Evaluation of pea protein concentrate as partial replacement of fish meal in practical diets for juvenile tench (Tinca tinca L.)

With the aim to evaluate different replacement levels of fish meal (FM) by pea protein concentrate (PPC) on survival, growth performance and body composition of juvenile tench (Tinca tinca), a 90‐day experiment was conducted with 6‐month‐old juveniles. Four practical diets (50% crude protein) differing in the level of replacement of FM protein by PPC protein were tested: 0% (control), 25%, 35% or 45%, corresponding to 0, 207.5, 290.4 or 373.3 g PPC kg^?1 diet respectively. Survival rates ranged from 96.4% to 98.5%. The 25% and 35% replacement diets resulted in similar growth values (P > 0.05) to those obtained with the control diet (average of the three feeding treatments: 57.57 mm total length, 2.48 g weight and 1.87% day^?1 specific growth rate). The 45% replacement diet had the lowest growth (P < 0.05). Fish with externally visible deformities ranged from 0% to 1.5%. The relation among amino acid profiles of the diets, body composition, growth performance of juveniles and amino acid requirements of other fish species is discussed. An amount of 290.4 g PPC kg^?1 diet (35% replacement of FM protein) can be included in juvenile tench diets without impairing growth performance.     

Evaluation of poultry by‐product meal as partial replacement of fish meal in practical diets for juvenile tench (Tinca tinca L.)

The aim of this study was to evaluate different replacement levels of fish meal (FM) by poultry by‐product meal (PBM) on survival, growth performance and body composition of juvenile tench (Tinca tinca). A 90‐day experiment was conducted with 5 month‐old juveniles (31.95 mm total length, 0.396 g weight). Eight practical diets (50% crude protein) differing in the level of replacement of FM protein by PMB protein were tested: 0% (control), 25%, 31%, 37%, 43%, 49%, 55% and 61% corresponding to 0, 184.8, 229.2, 273.5, 317.8, 362.1, 406.5 or 450.8 g PBM kg^?1 diet respectively. Significant differences were not found (P > 0.05) between 25% replacement of FM protein by PBM protein (184.8 g kg^?1 PBM in diet) and control diet. At higher replacement levels, fish had significantly lower growth, higher feed conversion ratio and lower protein productive value (P < 0.05). Fish with externally visible deformities ranged from 1.1% to 3.3%. The relation among amino acid profiles of the diets, body composition, growth performance of juveniles and amino acid requirements of other fish species is discussed. Up to 184.8 g PBM kg^?1 diet can be included in diets for juvenile tench without impairing growth performance.     

Response of juvenile tench (Tinca tinca L.) fed practical diets with different protein contents and substitution levels of fish meal by soybean meal

A basal practical diet for juvenile tench (Tinca tinca) was formulated and elaborated to test several protein contents and substitution possibilities of fish meal (FM) by soybean meal (SBM) in a 90‐day trial with 5‐month‐old juveniles (30.54 mm TL, 0.30 g W). A factorial design included nine feeding treatments: three protein contents (50%, 40% or 30%) and three levels of replacement (0%, 25% or 45%) of FM protein by SBM protein. In addition, a commercial carp feed was used as reference. Final survival ranged from 98.2% to 99.4%. The 50% dietary protein with 0% or 25% replacement and 40% dietary protein with 25% replacement diets enabled higher growth (P < 0.05) and lower FCR (P < 0.05) than the rest of practical diets. Fish fed 50% dietary protein had similar growth than those fed carp feed (63.8% protein). Deformed fish averaged 1% for the practical diets and 87.6% for the carp feed. The basal practical diet has showed to be feasible and levels of 40–50% dietary protein with 25% replacement of FM protein by SBM protein can be recommended for juvenile tench aged 5–8 months.     

Induced meiotic gynogenesis in tench, Tinca tinca (L.) using irradiated heterogenic sperm

Effects of the starting time and duration of cold shock, as well as the source of heterogenic sperm on the percentage of viable gynogenic larvae (PVGL) in tench were studied. The DNA in sperm of red common carp (Cyprinus carpio var singuonensis) was inactivated by ultraviolet radiation prior to use to induce meiotic gynogenetic development in tench. In experiment 1, tench eggs were cold-shocked for 30 min starting at 1, 3, 5, 7, 10 and 15 min post activation. In experiment 2, cold shock began 5 min after activation and lasted for 10, 20, 30, 40, 50 min, respectively. Each experiment was run in triplicate using 3 tench females, and one group not treated with cold shock was included in each experiment to serve as a control group. In experiment 3, sperm of bigmouth buffalo (Ictiobus cyprinellus), red common carp or grass carp (Ctenopharyngodon idella) was used to induce gynogenesis in tench with a 20-min cold shock starting 5 min post activation. The results showed that PVGL from the control group was very low (0.11–0.47%). In experiment 1, the highest average PVGL (9.60%) was observed when cold shock treatment was applied 5 min post activation. When cold shock treatment was started 5 min post activation, duration of cold shock affected PVGL. Cold shock lasting 20 min resulted in the highest average PVGL (12. 57%) among the selected duration of cold shock studied in experiment 2. The average PVGL was 2.3, 8.6, and 9.3%, respectively, for eggs induced by sperm of bigmouth buffalo, red common carp and grass carp. Average PVGL was significantly lower for eggs induced by sperm of bigmouth buffalo, compared with that for eggs induced by sperm of the other two species. However, average PVGL were similar for eggs induced by sperm of red common carp and grass carp. In summary, the optimal conditions for gynogenesis in tench include the use of irradiated sperm of grass carp to activate the eggs and cold shock of 20 min starting 5 min post activation. Since female tench grow much faster to a larger size than male tench, gynogenesis of tench holds great potential for production enhancement.     

Combined effects of water temperature and daily food availability period on the growth and survival of tench (Tinca tinca) larvae

A 16‐day experiment was designed to find the best combination of water temperature (27, 30, 33°C) and daily duration of food availability (12, 18, 24 h) for larval tench (Tinca tinca) growth and survival. Larvae with an initial mean size of 5.7 mm total length (TL) and 0.7 mg wet body weight (BW) were stocked at 15 L^?1. Larvae were fed in excess with live Artemia nauplii with the period of food availability lasting 12, 18 or 24 h daily. The largest final larval size was recorded at 27 and 30°C in groups fed for 24 h a day (17.7 and 17.9 mm TL, 76.1 and 77.7 mg BW, respectively). The combination of the highest temperature and the longest daily food availability was the only set of conditions under which final larval survival was affected (95.4% survival; 98.7–99.9% under all other conditions). The combination of water temperature of about 28.6°C and continuous food availability is recommended as the optimum combination for rearing T. tinca larvae under controlled conditions. Providing continuous food supply to fish larvae under aquaculture conditions was also advantageous in helping to mitigate the effects of slower growth relative to developmental progress, which can occur at high water temperatures. However, should one wish to limit the daily feeding period to 12 h per day, the use of a water temperature between 27.4 and 27.9°C would be the best solution.     

Egg oxidation status,antioxidant enzyme activities,lipid classes,fatty acid composition profile and embryo survival rates during in vitro oocyte ageing in tench Tinca tinca (Linnaeus, 1758)

The effect of in vitro storage of oocytes on embryos survival rate, egg oxidation status, antioxidant enzyme activities, lipid classes and fatty acid composition profile was investigated in common tench Tinca tinca. In order to identify the role of oxidative stress in the progress of oocyte ageing the levels of thiobarbituric acid reactive substances (TBARS) and carbonyls as indicators of lipid and protein oxidation were measured and the activity of antioxidant enzymes were examined. Stripped ova from six females were stored in cell culture plates at 20°C for up to 10 hr post‐stripping (HPS). The stored ova were fertilized and the embryo survival rates were assessed. The results indicated that tench eggs could be successfully stored in vitro for 4 hr after stripping at 20°C. Superoxide dismutase enzyme activity increased at 6 HPS, whereas glutathione peroxidase and glutathione reductase activities decreased in oocytes during in vitro storage (p < .05) at 4 and 8 HPSs, respectively. The level of malondialdehyde did not show any significant changes during the progress of oocyte ageing. Carbonyls increased up to 2 HPS and thereafter decreased significantly. However, ova ageing did not affect the main lipid class composition and the fatty acid composition of the eggs. Lower quality eggs exhibited lower levels of cholesterol but higher levels of triacylglycerol.     

Studies on sperm of diploid and triploid tench, Tinca tinca (L.)

The tench Tinca tinca is an interesting fish from the viewpoint of polyploidy and related atypical reproduction aspects. Triploid tench were produced artificially. Studies of spermiation as well as of sperm motility and structure were performed on several triploid and diploid males simultaneously with individual experimental crosses with diploid females to define their reproductive capacities. The testes of triploids visually looked less developed in the most of cases with lower sperm production (0.05 cm³ sperm per male), GSI and weight of testes compared to diploids (0.58 cm³ sperm per male). Analysis of variance showed significant influence of ploidy level on the percentage of motile spermatozoa. Triploidy did not change percentage of live spermatozoa and velocity of spermatozoa at the first time of sperm movement. The study of sperm structure by scanning electron microscopy revealed that most sperm cells were of normal structure with some anomalies. Sperm heads of triploid and diploid males were mostly round-shaped, 1.86±0.2 and 1.6±0.18 μm in diameter. The midpiece of triploid spermatozoa was slightly narrower than that of diploid ones with typical cylindrical shape. Flow cytometry revealed sperm cells of triploids to be largely aneuploid (1.47 n) with high mosaic DNA, oscillating from haploid DNA content (1.0 n) to diploid DNA content (1.9 n). Experimental crosses between triploid males and diploid females revealed that these males were capable to stimulate effective development with relatively high level of fertilization and hatching rates from 0 to 70%. In conclusion, triploidization does not seem to guarantee sterility of tench.     

Decapsulated Artemia cysts of different quality (high or low hatch‐rate) as direct food for tench (Tinca tinca L.) larvae

Three 30‐day experiments were conducted to evaluate decapsulated Artemia cysts with different quality (high or low hatch‐rate) as food for tench (Tinca tinca L.) larvae from the onset of exogenous feeding. In experiment 1, three diets were tested: Artemia nauplii‐only or cysts‐only for 30 days, and nauplii for the first 7 days and cysts thereafter. The cysts used had 86% hatching rate (high hatch‐rate cysts). The same feeding treatments were replicated in experiment 2 but with low hatch‐rate cysts (10% hatching rate). In experiment 3, five diets were tested: high hatch‐rate cysts only or low hatch‐rate cysts only for 30 days, and nauplii for the first 7, 4 or 2 days and low hatch‐rate cysts thereafter. In overall, survival was high, except with the low hatch‐rate cysts only diet. Feeding tench larvae with cysts resulted in higher growth and lower FCR compared to feeding with live nauplii only. High hatch‐rate Artemia cysts are a suitable food from the onset of exogenous feeding and low hatch‐rate cysts can be successfully used after 2–7 days feeding on nauplii.     

Efficiency of capture of tench, Tinca tinca L. by trammel nets of monofilament and multifilament net twine combinations

Efficiencies of different trammel net types on capture of tench, Tinca tinca L. were investigated in Lake Beyşehir in Turkey between April 1998 and August 1999. Nets with an inner wall of monofilament and outer walls of multifilament (type A) were more efficient than the other net types (type B, monofilament inner and outer walls; type C, multifilament inner wall and monofilament outer walls; and D, multifilament inner and outer walls). Type A nets were 2.08, 1.79 and 1.08 times more effective than the types D, C and B for catching tench. Monofilament inner walls of trammel nets were on average 1.85 times more effective than multifilament inner walls. No significant difference in catching efficiency of monofilament and multifilament outer walls was found ( P >0.05). The average size of tench caught in monofilament trammel nets was higher than those caught in multifilament trammel nets, although not significantly different.     

Analysis of growth, weight and relevant indices of diploid and triploid population of tench Tinca tinca (Linnaeus 1758)

This study determined biometric and weight parameters and relevant indices of diploid and triploid tench. Altogether, 137 siblings of tench were studied. The effect of ploidy level appeared in significantly better growth of triploids (P<0.001) as to biometric [total length (TL), standard length (SL), body height (BH), body width (BW)] and weight [fish weight (FW), carcass weight (CW)] parameters of T₃ of both sexes and of T₃₊ females. The effect of ploidy level also appeared as significantly higher dressing percentage (DP; P<0.001) of triploid T₃ females compared with other groups, significantly higher gonad weight (GW) and gonadosomatic index (GSI; P<0.001) of diploid T₃ females, as well as GSI and hepatosomatic index of diploid T₃₊ females. The effect of sex appeared in significantly higher (P<0.001) biometric (TL, SL, BH, BW) and weight (FW, CW) parameters of T₃ females of both ploidy levels, as well as of triploid T₃₊ females. The effect of sex also appeared as significantly higher DP (P<0.001) of males in diploid T₃ fish, as well as of males of both ploidy levels in T₃₊ fish and significantly higher GW and GSI (P<0.001) of females in diploid T₃ fish, as well as of females of both ploidy levels in T₃₊ fish. This study shows evidence for faster somatic growth and bigger final weight of triploid populations of tench compared with diploids in both age categories T₃ and T₃₊.     

Quantity, motility and fertility of tench Tinca tinca (L.) sperm in relation to LHRH analogue and carp pituitary treatments

The spermiation of tench males was stimulated with Supergestran containing mammalian LHRHa lecireline at the following doses: 5, 10, 20 and 40 g kg⁻¹ b.w.; then with carp pituitary suspension (CPS) at a dose of 2 mg kg⁻¹ b.w. and with a control of saline physiological solution. The following days, meaning 24, 48 and 72 h after injection, sperm was collected to evaluate volume and the number of sperm per male per kg body weight (B.W.) The percentage of motile sperm and velocity of spermatozoa were measured 48 h after hormonal injection, and 72 h after hormonal injection the sperm was evaluated for fertilization and hatching ability. All 42 males in experimental groups were diploid. Live weight did not differ significantly among experimental groups. The strongest stimulation of spermiation was achieved with LHRHa in dosage of 20 and 40 g kg⁻¹ b.w. and CPS compared to males of the control group and lower dosage of LHRHa. Analysis of variance showed no significant influence of the treatment on the velocity and percentage of motile spermatozoa. The effect of different treatment on the fertilization capacity (the number of spermatozoa per egg was equilibrated) was significant. Significantly the highest quality of sperm collected 72 h after injection expressed by percentage of fertilization and hatching (62–65% fertilization and 61–64% hatching rates, respectively) was found for LHRHa in dosage of 20 and 40 g kg⁻¹ b.w. Significantly the lowest parameters of fertilization and hatching were found for the control group, on the 12% level.     

Effect of feeding various levels of poultry by‐product meal on the blood parameters,filet composition and structure of female tenches (Tinca tinca)

Homogeneous background (age, sex, genetic lineage, culture conditions) was created to clearly demonstrate the impact of the tested dietary treatments. No feeds optimized for the rearing of the tench (Tinca tinca L. 1758) are available. Feeds are formulated to increase the growth rate or eliminate skeletal deformations. With the increasing prices of the basic components, fish meal (FM) and fish oil, poultry by‐product meal (PBM) can be used. The aim of the study was to evaluate the effects of substitution of FM with PBM on the tench blood parameters, body composition and structure of skeletal muscles. Cage‐reared female tenches (325 ± 18 g) were fed for 86 days with five types of feeds with 0% (control), 25.7%, 48.6%, 71.4% and 100% substitution of FM with PBM. No significant differences between the formulations were reported for weight, total length, fillet weight, visceral, liver, gonado‐somatic, proximate composition and biochemical blood parameters. However, significant differences were found in the fillet profiles of fatty acids—an increase in the PBM content correlated with an increase in saturated and monounsaturated fatty acids and a decrease in n‐3 PUFA and, generally, n‐6 PUFA. Significant differences were also observed in, for example, the content of intramuscular fatty tissue and the level of organ lipidosis between the control variant (0% PBM) and that with 100% substitution. A sensory assessment indicated a higher gustatory value of the fillets in the case of feeds with 48.6% and 71.4% substitution.     

Production, release and olfactory detection of sex steroids by the tench (Tinca tinca L.)

The present study is concerned with pheromone communication in tench (Tinca tinca L.), establishing firstly whether males have a high olfactory sensitivity to some typical teleost sex steroids and prostaglandins; and secondly whether males and females might be able to synthesise and release some of these steroids into the water. The C₂₁ steroid, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was found to give large electro-olfactogram responses with an estimated threshold of detection of 10⁻¹² M. The male tench were equally sensitive to glucuronidated 17,20β-P (10^−11.6 M) but 100 times less sensitive to sulphated 17,20β-P (11^−9.7 M). Preliminary data from cross-adaptation studies suggest that both the free and conjugated forms are detected by the same olfactory receptor(s). Male tench also had high olfactory sensitivity to prostaglandin F_2α (PGF_2α) and 15-keto PGF_2α (11^−11.5 and 10^−11.4 M). They were relatively insensitive, however, to testosterone (T), androstenedione (AD), 11-ketotestosterone (11-KT), 17β-oestradiol (E₂), 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) and 17,20α-dihydroxy-4-pregnen-3-one (17,20α-P). Radioimmunoassays were used to measure the steroids in plasma and water and all samples were processed for the measurement of free, sulphated and glucuronidated fractions. In females, free 17,20β-P, 17,20α-P, free and glucuronidated T, and AD in plasma showed the largest increases in response to injection with mammalian gonadotropin-releasing hormone analogue (GnRHa) or Ovaprim (a mixture of GnRHa and a dopamine inhibitor). Free 17,20β-P was released into the water at the greatest rate. Plasma concentrations of the two conjugated forms of 17,20β-P were also elevated 18 h after the administration of GnRHa, but not by as much as the free steroid. In males, AD and 11-KT showed the greatest increase in response to GnRHa and were moreover released into the water at a higher rate in the treated group than in the control. The data support a possible pheromonal role for free and glucuronidated 17,20β-P. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sperm cryopreservation of silver barb (Barbodes gonionotus): cryoprotectants,cooling rate and storage time on sperm quality

Effectiveness and efficiency of frozen sperm on fertilization and hatching success of eggs from silver barb was examined in relation to cryoprotectants, freezing rate and storage period. Sperm was diluted in calcium‐free Hank's balanced salt solution, equilibrated with dimethylsulphoxide (DMSO), propylene glycol, sucrose or methanol at 5%, 10%, 15% or 20% final concentrations, and frozen in 250‐μL straws using a one‐step freezing procedure (1, 5 and 8°C min^?1 from 25 to ?40°C). Highest post‐thaw sperm motility was found from a treatment using 10% DMSO and 5°C min^?1 (82.2 ± 2.1%), similar to that of 10% DMSO and 8°C min^?1 (87.8 ± 3.2%). Post‐thaw motility of sperm frozen at 5 or 8°C min^?1 was significantly higher than 1°C min^?1. Relative sperm motility declined significantly after 10 months of cryostorage while viability did not change during a 12‐month cryostorage. Average fertilization rates of sperm after 1 and 4 months of storage were 64.5 ± 4.6% and 61.3 ± 3.4%, respectively, similar to those of fresh sperm (69.6–72.3%). Hatching rates of cryopreserved sperm (45.4–51.2%) were similar to those of fresh sperm (51.8–57.8%). This study developed suitable methods for cryopreservation of silver barb sperm that can be used to facilitate hatchery operation.     

Seasonal changes in plasma gonadal steroid concentrations and gonadal morphology of male and female tench (Tinca tinca, L.)

In order to gain a better understanding of the reproductive cycles of male and female tench (Tinca tinca), gonadosomatic index, gonad histology and plasma concentrations of estradiol‐17β (E₂), testosterone, an drostenedione, 11‐ketotestosterone (11‐KT), 17,20β, 21‐trihydroxy‐4‐pregnen‐3‐one (17,20β,21‐P), 17,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) and 17,20α‐dihydroxy‐4‐pregnen‐3‐one (17,20α‐P) were measured at the four seasons of the year, plus a further sampling coincident with the peak of spawning in early July. As expected, in both males and females, the plasma concentrations of androgens (excluding 11‐KT in females – undetectable) and C₂₁ steroids were significantly more elevated in the spring and summer (when most gonadal development took place) than in the autumn and winter. The only unexpected finding was that 17,20β‐P and 17,20β,21‐P, the steroids that are normally associated with oocyte final maturation in females and spermiation in males, were found in substantial amounts in both pre‐vitellogenic, pre‐spermatogenic and post‐spawning fish. This suggests that these steroids may have other as yet unidentified roles in this species.     

Effects of four egg desticking procedures on hatching rate and further survival and growth of larvae in the tench (Tinca tinca L.)

Four desticking procedures for tench eggs (A: tannic acid solution (1 g L⁻¹) for 15 s; B: alcalase enzyme solution (8 mL L⁻¹) for 60 s; C: alcalase enzyme solution (15 mL L⁻¹) for 120 s; D: Woynarovich and Woynarovich (1980) solution for 58 min followed by tannic acid solution (1 g L⁻¹) for 15 s) were tested to obtain data about influence on embryo survival to hatching stage and further survival and growth of the larvae. In the tannic acid and Woynarovich and Woynarovich (1980) treatment (A and D) few eggs stuck together and some were adhered to the incubator walls, whereas in the alcalase treatments (B and C) eggs neither stuck together nor adhered to the incubator walls. Percentages of hatched larvae did not show significant differences (mean values ranged between 47.4% in treatment A to 37.0% in treatment C). Larvae deformities observed were <0.5% in all cases. There were no significant differences among survival and growth rates of the larvae from different egg desticking origin, reaching, after 30 days, mean survival values around 90% and total length and weight of 12.5 mm and 19 mg respectively.     

Short-term storage and cryopreservation of Urechis unicinctus (Echiura: Urechidae) sperm

In the present study, attempts were made to preserve Urechis unicinctus sperm at 4°C. Cryopreservation procedures were optimized for various cryoprotectants and freezing rates, equilibration times and dilution ratios. During short‐term storage, the motility of undiluted sperm was extended for 6 days of cold storage,and in 70% and 100% artificial seawater only persisted for 2 and 4 days respectively. The survival rate of undiluted sperm was maintained at a high level accordingly. After cryopreservation, the highest motility and survival rate (41.5±2.2%) were obtained in 15% dimethyl sulphoxide (Me₂SO) using a freezing rate of 30°C min^?1. After thawing the sperm cryopreserved in glycerol lost almost all motility. The motility and survival rate of post‐thawing sperm did not show significant differences after 8 and 15 min equilibration using 15% Me₂SO as cryoprotectant; the values were significantly higher than those of 2 min equilibration. Comparisons of motility and survival rate between treatments pooled by dilution ratio showed that the effect of 1:1 ratio (sperm volume to cryoprotectant volume) was best. There was no difference between 1:3 and 1:5, and other ratioswere significantly worse.     

Reciprocal hybrids of tench Tinca tinca (L.) × bream Abramis brama (L.), and tench × carp Cyprinus carpio L., and some characteristics of their early development

Reciprocal hybrids between tench Tinca tinca (L.) and carp Cyprinus carpio L., and tench and bream Abramis brama (L.) were produced artificially. The survival of all these hybrids during embryogenesis was quite high. The highest survival rate (over 60%) at the eyed stage was observed for tench and bream hybrids (both sex combinations). The hatching rates of these hybrids were also over 60%. The number of larvae with some abnormalities (i.e. deformed body) was low. In contrast, the hatching rates of tench and carp hybrids were very low (0.2%). From over 1000 fertilized eggs, only three specimens started swimming, and only one specimen survived to juvenile stage. Embryos of hybrids and their parental species differ in morphological features. These differences were also visible in the juveniles. Body parameters of juvenile hybrids produced from three species had intermediate values in comparison to parental fish.     

Cryopreservation of summer flounder,Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high‐value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose‐based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (?5, ?10 and ?15°C min^?1) were evaluated using differential staining to assess post‐thaw sperm survival. Seven combinations of the factors examined reduced post‐thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 ± 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at ?5°C min^?1 (38.4 ± 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.