RESPONSE OF PACIFIC WALRUSES TO DISTURBANCES FROM CAPTURE AND HANDLING ACTIVITIES AT A HAUL-OUT IN BRISTOL BAY, ALASKA

Observations were made on herds of the Pacific walrus ( Odobenus rosmarus divergens ) to study their response during the capturing and handling of adult males in summer 1995 at a haul-out at Cape Peirce in southwestern Alaska. Three behaviors (alertness, displacement, and dispersal) were quantified from 16 capture sessions. Herd sizes ranged from 622 to 5,289 walruses. Handling of an immobilized walrus consisted of attempts to attach telemetry devices to the tusks and collect various biological samples. Handling activities resulted in an average of about 10-fold or greater levels of behavior in alertness, displacement, and dispersal than during precapture and darting periods. High levels of behavior usually occurred within the first 45 min of handling. In 8 of 10 capture sessions, walruses returned to predisturbance levels of behavior within 40 min of cessation of the handling disturbance. Alertness and displacement were moderately and negatively correlated with herd size during the handling period, which may reflect an effect of a threshold distance from the point of disturbance to responding individuals. Observations of walruses tagged with VHF radio transmitters indicated that the activities from a given capture session did not preclude tagged walruses from using the haul-out over a subsequent 11 -wk monitoring period. Moreover, non-tagged walruses continued to extensively use the haul-out during and after the period in which capture sessions were conducted.     

Changes in accessibility of DNA to various fluorochromes during spermatogenesis

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4'6-diamidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permeabilized cells were either stained directly or after pretreatment with 0.06 N HCl. For histone-containing tetraploid, diploid, and round spermatid cells, HCl extraction of nuclear proteins caused an approximately sixfold increase of 7-amino-AMD stainability but had no significant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6- to 4.0-fold after HCl treatment. In sharp contrast, HCl treatment of vas sperm did not increase the staining level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3- to 1.5-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.     

Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea

The effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mg/kg body weight X 5 days and were sacrificed 28 days later. Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry. Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO. Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50%. Two-parameter (DNA, RNA) flow cytometry measurements showed a dose-response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals. Flow cytometric analysis of both heat-stressed and non-heat-stressed nuclei showed a relationship between dosage and the coefficient of variation of alpha t [red/(red + green fluorescence)] measurements of AO stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU. Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of single-stranded DNA induced by heat or acid treatment of chemically altered chromatin structure. The lowest daily dosage used (5 mg/kg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as alpha t. This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test.     

Sperm head morphology and nuclear chromatin structure evaluated by flow cytometry in a diallel cross in mice

Genetic factors affecting spermatogenesis, sperm morphology, and chromatin structure in mice were estimated using a diallel cross of the inbred lines C3H/HeJ, C57BL/6J, DBA/2J, and BALB/cByJ. Flow cytometry of acridine orange stained cells was used to evaluate proportions of testicular tetraploid, diploid, and haploid cells and nuclear chromatin structure of sperm, measured by resistance of chromatin to in situ acid denaturation, and quantified by the ratio of double- to single-stranded DNA (alpha t). Percent morphologically abnormal sperm was scored by light microscopy. Heterosis, line, maternal, and reciprocal effects, and general and specific combining abilities were estimated for body and testis weights, testicular cell proportions, sperm alpha t values, and percent abnormal sperm. Heterosis was important for testis weight, alpha t values, and percent abnormal sperm. Inbreds varied in body and testicular weights, alpha t values, and percent abnormal sperm. Significant maternal effects were noted for several traits but could be due to sex-linked (X or Y) factors, since maternal and sex-linked effects were confounded. Although a high positive correlation existed between alpha t values and percent abnormal sperm, the proportion of sperm with altered chromatin structure, measured by FCM, was generally much lower than proportion of morphologically abnormal cells.     

Heterogeneity of sperm nuclear chromatin structure and its relationship to bull fertility

The relationship between sperm nuclear chromatin structure and fertility was evaluated in two groups of Holstein bulls: Group 1, 49 mature bulls, and Group 2, 18 young bulls. Fertility ratings had been estimated for Group 1 and nonreturn rates were known for Group 2. Semen samples were measured by the sperm chromatin structure assay (SCSA): sperm were treated to induce partial in situ DNA denaturation, stained with acridine orange, and evaluated by flow cytometry. Acridine orange intercalated into double-stranded DNA emits green fluorescence upon excitation with 488 nm light, and red fluorescence when associated with single-stranded DNA. An index of DNA denaturation per cell is provided by alpha-t [alpha t = red/(red + green) fluorescence]. The standard deviation (SD alpha t), coefficient of variation (CV alpha t) and proportion of cells outside the main population (COMP alpha t) of the alpha t distribution quantify the extent of denaturation for a sample. Intraclass correlations of the alpha t values were high (greater than or equal to 0.70), based on four collections obtained over several years from Group 1 bulls. Negative correlations were obtained between fertility ratings and both SD alpha t (-0.58, p less than 0.01) and COMP alpha t (-0.40, p less than 0.01) in Group 1, and between nonreturn rates and both SD alpha t (-0.65, p less than 0.01) and COMP alpha t (-0.53, p less than 0.05) in Group 2. These data suggest that the SCSA will be of value for identification of low fertility sires and poor quality semen samples.     

Stress-related hormones and genetic diversity in sea otters (Enhydra lutris)

Sea otters ( Enhydra lutris ) once ranged throughout the coastal regions of the north Pacific, but were extirpated throughout their range during the fur trade of the 18th and 19th centuries, leaving only small, widely scattered, remnant populations. All extant sea otter populations are believed to have experienced a population bottleneck and thus have lost genetic variation. Populations that undergo severe population reduction and associated inbreeding may suffer from a general reduction in fitness termed inbreeding depression. Inbreeding depression may result in decreased testosterone levels in males, and reduced ability to respond to stressful stimuli associated with an increase in the stress-related adrenal glucocorticoid hormones, cortisol and corticosterone. We investigated correlations of testosterone, cortisol, and corticosterone with genetic diversity in sea otters from five populations. We found a significant negative correlation between genetic diversity and both mean population-level ( r ²= 0.27, P < 0.001) and individual-level ( r ²= 0.54, P < 0.001) corticosterone values, as well as a negative correlation between genetic diversity and cortisol at the individual level ( r ²= 0.17, P = 0.04). No relationship was found between genetic diversity and testosterone ( P = 0.57). The strength of the correlations, especially with corticosterone, suggests potential negative consequences for overall population health, particularly for populations with the lowest genetic diversity.