Accepted Manuscript

Title: A simple, inexpensive and successful freezing method

for curimba Prochilodus lineatus (Characiformes) semen
Authors: A.T.M. Viveiros, L.H. Orfao, A.N. Maria, I.B.
Allaman
PII:
DOI:
Reference:

S0378-4320(08)00155-3
doi:10.1016/j.anireprosci.2008.04.025
ANIREP 3614

To appear in:

Animal Reproduction Science

Received date:
Revised date:
Accepted date:

23-11-2007
3-4-2008
28-4-2008

Please cite this article as: Viveiros, A.T.M., Orfao, L.H., Maria, A.N., Allaman,
I.B., A simple, inexpensive and successful freezing method for curimba
Prochilodus lineatus (Characiformes) semen, Animal Reproduction Science (2007),
doi:10.1016/j.anireprosci.2008.04.025
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Manuscript Revised Editor 19 04 2008

A simple, inexpensive and successful freezing method for curimba Prochilodus lineatus

(Characiformes) semen

3
A.T.M. Viveiros*, L.H. Orfo, A.N. Maria, I.B. Allaman

Dept Animal Sciences, Federal University of Lavras, P.O. box 3037,

Lavras, MG, 37200-000, Brazil

*

ana.viveiros@ufla.br

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Corresponding author. Tel.: 55 35 38291223; fax: 55 35 38291231; E-mail address:

Page 1 of 18

Abstract
The cryopreservation of fish sperm provides a tool by which reproduction is

11

optimized and thereby larval production is increased. The aims of this study were to

12

evaluate the effects of cryosolutions, motility-activation media, straw volumes and

13

thawing temperatures on the post-thaw motility of curimba semen. Furthermore, semen

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cryopreserved in a simple and inexpensive cryosolution and that yielded excellent post-

15

thaw motility was tested for fertility. Semen was diluted in each of the eight

16

cryosolutions in a factorial of two cryoprotectants (DMSO and methylglycol) x four

17

extenders (0.9% NaCl, 5% glucose, BTS and M III). Diluted semen was frozen in

18

0.5-mL straws in a nitrogen vapor vessel. Sperm motility was evaluated after thawing

19

(60 C water bath for 8 s) and activation with a total of four different activation media

20

(distilled water, 0.15% NaCl, 0.29% NaCl or 1% NaHCO3). To evaluate straw volume

21

and thawing temperature, semen was diluted in 5% glucose and methylglycol and

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frozen in 0.5-mL and 4.0-mL straws. Half of the 0.5-mL straws were thawed in a water

23

bath at 60 C for 8 s and the other half at 30 C for 16 s. The 4.0-mL straws were thawed

24

at 60 C for 24 s only. In the last experiment, semen cryopreserved in 5% glucose and

25

methylglycol, 0.5-mL straws, and thawed at 60 C for 8 s was tested for fertility. The

26

results of these comparisons are presented and show that curimba semen can be

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successfully cryopreserved in a simple glucose solution combined with methylglycol as

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cryoprotectant, in 0.5-mL straws, yielding motility rates between 86 and 95% and

29

fertilization rates between 47 and 83%.

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Keywords: fish, Prochilodus lineatus, semen, cryopreservation, fertilization, sperm

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motility

Page 2 of 18

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1. Introduction
Many Brazilian fish species migrate up-stream to find clean water and spawn.

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This process is known as piracema and lasts from October to February. Hydroelectric

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dams, over-fishing and changes in river courses can disrupt reproductive cycles and

37

species survival. The curimba Prochilodus lineatus is an exemplary migratory species

38

and is native to South American rivers. Because curimba larvae are used as live food for

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endangered carnivorous species such as piracanjuba Brycon orbignyanus and ja

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Zungaro jahu, in addition to serving as a human food source, curimba are of great

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importance to the aquaculture industry.

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The cryopreservation of fish semen provides a tool by which to reproduction is

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optimized thereby larval production is increased. In addition, cryopreserved semen

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serves as a genetic bank, which may help ensure genetic diversity and reproductive

45

success for population management strategies. About 170 fish species have been

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documented to reside in the Grande River and some of these species have undergone

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semen cryopreservation studies, including the curimba. Typically, curimba semen is

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frozen in 0.5-mL straws in a vapor nitrogen vessel, and transferred to liquid nitrogen for

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storage. Glucose combined with egg yolk has been used as a fish semen extender,

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yielding 80% post-thaw motility (Cruz, 2001), and 68 to 77% fertility (Carolsfeld et al.,

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2003). A commercial extender of boar semen, Beltsville Thawing Solution (BTS;

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Minitub) was used as a curimba semen extender and produced 80% post-thaw

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motility (Murgas et al., 2007), and 37% fertility (Miliorini, 2006). Dimethyl sulphoxide

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(DMSO) is a greater quality cryoprotectant compared to ethylene glycol and

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propanediol (Cruz, 2001) and methanol (Miliorini, 2006).

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Recently, a study on cryopreservation of piracanjuba semen described the effects

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of another boar semen extender, Merck III (M III; Minitub) combined with egg

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yolk and methylglycol. This combination resulted in 63% post-thaw motility, 70% live

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spermatozoa, and 40% fertility (Maria et al., 2006a). Neither M III nor methylglycol

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has been tested on curimba semen. The aims of this study were to evaluate four different

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extenders (0.9% NaCl, 5% glucose, BTS and M III) combined with two

62

cryoprotectants (DMSO and methylglycol) as cryosolutions using four motility-

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activation media (Experiment 1) and two combinations of straw volume and thawing

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temperature (Experiment 2) on the cryopreservation of curimba semen. Finally, we

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show that semen cryopreserved in an inexpensive and simple manner produced

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excellent post-thaw motility and fertilization rates (Experiment 3).

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2. Materials and methods

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2.1. Animals and experimental conditions

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Curimba Prochilodus lineatus males were selected from earthen ponds at the

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Fish Culture Unit of the Hydroelectric Company of Minas Gerais (CEMIG) in Itutinga,

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Minas Gerais, Brazil, during the 2005 and 2006 spawning seasons. Males (n = 13, 1.2

73

0.4 kg BW) with detectable running semen under a soft abdominal pressure received a

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single intramuscular dose of carp pituitary extract (5 mg/kg BW). After 8 h, the

75

urogenital papilla was carefully dried and semen was hand stripped directly into test

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tubes. Immediately after collection, 5 L of each sample was placed on a slide and

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observed under a light microscope. Any samples showing signs of sperm motility (auto-

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activation) was discarded. In immotile samples, sperm motility was subjectively

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estimated at 400x magnification immediately following addition of 25 L of 0.29%

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Page 4 of 18

NaCl as activation medium (Maria et al., 2006a). Only samples containing at least 80%

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motile cells were used in subsequent studies. During manipulation, semen, extenders

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and cryoprotectants were maintained on ice (15 2 C). Samples were diluted in a

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proportion of 10% semen, 80% extender and 10% cryoprotectant, loaded into straws,

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and frozen in a vapor nitrogen vessel (Cryoporter LN2 dry vapor shipper) at -170 C.

85

Then cryopreserved semen was transported 50 km by car to the Animal Sciences

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Department at the Federal University of Lavras (UFLA), where semen was transferred

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to liquid nitrogen for storage within 20 to 24 h. Post-thaw motilities were analyzed

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within 1 to 2 weeks. For the fertilization trial, cryopreserved semen was transported

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again by car in the nitrogen vapor vessel from UFLA to Hydrobiology and Fish Culture

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Station of Furnas, So Jos da Barra, Minas Gerais, (approximately 215 km). After

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arriving in Furnas, semen was immediately thawed and used to fertilize fresh eggs.

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2.2. Experiment 1: Extenders, cryoprotectants and activation media

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Four semen extenders were prepared using deionized water: 0.9% NaCl (Maria

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et al., 2006b), 5% glucose (Carolsfeld et al., 2003), 5% BTS (Beltsville Thawing

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Solution - Minitub: 0.02% gentamycin sulfate, 4.0% glucose, 0.63% sodium citrate,

97

0.13% EDTA, 0.13% NaHCO3, 0.08% KCl; Maria et al., 2006a) and 6% M III

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(Merck III- Minitub, 0.03% gentamycin sulfate, 5.34% glucose, 0.18% sodium

99

citrate, 0.18% EDTA, 0.25% NaHCO3, Maria et al., 2006a). All extenders were adjusted

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to a pH of 7.6. DMSO ((CH3)2SO) and methylglycol (CH3O(CH2)2OH) were used as

101

cryoprotectants. Semen (n = 4 males) was added to each of eight cryosolutions,

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comprising combinations of four extenders and two cryoprotectants, and were

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immediately aspirated into 0.5-mL straws (n = 3 straws/cryosolution/male), and frozen

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Page 5 of 18

in nitrogen vapor as described above. Straws were thawed in a 60 C water bath for 8 s

105

(Maria et al., 2006a). Sperm motility was immediately estimated after dilution of 5 L

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of semen into 25 L of each of the four activation media (distilled water, 0.15% NaCl,

107

0.29% NaCl and 1% NaHCO3; Cruz, 2001).

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2.3. Experiment 2: Straw volume and thawing temperature

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Semen samples (n = 4 males) were collected and diluted into 5% glucose and

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methylglycol. Samples were then aspirated into 0.5-mL straws (n = 6 straws/male) and

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4.0-mL straws (n = 4 straws of pooled semen of the same males) and frozen. Three 0.5-

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mL straws were thawed in a water bath at 60 C for 8 s, and the remaining three straws

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were thawed at 30 C for 16 s. The 4.0-mL straws were thawed at 60 C for 24 s only,

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because of the small volume of semen. As there were no replicates of 4.0-mL straws

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with pooled semen of different males, these data were not analyzed statistically. Post-

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thaw sperm motility was activated with 0.29% NaCl and scored as described.

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2.4. Experiment 3: Fertilization rate of cryopreserved semen

Semen samples (n = 5 males) were diluted in 5% glucose and methylglycol,

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aspirated into 0.5-mL straws, frozen in nitrogen vapor and then stored in liquid nitrogen

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at UFLA. After 2 to 4 weeks, straws were placed back into the nitrogen vapor vessel,

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transported to Furnas and immediately thawed at 60 C for 8 s and used for fertilization.

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To harvest oocytes, females (n = 7; 2.1 0.7 kg BW) received two injections (0.5 and 5

125

mg/kg BW) of carp pituitary extract at 12 h intervals, and eggs were hand-stripped 5 h

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after the second injection. Eggs remained at room temperature (20-23 C) for a

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maximum of 1 h. Five aliquots of 0.5 g eggs from each female were fertilized with

Page 6 of 18

semen from one straw for each male (1 aliquot of eggs = 1 straw). Fertilization was

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initiated by addition of 1 mL tank water, and mixed for 1 min. Subsequently, 10 mL

130

tank water was added, and samples mixed for another 2 min. Finally eggs were

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transferred to a PVC basket, 10 cm in diameter, with a 0.5 mm mesh bottom (Viveiros

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et al., 2000), and incubated in a flow-through system at 26 C. The number of fertilized

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eggs, as percentage of total eggs was determined 16 h after addition of sperm samples.

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2.5. Statistical analysis

All data are expressed as mean standard deviation. Statistical analyses were

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carried out using SISVAR (Ferreira, 1999). Sperm motility and fertilization rate were

138

tested for normal distribution using the univariate procedure. If data did not fit the

139

normal distribution, an arcsin transformation was used. Data were tested for significant

140

differences by ANOVA, followed by Scott-Knott test when necessary.

141
3. Results

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3.1. Semen samples

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Fresh semen samples were collected from 13 males. These samples contained a

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mean concentration of 16.8 x 109 12.9 x 109 spermatozoa per mL, a mean volume of

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2.1 0.7 mL, and a mean motility of 100%.

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3.2. Experiment 1: Extenders, cryoprotectants and activation media

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A significant interaction among extenders, cryoprotectants and activation media

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was observed (Table 1). When methylglycol was tested as cryoprotectant, greatest post-

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thaw motilities (88-95%) were observed in semen cryopreserved in glucose, BTS or

Page 7 of 18

M III, and activated with any of the four activation media. However, when DMSO

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was tested as cryoprotectant, greatest post-thaw sperm motilities (80-89%) were

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observed only in semen cryopreserved in M III and activated with any of the four

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activation media, and in semen cryopreserved in BTS and activated with 0.15%

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NaCl, 0.29% NaCl, or 1% NaHCO3. All the other extender-cryoprotectant combinations

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yielded poor post-thaw sperm motility.

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3.3. Experiment 2: Straw volume and thawing temperature

There was no effect of thawing temperature on post-thaw sperm motility (Table

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2). Cryopreserved semen thawed at 30 or 60 C yielded 86 to 87% motility. Semen

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diluted in glucose-methylglycol and cryopreserved in 0.5-mL straws yielded 86%

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motility, while those cryopreserved in 4.0-mL straws yielded 95% motility.

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3.4. Experiment 3: Fertilization rate of cryopreserved semen

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Very acceptable fertilization rates among eggs fertilized with cryopreserved

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semen were observed (Table 3). Cryopreserved semen yielded mean fertilization rates

168

between 47% and 83%, depending on the female from which the eggs were collected.

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As mean sperm concentration of these five males was 5.4 x 109 spermatozoa per mL

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and 0.5 g of eggs contains about 583 eggs, the mean sperm: egg ratio estimated in this

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experiment was 5 x 105 spermatozoa per egg.

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4. Discussion

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This is the first study to test the use of the boar semen extender M III as

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semen extender and methylglycol as cryoprotectant of curimba sperm. Post-thaw semen

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quality was evaluated on the basis of sperm motility, and this was confirmed by

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fertilizing fresh eggs.

Four different solutions were tested as extenders of curimba semen: 0.9% NaCl,

179

5% glucose, BTS and M III. NaCl was the only extender that yielded poor

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motilities (less than 26%), regardless of the cryoprotectant and activation medium used.

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However, in bryconid species, 0.9% NaCl solution has been reported to be effective as a

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semen extender. In piracanjuba Brycon orbignyanus, post-thaw motility was poor (8%)

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when 0.9% NaCl was combined with DMSO and egg yolk, and acceptable (66%) when

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combined with methylglycol and egg yolk (Maria et al, 2006b). In pirapitinga Brycon

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nattereri, the combination of 0.9% NaCl with either DMSO or methylglycol produced

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post-thaw motilities above 51% (Oliveira et al, 2007).

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When glucose-based solutions (5% glucose, BTS and M III) were tested in

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combination with DMSO, post-thaw motilities exceeding 80% were observed only

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when samples were cryopreserved in M III (for all activation media) and BTS (for

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all activation media except for distilled water). Although the combination of glucose

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and DMSO was not the best cryosolution tested, this combination, supplemented with 5

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to 10% egg yolk has been extensively used as a semen cryosolution for tropical fish

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species (Cruz, 2001; Carolsfeld et al., 2003). When these glucose-based solutions were

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tested in combination with methylglycol, acceptable post-thaw motilities (above 88%)

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were observed for all samples, regardless of the activation medium used. Methylglycol,

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also known as 2-methoxyethanol or ethylene glycol monomethyl ether, is derived from

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methanol (CH3OH) and ethene oxide (CH2OCH2). Methylglycol is relatively nontoxic,

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and has been used as cryoprotectant for bovine embryos (Takagi et al., 1993), and has

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been previously tested for use as a fish sperm cryoprotectant (Maria et al, 2006a).

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10

Glucose provides a simple, inexpensive, and stable extender for curimba semen, and can

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be used in on-farm conditions without the need for laboratory equipment, because it can

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be purchased as a sterile, commercially available solution. During our fertilization trials,

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semen cryopreserved in a combination of glucose and methylglycol produced a mean

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fertilization rate of 74%. However, greater variation was observed among females (47%

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when eggs from female 7 were used and 83% when eggs from female 1 were used),

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underscoring the importance of egg quality for fertilization trials. Although the capacity

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of fertilized eggs to hatch is an important variable for evaluating fertility, the use of

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excess spermatozoa can complicate estimation of the quality of cryopreserved

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spermatozoa (Viveiros et al., 2000). In the present study, a ratio of 5.4 x 105 post-thaw

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spermatozoa per egg was used to ensure accurate estimation of fertilization efficiency.

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Further experiments are needed to evaluate the most effective number of post-thaw

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spermatozoa per egg for this species.

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When the four activation media (distilled water, 0.15% NaCl, 0.29% NaCl and

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1% NaHCO3) were tested with glucose-based solutions combined with methylglycol,

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post-thaw motilities were induced without significant differences. For the sake of

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creating a practical and standardized protocol in our lab with other fish species (Maria et

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al., 2006a; Oliveira et al., 2007), 0.29% NaCl was used as the activation medium in

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Experiment 2. During the fertilization trial (Experiment 3), however, tank water was

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used to induce sperm motility to reproduce on-farm working conditions. Furthermore,

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fertilization process is more effective when tank water is used to induce sperm motility,

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compared with use of 0.29% NaCl, or 1% NaHCO3 as activation medium (unpublished

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data).

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11

The 0.5-mL straws are commonly used by the bovine semen cryopreservation

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industry, and are available at lesser relative cost. Therefore, in most of the experiments,

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0.5-mL straws are used, because many replicates are needed and semen volume is

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frequently small. However, the use of 0.5-mL straws during the artificial reproduction

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in fish can be time consuming, as many straws are needed to fertilize eggs from a single

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female. Post-thaw sperm motilities for larger straws were evaluated, and these were

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apparently similar whether samples were cryopreserved in 0.5 or 4.0-mL straws,

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although statistical analysis among the data collected from 4.0-mL straws were not

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conducted. Similarly, no difference in sperm motility was reported for 0.5 and 4.0-mL

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straws from yam Brycon amazonicus (Velasco-Santamara et al., 2006) or on hatching

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rate of matrinx Brycon cephalus (Ninhaus-Silveira et al., 2006). Unfortunately,

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sufficient semen was not available to load 4.0-mL straws in duplicate for testing a range

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of thawing temperatures. According to studies of other fish species (Ninhaus-Silveira et

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al., 2006; Velasco-Santamara et al., 2006), it is possible that curimba semen

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cryopreserved in 4.0-mL straws would yield similar sperm motilities when thawed at

239

30 or 60 C, as was observed for 0.5-mL straws. Furthermore, as post-thaw motilities

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were apparently similar between semen cryopreservation in 0.5 or 4.0-mL straws,

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similar fertilization rates would be expected. During the next curimba spawning season,

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we will cryopreserve curimba semen in 4.0-mL straws, and test fertilization efficiency

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on a commercial scale.

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5. Conclusions

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Curimba semen can be successfully cryopreserved in a cryosolution containing

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5% glucose and methylglycol. Use of glucose as extender is recommended because it is

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12

widely available as a sterile commercial solution. Diluted semen can be loaded into 0.5-

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mL straws, cryopreserved in nitrogen vapor, and thawed at 60 C. Post-thaw semen

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produces acceptable motility and fertility and thus can be used in commercial hatcheries

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to facilitate artificial reproduction as only females have to be manipulated, injected and

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stripped.

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Acknowledgments
This research was supported by CNPq (n 471975/2004-4) and CAPES (MSc

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grant) and was part of a MSc project (L.H. Orfo). We thank Z.A. Isa, T.B. Amaral

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and J.F.A. Koch for assistance on experiments, CEMIG and Furnas for providing the

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broodfish and Minitub for providing boar semen extenders.

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References

260

Carolsfeld, J., Godinho, H.P., Zaniboni Filho, E., Harvey, B.J., 2003. Cryopreservation

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of sperm in Brazilian migratory fish conservation. J. Fish Biol. 63, 472-489.

Cruz, V.L.B., 2001. Criopreservao do smen de curimbat (Prochilodus lineatus). (In

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Portuguese, with English abstract). Masters Thesis in Vertebrate Zoology.

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Pontifcia Universidade Catlica de Minas Gerais, Belo Horizonte, MG, Brazil,

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59 pp.

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Ferreira, D.F., 1999. Analysis of variance system (SISVAR). Lavras: Department of

Statistics of UFLA. Version 4.3 (Build 43).

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Maria, A.N., Viveiros, A.T.M., Freitas, R.T.F., Oliveira, A.V., 2006a. Extenders and

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cryoprotectants for cooling and freezing of piracanjuba (Brycon orbignyanus)

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semen, an endangered Brazilian teleost fish. Aquaculture 260, 298-306.

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Maria, A.N., Viveiros, A.T.M., Orfo, L.H., Oliveira, A.V., Moraes, G.F., 2006b.

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Effects of cooling and freezing on sperm motility of the endangered fish

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piracanjuba Brycon orbignyanus (Characiformes, Characidae). Anim. Reprod. 3,

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55-60.

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Miliorini, A.B., 2006. Ativadores e concentraes de metanol e dimetilsulfxido na

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qualidade do smen criopreservado de curimba (Prochilodus lineatus). (In

277
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Portuguese, with English abstract). Masters Thesis in Veterinary Sciences.

Universidade Federal de Lavras, Lavras, MG, Brazil, 99 pp.

Murgas,

L.D.S.,

Miliorini,

A.B.,

Freitas,

R.T.F.,

Pereira,

G.J.M.,

2007.

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Cryopreservation of curimba (Prochilodus lineatus) semen after addition of

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different diluters, activators and cryoprotectants. R. Bras. Zootec. 36, 526-531.

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Ninhaus-Silveira, A., Foresti, F., Silveira, R.V., 2006. Seminal analysis, cryogenic

283

preservation, and fertility in matrinx fish, Brycon cephalus (Gnther, 1869).

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Braz. Arch. Biol. Technol. 49, 651-659.

Oliveira, A.V., Viveiros A.T.M., Maria, A.N., Freitas, R.T.F., Isa, Z.A., 2007. Sucesso

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do resfriamento e congelamento de smen de pirapitinga Brycon nattereri. Arq.

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Bras. Med. Vet. Zootec. 59, 1509-1515.

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Takagi, M., Boediono, A., Saha, S., Suzuki, T., 1993. Survival rate of frozen-thawed

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bovine IVF embryos in relation to exposure time using various cryoprotectants.

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Cryobiology 30, 306-312.

Velasco-Santamara,

Y.M.,

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Medina-Robles

V.M.,

Cruz-Casallas,

E.P.,

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Cryopreservation of yam (Brycon amazonicus) sperm for large scale

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fertilization. Aquaculture 256, 267-271.

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Viveiros, A.T.M., So, N., Komen, J., 2000. Sperm cryopreservation of African catfish

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(Clarias gariepinus): cryoprotectants, freezing rates and sperm: egg dilution

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ratio. Theriogenology 54, 1305-1308.

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Table Revised Editor 19 04 2008

Table 1

The effects of different cryosolutions and activation media on motility (expressed as

percentage; mean SD; n = 4 males) of cryopreserved curimba semen (Experiment 1)

Activation Media
distilled

Cryoprotectants
Extenders

NaCl 0.15%
(10%)

NaCl 0.29%

water
18 12 C a

16 13 B a

18 12 C a

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NaCl 0.9%

NaHCO3

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Cryosolutions

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1%

24 9 C a

Methyl-

95 4 A a

93 6 A a

95 5 A a

94 4 A a

BTS 5%

glycol

94 4 A a

93 3 A a

95 4 A a

92 4 A a

91 5 A a

91 5 A a

91 5 A a

88 7 A a

NaCl 0.9%

1 3 Cb

5 7 Cb

3 4 Db

26 7 C a

Glucose 5%

10 11 C c

25 16 B b

36 18 B a

50 17 B a

38 22 B b

80 10 A a

87 10 A a

88 6 A a

84 10 A a

86 8 A a

88 8 A a

89 6 A a

M III 6%

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Glucose 5%

DMSO

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p

te

M III 6%

BTS 5%

a-c; A-D

are different (Scott Knott; P<0.05)

5% BTS (%): 4.0 glucose, 0.63 sodium citrate, 0.13 EDTA, 0.13 NaHCO3, 0.08 KCl, 0.02

gentamycin sulfate, 95 deionized water

6% M III (%): 5.34 glucose, 0.18 sodium citrate, 0.18 EDTA, 0.25 NaHCO3, 0.03 gentamycin

10

Means followed by different superscripts (lowercase for lines and uppercase for columns)

sulfate, 94 deionized water

Page 16 of 18

Table 2

12

Post-thaw sperm motility (expressed as percentage; mean SD) of curimba (n = 4

13

males) semen cryopreserved in 0.5 and 4.0-mL straws and thawed in a water bath at 30

14

or 60 C (Experiment 2).
Straw volume (mL)

30

87 8

60

86 8

---

95 0

an

15

4.02

cr

0.51

us

Thawing temperature (C)

ip
t

11

Mean of 12 straws (three straws x four males); means are not different (ANOVA, p>0.05)

17

Mean of four straws filled with pooled semen of the same four males; data excluded from

18

statistical analysis

16

Ac
ce
p

te

19

Page 17 of 18

Table 3

21

Fertilization rate (expressed as percentage of fertilized eggs; mean SD) produced with

22

curimba semen cryopreserved in 5% glucose and methylglycol (Experiment 3).

Male ID

Fertilization

Female
1

(mean per

cr

ID

ip
t

20

us

female; %)

84

85

84

80

81

83 2

90

77

81

75

70

79 7

82

84

82

80

83

82 1

81

77

72

81

79

78 4

82

83

81

78

75

80 3

76

72

65

74

61

70 6

53

38

40

48

56

47 8 *

72 16

74 12

72 10

74 13

(mean per

d
te

Ac
ce
p

Fertilization

an

78 12

74 16

male; %)

23
24
25

* Mean lower compared to the others (Scott Knott; P<0.05)

Page 18 of 18