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The Influence of Dietary Lysine on Yellow PerchMaturation and the Quality of SpermKarolina Kwaseka, Konrad Dabrowskib, Joanna Nyncac, Michal Wojnod & Macdonald Wicke

a School of Environment and Natural Resources, Ohio State University, 2021 Coffey Road,Columbus, Ohio 43210, USA; and Department of Animal Sciences, Ohio State University, 2027Coffey Road, Columbus, Ohio 43210, USAb School of Environment and Natural Resources, Ohio State University, 2021 Coffey Road,Columbus, Ohio 43210, USAc School of Environment and Natural Resources, Ohio State University, 2021 Coffey Road,Columbus, Ohio 43210, USA; and Polish Academy of Sciences, Tuwina 10, 10-748 Olsztyn,Poland,d School of Environment and Natural Resources, Ohio State University, 2021 Coffey Road,Columbus, Ohio 43210, USAe Department of Animal Sciences, Ohio State University, 2027 Coffey Road, Columbus, Ohio43210, USAPublished online: 12 Feb 2014.

To cite this article: Karolina Kwasek, Konrad Dabrowski, Joanna Nynca, Michal Wojno & Macdonald Wick (2014) The Influenceof Dietary Lysine on Yellow Perch Maturation and the Quality of Sperm, North American Journal of Aquaculture, 76:2, 119-126,DOI: 10.1080/15222055.2013.856826

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North American Journal of Aquaculture 76:119–126, 2014C© American Fisheries Society 2014ISSN: 1522-2055 print / 1548-8454 onlineDOI: 10.1080/15222055.2013.856826

ARTICLE

The Influence of Dietary Lysine on Yellow Perch Maturationand the Quality of Sperm

Karolina KwasekSchool of Environment and Natural Resources, Ohio State University, 2021 Coffey Road, Columbus,Ohio 43210, USA; and Department of Animal Sciences, Ohio State University, 2027 Coffey Road,Columbus, Ohio 43210, USA

Konrad Dabrowski*School of Environment and Natural Resources, Ohio State University, 2021 Coffey Road, Columbus,Ohio 43210, USA

Joanna NyncaSchool of Environment and Natural Resources, Ohio State University, 2021 Coffey Road, Columbus,Ohio 43210, USA; and Polish Academy of Sciences, Tuwina 10, 10-748 Olsztyn, Poland

Michal WojnoSchool of Environment and Natural Resources, Ohio State University, 2021 Coffey Road, Columbus,Ohio 43210, USA

Macdonald WickDepartment of Animal Sciences, Ohio State University, 2027 Coffey Road, Columbus, Ohio 43210, USA

AbstractThe objective of the present study was to determine whether lysine (Lys) supplementation influences the maturation

and sperm quality of Yellow Perch Perca flavescens. Juveniles raised on a formulated commercial diet and weighingapproximately 75 g each were randomly distributed into six 400-L tanks. This experiment included two wheat-gluten-based diets in triplicate: (−) Lys (Lys-deficient) and (+) Lys (Lys-supplemented; 2.23% in dry feed) diets. In addition,16 control fish were kept under identical conditions and fed a commercial diet. The weight of males was larger inthe control group than in the (+) Lys and (−) Lys groups. The sperm concentration was significantly higher in thecontrol and (+) Lys groups than in the (−) Lys group. Sperm motility was lower in the (−) Lys group than in thecontrol and (+) Lys groups. The control group had significantly higher protein concentration in its seminal plasmathan did the (+) Lys and (−) Lys groups. The seminal plasma trypsin inhibitor activity showed the same trend. Theseminal plasma free amino acid concentrations of arginine, methionine, threonine, glutamine, alanine, and glycinediffered significantly among treatments. This is the first report demonstrating the negative effect of dietary lysinelevel in plant-protein-based diets on reproduction in fish.

The unpredictable and variable reproductive performance ofmany fish species is a limiting factor in the successful produc-tion of juveniles (Coward et al. 2002). Reproductive well-being

*Corresponding author: dabrowski.1@osu.eduReceived June 19, 2013; accepted October 13, 2013

is largely dependent on the nutritional status of the broodstockin which nutrient deficiency can reduce semen quality (Vassallo-Agius et al. 2001) and thus fertilization ability. Successful

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fertilization in fish depends greatly on the quality of the sperm(Ciereszko and Dabrowski 1994). Hence, improved knowledgeof sperm characteristics, such as motility, density, and seminalplasma proteins, is necessary to understand the reproductiveresponses of fish kept in captivity.

The role of seminal plasma is to create an optimal environ-ment for spermatozoa that consists of stable osmolality lev-els, ion concentrations, and pH. Seminal plasma also facilitatesthe synthesis of proteins involved in many of the physiologicalfunctions of semen, such as spermatozoa protection during theirstorage in the reproductive system. There are different spermparameters that contribute to sperm quality, such as the concen-tration of spermatozoa, sperm volume, motility, pH, and sem-inal plasma protein concentration (Lanes et al. 2010). Seminalplasma of teleost fish is characterized by a low protein concen-tration (1–3 g/L) when compared with protein levels in blood.For that reason, it is possible that antiproteinases may be oneof the main proteins in teleost seminal plasma. This suggestionis supported by the relationship between the inhibitory activ-ity and the mean protein concentration in fish seminal plasma(Dabrowski and Ciereszko 1994; Wojtczak et al. 2003).

The free amino acid (FAA) composition of seminal plasmavaries among fish species (Billard and Menezo 1984; Lahn-steiner 2009). These FAAs are assumed to originate from theepithelium of the spermatic cord or from the proteolysis of sem-inal plasma proteins (Lahnsteiner et al. 1994). The actual role ofFAAs in the seminal plasma of teleost fish is unclear. In mam-mals, seminal FAAs are involved in the enhanced metabolicactivity of spermatozoa and in detoxification and they affectsperm viability. It is possible that FAAs in fish seminal plasmacontribute to osmolality and have a positive effect on spermfertilizing ability (He and Woods 2003).

We have previously shown that lysine (Lys) directly affectsthe growth and blood plasma FAA pool in Yellow Perch Percaflavescens and that a wheat-gluten-based diet supplemented withLys improves fish performance on a diet containing plant protein(Kwasek et al. 2012). In the present study, which is a continua-tion of that previous study, we analyzed dietary effects on malematuration.

Sperm production in Yellow Perch can be affected by manyfactors, such as photoperiod, temperature (Dabrowski et al.1996), and broodstock diet. Understanding the effects of thesefactors on sperm quantity and sperm viability (motility, fertiliz-ing ability) is an important step for optimizing fertilization pro-tocols and methods of broodstock evaluation and management.Therefore, the objective of the present study was to determinewhether dietary Lys influences the maturation and sperm qualityof Yellow Perch.

METHODSTwo-year-old Yellow Perch weighing approximately 75 g

each were randomly distributed into six 400-L tanks at 32 ± 1fish per tank. All fish were individually marked with PIT tags(Biomark, Boise, Idaho; Baras et al. 2000). This experiment in-

cluded two diets: (−) Lys (Lys deficient) and (+) Lys (Lys sup-plemented; 2.23% free Lys; Bachem, New York) wheat-gluten-based (MP Biomedicals, Solon, Ohio) and fish-meal-based dietsin triplicate as previously described in Kwasek et al. 2012. Thetotal amount of Lys in the (−) Lys diet was approximately 1%(1.42% Lys in wheat gluten; 4.51% Lys in fish meal; NRC 1994).In addition, due to limited capacity and fish number, a total of16 control fish were kept under identical conditions in a sep-arate tank and fed a commercial diet (BioOregon, Westbrook,Maine). Fish were fed near to satiation at readjusted feedingrates that were equal across all treatments based on each day’sprojected change in weight. Fish size was checked periodicallyin order to monitor individual fish growth. The water tempera-ture during the experiment followed seasonal cycles and variedbetween 7.8◦C and 24.0◦C. The city water that was used was fil-tered through activated charcoal filters and additionally treatedwith sodium thiosulfate to keep chlorine levels < 0.1 mg/L. Thephotoperiod was 13 h light: 11 h dark.

In March 2008 Yellow Perch broodstock were evaluatedfor the advancement of maturation and sex based on size, ro-bustness, and sperm release. Males and females were injectedwith human chorionic gonadotropin (300 IU/kg; Sigma-Aldrich,Saint Louis, Missouri) (Dabrowski et al. 1994). Fish were anaes-thetized with tricaine methanesulfonate (50 mg/L), and spermfrom the males from the control (n = 9), the (+) Lys group (n =11), and the (−) Lys (n = 11) group was sampled by strippingfor fertility and biochemical analysis.

Sperm samples were centrifuged twice: at 3,000 × g for3 min and 10,000 × g for 10 min. The resulting seminal plasmawas stored at −80◦C. Sperm motility was evaluated after addinghatchery water (dechlorinated Columbus, Ohio, city water) andrecorded as the relative percent of motile sperm. Approximately50 µL of this water was placed on a glass slide, and approx-imately 10 µL of semen was added, mixed, and covered witha cover slip. The percentage of motile spermatozoa was im-mediately estimated using light microscopy at an initial mag-nification of 20× and subsequently at 40×. Prior to fertiliza-tion, the sperm from individuals with the highest motility (80–90%) was diluted 20 fold in ice-cold Moore extender (Rinchardet al. 2005). The sperm concentration was diluted for the fer-tilization to 50,000 spermatozoa per egg. Sperm concentrationwas measured according to Ciereszko and Dabrowski (1993).Protein concentration was determined by the method of Brad-ford (1976) using bovine serum albumin as a standard. Antit-rypsin activity of seminal plasma was evaluated by inhibitionof Atlantic Cod Gadus morhua trypsin (Sigma Chemical, SaintLouis, Missouri). Amidase activity was determined accordingto Geiger and Fritz (1983) with modification (Dabrowski andCiereszko 1994; Ciereszko et al. 1998). Trypsin inhibitor ac-tivity (APA) was expressed as U/L, where one inhibitory unit(U) corresponds to the apparent amount of inhibitor able toblock one unit of trypsin activity (defined as the hydrolysis of1 µM of N-Benzoyl-DL-arginine-4-nitroanilide hydrochlorideper minute at room temperature ∼25◦C).

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INFLUENCE OF DIETARY LYSINE 121

Fish seminal plasma samples (control, n = 9; both Lysgroups, n = 11) were extracted for FAA analysis with 0.1 M HClin 1:1 (v/v) containing 200 µmol/L norleucine internal standardaccording to Cohen et al. (1989). The norleucine recovery wasapproximately 104%. Samples were then spun at 12,000 × gfor 15 min at 4◦C. The supernatants were filtered (Millipore,10 kDa cutoff at 2,000 × g for 90 min at 4◦C). Blanks (0.1 MHCl + 200 µmol/L norleucine) (Terjesen et al. 2004) and ex-ternal standards (Sigma acid–neutral and basic amino acids)were prepared along with sample preparation. Samples, blanks,and external standards were stored at −80◦C until analysis. Thesame concentration of glutamine in 0.1 M HCl as external stan-dard was prepared on the day of analysis and added to the basicamino acids standard. Amino acids were precolumn derivatizedwith phenylisothiocyanate (Cohen et al. 1989). Sample precip-itates were removed by 10 min of centrifugation at 10,000 ×g (Terjesen et al. 2004). Free amino acids were quantified bya Waters Pico Tag reversed-phase HPLC (Waters Corporation,Milford, Massachusetts) equipped with an application-specificcolumn (3.9 mm × 30 cm), a Waters 717 autosampler, two Wa-ters 501 pumps, a Waters 441 absorbance detector at 254 nm,and a column heater set at 46◦C. Eluent 1 and Eluent 2, pur-chased from Waters Corporation, were used throughout the in-vestigation as mobile phases. Each amino acid was identified byspiking with known amino acid standards and retention times ofexternal standards. Free amino acid concentrations (expressedas µmol/L seminal plasma) were calculated using internal andexternal standards (Cohen et al. 1989).

Data are presented as mean ± standard deviation (SD). Thedata were analyzed by using a nonparametric two-sample testor a one-way analysis of variance (ANOVA) (SPSS version17.0; SPSS, Chicago, Illinois). A P < 0.05 was consideredsignificantly different.

RESULTSThe growth results (Kwasek et al. 2012) showed that the

mean weight of males was larger in the control group (131 ±26 g) (P < 0.05) than in the (+) Lys (66 ± 10 g) and (−) Lysgroups (54 ± 10 g) (Figure 1). The number of individuals withscoliosis and lordosis tended to be higher in the (−) Lys group(15 ± 10%) than in the (+) Lys group (2 ± 3%; mean ±SD for three replicate tanks). However, these numbers were notsignificantly different (P = 0.0809). There were no deformitiesobserved in the control group. Although not significant, thepercentage of identified (spermiating) males among all fish fedthe (+) Lys diet was 12%, whereas among those fed (−) Lysdiet it was 18% (P = 0.3827). The survival of the fish at the endof the spawning season (July 2008) reached approximately 86%for both (−) Lys and (+) Lys groups. The mortality was mainlydue to the sampling and handling of the fish.

Sperm concentration and motility were significantly higher incontrol and (+) Lys groups than in the (−) Lys group (Figure 2)(P < 0.05). The control group had significantly higher protein

FIGURE 1. The mean weight (whiskers indicate SD) of Yellow Perch malesfrom control (n = 9), (+) Lys (n = 12), and (−) Lys (n = 15) groups (fromKwasek et al. 2012). Different letters indicate a statistical difference at P <

0.05.

concentration and APA activity in seminal plasma than did the(+) Lys and (−) Lys groups (P < 0.05) (Figure 2).

The concentration of Lys in the seminal plasma of the (−)Lys group tended to be lower than in the control and (+) Lysgroups, but differences between treatments were not significant(P > 0.05) (Figure 3). The arginine (Arg) concentration wassignificantly lower in the (−) Lys group than in the control (P< 0.05), while there was no difference between the (−) Lysand (+) Lys treatments (P > 0.05). The methionine (Met) con-centration was different between (+) Lys and (−) Lys groups(P < 0.05) but not different from the control group (P > 0.05)(Figure 3). The tyrosine (Tyr) level was the highest in the (+)Lys group and differed from the level in the (−) Lys group (P <

0.05) but not from the control (P > 0.05). The threonine (Thr)concentration showed the same trend. Among the dispensableamino acids, the (+) Lys group had higher glutamine (Gln) lev-els that were significantly different from the (−) Lys group (P< 0.05) but not from the control groups (P > 0.05) (Figure 3).The control and (+) Lys groups had higher glycine (Gly) con-centrations than did the (−) Lys group (P < 0.05). There was nodifference in phenylalanine (Phe), valine (Val), aspartate (Asp),glutamate (Glu), asparagine (Asn), hydroxyproline (Hpro), ser-ine (Ser), taurine (Tau), histidine (His), proline (Pro), isoleucine(Ile), leucine (Leu), tryptophan (Trp), and ornithine (Orn) con-centrations among all three groups (P > 0.05) (Table 1). Therewere also no differences in the level of total FAA (P > 0.05).

DISCUSSIONBroodstock nutrition greatly affects the quality of sperm, and

a verification of sperm quality is an important step that allowsfor the selection of superior males for increased fertilizationand hatching rates. Dietary ascorbic acid was shown to improve

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TABLE 1. The concentration of free amino acids in Yellow Perch seminal plasma (µmol/L). The table shows average concentrations from control, (+) Lys, and(−) Lys groups with SDs. See the text for the definitions of the amino acid abbreviations.

Control (−) Lys (+) Lys

Free amino acids Average SD Average SD Average SD

Asp 163 185 66 130 37 20Glu 219 249 105 125 90 42Hpro 48 53 48 42 51 48Ser 2,135 479 1,586 662 2,034 820Asn 475 147 514 215 635 149Tau 528 274 487 166 667 239His 394 111 468 289 519 159Pro 457 1,198 2,441 3,639 1,181 1,602Val 609 161 622 289 716 236Ile 186 52 222 156 228 83Leu 432 141 416 215 541 232Phe 252 40 228 133 276 71Trp 47 9 51 23 51 12Orn 52 47 33 21 54 25

FIGURE 2. The quality of Yellow Perch sperm (sperm concentration, sperm motility, seminal plasma protein concentration, and seminal plasma trypsin inhibitoractivity) from control, (+) Lys, and (−) Lys groups. Bars are means and whiskers indicate SD. Different letters indicate a statistical difference at P < 0.05.

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INFLUENCE OF DIETARY LYSINE 123

FIGURE 3. Mean free amino acid concentration (whiskers indicate SD) in Yellow Perch seminal plasma from control, (+) Lys, and (−) Lys groups. See text forthe definitions of the amino acid abbreviations. Different letters above the whiskers indicate a statistical difference at P < 0.05. There was no statistical differencein the concentrations of Lys, Val, and indispensable amino acids (IDAA).

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sperm concentration, motility, and fertilizing ability in Rain-bow Trout Oncorhynchus mykiss (Canyurt and Akhan 2008).Ciereszko and Dabrowski (1995) showed that the nutrient con-centration, in this case ascorbic acid, in the seminal fluid closelyreflected the concentration of this vitamin in the broodstock diet.However, it did not affect semen quality at the beginning of thespawning season. A deficiency of ascorbic acid reduced spermconcentration and motility later during the spawning period. Inaddition, Lee and Dabrowski (2004) showed that dietary sup-plementation of vitamin C can increase sperm quality in YellowPerch.

Motility has been shown to be of predictive value in esti-mating reproductive success in fish (Ciereszko and Dabrowski,1994). The present study showed that two sperm quality parame-ters, sperm concentration and sperm motility, were significantlyhigher in the control and (+) Lys groups than in the (−) Lysgroup, suggesting that Lys deficiency has a significant effect onthe quality of sperm. The control and (+) Lys groups had signif-icantly higher protein concentration and APA in seminal plasmathan did the (−) Lys group. These findings are the first indica-tion, in fish, that reproductive performance can be impacted bydietary indispensable amino acid levels.

The highest protein concentration in seminal plasma wasfound in Yellow Perch when compared with Rainbow Troutand Lake Whitefish Coregonus clupeaformis (Dabrowski andCiereszko 1994). Since the protein concentration in fish semi-nal plasma is very low (1–2 mg/mL) in comparison with blood,it is possible that a species-specific system of proteins with an-titrypsin activity would constitute the main system of proteinsin this fluid (Dabrowski and Ciereszko 1994). Seminal plasmainhibitors are related to blood plasma inhibitors and differ intheir affinity toward serine proteinases, but their exact role stillremains unclear. It has been shown, however, that some seminalplasma protease inhibitors that originate from blood and skinmucus might indicate contamination of milt by proteases andtheir inhibitors (Kowalski et al. 2003). Mommens et al. (2008)suggested that a very high antiproteinase activity of AtlanticHalibut Hippoglossus hippoglossus seminal plasma might berelated to serine proteinase inhibitors participating in the an-tibacterial defense of sperm during storage.

The addition of 0.1% Trp to the diets of Ayu Plecoglossusaltivelis resulted in the rise of the serum testosterone levels andcaused earlier spermiation of males and induced maturation offemales (Akiyama et al. 1996). Kawabata et al. (1992) indicatedthat the sexual behavior and sperm release in the male RoseBitterling Rhodeus ocellatus could be induced by FAAs, suchas cysteine (Cys), Ser, alanine (Ala), Gly, and Lys, placed indialysis tubes suspended in tanks. Our study showed numeri-cally more advanced maturation (spermiation) of males in the(−) Lys group than in the (+) Lys group.

It is thus possible that dietary Lys deficiency induced stressin male Yellow Perch, which could be explained by resultsreported by Castranova et al. (2005). These authors indicatedthat low-stress-responding male Striped Bass Morone saxatilis

had plasma testosterone and 11-ketotestosterone levels lowerthan high-stress-responding males. Consequently, high-stress-sensitive fish began spermiating earlier and maintained a sper-miation response longer than low-stress-responding fish. Ourresults in Yellow Perch suggest that males that were under po-tential “nutritional stress” accelerated spermiation. This specu-lation, however, needs to be investigated further.

Lahnsteiner (2009) indicated that in vitro supplementation ofMet, Leu, and Ile (at 2,500 µmol/L) had a positive effect on thesperm viability of Rainbow Trout and Common Carp Cyprinuscarpio. He suggested that Met could play an important role inthe antioxidant defense, whereas Leu and Ile were assumed tobe sources of additional energy for spermatozoa during the pe-riod of testicular storage. The results of the present experimentwith Yellow Perch showed that the FAA composition of sem-inal plasma is tightly regulated and easily impacted by dietarydeficiencies of indispensable amino acids. For instance, the con-centration of Lys in the seminal plasma of the (−) Lys group wasnot different from that in the control and (+) Lys groups (P >

0.05). The Arg level, however, was significantly lower in the (−)Lys group than in the control (P < 0.05), but there was no dif-ference between the (−) Lys and (+) Lys treatments (P < 0.05).The Met concentrations were not different between treatmentgroups (P > 0.05). When our data are compared with resultsobtained by Billard and Menezo (1984) (Table 2), the (+) LysYellow Perch seminal plasma showed higher concentrations ofFAAs than did the Rainbow Trout (all FAA) and Common Carp(except Asp, Glu, Tau, Ala, Pro, and Gly) seminal plasmas. Theseminal plasma level of free Lys in the (+) Lys Yellow Perchwas 20-fold higher than in Rainbow Trout and 5-fold higherthan in Common Carp (Arg levels in Yellow Perch were 35-and 37-fold higher than in Rainbow Trout and Common Carp,respectively, and Met levels were 22-fold higher than in bothRainbow Trout and Common Carp). Furthermore, the level ofLys in the seminal plasma of European Perch Perca fluviatilis(also known as Eurasian Perch) was shown to be 20 µmol/L(Lahnsteiner 2010), which is a 22-fold lower concentration thanin Yellow Perch seminal plasma.

Yellow Perch blood plasma levels of FAAs (Kwasek et al.2012) are compared with the seminal plasma levels of FAAsin Table 2. The Lys level in the (+) Lys fed fish was approxi-mately two times higher in the seminal plasma than in the bloodplasma. However, the level of Lys in the (−) Lys group was fivetimes higher in the seminal plasma than in the blood plasma.Similarly, the blood plasma levels of Arg in the (+) Lys and (−)Lys groups (134 and 94 µmol/L, respectively) were almost fourtimes lower than the seminal plasma levels. An opposite patternwas observed with the amino acids Asp, Glu, Hpro, Tau, andPro in the (+) Lys group and Gly in the (−) Lys group. It is clearthat there is a barrier between blood and seminal plasma com-partments and amino acid metabolism and transport in thesebody fluids is regulated independently. Nutritional changesare indicated far more in the FAA levels in the blood; how-ever, the male reproductive system appears to be less impacted

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INFLUENCE OF DIETARY LYSINE 125

TABLE 2. Comparison of seminal plasma free amino acid concentrations (µmol/L) among Yellow Perch ([ + ] Lys treatment), Rainbow Trout, Common Carp(Billard and Menezo 1984), and European Perch (Lahnsteiner 2010). The table also shows blood plasma free amino acids of Yellow Perch from the (+) Lystreatment (as averages from small and large fish; please refer to Kwasek et al. [2012] for details). The Lys level in blood plasma in the (−) Lys group was onaverage 59 µmol/L. See the text for the definitions of the amino acid abbreviations. The abbreviation “nd” indicates the amino acid was not detected.

Yellow Perch Rainbow Trout Common Carp European Perch

Free amino acids Seminal plasma Blood plasma

Asp 37 347 20a 948a

Glu 90 158 23b 7,110b 960Hpro 51 87 ndSer 2,034 312 10 320 ndTau 667 4,553 82c 3,127c

His 519 323 9 276 ndAla 797 516 19 822 2,090Pro 1,181 3,955 5,413 ndTyr 778 163 355 ndIle 228 196 173 1,120Leu 541 397 408 ndTrp 51 29 23 70Orn 54 28 10 21Asn 635 75 1,120Lys 451 281 23 93 20Arg 485 134 14 13 7,790Gly 1,641 1,646 14 2,118 ndMet 371 226 17 17 1,600Thr 751 227 15 623 ndVal 716 387 13 336 530Phe 276 162 195 280Gln 1,415 901 nd

aIncluding Asp NH2.bIncluding Glu NH2.cIncluding phosphoethanoloamine and phosphoserine.

by diets and may also reflect the specific metabolic needs ofsperm.

In conclusion, this is the first report demonstrating the neg-ative effect of dietary Lys level in plant-protein-based diets onreproduction in fish.

ACKNOWLEDGMENTSThis project was supported by the U.S. Department of Agri-

culture Special Grant 600006883.

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