Sequence-specific isotope effects on the cleavage of DNA by bleomycin.

Bleomycin is a metal- and oxygen-dependent DNA cleaver. The chemistry of DNA damage has been proposed to involve rate-limiting abstraction of the 4'-hydrogen. A DNA fragment has been prepared that contains [4'-2H]thymidine residues of high isotopic content. Primary kinetic isotope effects have been directly observed at individual thymidine residues with DNA sequencing technology.

Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity.

Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.

Inhibitors of dihydrodipicolinate reductase, a key enzyme of the diaminopimelate pathway of Mycobacterium tuberculosis.

Tuberculosis (TB) remains a leading cause of infectious disease in the world today and therapies developed over the last forty years are becoming increasingly ineffective against resistant strains of Mycobacterium tuberculosis. In an effort to explore new mechanisms for drug development, we have investigated the enzymes of the diaminopimelate biosynthetic pathway as potential targets. Specifically, dihydrodipicolinate reductase, the essential gene product of dapB, was screened for novel inhibitors. Inhibitors were identified both by a molecular modeling approach which utilized the available crystal structure of the enzyme with an inhibitor bound at the active site as well as by more conventional screening strategies. The resulting compounds contain a number of structural motifs and were all found to be competitive with respect to the DHDP substrate. The K(i) values for the inhibitors range from 10 to 90 microM. The molecular modeling approach was very effective in identifying novel inhibitors of the enzyme. These compounds were obtained at a higher frequency based on the number of compounds analyzed than those inhibitors discovered via conventional screening. However, conventional screening proved beneficial in identifying compounds with greater structural diversity.

A model of the structure of HOO-Co.bleomycin bound to d(CCAGTACTGG): recognition at the d(GpT) site and implications for double-stranded DNA cleavage.

BACKGROUND: The bleomycins (BLMs) are a family of natural products used clinically as antitumor agents. In the presence of their required cofactors, iron and oxygen, BLMs bind to and mediate single-stranded and double-stranded DNA cleavage. Recently, two dimensional nuclear magnetic resonance (2D NMR) spectroscopic studies and molecular modeling have provided a picture of how the hydroperoxide form of cobalt BLM A2 (HOO-CoBLM), an analog of 'activated' iron BLM (HOO-FeBLM), binds to a d(GpC) motif and of the basis for both sequence specificity and chemical specificity of DNA cleavage. RESULTS: The solution structure of HOO-CoBLM bound to d(CCAGTACTGG) containing a 'hot spot' for double-stranded DNA cleavage at T5 and T15 is reported using constraints from 2D NMR spectroscopy. The mode of binding and basis for sequence specificity and chemical specificity of cleavage is almost identical to that of a d(GpC) motif. This structure has allowed formulation of a structural model for how a single molecule of FeBLM can mediate a double-stranded DNA cleavage event without dissociation from the DNA. CONCLUSIONS: The structural similarity of HOO-CoBLM bound to d(GpT) in d(CCAGTACTGG) compared to a d(GpC) motif suggests a general paradigm for the binding of HOO-CoBLM to DNA and, by analogy, for the binding of the biological significant entity HOO-FeBLM.

Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-beta-lactamase.

BACKGROUND: High level resistance to carbapenem antibiotics in gram negative bacteria such as Bacteroides fragilis is caused, in part, by expression of a wide-spectrum metallo-beta-lactamase that hydrolyzes the drug to an inactive form. Co-administration of metallo-beta-lactamase inhibitors to resistant bacteria is expected to restore the antibacterial activity of carbapenems. RESULTS: Biphenyl tetrazoles (BPTs) are a structural class of potent competitive inhibitors of metallo-beta-lactamase identified through screening and predicted using molecular modeling of the enzyme structure. The X-ray crystal structure of the enzyme bound to the BPT L-159,061 shows that the tetrazole moiety of the inhibitor interacts directly with one of the two zinc atoms in the active site, replacing a metal-bound water molecule. Inhibition of metallo-beta-lactamase by BPTs in vitro correlates well with antibiotic sensitization of resistant B. fragilis. CONCLUSIONS: BPT inhibitors can sensitize a resistant B. fragilis clinical isolate expressing metallo-beta-lactamase to the antibiotics imipenem or penicillin G but not to rifampicin.

The current status of equine embryo transfer.

The use of embryo transfer in the horse has increased steadily over the past two decades. However, several unique biological features as well as technical problems have limited its widespread use in the horse as compared with that in the cattle industry. Factors that affect embryo recovery include the day of recovery, number of ovulations, age of the donor and the quality of sire's semen. Generally, embryo recoveries are performed 7 or 8 d after ovulation unless the embryos are to be frozen, in which case recovery is performed 6 d after ovulation. Most embryos are recovered from single-ovulating mares. Because there is no commercially available hormonal preparation for inducing multiple ovulation in the horse, equine pituitary extract has been used to increase the number of ovulations in treated mares, but FSH of ovine or porcine origin is relatively ineffective in inducing multiple ovulation in the mare. Factors shown to affect pregnancy rates after embryo transfer include method of transfer, synchrony of the donor and recipient, embryo quality, and management of the recipient. One of the major improvements in equine embryo transfer over the last several years is the ability to store embryos at 5 degrees C and thus ship them to a centralized station for transfer into recipient mares. Embryos are collected by practitioners on the farm, cooled to 5 degrees C in a passive cooling unit and shipped to an embryo transfer station without a major decrease in fertility. However, progress in developing techniques for freezing equine embryos has been slow. Currently, only small, Day-6 equine embryos can be frozen with reasonable success. Additional studies are needed to refine the techniques for freezing embryos collected from mares 7 or 8 d after ovulation. Demand for the development of assisted reproductive techniques in the horse has increased dramatically. Collection of equine oocytes by transvaginal, ultrasound-guided puncture and the transfer of these oocytes into recipients is now being used to produce pregnancies from donors that had previously been unable to provide embryos. In vitro fertilization, however, has been essentially unsuccessful in the horse. One alternative to in vitro fertilization that has shown promise is intracytoplasmic sperm injection. However, culture conditions for in vitro-produced embryos appear to be inadequate. The continued demand for assisted reproductive technology will likely result in the further development of techniques that are suitable for use in the horse.

Effect of PGF2alpha and 13,14-dihydro-15-keto-PGF2alpha (PGFM) on corpora luteal function in nonpregnant mares.

The objective of this study was to determine if the primary circulating metabolite of PGF2alpha, 13,14-dihydro-15-keto-PGF2alpha (PGFM), is biologically active and would induce luteolysis in nonpregnant mares. On Day 9 after ovulation, mares (n = 7/group) were randomly assigned to receive: 1) saline control, 2) 10 mg PGF2alpha or 3) 10 mg PGFM in 5 mL 0.9% sterile saline i.m. On Days 0 through 16, blood was collected for progesterone analysis. In addition, blood was collected immediately prior to treatment, hourly for 6 h, and then at 12 and 24 h after treatment for progesterone and PGFM analysis; PGFM was measured to verify that equivalent amounts of hormone were administered to PGF2alpha- and PGFM-treated mares. Mares were considered to have undergone luteolysis if progesterone decreased to < or = 1.0 ng/mL within 24 h following treatment. Luteolysis was induced in 0/7 control, 7/7 PGF2alpha-treated, and 0/7 PGFM-treated mares. There was no difference (P>0.1) in the occurrence of luteolysis in control and PGFM-treated mares. More (P<0.001) PGF2alpha-treated mares underwent luteolysis than control or PGFM-treated mares. There was no difference (P>0.1) in progesterone concentrations between control and PGFM-treated mares on Days 10 through 16. Progesterone concentrations were lower (P<0.01) on Days 10 through 14 in PGF2alpha-treated compared with control and PGFM-treated mares. There was no difference (P>0.05) in PGFM concentrations between PGF2alpha- and PGFM-treated mares; PGFM concentrations in both groups were higher (P<0.001) than in control mares. These results do not support the hypothesis that PGFM is biologically active in the mare, since there was no difference in corpora luteal function between PGFM-treated and control mares.

Uterine transport of prostaglandin E(2)-releasing simulated embryonic vesicles in mares.

Transrectal ultrasonography was used to test the hypothesis that prostaglandin E(2) (PGE(2)) would increase the uterine transport of simulated embryonic vesicles in mares. Uterine transport of PGE(2)-releasing (PGE) vesicles, vehicle-releasing (sham) vesicles, and equine embryos was contrasted on Day 12 or Day 13 post ovulation. In Experiment 1, there was no difference (P>0.10) in transport of PGE vesicles, sham vesicles, Day-12 embryos, and Day-12 embryos after cervical manipulation (n = 3 per group). In Experiments 2 and 3, respectively, transport of PGE and sham vesicles was contrasted with transport of Day-13 embryos after the vesicles (1 vesicle per mare) were placed into the uterine lumen with the embryo, (n = 7 per group). In Experiment 2, PGE vesicles were transported less often (P<0.05) from horn to body and from segment to segment than Day-13 embryos before vesicle insertion. In Experiment 3, sham vesicles were transported less often from horn to body (P<0.10) and from segment to segment (P<0.01) than Day-13 embryos before vesicle insertion. However, there was no difference (P>0.10) in the transport of PGE vesicles and embryos (Experiment 2) or sham vesicles and embryos (Experiment 3) together in the uterine lumen. In Experiment 4, transport of PGE and sham vesicles was contrasted by placing them together into the uterine lumen of nonpregnant mares on Day 13 (n = 7). There was no difference (P>0.10) in the transport of PGE and sham vesicles together in the uterine lumen. These results do not support the hypothesis that PGE(2) increases uterine transport of simulated embryonic vesicles. In addition, these results do not support the hypothesis that equine embryos stimulate uterine transport.

Oviductal and uterine influence on the development of Day-2 equine embryos in vivo and in vitro.

The objective of this experiment was to contrast the influence of the oviductal and uterine environments on development of Day-2 embryos. Embryos were transferred to oviducts or uteri of synchronous recipient mares, or were incubated in oviductal co-culture, in uterine co-culture or in defined culture medium. Significantly more (P < 0.02) embryos transferred to the oviduct versus the uterus survived until Day 11 after ovulation (5 7 vs 0 7 , respectively). Significantly more (P < 0.001) embryos developed to expanded and hatched blastocysts in uterine co-culture than in culture medium (6 7 vs 0 7 , respectively). The rate of embryo development to expanded blastocysts was not significantly different (P > 0.1) in oviductal co-culture versus uterine co-culture (3 7 vs 6 7 , respectively), or in oviductal co-culture versus culture in medium (3 7 vs 0 7 , respectively). Three of 7 and 6 of 7 embryos developed to hatched blastocysts greater than 2000 mum in diameter during oviductal and uterine co-culture, respectively, while 0 of 7 embryos cultured in medium expanded to greater than 500 mum in diameter. Proportions of embryos that developed for at least 9 days.

Effect of homologous preovulatory follicular fluid on in vitro maturation of equine cumulus-oocyte complexes.

The objective of this study was to test the hypothesis that incubating equine cumulus-oocyte complexes (COCs) in medium containing 50% or 100% homologous preovulatory follicular fluid would improve cumulus expansion and nuclear maturation. Oocytes were incubated in one of three media: 1) supplemented TCM-199 (control), 2) 50% (v/v) follicular fluid in control medium or 3) 100% follicular fluid. Cumulus expansion was evaluated subjectively, and nuclear maturation was evaluated by staining oocytes with Hoechst 33258. The hypothesis that incubating COCs in medium containing follicular fluid would improve cumulus expansion was supported. More (P < 0.05) compact COCs incubated in 50% or 100% follicular fluid developed a moderately to completely expanded cumulus after 24 and 36 h of incubation and more (P < 0.05) expanded COCs incubated in 100% follicular fluid developed a moderately to completely expanded cumulus after 36 h of incubation compared to control medium. The hypothesis that incubating COCs in medium containing follicular fluid would improve nuclear maturation was not supported. Although more (P < 0.05) compact COCs incubated in 50% follicular fluid reached polar body-stage compared to those in control medium, the nuclear maturation rate in the control medium was lower than it was when the same medium was used in a preliminary experiment (described in main text); therefore, the apparent superiority of 50% follicular fluid must be interpreted cautiously. Based on these results, future studies are warranted to further address the value of adding preovulatory follicular fluid to equine IVM culture systems.

Corpus luteal function in nonpregnant mares following intrauterine administration of prostaglandin E(2) or estradiol-17beta.

The objective of this study was to test the hypothesis that intrauterine administration of prostaglandin E(2) (PGE(2)) or estradiol-17beta (E-17beta) would prolong CL function in nonpregnant mares. Nonpregnant mares were continuously infused with 240 mug/d of PGE(2), 6 mug/d of E-17beta, or vehicle (sham-treated) on Days 10 to 16 post ovulation (ovulation = Day 0), using osmotic minipumps surgically placed into the uterine lumen on Day 10 (n = 11 per group). Nonpregnant and pregnant mares served as negative and positive controls, respectively (n = 11 per group). Mares were defined as having prolonged CL function if plasma progesterone remained > 2.5 ng/ml and if ovulation did not occur on Days 9 to 30. Corpus luteal function was prolonged until Day 30 in 1 11 nonpregnant mares, 4 11 sham-treated mares, 6 11 E-17beta-treated mares, 8 11 PGE(2)-treated mares, and 11 11 pregnant mares. The incidence of prolonged CL function was similar (P=0.16) in the sham-treated and nonpregnant mares. The hypothesis that PGE(2) would prolong CL function in nonpregnant mares was supported, since the incidence of prolonged CL function was higher (P=0.003) in PGE(2)-treated versus nonpregnant mares, tended to be higher (P=0.09) in PGE(2)-versus sham-treated mares, and was not lower (P=0.11) in PGE(2)-treated versus pregnant mares. The hypothesis that E-17beta would prolong CL function in nonpregnant mares was not supported, since the incidence of prolonged CL function was not higher (P=0.34) in E-17beta-versus sham-treated mares, and was lower (P=0.02) in E-17beta-treated versus pregnant mares. These results demonstrate that intrauterine administration of a pharmacologic dose of PGE(2) initiated prolonged CL function in nonpregnant mares. Further experiments are needed to confirm the role of conceptus secretion of PGE(2) in CL maintenance, and to determine the mechanism of action of PGE(2) within the equine reproductive tract.

Ovarian follicles, ovulations and progesterone concentrations in aged versus young mares.

The objectives of this study were: 1) to document age-related ovulation failure in mares and 2) to contrast the number of ovarian follicles, occurrence of ovulations, and postovulatory concentrations of progesterone in aged versus young mares. In Experiment 1, 4 of 10 aged (25- to 33-years-old) mares were anovulatory between July 1 and September 1, 1989. In Experiment 2, two of 25 aged (20- to 30-years-old) and none of 21 young (3- to 12-years-old) mares were anovulatory between February 1 and June 30, 1990. The average (+/- SEM) day of the first ovulation was later (P<0.05) for aged versus young mares (May 9 +/- 7.1 vs April 25 +/- 7.4 days, respectively). There tended (P<0.10) to be fewer 11- to 20-mm ovarian follicles in aged versus young mares (2.8 +/- 0.2 vs 5.3 +/- 0.1, respectively), but there was no difference (P>0.10) in the total number of ovarian follicles in aged versus young mares (21.0 +/- 0.3 vs 26.1 +/- 0.2, respectively) during the pooled periovulatory period of the first and second (single) ovulations. The number of ovulatory cycles during the study period was less (P=0.01) for aged versus young mares (2.2 +/- 0.3 vs 3.2 +/- 0.3). Plasma progesterone concentrations on Days 10 and 15 of the first ovulatory cycle were higher (P<0.05) in aged versus young mares.

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer.

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.

Effect of spermatozoal concentration and number on fertility of frozen equine semen.

Information on the number of motile spermatozoa needed to maximize pregnancy rates for frozen-thawed stallion semen is limited. Furthermore, concentration of spermatozoa per 0.5-mL straw has been shown to affect post-thaw motility (7). The objectives of this study were 1) to compare the effect of increasing the concentration of spermatozoa in 0.5-mL straws from 400 to 1,600 x 10(6) spermatozoa/mL on pregnancy rate of mares, and 2) to determine whether increasing the insemination dose from approximately 320 to 800 million progressively motile spermatozoa after thawing would increase pregnancy rates. Several ejaculates from each of 5 stallions were frozen in a skim milk-egg yolk based freezing medium at 2 spermatozoal concentrations in 0.5-mL polyvinyl-chloride straws. Half of each ejaculate was frozen at 400 x 10(6) cells/mL and half at 1,600 x 10(6) cells/mL. Insemination doses were based on post-thaw spermatozoal motility and contained approximately 320 x 10(6) (320 to 400) motile spermatozoa or approximately 800 x 10(6) (800 to 900) motile spermatozoa. Sixty-three mares were assigned to 1 of 4 spermatozoal treatments (1--low spermatozoal number, low concentration; 2--low spermatozoal number, high concentration; 3--high spermatozoal number, low concentration; 4--high spermatozoal number, high concentration) and were inseminated daily. Post-thaw spermatozoal motility was similar for cells frozen at both spermatozoal concentrations (P > 0.1). One-cycle pregnancy rates were 15, 40, 28 and 33%, respectively, for Treatments 1, 2, 3 and 4. Packaging spermatozoa at the high concentration tended to increase pregnancy rates vs packaging at the low concentration (37 vs 22%; P = 0.095). Furthermore, when the lower spermatozoal number was used, there tended (P < 0.1) to be a higher pregnancy rate if spermatozoa were packaged at the higher concentration. There was no increase in pregnancy rates when higher numbers of motile spermatozoa were inseminated (27 vs 31%; P > 0.1). Based on these results, a single 0.5-mL straw dose containing 800 x 10(6) spermatozoa should be used and each insemination dose should contain approximately 320 x 10(6) motile spermatozoa. Fertility trials utilizing other freezing extenders are necessary before recommending a single 0.5-mL insemination dose for all freezing extenders.

Expression of the uterine Mx protein in cyclic and pregnant cows, gilts, and mares.

Pregnancy and interferon-tau (IFN tau) upregulate uterine Mx gene expression in ewes; however, the only known role for Mx is in the immune response to viral infection. We hypothesize that Mx functions as a conceptus-induced component of the anti-luteolytic mechanism and/or regulator of endometrial secretion or uterine remodeling during early pregnancy. This study was conducted to determine the effects of early pregnancy on uterine Mx expression in domestic farm species with varied mechanisms of pregnancy recognition. Endometrium from cows, gilts, and mares was collected during the first 20 d of the estrous cycle or pregnancy, and total messenger RNA (mRNA) and protein were analyzed for steady-state levels of Mx mRNA and protein. Northern blot analysis of Mx mRNA detected an approximately 2.5 Kb of mRNA in endometrium from each species. In pregnant cows, steady-state levels of Mx mRNA increased 10-fold (P < 0.05) above levels observed in cyclic cows by d 15 to 18. In cyclic gilts, slot blot analysis indicated that endometrial Mx mRNA levels did not change between d 5 and 18 of the cycle. However, in pregnant gilts, Mx levels tended (P = 0.06) to be elevated two-fold on d 16 only, and in situ hybridization indicated that this increase occurred in the stroma. In mares, Mx mRNA was low, but detectable, and did not change between ovulation (d 0) and d 20, regardless of reproductive status. Western blot analysis revealed multiple immunoreactive Mx protein bands in each species. One band was specific to pregnancy in cows. As in ewes, in situ hybridization analysis indicated that Mx mRNA was strongly expressed in the luminal epithelium, stroma, and myometrium by d 18 in cows. However, on d 14 in gilts, Mx was expressed primarily in the stroma, and on d 14 in mares, low levels of Mx expression were confined largely to the luminal epithelium. The uteruses of cows, gilts, and mares express Mx, and expression is upregulated during pregnancy in cows and gilts--animals whose conceptuses secrete interferons during early pregnancy, but that possess different mechanisms for pregnancy recognition.

Diagnosis and management of abnormal embryonic development characterized by formation of an embryonic vesicle without an embryo in mares.

OBJECTIVE: To determine the incidence, ultrasonographic characteristics, and risk factors associated with embryonic development characterized by formation of an embryonic vesicle without an embryo in mares. DESIGN: Prevalence survey. ANIMALS: 159 pregnant mares. PROCEDURES: From 1994 to 1998, mares between 11 and 40 days after ovulation with normal and abnormal embryonic development were examined ultrasonographically, and characteristics of each conceptus were recorded. RESULTS: The incidence of abnormal embryonic development in mares characterized by formation of an embryonic vesicle without an embryo was 7/159 (4.4%) during the 5 breeding seasons. Age and breed of mare or type of semen used did not differ for mares with normal and abnormal embryonic development. The percentage of mares in which the conceptus was undersized during > or = 1 examination was significantly higher for mares with abnormal conceptuses (5/7), compared with mares with normal conceptuses (2/147; 1.4%). The percentage of examinations during which the conceptus was undersized was significantly higher for abnormal conceptuses (12/27; 44.4%), compared with normal conceptuses (4/448; 0.9%). CONCLUSIONS AND CLINICAL RELEVANCE: To diagnose an embryonic vesicle without an embryo, mares should be examined by use of transrectal ultrasonography on day 25 after ovulation. When an embryo cannot be identified at that time, mares should be reexamined at intervals of 1 to 3 days until day 30. Because undersized conceptuses are more likely to be abnormal, development of undersized conceptuses should be monitored closely.

Prevalence (treatment days) and severity of illness in hypogammaglobulinemic and normogammaglobulinemic foals.

Serum samples for determination of IgG concentration were obtained between postpartum hours 18 and 48 from 132 Standardbred foals. Results of the IgG assay were not known to farm personnel. None of the foals was given plasma IV for treatment of hypogammaglobulinemia. Foal health records were examined retrospectively to determine prevalence of infectious-type illness (foal treatment days [FTD]), prevalence of life-threatening infectious illness (foal treatment days-serious condition [FTD-SC]), and number of diseases (NOD) per foal. Values for FTD, FTD-SC, and NOD per foal were compiled for the first 21 days of life and for the first 90 days of life. The FTD, FTD-SC, and NOD per foal values were compared for foals with less than 400 mg of IgG/dl and for foals with greater than or equal to 400 mg of IgG/dl; the same variables were compared for foals with less than 800 mg of IgG/dl and for foals with greater than or equal to 800 mg of IgG/dl. Statistical analysis indicated that IgG concentration was not associated with FTD, FTD-SC, or NOD in foals of any of the groups. Also, despite a large subpopulation of hypogammaglobulinemic foals (13.6% with less than 400 mg of IgG/dl and 44.7% with less than 800 mg of IgG/dl), the 21-day and 90-day overall survival rates were 100 and 99.2%, respectively. The data strongly suggest that serum IgG concentration was not related to prevalence or severity of illness or to survival rate in this population of foals.

Temporal and spatial associations of oestrogen receptor alpha and progesterone receptor in the endometrium of cyclic and early pregnant mares.

Uterine function is primarily controlled by the combined actions of oestrogen and progesterone working through their cognate nuclear receptors. The mechanism of establishment of pregnancy in the mare is of interest because it involves prolonged pre-attachment and conceptus migration phases, and both invasive and non-invasive placental cell types, and as such has been an important comparative model. This study characterised regulation of oestrogen (ER) and progesterone (PR) receptors in the endometrium of the mare during the oestrous cycle and early pregnancy. Endometrial tissues collected during the oestrous cycle and early pregnancy were analysed for steady-state levels of ER and PR mRNA and protein. Steady-state levels of ER and PR mRNA were highest on days 0, 17 and 20 in cyclic mares and lowest on days 11 and 14. A day-by-status interaction was detected, indicating that day 17 and day 20 pregnant mares exhibited low levels of ER and PR compared with the corresponding days of the oestrous cycle. In situ hybridisation analyses showed receptor mRNA localisation primarily in the luminal epithelium (LE), glandular epithelium (GE) and stroma around oestrus. During dioestrus and early pregnancy, receptors were not detected in the LE, and were lower in the stroma and deeper GE. Changes in hybridisation intensity in these cell types were consistent with changes in mRNA levels detected by slot-blot hybridisation. ER and PR proteins were detected in the nuclei of LE, GE and stromal cells. Consistent with results from in situ hybridisation, levels of ER and PR immunoreactivity were higher around oestrus, declined to low levels during dioestrus and remained low during early pregnancy. Results described here for temporal and spatial changes in steroid receptor gene expression in mares show the greatest similarities with those described for cattle and sheep.

Early embryonic development and evaluation of equine embryo viability.

Tremendous progress has been made in the development of assisted reproductive techniques that may enhance the reproductive efficiency of the horse. However, techniques that involve the manipulation of oocytes and/or embryos may themselves be detrimental to embryo viability and subsequent development. Therefore, an objective method of assessing viability of embryos before and/or after oocyte/embryo manipulation is desirable. At this time, morphologic evaluation is the most widely used method of determining the viability of equine embryos. Although morphologic assessment of embryo quality will not always accurately predict the survival of individual embryos, it is very useful for predicting the survival of groups of embryos. Other tests that have been used to evaluate equine embryo viability include (1) development during culture in vitro; (2) quantitating metabolism of the fluorescent substrate fluorescein diacetate; (3) quantitating uptake of the fluorescent stain DAPI; and (4) quantitating embryo metabolism. Although these tests offer potential advantages over morphologic assessment alone, their current limitations have prevented their routine use for embryo evaluation. It is likely that as improvements are made in these evaluation methods, they may offer advantages for use alone or in combination to more accurately assess the viability of equine embryos.