Developmental capacity of equine oocytes matured and cultured in equine trophoblast-conditioned media

The objective was to compare culture media for in vitro maturation of equine oocytes and for in vitro culture of zygotes produced from IVF of partially zona-removed oocytes. Cumulus-oocyte complexes from slaughterhouse-derived ovaries were washed in m-Dulbecco's PBS and cultured in TCM-199, F10-DMEM or c-F10-DMEM (50% F10-DMEM + 50% F10-DMEM conditioned medium from culture of an equine trophoblast monolayer for 3 or 4 days). All media included FSH, LH, E2, and 10% FCS. After 28 to 30 h maturation, cumulus expansion was scored from 0 (no expansion) to 4 (fully expanded). Oocytes with a 1st polar body were selected for manipulation after removing cumulus cells using hyaluronidase. About one-third of the zona pellucida was cut using a fragment of a razor blade. For fertilization, fresh stallion semen was washed twice in BGM3 (a modified Tyrode's medium) and capacitated with 0.5 mM c-AMP for 3.5 h and 100 microM ionomycin for 15 min and added to oocytes in fert-TALP at 10(6) spermatozoa/mL. After 20 h, some presumptive zygotes were stained, and the rest were cultured in 100% TCM-DMEM conditioned medium. Cumulus expansion in F10-DMEM and c-F10-DMEM was higher (P<0.05) than the TCM-199 control (3.2, 3.5 vs 1.3, on a scale of 0 to 4). However, polar body formation rates were not different among treatments (47, 52 and 50%). The fertilization rates of equine oocytes matured in TCM-199, F10-DMEM and c-F10-DMEM determined by fixing and staining were 41, 35 and 29%, with no significant differences. There were no significant differences among treatments in cleavage rates (36 to 40%), development to morula (3 to 10%), or blastocyst stages (3 to 5%). On Day 14 of culture in c-F10-DMEM treatment, one blastocyst had more than 500 nuclei, but no capsule was formed. In a further study, cleavage rates (46 to 50%) and development to morula (5 to 10%) and blastocyst stages (3 to 8%) were not different (P>0.1) between TCM-DMEM and 100% conditioned TCM-DMEM for culturing embryos. Six embryos (2 morulae and 4 blastocysts) were nonsurgically transferred to 4 recipient mares, but no pregnancy continued.     

Demonstration of a COOH-terminal extension on equine lutropin by means of a common acid-labile bond in equine lutropin and equine chorionic gonadotropin

The beta subunits of equine lutropin and equine chorionic gonadotropin were incubated in 0.013 N HCl for 30 min at 110 degrees C and separated into two fragments by reverse-phase high performance liquid chromatography. The amino acid and carbohydrate compositions of both fragments from each subunit were analyzed. The results demonstrated that equine lutropin-beta has a glycosylated COOH-terminal extension that differs only in carbohydrate composition from the COOH-terminal portion of equine chorionic gonadotropin-beta. This is the first demonstration of a glycosylated COOH-terminal extension in a pituitary glycoprotein hormone.     

Structural studies on equine glycoprotein hormones. Amino acid sequence of equine lutropin beta-subunit

The amino acid sequence was determined for equine lutropin beta (eLH beta). Large fragments were derived from reduced, carboxymethylated eLH beta by digestion with Staphylococcus aureus V8 protease, by cyanogen bromide cleavage, and by cleavage of acid-labile Asp-Pro bonds. The fragments were purified by gel filtration and high performance liquid chromatography (HPLC). The fragments were sequenced by automated Edman degradation to establish the primary structure of eLH beta. Some peptides were further digested with chymotrypsin and the resulting peptides purified by HPLC. In addition to sequencing by automated Edman degradation, these were also sequenced by the complementary 5-dimethylaminonaphthalene-1-sulfonyl-Edman procedure which enabled us to directly identify glycosylated amino acids. The eLH beta subunit is a glycoprotein of 149 amino acids containing both N- and O-linked oligosaccharides. It possesses a COOH-terminal extension similar to that seen in human chorionic gonadotropin. Carboxypeptidase Y digestions suggest that the COOH terminus is blocked by glycosylation. Interestingly, the amino acid sequence of eLH beta is identical to that of equine chorionic gonadotropin beta (Sugino, H., Bousfield, G. R., Moore, W. T., and Ward, D. N. (1987)J. Biol. Chem. 262, 8603-8609).     

Predisposition for venoconstriction in the equine laminar dermis: implications in equine laminitis.

Equine laminitis is a crippling condition associated with a variety of systemic diseases. Although it is apparent that the prodromal stages of laminitis involve microvascular dysfunction, little is known regarding the physiology of this vasculature. The aim of the present study was to determine the relative responses of equine laminar arteries and veins to the vasoconstrictor agonists phenylephrine (1 nM-10 microM), 5-HT (1 nM-10 microM), PGF2alpha (1 nM-100 microM), and endothelin-1 (1 pM-1 microM). We have determined that laminar veins were more sensitive, with respect to the concentration of agonist required to initiate a contractile response and to achieve EC(50), for all agonists tested. EC50 values, for veins and arteries, respectively, were 84+/-7 vs. 688+/-42 nM for phenylephrine, 35+/-6 vs. 224+/-13 nM for 5-HT, 496+/-43 nM vs. 3.0+/-0.6 microM for PGF2alpha, and 467+/-38 pM vs. 70.6+/-6.4 nM for endothelin-1. Moreover, when expressed as a percentage of the response to a depolarizing stimulus (80 mM potassium), the maximal contractile response of laminar veins exceeded that for the laminar arteries for each agonist. These results indicate that there may be a predisposition for venoconstriction within the vasculature of the equine digit. While this physiological predisposition for venoconstriction may be important in the regulation of blood flow during exercise, it also may help to explain why laminitis can result from a variety of pathological systemic conditions.     

Envelope glycoprotein mutations mediate equine amplification and virulence of epizootic venezuelan equine encephalitis virus

Epidemics of Venezuelan equine encephalitis (VEE) result from high-titer equine viremia of IAB and IC subtype viruses that mediate increased mosquito transmission and spillover to humans. Previous genetic studies suggest that mutations in the E2 envelope glycoprotein allow relatively viremia-incompetent, enzootic subtype ID strains to adapt for equine replication, leading to VEE emergence. To test this hypothesis directly, chimeric VEEV strains containing the genetic backbone of enzootic subtype ID strains and the partial envelope glycoprotein genes of epizootic subtype IC and IAB strains, as well as reciprocal chimeras, were used for experimental infections of horses. Insertion of envelope genes from two different, closely related enzootic subtype ID strains into the epizootic backbones resulted in attenuation, demonstrating that the epizootic envelope genes are necessary for the equine-virulent and viremia-competent phenotypes. The partial epizootic envelope genes introduced into an enzootic ID backbone were sufficient to generate the virulent, viremia-competent equine phenotype. These results indicate that a small number of envelope gene mutations can generate an equine amplification-competent, epizootic VEEV from an enzootic progenitor and underscore the limitations of small animal models for evaluating and predicting the epizootic phenotype.     

Seasonal differences in equine spermatocytogenesis

Spermatocytogenesis plays a pivotal role in regulation of spermatogenesis; however, its details remain relatively obscure in nonrodent species. The equine testis contains approximately 100% more spermatogonia in summer than in winter and appears to be a good model to identify the flexible components of spermatocytogenesis that cause seasonal changes in daily sperm production. Testes were taken from horses in the winter (n = 47) and in summer (n = 43). Tissues were fixed by glutaraldehyde-perfusion and submission in osmium, embedded in Epon or methacrylate, sectioned at 0.5 micron or 5 microns, stained with toluidine blue, and observed using bright-field microscopy. The combined total number of A1, A2, A3, and B1 (A plus B1) spermatogonia/testis and the numbers of B2 spermatogonia or early primary spermatocytes were determined by stereology of Epon sections involving testicular volume density and volume of spermatogonial nuclei. In a subset of horses, different spermatogonial subtypes (A1, A2, A3, and B1) were counted per 100 Sertoli cells in each of the 8 spermatogenic stages and expressed as percentage of all A plus B1 spermatogonia. The number of each spermatogonial subtype/testis for the large series of horses was calculated by multiplying the number of A plus B1 spermatogonia/testis (determined for each horse) by the percentage of that given spermatogonial subtype. Season did not significantly affect the number of any given subtype per 100 Sertoli cells in any stage or percentages of different subtypes of spermatogonia. Numbers of A1 (p less than 0.05), A2, A3, B1, or B2 spermatogonia (p less than 0.01) were greater in the breeding season.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrasonic anatomy of equine ovaries

A linear-array ultrasound scanner with a 5-MHz transducer was evaluated for studying follicular and luteal status in mares, and the ultrasonic properties of equine ovaries were characterized. Follicular diameters were estimated in vivo and after removing and slicing six ovaries. Correlation coefficients between the two kinds of determinations were 0.91 for number of follicles >/=2 mm in diameter and 0.95 for diameter of largest follicle. The ovaries of five mares were examined daily until all mares had been examined from three days before an ovulation to three days after the next ovulation. There was a significant difference among days for diameter of largest follicle and second largest follicle and for number of follicles 2-5 mm, 16-20 mm, and >20 mm. Differences seemed to be caused by the presence of many 2- to 5-mm follicles during early diestrus, initiation of growth of large follicles at mid-cycle, selective accelerated growth of an ovulatory follicle beginning five days before ovulation, and regression of large nonovulatory follicles a few days before ovulation. In one of the five mares, the corpus luteum was identified throughout the interovulatory interval, and the corresponding corpus albicans was identified for three days after the second ovulation. In the other four mares, the corpus luteum was last identified an average of 16 days after ovulation or five days before the next ovulation. In a blind study, the location of the corpus luteum (left or right ovary) as determined by ultrasonography agreed with a previous determination of side of ovulation by palpation in 88% of 40 mares on days 0-14. In the remaining 12% and in all of 12 estrous mares, the location was recorded as uncertain. The ultrasound instrument was judged effective for monitoring and evaluating follicles and corpora lutea.     

Hypoosmotic test in equine spermatozoa

The aim of the study was to evaluate equine sperm membrane integrity using the hypoosmotic swelling (HOS) test and to correlate this test with different sperm parameters in raw and frozen thawed semen. The HOS solutions were made with fructose, sucrose, lactose and sodium citrate each at 300, 150, 100, 50 and 25 mosm. Maximum numbers of swollen spermatozoa were observed in solutions of fructose, sucrose and lactose each at 100, 50 and 25 mosm. Correlations between progressive motility, morphologically normal spermatozoa and the HOS test were r = 0.75 and r = 0.51 in raw semen and r = 0.26 and r = -0.22 in frozen-thawed semen. The correlation between HOS and percentage of intact membranes with the fluorescent stain was r = 0.32 in frozen-thawed semen. The HOS test is a simple and accessible method which could be used as a complement to routine equine semen analysis. It has the added advantages of being less susceptible to the immediate effects of cold shock and of evaluating individual spermatozoa rather than the population as a whole, as does progressive motility.     

eFSH in clinical equine practice

Equine follicle stimulating hormone (eFSH) has been used to induce follicular development in transitional mares and problem acyclic mares, as well as superovulate cycling mares. The most efficacious protocol is to administer 12.5 mg eFSH, intramuscularly, twice daily beginning 5 to 7 days after ovulation when the diameter of the largest follicle is 20 to 25 mm. Prostaglandins are to be administered on the second day of eFSH therapy. Treatment with eFSH is continued for 3 to 5 days until follicle(s) are >or=35 mm in diameter. The mare is subsequently allowed to 'coast' for 36 h, after which human chorionic gonadotropin is administered to induce ovulation.     

Conformation of equine pituitary somatotropin

Equine pituitary somatotropin (growth hormone) has been studied by zero-order and second-order absorption spectroscopy, and by circular dichroism. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 16,050 +/- 330 M-1 cm-1 at 278.1 nm. Comparison of the conformations of equine somatotropin and somatotropins isolated from several other mammalian species indicates a close structural relationship between these molecules. With the increasing number of species which have been studied, it is becoming evident that with regard to conformation, the somatotropins can be subdivided into at least three major groups.     

Epizootic equine influenza in Tunisia

The authors describe an equine influenza epizootic that occurred in Tunisia during February and March 1998 in the regions of Tozeur, Sousse and Tunis. They relate the symptoms, the different stages of diagnosis and the serological results.     

Validation and usefulness of the Sperm Quality Analyzer V equine for equine semen analysis

Routine semen analysis includes evaluation of concentration combined with seminal volume, morphology and motility. Subjective analysis of these parameters is known to be inaccurate, imprecise and subject to variability. Automated semen analysis could lead to an increased standardization in and between laboratories but for that to happen automated devices need to be validated. A new device, the sperm quality analyzer V equine (SQA-Ve) version 1.00.43, was evaluated for its repeatability and agreement with light microscopy (LM), for raw and extended equine semen. Results were compared with computer assisted sperm analysis (CASA), which was also tested for its repeatability and agreement with LM. The SQA-Ve showed a good repeatability and fine agreement for assessing sperm concentration of raw semen based on scatter and Bland-Altman plots. This was in contrast with the motility parameters, which had a low repeatability. Morphology assessment with SQA-Ve was poorly repeatable as well as in poor agreement with LM. For extended semen, the findings were comparable. The SQA-Ve did well for concentration, whereas for the motility parameters repeatability was only just acceptable, with no agreement with LM. This sharply contrasted the CASA findings that were highly repeatable and almost in perfect agreement with LM. Based on these findings, the tested version of the SQA-Ve is insufficiently accurate to be used for analyzing raw or extended equine semen.     

Trinitrophenylation of equine growth hormone

The reactivity of the amino groups in equine growth hormone towards trinitrophenylation with picryl sulfonic acid was investigated, and the localization in the molecule of the various kinetically distinguishable amino groups was achieved. The N-terminal residue provides the most reactive amino group followed by the ?-amino groups of lysines 179 and 156 and lysines 63, 143, and 165 in decreasing order. Total trinitrophenylation of equine growth hormone brings about a complete loss of the growth-promoting capacity of the protein, but half-maximal potency is still present when there is more than 80% substitution in lysine 179, about 50% in lysine 156, and approximately 20% in each of lysines 63, 143, and 165 besides 100% reaction of the N-terminal group. On the other hand, the immunological properties, as measured in this work, are practically unmodified, even in completely trinitrophenylated equine growth hormone.