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.
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 ...
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 ...
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...
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...
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...
Pirhonen A, Linnala-Kankkunen A, Kenney RM, Mäenpää PH.Protamine P1 amino acid sequences were determined from semen samples of the Przewalski horse, donkey, Somali wild ass, Grevy's zebra, and Grant's zebra (odd-toed perissodactyls), and compared with those of the domestic horse. Although the rate of amino acid variation of protamine P1 is known to be among the most rapidly diverging polypeptides, the equid sequences revealed only little variation. The sequence from the Przewalski horse was identical with that from the domestic horse. The other sequences differed from the corresponding sequences of the domestic and Przewalski horses in two positio...
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...
Hinrichs K, Love CC, Choi YH, Varner DD, Wiggins CN, Reinoehl C.Germinal vesicle (GV)-stage horse oocytes with diffuse chromatin are meiotically incompetent and degenerate in culture, whereas horse oocytes having condensed chromatin within the GV are meiotically competent. Degeneration of incompetent oocytes in culture may be related to premature GV breakdown, which could possibly be prevented by inhibition of m-phase protein activity. We examined the effects of 6-dimethylaminopurine (6-DMAP), butyrolactone and roscovitine on GV-stage horse oocytes. Culture in the presence of 2 mM 6-DMAP for 24 h suppressed meiosis (2% MI or MII compared with 38% for untre...
Bézard J, Bøgh IB, Duchamp G, Hyttel P, Greve T.Nuclear maturation of equine oocytes was assessed immediately after in vivo collection. A double-staining technique (Hoechst and orcein) was used on the same oocytes to visualize nuclear morphology, i.e. to evaluate the chromatin configurations of each oocyte after Hoechst in relation to the nuclear morphology after orcein staining. The proportion of oocytes evaluated as germinal vesicle stages was significantly (p < 0.02) lower after Hoechst (14.5%) than after orcein staining (29.0%), while the incidence of the so-called dense chromatin stage was assessed to be higher (p < 0.05) after H...
Tremoleda JL, Schoevers EJ, Stout TA, Colenbrander B, Bevers MM.Meiotic maturation of mammalian oocytes is a complex process during which microfilaments and microtubules provide the framework for chromosomal reorganisation and cell division. The aim of this study was to use fluorescence and confocal laser scanning microscopy to examine changes in the distribution of these important cytoskeletal elements and their relationship to chromatin configuration during the maturation of horse oocytes in vitro. Oocytes were cultured in M199 supplemented with pFSH and eLH and, at 0, 12, 24, and 36 hr after the onset of culture, they were fixed for immunocytochemistry ...
Deriusheva SE, Loginova IuA, Chiriaeva OG, Iasinetskaia NI, Efimov AM.Restriction endonuclease in situ digestion of metaphase chromosomes gives an opportunity to reveal strips with different structure within GC-rich pericentric heterochromatin of the domestic horse and the wild Przewalski horse. Blocks of heterochromatin, which are insensitive to HaeIII and brightly stained with chromomycin A3 after restriction enzyme digestion, are localized on the border with euchromatin in the majority of chromosomes of Equus caballus and E. przewalskii. In contrast to chromosome 5 of E. caballus, acrocentric chromosomes of E. prezewalskii which are homologous to this chromos...
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...
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...
Evenson DP, Jost LK, Varner DD.Data from the sperm chromatin structure assay (SCSA), a flow cytometric measurement of susceptibility of sperm nuclear DNA to denaturation, show strong correlation with the fertility potential of bulls, boars, men and stallions. Previous studies showed a strong relationship between stallion spermatozoa with denatured DNA and the presence of DNA strand breaks. In the present study, the relationship between stallion sperm DNA denaturation and the redox status of -SH groups on the cysteine residues of sperm nuclear protamines that are thought to stabilize chromatin was investigated. Semen samples...
Pedersen HG, Watson ED, Telfer EE.During the oestrous cycle follicles grow and either ovulate or regress. Regressing follicles undergo atresia and in many species apoptosis has been identified as the underlying mechanism in this process. The aims of this study were to establish whether equine granulosa cells degenerate via an apoptotic mechanism and whether the presence of apoptotic cell death in granulosa cells is correlated with oocyte quality. Ovaries from mares at unknown stages of the oestrous cycle were obtained from an abattoir. In Expt 1, follicles (n=352) from 37 mares were processed. DNA was extracted from granulosa ...
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...
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...
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...
Rodriguez H, Bustos Obregon E.The fertilizing ability of stallion semen was analyzed using fresh and frozen samples, obtained before (June-July) or during (October-November) the breeding season. Thirty ejaculates obtained from 4 stallions were used. The analysis comprises routine seminogram; ATP concentration (Comhaire et al., 1983); subjective and objective motility and sperm velocity (Makler, 1980). Freezing was done following the technique of Martin et al. (1979). Sperm velocity, ATP content and objective motility in ejaculates of subjective motility >50% show values of 14.0 + or - 0.84 mu m s(-1); 4.8 + or - 2.7x10(...
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...
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 ...
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...
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...
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...
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...
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 ...
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...
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...
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...
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...
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...
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...
Rodriguez H, Bustos Obregon E.The fertilizing ability of stallion semen was analyzed using fresh and frozen samples, obtained before (June-July) or during (October-November) the breeding season. Thirty ejaculates obtained from 4 stallions were used. The analysis comprises routine seminogram; ATP concentration (Comhaire et al., 1983); subjective and objective motility and sperm velocity (Makler, 1980). Freezing was done following the technique of Martin et al. (1979). Sperm velocity, ATP content and objective motility in ejaculates of subjective motility >50% show values of 14.0 + or - 0.84 mu m s(-1); 4.8 + or - 2.7x10(...
Pedersen HG, Watson ED, Telfer EE.During the oestrous cycle follicles grow and either ovulate or regress. Regressing follicles undergo atresia and in many species apoptosis has been identified as the underlying mechanism in this process. The aims of this study were to establish whether equine granulosa cells degenerate via an apoptotic mechanism and whether the presence of apoptotic cell death in granulosa cells is correlated with oocyte quality. Ovaries from mares at unknown stages of the oestrous cycle were obtained from an abattoir. In Expt 1, follicles (n=352) from 37 mares were processed. DNA was extracted from granulosa ...
While the centromeric function is conserved and epigenetically specified by CENP-A, centromeric DNA, typically composed of satellite repeats, is highly divergent and rapidly evolving. In the species of the genus Equus (horses, asses and zebras), also known as equids, the numerous centromeres devoid of satellite repeats enabled us to carry out molecular analysis of centromeric chromatin establishing a unique model system for mammalian centromere biology. In this review, after a brief description of the rapid evolution of equids, we outline one of our most relevant initial discoveries: the posit...
Conn KL.Equid alphaherpesvirus 1 (EHV1) is a DNA virus that causes severe disease in equids. Some strains are neurotropic and cause disease in the central nervous system, whereas others are non-neurotropic and can cause negative reproductive outcomes. Mechanisms governing EHV1 pathotype are not understood. However, EHV1 pathotypes have different infection efficiencies and replication kinetics in various cell types. They are also differentially susceptible to epigenetic inhibitors. Aside from this observation little is known about EHV1 chromatin or how its regulation influences infection. To build know...
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.
Brownlow MA, Hutchins DR, Johnston KG.Cells in the peritoneal fluid from 159 horses were examined in Giemsa stained preparations using light microscopy. Normal mesothelial cells showed an oval nucleus with finely reticular chromatin and pale blue cytoplasm. Activated mesothelial cells occurred in fluids derived from mesothelium under acute or subacute, non-septic stimulus and were remarkable for their pleomorphism and intense basophilia which may mimic neoplasia. Transformed mesothelial cells seen in chronic inflammatory fluids were sometimes phagocytic and showed conspicuous cytoplasmic vacuolation.
Pirhonen A, Linnala-Kankkunen A, Kenney RM, Mäenpää PH.Protamine P1 amino acid sequences were determined from semen samples of the Przewalski horse, donkey, Somali wild ass, Grevy's zebra, and Grant's zebra (odd-toed perissodactyls), and compared with those of the domestic horse. Although the rate of amino acid variation of protamine P1 is known to be among the most rapidly diverging polypeptides, the equid sequences revealed only little variation. The sequence from the Przewalski horse was identical with that from the domestic horse. The other sequences differed from the corresponding sequences of the domestic and Przewalski horses in two positio...
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...
Hinrichs K, Love CC, Choi YH, Varner DD, Wiggins CN, Reinoehl C.Germinal vesicle (GV)-stage horse oocytes with diffuse chromatin are meiotically incompetent and degenerate in culture, whereas horse oocytes having condensed chromatin within the GV are meiotically competent. Degeneration of incompetent oocytes in culture may be related to premature GV breakdown, which could possibly be prevented by inhibition of m-phase protein activity. We examined the effects of 6-dimethylaminopurine (6-DMAP), butyrolactone and roscovitine on GV-stage horse oocytes. Culture in the presence of 2 mM 6-DMAP for 24 h suppressed meiosis (2% MI or MII compared with 38% for untre...
Bézard J, Bøgh IB, Duchamp G, Hyttel P, Greve T.Nuclear maturation of equine oocytes was assessed immediately after in vivo collection. A double-staining technique (Hoechst and orcein) was used on the same oocytes to visualize nuclear morphology, i.e. to evaluate the chromatin configurations of each oocyte after Hoechst in relation to the nuclear morphology after orcein staining. The proportion of oocytes evaluated as germinal vesicle stages was significantly (p < 0.02) lower after Hoechst (14.5%) than after orcein staining (29.0%), while the incidence of the so-called dense chromatin stage was assessed to be higher (p < 0.05) after H...
Evenson DP, Jost LK, Varner DD.Data from the sperm chromatin structure assay (SCSA), a flow cytometric measurement of susceptibility of sperm nuclear DNA to denaturation, show strong correlation with the fertility potential of bulls, boars, men and stallions. Previous studies showed a strong relationship between stallion spermatozoa with denatured DNA and the presence of DNA strand breaks. In the present study, the relationship between stallion sperm DNA denaturation and the redox status of -SH groups on the cysteine residues of sperm nuclear protamines that are thought to stabilize chromatin was investigated. Semen samples...
Deriusheva SE, Loginova IuA, Chiriaeva OG, Iasinetskaia NI, Efimov AM.Restriction endonuclease in situ digestion of metaphase chromosomes gives an opportunity to reveal strips with different structure within GC-rich pericentric heterochromatin of the domestic horse and the wild Przewalski horse. Blocks of heterochromatin, which are insensitive to HaeIII and brightly stained with chromomycin A3 after restriction enzyme digestion, are localized on the border with euchromatin in the majority of chromosomes of Equus caballus and E. przewalskii. In contrast to chromosome 5 of E. caballus, acrocentric chromosomes of E. prezewalskii which are homologous to this chromos...
Yi M, Asgenbaatar N, Wang X, Ulaangerel T, Shen Y, Wen X, Du M, Dong X, Dugarjav M, Bou G.DNA methyltransferases (DNMTs) are important epigenetic modification during spermatogenesis. To further evaluate the pattern of DNMTs in horse testes during development, we investigated the expression and localization of DNMT1, DNMT3a and DNMT3b at different time points. The qRT-PCR results showed that DNMT1 expression was maintained in testes tissue from 6-month-old (0.5y) to 2-year-old (2y) of age and decreased after 3-year-old (3y) (P < 0.01). The expression levels of DNMT3a and DNMT3b peaked in testes tissue at 3y (P < 0.01). At 4-year-old (4y), the expression of DNMT3a and DNMT3b was ...
