The effect of the breeding season, cryopreservation and physiological extender on selected sperm and semen parameters of four ferret species: implications for captive breeding in the endangered black-footed ferret.

In the present investigation, comparative baseline information on selected sperm characteristics of ejaculate spermatozoa of the domestic (Mustela putorius furo), fitch (Mustela sp.) and black-footed ferrets (Mustela nigripes) and the Siberian polecat (Mustela eversmanni) are presented. The main emphasis was to establish differences and similarities among these species in relation to semen and sperm quality during the breeding season, in cryopreservation success and in supporting sperm motility in different extenders or physiological media. The results confirm that most sperm morphology abnormalities were evident during the beginning of the breeding cycle in all four species. No significant interspecies differences were apparent in the sperm attributes examined, for all sampling months during the breeding season. Moreover, all species exhibited comparable patterns of reproductive seasonality. Cryopreservation suppressed sperm characteristics equally in all species studied. Ejaculate spermatozoa of closely related ferret species shared many similar motion characteristics using computer-aided sperm motility analysis. These results suggest that the basic sperm physiology of the ferret species under examination is very similar. Disparate to the interspecies comparisons, there were significant differences for most sperm motion parameters when spermatozoa of any of the ferrets were compared in different extenders. Assisted reproductive technologies developed for use in domestic ferret, fitch ferret or Siberian polecat may be successfully applied to captive breeding of the black-footed ferret using semen during any of the functional breeding months.

Evaluation of the genotoxic potential of selected anti-AIDS treatment drugs at clinical doses in vivo in mice.

Six anti-AIDS drugs were assessed for in vivo genotoxicity and cytotoxicity at human clinical doses with the mouse bone marrow micronucleus assay. These included four dideoxynucleosides (azidothymidine, dideoxycytidine, dideoxyadenosine, and dideoxyinosine), an anthracycline antibiotic (doxorubicin), and a chelating agent (D-penicillamine). Cytological analysis of the mouse bone marrow cells revealed: (i) The dideoxynucleosides and D-penicillamine failed to induce significant number of micronuclei, and except for one of the five doses of dideoxyinosine, none of the dideoxynucleosides were cytotoxic at the doses tested. (ii) Doxorubicin induced micronuclei in a dose-dependent manner which was statistically significant at 4-times the clinical dose but was not cytotoxic at any of the doses tested.

Assessment of genotoxicity of two anti-parkinsonian drugs (selegiline hydrochloride and bromocriptine mesylate) in vivo in mouse bone marrow cells.

Selegiline hydrochloride (1-deprenyl) and bromocriptine mesylate (2-bromo-alpha-ergocryptine) are two drugs that have shown considerable promise in the treatment of Parkinson's disease. The in vivo mouse bone marrow micronucleus assay was used to examine their clastogenic and cytotoxic potential in human clinical dose range. Our results indicate that both drugs failed to induce significant number of micronuclei and were not cytotoxic at any of the doses tested, in vivo in mouse bone marrow cells, at doses as high as 16-times the clinical dose used in humans.

Quantitative light and scanning electron microscopy of ferret sperm.

Sperm were obtained via electroejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black-footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black-footed ferret.

A comparison of two simple hazard ratio estimators based on the logrank test.

Two hazard ratio estimators based on the logrank test are investigated using a simulation study. The Pike estimator (ratio of relative death rates) was shown to be consistently less biased than the Peto (1-step) estimator. The latter has recently been advocated as the method of choice for point and interval estimation. Both estimators exhibited bias with increasing hazard ratios, although the bias was minimal for effects less than 3. The confidence intervals also did not achieve the nominal coverage with increasing hazard ratios, but again the Pike estimator was superior. The coverage could be improved by recalculation of the variance incorporating the point estimate. For a hazard ratio of less than 3 we recommend the use of the Pike estimator, otherwise it is necessary to use a more complex method of estimation.

The lack of mutagenicity of aryl 4-guanidinobenzoates in the transplacental micronucleus assay in mice.

Two acrosin inhibitors, 4'-methylumbelliferyl 4-guanidinobenzoate and 2'-carbomethoxyphenyl 4-guanidinobenzoate, were tested for mutagenicity in the transplacental micronucleus assay and the mouse bone marrow micronucleus assay. The compounds were administered intraperitoneally at doses of 125 mg/kg and 250 mg/kg to pregnant mice. Fetal peripheral blood and maternal bone marrow cells were examined at 36 h for the frequency of micronucleated polychromatic erythrocytes. Neither compound induced micronuclei in maternal or fetal tissues. The ratio of polychromatic erythrocytes to normochromatic erythrocytes was not affected by the drug treatments indicating that the compounds had no effect on the cell cycle or mitosis in these tissues and that they were not cytotoxic. Both compounds, which show promise as vaginal contraceptives, were not mutagenic in this study.

Use of membrane filters and osmium tetroxide etching in the preparation of sperm for scanning electron microscopy.

A new method was developed which is suitable for the preparation of mammalian sperm for scanning electron microscopy under either laboratory or field conditions. Samples of ejaculates from humans, two ferret species, and epididymal sperm from the African elephant were diluted in Millonig phosphate buffer and then fixed in glutaraldehyde solution. A small sample of the fixed sperm suspension was diluted in the same buffer, withdrawn with a syringe, and injected very slowly onto either a cellulose acetate or a polycarbonate membrane filter. This step was essential to concentrate the dilute sperm samples. During the various dilution steps most of the granular prostatic secretions were lost. However, a protein-like sheath, which remained attached to most sperm, obscured the surface features and had to be removed for SEM studies. It was removed by prolonged fixation/etching in 1% osmium tetroxide. Membrane filters containing sperm on their surfaces then were dehydrated, dried by the critical point drying method, and sputter coated with gold. Polycarbonate filters were superior to cellulose acetate filters in producing a flat and homogeneous background.

A comparison of sperm morphology and silver nitrate staining characteristics in the domestic ferret and the black-footed ferret.

Ejaculated sperm from the domestic ferret (Mustela putorius furo) and the black-footed ferret (Mustela nigripes) were compared for differences in morphological abnormalities and argentophilic protein distribution. Thawed domestic ferret sperm was also compared to fresh sperm to determine whether there were any effects on cell morphology due to cryopreservation. There were statistically significant differences between the two species of ferret in two of the categories scored. The domestic ferret had a higher frequency of cells that were bent in the midpiece and in the principal piece, and a higher frequency of headless and tailless cells when compared to the black-footed ferret. There were no statistically significant differences in cell morphology between the fresh and cryopreserved ejaculates of the domestic ferret employing a standard egg yolk cryoextender. Silver nitrate staining distribution was different between the two species in both the head and tail region.

The structure-function relationships of nitrofluorenes and nitrofluorenones in the Salmonella mutagenicity and CHO sister-chromatid exchange assays.

Nitrofluorenes and nitrofluorenones are bacterial mutagens and are detected in a variety of environmental pollution sources. We tested a series of nitrosubstituted fluorenes and fluorenones for their genotoxicity using both Salmonella bacteria and Chinese hamster ovary (CHO) cells to determine if structure-function relationships observed in bacteria for mutation induction are similar to those for mutations and SCE induction in mammalian (CHO) cells. The compounds studied were 2-nitrofluorene (2-NF), 2,7-dinitrofluorene (2,7-DNF), 3-nitrofluorenone (3-NFone), 2-nitrofluorenone (2-NFone), 2,7-dinitrofluorenone (2,7-DNFone), 2,4,7-trinitrofluorenone (2,4,7-TNFone), and 2,4,5,7-tetranitrofluorenone (2,4,5,7-TNFone). In bacteria, the presence of carbonyl group to convert mono-nitrofluorenes to nitrofluorenones and the addition of a second nitro group to either mono-nitrofluorene or fluorenone to form the dinitro compounds increased mutagenic activity in the Ames test. Location of the nitro group relative to the carbonyl group was important in enhancing mutagenic activity as 2-nitrofluorenone was more mutagenic than 3-nitrofluorenone. In CHO cells, the di-, tri- and tetra-nitrofluorenones were cytotoxic and delayed the progression of CHO cells through the cell cycle. The degree of the cytotoxicity could be decreased by the addition of S9. None of the compounds produced mutations when tested in the CHO/HGPRT mutation assay with the addition of S9. Nonetheless, the current study did show that these compounds, both with and without the activation by S9, can interact with the DNA and produce SCE in CHO cells. The addition of a carbonyl group had no influence on SCE frequency since both 2-nitrofluorene and 2-nitrofluorenone induced a similar frequency of SCE either with or without S9. Additional nitro groups, forming di-, tri- or tetra-nitrofluorenones, increased the frequency of SCE induced, especially when tested with S9 which limits cytotoxicity. The addition of a single nitro group to 2-nitrofluorenone did not change the SCE frequency but did cause a large increase in the frequency of mutations in bacteria. In contrast, 2,4,7-TNFone and 2,4,5,7-TNFone were less mutagenic than the 2,7-DNFone in bacteria but were more effective in production of SCE in CHO cells. This study illustrates that structure-function relationships are dependent on both the compounds tested and the type of genetic change induced.

Induction of sister-chromatid exchanges in vivo in mice by the mycotoxins sterigmatocystin and griseofulvin.

Two naturally occurring fungal mycotoxins, sterigmatocystin and griseofulvin, were tested for induction of sister-chromatid exchanges (SCEs) in bone marrow cells of female Swiss albino mice. Sterigmatocystin gave elevated SCE frequencies at all doses tested (0.06-6.0 mg/kg). In contrast, griseofulvin, tested from 0.4 to 200 mg/kg, elevated the SCE frequency only in those mice which received doses of 100 or 200 mg/kg body weight. These results indicate that both fungal mycotoxins induce SCE in vivo and are potentially mutagenic.

Genetic analysis of tumorigenesis: XII. Genetic control of the anchorage requirement in CHEF cells.

Chinese hamster somatic cell hybrids between diploid anchorage-independent CHEF/204Bu50 cells and diploid anchorage dependent CHEF/205-30 cells are anchorage dependent but can segregate subclones at low frequency which reexpress anchorage independence. Thus, anchorage independence, like other characteristics of the transformed phenotype, is suppressed in these hybrids. Anchorage-independent subclones were recovered from the anchorage-dependent hybrids under conditions which favored the retention of most chromosomes. Karyotype analysis of suppressed hybrids and their anchorage-independent subclones showed that segregation of anchorage dependence was correlated with the loss of one copy of chromosome 1 in CHEF Chinese hamster hybrids. Thus, suppression of anchorage independence has a chromosomal basis. Several genetic models are considered for the origin of anchorage-independent subclones from suppressed Chinese hamster hybrids.

Genetic analysis of tumorigenesis: X. Chromosome studies of transformed mutants and tumor-derived CHEF/18 cells.

Chinese hamster embryo fibroblast cell line CHEF/18 is stably diploid, anchorage-dependent, has a high serum requirement, and a does not form tumors in nude mice. The chromosome constitutions of spontaneous and chemically induced anchorage-independent and/or low-serum CHEF/18 mutants and tumors produced in nude mice by some of these mutants are compared. We find a correlation between diploidy and nontumorigenicity among the anchorage-independent mutants but not in the low-serum mutants. One of the four spontaneous and six of the 15 chemically induced anchorage mutants have remained diploid. The remaining 12 mutants are pseudodiploid or aneuploid, and seven of them contain changes in chromosome 1, either a translocation or a deletion involving breakage at the same position (1q11-12). Each of the tumors induced by six mutants has a unique pattern of rearrangements; however five of the six have changes involving chromosome 3. This chromosome was also frequency rearranged in tumor-derived cells previously investigated.

Genetic analysis of tumorigenesis: VI. Chromosome rearrangements in tumors derived from diploid premalignant Chinese hamster cells in nude mice.

The chromosome constitution of CHEF/16 clones recovered from methylcellulose and of uncloned, tumor-derived CHEF/16 populations are compared. Nine of 11 clones recovered from methycellulose were initiated by diploid cells. Moreover, chromosomally diploid cells were still present in most CHEF/16 clones even after growth in anchorage-independent conditions. In contrast, none of the CHEF/16 cells recovered from tumors were diploid. Nonrandom chromosome changes were observed, but no specific chromosome alterations were consistently found in tumor-derived CHEF cells. Although CHEF/16 cells are uniformly tumorigenic in nude mice, each of 10 uncloned tumor-derived populations from inocula of 10(2), 10(4), and 10(6) CHEF/16 cells consisted of only 1-3 stemlines. Our results show that diploid CHEF/16 cells are premalignant and undergo karyotypic changes leading to successful and usually clonal establishment of tumors in nude mice.

Genetic analysis of tumorigenesis: V. Chromosomal analysis of tumorigenic and nontumorigenic diploid chinese hamster cell lines.

The chromosomal constitution of four established diploid Chinese hamster embryo fibroblast (CHEF) cell lines is described. CHEF/18 exhibits anchorage-dependent growth and is not tumorigenic in nude mice. CHEF/16 has a high plating efficiency in methylcellulose and is highly tumorigenic in nude mice. Both CHEF/8 and CHEF/16 have a normal Giemsa banding pattern and constitutive heterochromatin distribution characteristic of normal diploid Chinese hamster cells and exhibit relatively little chromosomal variation within their cell populations. These results suggest that the nuclear changes responsible for tumorigenicity of CHEF/16 involve alterations below the level detectable by Giemsa banding analysis. Chromosome rearrangements were detected in two other CHEF cell lines; CHEF/205-30, a diploid, thioguanine-resistant derivative of CHEF/18, and CHEF/204-Bu 50, a diploid, 5-bromodeoxyuridine-resistant derivative of CHEF/16, which are being used as genetic markers in intraspecific somatic cell hybrids.

A correlation between a spectrophotometric quantitation of rabbit spermatozoan motility and velocity.

Rabbit sperm motility was measured spectrophotometrically and a sperm motility index (SMI) was obtained. A comparative analysis between the SMI values and the velocity of motile sperm (obtained by time lapse photography of sperm tracks) was performed. The SMI specifically was compared to the mean velocity of the first 300 sperm cells observed and to the mean velocity of just the first 300 progressively motile sperm cells. In both comparisons, the SMI was highly correlated to the sperm track length (sperm velocity). In addition, a modification of the spectrophotometric procedure is described which allows measurements of the SMI to be made on semen extended into egg-yolk glycerine cryopreservation agents.