Cryopreservation and in vitro culture of white-tailed deer ovarian tissue.

The aims of this study were to evaluate (1) the survivability of white-tailed deer ovarian tissue after cryopreservation by slow-freezing (SF) and vitrification (VIT) techniques and in vitro culture (IVC) for up to 7 days, and (2) the effects of cryopreservation techniques on protein expression of proliferative and apoptotic markers of ovarian tissue pre- and post-in vitro culture. Ovaries (n = 14) of seven white-tailed deer fawns (<1.5 years old) were used. Ovarian cortexes were cut into fragments (2 × 2 × 0.5 mm) and split into nine treatment groups: (1) fresh noncultured control, (2) fresh-IVC 1 day, (3) fresh-IVC 7 days, (4) SF noncultured, (5) SF-IVC 1 day, (6) SF-IVC 7 days, (7) VIT noncultured, (8) VIT-IVC 1 day, and (9) VIT-IVC 7 days. Preantral follicle morphology, class distribution, and density; stromal cell density; EGFR, Ki-67, Bax, and Bcl-2 protein expression; and DNA fragmentation were assessed. Results showed that: (i) white-tailed deer fresh ovarian tissue can be cultured for up to 7 days, preserving the tissue integrity and 50% of morphologically normal preantral follicles; (ii) cryopreservation of white-tailed deer ovarian tissue by either slow-freezing or vitrification does not disrupt markers of proliferation and apoptosis after thawing; (iii) ovarian fragments cryopreserved by the vitrification method had greater follicle viability during in vitro culture than the slow-freezing method; and (iv) fragments cryopreserved by slow-freezing suffered apoptosis earlier than those preserved by vitrification. The findings herein reported advance knowledge towards development of adequate cryopreservation protocols for long-term banking programs for Cervidae species.

Tracking multiple objects is limited only by object spacing, not by speed, time, or capacity.

In dealing with a dynamic world, people have the ability to maintain selective attention on a subset of moving objects in the environment. Performance in such multiple-object tracking is limited by three primary factors-the number of objects that one can track, the speed at which one can track them, and how close together they can be. We argue that this last limit, of object spacing, is the root cause of all performance constraints in multiple-object tracking. In two experiments, we found that as long as the distribution of object spacing is held constant, tracking performance is unaffected by large changes in object speed and tracking time. These results suggest that barring object-spacing constraints, people could reliably track an unlimited number of objects as fast as they could track a single object.

Inhibition of glutathione synthesis with propargylglycine enhances N-acetylmethionine protection and methylation in bromobenzene-treated Syrian hamsters.

The finding that liver necrosis caused by the environmental glutathione (GSH)-depleting chemical, bromobenzene (BB) is associated with marked impairment in O- and S-methylation of BB metabolites in Syrian hamsters raises questions concerning the role of methyl deficiency in BB toxicity. N-Acetylmethionine (NAM) has proven to be an effective antidote against BB toxicity when given after liver GSH has been depleted extensively. The mechanism of protection by NAM may occur via a replacement of methyl donor and/or via an increase of GSH synthesis. If replacement of the methyl donor is an important process, then blocking the resynthesis of GSH in the methyl-repleted hamsters should not decrease NAM protection. This hypothesis was examined in this study. Propargylglycine (PPG), an irreversible inhibitor of cystathionase, was used to inhibit the utilization of NAM for GSH resynthesis. Two groups of hamsters were pretreated with an intraperitoneal (ip) dose of PPG (30 mg/kg) or saline 24 h before BB administration (800 mg/kg, ip). At 5 h after BB treatment, an ip dose of NAM (1200 mg/kg) was given. Light microscopic examinations of liver sections obtained 24 h after BB treatment indicated that NAM provided better protection (P < 0.05) in the PPG + BB + NAM group than in the BB + NAM group. Liver GSH content, however, was lower in the PPG + BB + NAM group than in the BB + NAM group. The Syrian hamster has a limited capability to N-deacetylated NAM. The substitution of NAM with methionine (Met; 450 mg/kg) resulted in a higher level of GSH in the BB + Met group than in the BB + NAM group (P < 0.05). The enhanced protection by PPG in the PPG + BB + NAM group was accompanied by higher (P < 0.05) urinary excretions of specificO- and S-methylated bromothiocatechols than in the BB + NAM group. The results suggest that NAM protection occurs primarily via a replacement of the methyl donor and that methyl deficiency occurring in response to GSH repletion plays a potential role in BB toxicity.

Glutathione depletion-induced thymidylate insufficiency for DNA repair synthesis.

Dietary methionine (Met) deficiency is known to divert folate away from de novo biosyntheses of purines and the pyrimidine, thymidylate, to the resynthesis of Met resulting in deoxynucleoside triphosphate imbalance. We have recently shown that Met can easily be depleted and methylation can be impaired by exposure to a model glutathione (GSH)-depleting agent, bromobenzene (BB). GSH depletion-induced Met depletion, therefore, could cause thymidylate insufficiency for DNA repair synthesis. The administration of thymidine (Thy) should repair this impairment. When this hypothesis was examined in the present study, several interesting results were found. The administration of Thy labeled with [2-14C]Thy to BB-treated Syrian hamsters at either 1, 5, 7 or 9 h after BB resulted in an attenuation of liver toxicity. Intrahepatic hemorrhage, which is a typical characteristic of BB toxicity in the Syrian hamster, was decreased in the BB + Thy groups. The attenuation of liver toxicity was accompanied by a progressive increase of Thy incorporation into liver genomic DNA at 24 h after BB. With respect to the time points chosen for Thy administration, Thy incorporation found in the BB + Thy groups were 2-, 2-, 3- and 4-fold of the controls that received only Thy. The results provide evidence that BB causes a progressive increase of thymidylate insufficiency in liver cells. Thymidylate insufficiency is due to Met depletion, a depletion that occurs as a result of GSH depletion.

Methyl-donor deficiency due to chemically induced glutathione depletion.

Dietary deficiency of methionine (Met) is known to deplete cellular Met and cause DNA hypomethylation, but depletion of Met and impairment in methylation due to chemically induced glutathione (GSH) depletion has escaped recognition. In this study, the effect of GSH depletion on the Met pool and methylation capability was examined after bromobenzene (BB), a model GSH-depleting hepatotoxin, was administered to the Syrian hamster. An i.p. dose of BB (800 mg/kg) caused a rapid and extensive depletion of liver GSH; approximately 68% of the initial concentration was depleted during the first hour. The lowest level of GSH, only 4% of the control, was detected at 5 h. GSH depletion was accompanied by a prompt increase in liver Met during the first hour. This initial increase was followed by an extensive depletion during the next 4 h. At 5 h after BB, liver Met was 12% below the control value, and it remained around this concentration throughout the 24-h experiment. To further confirm these results, the endogenous Met pool was labeled with deuterated Met. The administration of (L)- Met-methyl-d(3) to the Syrian hamster after GSH had been depleted by BB resulted in a significant protection of the liver against necrosis. The protection was accompanied by a marked incorporation of deuterated Met into the liver Met pool. The incorporation, which was determined by gas chromatography-mass spectrometry, shows BB dose dependence. Approximately 53% of the liver Met was labeled when a toxic BB dose (800 mg/kg) was used, while only 25% incorporation was found for the nontoxic dose (100 mg/kg). These results were different from the controls, where only 15% incorporation was found. The differences in the incorporation indicate that there are differences in the degree of utilization and/or depletion of Met in these hamsters, and these differences apparently are dependent upon the degree of toxicity and GSH depletion. The marked incorporation of deuterated Met in the high-dose group was accompanied with a striking increase in the methylation capability. Urinary excretion of the O- and S-methylated 4- and 5-bromo-2-hydroxythiophenols and S-methylated 4- and 5-bromo-2-hydroxy-1,2-dihydrobenzenethiols was significantly increased when compared with the BB treated alone. Approximately 40-45% of the methyl groups in these methylated BB metabolites were methyl-d(3). These results provide direct evidence that depletion of GSH leads to Met depletion and also injures the methylation processes.

In vivo reduction of bacterial populations in the urinary tract of catheterized sheep by iontophoresis.

PURPOSE: Iontophoresis kills microbes in vitro and, therefore, may be a useful method for eliminating microbial populations associated with catheter-induced urinary tract infections in vivo. MATERIALS AND METHODS: Catheters were modified to deliver current to platinum electrodes in the catheter tip. Female sheep were catheterized with this iontophoretic catheter and left ambulatory. In 5 sheep (experimental group) 400 microA was applied to the catheter and withheld in 4 sheep (control group) for 20 to 21 days. The animals were then sacrificed. During the study, types and concentrations of bacteria, and physical and chemical characteristics of the urine samples were determined. RESULTS: Throughout the study, bacteria levels were reduced in urinary tracts of the experimental group (10(3) to 10(4) microbes per ml.) compared with the control group (10(7) microbes per ml.), without extensive alterations to urine chemistry or the sheep urinary tract. CONCLUSIONS: Since iontophoresis safely reduced bacterial populations in catheterized sheep, this technology may reduce or eliminate nosocomial, catheter-induced urinary tract infections in humans.

Prevention of bromobenzene toxicity by N-acetylmethionine: correlation between toxicity and the impairment in O- and S-methylation of bromothiocatechols.

Bromobenzene (800 mg/kg, ip) caused severe liver necrosis with massive hemorrhage in the golden Syrian hamster within the first 24 hr. Kidney injury was also observed. Treatment with N-acetylmethionine (NAM) at an ip dose of 1200 mg/kg at 5 hr after bromobenzene administration significantly protected the liver and kidney against injuries. Plasma glutamate pyruvate transaminase and blood urea nitrogen levels were substantially decreased in the NAM-treated animals. Histological evaluations confirmed these results. When the urinary neutral and phenolic metabolites of bromobenzene from NAM-treated and untreated hamsters were isolated and compared by GC and GC/MS, a striking result was observed in terms of O- and S-methylated thiol-containing metabolite formation. The NAM-treated animals showed approximately a 8- to 14-fold increase in the excretion of the four isomeric O- and S-methylated bromothiocatechols. These thiocatechols, which are now known to be the 3,4-series metabolites of bromobenzene, can undergo methylation at either the thiol or the hydroxyl functional group. The excretion of 3-S- and 4-S-methylated bromodihydrobenzene thiolols was also increased significantly in the NAM-treated hamster, but other neutral and phenolic metabolites were relatively unchanged. These results suggest that bromobenzene toxicity in the Syrian hamster may be associated with impaired methylation capabilities, an impairment that could be due to methionine and glutathione depletion.

Laboratory survival, growth, and digestive gland histologic features of squids reared on living and non-living fish diets.

The effects of non-living diets on the survival, growth, and digestive gland histologic features of the bigfin reef squid (Sepioteuthis lessoniana Lesson, 1830) cultured in the laboratory were evaluated during one-half of their life cycle (95 days). Two groups of squids (n = 16 per group) were held in closed seawater systems with similar water volume, temperature, salinity, water filtration, and water flow velocities. Food for the control group consisted of live, freely swimming fish (Cyprinodon variagatus); the test group was trained to grab freshly dead fish (days 1 to 45) and then thawed, frozen fish (days 46 to 95). The two groups were evaluated for differences in (1) food intake, (2) survival, (3) growth (wet weight, mantle length, instantaneous growth rate), (4) morphologic (mantle thickness in four locations, digestive gland weight), and (5) digestive gland histologic features (indices for nuclear density and relative vacuolar density). Unexpectedly, no significant differences were found between the two groups. Mean wet weight increased from 32.1 g to 342.9 g for the control group and from 58.6 g to 372.0 g for the group fed dead food. The results demonstrate that laboratory-cultured squids can survive and grow when fed dead fish (fresh or frozen) as well as live fish without adverse effects on growth, survival, or digestive gland histologic features.

Developmental changes in ECG associated with heart rate are similar in squirrel monkey and human infants.

Interest in refining noninvasive methods of diagnosis and further characterization of squirrel monkeys (Saimiri sp.) as a model for pediatric cardiology studies led to this investigation of electrocardiogram (ECG) changes associated with changes in age and position. During a single delivery season, ECGs were performed at 1 day, 1 month, and 1 year of age. For each age group, ECGs were recorded with animals in dorsal, ventral, and right lateral recumbency. The 1-day-old group had the lowest heart rates (271 +/- 10, right lateral recumbency, mean +/- SEM) relative to the other age groups. One-year-old monkeys had heart rates of 333 +/- 18. One-month-old infants had rates significantly higher than the other two age groups (366 +/- 4). The QRS frontal-plane axis showed an age-related leftward change from 1 day (151 +/- 28 degrees) to 1 year of age (121 +/- 44 degrees) while the P-wave frontal plane axis remained nearly constant over a narrow range at all ages. The pattern of heart rate changes with age were similar to those in humans, although the ranges of absolute heart rates were markedly different. These data suggest that factors that influence maturational changes in heart rate, conduction time (as reflected by ECG intervals) and cardiac chamber size and position (inferred from axis and voltage) are similar among primates of widely variant body sizes.

Quantitation of purified monoclonal antibody needed to prevent HSV-1 induced stromal keratitis in mice.

A purified IgG2a monoclonal antibody with a neutralizing titer of 10(4) and specificity for gD was evaluated for its therapeutic potential in a murine ocular infection model. BALB/c mice, infected on the scarified cornea with 10 times the HSV-1 strain RE concentration needed to produce severe and persistent stromal opacity, were given a single inoculation of antibody intraperitoneally 24 hours later. The animals were then followed for corneal disease development. Antibody, at concentrations as low as 10 micrograms per mouse, was strikingly effective at preventing corneal opacity. Furthermore, the corneas, once clear, remained clear whereas the controls developed +3 to +5 stromal disease which was still present 60 days post-infection. Animals that had been treated and recovered from infection were resistant to subsequent HSV-1 challenge on the opposite cornea. These results demonstrate the therapeutic potential of systemically administered microgram quantities of anti-gD antibody.

Intestinal trichomonads (Tritrichomonas mobilensis) in the natural host Saimiri sciureus and Saimiri boliviensis.

A retrospective study of cecal and colonic tissues from 28 squirrel monkeys (Saimiri sciureus and Saimiri boliviensis) demonstrated enteric trichomonads within luminal crypts. Twenty-one of 28 (75%) had trichomonads in the mucosal epithelium either in cup-like depressions or intraepithelial vacuoles. Organisms were also beneath the superficial luminal mucosal epithelium and between the basement membrane and crypt epithelial cells. Immunoperoxidase staining also identified organisms within the lamina propria and submucosa. Additional histologic changes included mucosal ulceration, multifocal cryptitis, and focal epithelial necrosis. Most areas containing trichomonads did not have an associated inflammatory response.

Pathogenicity of Tritrichomonas mobilensis: subcutaneous inoculation in mice.

The standard "subcutaneous mouse assay" was used to investigate the inherent pathogenicity of Tritrichomonas mobilensis, an intestinal parasite of squirrel monkeys. C57B1/6 mice given subcutaneous bilateral inocula of T. mobilensis died by day 4 postinoculation with lesions too small to be measured. Control mice similarly inoculated with pathogenic and nonpathogenic strains of Trichomonas vaginalis survived the challenge and produced lesions on day 6 with mean volumes in agreement with previous reports. CD1 mice similarly inoculated with standard and double doses of trichomonads (T. mobilensis) again produced small lesions. CD1 mice inoculated at double dosage were moribund or dead on days 5 and 6, respectively, postinoculation. Necropsies were performed on dead and sacrificed mice. Tissues were obtained from internal organs for histology and culture. Unexpectedly, trichomonads were cultured from liver and lung of C57B1/6 mice at the standard level of inoculation and liver, lung, and spleen of CD1 mice at the higher level of inoculation. Although trichomonads are normally considered surface-dwelling noninvasive organisms, the penetration of trichomonads to deep tissues is not without precedent. Tritrichomonas foetus and Trichomonas gallinae are known to invade tissues of their respective hosts. Trichomonas vaginalis has been demonstrated in subepithelial areas of both the prostate gland and cervix of humans. The ability of several species of trichomonads to invade tissues and/or migrate to other sites in their hosts suggests a need for revision of the concept of trichomonads as strictly lumen or surface-dwelling parasites.

Intraprostatic spermatozoa in the squirrel monkey.

The recent observation of spermatozoa within the human prostate prompted this study to determine if spermatozoa were present in prostates of nonhuman primates. Six of 32 squirrel monkey prostates contained intraglandular spermatozoa. No associated pathologic lesions were found. Spermiophages were seen in one case. Although intraprostatic spermatozoa have been postulated to play a role in various prostatic diseases, a normal physiologic phenomenon cannot be excluded.

Experimental genital trichomoniasis in the squirrel monkey (Saimiri sciureus).

The squirrel monkey (Saimiri sciureus) has been proposed as a model for urogenital trichomoniasis in man, but has not been accepted as such because of the purported presence of naturally occurring vaginal trichomonads in this animal. The study published here shows that these are easily eradicated organisms of intestinal origin, which eliminates the potential confusion created by them. In addition, our experiments have shown that the hormonal status of primates seems to be a determinant in successfully establishing experimental trichomoniasis. This experimental infection recapitulates the clinical observations sufficiently to warrant the use of this model for studies of vaginal trichomoniasis.