Cryopreservation of yamú (Brycon amazonicus) sperm for large scale fertilization

Aquaculture 256 (2006) 264 – 271 www.elsevier.com/locate/aqua-online

Cryopreservation of yamú (Brycon amazonicus) sperm for large scale fertilization Yohana M. Velasco-Santamaría, Víctor M. Medina-Robles, Pablo E. Cruz-Casallas ⁎ Instituto de Acuicultura, Universidad de los Llanos, Apartado Aéreo 110, km 4 via Puerto López, Villavicencio, Meta, Colombia Received 18 January 2005; received in revised form 26 January 2006; accepted 10 February 2006

Abstract To determine the effect of straw size and thawing temperature on cryopreserved sperm quality of yamú (Brycon amazonicus), ovulation and spermiation were induced in sexually mature broodstock using Carp Pituitary Extract. Sperm quality was evaluated by motility, activation time and fertility. Sperm was diluted (1:4) in a solution of glucose, egg yolk and dimethyl sulfoxide (DMSO). Sperm concentration was determined using a Neubauer chamber, and motility evaluated after activation with 1% NaHCO3. In the laboratory, four sizes of straw (0.5, 1.8, 2.5 and 4.0 mL) and two thawing temperatures (35 °C or 80 °C water bath) were evaluated. To assess fertility, 2 g of eggs (ca. 2800) were inseminated with 500 μL of frozen-thawed sperm (ca. 75,000 motile spermatozoa/egg) from each straw thawed at 35 °C or 80 °C, or 160 μL (ca. 50,000 motile spermatozoa/egg) of fresh sperm. Large scale fertility assays consisted of 40 g eggs inseminated with approximately 5.0 mL (ca. 75,000 motile spermatozoa/egg) of cryopreserved sperm in large straws thawed at 35 °C. The fertilization rate was estimated 6 h post-insemination. In all straws, postthaw motility was significantly lower than for fresh sperm (p b 0.05). In laboratory trials, fertility of fresh sperm was higher (67 ± 4%) than frozen-thawed sperm (p b 0.05). For all types of straw, semen thawed at 35 °C had a higher percentage of fertility (p b 0.05) than semen thawed at 80 °C; sperm cryopreserved in 1.8-, 2.5- and 4.0-mL straws had similar fertility percentages (p N 0.05) to sperm frozen in 0.5-mL straws (48 ± 2%, 51 ± 2%, 52 ± 2% and 54 ± 3%, respectively). In large scale fertilization trials, fresh sperm showed a higher (p b 0.05) fertilization rate (83 ± 1%) than frozen-thawed sperm (68 ± 1%). Although the fertility percentage with fresh sperm was significantly higher than with frozen-thawed sperm in large straws, the fertilization rate of the latter is considered acceptable and profitable in a commercial setting. © 2006 Elsevier B.V. All rights reserved. Keywords: Brycon amazonicus; Cryopreservation; Fertility; Large straws; Sperm; Yamú

1. Introduction In Colombia, sperm cryopreservation is a relatively new field of investigation, in which little practical ⁎ Corresponding author. Tel.: +57 98 6698701; fax: +57 98 6698700. E-mail addresses: [email protected] (Y.M. Velasco-Santamaría), [email protected] (P.E. Cruz-Casallas). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.02.039

progress has been achieved on a commercial scale. To date, no national hatchery has used cryopreserved sperm in its fertilization process and fish farms always use broodstock kept in captivity, or usually wild adults fished from the environment. Yamú (Brycon amazonicus—Spix and Agassiz 1829) is a native freshwater fish of the Orinoco River. It is an omnivorous fast-growing fish with excellent meat quality that has become a species of great commercial importance for South American pisciculture. The reproduction is seasonal in

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this characid Brycon (February to May) (Arias, 2001); therefore, it is necessary to implement procedures to improve artificial reproduction. Cruz-Casallas et al. (2005) described the sperm characteristics and spermatozoa morphology of yamú without hormonal induction: volume 1.8 ± 1.2 mL, sperm concentration 13.9 ± 4.0 × 109 spermatozoa/mL, sperm motility 88 ± 9% and fertilization rate 84 ± 8%, and they concluded that this species is able to fertilize a considerable amount of eggs successfully. Cruz-Casallas et al. (2004) also reported that a simple medium consisting of 5.5% glucose, 12% egg yolk, and 10% DMSO as a cryoprotectant was effective for the cryopreservation of yamú spermatozoa, with fertilization rates near to 60%. In spite of these satisfactory results, further research is necessary to improve the effectiveness of yamú spermatozoa cryopreservation on large scale. Protocols at laboratory scale for sperm cryopreservation of Brazilian migratory characids using only 0.5-mL French straws have been carried out by Carolsfeld et al. (2003); although they reported satisfactory fertilization rates for some of these species, the fertility of cryopreserved Brycon orbignyanus spermatozoa was not reported. During the last years, laboratory protocols for cryopreserved fish sperm have been standardized for some species, such as rainbow trout (Oncorhynchus mykiss) (Wheeler and Thorgaard, 1991), striped trumpeter (Latris lineate) (Ritar, 1999), yellowtail flounder (Pleuronectes ferrugineus) (Richardson et al., 1999), seabream (Sparus aurata) (Fabbrocini et al., 2000), cyprinid fish (Lahnsteiner et al., 2000), common carp (Cyprinus carpio) (Linhart et al., 2000; Horváth et al., 2003), catfish (Bart et al., 1998; Viveiros et al., 2000), characid Brazilian fish (Carolsfeld et al., 2003), yamú (B. amazonicus) (CruzCasallas et al., 2004) and cachama blanca (Piaractus brachypomus) (Navarro et al., 2004). However, there are few reports about the efficiency of cryopreserved sperm in large straws on a commercial scale. Some of these studies have been carried out in species such as rainbow trout (O. mykiss) (Wheeler and Thorgaard, 1991; Cabrita et al., 2001; Lahnsteiner et al., 2002), yellowtail flounder (P. ferrugineus) (Richardson et al., 1999), ocean pout (Macrozoarces americanus L.) (Yao et al., 2000) and cyprinid fish (Lahnsteiner et al., 2003). Semen cryopreserved in small straws (0.25 or 0.5 mL) has been successfully used in several fish species to fertilize small egg batches (Lahnsteiner et al., 1995, 1996; Carolsfeld et al., 2003; CruzCasallas et al., 2004). This packaging is useful for laboratory purposes such as gene banking, or for

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commercial use in species in which small quantities of sperm are required. However, its application on a commercial scale requires a large number of straws because it is necessary to fertilize simultaneously large numbers of eggs. For this reason, sperm cryopreservation using large straws (1.8 to 5.0 mL) could reduce the time required for semen packaging and thawing, facilitating sperm handling during the fertilization process (Cabrita et al., 2001). Several authors have reported low fertility rates in eggs inseminated with cryopreserved sperm when large straws were used (Wheeler and Thorgaard, 1991; Yao et al., 2000). However, other studies reported fertilization rates using cryopreserved sperm in straws with volume greater than 1.2 mL, similar to the percentage obtained with fresh sperm (Richardson et al., 1999; Cabrita et al., 2001; Lahnsteiner et al., 2002). Therefore, the aim of this study was to evaluate the effect of straw size and thawing conditions of cryopreserved yamú (B. amazonicus) sperm on motility, activation time and fertilization rate of the sperm, and the application of these conditions on a commercial scale. 2. Materials and Methods 2.1. Fish Sexually mature female and male yamú reared and maintained in the hatchery at the Instituto de Acuicultura de los Llanos of the Universidad de los Llanos were used. The female and male fish weighed 2.3 ± 0.2 kg and 2.0 ± 0.1 kg and had a total length of 51.4 ± 1.7 cm and 49.5 ± 0.8 cm, respectively. The experiments were carried out during the breeding season from March to June in 2004 and 2005, at a mean temperature of 26 °C. Final maturation and ovulation in the females was induced by three injections into the dorsal musculature (IM) of a total of 5.75 mg/kg of Carp Pituitary Extract (CPE-Stoller Fisheries, USA) administered at 0, 24, and 36 h, and spermiation was induced with one injection of 4.0 mg/kg of CPE administered at 24 h (at the time of the second injection of the female). Before the collection of gametes, the fish were anaesthetized by immersion in 2phenoxyethanol (300 ppm, Sigma Chemical Co., St. Louis, Missouri, USA). The gametes were obtained after wiping the fish dry and stripping them by anteriorposterior abdominal massage, from 6 to 7 h and from 18 to 20 h after the last injection for females and males, respectively. The bladders were cleared of urine by abdominal pressure and special care was taken to avoid sperm contamination with water, blood, urine, bile or faeces.

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2.2. Gamete handling Semen was collected individually in sterile graduated glass tubes and eggs were collected in dry plastic containers. Semen was covered and maintained at a controlled temperature (25 ± 2 °C). Motility was estimated as the percentage of motile spermatozoa observed under a light microscope (10 ×), and activation time (expressed in seconds) was measured immediately after sperm activation, and until the sperm movements stopped. These analyses were carried out by placing a drop of semen on an excavated glass microscope slide (1.0 to 1.2 mm depth, Premiere, China) and activating with distilled water at a ratio of 1:10. Samples with less than 80% motile spermatozoa were discarded. Sperm concentration was determined in a Neubauer Improved counting chamber, using a dilution of 1:1200 with physiological saline solution (0.9% NaCl). Before spermatozoa counting, the Neubauer chamber was kept in a humid atmosphere for at least 10 min approximately. The numbers of eggs per gram were determined by direct counting. The eggs were recovered from individual females whereas semen samples were pooled (2 to 4 males). 2.3. Sperm cryopreservation and freezing and thawing conditions Suitable semen for cryopreservation was diluted at a ratio of 1:4 in a solution of glucose (5.5% w/v) as external cryoprotectant (Merck, Darmstadt, Germany), egg yolk (12% v/v) as membrane stabilizer, and dimethyl sulfoxide (DMSO) (10% v/v) (Merck Schuchardt, Hohenbrunn, Germany) as internal cryoprotectant, as a described by Cruz-Casallas et al. (2004). In all assays, the spermatozoas were immotile after dilution with extenders. Thereafter, diluted sperm was aspirated with precision micropipettes (Brand, GmbH and Co., Wertheim, Germany) and packaged in 1.8-, 2.5- or 4.0-mL macrotubes (120 × 5 mm, 140 × 5 mm or 240 × 5 mm, respectively, Minitub, Abfül-und Labortechnik GmbH and Co., Germany). The 0.5-mL French straws (130 × 3 mm, Instruments de Médecine Vétérinaire, Minneapolis, USA) were filled through manual aspiration. The open end of 0.5mL French straws and one end of each macrotube were sealed with polyvinyl powder and the other end of each macrotube was sealed with cotton and polyvinyl powder. The straws were placed vertically in a custom-made support and frozen in nitrogen vapor in a dry shipper (CP100, Taylor Wharton, Theodore, AL, USA) for 30 min.

To determine the freezing rate, the temperature inside the filled straws was measured with a thermocouple probe (precision 0.01 °C Thermometer; WBrand, USA) and the temperature was recorded every 5 s for the first 60 s, every 10 s until 300 s, and finally every 15 s until 30 min. The thawing temperature was registered every 5 s. The thermocouple probe was kept inside the frozen straws until thawing, and then the thawing rates were recorded. Finally, the straws were transferred and submerged in liquid nitrogen storage containers at − 196 °C (35HC, Taylor Wharton, Theodore, AL, USA) for 15 to 30 days until evaluated. For these experiments we used three straws of each size and thawing temperature. Freezing rates were analyzed as gradients from 28 °C to − 20 °C, − 20 °C to − 40 °C, − 40 °C to − 100 °C and from − 100 °C to − 140 °C, and thawing rates were analyzed from − 196 °C to 0 °C and from 0 °C to 35 °C or 25 °C, for straws thawed at 35 °C or 80 °C, respectively. The overall freezing rate (°C/min) was determined from 28 °C to − 140 °C. 2.4. Experimental design 2.4.1. Experiment I—effect of thawing temperature To evaluate the effect of two thawing temperatures, straws of each size (0.5, 1.8, 2.5 and 4.0 mL) were thawed in a 35 °C or 80 °C water bath for 90 or 25 s, respectively, except for 0.5-mL straws which were thawed at 80 °C for only 10 s. At the end of the thawing period, the temperature inside each straw was about 35 °C and 25 °C, for straws thawed at 35 °C and 80 °C, respectively. To assess post-thaw semen quality, the motility, activation time and fertilization rate were evaluated. The motility was induced with 1% sodium hydrogen carbonate (NaHCO3) and tested visually using a scale from 0 to 100% motility, and activation time was registered in seconds (n = 13 to 20 straws per treatment). To evaluate the effect on fertility, the experiment consisted of a 2 × 4 factorial design (two thawing temperatures and four straw sizes) (n = 15 to 25 per treatment). Diluted sperm from each straw was emptied into a previously labeled glass tube. Two grams of eggs (ca. 2800) were inseminated with 500 μL of frozenthawed sperm (ca. 75,000 motile spermatozoa per egg) and 5 mL of 1% NaHCO3 was immediately added to activate the spermatozoa. After 1 min, the eggs were washed several times with tap water in order to hydrate the eggs and remove dead sperm. The fertilized eggs were incubated in vertical incubators (1.5 L conic plastic container) with a continuous flow of water at 26 ± 1 °C. The same amount of eggs were used as control and inseminated with 160 μL of fresh sperm (ca. 50,000

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40

0

1

2

3

4

-35

5

6

Time (min.)

-70 -105

Temperature (°C)

Temperature (°C)

35

267

0

10

20

30

40

50

60

70

-40

80

90

Time (s)

-80 -120 -160 -200

-140 0.5 mL

1.8 mL

2.5 mL

1.8 mL

0.5 mL

4.0 mL

2.5 mL

4.0 mL

Fig. 1. Freezing curves of extended yamú semen in 0.5-, 1.8-, 2.5- or 4.0-mL straws frozen in nitrogen vapor in a dry shipper for 30 or 6 min. The values correspond to the mean (n = 3).

Fig. 2. Thawing curves of cryopreserved yamú semen in 0.5-, 1.8-, 2.5or 4.0-mL straws thawed in a 35 °C water bath. The values correspond to the mean (n = 3).

motile spermatozoa per egg) and activated with 5 mL of 1% NaHCO3. The procedure after sperm activation was the same as described for frozen-thawed sperm. The fertilization rate was estimated in triplicate at 6 h postinsemination (ca. 100 eggs per sample), approximately 6 h before hatching, in a stage of blastopore closure. The fertility was determined as the percentage of viable embryos defined as perfectly spherical, translucent and with a normal embryonic development, as described by Cruz-Casallas et al. (2004, 2005).

all large scale fertilization experiments (2 to 3 replicates per experiment). Each experiment consisted of the insemination of 40 g of eggs (ca. 1400 eggs/g) with 5 mL of cryopreserved sperm from a pool of two 4.0 mL large straws thawed at 35 °C for 90 s. The motile spermatozoa/egg ratio was ca. 75,000:1. The control consisted of a similar number of eggs fertilized with fresh sperm obtained from mature males of each farm, adjusting the insemination ratio at 50,000 motile spermatozoa per egg. The inseminated eggs were maintained in commercial incubators with a vertical ascendant flow (Woynarovich or Nielsen) and fertilization rate was evaluated 6 h postinsemination.

Table 1 Freezing rates (°C/min) of extended yamú semen in 0.5-, 1.8-, 2.5- or 4.0-mL straws (n = 3) Range (°C)

Straw size (mL) 0.5

28 to −20 116.2 ± 0.15a − 20 to − 40 130.5 ± 25.2a − 40 to − 100 155.4 ± 22a − 100 to − 140 37.1 ± 2.8b Overall freezing rate 93.1 ± 25.7 ⁎ a,b

1.8

2.5

35.3 ± 1.2b 45.7 ± 5.1b 43.1 ± 5.3b 29.1 ± 3.8b 36.7 ± 3.3

35.2 ± 1.2b 30.5 ± 0.9b 46.3 ± 3.1b 33.9 ± 2b 43.5 ± 1.3b 31.3 ± 1.8b 28.6 ± 0b 22.6 ± 1.4b 36.7 ± 0.5 28.7 ± 1

4.0

Means with different superscripts are significantly different (p b 0.05). ⁎ Value different (p b 0.05) from the other overall freezing rates.

2.5. Statistical analysis All values are expressed as mean ± S.E.M. The activation time was analyzed by two-way analysis of variance (ANOVA) with subsequent Tukey–Kramer test.

Temperature (°C)

2.4.2. Experiment II—fertility assays with large straws on a large scale The experiments were carried out at four hatcheries in the Meta Department, Colombia. Sexually mature broodstock of yamú, reared and maintained in captivity, were used. Final maturation, ovulation and spermiation were induced with CPE as described above. Likewise, the procedures of gamete handling and evaluation were as described for Experiment I. Two experiments were performed per farm using two different semen pools for

25 0 -25 -50 -75 -100 -125 -150 -175 -200

5

10

15

20

25

Time (s)

0.5 mL

1.8 mL

2.5 mL

4.0 mL

Fig. 3. Thawing curves of cryopreserved yamú semen in 0.5-, 1.8-, 2.5or 4.0-mL straws thawed in a 80 °C water bath. The values correspond to the mean (n = 3).

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Table 2 Thawing rates (°C/min) of cryopreserved yamú semen in 0.5-, 1.8-, 2.5- or 4.0-mL straws, thawed in a 35 °C or 80 °C water bath (n = 3) Range (°C)

Straw size (mL) 35 °C

80 °C

0.5 − 196 to 0 0 to 351 − 196 to 351

1.8 b

2.5 c

824.0 152.9a,b,c 552.4b

4.0 d

593.0 35.3b,c 173.8c

336.4 23.9c 111.1c

0.5 d

1.8 a

388.8 20.0c 99.2c

2.5 b

1205.3 282.7a 910.0a

4.0 b

781.0 166.7a,b,c 509.7b

671.0b 218.0a,b 499.0b

681.0 169.5a,b,c 495.5b

1

For straws thawed at 80 °C, the thawing rate was analyzed until 25 °C. For the same range, means with different superscripts are significantly different (p b 0.05).

a,b,c,d

3. Results 3.1. Freezing and thawing rates In all straws, the freezing curve descended rapidly until − 140 °C. Semen in the 1.8-, 2.5- and 4.0-mL macrotubes had a similar freezing rate (p N 0.05) for each interval evaluated (Fig. 1, Table 1). The freezing rates until − 100 °C in 0.5-mL straws were significantly different (p b 0.05) compared with the other straws (Table 1). After this range, all straws had similar freezing rate (p N 0.05). The lower freezing rate was observed in 4.0-mL straws from − 100 °C to − 140 °C (Fig. 1). The overall freezing rate was higher (p b 0.05) in 0.5-mL straws than the other macrotubes (Table 1). Thawing curves for 35 °C and 80 °C in each straw are illustrated in Figs. 2 and 3. Thawing rates at 80 °C in all straws were faster than at 35 °C, with 0.5-mL straws

having the highest rate (p b 0.05). Until 0 °C, straws with the same size thawed at 35 °C had a different thawing rate compared with the same straws thawed at 80 °C (p b 0.05). In the range from 0 °C to 35 °C and from − 196 °C to 35 °C, all macrotubes thawed at 35 °C showed the slowest (p b 0.05) thawing rates (Table 2). 3.2. Experiment I—effect of thawing temperature Table 3 shows the post-thaw motility for each straw size after thawing at 35 °C or 80 °C. Thawing temperature did not affect the post-thaw sperm motility in most sizes of straw (p N 0.05). However, sperm from 0.5-mL straws thawed in a 80 °C water bath had lower percentage motility than sperm from all other treatments (p b 0.05). On the other hand, in semen of straws thawed at 35 °C or 80 °C significant differences in the activation time (p N 0.05), with a mean value of 52.2 ± 1.3 s (n = 92; date not shown) were not detected. Significant differences (p b 0.05) in the activation time between fresh sperm 80

a

70 60

Fertility (%)

The motility and fertilization rates were arcsine transformed and these values were analyzed by twoway analysis of variance (ANOVA) followed by Tukey– Kramer test. For all data, Brown and Forsythe's Test for homogeneity of variance was made. Differences among treatments were tested at a probability level of 0.05. For all statistical analysis, the SAS system for Windows software version 8.02 (1999–2001 by SAS Institute Inc., Cary, NC, USA) was used.

b

40

b

b

bc

50

d

d

cd

d

30 20

Table 3 Percentage of post-thaw motility of yamú sperm cryopreserved in 0.5-, 1.8-, 2.5- or 4.0-mL straws and thawed in a 35 °C or 80 °C water bath Thawing Straw size (mL) temperature (°C) 0.5 1.8 35 80

a

2.5 a

4.0 a

35 ± 2 (13) 39 ± 2 (20) 33 ± 2 (20) 39 ± 3a (20) 7 ± 2b (13) 38 ± 2a (19) 37 ± 2a (18) 35 ± 2a (19)

Values in parentheses correspond to number of evaluated straws. a,b Means with different superscripts are significantly different (p b 0.05).

10 0

0.5

1.8

2.5

4.0

Straw size (mL) 35 ºC

Fresh Sperm

80 °C

Fig. 4. Effect of thawing temperature on cryopreserved yamú sperm in 0.5-, 1.8-, 2.5- or 4.0-mL straws on fertility. The values correspond to the mean ± S.E.M. Bars with different letters indicate significant differences (p b 0.05). n = 15 to 25.

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100 a

a

b

80

a b

Fertility (%)

b 60 40 20 0

1

2

3

4

Fish farms Fresh sperm

Cryopreserved sperm

Fig. 5. Fertilization percentages with fresh and cryopreserved yamú sperm in 4.0-mL straws. The values correspond to the mean ± S.E.M. Bars from the same fish farm with different letters are significantly different (p b 0.05), n = 20. In the fourth farm, fresh semen was not available.

(38.6 ± 1.3 s) and frozen-thawed sperm were observed. In all cases, the motility of fresh sperm was N90%, being distilled water as efficient as NaHCO3 to activate fresh spermatozoa motility (unpublished results). The effects of straw size and thawing temperature on fertility are illustrated in Fig. 4. The fertility level with fresh sperm was significantly higher (67 ± 4%) than for frozenthawed sperm. The highest fertilization rate was obtained with 0.5-mL straws thawed at 35 °C (54 ± 3%); however, sperm frozen in 1.8-, 2.5- and 4.0-mL macrotubes thawed at 35 °C had high fertilization rates (48± 2%, 51± 2% and 52 ± 2%, respectively) without significant differences from sperm frozen in 0.5-mL straws. In general, the high thaw temperature (80 °C) significantly decreased the percentage fertilization of the eggs (p b 0.05). 3.3. Experiment II—fertility assays with large straws on a large scale Eggs inseminated with cryopreserved sperm in 4.0-mL macrotubes showed a fertilization rate of 67 ± 2%, 75 ± 2%, 71 ± 1% and 51 ± 2% in the first, second, third and fourth farm, respectively. Eggs inseminated with fresh sperm showed a higher (p b 0.05) percentage of fertilization (85 ± 2%, 86 ± 1% and 77± 2%) when compared with percentages of cryopreserved sperm (Fig. 5). In the fourth farm, mature males were not available to provide fresh semen for control. 4. Discussion The use of 0.5-mL straws for semen cryopreservation has been extensively used for experimental purposes;

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however, on a commercial scale, insemination of eggs with this volume makes the fertilization process extremely costly. For this reason, the use of large straws would facilitate the insemination procedure when it is necessary to fertilize large quantities of eggs. Additionally, the use of macrotubes in the cryopreservation of large sperm quantities has been useful when males are treated with hormones to increase the seminal volume (Richardson et al., 1999). Wheeler and Thorgaard (1991) used 4.5-mL straws for the cryopreservation of rainbow trout sperm in 5.4% glucose, 10% egg yolk and 9% DMSO, and thawed at 5 °C. They obtained average fertilization levels of 49% of the control. In contrast, the use of 1.7-mL straws with 10% propylene glycol in sperm cryopreservation of P. ferrugineus has shown fertilization percentages similar to the ones obtained in the present study with 1.8-, 2.5- and 4.0-mL straws (Richardson et al., 1999). Additionally, Fauvel et al. (1998) concluded that the use of 1.5-mL cryotubes in sperm cryopreservation of sea bass (Dicentrarchus labrax) could be used successfully as a sperm container for cryobank purposes. In the freezing curves of large straws, a small rise in temperature (freezing point plateau) was observed between − 10 °C and − 15 °C as a result of release of the latent heat of fusion. This phenomenon is known as supercooling point or temperature of crystallization, in which aqueous solutions remain in the liquid state when cooled below the freezing point (Zachariassen and Kristiansen, 2000). Watson and Martin (1974) reported that this interval of temperature is one of the most important in the cryopreservation process. There is a direct association between the duration of the freezing point plateau and the straw diameter on the viability of frozen-thawed spermatozoa (Bwanga et al., 1991). Several authors showed that reduction in the duration of this plateau improved the fertility of bull spermatozoa (Parkinson and Whitfield, 1987) and boar sperm (Bwanga et al., 1991). Fauvel et al. (1998) reported a longer period of crystallization in samples packaged in a large volume and concluded that for this reason the latency before cooling was longer. Although in the present study a long plateau was observed in large straws, the length of heat of fusion and the straw diameter (5 mm) apparently did not affect the fertilization rates in comparison with semen frozen in straws that had a smaller diameter (0.5 mL). The low fertility rates obtained with straws thawed at 80 °C is in agreement with results obtained by Lahnsteiner et al. (1997) and Cabrita et al. (2001), who concluded that high thaw temperatures can lead to denaturation of sperm enzymes in the peripheral area of the straws before the

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inside was thawed, as well as non-homogeneity in the thawing process and reduced viability. Additionally, Holt (2000) reported that increases in post-thaw temperature cause physical disruption of the plasma membrane due to subjecting it to structural rearrangements involving lipids and proteins. At this respect, Lahnsteiner et al. (1996) reported that the thawing curve is as important as freezing process; for this reason, both protocols must be standardized properly. Reported satisfactory freezing rates for cryopreserving semen vary considerably among species. They range from 1 to 30 °C/min (Conget et al., 1996) and from 20.8 to 63 °C/min (Cabrita et al., 2001) for rainbow trout, from 61 to 64.9 °C/min for sea bass (D. labrax) (Fauvel et al., 1998), ca. 30 °C/min in characidae Brazilian fish (Carolsfeld et al., 2003), and from 5.5 to 19.5 °C/min in yellowtail flounder (Richardson et al., 1999). In all cases, the freezing rate depended of the straw size of and the range of the freezing curve. In the present study, the mean of the overall freezing rate was 34.4 °C/min and of 93.1 °C/min for macrotubes and 0.5-mL straws, respectively. Post-thaw sperm motility is widely used for frozenthawed sperm evaluation and is a useful guide to estimate survival of spermatozoa (Holt, 2000); however, in the present study, there were no significant differences in the percentage motility of semen in all sizes of straw thawed at 35 °C and 80 °C (except 0.5-mL thawed at 80 °C). For this reason, the determination of fertilization rate is recommended to evaluate frozen-thawed sperm viability. We have shown here that cryopreservation of yamú sperm in 2.5- and 4.0-mL straws thawed at 35 °C was effective for artificial reproduction in yamú, obtaining results comparable to those obtained with straws of smaller volume (0.5 mL). Despite the fact that the fertilization percentages observed with fresh sperm were significantly higher than those of cryopreserved sperm, the percentage of fertilization reached using semen frozen in 2.5- and 4.0-mL straws is considered acceptable on a commercial scale. 5. Conclusion The process of artificial insemination in tropical native fish species demands the simultaneous fertilization of large batches of eggs. However, the reproductive seasonal behavior and the asynchrony of gonadal maturation between males and females in these species make the use of sperm cryopreservation necessary to solve this problem. The results obtained in this study suggest that 2.5- and 4.0-mL straws thawed at 35 °C are effective for fertilization processes of yamú (B. amazonicus) on a commercial scale.

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