Rolf L. Ingermann

Altered behaviors in male mice, male quail, and salamander larvae following early exposures to the estrogenic pesticide methoxychlor.

Numerous publications show that methoxychlor (MXC), in use today as a DDT substitute, is a reproductive toxicant; it produces deleterious effects on the structure and function of the reproductive organs in exposed species. Exposure of mice (33 mg/kg body weight) to purified (95%) MXC at the time of implantation, or injection, of 5 mg MXC into freshly laid quail eggs prior to artificial incubation, altered sexual arousal and sexual behavior in adult males of both species. When placed near a plastic partition with an estrus female behind it, the MXC-exposed male mice showed no sexual arousal, spent less time near the partition, and exhibited lower testosterone levels. Similarly, adult quail males that were exposed to MXC during incubation showed a lack of sexual interest and copulatory behavior when presented with a receptive female. Some males showed a longer latency period before mounting the female, while others did not show any sexual interest at all. Exposure of salamander embryos to purified MXC at or above 0.3 microM (0.1 mg/l) induced precocial hatching of embryos and reduced the startle response and the distance traveled in response to startle. Exposing hatched larvae to MXC for 3 days also resulted in a blunted startle response. Due to the blunted startle response and decreased avoidance travel, the exposed salamander larvae appear more susceptible to predation and these effects may contribute to amphibian population declines. The results of these studies indicate that developmental exposures to environmental chemicals with hormonal activities produce undesirable behaviors that may affect population dynamics and survivability of exposed species.

Influence of osmolality and ions on the activation and characteristics of zebrafish sperm motility

Despite the prevalence of zebrafish as a model scientific organism, understanding sperm function in this species is essentially limited to observations that osmotic shock initiates motility. During natural spawning, sperm encounter a range of environmental salinities as well as freshwater mixed with egg-associated ovarian fluid (OF), thus sperm are likely to be exposed to saline prior to egg contact. Effects of saline on sperm function in this model species are unknown, but likely to be important. Using computer assisted sperm analysis, this study addressed the effects of osmolality of spawning media and ionic composition and pH on the proportion of sperm becoming motile at activation (motility), as well as sperm velocity and path. When activated with tap water, motility was maximal (80%) at 10s (earliest time measured), declining to 5% by 87 s postactivation. With activation at moderate osmolalities ( approximately 160-200 mmol/kg) initial motility was decreased relative to low osmolality, increased from 10 to 30s, and subsequently declined less rapidly (motility in 80 mM NaCl was 35%, 80%, and 60% at 10, 30 and 147 s, respectively). Thus, moderate osmolality increased duration, but introduced a temporal lag in motility onset. With moderate osmolalities, the rate of velocity decay was less than that with tap water activation. Sodium chloride and sucrose similarly impacted both motility and velocity. Replacement of NaCl with KCl, pH values ranging from 6.8 to 8.4, or the presence of gadolinium were without effect. Motility, but not velocity, was slightly supressed by Ca(2+). Therefore, whereas pH and concentrations of Ca(2+) or K(+) of OF are unlikely to impact fertility via sperm motility, the OF contribution to spawning media osmolality may have pronounced effects on motility and velocity of sperm, factors previously correlated with fertility in other species.

Low seminal plasma buffering capacity corresponds to high pH sensitivity of sperm motility in salmonids

The buffering capacity of air-equilibrated seminal plasma from the chinook salmon Oncorhynchus tshawytscha and steelhead Oncorhynchus mykiss were determined. Buffering capacities were significantly less at pH values at and above 7.3 than over the range 6.0 to 7.0 and were particularly low between pH values of approximately 7.5 to 8.5. The potential of chinook salmon sperm to become motile upon addition to water was highly sensitive to pH. Only very low levels of motility were demonstrable when sperm had been maintained at pH values at or below 7.5 prior to water exposure while maximum motility was noted when sperm had been maintained at pH values of about 8.0 and above. Thus, the potential for motility appeared especially sensitive to that pH range over which seminal plasma exhibited its lowest buffering capacity. Buffering capacity, protein content and inorganic phosphate content of chinook salmon and steelhead seminal plasmas were less than those of corresponding blood plasma or ovarian fluid suggesting that seminal plasma is unique among salmonid body fluids in its composition and low buffering capacity. This low buffering capacity is likely to facilitate the role of the sperm duct epithelium in semen pH regulation and thus, in the sperms acquisition of the potential for motility upon exposure to water.

Effect of Temperature on Clove Oil Anesthesia in Steelhead Fry

Abstract The effects of temperature (11, 15, and 20°C) on clove-oil-induced anesthesia in fry of steelhead Oncorhynchus mykiss (0.18 ± 0.01 g [mean ± SE]) were evaluated at 25, 50, and 100 mg clove oil/L. Induction time decreased significantly with increasing temperature and dose. Recovery time after removal from anesthesia decreased significantly with increasing temperature. Mortality at 24 h postanesthesia increased significantly with increasing temperature and dose. Although clove oil appears to be an effective general anesthetic for salmonid fry, the data indicate that care must be taken in determining the appropriate dose to minimize temperature-associated mortality.

Short-Term Storage of Salmonid Sperm in Air versus Oxygen

Abstract We evaluated the effects of different gaseous environments on the short-term storage of semen samples from hatchery-reared chinook salmon Oncorhynchus tshawytscha, commercial rainbow trout O. mykiss, and steelhead (anadromous rainbow trout). Within each species examined, sperm motility declined steadily over time and was generally about 50% of the initial value after 72 h; however, there was no difference in motility between samples maintained under either ambient air or 100% O2. The motility of samples incubated under either exhaled air or 95% O2 plus 5% CO2, as well as samples incubated under 100% N2, was significantly decreased after 24 h. Sperm samples demonstrating a significant loss of motility following incubation under exhaled air recovered much of the motility after subsequent incubation under ambient air for 24 h. Sperm viability was not altered after 24 h under any incubation condition but was decreased significantly after 72 h under 100% N2. Finally, the spermatocrits of samples maint...

Does Co2 enhance short-term storage success of chinook salmon ( Oncorhynchus tshawytscha ) milt?

Successful short-term storage of salmonid milt depends on numerous factors, including temperature, fluid volume, and gaseous environment, with storage at low temperatures under an atmosphere of 100% O2 being the most common method. Salmonid sperm maintained in a storage environment with elevated carbon dioxide (CO2) levels, such as the approximately 4% CO2 in exhaled air, are not motile when activated. While these modest levels of CO2 inhibit sperm motility, the effect is reversible within hours after exposure to a CO2-free oxygenated environment. Therefore, the effect of CO2 (as a component gas in the storage environment) on chinook salmon (Oncorhynchus tshawytscha) sperm motility and viability was examined. The hypothesis of the current investigation was that CO2-exposure with subsequent CO2 removal would be beneficial during short-term chinook salmon milt storage. Milt samples were collected from mature (adult) and precocious (jack) male chinook salmon and stored under various CO2 and O2 levels at 3 to 4 degrees C for up to 14 days. Milt samples were then removed from the incubation environments and maintained under CO2-free humidified air with continuous mixing for 4 h at 10 degrees C before analysis of motility. The resultant motility of samples incubated under 3.5% or less CO2 was not different than controls during the 14 d incubation period; motility of samples stored under higher CO2 tensions were significantly lower. The motility of samples incubated under 3.5% CO2 reached the maximum recovered motility after 2 h exposure to CO2-free humidified air, while the motility of sperm incubated under 13.4% CO2 levels recovered no motility even after 6 h exposure to CO2-free humidified air. The motility of samples incubated under normoxia was significantly greater than that of samples incubated under hyperoxia (approximately 90% O2) at both 7 and 14 d, regardless of the CO2 level. Sperm viability was relatively unaltered by any of the incubation conditions examined. The results of this investigation suggest that there is no apparent advantage to storage of chinook salmon sperm in the presence of CO2 and that storage under hyperoxia negatively affects sperm function compared to storage under normoxia.

Effect of aging on male reproduction in zebrafish (Danio rerio).

The study was designed to test the hypothesis that male aging is associated with a change in reproductive function in the zebrafish. Young (290 ± 37 d) and older (911 ± 48 d) males were combined with females (604 ± 24 d) to test the effect of male age on the number and fertility of eggs laid by their mates. 48% of breeding trials with young males and 25% of the trails with older males resulted in egg deposition. Although young males were associated with significantly more successful breeding attempts than older males, number of eggs laid per clutch, number and percent of fertilized eggs and the number and percent living embryos were not statistically different between young and older males. These data suggest that male aging is associated with altered reproductive behavior and/or female response but not in sperm quality per se. Consistent with this interpretation were the findings that percent motility and sperm motility characteristics did not differ between sperm from young and older males as assessed by computer-assisted sperm analysis. However, older males contained higher quantities of extractable sperm than did young males, perhaps associated with fewer successful breeding attempts. Age-related effects on male reproductive in the zebrafish may therefore be a consequence of behavioral or morphological features that play a role in female mate choice and/ or male sexual response.

Ecto‐ATPase Activity of Vertebrate Blood Cells

Ecto‐ATPase activity was measured for red blood cells, white blood cells, and whole blood from a variety of vertebrates. A large range of red blood cell ecto‐ATPase activity was observed; for example, at 10°C, red blood cells from a catastomid fish (Catostomus macrocheilus) and a newt (Taricha rivularis) had activities of 56 ± 9 and 25,000,000 ± 14,000,000 pmol ATP per 106 red blood cells per hour, respectively (mean ± SD). Several control experiments verified that the measured ATPase activity was not the result of intracellular ATPases released due to cell damage or lysis nor due to the release of intracellular nucleoside triphosphate or uptake of extracellular ATP. Red blood cell ecto‐ATPase activity was relatively low within the teleosts, was high within the reptiles, and had the greatest range and single highest value within the amphibians. Within the endotherms, avian red blood cell ecto‐ATPase activities were greater than mammalian red blood cell ecto‐ATPase activities, which were the lowest for all vertebrates examined. The lowest ecto‐ATPase activities measured were for human and skunk red blood cells, which had activities of 13 ± 1 and 11 ± 2 pmol ATP per 106 red blood cells per hour, respectively, at 35°C. Ecto‐ATPase activity was measured in white blood cells of several vertebrate species and appeared generally high and less variable than red blood cell ecto‐ATPase activity. Measured whole blood ecto‐ATPase activity showed a range of three orders of magnitude and correlated positively with red blood cell ecto‐ATPase activities. Ecto‐ATPase activity was also determined for red blood cells from fetal, 1–3 d old neonatal, and pregnant garter snakes (Thamnophis elegans); these activities were not significantly different from the activity of red blood cells from nonpregnant adult females. Overall, the data from the present study demonstrate a wide range of red blood cell and whole blood ecto‐ATPase activities among vertebrates and include some of the highest ecto‐ATPase activities reported to date.

Initiation of motility by steelhead (Oncorhynchus mykiss) sperm: Membrane ion exchangers and pH sensitivity

Initiation of motility in salmonid sperm is sensitive to the pH of the extracellular medium, however, the basis of this sensitivity is not clear. Sperm incubated in an immobilization buffer (SI) at low pH ( approximately 7.1-7.2) become motile when diluted with activating medium (AM) at high ( approximately 8.5) but not low pH. Based on this observation, various agents were tested to determine whether the onset of steelhead sperm motility upon activation with high pH AM, following incubation with low pH SI, could be blocked by inhibiting membrane exchangers postulated to be important in intracellular pH (pHi) regulation. Amiloride (inhibitor of proton:sodium exchange), SITS and DIDS (inhibitors of anion exchange) and bafilomycin A 1 (inhibitor of H(+)-ATPase activity) were not effective in this experimental design. However, regardless of SI pH, DIDS was effective in blocking motility as was replacing chloride with thiocyanate or including the chloride channel blocker, niflumic acid, in SI suggesting that chloride efflux plays a key role in motility initiation. Nonetheless, the results of this study suggest that the rapid onset of sperm motility with activation at high pH following incubation at low pH is probably not based on rapid adjustment of pHi via membrane exchangers/transporters but rather due to an effect of pH on motility-associated processes at the extracellular surface of the sperm.

Subpopulation distribution of motile sperm relative to activation medium in steelhead (Oncorhynchus mykiss)

In the present study, steelhead sperm were activated in artificial tap water, ovarian fluid, activating saline, or in combinations of these media, and motility characteristics were determined using computer-assisted sperm analysis. Motility characteristics of individual sperm were then assessed to test the hypothesis that motile sperm are distributed among discrete subpopulations and that their distribution is influenced by the activation medium. Analysis with k-means clustering detected three discrete motile sperm subpopulations in steelhead semen, regardless of the activation medium. Based on multivariate analysis of variance, proportions of these subpopulations did not differ between sperm activated with ovarian fluid and activating saline, or any combination of these two media. However, subpopulation distributions for sperm activated with either ovarian fluid or activating saline were influenced by the level of dilution of these media in artificial tap water. There was an increase in the number of sperm in high velocity (curvilinear), high straightness, and high wobble subpopulation with increased levels of ovarian fluid or activating saline. The change in sperm motility characteristics with a change in activation medium may play a role in normal fertilization, as discharged sperm pass from seminal plasma and water through ovarian fluid en route to the egg.