while H1t was detected at 29kDa. H1 protein was found to be expressed in all germ cells up to elongating spermatids (Sc) at stage IV. In peripubertal animals, there was a prolonged express...
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...
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...
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 ...
Gutiérrez-Cepeda L, Fernández A, Crespo F, Ramírez MÁ, Gosálvez J, Serres C.Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of e...
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...
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...
Cortés-Gutiérrez EI, Dávila-Rodríguez MI, López-Fernández C, Fernández JL, Crespo F, Gosálvez J.The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the AL...
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...
Johannisson A, Lundgren A, Humblot P, Morrell JM.The decrease in foaling rates after artificial insemination with cooled semen warrants the search for new predictors of fertility. The objectives were to investigate levels of naturally occurring reactive oxygen species (ROS) in cooled, stored stallion semen doses for artificial insemination (AI), and their relationship with parameters of semen quality and with pregnancy rate. Semen was collected from warmblood stallions (n=15) and used to prepare commercial semen doses for AI. Sperm quality was evaluated after cooled transport to the laboratory overnight. The results were correlated with obse...
Oldenhof H, Friedel K, Akhoondi M, Gojowsky M, Wolkers WF, Sieme H.Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures...
Cortés-Gutiérrez EI, Crespo F, Serres-Dalmau C, Gutiérrez de las Rozas AL, Dávila-Rodríguez MI, López-Fernández C, Gósalvez J.Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present....
Blöcher S, Behr R, Weinbauer GF, Bergmann M, Steger K.Histone-to-protamine exchange causes chromatin condensation ceasing gene expression in elongating spermatids. Gene expression of protamines is regulated by the transcription factor cAMP-responsive element modulator (CREM). Altered CREM expression results in male infertility, as shown by CREM-knock-out mice being sterile due to round spermatid maturation arrest and patients exhibiting round spermatid maturation arrest revealing a lack or substantial reduction of both CREM-mRNA and CREM-protein. Similar defects in histone-to-protamine exchange have been suggested in infertile stallions exhibitin...
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...
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...
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...
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 ...
Martino NA, Dell'Aquila ME, Filioli Uranio M, Rutigliano L, Nicassio M, Lacalandra GM, Hinrichs K.Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle's/Hank's' M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic su...
Morrell JM, Stuhtmann G, Meurling S, Lundgren A, Winblad C, Macias Garcia B, Johannisson A.Many attempts have been made to identify laboratory tests that are predictive of sperm fertility, both to improve the quality of stallion semen doses for artificial insemination (AI) and to identify potential breeding sires if no fertility data are available. Sperm quality at the stud is mostly evaluated by assessing subjective motility, although this parameter can be poorly indicative of fertility. Sperm morphology and chromatin integrity in Swedish stallions are correlated to pregnancy rate after AI. Because single layer centrifugation (SLC) selects for spermatozoa with normal morphology and...
Love CC, Love LB, Varner DD, Hinrichs K.The relationship of holding time in media at room temperature (approximately 22 degrees C) to initial chromatin configuration and rate of in vitro maturation (IVM) of equine oocytes was determined. Only oocytes having a complete, compact cumulus were used in this study. Oocytes were removed from ovaries 3.5-8 h after slaughter and were put into one of four treatment groups: (1) immediate/fix (IF) = immediate fixation following removal from the ovary; (2) delay/fix (DF) = fixation after oocytes were held 1-4 h in medium at room temperature; (3) immediate/mature (IM) = immediate placement into m...
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...
Mäkelä O, Gustavsson I, Hollmén T.Chromosomal analysis is not a routine examination in equine
practice. It is indicated, however, in infertile or subfertile
mares with small, inactive ovaries (Chandley et a/. 1975;
Power 1986).
The most commonly reported abnormalities in mares
concern sex chromosomes such as 63,XO and mosaic
63,XO/64,XX giving sterile mares and 64,XY resulting in
gonadal dysgenesis, sex reversal and testicular feminisation
(Power 1990). The phenotypic manifestations of a horse with
these karyotypic abnormalities are usually quite mild
compared with the corresponding abnormalities in man. These
mild ...
Kanitz W, Alm H, Becker F, Nürnberg G, Kurth J, Hinrichs K.Cumulus-oocyte complexes (COCs) recovered from ovaries of mares killed at abattoirs or after in vivo collection have heterogeneous morphologies and meiotic competence as follicles of variable quality are used. It is thought that it should be possible to recover more uniform COCs, with respect to morphology and nuclear maturation, by repeated follicle aspiration. Therefore, the influence of repeated follicle aspiration on the number and diameter of follicles > or =5 mm in diameter, the morphology and recovery rate of COCs, and the chromatin configuration in oocytes was investigated. Repeated...
