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a School of Natural Resources, The Ohio State University, Columbus, Ohio 43210
b Piketon Research and Extension Center, Piketon, Ohio 45661
c Department of Molecular Andrology, Polish Academy of Sciences, 10-957 Olsztyn-Kortowo, Poland
d Department of Animal Sciences, The Ohio State University, Columbus, Ohio 43210
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a recent study, Robinson and Tiersch [8] found that mature catfish fed diets containing 37.5% or 52% of CSM (300 or 400 mg of free gossypol per kilogram of feed), gained less weight than fish fed either control diet (no CSM) or a diet with 25% CSM. Gossypol concentrations in the liver were proportional to dietary CSM levels. Surprisingly, replacing soybean meal with CSM had a positive effect on sperm motility in catfish. This would be in major contrast to mammals, in which gossypol administered orally inhibited the fertility of male rats [9,10], birds [11], and humans [12]. Spermicidal effects were also demonstrated in vitro on human, monkey, and mouse spermatozoa. Circulating steroid hormones in both in vivo-treated animals and in in vitro testis tissues incubated with gossypol have shown decreased testosterone production [9]. Gossypol's major metabolite, gossypolone, suppressed progesterone synthesis in bovine luteal cells [13]. The gossypol antifertility effect is well documented in mammals and birds; however, very few studies have been completed with fish [8]. To the best of our knowledge, no study of gossypol effect on fertility of salmonid fishes has been carried out. It has been claimed that tilapia are able to withstand 1800 mg/kg gossypol in the diet without growth depression. In comparing this to the case in monogastric mammals, Martin [14] concluded that fish have a high gossypol tolerance.
Most of the analyses of gossypol in fish tissues were accomplished with a colorimetric method that includes anisidine. This method showed values of gossypol concentrations 2–5 times higher than did high-pressure chromatographic techniques because of coloring interferences [15]. Therefore, it is possible that the estimates of gossypol levels in fish diets have been exaggerated. Thus, we have evaluated the effect of diets with graded levels of CSM (and consequently gossypol) on growth, hematological parameters, and blood plasma steroid hormones of rainbow trout (Oncorhynchus mykiss). Special attention was focused on males, in which fertility indicators were studied. We examined for the first time blood and seminal plasma concentrations of protein-bound and free gossypol to better understand mechanisms of gossypol toxicity on reproductive tissues of fish.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiment was performed with 1.5-yr-old rainbow trout obtained from the Ohio Department of Natural Resources, London State Fish Hatchery (London, Ohio). Fish were brought into Piketon Research and Extension Center aquaculture facility. After several weeks of acclimation, fish were randomly distributed among 15 circular tanks (400 L) at an initial density of 40 animals per tank. Each tank was supplied with 8–10 L/min of well water and was equipped with airlifts to maintain continuous circular water flow. Ambient water temperature ranged from 11°C in March to 16°C in July.
Experimental Diets
In the experimental diets, 0%, 25%, 50%, 75%, and 100% of fish meal protein (herring and menhaden meals) was replaced by solvent-extracted CSM protein (Table 1). CSM was obtained from Southern Cotton Oil Co. (Memphis, TN). Diets were prepared with steam pelleting at the Ohio State University experimental feed mill, Wooster, OH, and were stored at -15°C before feeding. The fish were fed at a ratio of 1.5% of body weight, and feed was readjusted after monthly weighing. Feeding was stopped 24 h before sampling.
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Data Collection
The dietary treatments began on March 10 (Day 0). The fish were weighed at monthly intervals and/or at the time of blood sampling. Blood was sampled on Days 0, 64, 112, and 131; sperm were collected on Day 71. Two fish were randomly selected per tank (6 fish/treatment) at each sampling date. Blood and sperm were transported on ice and centrifuged in the laboratory at 4°C at 1500 and 7280 x g, respectively, to separate blood and seminal plasma. Samples of plasma were retained at -83°C for later analysis. On Day 131, fish were killed. Gonads were removed and weighed to calculate the gonadosomatic index (GSI; gonad weight x 100/body weight). Fish were then bled and transported on ice to a commercial processor (Freshwater Farms of Ohio, Urbana, OH). These fish were filleted and weighed. Skinned fillet color was compared to Salmo Fan standards (F. Hoffmann-La Roche, Nutley, NJ). All animal procedures and handling were conducted with special care in compliance with the guidelines of the Institutional Animal Care and Use Committee, The Ohio State University.
Hematological Analysis
Hemoglobin and hematocrit were analyzed with standard procedures 3–4 h after storage on ice. Hematocrit was determined by the microhematocrit method [19]. Hemoglobin was measured by the cyanmethemoglobin procedure using Drabkin's solution (Sigma Chemical Co., St. Louis, MO). Hemoglobin standard prepared from human blood (Sigma Chemical Co.) was used. Protein concentrations in blood and seminal plasma were determined by a protein-dye binding method [20].
Gossypol Analysis
Gossypol concentration in diets and body fluids in the present study was determined according to the method described by Hron et al. [21] with some modifications. This method will be referred to as HPLC (3-amino-1-propanol); it was chosen because of its accuracy and recovery of internal standards as tested in our laboratory. Plasma samples were mixed with 70% acetone (1:3 v:v), vortexed, and centrifuged. The supernatant extract, which contained the free gossypol, was separated. The supernatant was then reconstituted with an equal volume of complexing reagent made of 4 ml of 3-amino-1-propanol, 20 ml of glacial acetic acid, and diluted to 200 ml with N,N-dimethylformamide. The mixture was heated at 95°C for 30 min. For total gossypol, the whole slurry (sample vortexed with 70% acetone) was used for a complexing reaction. Working standards of 0.05, 0.1, 0.2, 0.4, and 1 µg/ml of gossypol acetic acid (Sigma Chemical Co.) were used to prepare standard curves for each batch of assay. Recovery rate for both free and total gossypol extraction was 91.3 ± 3.10% (n = 4). A solution of gossypol with complexing reagent was diluted with 87% methanol and 13% water containing 0.1% phosphoric acid (mobile phase) to serve as internal standard. Gossypol-aminopropanol complex was determined by HPLC using a Beckman 506A (Beckman Instruments, Inc., Fullerton, CA) solvent delivery system equipped with a 20-µl injection loop with a reverse-phase C 18 column (Shodex, Showa Denko America, Inc., New York, NY) and an electrochemical detector (Model LC-4C; BAS, West Lafayette, IN). The detection level was 3 ng/20 µl of injection volume with a sample to noise ratio of 3 in the chromatogram. Additionally, diets were analyzed by two different methods [22,23] by Dr. M. Calhoun, Texas Agricultural Experimental Station, San Angelo, TX, referred to as HPLC (2-amino-1-propanol) and AOCS (the American Oil Chemists' Society colorimetric method).
Plasma Sex Steroids Analysis
Plasma steroid hormones (testosterone [T], estradiol-17ß [E2], 11-ketotestosterone [11-kT], and 17,20ß-dihydroxy-4-pregnen-3-one [17,20ßP]) were determined by RIA according to methods similar to those of Ottobre et al. [24]. The steroids were previously extracted with ethyl ether. [1,2,6,7-3H]T (96.5 Ci/mmol) and [2,4,6,7,16,17-3H]E2 (141 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). [3H]11-kT and [3H]17,20ßP were a gift from Dr. C.B. Schreck (Oregon State University, OR) and Dr. A. Fostier (INRA, Rennes, France). Unlabeled steroids were purchased from ICN Pharmaceuticals (Costa Mesa, CA), Sigma Chemical Co. and Steraloids (Wilton, NH). The T antiserum was provided by Dr. R.E. Ciereszko (Institute of Animal Physiology, University of Agriculture and Technology, Olsztyn, Poland), the E2 antiserum by Dr. R.L. Butcher (West Virginia University, WV), the 11-kT antiserum by Dr. D.E. Kime (University of Sheffield, UK), and the 17,20ßP antiserum by Dr. A. Fostier. The characteristics of these antisera have been reported previously [16,25–27].
Sperm Characteristics and Fertility
On Day 71, sperm density and motility were evaluated using methods described previously [28]. The sperm were also used to test male fertility. The unfertilized eggs of the cutthroat trout (O. clarki) were shipped overnight from Egan State Hatchery in Utah and used within 24 h after stripping. Hybridization between O. mykiss and O. clarki does not result in reduced hatchability or viability of the eggs [29]. Two subsamples of approximately 200 eggs from 3 females were fertilized with 300 x 105 sperm/egg per each male. Eggs were incubated in compartmentalized California hatching trays at approximately 15°C. The percentage of survival was determined at the eyed embryo stage and at the time of hatching. On one occasion, water temperature rose to 18°C for several hours and consequently a large percentage of twin embryos was observed in hatching from all three females.
Statistical Analysis
All data are expressed as means ± SD or SE. Average values per tank were used as the statistical unit. Significance of differences among treatments was determined by ANOVA followed by Scheffé's test. Normality and homogeneity of variance tests were performed on raw data. Sample distributions violating assumptions were log-transformed before analysis. Data expressed as percentages were arc sine-transformed before analysis. All differences were regarded as significant at P < 0.05. Data were also fitted according to an exponential curve or linear regression (Sigma-plot; Jandel Scientific, Corte Madera, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Krill meal was used as attractant in diet formulation, and two essential amino acids were supplemented to account for possible limitation in nutrients. Proximate composition of diets indicated uniform amount of protein and lipids across treatments (Table 1). No differences in the acceptance of diets was noticed during the study. Table 2 shows results of three different analytical procedures to establish concentrations of gossypol, its nature (+ and - isomers), and association with dietary proteins (free, i.e., acetone-soluble, and total, including protein-bound). Although values of gossypol concentration vary across methods, the relative increases across diet and within method parallel the increases in percentage of CSM in the diet. These differences in absolute values among the methods are not unusual in light of the literature [15]. Concentrations of free gossypol in the diet with the CSM substitution tend to be underestimated by the colorimetric method. The value of free gossypol as a measure of dietary effective concentration becomes less apparent if a large portion of gossypol is being released from total pool during stomach digestion in fish or other monogastric animals.
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Fish in all groups showed normal growth for the duration of the experiment (Table 3), although fish in treatment 5 showed a trend of slightly smaller increases relative to the others. A significant decrease in hemoglobin and hematocrit was found in fish fed the diet with 100% of CSM protein (Table 4). Hemoglobin and plasma protein concentrations were significantly but weakly correlated (r = 0.57, n = 16, P < 0.05). No differences were observed in fillet relative weight and fillet color at the end of the study (Table 5).
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GSI and Steroid Concentrations in Blood Plasma
Females All of the females were 1.5 yr old, and the majority were not expected to mature until the subsequent season. At the end of the experiment (Day 131), relative ovarian weight was not significantly different among treatments and generally was below 1% body weight (Fig. 1A). Incidentally, a few females were found with overripened eggs in July; they were not included in this analysis. Concentrations of plasma sex steroids (T, E2, 17,20ßP) were low or below the detection limit at the beginning of the study (Day 0) and amounted to 0.12 ± 0.02 ng/ml, 0.03 ± 0.01 ng/ml, and 240 ± 160 pg/ml, respectively. At the end of the experiment (Day 131), concentrations of T and E2 increased among treatments to reach 2–5 ng/ml, whereas the levels of 17,20ßP decreased further (< 70 pg/ml; Fig. 1B). Although higher concentrations of T and E2 were observed in treatment 3, they were not significantly different from those found in females from other treatments.
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Males GSI at the end of the experiment (Day 131) was variable among treatments (Fig. 1A). Concentration of plasma T in males at the beginning of the study (Day 0) reached 7 ± 2.5 ng/ml. It increased to 10–20 ng/ml in May (Day 64) and decreased at the time of the termination of the experiment (Fig. 2, A–C). A similar trend was observed in 11-kT and 17,20ßP concentrations. The levels of both of these steroids were low at the beginning of the experiment (Day 0), ranging from 3 to 29 ng/ml and from 0.01 to 2.9 ng/ml, respectively. They increased to reach their highest values in May (Day 64; Fig. 2A) and decreased progressively in July (Days 112 and 131; Fig. 2, B and C). A significant effect of dietary treatment on plasma T and 11-kT was observed at the end of the experiment (Fig. 2C), whereas on Day 112 only plasma T concentration was significantly affected by the dietary treatment (Fig. 2B). There were no significant differences in steroid concentrations between Days 112 and 131. At the end of the experiment (Day 131), plasma levels of T were significantly correlated with GSI (Fig. 3A) and plasma levels of 11-kT (Fig. 3B).
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Tissue Concentrations of Gossypol
Blood plasma concentrations of free gossypol differed significantly among treatments, and percentages of free to total (free plus protein-bound) gossypol gradually increased, indicating that carrying capacity of plasma proteins had not been reached (Table 6). In seminal plasma, gossypol levels were approximately 100 times lower than in blood. In addition, there seemed to be a predominance of free gossypol in seminal plasma. At the end of the experiment, plasma concentrations of free and total gossypol in males were inversely correlated with the plasma concentrations of T (Fig. 3C; r = -0.69, n = 13, P < 0.01 and r = -0.70, n = 13, P < 0.01, respectively). A significant correlation between hemoglobin and plasma free gossypol concentration was also observed at this period in males (r = -0.66, n = 13, P < 0.05).
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Sperm Characteristics and Fertility
There was no measurable effect of feeding rainbow trout with CSM protein-replacement diets on sperm characteristics and fertility (Fig. 4). Sperm concentrations and motilities were in the range encountered in this fish species during the reproductive period [30], although semen samples in the present study were collected from fish at least 3 mo into the spermiation period. Sperm fertility was low when estimated on the basis of absolute values of embryo survival. Various factors may have contributed to poor fertility. First, the eggs may have been of low quality, since they were collected at the end of the spawning season. Unfortunately, we were not able to assess this, since there is no method available to unequivocally determine egg quality before a fertilization test in salmonid fishes. In addition, a low sperm:egg ratio was used. If excessive numbers of spermatozoa were used, fertility could be high even if many spermatozoa were damaged, and this would jeopardize our evaluation. This low fertility may have limited the sensitivity for assessment of the effects of gossypol on the ability of sperm to fertilize eggs. However, lack of effect of gossypol on sperm characteristics is consistent with a lack of effect on fertility.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Growth depression was also demonstrated in cows fed whole cottonseed, which resulted in a free gossypol dose of approximately 30 mg/kg body weight per day [31]. Cytotoxic effects of gossypol are frequently associated with decreased hematocrit and hemoglobin, and increased red blood cell fragility [31,32]. Herman [6] observed a nearly 50% decrease in hematocrit and blood plasma protein concentrations in rainbow trout fed a casein-gelatin diet supplemented with 1000 ppm of free gossypol. The first signs of liver damage appeared in trout within 10 days, and hematological parameters might have been affected in Herman's [6] experiment much earlier than observed in the current study. In the present study, the decline in both hematocrit and hemoglobin probably reflected the increase in gossypol concentration in food and consequently in blood plasma (Table 4). Possibly, decreased hemoglobin limited the ability of fish to grow.
Bulls fed a cottonseed-containing diet were exposed to 16 mg of free gossypol/kg body weight per day, and this resulted in 2.2–6.0 µg of total gossypol per milliliter of plasma ([33]; personal communication with M. Calhoun in 1998). In the present study, the highest level of CSM corresponded to a dose of 15 mg of free gossypol/kg body weight. This dose resulted in circulating concentrations of total gossypol that were 29.94 µg/ml. Thus, fish in the current study were fed levels of gossypol similar to those fed to bulls [33]. The resulting circulating concentrations of total gossypol were higher in fish. Oral administration of gossypol to rats at the dose of 15 mg of free gossypol per kilogram body weight daily for 30 days resulted in plasma concentrations of free gossypol that were 1.04 µg/ml [34]. In the current experiment, plasma concentrations of free gossypol were 5.56 µg/ml after the feeding of similar concentrations of gossypol. Thus, on the basis of these examples, the fish in our experiment had 5-fold higher circulating concentrations of gossypol compared to those of mammals fed similar amounts of gossypol in relation to body weight. Differences between the studies in mammals and the present ones in fish may reflect better absorption of dietary gossypol in cold-blooded animals or a slower depuration rate.
The antifertility effect of gossypol is related to how efficiently gossypol crosses a general circulation-gonadal barrier. Most of the seminal fluid in mammals comes from the accessory sex glands. Rainbow trout do not have separate accessory sex glands outside of the gonad. Ciereszko et al. [35] demonstrated that several proteins in blood and seminal plasma of rainbow trout are electrophoretically identical, although protein concentration in seminal fluid was 100-fold lower than in blood plasma (Table 4). Therefore, we can assume that a blood-gonadal barrier accounts for differences in gossypol concentrations between these fluids. There are no comparable data in the literature with respect to mammals. The understanding of the mechanism of this transfer would perhaps make it possible to modulate the process by either facilitating the entry of gossypol into the gonads (sterility) or blocking this transport. It is apparent that this barrier is extremely effective in mature rainbow trout, in which differences in gossypol concentrations in blood plasma and seminal fluid were 100-fold (Table 6). Although concentrations of gossypol in plasma and seminal fluid were determined at different dates, we confirmed this relationship in an extension of the same experiment after 9 mo of feeding (unpublished data). These differences are largely related to concentrations of protein and carrying capacity of protein-bound gossypol (Table 7). Further studies should elucidate whether fish seminal plasma proteins include those of high affinity to gossypol as described in humans [36]. Wang et al. [37] demonstrated that after intravenous infusion of gossypol in rats, concentrations in rete testis fluid were 1000-fold lower (0.023–0.094 µg free gossypol/ml) than in blood plasma. This may represent differences between species as well as the route of administration (oral versus infusion).
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Chenoweth et al. [33] found that in bulls fed CSM, significant differences in midpiece sperm abnormalities appeared after 3 wk at the rate of 32% vs. 8.3% in controls. Sperm motility decreased significantly after 5 wk of dietary treatment with 16 mg of free gossypol per kilogram of body weight per day. In rats given 15 mg/kg orally per day for 3 wk, sperm were found to be immotile in the cauda epididymidis [34]. There were no studies found of in vivo assessment in mammals of sperm motility or fertilizing ability with simultaneous measurements of concentration of gossypol in reproductive tissues. In rainbow trout, there was no correlation between gossypol concentrations in seminal plasma (Table 4) and fertility (Fig. 4). Brocas et al. [32] concluded that in vitro exposure of bull spermatozoa to 10–50 µg/ml gossypol did not affect cleavage rate of fertilized oocytes. In the present study, no detrimental effects of the cottonseed-supplemented diets on rainbow trout sperm concentrations, motility, or fertilizing ability could be demonstrated (Fig. 4). The depression of GSI and steroid hormone concentrations in blood plasma had no measurable impact on reproductive performance of male trout in the late phase of reproductive cycle. Chenoweth et al. [33] observed a relative equilibrium of gossypol spermatotoxicity in bulls between weeks 5 and 11 of exposure as indicated by sperm motility. This corresponds to depressed growth in catfish males fed diets high in CSM and the counterintuitive enhanced motility of their sperm [8]. That may have been due to a postponement in gonad maturation and higher GSI in July (end of catfish spawning season) in fish treated with gossypol.
Indeed, gossypol concentrations in seminal plasma of trout (~0.3 µM) compare favorably with gossypol levels found to immobilize mammalian sperm (2 mM, Kim et al. [38]; 10 µM gossypol plus 25 mM theophylline, Fornes et al. [39]). Therefore, it seems unlikely that gossypol would affect the motility and fertilizing ability of spermatozoa in the sperm ducts of trout that are ready to spawn. This is the first study to show the concentration of gossypol in seminal fluid and sperm characteristics simultaneously. An important finding is that sperm motility and fertility was not diminished because of extremely low concentrations of gossypol in seminal plasma. Further studies are required to identify acceptable proportions of CSM in diets of fish. In addition, it is necessary to examine the effects of gossypol exposure during the entire process of spermatogenesis, which takes place in rainbow trout from August to November. Sperm characteristics did not indicate depressed fertility. This lack of depressed fertility is associated with restricted transfer of gossypol into reproductive tissues after completion of spermatogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by the National Cottonseed Products Association, and Cotton Foundation, Memphis, TN, and Pukyong National University, Pusan, S. Korea. Salaries were partly provided by state and federal funds appropriated to Ohio Agriculture Research and Development Center, Wooster, OH. 
2 Correspondence: Konrad Dabrowski, School of Natural Resources, The Ohio State University, 2021 Coffey Road, Columbus, OH 43210-1085. FAX: 614 292 7432; dabrowski.1{at}osu.edu
Accepted: September 13, 1999.
Received: April 6, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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