Goldfish urine

Visualization of Pheromone Pulses in Goldfish Urine with Isosulfan Blue... 

Alcohol sulfates are easily metabolized by mammals and fishes either by oral or intraperitoneal and intravenous administration. Several labeled 35S and 14C alcohol sulfates have been used to determine their metabolism in experiments with rats [336-340], dogs [339], swines [341], goldfish [342], and humans [339]. From all of these studies it can be concluded that alcohol sulfates are absorbed in the intestine of mammals and readily metabolized by to and p oxidation of the alkyl chain and excreted in the urine and feces, but are also partially exhaled as carbon dioxide. Fishes absorb alcohol sulfates through their gills and metabolize them in a similar way to that of mammals. 

An experiment has been done to confirm the participation of renal route in PCP excretion by goldfish, using a urine collecting apparatus shown in Figure 1. 

After 24-h exposure to 0.1 ppm PCP, goldfish(av. 70 g) were transferred into the urine collecting apparatus and a catheter inserted Into the urinary bladder from the urogenital cavity. Approximately 13 ml of urine was collected from each fish for 24 h. The urine collected from 14 fish was pooled and used for the determination of PCP. 

It is very interesting from the comparative biochemical view point that goldfish excrete PCP in the urine as the sulfate and in the bile as the glucuronide, while rabbits which were orally administered with PCP-Na excreted PCP-glucuronide accompanied by a large amount of free-PCP in the urine, Tashiro et al. (11). 

Metabolites in urine or feces provide the energetically least expensive, and evolutionarily probably the original, chemical signals in vertebrates. Much of history of evolution has concerned the development by living things of responses to metabolites, sometimes their own and sometimes produced by others. Those organisms which developed satisfactory responses succeeded, and those which did not, failed. (Lucas, 1944). Interested parties, such as members of the opposite sex, can then spy and read pertinent information about sexual and dominance status, health and body condition, quality of diet, and more. For instance, female goldfish release sex pheromones in their urine that... 

Thresholds of the olfactory receptors of male goldfish are 35 pg/ml water for the prostaglandin F2a, and 100 times less for its 15-keto-derivative. The males receptor threshold for 17,20-progesterone (from females) is a tiny fraction of 1 pg/ml water. Three grams (one teaspoonful) would provide an abovethreshold stimulus when diluted in 500 x 500 x 500 m water (Bjerselius and Olsen, 1993). In lampreys, testosterone from males attracts females at a concentration of 29 pg/ml water but urine with a testosterone concentration of 29 X 10 pg/ml is active (Adams etal., 1987). 

All male fish pheromones are steroidal (Sorensen and Stacey, 1990). Many male fish release chemicals that attract females, stimulate them to spawn, and inhibit their aggression. As early as 1982, Liley compiled a long list of examples testes, as in goldfish (Stacey and Hourston, 1982), urogenital fluid, glands on the caudal peduncle (an anal fin appendage), mucus and urine, can be sources of male pheromones. 

Female rainbow trout, Oncorhynchusmykiss, also release in their urine 17,20jSP. As in goldfish, this pheromone increases the plasma levels of gonadotropin II and testosterone in spermiating males Scott etal, 1994). Levels of 17,20jSP rises within 1 hour of exposure and peak at 3-4 hours. Milt production also increases (Vermeirssenetfl /., 1997). 

Female Goldfish release a spawning pheromone in their urine. They release urine in pulses every 2-4 min. These pulses can be made visible by a blue dye in the urine (Appelt and Sorensen 1999). 

The primary objective of this study was to characterize the temporal nature of postovulatory PGF pheromone release in goldfish. To do this, we had to develop a new technique for observing urinary release in aquatic animals. Developing this technique was a secondary objective of this study. We asked the following questions 1) Is urinary release in goldfish pulsatile, and if so, can it be measured reliably 2) Do ovulated goldfish release urine in pulses 3) Are the temporal characteristics of urinary release sexually dimorphic 4) Is urinary release pulsatile in another species of freshwater fish, and if so, does its pattern of release differ from that of the goldfish A portion of the data presented in this paper is also described in a recent unpublished Masters thesis (Appelt 1997). 

Our objectives here were to 1) assess whether urine is released in a pulsatile and predictable manner by sexually inactive goldfish, and 2) determine whether pulsatile urinary release can be measured reliably in sexually-active females. To address these objectives, we injected female goldfish with either a dye or a radio-label, both known to be released in urine, and compared the results. The use of this dye to measure urine release in an aquatic organism has not been described before. 

Experiment 1 Assessing Whether Urine Is Released in a Pulsatile and Predictable Manner by Sexually-lnactive Goldfish... 

Results. Goldfish injected with the dye released urine in visible pulses within 15 min of injection, and casual observation revealed that urine release was still noticeable approximately 2 h after the experiment ended (i.e. 3 h after injection). The rate of urinary release per 5 min interval did not change over the 60 min experimental period (Figure 1), and thus permitted us to conduct dye injection experiments over this time period with confidence. 

Figure 1. Mean number ( SEM) of urinations per 5 min interval over a 60 min period (i.e. 15-20, 20—25,. .., 70-75 min after injection) for six female goldfish injected with dye. No significant differences (p >0.10) were observed between the intervals.

To begin the experiment, each female (18—25 g) was removed, injected with tritiated PGp2 (Dupont NEN, Boston, MA 3.6 pCi or -3,600,000 cpm, 20 pg PGF2 ), briefly dipped into another bucket of water to rinse off excess radiation, and returned to the experimental apparatus. The pump withdrew water at 5 ml/10 s, and 5 ml samples were collected every minute from 15-45 min after returning the female to the apparatus. We chose 1 min intervals because it was the fastest rate at which we could sample reliably and because Experiment 1 demonstrated that goldfish typically urinate less than once per minute. Water samples were mixed with scintillation fluid in a 1 3 ratio, and radiation levels of samples were measured in a scintillation counter. Each sample was counted three times, and the average for each sample was used to calculate (see below) whether or not an increase in radiation was attributable to urinary release. 

EXPERIMENT 3 DO OVULATED SEXUALLY-ACTIVE GOLDFISH ALSO RELEASE URINE AS PULSES ... 

We next sought to demonstrate whether ovulated females release urine in a pulsatile manner similar to that shown by PGp2jj-injected females. To determine this, we injected ovulated female goldfish with the dye and recorded urinary release. 

Ovulated goldfish also released urine in a pulsatile manner and at a rate similar to that seen in non-ovulated fish (Figure 2). Furthermore, ovulated fish appeared to increase their urination rate slightly when they were sexually active (male present) in all instances they released urine more frequently when sexually active (4.3 0.3 urinations/15 min) than when they when sexually inactive (3.0 0.6 urinations/15 min). Further possible evidence of an association between urination and sexual activity is that fish tended to urinate within a short time of either rising or spawning (median time between each urination and the nearest rise or spawn = 2.7 s range = 0.5—107.4 s n=16). 

Here, we sought to determine whether mature male and female goldfish release urine with different temporal characteristics. 

We compared urine release of eight male (20—26 g) and eight female (20-33 g) goldfish using a protocol modified from Experiments 1 and 2a. We eliminated the acclimation periods and conducted experiments in 8 L glass aquaria with three sides covered in translucent, white plastic. Fish were held in 75 L aquaria with 4—5 individuals of the same sex... 

Figure 2. Patterns of urination and female sexual behavior (rising and oviposition behavior) in three ovulated female goldfish for a 15 min period before a male was added and for a 15 min period while a male was present and females were exhibiting sexual activity.

Ruffe released urine in pulses but with a different temporal pattern than that of goldfish (Table 1). Notably, the number of urinations and time between urinations differed significantly between ruffe and goldfish (p < 0.05). However, the mean duration of each urination for ruffe and goldfish was not different (p > 0.10). The total duration of all urinations differed (p < 0.001) between ruffe (26.5 6.3 s) and goldfish (81.4 7.8 s). 

We describe a new technique that can be used to reliably and effectively record the temporal components of urinary pheromone release in fish (and possibly other aquatic vertebrates). We used this technique to demonstrate that both ovulated and PGp2 -injected goldfish release urine and the pheromone it contains in a pulsatile manner. Although the pattern of urinary release does not appear to be sexually dimorphic in goldfish, the possi-... 

We found differences in the temporal nature of urinary release in two fish species. Ruffe release urine less often (different pulse frequency and inter-pulse interval) than goldfish, but the duration of each urination is the same as goldfish. As a result, ruffe spend considerably less total time urinating than goldfish (i.e. likely release less urine). This conclusion is supported by data from catheterized fish demonstrating that goldfish typically produce 0.75 ml of urine in 1.5 h while the same volume is produced in 6 h by ruffe. Future studies should closely examine urinary release in pheromone-releasing females of both species in association with female behavior and the effect of these patterns on male behavior. 

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